query: what is known about an mRNA vaccine for the SARS-CoV-2 virus?

positive: Fighting COVID-19: a quick review of diagnoses, therapies, and vaccines The COVID-19 pandemic caused by a novel coronavirus, SARS-CoV-2, has infected more than 4.9 million individuals and resulted in over 300,000 deaths globally. The rapid spread of the virus and the precipitously increasing numbers of cases necessitate the urgent development of accurate diagnostic methods, effective treatments, and vaccines. Here, we review the progress of developing diagnostic methods, therapies, and vaccines for SARS-CoV-2 with a focus on current clinical trials and their challenges. For diagnosis, nucleic acid amplification tests remain the mainstay diagnostics for laboratory confirmation of SARS-CoV-2 infection, while serological antibody tests are used to aid contact tracing, epidemiological, and vaccine evaluation studies. Viral isolation is not recommended for routine diagnostic procedures due to safety concerns. Currently, no single effective drug or specific vaccine is available against SARS-CoV-2. Some candidate drugs targeting different levels and stages of human responses against COVID-19 such as cell membrane fusion, RNA-dependent RNA polymerase, viral protease inhibitor, interleukin 6 blocker, and convalescent plasma may improve the clinical outcomes of critical COVID-19 patients. Other supportive care measures for critical patients are still necessary. Advances in genetic sequencing and other technological developments have sped up the establishment of a variety of vaccine platforms. Accordingly, numerous vaccines are under development. Vaccine candidates against SARS-CoV-2 are mainly based upon the viral spike protein due to its vital role in viral infectivity, and most of these candidates have recently moved into clinical trials. Before the efficacy of such vaccines in humans is demonstrated, strong international coordination and collaboration among studies, pharmaceutical companies, regulators, and governments are needed to limit further damage due the emerging SARS-CoV-2 virus.

positive: Towards effective COVID-19 vaccines: Updates, perspectives and challenges (Review) In the current context of the pandemic triggered by SARS-COV-2, the immunization of the population through vaccination is recognized as a public health priority. In the case of SARS-COV-2, the genetic sequencing was done quickly, in one month. Since then, worldwide research has focused on obtaining a vaccine. This has a major economic impact because new technological platforms and advanced genetic engineering procedures are required to obtain a COVID-19 vaccine. The most difficult scientific challenge for this future vaccine obtained in the laboratory is the proof of clinical safety and efficacy. The biggest challenge of manufacturing is the construction and validation of production platforms capable of making the vaccine on a large scale.

positive: Cooperation of an RNA packaging signal and a viral envelope protein in coronavirus RNA packaging. Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA packaging signal is necessary and sufficient for packaging RNA into MHV particles. The present study tested the possibility that the selective M protein-mRNA 1 interaction is due to the packaging signal in mRNA 1. Regardless of the presence or absence of the packaging signal, N protein bound to MHV defective interfering RNAs and intracellularly expressed non-MHV RNA transcripts to form ribonucleoprotein complexes; M protein, however, interacted selectively with RNAs containing the packaging signal. Moreover, only the RNA that interacted selectively with M protein was efficiently packaged into MHV particles. Thus, it was the packaging signal that mediated the selective interaction between M protein and viral RNA to drive the specific packaging of RNA into virus particles. This is the first example for any RNA virus in which a viral envelope protein and a known viral RNA packaging signal have been shown to determine the specificity and selectivity of RNA packaging into virions.

positive: The Challenges of Vaccine Development against a New Virus during a Pandemic The rapid emergence of a highly pathogenic, readily transmissible coronavirus has resulted in a global pandemic, affecting millions and destabilizing economies. This catastrophe triggered a clarion call for the immediate deployment of a protective vaccine. We describe the unique challenges of developing a vaccine against SARS-CoV-2 in a pandemic setting.

positive: SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

positive: The vaccine journey for COVID-19: a comprehensive systematic review of current clinical trials in humans. INTRODUCTION Since December 2019, there has been an outbreak of a novel beta-coronavirus (SARS-CoV-2) in Wuhan, China. On March the 11th the World Health Organization (WHO) declared COVID-19 as a pandemic, with over 118,000 cases in more than 110 countries around the world. In response to the global coronavirus disease 2019 (COVID-19) emergency, clinical trial research assessing the efficacy and safety of experimental vaccines to prevent COVID-19 are emerging at an unprecedented rate. The aim of this systematic review is to summarize the preliminary experiences and ongoing clinical trials of the major candidates and challenges of the vaccine strategies in humans. EVIDENCE ACQUISITION After a priori protocol registration with PROSPERO (181483), a systematic research of the published literature was conducted on 24 April 2020 using Medline (via PubMed), Embase (via Ovid), and WHO databases. Moreover, to explore the more recent literature we also searched the preprint server medRxiv. Finally, we scrutinized the Cochrane COVID-19 study register and the COVID-19 section of ClinicalTrials.gov database for identifying relevant ongoing clinical trials. Thereafter we selected the articles according to the PRISMA guidelines. Animal or in-vitro experimental studies were excluded. Moreover editorials, commentaries, abstracts, reviews, book chapters, and articles not in English were not included. EVIDENCE SYNTHESIS Our search identified 1359 published papers, 478 pre-print articles and 367 ongoing clinical trials. Finally, only ten ongoing clinical trials met the inclusion criteria. Specifically, seven developed vaccines for the S protein of SARS-CoV-2 and three clinical trials assessed the protective role of BCG vaccine against COVID-19. The first group included phase I/II trials with different types of molecules (DNA or mRNA vaccine, bacterial plasmid or viral vectors), the latter were phase III/IV trials designed on the basis of a heterologous lymphocyte activation by the BCG vaccine. CONCLUSIONS This new disease is pushing the scientific community to develop swiftly a safe and effective vaccine. Notwithstanding the limitations of our analysis, given by the absence of available results, we try to provide a comprehensive view of the ongoing clinical trials in humans. Our analysis reveals a worldwide effort of both scientists and enterprises to achieve one of the most challenging goals of our century.

positive: The COVID-19 pandemic: catching up with the cataclysm Infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the Coronaviridae family and is a positive-sense single-stranded RNA virus originating from Wuhan, China, was declared a global public health emergency on 11 March 2020. SARS-CoV-2 infection in humans is characterized by symptoms such as fever and dyspnea accompanied by infrequent incidence of lymphopenia, gastrointestinal complications such as elevated hepatic aminotransferases, and diarrhea. Originating in bats, the SARS-CoV-2 virus has been transmitted to humans likely via an intermediate host that is yet to be discovered. Owing to the absence of any vaccines or definite anti-viral drugs alongside the greater mobility of people across the globe, international and national efforts in containing and treating SARS-CoV-2 infection are experiencing severe difficulties. In this review, we have provided a picture of SARS-CoV-2 epidemiological characteristics, the clinical symptoms experienced by patients of varying age groups, the molecular virology of SARS-CoV-2, and the treatment regimens currently employed for fighting SARS-CoV-2 infection as well as their outcomes.

positive: Current Status of Epidemiology, Diagnosis, Therapeutics, and Vaccines for Novel Coronavirus Disease 2019 (COVID-19) Coronavirus disease 2019 (COVID-19), which causes serious respiratory illness such as pneumonia and lung failure, was first reported in Wuhan, the capital of Hubei, China. The etiological agent of COVID-19 has been confirmed as a novel coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is most likely originated from zoonotic coronaviruses, like SARS-CoV, which emerged in 2002. Within a few months of the first report, SARS-CoV-2 had spread across China and worldwide, reaching a pandemic level. As COVID-19 has triggered enormous human casualties and serious economic loss posing global threat, an understanding of the ongoing situation and the development of strategies to contain the virus's spread are urgently needed. Currently, various diagnostic kits to test for COVID-19 are available and several repurposing therapeutics for COVID-19 have shown to be clinically effective. In addition, global institutions and companies have begun to develop vaccines for the prevention of COVID-19. Here, we review the current status of epidemiology, diagnosis, treatment, and vaccine development for COVID-19.

positive: Highly pathogenic coronaviruses: thrusting vaccine development in the spotlight Coronaviruses (CoVs) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has caused major public health crises. There have been more than 4,400,000 reported cases of COVID-2019 and more than 300,000 reported deaths to date (16/05/2020). SARS-CoV, MERS-CoV and SARS-CoV-2 have attracted widespread global attention due to their high infectivity and pathogenicity. To date, there is no specific treatment proven effective against these viral infectious diseases. Vaccination is considered one of the most effective strategies to prevent viral infections. Therefore, the development of effective vaccines against highly pathogenic coronaviruses is essential. In this review, we will briefly describe coronavirus vaccine design targets, summarize recent advances in the development of coronavirus vaccines, and highlight current adjuvants for improving the efficacy of coronavirus vaccines.

positive: Will the coronavirus help mRNA and DNA vaccines prove their worth? Biotech companies, pharma firms, universities, and government agencies are all rushing to start clinical trials of newly created gene-based vaccines for COVID-19 Developer;Technology;Phase I clinical trial Moderna and National Institutes of Health;mRNA vaccine;Began in March 2020 CanSino Biologics;Adenoviral vector vaccine;Began in March 2020 BioNTech, Shanghai Fosun Pharmaceutical, and Pfizer;mRNA vaccine;April 2020 Inovio Pharmaceuticals and Wistar Institute;DNA vaccine;April 2020 University of Oxford;Adenoviral vector vaccine;April 2020 CureVac;mRNA vaccine;By early summer Imperial College London;mRNA vaccine;Early summer Arcturus Therapeutics and Duke-NUS Medical School;mRNA vaccine;Second half of 2020 Johnson & Johnson and Biomedical Advanced Research and Development Authority;Adenoviral vector vaccine;By September Altimmune;Adenoviral vector vaccine;Q3 2020 Translate Bio and Sanofi Pasteur;mRNA vaccine;By end of 2020 eTheRNA Immunotherapies;mRNA vaccine;2021 Karolinska Institute;DNA vaccine;2021 University of Pennsylvania and Duke Human Vaccine Institute;mRNA vaccine;Unknown

positive: A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells The outbreak of pneumonia caused by SARS-CoV-2 posed a great threat to global human health, which urgently requires us to understand comprehensively the mechanism of SARS-CoV-2 infection. Angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for SARS-CoV-2, distribution of which may indicate the risk of different human organs vulnerable to SARS-CoV-2 infection. Previous studies investigating the distribution of ACE2 mRNA in human tissues only involved a limited size of the samples and a lack of determination for ACE2 protein. Given the heterogeneity among humans, the datasets covering more tissues with a larger size of samples should be analyzed. Indeed, ACE2 is a membrane and secreted protein, while the expression of ACE2 in blood and common blood cells remains unknown. Herein, the proteomic data in HIPED and the antibody-based immunochemistry result in HPA were collected to analyze the distribution of ACE2 protein in human tissues. The bulk RNA-seq profiles from three separate public datasets including HPA tissue Atlas, GTEx, and FANTOM5 CAGE were also obtained to determine the expression of ACE2 in human tissues. Moreover, the abundance of ACE2 in human blood and blood cells was determined by analyzing the data in the PeptideAtlas and the HPA Blood Atlas. We found that the mRNA expression cannot reflect the abundance of ACE2 factor due to the strong differences between mRNA and protein quantities of ACE2 within and across tissues. Our results suggested that ACE2 protein is mainly expressed in the small intestine, kidney, gallbladder, and testis, while the abundance of which in brain-associated tissues and blood common cells is low. HIPED revealed enrichment of ACE2 protein in the placenta and ovary despite a low mRNA level. Further, human secretome shows that the average concentration of ACE2 protein in the plasma of males is higher than those in females. Our research will be beneficial for understanding the transmission routes and sex-based differences in susceptibility of SARS-CoV-2 infection.

positive: mRNA Vaccines: Possible Tools to Combat SARS-CoV-2

positive: Questions remain following first COVID-19 vaccine results

positive: Minus-strand copies of replicating coronavirus mRNAs contain antileaders. The 5' leader sequence on mRNAs of the porcine transmissible gastroenteritis coronavirus was determined and found to be 90 nucleotides in length. An oligodeoxynucleotide with a sequence from within the leader was used as a probe in Northern analysis on RNA from infected cells, and an antileader (a minus-strand copy of the leader sequence) was shown to be present on all mRNA minus-strand species. RNase protection analysis showed the antileader to be approximately the same length as the leader. The kinetics of antileader appearance was the same as that for the appearance of minus-strand RNA species. This, along with a demonstration that viral mRNAs become packaged, gives further support to the idea that coronavirus mRNAs can undergo replication via subgenomic mRNA-length replicative intermediates, and that input mRNAs from infecting virions may serve as initial templates for their own replication. In this sense, then, coronaviruses behave in part like RNA viruses with segmented genomes.

positive: COVID-19 vaccines: breaking record times to first-in-human trials The twenty-first century has come with a new era in vaccinology, in which recombinant genetic technology has contributed to setting an unprecedented fast pace in vaccine development, clearly demonstrated during the recent COVID-19 pandemic.

positive: [RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells]. SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.

positive: An Evidence Based Perspective on mRNA-SARS-CoV-2 Vaccine Development The first outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in Wuhan, Hubei Province, China, in late 2019. The subsequent COVID-19 pandemic rapidly affected the health and economy of the world. The global approach to the pandemic was to isolate populations to reduce the spread of this deadly virus while vaccines began to be developed. In March 2020, the first phase I clinical trial of a novel lipid nanoparticle (LNP)-encapsulated mRNA-based vaccine, mRNA-1273, which encodes the spike protein (S protein) of SARS-CoV-2, began in the United States (US). The production of mRNA-based vaccines is a promising recent development in the production of vaccines. However, there remain significant challenges in the development and testing of vaccines as rapidly as possible to control COVID-19, which requires international collaboration. This review aims to describe the background to the rationale for the development of mRNA-based SARS-CoV-2 vaccines and the current status of the mRNA-1273 vaccine.

positive: SARS-CoV-2 Vaccines: Status Report SARS-CoV-2, the causal agent of COVID-19, first emerged in late 2019 in China. It has since infected more than 870,000 individuals and caused more than 43,000 deaths globally. Here, we discuss therapeutic and prophylactic interventions for SARS-CoV-2 with a focus on vaccine development and its challenges. Vaccines are being rapidly developed but will likely come too late to affect the first wave of a potential pandemic. Nevertheless, critical lessons can be learned for the development of vaccines against rapidly emerging viruses. Importantly, SARS-CoV-2 vaccines will be essential to reducing morbidity and mortality if the virus establishes itself in the population.

positive: Emerging Prevention and Treatment Strategies to Control COVID-19 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has now become a serious global threat after inflicting more than 8 million infections and 425,000 deaths in less than 6 months. Currently, no definitive treatment or prevention therapy exists for COVID-19. The unprecedented rise of this pandemic has rapidly fueled research efforts to discover and develop new vaccines and treatment strategies against this novel coronavirus. While hundreds of vaccines/therapeutics are still in the preclinical or early stage of clinical development, a few of them have shown promising results in controlling the infection. Here, in this review, we discuss the promising vaccines and treatment options for COVID-19, their challenges, and potential alternative strategies.

positive: From SARS-CoV to SARS-CoV-2: safety and broad-spectrum are important for coronavirus vaccine development The global pandemic of COVID-19 caused by SARS-CoV-2 (also known as 2019-nCoV and HCoV-19) has posed serious threats to public health and economic stability worldwide, thus calling for development of vaccines against SARS-CoV-2 and other emerging and reemerging coronaviruses. Since SARS-CoV-2 and SARS-CoV have high similarity of their genomic sequences and share the same cellular receptor (ACE2), it is essential to learn the lessons and experiences from the development of SARS-CoV vaccines for the development of SARS-CoV-2 vaccines. In this review, we summarized the current knowledge on the advantages and disadvantages of the SARS-CoV vaccine candidates and prospected the strategies for the development of safe, effective and broad-spectrum coronavirus vaccines for prevention of infection by currently circulating SARS-CoV-2 and other emerging and reemerging coronaviruses that may cause future epidemics or pandemics.

positive: Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo SARS-CoV-2 has rapidly become a pandemic worldwide; therefore, an effective vaccine is urgently needed. Recently, messenger RNAs (mRNAs) have emerged as a promising platform for vaccination. Here, we systematically investigated the untranslated regions (UTRs) of mRNAs in order to enhance protein production. Through a comprehensive analysis of endogenous gene expression and de novo design of UTRs, we identified the optimal combination of 5’ and 3’ UTR, termed as NASAR, which was five to ten-fold more efficient than the tested endogenous UTRs. More importantly, NASAR mRNAs delivered by lipid-derived nanoparticles showed dramatic expression of potential SARS-CoV-2 antigens both in vitro and in vivo. These NASAR mRNAs merit further development as alternative SARS-CoV-2 vaccines.

positive: Phase 1/2 Study to Describe the Safety and Immunogenicity of a COVID-19 RNA Vaccine Candidate (BNT162b1) in Adults 18 to 55 Years of Age: Interim Report Abstract In March 2020, the WHO declared a pandemic of coronavirus disease 2019 (COVID-19), due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With >8.8 million cases and >450,000 deaths reported globally, a vaccine is urgently needed. We report the available safety, tolerability, and immunogenicity data from an ongoing placebo-controlled, observer-blinded dose escalation study among healthy adults, 18-55 years of age, randomized to receive 2 doses, separated by 21 days, of 10 g, 30 g, or 100 g of BNT162b1, a lipid nanoparticle-formulated, nucleoside-modified, mRNA vaccine that encodes trimerized SARS-CoV-2 spike glycoprotein RBD. Local reactions and systemic events were dose-dependent, generally mild to moderate, and transient. RBD-binding IgG concentrations and SARS-CoV-2 neutralizing titers in sera increased with dose level and after a second dose. Geometric mean neutralizing titers reached 1.8- to 2.8-fold that of a panel of COVID-19 convalescent human sera. These results support further evaluation of this mRNA vaccine candidate.

positive: Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

positive: Preclinical data from SARS-CoV-2 mRNA vaccine

positive: [Progress and challenge of vaccine development against 2019 novel coronavirus (2019-nCoV)] The outbreak of 2019 novel coronavirus (2019-nCoV) infection poses a serious threat to global public health. Vaccination is an effective way to prevent the epidemic of the virus. 2019-nCoV along with severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) belong to the same ß-genus of coronavirus family. Base on the previous experience and the technical platform of developing SARS-CoV and MERS-CoV vaccines, scientists from all over the world are working hard and quickly on the related fields. There are substantial progress in these fields including the characterizing the 2019-nCoV virus, identification of candidate antigens and epitopes, establishment of animal models, characterizing the immune responses, and the design of vaccines. The development of 2019-nCoV vaccines cover all types: inactivated virus vaccine, recombinant protein vaccine, viral vector-based vaccine, mRNA vaccine, and DNA vaccine, et al. As of March 2020, two 2019-nCoV vaccines have entered phase I clinical trials. One is named as Ad5-nCoV developed by the Chinese Institute of Biotechnology of the Academy of Military Medical Sciences and Tianjin Cansino Biotechnology Inc. Ad5-nCoV is based on the replication-defective adenovirus type 5 as the vector to express 2019-nCoV spike protein. The another vaccine is mRNA-1273 developed by the National Institute of Allergy and Infectious Diseases and Moderna, Inc.. RNA-1273 is an mRNA vaccine expressing 2019-nCoV spike protein. Although the rapid development of 2019-nCoV vaccine, it still faces many challenges with unknown knowledge, including the antigenic characteristics of the 2019-nCoV, the influence of antigenic variation, the protective immune response of host, the protection of the elderly population, and the downstream manufacturing process of the new vaccine. The safety and efficacy of vaccines are the first priority for vaccine development and should be carefully evaluated.

positive: Vaccine development and therapeutic design for 2019‐nCoV/SARS‐CoV‐2: Challenges and chances The ongoing outbreak of the recently emerged 2019 novel coronavirus (nCoV), which has seriously threatened global health security, is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) with high morbidity and mortality. Despite the burden of the disease worldwide, still, no licensed vaccine or any specific drug against 2019‐nCoV is available. Data from several countries show that few repurposed drugs using existing antiviral drugs have not (so far) been satisfactory and more recently were proven to be even highly toxic. These findings underline an urgent need for preventative and therapeutic interventions designed to target specific aspects of 2019‐nCoV. Again the major factor in this urgency is that the process of data acquisition by physical experiment is time‐consuming and expensive to obtain. Scientific simulations and more in‐depth data analysis permit to validate or refute drug repurposing opportunities predicted via target similarity profiling to speed up the development of a new more effective anti‐2019‐nCoV therapy especially where in vitro and/or in vivo data are not yet available. In addition, several research programs are being developed, aiming at the exploration of vaccines to prevent and treat the 2019‐nCoV. Computational‐based technology has given us the tools to explore and identify potentially effective drug and/or vaccine candidates which can effectively shorten the time and reduce the operating cost. The aim of the present review is to address the available information on molecular determinants in disease pathobiology modules and define the computational approaches employed in systematic drug repositioning and vaccine development settings for SARS‐CoV‐2.

positive: Dynamics of B-cell repertoires and emergence of cross-reactive responses in COVID-19 patients with different disease severity COVID-19 patients show varying severity of the disease ranging from asymptomatic to requiring intensive care. Although a number of monoclonal antibodies against SARS-CoV-2 have been identified, we still lack an understanding of the overall landscape of B-cell receptor (BCR) repertoires in COVID-19 patients. Here, we used high-throughput sequencing of BCR repertoires collected over multiple time points during an infection to characterize statistical and dynamical signatures of the B-cell response to SARS-CoV-2 in 19 patients with different disease severities. Based on principled statistical approaches, we determined differential sequence features of BCRs associated with different disease severity. We identified 34 significantly expanded rare clonal lineages shared among patients as candidates for a specific response to SARS-CoV-2. Moreover, we identified natural emergence of a BCR with cross-reactivity to SARS-CoV and SARS-CoV-2 in a number of patients. Overall, our results provide important insights for development of rational therapies and vaccines against COVID-19.

positive: Vaccination strategies to combat novel corona virus SARS-CoV-2 The 2019-novel coronavirus disease (COVID-19) is caused by SARS-CoV-2 is transmitted from human to human has recently reported in China. Now COVID-19 has been spread all over the world and declared epidemics by WHO. It has caused a Public Health Emergency of International Concern. The elderly and people with underlying diseases are susceptible to infection and prone to serious outcomes, which may be associated with acute respiratory distress syndrome (ARDS) and cytokine storm. Due to the rapid increase of SARS-CoV-2 infections and unavailability of antiviral therapeutic agents, developing an effective SAR-CoV-2 vaccine is urgently required. SARS-CoV-2 which is genetically similar to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) is an enveloped, single and positive-stranded RNA virus with a genome comprising 29,891 nucleotides, which encode the 12 putative open reading frames responsible for the synthesis of viral structural and nonstructural proteins which are very similar to SARS-CoV and MERS-CoV proteins. In this review we have summarized various vaccine candidates i.e., nucleotide, subunit and vector based as well as attenuated and inactivated forms, which have already been demonstrated their prophylactic efficacy against MERS-CoV and SARS-CoV, so these candidates could be used as a potential tool for the development of a safe and effective vaccine against SARS-CoV-2.

positive: Coronavirus vaccine trials have delivered their first results - but their promise is still unclear.

positive: Immunoinformatics-guided designing of epitope-based subunit vaccines against the SARS Coronavirus-2 (SARS-CoV-2) SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus. So far, the pandemic has caused the death of hundreds of thousands of people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. Therefore, this study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation, three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against the SARS-CoV-2. Thereafter, the molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive preventative measure against this lethal disease.

positive: Sanofi, BARDA team for coronavirus vaccine Sanofi Pasteur is working with the US Biomedical Advanced Research and Development Authority (BARDA) to develop a vaccine against the novel coronavirus, known as severe acute respiratory syndrome-CoV-2, which as of Feb 20 had infected more than 75,000 people and killed more than 2,100 Sanofi will build on work on a vaccine against SARS, another coronavirus that circulated in the early 2000s In December 2019, BARDA awarded the firm a $226 million contract to expand manufacturing capacity for use in a potential flu pandemic Sanofi says the new vaccine will use the same technology

positive: Structural Basis of RNA Cap Modification by SARS-CoV-2 Coronavirus The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5’-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2’-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2’-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.

positive: Balancing Expediency and Scientific Rigor in Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine Development

positive: Immune-mediated approaches against COVID-19 The coronavirus disease-19 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The long incubation period of this new virus, which is mostly asymptomatic yet contagious, is a key reason for its rapid spread across the world. Currently, there is no worldwide-approved treatment for COVID-19. Therefore, the clinical and scientific communities have joint efforts to reduce the severe impact of the outbreak. Research on previous emerging infectious diseases have created valuable knowledge that is being exploited for drug repurposing and accelerated vaccine development. Nevertheless, it is important to generate knowledge on SARS-CoV-2 mechanisms of infection and its impact on host immunity, to guide the design of COVID-19 specific therapeutics and vaccines suitable for mass immunization. Nanoscale delivery systems are expected to play a paramount role in the success of these prophylactic and therapeutic approaches. This Review provides an overview of SARS-CoV-2 pathogenesis and examines immune-mediated approaches currently explored for COVID-19 treatments, with an emphasis on nanotechnological tools.

positive: Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

positive: Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.

positive: A review on Promising vaccine development progress for COVID-19 disease Abstract The emergence of the strain of coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) that causes corona virus disease 2019 (COVID-19) and its impact on in the world have made imperative progress to develop an effective and safe vaccine. Despite several measures undertaken, the spread of this virus is ongoing. So far, more than 1,560,000 cases and 1000,000 deaths occurred in the world. Efforts have been made to develop vaccines against human coronavirus (CoV) infections such as MERS and SARS. However, currently, no approved vaccine exists for these coronavirus strains. Such Previous research efforts to develop a coronavirus vaccine in the years following the 2003 pandemic have opened the door for the scientist to design a new vaccine for the COVID-19. Both SARS-CoV and SARS-CoV-2 has a high degree of genetic similarity and bind to the same host cell ACE2 receptor. By using different vaccine development platforms including whole virus vaccines, recombinant protein subunit vaccines, and nucleic acid vaccines several candidates displayed efficacy in vitro studies but few progressed to clinical trials. This review provides a brief introduction of the general features of SARS-CoV-2 and discusses the current progress of ongoing advances in designing vaccine development efforts to counter COVID-19.

positive: Progress and Prospects on Vaccine Development against SARS-CoV-2 In December 2019, the outbreak of pneumonia caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a serious pandemic in China and other countries worldwide. So far, more than 460,000 confirmed cases were diagnosed in nearly 190 countries, causing globally over 20,000 deaths. Currently, the epidemic is still spreading and there is no effective means to prevent the infection. Vaccines are proved to be the most effective and economical means to prevent and control infectious diseases. Several countries, companies, and institutions announced their programs and progress on vaccine development against the virus. While most of the vaccines are under design and preparation, there are some that have entered efficacy evaluation in animals and initial clinical trials. This review mainly focused on the progress and our prospects on field of vaccine development against SARS-CoV-2.

positive: Pharmacological and non-pharmacological efforts at prevention, mitigation, and treatment for COVID-19. A global outbreak of the SARS-CoV-2 virus has infected millions of people over a short period of time. The communicability and increased mortality from the SARS-CoV-2 infection mandated the WHO to declare COVID-19 a worldwide pandemic. The virus outbreak has spread when there are no approved vaccines, treatments, or prophylactic therapies available. Researchers from all over the world have prioritized development of vaccines and antivirals. Several vaccine projects have seen successes in preclinical, phase I, and phase II clinical trials using recombinant DNA, mRNA, live attenuated virus, S-protein subunits, virus like particles, and viral vectors. Initial findings from antivirals such as remdesivir, favipiravir, danoprevir or lopinavir with ritonavir are presented. Immunomodulatory molecules such as sarilumab, tocilizumab, janus kinase inhibitors, and hyperimmune convalescent plasma have mixed outcomes from initial clinical findings; however, pending randomized controlled trials will assist national health institutions to make treatment recommendations for COVID-19. Where compassionate use of remdesivir has shown some benefits, therapies such as hydroxychloroquine have proven harmful due to their toxicities. This review discusses pharmacological interventions at play and evidence-based successes and limitations of non-pharmacological therapies such as social distancing, personal protective equipment, and ventilator support associated with the prevention and treatment of COVID-19.

positive: On setting expectations for a SARS-CoV-2 Vaccine The global coronavirus pandemic is unlike any other since 1918. A century of dramatic medical advances has produced a public expectation that the medical field will rapidly provide solutions to restore normalcy. In under 6 months, since SARS-CoV-2 was identified, the massive international effort to develop a SARS-CoV-2 vaccine has generated more than 140 vaccines in different stages of development with 9 already recruiting into clinical trials posted on clinicaltrials.gov. The long-term strategy to handle COVID-19 will almost certainly rely on vaccines. But, what type of protection can we realistically expect to achieve from vaccines and when?

positive: Logistical challenges for potential SARS-CoV-2 vaccine and a call to research institutions, developers and manufacturers

positive: Vaccines for COVID-19: the current state of play There is a strong consensus globally that a COVID-19 vaccine is likely the most effective approach to sustainably controlling the COVID-19 pandemic. An unprecedented research effort and global coordination has resulted in a rapid development of vaccine candidates and initiation of trials. Here, we review vaccine types, and progress with 10 vaccine candidates against SARS-CoV-2 – the virus that causes COVID-19 - currently undergoing early phase human trials. We also consider the many challenges of developing and deploying a new vaccine on a global scale, and recommend caution with respect to our expectations of the timeline that may be ahead.

positive: SARS-CoV-2 and Coronavirus Disease 2019: What We Know So Far In December 2019, a cluster of fatal pneumonia cases presented in Wuhan, China. They were caused by a previously unknown coronavirus. All patients had been associated with the Wuhan Wholefood market, where seafood and live animals are sold. The virus spread rapidly and public health authorities in China initiated a containment effort. However, by that time, travelers had carried the virus to many countries, sparking memories of the previous coronavirus epidemics, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and causing widespread media attention and panic. Based on clinical criteria and available serological and molecular information, the new disease was called coronavirus disease of 2019 (COVID-19), and the novel coronavirus was called SARS Coronavirus-2 (SARS-CoV-2), emphasizing its close relationship to the 2002 SARS virus (SARS-CoV). The scientific community raced to uncover the origin of the virus, understand the pathogenesis of the disease, develop treatment options, define the risk factors, and work on vaccine development. Here we present a summary of current knowledge regarding the novel coronavirus and the disease it causes.

positive: Vaccine Technologies: From Whole Organisms to Rationally Designed Protein Assemblies Vaccines have been the single most significant advancement in public health, preventing morbidity and mortality in millions of people annually. Vaccine development has traditionally focused on whole organism vaccines, either live attenuated or inactivated vaccines. While successful for many different infectious diseases whole organisms are expensive to produce, require culture of the infectious agent, and have the potential to cause vaccine associated disease in hosts. With advancing technology and a desire to develop safe, cost effective vaccine candidates, the field began to focus on the development of recombinantly expressed antigens known as subunit vaccines. While more tolerable, subunit vaccines tend to be less immunogenic. Attempts have been made to increase immunogenicity with the addition adjuvants, either immunostimulatory molecules or an antigen delivery system that increases immune responses to vaccines. An area of extreme interest has been the application of nanotechnology to vaccine development, which allows for antigens to be expressed on a particulate delivery system. One of the most exciting examples of nanovaccines are rationally designed protein nanoparticles. These nanoparticles use some of the basic tenants of structural biology, biophysical chemistry, and vaccinology to develop protective, safe, and easily manufactured vaccines. Rationally developed nanoparticle vaccines are one of the most promising candidates for the future of vaccine development.

positive: An overview on COVID-19: reality and expectation Recently, severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), commonly known as coronavirus disease-2019 (COVID-19) has rapidly spread across China and around the world. By the declaration of WHO, COVID-19 outbreak considered as a public health problem of international concern. The aim of this study is to provide a comprehensive view on COVID-19 and the future expectations to control virus progression. Patients with liver disease, diabetes, high blood pressure, and obesity are more susceptible to the incidence of COVID-19 infection. So, there is a rapid need for disease diagnosis, vaccine development, and drug discovery to detect, prevent, and treat this sudden and lethal virus. Real-time polymerase chain reaction (RT-PCR) is considered as a rapid, accurate, and specific tool for disease diagnosis. Under this emergency situation that the world facing against COVID-19, there are about 15 potential vaccine candidates tested globally based on messenger RNA, DNA-based, nanoparticle, synthetic, and modified virus-like particle. Certain drugs that are clinically approved for other diseases were tested against COVID-19 as chloroquine, hydroxychloroquine, ivermectin, favipiravir, ribavirin, and remdesivir. Convalescent plasma transfusion and traditional herbal medicine were also taken into consideration. Due to the absence of effective treatment or vaccines against COVID-19 so far, the precautionary measures according to WHO’s strategic objectives are the only way to confront this crisis. Governments should adopt national medical care programs to reduce the risk of exposure to any future viral outbreaks especially to patients with pre-existing medical conditions.

positive: An insight into the epitope-based peptide vaccine design strategy and studies against COVID-19 SARS-CoV-2 is a new member of the coronavirus family and caused the pandemic of coronavirus disease 2019 (COVID-19) in 2020. It is crucial to design and produce an effective vaccine for the prevention of rapid transmission and possible deaths wcaused by the disease. Although intensive work and research are being carried out all over the world to develop a vaccine, an effective and approved formulation that can prevent the infection and limit the outbreak has not been announced yet. Among all types of vaccines, epitope-based peptide vaccines outshine with their low-cost production, easy modification in the structure, and safety. In this review, vaccine studies against COVID-19 have been summarized and detailed information about the epitope-based peptide vaccines against COVID-19 has been provided. We have not only compared the peptide vaccine with other types of vaccines but also presented comprehensive literature information about development steps for an effective and protective formulation to give an insight into on-going peptide vaccine studies against SARS-CoV-2.

positive: Covid-19: What do we know so far about a vaccine?

positive: The Current and Future State of Vaccines, Antivirals and Gene Therapies Against Emerging Coronaviruses Emerging coronaviruses (CoV) are constant global public health threats to society. Multiple ongoing clinical trials for vaccines and antivirals against CoVs showcase the availability of medical interventions to both prevent and treat the future emergence of highly pathogenic CoVs in human. However, given the diverse nature of CoVs and our close interactions with wild, domestic and companion animals, the next epidemic zoonotic CoV could resist the existing vaccines and antivirals developed, which are primarily focused on Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS CoV). In late 2019, the novel CoV (SARS-CoV-2) emerged in Wuhan, China, causing global public health concern. In this review, we will summarize the key advancements of current vaccines and antivirals against SARS-CoV and MERS-CoV as well as discuss the challenge and opportunity in the current SARS-CoV-2 crisis. At the end, we advocate the development of a “plug-and-play” platform technologies that could allow quick manufacturing and administration of broad-spectrum countermeasures in an outbreak setting. We will discuss the potential of AAV-based gene therapy technology for in vivo therapeutic antibody delivery to combat SARS-CoV-2 outbreak and the future emergence of severe CoVs.

positive: Vaccines and Therapies in Development for SARS-CoV-2 Infections. The current COVID-19 pandemic is caused by the novel coronavirus SARS-CoV-2. The virus causes severe respiratory symptoms which manifest disproportionately in the elderly. Currently, there are over 6.5 million cases and 380,000 deaths reported. Given the current severity of the outbreak, there is a great need for antiviral therapies and vaccines to treat and prevent COVID-19. In this review, we provide an overview of SARS-CoV-2 and discuss the emerging therapies and vaccines that show promise in combating COVID-19. We also highlight potential viral targets that could be exploited by researchers and drug manufacturers.

positive: [At least 68 vaccine candidates under development]. The development of vaccines against SARS-CoV-2 is progressing at an unparalleled speed. As of the 29th of March, there were at least 68 vaccine candidates comprising several different vaccine designs, including whole killed virus, subunit, attenuated, viral vector, DNA and mRNA vaccines. Whilst it usually takes 10-15 years to develop a vaccine, it has only taken just over 9 weeks from the publication of the viral genetic sequence for the first vaccine candidate to reach clinical testing. Development has been expediated by using existing technological platforms and by performing preclinical and clinical testing simultaneously.

positive: Coronavirus vaccines: five key questions as trials begin.

positive: COVID-19 Clinical Research INTRODUCTION: While the global COVID-19 pandemic has challenged the entire humanity and health systems, it also triggered researchers to urgently perform clinical trials to assess the safety and efficacy of many agents and modalities to combat COVID-19. As of April 22, over 650 clinical studies have been registered both in USA and internationally. Results from these studies are also coming at a brisk pace in this unprecedented emergency. AREAS COVERED: We searched the NCI website and Medline and summarize various national and international clinical trials and summarize few of the pivotal ones in this paper, including those specific to oncology population. Two hundred and eighty four studies are actively recruiting adults and children with confirmed COVID-19, including 25 are early-phase I/phase I, 72 phase II, 58 phase III, 12 phase IV, and 31 other trials. They can be categorized into four groups: drugs that combat SARS-CoV-2, immunomodulatory agents to counteract cytokine storm, convalescence plasma therapies and vaccines trials. EXPERT OPINION: It is hoped that these efforts will results in a successful treatment to COVID-19, especially in a timely fashion before the second pandemic expected in fall. It is essential to acknowledge the devotion and hard work of the clinical research team and clinical research volunteers.

positive: SARS-CoV-2/COVID-19: Viral Genomics, Epidemiology, Vaccines, and Therapeutic Interventions The COVID-19 pandemic is due to infection caused by the novel SARS-CoV-2 virus that impacts the lower respiratory tract. The spectrum of symptoms ranges from asymptomatic infections to mild respiratory symptoms to the lethal form of COVID-19 which is associated with severe pneumonia, acute respiratory distress, and fatality. To address this global crisis, up-to-date information on viral genomics and transcriptomics is crucial for understanding the origins and global dispersion of the virus, providing insights into viral pathogenicity, transmission, and epidemiology, and enabling strategies for therapeutic interventions, drug discovery, and vaccine development. Therefore, this review provides a comprehensive overview of COVID-19 epidemiology, genomic etiology, findings from recent transcriptomic map analysis, viral-human protein interactions, molecular diagnostics, and the current status of vaccine and novel therapeutic intervention development. Moreover, we provide an extensive list of resources that will help the scientific community access numerous types of databases related to SARS-CoV-2 OMICs and approaches to therapeutics related to COVID-19 treatment.

positive: The current update of vaccines for SARS-COV-2 Late 2019 witnessed the appearance of coronavirus disease 2019 (COVID-19) outbreak triggered by a new coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 spread across China from Wuhan and has been circulated to a broader variety of countries. Seeing that COVID-19 has caused tremendous human casualties posing a global danger, an awareness of the current situation and the strategies to curtail the spread of the virus are desperately required. As the most natural way to preserve public wellbeing, preventive and therapeutic vaccinations must have essential roles. Large organizations and companies have started implementing SARS-CoV-2 preventive vaccinations. Hence, in this study, we aimed to update the vaccine evolution for SARS-CoV-2.

positive: Expected immune recognition of COVID-19 virus by memory from earlier infections with common coronaviruses in a large part of the world population SARS-CoV-2 is the coronavirus agent of the COVID-19 pandemic causing high mortalities. In contrast, the widely spread human coronaviruses OC43, HKU1, 229E, and NL63 tend to cause only mild symptoms. The present study shows, by in silico analysis, that these common human viruses are expected to induce immune memory against SARS-CoV-2 by sharing protein fragments (antigen epitopes) for presentation to the immune system by MHC class I. A list of such epitopes is provided. The number of these epitopes and the prevalence of the common coronaviruses suggest that a large part of the world population has some degree of specific immunity against SARS-CoV-2 already, even without having been infected by that virus. For inducing protection, booster vaccinations enhancing existing immunity are less demanding than primary vaccinations against new antigens. Therefore, for the discussion on vaccination strategies against COVID-19, the available immune memory against related viruses should be part of the consideration.

positive: Pharmacological and non-pharmacological efforts at prevention, mitigation, and treatment for COVID-19 A global outbreak of the SARS-CoV-2 virus has infected millions of people over a short period of time. The communicability and increased mortality from the SARS-CoV-2 infection mandated the WHO to declare COVID-19 a worldwide pandemic. The virus outbreak has spread when there are no approved vaccines, treatments, or prophylactic therapies available. Researchers from all over the world have prioritized development of vaccines and antivirals. Several vaccine projects have seen successes in preclinical, phase I, and phase II clinical trials using recombinant DNA, mRNA, live attenuated virus, S-protein subunits, virus like particles, and viral vectors. Initial findings from antivirals such as remdesivir, favipiravir, danoprevir or lopinavir with ritonavir are presented. Immunomodulatory molecules such as sarilumab, tocilizumab, janus kinase inhibitors, and hyperimmune convalescent plasma have mixed outcomes from initial clinical findings; however, pending randomized controlled trials will assist national health institutions to make treatment recommendations for COVID-19. Where compassionate use of remdesivir has shown some benefits, therapies such as hydroxychloroquine have proven harmful due to their toxicities. This review discusses pharmacological interventions at play and evidence-based successes and limitations of non-pharmacological therapies such as social distancing, personal protective equipment, and ventilator support associated with the prevention and treatment of COVID-19.

positive: Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM(®)) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (T(CM)) and effector memory (T(EM)) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM(®) platform as another promising strategy for the development of broad-spectrum universal influenza vaccines.

positive: The 2019-2020 novel coronavirus (severe acute respiratory syndrome coronavirus 2) pandemic: A joint american college of academic international medicine-world academic council of emergency medicine multidisciplinary COVID-19 working group consensus paper What started as a cluster of patients with a mysterious respiratory illness in Wuhan, China, in December 2019, was later determined to be coronavirus disease 2019 (COVID-19). The pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel Betacoronavirus, was subsequently isolated as the causative agent. SARS-CoV-2 is transmitted by respiratory droplets and fomites and presents clinically with fever, fatigue, myalgias, conjunctivitis, anosmia, dysgeusia, sore throat, nasal congestion, cough, dyspnea, nausea, vomiting, and/or diarrhea. In most critical cases, symptoms can escalate into acute respiratory distress syndrome accompanied by a runaway inflammatory cytokine response and multiorgan failure. As of this article's publication date, COVID-19 has spread to approximately 200 countries and territories, with over 4.3 million infections and more than 290,000 deaths as it has escalated into a global pandemic. Public health concerns mount as the situation evolves with an increasing number of infection hotspots around the globe. New information about the virus is emerging just as rapidly. This has led to the prompt development of clinical patient risk stratification tools to aid in determining the need for testing, isolation, monitoring, ventilator support, and disposition. COVID-19 spread is rapid, including imported cases in travelers, cases among close contacts of known infected individuals, and community-acquired cases without a readily identifiable source of infection. Critical shortages of personal protective equipment and ventilators are compounding the stress on overburdened healthcare systems. The continued challenges of social distancing, containment, isolation, and surge capacity in already stressed hospitals, clinics, and emergency departments have led to a swell in technologically-assisted care delivery strategies, such as telemedicine and web-based triage. As the race to develop an effective vaccine intensifies, several clinical trials of antivirals and immune modulators are underway, though no reliable COVID-19-specific therapeutics (inclusive of some potentially effective single and multi-drug regimens) have been identified as of yet. With many nations and regions declaring a state of emergency, unprecedented quarantine, social distancing, and border closing efforts are underway. Implementation of social and physical isolation measures has caused sudden and profound economic hardship, with marked decreases in global trade and local small business activity alike, and full ramifications likely yet to be felt. Current state-of-science, mitigation strategies, possible therapies, ethical considerations for healthcare workers and policymakers, as well as lessons learned for this evolving global threat and the eventual return to a 'new normal' are discussed in this article.

positive: Vaccines for COVID-19: The current state of play There is a strong consensus globally that a COVID-19 vaccine is likely the most effective approach to sustainably controlling the COVID-19 pandemic. An unprecedented research effort and global coordination has resulted in a rapid development of vaccine candidates and initiation of trials. Here, we review vaccine types, and progress with 10 vaccine candidates against SARS-CoV-2 - the virus that causes COVID-19 - currently undergoing early phase human trials. We also consider the many challenges of developing and deploying a new vaccine on a global scale, and recommend caution with respect to our expectations of the timeline that may be ahead.

positive: Current global vaccine and drug efforts against COVID-19: Pros and cons of bypassing animal trials. COVID-19 has become one of the biggest health concern, along with huge economic burden. With no clear remedies to treat the disease, doctors are repurposing drugs like chloroquine and remdesivir to treat COVID-19 patients. In parallel, research institutes in collaboration with biotech companies have identified strategies to use viral proteins as vaccine candidates for COVID-19. Although this looks promising, they still need to pass the test of challenge studies in animal models. As various models for SARS-CoV-2 are under testing phase, biotech companies have bypassed animal studies and moved to Phase I clinical trials. In view of the present outbreak, this looks a justified approach, but the problem is that in the absence of animal studies, we can never predict the outcomes in humans. Since animal models are critical for vaccine development and SARS-CoV-2 has different transmission dynamics, in this review we compare different animal models of SARS-CoV-2 with humans for their pathogenic, immune response and transmission dynamics that make them ideal models for vaccine testing for COVID-19. Another issue of using animal model is the ethics of using animals for research; thus, we also discuss the pros and cons of using animals for vaccine development studies.

positive: Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine. We observe remarkably high and dose-dependent SARS-CoV-2 specific antibody titers in mouse sera, as well as robust neutralization of both a pseudo-virus and wild-type virus. Upon further characterization we find that the neutralization is proportional to the quantity of specific IgG and of higher magnitude than recovered COVID-19 patients. saRNA LNP immunizations induce a Th1-biased response in mice, and there is no antibody-dependent enhancement (ADE) observed. Finally, we observe high cellular responses, as characterized by IFN-Î³ production, upon re-stimulation with SARS-CoV-2 peptides. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.

positive: A Snapshot of the Global Race for Vaccines Targeting SARS-CoV-2 and the COVID-19 Pandemic A novel coronavirus SARS-CoV-2 causing Coronavirus disease 2019 (COVID-19) has entered the human population and has spread rapidly around the world in the first half of 2020 causing a global pandemic. The virus uses its spike glycoprotein receptor-binding domain to interact with host cell angiotensin-converting enzyme 2 (ACE2) sites to initiate a cascade of events that culminate in severe acute respiratory syndrome in some individuals. In efforts to curtail viral spread, authorities initiated far-reaching lockdowns that have disrupted global economies. The scientific and medical communities are mounting serious efforts to limit this pandemic and subsequent waves of viral spread by developing preventative vaccines and repurposing existing drugs as potential therapies. In this review, we focus on the latest developments in COVID-19 vaccine development, including results of the first Phase I clinical trials and describe a number of the early candidates that are emerging in the field. We seek to provide a balanced coverage of the seven main platforms used in vaccine development that will lead to a desired target product profile for the “ideal” vaccine. Using tales of past vaccine discovery efforts that have taken many years or that have failed, we temper over exuberant enthusiasm with cautious optimism that the global medical community will reach the elusive target to treat COVID-19 and end the pandemic.

positive: Preparing for Pandemics: RNA Vaccines at the Forefront

positive: Vaccines and Drug Therapeutics to Lock Down Novel Coronavirus Disease 2019 (COVID-19): A Systematic Review of Clinical Trials The ongoing novel coronavirus disease 2019 (COVID-19) pandemic has been responsible for millions of infections and hundreds of thousands of deaths. To date, there is no approved targeted treatment, and many investigational therapeutic agents and vaccine candidates are being considered for the treatment of COVID-19. To extract and summarize information on potential vaccines and therapeutic agents against COVID-19 at different stages of clinical trials from January to March 2020, we reviewed major clinical trial databases such as ClinicalTrials.gov, WHO International Clinical Trials Registry Platform (ICTRP), and other primary registries between January and March 15, 2020. Interventional studies at different phases under the COVID-19 pipeline were included. A total of 249 clinical trials were identified between January to March 15, 2020. After filtering observational studies (194 studies), a total of 56 interventional trials were considered. The majority of clinical trials have been conducted on chloroquine (n=10) and traditional Chinese medications (TCMs; n=10), followed by antivirals (n=8), anti-inflammatory/immunosuppressants (n=9), cellular therapies (n=4), combinations of different antivirals therapies (n=3), antibacterial (n=1), and other therapies (n=5). Five vaccines are under phase I, and there are a couple of phase III trials on the Bacillus Calmette-Guérin (BCG) vaccine under investigation among healthcare workers. Many novel compounds and vaccines against COVID-19 are currently under investigation. Some candidates have been tested for other viral infections and are listed for clinical trials against the COVID-19 pipeline. Currently, there are no effective specific antivirals or drug combinations available for the treatment of COVID-19.

positive: A single dose SARS-CoV-2 simulating particle vaccine induces potent neutralizing activities Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for which a vaccine is urgently needed to control its spreading. To facilitate the representation of a native-like immunogen without being infectious, here, we reported a SARS-CoV-2 vaccine candidate (designated ShaCoVacc) by incorporating spike-encoding mRNA inside and decorating spike protein on the surface of the virus simulating particles (VSPs) derived from lentiviral particles. We characterized the mRNA copy number, glycosylation status, transduction efficiency, and innate immune property of the new vaccine platform. Importantly, we showed the ShaCoVacc induced strong spike-specific humoral immune responses and potent neutralizing activities by a single injection. Additionally, we disclosed the epitopes of spike-specific antibodies using peptide microarray and revealed epitopes susceptible to specific neutralizing antibodies. These results support further development of ShaCoVacc as a candidate vaccine for COVID-19 and VSP may serve as a new vaccine platform for emerging infectious diseases.

positive: Designing a novel mRNA vaccine against SARS-CoV-2: An immunoinformatics approach SARS-CoV-2 is the deadly virus behind COVID-19, the disease that went on to ravage the world and caused the biggest pandemic 21st century has witnessed so far. On the face of ongoing death and destruction, the urgent need for the discovery of a vaccine against the virus is paramount. This study resorted to the emerging discipline of immunoinformatics in order to design a multi-epitope mRNA vaccine against the spike glycoprotein of SARS-CoV-2. Various immunoinformatics tools were utilized to predict T and B lymphocyte epitopes. The epitopes were channeled through a filtering pipeline comprised of antigenicity, toxicity, allergenicity, and cytokine inducibility evaluation with the goal of selecting epitopes capable of generating both T and B cell-mediated immune responses. Molecular docking simulation between the epitopes and their corresponding MHC molecules was carried out. 13 epitopes, a highly immunogenic adjuvant, elements for proper sub-cellular trafficking, a secretion booster, and appropriate linkers were combined for constructing the vaccine. The vaccine was found to be antigenic, almost neutral at physiological pH, non-toxic, non-allergenic, capable of generating a robust immune response and had a decent worldwide population coverage. Based on these parameters, this design can be considered a promising choice for a vaccine against SARS-CoV-2.

positive: A SARS-CoV-2 Vaccination Strategy Focused on Population-Scale Immunity Here we propose a vaccination strategy for SARS-CoV-2 based on identification of both highly conserved regions of the virus and newly acquired adaptations that are presented by MHC class I and II across the vast majority of the population, are highly dissimilar from the human proteome, and are predicted B cell epitopes. We present 65 peptide sequences that we expect to result in a safe and effective vaccine which can be rapidly tested in DNA, mRNA, or synthetic peptide constructs. These include epitopes that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor, and within a novel furin cleavage site thought to increase membrane fusion. This vaccination strategy specifically targets unique vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the human population.

positive: Vaccines for SARS-CoV-2: Lessons from Other Coronavirus Strains. The emergence of the strain of coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and its impact on global health have made imperative the development of effective and safe vaccines for this lethal strain. SARS-CoV-2 now adds to the list of coronavirus diseases that have threatened global health, along with the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) coronaviruses that emerged in 2002/2003 and 2012, respectively. As of April 2020, no vaccine is commercially available for these coronavirus strains. Nevertheless, the knowledge obtained from the vaccine development efforts for MERS and SARS can be of high value for COVID-19 (coronavirus disease 2019). Here, we review the past and ongoing vaccine development efforts for clinically relevant coronavirus strains with the intention that this information helps in the development of effective and safe vaccines for COVID-19. In addition, information from naturally exposed individuals and animal models to coronavirus strains is described for the same purpose of helping into the development of effective vaccines against COVID-19.

positive: The race for coronavirus vaccines: a graphical guide

positive: New Vaccine Technologies to Combat Outbreak Situations Ever since the development of the first vaccine more than 200 years ago, vaccinations have greatly decreased the burden of infectious diseases worldwide, famously leading to the eradication of small pox and allowing the restriction of diseases such as polio, tetanus, diphtheria, and measles. A multitude of research efforts focuses on the improvement of established and the discovery of new vaccines such as the HPV (human papilloma virus) vaccine in 2006. However, radical changes in the density, age distribution and traveling habits of the population worldwide as well as the changing climate favor the emergence of old and new pathogens that bear the risk of becoming pandemic threats. In recent years, the rapid spread of severe infections such as HIV, SARS, Ebola, and Zika have highlighted the dire need for global preparedness for pandemics, which necessitates the extremely rapid development and comprehensive distribution of vaccines against potentially previously unknown pathogens. What is more, the emergence of antibiotic resistant bacteria calls for new approaches to prevent infections. Given these changes, established methods for the identification of new vaccine candidates are no longer sufficient to ensure global protection. Hence, new vaccine technologies able to achieve rapid development as well as large scale production are of pivotal importance. This review will discuss viral vector and nucleic acid-based vaccines (DNA and mRNA vaccines) as new approaches that might be able to tackle these challenges to global health.

positive: Vaccines for SARS-CoV-2: Lessons from Other Coronavirus Strains The emergence of the strain of coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and its impact on global health have made imperative the development of effective and safe vaccines for this lethal strain. SARS-CoV-2 now adds to the list of coronavirus diseases that have threatened global health, along with the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) coronaviruses that emerged in 2002/2003 and 2012, respectively. As of April 2020, no vaccine is commercially available for these coronavirus strains. Nevertheless, the knowledge obtained from the vaccine development efforts for MERS and SARS can be of high value for COVID-19 (coronavirus disease 2019). Here, we review the past and ongoing vaccine development efforts for clinically relevant coronavirus strains with the intention that this information helps in the development of effective and safe vaccines for COVID-19. In addition, information from naturally exposed individuals and animal models to coronavirus strains is described for the same purpose of helping into the development of effective vaccines against COVID-19.

positive: The SARS-CoV-2 Vaccine Pipeline: an Overview PURPOSE OF REVIEW: The goal of this review is to provide a timely overview on efforts to develop a vaccine for the 2019 novel coronavirus SARS-CoV-2, the causative agent of coronavirus disease (COVID-19). RECENT FINDINGS: Previous research efforts to develop a severe acute respiratory syndrome coronavirus (SARS-CoV) vaccine in the years following the 2003 pandemic have opened the door for investigators to design vaccine concepts and approaches for the COVID-19 epidemic in China. Both SARS-CoV and SARS-CoV-2 exhibit a high degree of genetic similarity and bind to the same host cell ACE2 receptor. Based on previous experience with SARS-CoV vaccines, it is expected that all COVID-19 vaccines will require careful safety evaluations for immunopotentiation that could lead to increased infectivity or eosinophilic infiltration. Besides this, a COVID-19 vaccine target product profile must address vaccinating at-risk human populations including frontline healthcare workers, individuals over the age of 60, and those with underlying and debilitating chronic conditions. Among the vaccine technologies under evaluation are whole virus vaccines, recombinant protein subunit vaccines, and nucleic acid vaccines. SUMMARY: Each current vaccine strategy has distinct advantages and disadvantages. Therefore, it is paramount that multiple strategies be advanced quickly and then evaluated for safety and efficacy. Ultimately, the safety studies to minimize undesired immunopotentiation will become the most significant bottleneck in terms of time.

positive: Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo. mRNA has broad potential for treating diseases requiring protein expression. However, mRNA can also induce an immune response with associated toxicity. Replacement of uridine bases with pseudouridine has been postulated to modulate both mRNA immunogenicity and potency. Here, we explore the immune response and activity of lipid nanoparticle-formulated unmodified and pseudouridine-modified mRNAs administered systemically in vivo. Pseudouridine modification to mRNA had no significant effect on lipid nanoparticle physical properties, protein expression in vivo, or mRNA immunogenicity compared to unmodified mRNA when delivered systemically with liver-targeting lipid nanoparticles, but reduced in vitro transfection levels. Indicators of a transient, extracellular innate immune response to mRNA were observed, including neutrophilia, myeloid cell activation, and up-regulation of four serum cytokines. This study provides insight into the immune responses to mRNA lipid nanoparticles, and suggests that pseudouridine modifications may be unnecessary for therapeutic application of mRNA in the liver.

positive: Emerging Manufacturers engagements in the COVID -19 vaccine research, development and supply The World Health Organization declared the COVID-19 disease as a pandemic requiring a rapid response. Through online search, direct communication with network members and an internal survey, engagements of developing countries’ vaccine manufacturers’ network members in the research and development of COVID-19 vaccines and their capacities in the manufacturing, fill-finish and distribution of vaccines were assessed. Currently, 19 network members engaged in research and development of COVID-19 vaccines, using six principal technology platforms. In addition, an internal survey showed that the number of vaccines supplied collectively by 37 members, in 2018-19, was about 3.5 billion doses annually. Almost a third of network members having vaccines prequalified by the World Health Organization comply with international regulations and mechanisms to distribute vaccines across borders. The use of existing manufacturing, fill-finish and distribution capabilities can support an efficient roll-out of vaccines against COVID-19, while maintaining supply security of existing vaccines for on-going immunization programmes.

positive: Global efforts on vaccines for COVID-19: Since, sooner or later, we all will catch the coronavirus. COVID-19 is an emerging infectious disease that has turned into a pandemic. It spreads through droplet transmission of the new coronavirus SARS-CoV-2. It is an RNA virus displaying a spike protein as the major surface protein with significant sequence similarity to SARS-CoV which causes severe acute respiratory syndrome. The receptor binding domain of the spike protein interacts with the human angiotensin converting enzyme 2 and is considered as the antigenic determinant for stimulating an immune response. While multiple candidate vaccines are currently under different stages of development, there are no known therapeutic interventions at the moment. This review describes the key genetic features that are being considered for generating vaccine candidates by employing innovative technologies. It also highlights the global efforts being undertaken to deliver vaccines for COVID-19 through unprecedented international cooperation and future challenges post development.

positive: The race for coronavirus vaccines: a graphical guide.

positive: [Progress and analysis on the development of 2019-nCoV vaccine]. As the COVID-19 pandemic is intensifying globally, more and more people are pinning their hopes on the development of vaccines. At present, there are many research teams who have adopted different vaccine technology routes to develop 2019-nCoV vaccines. This article reviews and analyzes the current development and research status of 2019-nCoV vaccines in different routes, and explores their possible development in the future.

positive: Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site Direct RNA sequencing using an Oxford Nanopore MinION characterised the transcriptome of SARS-CoV-2 grown in Vero E6 cells. This cell line is being widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. This revealed the pattern of viral transcripts, (i.e. subgenomic mRNAs), generally fitted the predicted replication and transcription model for coronaviruses. A 24 nt in-frame deletion was detected in subgenomic mRNAs encoding the spike (S) glycoprotein. This feature was identified in over half of the mapped transcripts and was predicted to remove a proposed furin cleavage site from the S glycoprotein. This motif directs cleavage of the S glycoprotein into functional subunits during virus entry or exit. Cleavage of the S glycoprotein can be a barrier to zoonotic coronavirus transmission and affect viral pathogenicity. Allied to this transcriptome analysis, tandem mass spectrometry was used to identify over 500 viral peptides and 44 phosphopeptides, covering almost all of the proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein reinforces the point that this and other regions of SARS-CoV-2 proteins may readily mutate. This is of clear significance given the interest in the S glycoprotein as a potential vaccine target and the observation that the furin cleavage site likely contributes strongly to the pathogenesis and zoonosis of this virus. The viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.

positive: Vaccines and Therapies in Development for SARS-CoV-2 Infections The current COVID-19 pandemic is caused by the novel coronavirus SARS-CoV-2. The virus causes severe respiratory symptoms which manifest disproportionately in the elderly. Currently, there are over 6.5 million cases and 380,000 deaths reported. Given the current severity of the outbreak, there is a great need for antiviral therapies and vaccines to treat and prevent COVID-19. In this review, we provide an overview of SARS-CoV-2 and discuss the emerging therapies and vaccines that show promise in combating COVID-19. We also highlight potential viral targets that could be exploited by researchers and drug manufacturers.

positive: COVID-19 vaccines: A race against time in the middle of death and devastation! The COVID-19 has turned in to a global human tragedy and economic devastation. Governments have implemented lockdown measures, blocked international travel, and enforced other public containment measures to mitigate the virus morbidity and mortality. As of today, no drug has the power to fight the infection and bring normalcy to the utter chaos. This leaves us with only one choice namely an effective and safe vaccine that shall be manufactured as soon as possible and available to all countries and populations affected by the pandemic at an affordable price. There has been an unprecedented fast track path taken in Research & Development (R&D) by the World community for developing an effective and safe vaccine. Platform technology has been exploited to develop candidate vaccines in a matter of days to weeks and as of now, 108 such vaccines are available. Six of these vaccines have entered clinical trials. As clinical trials are ‘rate-limiting’ and ‘time-consuming’, many innovative methods are in practice for a fast track. These include parallel phase I-II trials and obtaining efficacy data from phase IIb trials. Human ‘challenge experiments’ to confirm efficacy in humans is under serious consideration. The availability of the COVID-19 vaccine has become a race against time in the middle of death and devastation. There is an atmosphere of tremendous hype around the COVID-19 vaccine and developers are using every moment to make claims, which remain unverified. However, concerns are raised about a rush to deploy a COVID-19 vaccine. Applying ‘Quick fix’ and ‘short cuts’ can lead to errors with disastrous consequences.

positive: LinearDesign: Efficient Algorithms for Optimized mRNA Sequence Design A messenger RNA (mRNA) vaccine has emerged as a promising direction to combat the current COVID-19 pandemic. This requires an mRNA sequence that is stable and highly productive in protein expression, features which have been shown to benefit from greater mRNA secondary structure folding stability and optimal codon usage. However, sequence design remains a hard problem due to the exponentially many synonymous mRNA sequences that encode the same protein. We show that this design problem can be reduced to a classical problem in formal language theory and computational linguistics that can be solved in O(n^3) time, where n is the mRNA sequence length. This algorithm could still be too slow for large n (e.g., n = 3, 822 nucleotides for the spike protein of SARS-CoV-2), so we further developed a linear-time approximate version, LinearDesign, inspired by our recent work, LinearFold. This algorithm, LinearDesign, can compute the approximate minimum free energy mRNA sequence for this spike protein in just 11 minutes using beam size b = 1, 000, with only 0.6% loss in free energy change compared to exact search (i.e., b = +infinity, which costs 1 hour). We also develop two algorithms for incorporating the codon optimality into the design, one based on k-best parsing to find alternative sequences and one directly incorporating codon optimality into the dynamic programming. Our work provides efficient computational tools to speed up and improve mRNA vaccine development.

positive: Planning for COVID-19 Vaccines Safety surveillance

positive: Recent insights for the emerging COVID-19: drug discovery, therapeutic options and vaccine development SARS-CoV-2 has been marked as a highly pathogenic coronavirus of COVID-19 disease into the human population, causing over 5.5 million confirmed cases worldwide. As COVID-19 has posed a global threat with significant human casualties and severe economic losses, there is a pressing demand to further understand the current situation and develop rational strategies to contain the drastic spread of the virus. Although there are no specific antiviral therapies that have proven effective in randomized clinical trials, currently, the rapid detection technology along with several promising therapeutics for COVID-19 have mitigated its drastic transmission. Besides, global institutions and corporations have commenced to parse out effective vaccines for the prevention of COVID-19. Herein, the present review will give exhaustive details of extensive researches concerning the drug discovery and therapeutic options for COVID-19 as well as some insightful discussions of the status of COVID-19.

positive: Screening and testing for a suitable untransfected cell line for SARS-CoV-2 studies At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus. Author summary In the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.

positive: A recombinant Lactobacillus plantarum strain expressing the spike protein of SARS-CoV-2 Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic in the past four months and causes respiratory disease in humans of almost all ages. Although several drugs have been announced to be partially effective treatments for this disease, no approved vaccine is available. Here, we described the construction of a recombinant Lactobacillus plantarum strain expressing the SARS-CoV-2 spike protein. The results showed that the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity. The highest protein yield was obtained under the following conditions: cells were induced with 50 ng/mL SppIP at 37 °C for 6–10 h. The recombinant spike (S) protein was stable under normal conditions and at 50 °C, pH = 1.5, or a high salt concentration. Recombinant L. plantarum may provide a promising food-grade oral vaccine candidate against SARS-CoV-2 infection.

positive: Countdown to a vaccine More than 100 coronavirus vaccines are in development, but will any of them work?

positive: Development of a COVID-19 vaccine based on the receptor binding domain displayed on virus-like particles The recently ermerging disease COVID-19 is caused by the new SARS-CoV-2 virus first detected in the city of Wuhan, China. From there it has been rapidly spreading inside and outside China. With initial death rates around 4%, COVID-19 patients at longer distances from Wuhan showed reduced mortality as was previously observed for the SARS coronavirus. However, the new coronavirus spreads more strongly, as it sheds long before onset of symptoms or may be transmitted by people without symptoms. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrate that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro.

positive: Designing a multi-epitope peptide-based vaccine against SARS-CoV-2 COVID-19 pandemic has resulted so far 14,395,16 confirmed cases with 85,711 deaths from the 212 countries, or territories. Due to multifacet issues and challenges in implementation of the safety & preventive measures, inconsistent coordination between societies-governments and most importanly lack of specific vaccine to SARS-CoV-2, the spread of Wuhan originated virus is still uprising after taking a heavy toll on human life. In the present study, we mapped several immunogenic epitopes (B-cell, T-cell, and IFN-gamma) over the entire structural proteins of SARS-CoV-2 and by applying various computational and immunoinformatics approaches, we designed a multi-epitope peptide based vaccine that predicted high immunogenic response in the largest proportion of world’s human population. To ensure high expression of the recombinant vaccine in E. coli, codon optimization and in-silico cloning were also carried out. The designed vaccine with high molecular affinity to TLR3 and TLR4, was found capable to initiate effective innate and adaptive immune response. The immune simulation also suggested uprising high levels of both B-cell and T-cell mediated immunity which on subsequent exposure cleared antigen from the system. The proposed vaccine found promising by yielding desired results and hence, should be tested by practical experimentations for its functioning and efficacy to neutralize SARS-CoV-2.

positive: COVID-19 Vaccine Candidates: Prediction and Validation of 174 SARS-CoV-2 Epitopes The recent outbreak of SARS-CoV-2 (2019-nCoV) virus has highlighted the need for fast and efficacious vaccine development. Stimulation of a proper immune response that leads to protection is highly dependent on presentation of epitopes to circulating T-cells via the HLA complex. SARS-CoV-2 is a large RNA virus and testing of all overlapping peptides in vitro to deconvolute an immune response is not feasible. Therefore HLA-binding prediction tools are often used to narrow down the number of peptides to test. We tested 19 epitope-HLA-binding prediction tools, and using an in vitro peptide MHC stability assay, we assessed 777 peptides that were predicted to be good binders across 11 MHC allotypes. In this investigation of potential SARS-CoV-2 epitopes we found that current prediction tools vary in performance when assessing binding stability, and they are highly dependent on the MHC allotype in question. Designing a COVID-19 vaccine where only a few epitope targets are included is therefore a very challenging task. Here, we present 174 SARS-CoV-2 epitopes with high prediction binding scores, validated to bind stably to 11 HLA allotypes. Our findings may contribute to the design of an efficacious vaccine against COVID-19.

positive: Quantitative measurement of activity of JAK-STAT signaling pathways in blood samples and immune cells to predict innate and adaptive cellular immune response to viral infection and accelerate vaccine development The host immune response determines the clinical course of a viral infection, for example in case of COVID-19 infection. The effectiveness of vaccination also depends on the induced immune response. Currently there is no method to measure the cellular immune response in blood samples. The functional activity of cells of innate and adaptive immune system is determined by coordinated activity of signaling pathways, especially the JAK-STAT pathways. Using a previously described approach we developed mRNA-based tests to measure activity of these signaling pathways, and show that they can be used to measure in a quantitative manner the cellular innate and adaptive immune response to a viral infection or vaccine in whole blood, PBMC, and specific immune cell type samples. Pathway activity level and range in healthy individuals was established, enabling interpretation of a pathway activity score on a patient sample without the need for a reference sample. Evidence is presented that the pathway activity analysis may also be useful for in vitro vaccine development and assessment of vaccine immunogenicity. Other envisioned applications lie in development of immunomodulatory drugs and drug response prediction and monitoring. Tests are expected to be of value in the COVID-19 crisis. In addition to the described Affymetrix microarray-based pathway tests for measuring host immune response, qPCR-based versions are in development; the latter can in principle be performed within three hours in routine hospital labs.

positive: Single-dose replicating RNA vaccine induces neutralizing antibodies against SARS-CoV-2 in nonhuman primates The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.

positive: Vaccines against Coronaviruses: The State of the Art The emerging epidemic caused by the new coronavirus SARS-CoV-2 represents the most important socio-health threat of the 21st century. The high contagiousness of the virus, the strong impact on the health system of the various countries and the absence to date of treatments able to improve the prognosis of the disease make the introduction of a vaccine indispensable, even though there are currently no approved human coronavirus vaccines. The aim of the study is to carry out a review of the medical literature concerning vaccine candidates for the main coronaviruses responsible for human epidemics, including recent advances in the development of a vaccine against COVID-19. This extensive review carried out on the vaccine candidates of the main epidemic coronaviruses of the past has shown that the studies in animal models suggest a high efficacy of potential vaccines in providing protection against viral challenges. Similar human studies have not yet been carried out, as the main trials are aimed at assessing mainly vaccine safety and immunogenicity. Whereas the severe acute respiratory syndrome (SARS-CoV) epidemic ended almost two decades ago and the Middle East respiratory syndrome (MERS-CoV) epidemic is now better controlled, as it is less contagious due to the high lethality of the virus, the current SARS-CoV-2 pandemic represents a problem that is certainly more compelling, which pushes us to accelerate the studies not only for the production of vaccines but also for innovative pharmacological treatments. SARS-CoV-2 vaccines might come too late to affect the first wave of this pandemic, but they might be useful if additional subsequent waves occur or in a post-pandemic perspective in which the virus continues to circulate as a seasonal virus.

positive: Genomic diversity and hotspot mutations in 30,983 SARS-CoV-2 genomes: moving toward a universal vaccine for the “confined virus”? The Coronavirus disease 19 (COVID-19) pandemic has been ongoing since its onset in late November 2019 in Wuhan, China. To date, the SARS-CoV-2 virus has infected more than 8 million people worldwide and killed over 5% of them. Efforts are being made all over the world to control the spread of the disease and most importantly to develop a vaccine. Understanding the genetic evolution of the virus, its geographic characteristics and stability is particularly important for developing a universal vaccine covering all circulating strains of SARS-CoV-2 and for predicting its efficacy. In this perspective, we analyzed the sequences of 30,983 complete genomes from 80 countries located in six geographical zones (Africa, Asia, Europe, North & South America, and Oceania) isolated from December 24, 2019 to May 13, 2020, and compared them to the reference genome. Our in-depth analysis revealed the presence of 3,206 variant sites compared to the reference Wuhan-Hu-1 genome, with a distribution that is largely uniform over all continents. Remarkably, a low frequency of recurrent mutations was observed; only 182 mutations (5.67%) had a prevalence greater than 1%. Nevertheless, fourteen hotspot mutations (> 10%) were identified at different locations, seven at the ORF1ab gene (in regions coding for nsp2, nsp3, nsp6, nsp12, nsp13, nsp14 and nsp15), three in the nucleocapsid protein, one in the spike protein, one in orf3a, and one in orf8. Moreover, 35 non-synonymous mutations were identified in the receptor-binding domain (RBD) of the spike protein with a low prevalence (<1%) across all genomes, of which only four could potentially enhance the binding of the SARS-CoV-2 spike protein to the human receptor ACE2. These results along with the phylogenetic analysis demonstrate that the virus does not have a significant divergence at the protein level compared to the reference both among and within different geographical areas. Unlike the influenza virus or HIV viruses, the slow rate of mutation of SARS-CoV-2 makes the potential of developing an effective global vaccine very likely.

positive: SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in ‘lasso’ for the multi-faced virus During the current COVID-19 pandemic, the global ratio between the dead and the survivors is approximately 1 to 10, which has put humanity on high alert and provided strong motivation for the intensive search for vaccines and drugs. It is already clear that if we follow the most likely scenario, which is similar to that used to create seasonal influenza vaccines, then we will need to develop improved vaccine formulas every year to control the spread of the new, highly mutable coronavirus SARS-CoV-2. In this article, using well-known RNA viruses (HIV, influenza viruses, HCV) as examples, we consider the main successes and failures in creating primarily highly effective vaccines. The experience accumulated dealing with the biology of zoonotic RNA viruses suggests that the fight against COVID-19 will be difficult and lengthy. The most effective vaccines against SARS-CoV-2 will be those able to form highly effective memory cells for both humoral (memory B cells) and cellular (cross-reactive antiviral memory T cells) immunity. Unfortunately, RNA viruses constantly sweep their tracks and perhaps one of the most promising solutions in the fight against the COVID-19 pandemic is the creation of 'universal' vaccines based on conservative SARS-CoV-2 genome sequences (antigen-presenting) and unmethylated CpG dinucleotides (adjuvant) in the composition of the phosphorothioate backbone of single-stranded DNA oligonucleotides (ODN), which can be effective for long periods of use. Here, we propose a SARS-CoV-2 vaccine based on a lasso-like phosphorothioate oligonucleotide construction containing CpG motifs and the antigen-presenting unique ACG-containing genome sequence of SARS-CoV-2. We found that CpG dinucleotides are the least rare dinucleotides in the genomes of SARS-CoV-2 and other known human coronaviruses, and hypothesized that their higher frequency could be responsible for the unwanted increased lethality to the host, causing a ‘cytokine storm’ in people who overexpress cytokines through the activation of specific Toll-like receptors in a manner similar to TLR9-CpG ODN interactions. Interestingly, the virus strains sequenced in China (Wuhan) in February 2020 contained on average one CpG dinucleotide more in their genome than the later strains from the USA (New York) sequenced in May 2020. Obviously, during the first steps of the microevolution of SARS-CoV-2 in the human population, natural selection tends to select viral genomes containing fewer CpG motifs that do not trigger a strong innate immune response, so the infected person has moderate symptoms and spreads SARS-CoV-2 more readily. However, in our opinion, unmethylated CpG dinucleotides are also capable of preparing the host immune system for the coronavirus infection and should be present in SARS-CoV-2 vaccines as strong adjuvants.

positive: Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin. UNLABELLED The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) represent highly pathogenic human CoVs that share a property to inhibit host gene expression at the posttranscriptional level. Similar to the nonstructural protein 1 (nsp1) of SARS-CoV that inhibits host gene expression at the translational level, we report that MERS-CoV nsp1 also exhibits a conserved function to negatively regulate host gene expression by inhibiting host mRNA translation and inducing the degradation of host mRNAs. Furthermore, like SARS-CoV nsp1, the mRNA degradation activity of MERS-CoV nsp1, most probably triggered by its ability to induce an endonucleolytic RNA cleavage, was separable from its translation inhibitory function. Despite these functional similarities, MERS-CoV nsp1 used a strikingly different strategy that selectively targeted translationally competent host mRNAs for inhibition. While SARS-CoV nsp1 is localized exclusively in the cytoplasm and binds to the 40S ribosomal subunit to gain access to translating mRNAs, MERS-CoV nsp1 was distributed in both the nucleus and the cytoplasm and did not bind stably to the 40S subunit, suggesting a distinctly different mode of targeting translating mRNAs. Interestingly, consistent with this notion, MERS-CoV nsp1 selectively targeted mRNAs, which are transcribed in the nucleus and transported to the cytoplasm, for translation inhibition and mRNA degradation but spared exogenous mRNAs introduced directly into the cytoplasm or virus-like mRNAs that originate in the cytoplasm. Collectively, these data point toward a novel viral strategy wherein the cytoplasmic origin of MERS-CoV mRNAs facilitates their escape from the inhibitory effects of MERS-CoV nsp1. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human CoV that emerged in Saudi Arabia in 2012. MERS-CoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of MERS-CoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. The nonstructural protein 1 (nsp1) of CoVs is one such protein that inhibits host gene expression and is a major virulence factor. This study presents evidence for a strategy used by MERS-CoV nsp1 to inhibit host gene expression that has not been described previously for any viral protein. The present study represents a meaningful step toward a better understanding of the factors and molecular mechanisms governing the virulence and pathogenesis of MERS-CoV.

positive: RNA to the rescue: RNA is one of the most promising targets for drug development given its wide variety of uses The race for a vaccine against SARS‐CoV‐2 has accelerated research on RNA‐based therapeutics. Beyond vaccines, RNA also shows great potential for cancer therapies.[Image: see text]

positive: Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine Abstract The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5α and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8+ CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS.

positive: Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza The outbreak of the coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

positive: In silico approach for designing of a multi-epitope based vaccine against novel Coronavirus (SARS-COV-2) A novel Coronavirus (SARS-COV-2) has now become a global pandemic. Considering the severity of infection and the associated mortalities, there is an urgent need to develop an effective preventive measure against this virus. In this study, we have designed a novel vaccine construct using computational strategies. Spike (S) glycoprotein is the major antigenic component that trigger the host immune responses. Detailed investigation of S protein with various immunoinformatics tools enabled us to identify 5 MHC I and 5 MHC II B-cell derived T-cell epitopes with VaxiJen score > 1 and IC50 value < 100nM. These epitopes were joined with a suitable adjuvant and appropriate linkers to form a multi-epitope based vaccine construct. Further, in silico testing of the vaccine construct for its antigenicity, allergenicity, solubility, and other physicochemical properties showed it to be safe and immunogenic. Suitable tertiary structure of the vaccine protein was generated using 3Dpro of SCRATCH suite, refined with GalaxyRefine, and validated with ProSA, PROCHECK, and ERRAT server. Finally, molecular docking studies were performed to ensure a favorable binding affinity between the vaccine construct and TLR3 receptor. The designed multi-epitope vaccine showed potential to elicit specific immune responses against the SARS-COV-2. However, further wet lab validation is necessary to confirm the actual effectiveness, safety and immunogenic potency of the vaccine construct against derived in this study.

positive: Revisiting potential druggable targets against SARS‐CoV‐2 and repurposing therapeutics under preclinical study and clinical trials: A comprehensive review Coronavirus disease‐19 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is one of the most contagious diseases in human history that has already affected millions of lives worldwide. To date, no vaccines or effective therapeutics have been discovered yet that may successfully treat COVID‐19 patients or contain the transmission of the virus. Scientific communities across the globe responded rapidly and have been working relentlessly to develop drugs and vaccines, which may require considerable time. In this uncertainty, repurposing the existing antiviral drugs could be the best strategy to speed up the discovery of effective therapeutics against SARS‐CoV‐2. Moreover, drug repurposing may leave some vital information on druggable targets that could be capitalized in target‐based drug discovery. Information on possible drug targets and the progress on therapeutic and vaccine development also needs to be updated. In this review, we revisited the druggable targets that may hold promise in the development of the anti‐SARS‐CoV‐2 agent. Progresses on the development of potential therapeutics and vaccines that are under the preclinical studies and clinical trials have been highlighted. We anticipate that this review will provide valuable information that would help to accelerate the development of therapeutics and vaccines against SARS‐CoV‐2 infection.

positive: Emerging vaccine delivery systems for COVID-19: Functionalised silica nanoparticles offer a potentially safe and effective alternative delivery system for DNA/RNA vaccines and may be useful in the hunt for a COVID-19 vaccine

positive: The vaccine journey for COVID-19: a comprehensive systematic review of current clinical trials in humans INTRODUCTION: Since December 2019, there has been an outbreak of a novel beta-coronavirus (SARS-CoV-2) in Wuhan, China. On March the 11th the World Health Organization (WHO) declared COVID-19 as a pandemic, with over 118,000 cases in more than 110 countries around the world. In response to the global coronavirus disease 2019 (COVID-19) emergency, clinical trial research assessing the efficacy and safety of experimental vaccines to prevent COVID-19 are emerging at an unprecedented rate. The aim of this systematic review is to summarize the preliminary experiences and ongoing clinical trials of the major candidates and challenges of the vaccine strategies in humans. EVIDENCE ACQUISITION: After a priori protocol registration with PROSPERO (181483), a systematic research of the published literature was conducted on 24 April 2020 using Medline (via PubMed), Embase (via Ovid), and WHO databases. Moreover, to explore the more recent literature we also searched the preprint server medRxiv. Finally, we scrutinized the Cochrane COVID-19 study register and the COVID-19 section of ClinicalTrials.gov database for identifying relevant ongoing clinical trials. Thereafter we selected the articles according to the PRISMA guidelines. Animal or in-vitro experimental studies were excluded. Moreover editorials, commentaries, abstracts, reviews, book chapters, and articles not in English were not included. EVIDENCE SYNTHESIS: Our search identified 1359 published papers, 478 pre-print articles and 367 ongoing clinical trials. Finally, only ten ongoing clinical trials met the inclusion criteria. Specifically, seven developed vaccines for the S protein of SARS-CoV-2 and three clinical trials assessed the protective role of BCG vaccine against COVID-19. The first group included phase I/II trials with different types of molecules (DNA or mRNA vaccine, bacterial plasmid or viral vectors), the latter were phase III/IV trials designed on the basis of a heterologous lymphocyte activation by the BCG vaccine. CONCLUSIONS: This new disease is pushing the scientific community to develop swiftly a safe and effective vaccine. Notwithstanding the limitations of our analysis, given by the absence of available results, we try to provide a comprehensive view of the ongoing clinical trials in humans. Our analysis reveals a worldwide effort of both scientists and enterprises to achieve one of the most challenging goals of our century.

positive: COVID-19 Coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles’ heel conserved region to minimize probability of escape mutations and drug resistance Abstract This paper continues a recent study of the spike protein sequence of the COVID-19 virus (SARS-CoV-2). It is also in part an introductory review to relevant computational techniques for tackling viral threats, using COVID-19 as an example. Q-UEL tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. Subsequence KRSFIEDLLFNKV of the S2′ spike glycoprotein proteolytic cleavage site continues to appear important. Here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. Its function or functions thus seem important to the coronaviruses. It might represent SARS-CoV-2 Achilles’ Heel, less likely to acquire resistance by mutation, as has happened in some early SARS vaccine studies discussed in the previous paper. Preliminary conformational analysis of the receptor (ACE2) binding site of the spike protein is carried suggesting that while it is somewhat conserved, it appears to be more variable than KRSFIEDLLFNKV. However compounds like emodin that inhibit SARS entry, apparently by binding ACE2, might also have functions at several different human protein binding studies. The enzyme 11β-hydroxysteroid dehydrogenase type 1 is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. Perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets.

positive: Current Status of Multiple Drug Molecules, and Vaccines: An Update in SARS-CoV-2 Therapeutics The coronavirus disease of 2019 (COVID-19) is a pandemic disease that has taken the lives of many around the world. It is caused by severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2). To date, the USA, Italy, Spain, France, Russia, and the UK have been hit the hardest by the virus. However, death counts are still rising. Some nations have managed to “flatten” the death rate via protective measures such physical distancing, quarantine measures, and therapeutic management. The structure of the SARS-CoV-2 virus comprises of S proteins, M proteins, E proteins, hemagglutinin esterases, nucleocapsid proteins, and a 30-kb RNA genome. Viral proteases cleave these polyproteins and RNA-dependent polymerases replicate the genome. Currently, there are no effective therapies against this new disease. Numerous investigators are developing novel protease inhibitors, some of which have made it into clinical trials. Researchers are also attempting to develop a vaccine. In this review paper, we discuss the latest therapeutic developments against COVID-19. [Figure: see text]

positive: SARS-CoV-2 vaccines: 'Warp Speed' needs mind melds not warped minds. In this review, we address issues that relate to the rapid "Warp Speed" development of vaccines to counter the COVID-19 pandemic. We review the antibody response that is triggered by SARS-CoV-2 infection of humans, and how it may inform vaccine research. The isolation and properties of neutralizing monoclonal antibodies from COVID-19 patients provide additional information on what vaccines should try to elicit. The nature and longevity of the antibody response to coronaviruses are relevant to the potency and duration of vaccine-induced immunity. We summarize the immunogenicity of leading vaccine candidates tested to date in animals and humans, and discuss the outcome and interpretation of virus-challenge experiments in animals. By far the most immunogenic vaccine candidates for antibody responses are recombinant proteins, which are not included in the initial wave of "Warp Speed" immunogens. A substantial concern for SARS-CoV-2 vaccines is adverse events, which we review by considering what was seen in studies of SARS-CoV-1 and MERS-CoV vaccines. We conclude by outlining the possible outcomes of the "Warp Speed" vaccine program, which range from the hoped-for rapid success to a catastrophic adverse influence on vaccine uptake generally.

positive: Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development Abstract Background Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. Methods We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. Findings Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. Interpretation MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.

positive: SARS-CoV-2 induces transcriptional signatures in human lung epithelial cells that promote lung fibrosis BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV-2-induced coronavirus disease-2019 (COVID-19) is a pandemic disease that affects > 2.8 million people worldwide, with numbers increasing dramatically daily. However, there is no specific treatment for COVID-19 and much remains unknown about this disease. Angiotensin-converting enzyme (ACE)2 is a cellular receptor of SARS-CoV-2. It is cleaved by type II transmembrane serine protease (TMPRSS)2 and disintegrin and metallopeptidase domain (ADAM)17 to assist viral entry into host cells. Clinically, SARS-CoV-2 infection may result in acute lung injury and lung fibrosis, but the underlying mechanisms of COVID-19 induced lung fibrosis are not fully understood. METHODS: The networks of ACE2 and its interacting molecules were identified using bioinformatic methods. Their gene and protein expressions were measured in human epithelial cells after 24 h SARS-CoV-2 infection, or in existing datasets of lung fibrosis patients. RESULTS: We confirmed the binding of SARS-CoV-2 and ACE2 by bioinformatic analysis. TMPRSS2, ADAM17, tissue inhibitor of metalloproteinase (TIMP)3, angiotensinogen (AGT), transformation growth factor beta (TGFB1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) A and fibronectin (FN) were interacted with ACE2, and the mRNA and protein of these molecules were expressed in lung epithelial cells. SARS-CoV-2 infection increased ACE2, TGFB1, CTGF and FN1 mRNA that were drivers of lung fibrosis. These changes were also found in lung tissues from lung fibrosis patients. CONCLUSIONS: Therefore, SARS-CoV-2 binds with ACE2 and activates fibrosis-related genes and processes to induce lung fibrosis.

positive: Immunization with the receptor–binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement Recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Currently, there is no vaccine available for preventing SARS-CoV-2 infection. Like closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses its receptor-binding domain (RBD) on the spike (S) protein to engage the host receptor, human angiotensin-converting enzyme 2 (ACE2), facilitating subsequent viral entry. Here we report the immunogenicity and vaccine potential of SARS-CoV-2 RBD (SARS2-RBD)-based recombinant proteins. Immunization with SARS2-RBD recombinant proteins potently induced a multi-functional antibody response in mice. The resulting antisera could efficiently block the interaction between SARS2-RBD and ACE2, inhibit S-mediated cell-cell fusion, and neutralize both SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection. In addition, the anti-RBD sera also exhibited cross binding, ACE2-blockade, and neutralization effects towards SARS-CoV. More importantly, we found that the anti-RBD sera did not promote antibody-dependent enhancement of either SARS-CoV-2 pseudovirus entry or authentic virus infection of Fc receptor-bearing cells. These findings provide a solid foundation for developing RBD-based subunit vaccines for SARS-CoV2.

positive: Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus Middle East respiratory syndrome (MERS) is an emerging infectious disease caused by MERS coronavirus (MERS-CoV). The continuous increase of MERS cases has posed a serious threat to public health worldwide, calling for development of safe and effective MERS vaccines. We have previously shown that a recombinant protein containing residues 377–588 of MERS-CoV receptor-binding domain (RBD) fused with human Fc (S377-588-Fc) induced highly potent anti-MERS-CoV neutralizing antibodies in the presence of MF59 adjuvant. Here we optimized the doses of S377-588-Fc using MF59 as an adjuvant in order to elicit strong immune responses with minimal amount of antigen. Our results showed that S377-588-Fc at 1 μg was able to induce in the immunized mice potent humoral and cellular immune responses. Particularly, S377-588-Fc at 1 μg elicited strong neutralizing antibody responses against both pseudotyped and live MERS-CoV similar to those induced at 5 and 20 μg, respectively. These results suggest that this RBD-based subunit MERS vaccine candidate at the dose as low as one μg is sufficiently potent to induce strong humoral and cellular immune responses, including neutralizing antibodies, against MERS-CoV infection, thus providing guidance for determining the optimal dosage of RBD-based MERS vaccines in the future clinical trials and for applying the dose-sparing strategy in other subunit vaccine trials.

positive: Immunoinformatic identification of B cell and T cell epitopes in the SARS-CoV-2 proteome A novel coronavirus (SARS-CoV-2) emerged from China in late 2019 and rapidly spread across the globe, infecting millions of people and generating societal disruption on a level not seen since the 1918 influenza pandemic. A safe and effective vaccine is desperately needed to prevent the continued spread of SARS-CoV-2; yet, rational vaccine design efforts are currently hampered by the lack of knowledge regarding viral epitopes targeted during an immune response, and the need for more in-depth knowledge on betacoronavirus immunology. To that end, we developed a computational workflow using a series of open-source algorithms and webtools to analyze the proteome of SARS-CoV-2 and identify putative T cell and B cell epitopes. Using increasingly stringent selection criteria to select peptides with significant HLA promiscuity and predicted antigenicity, we identified 41 potential T cell epitopes (5 HLA class I, 36 HLA class II) and 6 potential B cell epitopes, respectively. Docking analysis and binding predictions demonstrated enrichment for peptide binding to HLA-B (class I) and HLA-DRB1 (class II) molecules. Overlays of predicted B cell epitopes with the structure of the viral spike (S) glycoprotein revealed that 4 of 6 epitopes were located in the receptor-binding domain of the S protein. To our knowledge, this is the first study to comprehensively analyze all 10 (structural, non-structural and accessory) proteins from SARS-CoV-2 using predictive algorithms to identify potential targets for vaccine development. Significance Statement The novel coronavirus SARS-CoV-2 recently emerged from China, rapidly spreading and ushering in a global pandemic. Despite intensive research efforts, our knowledge of SARS-CoV-2 immunology and the proteins targeted by the immune response remains relatively limited, making it difficult to rationally design candidate vaccines. We employed a suite of bioinformatic tools, computational algorithms, and structural modeling to comprehensively analyze the entire SARS-CoV-2 proteome for potential T cell and B cell epitopes. Utilizing a set of stringent selection criteria to filter peptide epitopes, we identified 41 T cell epitopes (5 HLA class I, 36 HLA class II) and 6 B cell epitopes that could serve as promising targets for peptide-based vaccine development against this emerging global pathogen.

positive: COVID-19 vaccine development pipeline gears up

positive: Update on therapeutic approaches and emerging therapies for SARS-CoV-2 virus The global pandemic of coronavirus disease 2019 (COVID-19), caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in over 7,273,958 cases with almost over 413,372 deaths worldwide as per the WHO situational report 143 on COVID-19. There are no known treatment regimens with proven efficacy and vaccines thus far, posing an unprecedented challenge to identify effective drugs and vaccines for prevention and treatment. The urgency for its prevention and cure has resulted in an increased number of proposed treatment options. The high rate and volume of emerging clinical trials on therapies for COVID-19 need to be compared and evaluated to provide scientific evidence for effective medical options. Other emerging non-conventional drug discovery techniques such as bioinformatics and cheminformatics, structure-based drug design, network-based methods for prediction of drug-target interactions, artificial intelligence (AI) and machine learning (ML) and phage technique could provide alternative routes to discovering potent Anti-SARS-CoV2 drugs. While drugs are being repurposed and discovered for COVID-19, novel drug delivery systems will be paramount for efficient delivery and avoidance of possible drug resistance. This review describes the proposed drug targets for therapy, and outcomes of clinical trials that have been reported. It also identifies the adopted treatment modalities that are showing promise, and those that have failed as drug candidates. It further highlights various emerging therapies and future strategies for the treatment of COVID-19 and delivery of Anti-SARS-CoV2 drugs.

positive: Universal coronavirus vaccines: the time to start is now The continued explosive spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) despite aggressive public health measures has triggered an unprecedented international vaccine effort. However, correlates of protection, which can help guide intelligent vaccine design, are not known for SARS-CoV-2. Research on influenza immunity and vaccine development may provide valuable lessons for coronavirus efforts, especially considering similarities in rapid evolutionary potential. The apparent inevitability of future novel coronavirus outbreaks must prompt work on a universal coronavirus vaccine.

positive: The crystal structure of nsp10-nsp16 heterodimer from SARS-CoV-2 in complex with S-adenosylmethionine SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2’-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 Å. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

positive: COVID-19: immunopathology and its implications for therapy Severe coronavirus disease 2019 (COVID-19) is characterized by pneumonia, lymphopenia, exhausted lymphocytes and a cytokine storm. Significant antibody production is observed; however, whether this is protective or pathogenic remains to be determined. Defining the immunopathological changes in patients with COVID-19 provides potential targets for drug discovery and is important for clinical management.

positive: Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2). About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

positive: The Potential of Various Nanotechnologies for Coronavirus Diagnosis/Treatment Highlighted through a Literature Analysis [Image: see text] With the current COVID-19 outbreak, it has become essential to develop efficient methods for the treatment and detection of this virus. Among the new approaches that could be tested, that relying on nanotechnology finds one of its main grounds in the similarity between nanoparticle (NP) and coronavirus (COV) sizes, which promotes NP–COV interactions. Since COVID-19 is very recent, most studies in this field have focused on other types of coronavirus than COVID-19, such as those involved in MERS or SARS diseases. Although their number is limited, they have led to promising results on various COV using a wide range of different types of nanosystems, e.g., nanoparticles, quantum dos, or nanoassemblies of polymers/proteins. Additional efforts deserve to be spent in this field to consolidate these findings. Here, I first summarize the different nanotechnology-based methods used for COV detection, i.e., optical, electrical, or PCR ones, whose sensitivity was improved by the presence of nanoparticles. Furthermore, I present vaccination methods, which comprise nanoparticles used either as adjuvants or as active principles. They often yield a better-controlled immune response, possibly due to an improved antigen presentation/processing than in non-nanoformulated vaccines. Certain antiviral approaches also took advantage of nanoparticle uses, leading to specific mechanisms such as the blocking of virus replication at the cellular level or the reduction of a COV induced apoptotic cellular death.

positive: Drug Repurposing to find Inhibitors of SARS-CoV-2 Main Protease Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 pandemic. Currently there is no known vaccine or specific antiviral treatment for COVID-19 and so, there is an urgent need for expedite discovery of new therapeutics to combat the disease until a vaccine will be available worldwide. Drug repurposing is a strategy for identifying new uses for approved drugs that has the advantage (over conventional approaches that attempt to develop a drug from scratch) that time frame of the overall process can be significantly reduced because of the few number of clinical trial required. In this work, a virtual screening of FDA-approved drugs was performed for repositioning as potential inhibitors of the main protease Mpro of SARS-CoV-2. As a result of this study, 12 drugs are proposed as candidates for inhibitors of the Mpro enzyme. Some of the selected compounds are antiviral drugs that are already being tested in COVID-19 clinical trials (i.e. ribavirin) or are used to alleviate symptoms of the disease (i.e. codeine). Surprisingly, the most promising candidate is the naturally occurring broad spectrum antibiotic oxytetracycline. This compound has largely outperformed the remaining selected candidates along all filtering steps of our virtual screening protocol. If the activity of any of these drugs is experimentally corroborated, they could be used directly in clinical trials without the need for pre-clinical testing or safety evaluation since they are already used as drugs for other diseases.

positive: Influenza Vaccination and COVID19 Mortality in the USA COVID-19 mortality rate is higher in the elderly and in those with preexisting chronic medical conditions. The elderly also suffer from increased morbidity and mortality from seasonal influenza infection, and thus annual influenza vaccination is recommended for them. In this study, we explore a possible area-level association between influenza vaccination coverage in people aged 65 years and older and the number of deaths from COVID-19. To this end, we used COVID-19 data until June 10, 2020 together with population health data for the United States at the county level. We fit quasi-Poisson regression models using influenza vaccination coverage in the elderly population as the independent variable and the number of deaths from COVID-19 as the outcome variable. We adjusted for a wide array of potential confounding variables using both county-level generalized propensity scores for influenza vaccination rates, as well as direct adjustment. Our results suggest that influenza vaccination coverage in the elderly population is negatively associated with mortality from COVID-19. This finding is robust to using different analysis periods, different thresholds for inclusion of counties, and a variety of methodologies for confounding adjustment. In conclusion, our results suggest a potential protective effect of the influenza vaccine on COVID-19 mortality in the elderly population. The significant public health implications of this possibility point to an urgent need for studying the relationship between influenza vaccination and COVID-19 mortality at the individual level, to investigate both the epidemiology and any underlying biological mechanism.

positive: Global efforts on vaccines for COVID-19: Since, sooner or later, we all will catch the coronavirus COVID-19 is an emerging infectious disease that has turned into a pandemic. It spreads through droplet transmission of the new coronavirus SARS-CoV-2. It is an RNA virus displaying a spike protein as the major surface protein with significant sequence similarity to SARS-CoV which causes severe acute respiratory syndrome. The receptor binding domain of the spike protein interacts with the human angiotensin converting enzyme 2 and is considered as the antigenic determinant for stimulating an immune response. While multiple candidate vaccines are currently under different stages of development, there are no known therapeutic interventions at the moment. This review describes the key genetic features that are being considered for generating vaccine candidates by employing innovative technologies. It also highlights the global efforts being undertaken to deliver vaccines for COVID-19 through unprecedented international cooperation and future challenges post development.

positive: Identification of a Noncanonical Signal for Transcription of a Novel Subgenomic mRNA of Mouse Hepatitis Virus: Implication for the Mechanism of Coronavirus RNA Transcription Abstract Subgenomic RNA transcription of coronaviruses involves the interaction between the leader (or antileader) and the intergenic (IG) sequences. However, it is not clear how these two sequences interact with each other. In this report, a previously unrecognized minor species of subgenomic mRNA, termed mRNA5–1, was identified in cells infected with mouse hepatitis virus (MHV) strains JHM2c, JHM(2), JHM(3), A59, and MHV-1. Sequence analysis revealed that the leader-body fusion site of the mRNA is located at approximately 150 nucleotides (nt) downstream of the consensus IG sequence for mRNA 5 and did not have sequence homology with any known IG consensus sequences. To determine whether this sequence functions independently as a promoter, we cloned a 140-nt sequence (from ≈70 nt upstream to ≈70 nt downstream of the fusion site) from viral genomic RNA and placed it in front of a reporter gene in the defective-interfering (DI) RNA-chloramphenicol acetyltransferase (CAT) reporter vector. Transfection of the reporter RNA into MHV-infected cells resulted in synthesis of a CAT-specific subgenomic mRNA detected by reverse transcription-polymerase chain reaction (RT-PCR). The strength of this promoter was similar to that of the IG7 (for mRNA 7) as measured by the CAT activity. Deletion analysis showed that the sequence as few as 13 nt was sufficient to initiate mRNA transcription, while mutations within the 13-nt abolished mRNA transcription. In vitro translation study confirmed that the envelope (E) protein was translated from mRNA5–1, which encodes the open reading frame (ORF) 5b at its 5′-end, indicating that mRNA5–1 is a functional message. Furthermore, when the ORF5b was replaced with the CAT gene and placed in the DI in the context of viral mini-genome, CAT was expressed not only from the first ORF of mRNA5–1 but also from the second and third ORF of mRNA5 and genomic DI RNA, respectively, suggesting that more than one mechanism is involved in regulation of ORF5b expression. Our findings thus support the notion that base-pairing between the leader (or antileader) and the IG is not the sole mechanism in subgenomic RNA transcription.

positive: Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses. The demand for rapid and simple development of a vaccine against a newly emerging infectious disease is increasing worldwide. We previously revealed that UV-inactivated severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) virions (UV-V) elicited high levels of humoral immunity and a weak Th0 response in mice immunized subcutaneously. To ensure the safety of such a whole inactivated SARS-CoV vaccine, we additionally treated the UV-V vaccine with formalin, resulting in the UV-F-V vaccine. Analysis of the immunogenicity of the UV-F-V+alum vaccine in mice revealed that it generated comparable neutralizing serum anti-SARS-CoV IgG antibody levels as the UV-V+alum vaccine. Moreover, both vaccines induced similar frequencies of anti-SARS-CoV IgG antibody-producing cells in bone marrow. Interestingly, the UV-F-V vaccine induced fewer IgG(2a) subtype antibodies and higher interleukin-4 production in vaccinated mice than did UV-V. Thus, UV-F-V imposes a Th2-type bias on the immune response, unlike UV-V. We propose here that doubly-inactivated SARS-CoV virions by UV and formalin constitute a safe vaccine that may effectively induce neutralizing antibodies in humans.

positive: Are genetic vaccines the right weapon against Covid-19?

positive: Discovery of Synergistic and Antagonistic Drug Combinations against SARS-CoV-2 In Vitro COVID-19 is undoubtedly the most impactful viral disease of the current century, afflicting millions worldwide. As yet, there is not an approved vaccine, as well as limited options from existing drugs for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro. Overall, we identified 16 synergistic and 8 antagonistic combinations, 4 of which were both synergistic and antagonistic in a dose-dependent manner. Among the 16 synergistic cases, combinations of nitazoxanide with three other compounds (remdesivir, amodiaquine and umifenovir) were the most notable, all exhibiting significant synergy against SARS-CoV-2. The combination of nitazoxanide, an FDA-approved drug, and remdesivir, FDA emergency use authorization for the treatment of COVID-19, demonstrate a strong synergistic interaction. Notably, the combination of remdesivir and hydroxychloroquine demonstrated strong antagonism. Overall, our results emphasize the importance of both drug repurposing and preclinical testing of drug combinations for potential therapeutic use against SARS-CoV-2 infections.

positive: Vaccines against Coronaviruses: The State of the Art. The emerging epidemic caused by the new coronavirus SARS-CoV-2 represents the most important socio-health threat of the 21st century. The high contagiousness of the virus, the strong impact on the health system of the various countries and the absence to date of treatments able to improve the prognosis of the disease make the introduction of a vaccine indispensable, even though there are currently no approved human coronavirus vaccines. The aim of the study is to carry out a review of the medical literature concerning vaccine candidates for the main coronaviruses responsible for human epidemics, including recent advances in the development of a vaccine against COVID-19. This extensive review carried out on the vaccine candidates of the main epidemic coronaviruses of the past has shown that the studies in animal models suggest a high efficacy of potential vaccines in providing protection against viral challenges. Similar human studies have not yet been carried out, as the main trials are aimed at assessing mainly vaccine safety and immunogenicity. Whereas the severe acute respiratory syndrome (SARS-CoV) epidemic ended almost two decades ago and the Middle East respiratory syndrome (MERS-CoV) epidemic is now better controlled, as it is less contagious due to the high lethality of the virus, the current SARS-CoV-2 pandemic represents a problem that is certainly more compelling, which pushes us to accelerate the studies not only for the production of vaccines but also for innovative pharmacological treatments. SARS-CoV-2 vaccines might come too late to affect the first wave of this pandemic, but they might be useful if additional subsequent waves occur or in a post-pandemic perspective in which the virus continues to circulate as a seasonal virus.

positive: Characterization of RNA in Saliva Background: We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA. Methods: We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivary β-actin mRNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA–macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR. Results: In most cases, we detected partial- to full-length salivary mRNAs and smaller amounts of middle and 3′ gene amplicons compared with the 5′. RNA was present in all oral fluids examined. Endogenous salivary β-actin mRNA degraded more slowly than exogenous β-actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not pass through 0.22 or 0.45 μm pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA. Conclusions: Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.

positive: The outbreak of SARS-CoV-2 pneumonia calls for viral vaccines The outbreak of 2019-novel coronavirus disease (COVID-19) that is caused by SARS-CoV-2 has spread rapidly in China, and has developed to be a Public Health Emergency of International Concern. However, no specific antiviral treatments or vaccines are available yet. This work aims to share strategies and candidate antigens to develop safe and effective vaccines against SARS-CoV-2.

positive: Current pharmacological treatments for SARS-COV-2: A narrative review The novel coronavirus, later identified as SARS-CoV-2, originating from Wuhan in China in November 2019, quickly spread around the world becoming a pandemic. Despite the knowledge of previous coronaviruses, such as those responsible for the SARS and MERS-CoV epidemic, there is no drug or prophylaxis treatment to this day. The rapid succession of scientific findings on SARS-CoV-2 provides a significant number of potential drug targets. Nevertheless, at the same time, the high quantity of clinical data, generated by a large number of rapidly infected people, require accurate tests regarding effective medical treatments. Several in vitro and in vivo studies were rapidly initiated after the outbreak of the pandemic COVID-19. Initial clinical studies revealed the promising potential of remdesivir that demonstrated a powerful and specific in vitro antiviral activity for COVID-19. Promising effects appear to be attributable to hydroxychloroquine. Remdesivir and hydroxychloroquine are being tested in ongoing randomized trials. In contrast, oseltamivir was not effective and corticosteroids are not currently recommended. However, few data from ongoing clinical trials are identifying low molecular weight heparins, innate immune system stimulating agents, and inflammatory modulating agents as potential effective agents. The authors assume that the current pandemic will determine the need for a systematic approach based on big data analysis for identifying effective drugs to defeat SARS-Cov-2. This work is aimed to be a general reference point and to provide an overview as comprehensive as possible regarding the main clinical trials in progress at the moment.

positive: Emerging Technologies for Use in the Study, Diagnosis, and Treatment of Patients with COVID-19 INTRODUCTION: The COVID-19 pandemic has caused an unprecedented health and economic worldwide crisis. Innovative solutions are imperative given limited resources and immediate need for medical supplies, healthcare support and treatments. AIM: The purpose of this review is to summarize emerging technologies being implemented in the study, diagnosis, and treatment of COVID-19. RESULTS: Key focus areas include the applications of artificial intelligence, the use of Big Data and Internet of Things, the importance of mathematical modeling for predictions, utilization of technology for community screening, the use of nanotechnology for treatment and vaccine development, the utility of telemedicine, the implementation of 3D-printing to manage new demands and the potential of robotics. CONCLUSION: The review concludes by highlighting the need for collaboration in the scientific community with open sharing of knowledge, tools, and expertise.

positive: Antiviral RNAi therapy: emerging approaches for hitting a moving target The field of directed RNA interference (RNAi) has rapidly developed into a highly promising approach for specifically downregulating genes to alleviate disease pathology. This technology is especially well-suited to treating viral infections, and numerous examples now illustrate that a wide range of viruses can be inhibited with RNAi, both in vitro and in vivo. One principle that has arisen from this work is that antiviral RNAi therapies must be tailored to the unique life cycle of each pathogen, including the choice of delivery vehicle, route of administration, gene(s) targeted and regulation and duration of RNAi induction. Although effective strategies will be customized to each virus, all such therapies must overcome similar challenges. Importantly, treatment strategies must compensate for the inevitable fact that viral genome sequences evolve extremely rapidly, and computational and bioinformatics approaches may aid in the development of therapies that resist viral escape. Furthermore, all RNAi strategies involve the delivery of nucleic acids to target cells, and all will therefore benefit from the development of enhanced gene design and delivery technologies. Here, we review the substantial progress that has been made towards identifying effective antiviral RNAi targets and discuss strategies for translating these findings into effective clinical therapies.

positive: Another Set of Coronavirus Vaccine Candidates I surveyed the coronavirus vaccine landscape in this post, and then detailed some of the larger efforts in the field here (several updates have been added to that one since its initial posting) Now it’s time to look at several programs that aren’t in either of those, but still have plenty of serious science behind them For an example of a relatively new technology that’s now in use for human patients, there’s the VSV (vesicular stomatitis virus) platform, which was used to produce the Ebola vaccine now manufactured by Merck Stat has an excellent long-form article from earlier this year about how this came about, and it’s well worth a read, both for the history itself and as a look into the ups and downs of vaccine research in general The Yale team behind that one had developed a promising vaccine candidate for the SARS coronavirus during its epidemic, and they’re using those lessons in their current work If you look at that second link above on vaccine candidates, you will note that there are several using adenovirus vectors – this is conceptually the same sort of thing, but using a livestock virus (VSV) instead of human or primate-associated adenoviruses I don’t know if the Yale team has partnered with anyone yet, but I should also mention another connection of theirs, a spinoff company called CaroGen that has another engineered virus platform that is also being put to use against SARS-CoV-2 These projects are aiming at FDA approval for Phase I trials, but there’s no word yet on what such an application might go in

positive: The global and local distribution of RNA structure throughout the SARS-CoV-2 genome SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ~30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to locate regions of high base pair content across this long genome and also identify well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity to stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most highly structured viral RNAs in nature. Furthermore, our analysis reveals varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts. The conclusions reported in this study provide a foundation for structure-function hypotheses in SARS-CoV-2 biology, and in turn, may guide the 3D structural characterization of potential RNA drug targets for COVID-19 therapeutics.

positive: Four vaccine manufacturers have been asked by the US HHS to test their vaccines against coronavirus,

negative: The Father-Daughter Dinner Dance: A Waltz With Ethics and COVID-19 Commentary on "The Father-Daughter Dinner Dance: A Waltz With Ethics and COVID-19"

negative: In silico multi-epitope vaccine against covid19 showing effective interaction with HLA-B*15:03 The recent outbreak of severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 (SARS-CoV-2) causing coronavirus disease (covid19) has posed a great threat to human health. Previous outbreaks of SARS-CoV and Middle East respiratory Syndrome CoV (MERS-CoV) from the same CoV family had posed similar threat to human health and economic growth. To date, not even a single drug specific to any of these CoVs has been developed nor any anti-viral vaccine is available for the treatment of diseases caused by CoVs. Subunits present in spike glycoproteins of SARS-CoV and SARS-CoV-2 are involved in binding to human ACE2 Receptor which is the primary method of viral invasion. As it has been observed in the previous studies that there are very minor differences in the spike glycoproteins of SARS-CoV and SARS-CoV-2. SARS-CoV-2 has an additional furin cleavage site that makes it different from SARS-CoV (Walls et al., 2020). In this study, we have analyzed spike glycoproteins of SARS-CoV-2 and SARS-CoV phylogenetically and subjected them to selection pressure analysis. Selection pressure analysis has revealed some important sites in SARS-CoV-2 and SARS-CoV spike glycoproteins that might be involved in their pathogenicity. Further, we have developed a potential multi-epitope vaccine candidate against SARS-CoV-2 by analyzing its interactions with HLA-B*15:03 subtype. This vaccine consists of multiple T-helper (TH) cells, B-cells, and Cytotoxic T-cells (CTL) epitopes joined by linkers and an adjuvant to increase its immunogenicity. Conservation of selected epitopes in SARS, MERS, and human hosts, suggests that the designed vaccine could provide cross-protection. The vaccine is designed in silico by following a reverse vaccinology method acknowledging its antigenicity, immunogenicity, toxicity, and allergenicity. The vaccine candidate that we have designed as a result of this work shows promising result indicating its potential capability of simulating an immune response.

negative: Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M(pro)). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC(50) values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC(50) values ranging from 0.49 to 3.37 μM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known M(pro) inhibitors. A complex crystal structure of SARS-CoV-2 M(pro) with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M(pro). Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

negative: Current Therapies Under Investigation for COVID-19 In response to the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), researchers are expeditiously searching for antiviral treatments able to alleviate the symptoms of infection, which can be life-threatening. Here, we provide a general overview of what is currently known about the structure and characteristic features of SARS-CoV-2, some of which could potentially be exploited for the purposes of antiviral therapy and vaccine development. This mini-review also covers selected and noteworthy antiviral agents/supportive therapy out of hundreds of drugs that are being repurposed or tested as potential treatments for COVID-19, the disease caused by SARS-CoV.

negative: RNA-based drugs and vaccines RNA-based approaches have provided novel alternatives for modern drug discovery. The application of RNA as therapeutic agents has, until recently, been hampered by issues related to poor delivery and stability, but chemical modifications and new delivery approaches have increased progress. Moreover, the discovery of the importance of RNA in gene regulation and gene silencing has revealed new drug targets, especially related to treatment of cancer and other diseases. Recent engineering of small molecules designed from RNA sequences to target miRNAs opens up new possibilities in drug development. Furthermore, RNA-based vaccines have been engineered applying RNA virus vectors and non-viral delivery for vaccine development.

negative: Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells. The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.

negative: Bioinformatic characterization of angiotensin-converting enzyme 2, the entry receptor for SARS-CoV-2 The World Health Organization declared the COVID-19 epidemic a public health emergency of international concern on March 11th, 2020, and the pandemic is rapidly spreading worldwide. COVID-19 is caused by a novel coronavirus SARS-CoV-2, which enters human target cells via angiotensin converting enzyme 2 (ACE2). We used a number of bioinformatics tools to computationally characterize ACE2 by determining its cell-specific expression in trachea, lung, and small intestine, derive its putative functions, and predict transcriptional regulation. The small intestine expressed higher levels of ACE2 than any other organ. The large intestine, kidney and testis showed moderate signals, whereas the signal was weak in the lung. Single cell RNA-Seq data from trachea indicated positive signals along the respiratory tract in key protective cell types including club, goblet, proliferating, and ciliary epithelial cells; while in lung the ratio of ACE2-expressing cells was low in all cell types (<2.6%), but was highest in vascular endothelial and goblet cells. Gene ontology analysis suggested that, besides its classical role in renin-angiotensin system, ACE2 may be functionally associated with angiogenesis/blood vessel morphogenesis. Using a novel tool for the prediction of transcription factor binding sites we identified several putative binding sites within two tissue-specific promoters of the ACE2 gene. Our results also confirmed that age and gender play no significant role in the regulation of ACE2 mRNA expression in the lung. IMPORTANCE Vaccines and new medicines are urgently needed to prevent spread of COVID-19 pandemic, reduce the symptoms, shorten the duration of disease, prevent virus spread in the body, and most importantly to save lives. One of the key drug targets could be angiotensin-converting enzyme 2 (ACE2), which is a crucial receptor for the corona virus (SARS-CoV-2). It is known that SARS coronavirus infections lead to worse outcome in the elderly and in males. Therefore, one aim of the present study was to investigate whether age or sex could contribute to the regulation of ACE2 expression. We also decided to explore the transcriptional regulation of ACE2 gene expression. Since data on ACE2 distribution is still conflicting, we aimed to get a more comprehensive view of the cell types expressing the receptor of SARS-CoV-2. Finally, we studied the coexpression of ACE2 with other genes and explored its putative functions using gene ontology enrichment analysis.

negative: The quest to find an effective vaccine for COVID-19 Professor Alan Glasper, from the University of Southampton, delves into history and discusses the international quest to develop an effective vaccine to tackle COVID-19.

negative: Respiratory viral diseases: access to RNA interference therapy This review summarizes recent experimental achievements in the area of the development of new RNA interference (RNAi) therapeutics for the treatment of viral respiratory diseases. Delivery of siRNA to their intended target tissue remains the biggest problem for most therapeutic applications of these compounds. Appropriate formulations and chemical modifications for improved stability will boost the probability of utilization of RNAi drugs in the clinical applications.

negative: A Comparison of Plasmid DNA and mRNA as Vaccine Technologies This review provides a comparison of the theoretical issues and experimental findings for plasmid DNA and mRNA vaccine technologies. While both have been under development since the 1990s, in recent years, significant excitement has turned to mRNA despite the licensure of several veterinary DNA vaccines. Both have required efforts to increase their potency either via manipulating the plasmid DNA and the mRNA directly or through the addition of adjuvants or immunomodulators as well as delivery systems and formulations. The greater inherent inflammatory nature of the mRNA vaccines is discussed for both its potential immunological utility for vaccines and for the potential toxicity. The status of the clinical trials of mRNA vaccines is described along with a comparison to DNA vaccines, specifically the immunogenicity of both licensed veterinary DNA vaccines and select DNA vaccine candidates in human clinical trials.

negative: Covid-19 pandemic: Will a vaccine be available soon?

negative: A Guide to COVID‐19: a global pandemic caused by the novel coronavirus SARS‐CoV‐2 The emergence of the SARS‐CoV‐2 strain of the human coronavirus has thrown the world into the midst of a new pandemic. In the human body, the virus causes COVID‐19, a disease characterized by shortness of breath, fever, and pneumonia, which can be fatal in vulnerable individuals. SARS‐CoV‐2 has characteristics of past human coronaviruses, with close genomic similarities to SARS‐CoV, the virus that causes the disease SARS. Like these related coronaviruses, SARS‐CoV‐2 is transmitted through the inhalation of droplets and interaction with contaminated surfaces. Across the world, laboratories are developing candidate vaccines for the virus – with vaccine trials underway in the US and the United Kingdom ‐ and considering various drugs for possible treatments and prophylaxis. Here, we provide an overview of SARS‐CoV‐2 by analyzing its virology, epidemiology, and modes of transmission while examining the current progress of testing procedures and possible treatments through drugs and vaccines.

negative: Current Status of Treatment Options, Clinical Trials, and Vaccine Development for SARS-CoV-2 Infection The severe acute respiratory syndrome virus (SARS-CoV-2), a novel coronavirus first discovered in Wuhan, China in December 2019 causes the Coronavirus Disease 19 (COVID-19), which presents with a wide range of clinical symptoms from mild or moderate to severe and critical illnesses With the continuing transmission of the virus worldwide and the rapidly evolving situation globally, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic in March Currently, there is no proven specific treatment for this potentially deadly disease beyond supportive care However, a massive effort has been put globally into the investigation of medications and other interventional measures to fight COVID-19 Convalescent plasma therapy from recovered patients has recently drawn considerable interest Several alternative medical treatments, although evidence of their efficacy still lacking, have also gained popularity, especially in countries with such traditions such as India and China Rapid repurposing of drugs for COVID-19 has revealed a few promising candidate antiviral agents, but further research, especially high quality randomized controlled trials, will be needed to prove their efficacy and safety in the clinical use to treat COVID-19 Vaccine development has been the imperative task in the battle against SARS-CoV-2 While clinical trials have been launched for several candidate vaccines, research on COVID-19 vaccines is still at an early stage So far, optimized supportive care remains the best practice against COVID-19

negative: Structural features of coronavirus SARS-CoV-2 spike protein: Targets for vaccination Various human pathogenic viruses employ envelope glycoproteins for host cell receptor recognition and binding, membrane fusion and viral entry. The spike (S) glycoprotein of betacoronavirus SARS-CoV-2 is a homotrimeric class I fusion protein that exists in a metastable conformation for cleavage by host cell proteases furin and TMPRSS2, thereby undergoing substantial structural rearrangement for ACE2 host cell receptor binding and subsequent viral entry by membrane fusion. The S protein is densely decorated with N-linked glycans protruding from the trimer surface that affect S protein folding, processing by host cell proteases and the elicitation of humoral immune response. Deep insight into the sophisticated structure of SARS-CoV-2 S protein may provide a blueprint for vaccination strategies, as reviewed herein.

negative: An integrated in silico immuno-genetic analytical platform provides insights into COVID-19 serological and vaccine targets Background The COVID-19 pandemic is causing a major global health and socio-economic burden, instigating the mobilisation of resources into the development of control tools, such as diagnostics and vaccines. The poor performance of some diagnostic serological tools has emphasised the need for up to date immune-informatic analyses to inform the selection of viable targets for further study. This requires the integration and analysis of genetic and immunological data for SARS-CoV-2 and its homology with other human coronavirus species to understand cross-reactivity. Methods We have developed an online tool for SARS-CoV-2 research, which combines an extensive epitope mapping and prediction meta-analysis, with an updated variant database (55,944 non-synonymous mutations) based on 16,087 whole genome sequences, and an analysis of human coronavirus homology. To demonstrate its utility, we present an integrated analysis of the SARS-CoV-2 spike and nucleocapsid proteins, which are candidate vaccine and serological diagnostic targets. Results Our analysis reveals that the nucleocapsid protein in its native form appears to be a sub-optimal target for use in serological diagnostic platforms. Whilst, a further analysis suggests that orf3a proteins may be a suitable alternative target for diagnostic assays. Conclusions The tool can be accessed online (http://genomics.lshtm.ac.uk/immuno) and will serve as a useful tool for biological discovery in the fight against SARS-CoV-2. Further, it may be adapted to inform on biological targets in future outbreaks, including new human coronaviruses that spill over from animal hosts.

negative: Lipid nanoparticle technology for therapeutic gene regulation in the liver Hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. Most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. Targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs s holds great promise as a therapeutic approach. However, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. Lipid nanoparticle (LNP) delivery technology is a revolutionary development that has enabled clinical translation of gene (editing) therapies. LNPs can deliver siRNA, mRNA, DNA, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. Here we discuss the state-of-the-art LNP technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation.

negative: Is the production of a Covid-19 vaccine using transformed Pasteurella plausible?

negative: Decrease in ACE2 mRNA expression in aged mouse lung Angiotensin-converting enzyme 2 (ACE2) has been identified as a critical receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has led to extensive speculation on the role of ACE2 in disease severity, and in particular, whether variation in its expression can explain higher mortality in older individuals. We examine this question in mouse lung and show that 24-month old mice have significantly reduced ACE2 mRNA expression relative to 3-month old mice. The differences appear to be localized to ciliated cells.

negative: A candidate multi-epitope vaccine against SARS-CoV-2 In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.

negative: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples COVID-19 pandemic has caused tens of thousands of deaths and is now a severe threat to global health. Clinical practice has demonstrated that the SARS-CoV-2 S1 specific antibodies and viral antigens can be used as diagnostic and prognostic markers of COVID-19. However, the popular point-of-care biomarker detection technologies, such as the lateral-flow test strips, provide only yes/no information and have very limited sensitivities. Thus, it has a high false negative rate and cannot be used for the quantitative evaluation of patient’s immune response. Conventional ELISA (enzyme-linked immunosorbent assay), on the other hand, can provide quantitative, accurate, and sensitive results, but it involves complicated and expensive instruments and long assay time. In addition, samples need to be sent to centralized labs, which significantly increases the turn-around time. Here, we present a microfluidic ELISA technology for rapid (15-20 minutes), quantitative, sensitive detection of SARS-CoV-2 biomarkers using SARS-CoV-2 specific IgG and viral antigen – S protein in serum. We also characterized various humanized monoclonal IgG, and identified a candidate with a high binding affinity towards SARS-CoV-2 S1 protein that can serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. Furthermore, we demonstrated that our microfluidic ELISA platform can be used for rapid affinity evaluation of monoclonal anti-S1 antibodies. The microfluidic ELISA device is highly portable and requires less than 10 μL of samples for each channel. Therefore, our technology will greatly facilitate rapid and quantitative analysis of COVID-19 patients and vaccine recipients at point-of-care.

negative: The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations. Murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS) coronavirus (CoV) are two of the best-studied representatives of the family Coronaviridae. During CoV infection, numerous cytokines and chemokines are induced in vitro and in vivo. Human interleukin 8 and its mouse functional counterpart, CXCL2, are early-expressed chemokines. Here we show that SARS-CoV and MHV induce endoplasmic reticulum (ER) stress and Cxcl2 mRNA transcription during infection in vitro. Expression of the viral spike protein significantly induced ER stress and Cxcl2 mRNA upregulation, while expression of the other structural genes did not. Additional experiments with UV-inactivated virus, cell-cell fusion-blocking antibodies, and an MHV mutant with a defect in spike protein maturation demonstrated that spike-host interactions in the ER are responsible for the induction of ER stress and subsequent Cxcl2 mRNA transcription. Despite significant increases in levels of Cxcl2 mRNA and functional nucleus-to-cytoplasm RNA transport, no CXCL2 protein was released into the medium from MHV-infected cells. Yet Sendai virus-infected cells showed substantial Cxcl2 mRNA induction and a simultaneous increase in levels of secreted CXCL2 protein. Our results demonstrate that expression of CoV spike proteins induces ER stress, which could subsequently trigger innate immune responses. However, at that point in infection, translation of host mRNA is already severely reduced in infected cells, preventing the synthesis of CXCL2 and ER stress proteins despite their increased mRNA concentrations.

negative: Bacillus Calmette–Guérin vaccination and clinical characteristics and outcomes of COVID-19 in Rhode Island, United States: a cohort study Coronavirus disease 2019 (COVID-19) has resulted in a global pandemic, and there is limited data on effective therapies. Bacillus Calmette–Guérin (BCG) vaccine, a live-attenuated strain derived from an isolate of Mycobacterium bovis and originally designed to prevent tuberculosis, has shown some efficacy against infection with unrelated pathogens. In this study, we reviewed 120 consecutive adult patients (≥18 years old) with COVID-19 at a major federally qualified health centre in Rhode Island, United States from 19 March to 29 April 2020. Median age was 39.5 years (interquartile range, 27.0–50.0), 30% were male and 87.5% were Latino/Hispanics. Eighty-two (68.3%) patients had BCG vaccination. Individuals with BCG vaccination were less likely to require hospital admission during the disease course (3.7% vs. 15.8%, P = 0.019). This association remained unchanged after adjusting for demographics and comorbidities (P = 0.017) using multivariate regression analysis. The finding from our study suggests the potential of BCG in preventing more severe COVID-19.

negative: COVID-19 pandemic predictions using the modified Bateman SIZ model and observational data for Heidelberg, Germany: Effect of vaccination with a SARS-CoV-2 vaccine, coronavirus testing and application of the Corona-Warn-App AIMS OF THE STUDY: Published data show that the current progression of the COVID-19 pandemic in Heidelberg, Germany, despite the current lockdown, could continue into 2021 and become more severe. We have used the modified Bateman SIZ algorithm to predict the effects of interventional measures to control the COVID-19 pandemic. MATERIALS AND METHODS: Model parameters, e.g., doubling time and rate of decrease in the number of infectious persons were obtained from published reports. Predictions were made for the status quo on June 1, 2020, and for interventional measures obtained for 4 scenarios. These included vaccination of the whole population using a SARS-CoV-2 vaccine having an efficacy of 50% and 100%, mass-testing for COVID-19 coronavirus and application of the Corona-Warn-App. RESULTS: The principle findings were 1) without new measures to control the pandemic, the daily number of infectious persons could reach a peak of > 4,500 daily within 18 months when > 67,000 persons would have been infected. This could be prevented by using a vaccine with 50% efficacy which was almost equally effective as a vaccine with 100% efficacy. Application of the Corona-Warn-App was the most effective method and more effective than testing for COVID-19. The methodology used has been described in detail to enable other researchers to apply the modified Bateman SIZ model to obtain predictions for COVID-19 outbreaks in other regions. Application of the model has been verified by independent investigators using different commercial software packages. CONCLUSION: The modified Bateman SIZ model has been verified and used to predict the course of the COVID-19 pandemic in Heidelberg. Lockdown measures alone are insufficient to control the pandemic during 2021. Vaccination, diagnostic tests, and use of the Corona-Warn-App with quarantine could successfully control the spread of the coronavirus infection in the community. The Corona-Warn-App applied correctly may be the most effective. The model showed that vaccination with 50% efficacy is almost as effective as vaccination with 100% efficacy.

negative: The SARS-CoV-2 receptor, Angiotensin converting enzyme 2 (ACE2) is required for human endometrial stromal cell decidualization STUDY QUESTION Is SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE 2) expressed in the human endometrium during the menstrual cycle, and does it participate in endometrial decidualization? SUMMARY ANSWER ACE2 protein is highly expressed in human endometrial stromal cells during the secretory phase and is essential for human endometrial stromal cell decidualization. WHAT IS KNOWN ALREADY ACE2 is expressed in numerous human tissues including the lungs, heart, intestine, kidneys and placenta. ACE2 is also the receptor by which SARS-CoV-2 enters human cells. STUDY DESIGN, SIZE, DURATION Proliferative (n = 9) and secretory (n = 6) phase endometrium biopsies from healthy reproductive-age women and primary human endometrial stromal cells from proliferative phase endometrium were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS ACE2 expression and localization were examined by qRT-PCR, Western blot, and immunofluorescence in both human endometrial samples and mouse uterine tissue. The effect of ACE2 knockdown on morphological and molecular changes of human endometrial stromal cell decidualization were assessed. Ovariectomized mice were treated with estrogen or progesterone to determine the effects of these hormones on ACE2 expression. MAIN RESULTS AND THE ROLE OF CHANCE In human tissue, ACE2 protein is expressed in both endometrial epithelial and stromal cells in the proliferative phase of the menstrual cycle, and expression increases in stromal cells in the secretory phase. The ACE2 mRNA (P < 0.0001) and protein abundance increased during primary human endometrial stromal cell (HESC) decidualization. HESCs transfected with ACE2-targeting siRNA were less able to decidualize than controls, as evidenced by a lack of morphology change and lower expression of the decidualization markers PRL and IGFBP1 (P < 0.05). In mice during pregnancy, ACE2 protein was expressed in uterine epithelial and stromal cells increased through day six of pregnancy. Finally, progesterone induced expression of Ace2 mRNA in mouse uteri more than vehicle or estrogen (P < 0.05). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Experiments assessing the function of ACE2 in human endometrial stromal cell decidualization were in vitro. Whether SARS-CoV-2 can enter human endometrial stromal cells and affect decidualization have not been assessed. WIDER IMPLICATIONS OF THE FINDINGS Expression of ACE2 in the endometrium allow SARS-CoV-2 to enter endometrial epithelial and stromal cells, which could impair in vivo decidualization, embryo implantation, and placentation. If so, women with COVID-19 may be at increased risk of early pregnancy loss. STUDY FUNDINGS/COMPETING INTEREST(S) This study was supported by National Institutes of Health / National Institute of Child Health and Human Development grants R01HD065435 and R00HD080742 to RK and Washington University School of Medicine start-up funds to RK. The authors declare that they have no conflicts of interest.

negative: Epitope-based chimeric peptide vaccine design against S, M and E proteins of SARS-CoV-2 etiologic agent of global pandemic COVID-19: an in silico approach Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19), a public health emergency of international concern declared by the World Health Organization (WHO). An immuno-informatics approach along with comparative genomic was applied to design a multi-epitope-based peptide vaccine against SARS-CoV-2 combining the antigenic epitopes of the S, M and E proteins. The tertiary structure was predicted, refined and validated using advanced bioinformatics tools. The candidate vaccine showed an average of ≥ 90.0% world population coverage for different ethnic groups. Molecular docking of the chimeric vaccine peptide with the immune receptors (TLR3 and TLR4) predicted efficient binding. Immune simulation predicted significant primary immune response with increased IgM and secondary immune response with high levels of both IgG1 and IgG2. It also increased the proliferation of T-helper cells and cytotoxic T-cells along with the increased INF-γ and IL-2 cytokines. The codon optimization and mRNA secondary structure prediction revealed the chimera is suitable for high-level expression and cloning. Overall, the constructed recombinant chimeric vaccine candidate demonstrated significant potential and can be considered for clinical validation to fight against this global threat, COVID-19.

negative: Infectious Bronchitis Virus Population Structure Defines Immune Response and Protection. A commercial Arkansas (Ark) Delmarva Poultry Industry (DPI)-type vaccine and a more homogeneous population of that vaccine obtained previously through adaptation to chicken embryo kidney (CEK) cells (CEK-ArkDPI) were used as a model to further understand the impact of population genetic structure on generation of immune responses and protection. In a first experiment, vaccinated chickens were challenged with an IBV Ark99-type virulent strain (AL/4614/98). Despite extensive sequence similarity between the vaccines, the more heterogeneous commercial ArkDPI was more efficient at reducing viral loads in challenged chickens, while respiratory signs and tracheal lesions were reduced similarly by either vaccine. A distinct subpopulation of the Ark challenge virus showing asparagine at S1 position 56 was consistently negatively selected by immune pressure originating from vaccination with either vaccine. Antibody levels and antibody avidity to Ark-type S1 protein were greater in CEK-ArkDPI-vaccinated chickens compared to chickens vaccinated with the more diverse commercial ArkDPI vaccine. Synchronous replication of a homogeneous virus population likely elicits clonal expansion and affinity maturation of a greater number of responding B cells compared to a diverse virus population continuously changing its proportion of phenotypes during replication. The results of a second experiment showed that during initial vaccine virus replication (24 and 48 hr postvaccination), the virus population showing increased diversity (commercial ArkDPI) achieved higher concentrations of IBV RNA in the trachea compared to the more homogenous virus. mRNA expression of genes associated with innate immune responses in the trachea 48 hr postvaccination generally showed greater upregulation in chickens vaccinated with the heterogeneous commercial ArkDPI vaccine compared to the CEK-adapted virus. The greater upregulation of these genes is likely associated with higher virus replication achieved by the heterogeneous commercial vaccine. Thus, while the adaptive antibody response was favored by the more homogenous structure of the CEK-ArkDPI vaccine population (higher antibody levels and antibody avidity), the innate immune response was favored by the more diverse viral population of the commercial ArkDPI. We confirmed previous results that distinct subpopulations in wild Ark challenge virus become selected by immune pressure originating from vaccination, and we concluded that the population structure of IBV vaccines impacts innate immune response, antibody avidity, and protection.

negative: [SARS-CoV-2 infection (COVID-19): what can we expect?] - Case numbers in China are clearly declining, case numbers in many European regions are no longer increasing exponentially.- Data on mortality from SARS-CoV-2 infection are contradictory; mortality is certainly lower than for SARS and MERS, but probably higher than for most seasonal flu outbreaks in recent years- The main complication of SARS-CoV-2 infection is pneumonia with development of acute respiratory distress syndrome (ARDS)- Asymptomatic and oligosymptomatic courses with virus shedding are not uncommon; they may be more frequent in children than in adults. Virus excretion in asymptomatic people and in the pre-symptomatic phase of an infection is relevant for transmission- An effective antiviral therapy has not yet been established. Steroids for anti-inflammatory therapy are not recommended- It is very important to prepare all actors in the health care system for a longer-term burden of inpatients and complications and to create the necessary capacities. Low-threshold diagnostic testing and rapid detection of infection chains remain essential for better control of the pandemic. An effective vaccine is urgent.

negative: Genotypic and antigenic study of SARS-CoV-2 from an Indian isolate Coronaviruses (CoVs) are one of the largest groups of positive-sense RNA virus families within the Nidovirales order, which are further classified into four genera: alpha, beta, gamma, and delta. Coronaviruses have an extensive range of natural hosts and are known to be responsible for a broad spectrum of diseases in multiple species. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) that has unleashed a global threat to public health and the economy. Coronaviruses are extensively present in birds and mammals, with horseshoe bats (Rhinolophus affinis), being the reservoir for the ongoing SARS-CoV-2 that seems to have resulted from a zoonotic spillover to the human host, causing respiratory infections, lung injury and Acute Respiratory Distress Syndrome(ARDS). About six coronavirus serotypes are linked with the disease in humans, namely HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, SARS-CoV-2, and MERS-CoV. SARS-CoV-2 is the seventh CoV to infect humans. We analyzed the genome sequence of CoV-2 from isolates derived from China as well from India and encountered minute variations in their sequence. A cladogram analysis revealed the predominant strain circulating in India belongs to the A2a clad. We took one such strain (MT012098) and performed a rigorous in-silico genotypic and antigenic analysis to identify its relatedness to other strains. Further, we also performed a detailed prediction for B and T cell epitopes using BepiPred 2.0 server and NetCTL 1.2 server (DTU Bioinformatics), respectively. We hope this information may assist in an effective vaccine designing program against SARS-CoV-2.

negative: Computers and viral diseases. Preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the SARS-CoV-2 (2019-nCoV, COVID-19) coronavirus Abstract This paper concerns study of the genome of the Wuhan Seafood Market isolate believed to represent the causative agent of the disease COVID-19. This is to find a short section or sections of viral protein sequence suitable for preliminary design proposal for a peptide synthetic vaccine and a peptidomimetic therapeutic, and to explore some design possibilities. The project was originally directed towards a use case for the Q-UEL language and its implementation in a knowledge management and automated inference system for medicine called the BioIngine, but focus here remains mostly on the virus itself. However, using Q-UEL systems to access relevant and emerging literature, and to interact with standard publically available bioinformatics tools on the Internet, did help quickly identify sequences of amino acids that are well conserved across many coronaviruses including 2019-nCoV. KRSFIEDLLFNKV was found to be particularly well conserved in this study and corresponds to the region around one of the known cleavage sites of the SARS virus that are believed to be required for virus activation for cell entry. This sequence motif and surrounding variations formed the basis for proposing a specific synthetic vaccine epitope and peptidomimetic agent. The work can, nonetheless, be described in traditional bioinformatics terms, and readily reproduced by others, albeit with the caveat that new data and research into 2019-nCoV is emerging and evolving at an explosive pace. Preliminary studies using molecular modeling and docking, and in that context the potential value of certain known herbal extracts, are also described.

negative: Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine [Image: see text] Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.

negative: SARS-CoV-2 SPIKE PROTEIN: an optimal immunological target for vaccines COVID-19 has rapidly spread all over the world, progressing into a pandemic. This situation has urgently impelled many companies and public research institutes to concentrate their efforts on research for effective therapeutics. Here, we outline the strategies and targets currently adopted in developing a vaccine against SARS-CoV-2. Based on previous evidence and experience with SARS and MERS, the primary focus has been the Spike protein, considered as the ideal target for COVID-19 immunotherapies.

negative: 3CL Hydrolase Based Multi Epitope Peptide Vaccine Against Sars-CoV-2 Using Immunoinformatics. The present study provides the first multi-epitope vaccine construct using 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This research was based on immunoinformatics and structural vaccinology strategies. The design of the multi epitope vaccine was built using HTLs and CTLs epitopes from 3CL hydrolase protein along with adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and Interferon-γ inducing epitopes. Epitopes of the constructed multi epitope vaccine was found to be antigenic, nonallergic, nontoxic, and cover large human population worldwide. The vaccine construct was modeled, validated and refined by different programs to achieve high-quality 3D structure. The resulting high-quality model was applied for conformational B cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of vaccine to elicit an immune response. Insilico cloning and Codon adaptation were also performed into the pET-19b plasmid vector. The designed multi-epitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection. This article is protected by copyright. All rights reserved.

negative: Direct High‐Throughput Screening Assay for mRNA Cap Guanine‐N7 Methyltransferase Activity In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5′ cap biosynthesis, involving RNA cap guanine‐N7 methyltransferase (N7‐MTase). N7‐MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7‐MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7‐MTase activity assay based on small‐molecule fluorescent probes. We synthesized 12 fluorescent substrate analogs (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3′‐O position of adenosine acted as an artificial substrate with properties of a turn‐off probe for all three tested N7‐MTases (human, parasite, and viral). Using this compound, a high‐throughput N7‐MTase inhibitor assay was developed and used for screening of synthetic substrate analogs and a commercial library. Several inhibitors with nanomolar activities were identified.

negative: Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis We developed a label-free, real-time, and highly sensitive nucleic acid biosensor based on fiber optic particle plasmon resonance (FOPPR). The biosensor employs a single-strand deoxyoligonucleotides (ssDNA) probe, conjugated to immobilized gold nanoparticles on the core surface of an optical fiber. We explore the steric effects on hybridization affinity and limit of detection (LOD), by using different ssDNA probe designs and surface chemistries, including diluent molecules of different lengths in mixed self-assembled monolayers, ssDNA probes of different oligonucleotide lengths, ssDNA probes in different orientations to accommodate target oligonucleotides with a hybridization region located unevenly in the strand. Based on the optimized ssDNA probe design and surface chemistry, we achieved LOD at sub-nM level, which makes detection of target oligonucleotides as low as 1 fmol possible in the 10-μL sensor chip. Additionally, the FOPPR biosensor shows a good correlation in determining HLA-B27 mRNA, in extracted blood samples from patients with ankylosing spondylitis (AS), with the clinically accepted real-time reverse transcription-polymerase chain reaction (RT-PCR) method. The results from this fundamental study should guide the design of ssDNA probe for anti-sense sensing. Further results through application to HLA-B27 mRNA detection illustrate the feasibility in detecting various nucleic acids of chemical and biological relevance.

negative: Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine. We observe remarkably high and dose-dependent SARS-CoV-2 specific antibody titers in mouse sera, as well as robust neutralization of both a pseudo-virus and wild-type virus. Upon further characterization we find that the neutralization is proportional to the quantity of specific IgG and of higher magnitude than recovered COVID-19 patients. saRNA LNP immunizations induce a Th1-biased response in mice, and there is no antibody-dependent enhancement (ADE) observed. Finally, we observe high cellular responses, as characterized by IFN-γ production, upon re-stimulation with SARS-CoV-2 peptides. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.

negative: Origin, Potential Therapeutic Targets and Treatment for Coronavirus Disease (COVID-19) The ongoing episode of coronavirus disease 19 (COVID-19) has imposed a serious threat to global health and the world economy. The disease has rapidly acquired a pandemic status affecting almost all populated areas of the planet. The causative agent of COVID-19 is a novel coronavirus known as SARS-CoV-2. The virus has an approximate 30 kb single-stranded positive-sense RNA genome, which is 74.5% to 99% identical to that of SARS-CoV, CoV-pangolin, and the coronavirus the from horseshoe bat. According to available information, SARS-CoV-2 is inferred to be a recombinant virus that originated from bats and was transmitted to humans, possibly using the pangolin as the intermediate host. The interaction of the SARS-CoV-2 spike protein with the human ACE2 (angiotensin-converting enzyme 2) receptor, and its subsequent cleavage by serine protease and fusion, are the main events in the pathophysiology. The serine protease inhibitors, spike protein-based vaccines, or ACE2 blockers may have therapeutic potential in the near future. At present, no vaccine is available against COVID-19. The disease is being treated with antiviral, antimalarial, anti-inflammatory, herbal medicines, and active plasma antibodies. In this context, the present review article provides a cumulative account of the recent information regarding the viral characteristics, potential therapeutic targets, treatment options, and prospective research questions.

negative: Towards a Coronavirus-Based HIV Multigene Vaccine Human immunodeficiency virus (HIV) infection represents one of the major health threats in the developing world. The costly treatment of infected individuals with multiple highly efficient anti-HIV drugs is only affordable in industrialized countries. Thus, an efficient vaccination strategy is required to prevent the further spread of the infection. The molecular biology of coronaviruses and particular features of the human coronavirus 229E (HCoV 229E) indicate that HCoV 229E-based vaccine vectors can become a new class of highly efficient vaccines. First, the receptor of HCoV 229E, human aminopeptidase N (hAPN or CD13) is expressed mainly on human dendritic cells (DCs) and macrophages indicating that targeting of HCoV 229E-based vectors to professional antigen presenting cells can be achieved by receptor-mediated transduction. Second, HCoV 229E structural genes can be replaced by multiple transcriptional units encoding various antigens. These virus-like particles (VLPs) containing HCoV 229E-based vector RNA have the ability to transduce human DCs and to mediate heterologous gene expression in these cells. Finally, coronavirus infections are associated with mainly respiratory and enteric diseases, and natural transmission of coronaviruses occurs via mucosal surfaces. In humans, HCoV 229E causes common cold by infecting the upper respiratory tract. HCoV 229E infections are mainly encountered in children and re-infection occurs frequently in adults. It is thus most likely that pre-existing immunity against HCoV 229E will not significantly impact on the vaccination efficiency if HCoV 229E-based vectors are used in humans.

negative: The SARS-CoV-2 receptor ACE2 expression of maternal-fetal interface and fetal organs by single cell transcriptome study The new type of pneumonia caused by the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has been declared as a global public health concern by WHO. Thousands of human infections have been diagnosed in China along with many other countries, which exhibited apparent person-to-person transmission characteristics of this virus. The capacity of vertical transmission in SARS-CoV-2 remains controversial recently. Angiotensin-converting enzyme 2 (ACE2) is now confirmed as the receptor of SARS-CoV-2 and plays essential roles in human infection and transmission. In present study, we collected the online available single-cell RNA sequencing (scRNA-seq) data to evaluate the cell specific expression of ACE2 in maternal-fetal interface as well as in multiple fetal organs. Our results revealed that ACE2 was highly expressed in maternal-fetal interface cells including stromal cells and perivascular cells of decidua, and cytotrophoblast and syncytiotrophoblast in placenta. Meanwhile, ACE2 was also expressed in specific cell types of human fetal heart, liver and lung, but not in kidney. And in a study containing series fetal and post-natal mouse lung, we observed ACE2 was dynamically changed over the time, and ACE2 was extremely high in neonatal mice at post-natal day 1~3. In summary, this study revealed that the SARS-CoV-2 receptor ACE2 was widely spread in specific cell types of maternal-fetal interface and fetal organs. Although previous clinical studies have not observed vertical transmission of SARS-COV-2 among limited cases, this phenomenon still needs to be further carefully investigated in clinical practice.

negative: Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2 SARS-CoV-2 is the causative agent of the current COVID-19 pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1) which suppresses host gene expression by ribosome association via an unknown mechanism. Here, we show that Nsp1 from SARS-CoV-2 binds to 40S and 80S ribosomes, resulting in shutdown of capped mRNA translation both in vitro and in cells. Structural analysis by cryo-electron microscopy (cryo-EM) of in vitro reconstituted Nsp1-40S and of native human Nsp1-ribosome complexes revealed that the Nsp1 C-terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks RIG-I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

negative: A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

negative: Novel Coronavirus (nCoV): a Bitter Old Enemy in a New Avatar Currently, pandemic coronavirus disease 2019 (COVID-19) is the biggest threat to all human beings globally. Till June 8, 2020, it has infected 6,931,000 people and caused 400,857 deaths worldwide. The first case was identified in a patient with influenza-like symptoms along with severe acute respiratory syndrome in Wuhan, China, in December 2019 and now it has spread in more than 200 countries. Since there is no approved cure for this disease until now, there is a lot of mass fear, apprehensions, and questions globally regarding (i) genetic origin and history of the novel coronavirus, (ii) what are the first-line therapies for those who contract this disease, and (iii) what could be the potential vaccine targets. In this short review, we have tried to address these queries in the simplest manner and compiled the history of previous coronaviruses, recent developments in the COVID-19 research, potential future therapeutics, and possible targets to cure the disease.

negative: Identification of peptide candidate against COVID-19 through reverse vaccinology: An immunoinformatics approach Novel corona virus disease 2019 (COVID-19) is emerging as a pandemic situation and declared as a global health emergency by WHO. Due to lack of specific medicine and vaccine, viral infection has gained a frightening rate and created a devastating state across the globe. Here the authors have attempted to design epitope based potential peptide as a vaccine candidate using immunoinformatics approach. As of evidence from literatures, SARS-CoV-2 Spike protein is a key protein to initiate the viral infection within a host cell thus used here as a reasonable vaccine target. We have predicted a 9-mer peptide as representative of both B-cell and T-cell epitopic region along with suitable properties such as antigenic and non-allergenic. To its support, strong molecular interaction of the predicted peptide was also observed with MHC molecules and Toll Like receptors. The present study may helpful to step forward in the development of vaccine candidates against COVID-19.

negative: Review of COVID-19 Antibody Therapies Under the global health emergency caused by coronavirus disease 2019 (COVID-19), efficient and specific therapies are urgently needed. Compared with traditional small-molecular drugs, antibody therapies are relatively easy to develop and as specific as vaccines in targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and thus attract much attention in the past few months. This work reviews seven existing antibodies for SARS-CoV-2 spike (S) protein with three-dimensional (3D) structures deposited in the Protein Data Bank. Five antibody structures associated with SARS-CoV are evaluated for their potential in neutralizing SARS-CoV-2. The interactions of these antibodies with the S protein receptor-binding domain (RBD) are compared with those of angiotensin-converting enzyme 2 (ACE2) and RBD complexes. Due to the orders of magnitude in the discrepancies of experimental binding affinities, we introduce topological data analysis (TDA), a variety of network models, and deep learning to analyze the binding strength and therapeutic potential of the aforementioned fourteen antibody-antigen complexes. The current COVID-19 antibody clinical trials, which are not limited to the S protein target, are also reviewed.

negative: Recombinant Live Vaccines to Protect Against the Severe Acute Respiratory Syndrome Coronavirus The severe acute respiratory syndrome (SARS) coronavirus (CoV) was identified as the etiological agent of an acute respiratory disease causing atypical pneumonia and diarrhea with high mortality. Different types of SARS-CoV vaccines, including nonreplicative and vectored vaccines, have been developed. Administration of these vaccines to animal model systems has shown promise for the generation of efficacious and safe vaccines. Nevertheless, the identification of side effects, preferentially in the elderly animal models, indicates the need to develop novel vaccines that should be tested in improved animal model systems. Live attenuated viruses have generally proven to be the most effective vaccines against viral infections. A limited number of SARS-CoV attenuating modifications have been described, including mutations, and partial or complete gene deletions affecting the replicase, like the nonstructural proteins (nsp1 or nsp2), or the structural genes, and drastic changes in the sequences that regulate the expression of viral subgenomic mRNAs. A promising vaccine candidate developed in our laboratory was based on deletion of the envelope E gene alone, or in combination with the removal of six additional genes nonessential for virus replication. Viruses lacking E protein were attenuated, grew in the lung, and provided homologous and heterologous protection. Improvements of this vaccine candidate have been directed toward increasing virus titers using the power of viruses with mutator phenotypes, while maintaining the attenuated phenotype. The safety of the live SARS-CoV vaccines is being increased by the insertion of complementary modifications in genes nsp1, nsp2, and 3a, by gene scrambling to prevent the rescue of a virulent phenotype by recombination or remodeling of vaccine genomes based on codon deoptimization using synthetic biology. The newly generated vaccine candidates are very promising, but need to be evaluated in animal model systems that include young and aged animals.

negative: COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses The coronavirus disease 19 (COVID-19) is a highly transmittable and pathogenic viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in Wuhan, China and spread around the world. Genomic analysis revealed that SARS-CoV-2 is phylogenetically related to severe acute respiratory syndrome-like (SARS-like) bat viruses, therefore bats could be the possible primary reservoir. The intermediate source of origin and transfer to humans is not known, however, the rapid human to human transfer has been confirmed widely. There is no clinically approved antiviral drug or vaccine available to be used against COVID-19. However, few broad-spectrum antiviral drugs have been evaluated against COVID-19 in clinical trials, resulted in clinical recovery. In the current review, we summarize and comparatively analyze the emergence and pathogenicity of COVID-19 infection and previous human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). We also discuss the approaches for developing effective vaccines and therapeutic combinations to cope with this viral outbreak.

negative: SARS-CoV-2 neutralizing antibody development strategies In December 2019 a novel coronavirus was detected in Wuhan City of Hubei Province-China. Owing to a high rate of transmission from human to human, the new virus called SARS-CoV-2 differed from others by its unexpectedly rapid spread. The World Health Organization (WHO) described the most recent coronavirus epidemic as a global pandemic in March 2020. The virus spread triggered a health crisis (the COVID-19 disease) within three months, with socioeconomic implications. No approved targeted-therapies are available for COVID-19, yet. However, it is foreseen that antibody-based treatments may provide an immediate cure for patients. Current neutralizing antibody development studies primarily target the S protein among the structural elements of SARS-CoV-2, which mediates the cell entry of the virus through the angiotensin converting enzyme 2 (ACE2) receptor of host cells. This review aims to provide some of the neutralizing antibody development strategies for SARS-CoV-2 and in vitro and in vivo neutralization assays.

negative: Can existing live vaccines prevent COVID-19?

negative: Rapid and robust bioanalytical assays are critical for SARS-CoV-2 therapeutic and vaccine development and beyond

negative: SARS-CoV-2, from its current highly contagious spreading toward the global development of an effective and safe vaccine: challenges and uncertainties

negative: Expression of the COVID‐19 receptor ACE2 in the human conjunctiva SARS‐CoV‐2 is assumed to use angiotensin‐converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory‐confirmed SARS‐CoV‐2, and there has been speculation that SARS‐CoV‐2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctiva, 12 melanoma, 7 squamous cell carcinoma and 7 papilloma samples, were analyzed using high‐throughput RNA sequencing to assess mRNA expression of the SARS‐CoV‐2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS‐CoV‐2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median 0.0 transcripts per million (TPM), min 0.0 TPM, max 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS‐CoV‐2 via these mediators unlikely. This article is protected by copyright. All rights reserved.

negative: Does the Bacillus Calmette–Guérin vaccine provide protection from COVID-19? OBJECTIVE: The novel coronavirus pandemic is ravaging throughout the world. It has infected more than 1.2 million people and killed more than 64,000. Frantic research is underway to find prevention and cure. Of late, Bacillus Calmette–Guérin (BCG) has been speculated as a possible protection from COVID-19. We sought to investigate the evidence behind the claim. MATERIAL AND METHODS: Data were collected regarding the total number of COVID-19 cases per million and total number of COVID-19 deaths per million in various countries. The BCG vaccination policies of these countries were also obtained. RESULTS: It was seen that the countries with no universal BCG policy had a mean 1272.9 (median 795) cases per million and 80.7 deaths (median 18) per million population. On the contrary, the countries with a universal BCG vaccination policy had a mean 131.2 (median 40) cases per million and 4 deaths (median 1) per population. The difference is highly significant (P < 0.001). CONCLUSION: The data strongly support the hypothesis that BCG may offer protection from COVID-19. Heterologous protection offered by BCG through production of trained immunity, epigenetic reprogramming of monocytes, non-specific activation of NK cells, and increase of pro-inflammatory cytokines (particularly, tumor necrosis factor [TNF]-alpha and interleukin 1 beta) production may be the mechanism behind its cross- protection against the novel coronavirus.

negative: Opportunities, Challenges and Directions in Science and Technology for Tackling COVID-19 The ongoing global crisis due to Coronavirus disease-2019 (COVID-19) pandemic has caused an enormous socioeconomic burden. A novel coronavirus causing severe acute respiratory syndrome (SARS-CoV-2) that evolved from a virus infecting bats is responsible for COVID-19, first reported in the Chinese city of Wuhan. In the absence of any specific scientifically proven and clinically tested drug or vaccine against SARS-CoV-2, the virus is wreaking havoc across the world, claiming more than 2,50,000 lives in less than 5 months, and posed a global health emergency. The scientific community is relentlessly working on the design and testing of vaccines and antiviral drugs against the novel coronavirus, several of which have reached advanced stages of testing and are undergoing clinical trials. Here we discuss the recent advances and developments in understanding the etiology and epidemiology of the COVID-19 pandemic, the factors influencing the disease transmission, and the countermeasures adopted to combat and stop further spread of the disease.

negative: Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates The COVID-19 outbreak has become a global pandemic responsible for over 2,000,000 confirmed cases and over 126,000 deaths worldwide. In this study, we examined the immunogenicity of CHO-expressed recombinant SARS-CoV-2 S1-Fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a COVID-19 vaccine. We demonstrate that the S1-Fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day 7. Strong virus neutralizing activity was observed on day 14 in rabbits immunized with the S1-Fc fusion protein using a pseudovirus neutralization assay. Most importantly, in <20 days and three injections of the S1-Fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered COVID-19 patient in a live SARS-CoV-2 infection assay. Our data strongly suggests that the CHO-expressed SARS-CoV-2 S1-Fc recombinant protein could be a strong candidate for vaccine development against COVID-19.

negative: In silico identification of vaccine targets for 2019-nCoV Background: The newly identified coronavirus known as 2019-nCoV has posed a serious global health threat. According to the latest report (18-February-2020), it has infected more than 72,000 people globally and led to deaths of more than 1,016 people in China. Methods: The 2019 novel coronavirus proteome was aligned to a curated database of viral immunogenic peptides. The immunogenicity of detected peptides and their binding potential to HLA alleles was predicted by immunogenicity predictive models and NetMHCpan 4.0. Results: We report in silico identification of a comprehensive list of immunogenic peptides that can be used as potential targets for 2019 novel coronavirus (2019-nCoV) vaccine development. First, we found 28 nCoV peptides identical to Severe acute respiratory syndrome-related coronavirus (SARS CoV) that have previously been characterized immunogenic by T cell assays. Second, we identified 48 nCoV peptides having a high degree of similarity with immunogenic peptides deposited in The Immune Epitope Database (IEDB). Lastly, we conducted a de novo search of 2019-nCoV 9-mer peptides that i) bind to common HLA alleles in Chinese and European population and ii) have T Cell Receptor (TCR) recognition potential by positional weight matrices and a recently developed immunogenicity algorithm, iPred, and identified in total 63 peptides with a high immunogenicity potential. Conclusions: Given the limited time and resources to develop vaccine and treatments for 2019-nCoV, our work provides a shortlist of candidates for experimental validation and thus can accelerate development pipeline.

negative: Feasibility of Known RNA Polymerase Inhibitors as Anti-SARS-CoV-2 Drugs Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.

negative: If a coronavirus vaccine arrives, can the world make enough?

negative: Priming with rAAV encoding RBD of SARS-CoV S protein and boosting with RBD-specific peptides for T cell epitopes increase T cell responses and provide protection against SRAS-CoV infection Development of vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is crucial in the prevention of SARS reemergence. The receptor-binding domain (RBD) of SARS-CoV spike (S) protein is an important target in developing safe and effective SARS vaccines. Our previous study has demonstrated that vaccination with adeno-associated virus encoding RBD (RBD-rAAV) induces high titer of neutralizing antibodies. In this study, we further assessed the immune responses and protective effect of the immunization with RBD-rAAV prime/RBD-specific T cell peptide boost, as compared to those of vaccinations with RBD-rAAV and RBD-peptides alone. Our results indicated that RBD-rAAV prime/RBD-peptide boost induced similar Th1 and neutralizing antibody responses, but stronger Th2 and CTL responses than RBD-rAAV prime/boost. The resulting immune responses protected the vaccinated mice from subsequent SARS-CoV challenge, which was evidenced by lower level of viral replication in mouse lung tissues. However, no significant immune responses and protective effect were detected in mice vaccinated with RBD-peptides or blank AAV alone. Since T cell epitopes are highly conserved and boosting with peptides may induce the production of effector memory T cells, our results suggest that the vaccination protocol used may be ideal for providing effective, universal and long-term protection against SARS-CoV infection.

negative: The line starts to form for a coronavirus vaccine

negative: SARS-CoV-2 is not detectable in the vaginal fluid of women with severe COVID-19 infection BACKGROUND: Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is mainly spread through respiratory droplets or direct contact. But the infection condition of genital system is still unknown. This study aimed to evaluate whether or not SARS-CoV-2 is found in the vaginal fluid of women with COVID-19 illness. METHODS: 10 women with confirmed severe COVID-19 pneumonia admitted to in Tongji Zhongfa Hospital Intensive care union(ICU) ward from Feb 4, 2020 to Feb 24, 2020 were included. Clinical records, laboratory results, and computer tomography(CT)-scan examination were retrospectively reviewed. The evidence of genital infection potential was accessed by testing for the presence of SARS-CoV-2 in vaginal fluids obtained from vaginal swab samples. Reverse transcriptase polymerase chain reaction(RT-PCR) was used to confirm the SARS-CoV-2 infection in vaginal fluids. RESULTS: The clinical characteristics of these ten women were similar to those reported severe COVID-19 patients. All ten patients were tested for SARS-CoV-2 in vaginal fluid, and all samples tested negative for the virus. CONCLUSION: Findings from this small group of cases suggest that no SARS-CoV-2 virus existing in the vaginal fluids of severe COVID-19 patients.

negative: Elucidation of Cellular Targets and Exploitation of the Receptor Binding Domain of SARS‐CoV‐2 for vaccine and monoclonal antibody synthesis The pandemic caused by novel severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) has resulted in over 452,822 deaths in the first twenty days of June 2020 due to the coronavirus virus disease 2019 (COVID‐19). The SARS‐CoV‐2 uses the host angiotensin‐converting enzyme 2 (ACE2) receptor to gain entry inside the human cells where it replicates by using the cell protein synthesis mechanisms. The knowledge of the tissue distribution of ACE2 in human organs is therefore important to predict the clinical course of the COVID‐19. Also important is the understanding of the viral receptor‐binding domain (RBD) a region within the spike (S) proteins that enable the entry of the virus into the host cells to synthesize vaccine and monoclonal antibodies (mAbs). We performed an exhaustive search of human protein databases to establish the tissues that express ACE2 and performed an in‐depth analysis like sequence alignments and homology modeling of the spike protein (S) of the SARS‐CoV‐2 to identify antigenic regions in the RBD that can be exploited to synthesize vaccine and mAbs. Our results show that ACE2 is widely expressed in human organs that may explain the pulmonary, systemic, and neurological deficits seen in COVID‐19 patients. We show that though the S protein of the SARS‐CoV‐2 is a homolog of S protein of SARS‐CoV‐1, it has regions of dissimilarities in the RBD and transmembrane segments. We show peptide sequences in the RBD of SARS‐CoV‐2 that can bind to the MHC alleles and serve as effective epitopes for vaccine and mAbs synthesis. This article is protected by copyright. All rights reserved.

negative: Public Health Aspects of COVID-19 Infection with Focus on Cardiovascular Diseases INTRODUCTION: COVID-19 is the disease caused by an infection of the SARS-CoV-2 virus, first identified in the city of Wuhan, in China’s Hubei province in December 2019. COVID-19 was previously known as 2019 Novel Coronavirus (2019-nCoV) respiratory disease before the World Health Organization (WHO) declared the official name as COVID-19 in February 2020. AIM: The aim of this study is to search scientific literature in the biomedicine and analyzed current results of investigations regarding morbidity and mortality rates as consequences of COVID-19 infection of Cardiovascular diseases (CVD), and other most common chronic diseases which are on the top mortality and morbidity rates in almost all countries in the world. Also, to propose most useful measures how to prevent patients to keep themselves against COVID-19 infection. METHODS: We used method of descriptive analysis of the published papers with described studies about Corona virus connected with CVD, and, also, Guidelines proposed by World Health Organization (WHO) and European Society of Cardiology (ESC), and some other international associations which are included in global fighting against COVID-19 infection. RESULTS: After searching current scientific literature we have acknowledged that not any Evidence Based Medicine (EBM) study in the world during last 5 months from the time when first cases of COVID-10 infection was detected. Also, there is no unique proposed ways of treatments and drugs to protect patients, especially people over 65 years old, who are very risk group to be affected with COVID-19. Expectations that vaccine against COVID-19 will be produced optimal during at least 10 months to 2 years, and in all current Guidelines most important proposed preventive measures are the same like which one described in Strategic documents of WHO, in statements of Declaration of Primary Health Care in Alma Ata in 1978. CONCLUSION: WHO proposed preventive measures can be helpful to everybody. Physicians who work at every level of Health Care Systems, but especially at primary health care level, must follow those recommendations and teach their patients about it. But, the fact is that current focus of COVID-19 epidemic has targeted on protection of physical health of population in global, however, the influence on mental health which will be one of the important consequences of COVID-19 pandemic in the future, and which could be declared as «Post-coronavirus Stress Syndrome„ (PCSS) could be bigger challenge for Global Public Health.

negative: Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.

negative: Could sudden sensorineural hearing loss be the sole manifestation of COVID-19? An investigation into SARS-COV-2 in the etiology of sudden sensorineural hearing loss Abstract Objective The aim of this study is to investigate the presence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in patients presenting with only sudden sensorineural hearing loss (SSNHL) during the pandemic. Methods The study included 5 male patients who presented with the sole complaint of unilateral SSNHL to the otolaryngology outpatient clinic between April 3 and April 12, 2020. The patients were referred to the infectious diseases clinic to be evaluated for SARS-CoV-2 by using real time polymerase chain reaction (RT-PCR) testing. Results RT-PCR testing for SARS-CoV-2 were positive in one of the patient and negative in rest four patients. We also noted positive response to Coronavirus Disease-19 (COVID-19) specific treatment in SARS-CoV-2 positive SSNHL patient. Conclusion It should be kept in mind that non-specific symptoms such as SSNHL could be the only sign to recognize a COVID-19 case. Awareness of such a non-specific presentation of COVID-19 patients is crucial during this pandemic period for the prevention of infectious spread through isolation and early initiation of COVID-19 targeted treatment.

negative: Humoral and cellular immune responses induced by 3a DNA vaccines against severe acute respiratory syndrome (SARS) or SARS-like coronavirus in mice. Vaccine development for severe acute respiratory syndrome coronavirus (SARS-CoV) has mainly focused on the spike (S) protein. However, the variation of the S gene between viruses may affect the efficacy of a vaccine, particularly for cross-protection against SARS-like CoV (SL-CoV). Recently, a more conserved group-specific open reading frame (ORF), the 3a gene, was found in both SARS-CoV and SL-CoV. Here, we studied the immunogenicity of human SARS-CoV 3a and bat SL-CoV 3a DNA vaccines in mice through electroporation immunization followed by enzyme-linked immunosorbent, enzyme-linked immunospot, and flow cytometry assays. Our results showed that high levels of specific humoral responses were induced by SARS-CoV 3a and SL-CoV 3a DNA vaccines. Furthermore, a strong Th1-based cellular immune response was stimulated by both DNA vaccines. The vaccines stimulated gamma interferon production mainly by CD8(+) T cells and interleukin-2 (IL-2) mainly by CD4(+) T cells. Of interest, the frequency of IL-2-positive cells elicited by the SARS-CoV 3a DNA vaccine was significantly higher than that elicited by the SL-CoV 3a DNA vaccine. In summary, our study provides a reference for designing cross-protective DNA vaccines based on the group-specific ORFs of CoVs.

negative: Off-label therapy targeting pathogenic inflammation in COVID-19

negative: Novel decoy cellular vaccine strategy utilizing transgenic antigen-expressing cells as immune presenter and adjuvant in vaccine prototype against SARS-CoV-2 virus Abstract A novel approach modifying cells to express viral markers to elicit protective immunity responses (decoy cellular vaccination) in the prevention of COVID-19 disease is currently being explored. Our approach entails utilizing SARS-CoV-2 Spike antigen-expressing, non-replicating cells as carriers and presenters of immunogenic antigens, so called “I-cells”. By using irradiated cells as presenting vehicles of SARS-CoV-2 viral antigens(s) in a cellular context, these presented viral proteins can be recognized by the host immune system, thus, an efficient protective immune response might be elicited. Another advantage of this strategy is that the manufacturing process is scalable and yields uniform cell products allowing for “off-the-shelf” frozen supply availability. To prevent engraftment and proliferation of the cells after administration, the cells will be irradiated post-harvesting abolishing in vivo replication potential. Specifically, immunoreactive Spike-1 proteins from SARS-CoV-2 are expressed on the surface of irradiated target I-cells. Utilizing this innovative strategy, these viral antigen-displaying decoy cells will be developed as a vaccine to protect against COVID-19 disease.

negative: Hypothesis for the management and treatment of the COVID-19-induced acute respiratory distress syndrome and lung injury using mesenchymal stem cell-derived exosomes Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the coronaviridae that causes respiratory disorders. After infection, large amounts of inflammatory cytokines are secreted, known as the cytokine storm. These cytokines can cause pulmonary damage induced by inflammation resulting in acute respiratory distress syndrome (ARDS), and even death. One of the therapeutic approaches for treatment of ARDS is a mesenchymal stem cell (MSC). MSCs suppress inflammation and reduce lung injury through their immunomodulatory properties. MSCs also have the potential to prevent apoptosis of the lung cells and regenerate them. But our suggestion is using MSCs-derived exosomes. Because these exosomes apply the same immunomodulatory and tissue repair effects of MSCs and they don’t have problems associated to cell maintenance and injections. For investigation the hypothesis, MSCs should be isolated from tissues and characterized. Then, the exosomes should be isolated from the supernatants and characterized. These exosomes should be injected into a transgenic animal for COVID-19. In the final section, lung function assessment, histological examination, micro-CT, differential leukocyte, viral load analysis, cytokine assay, and CRP level analysis can be investigated. COVID-19 treatment is currently focused on supportive therapies and no vaccine has not been developed for it, so, numerous research studies are needed to find potential. Since the pathogenesis of this disease was identified in previous studies and can cause lung injury with ARDS, so, investigation of the therapeutic approaches that can suppress inflammation, cytokine storm and ARDS can be helpful in finding a novel therapeutic approach for this disease.

negative: Linear epitope landscape of SARS-CoV-2 Spike protein constructed from 1,051 COVID-19 patients Neutralization antibodies and vaccines for treating COVID-19 are desperately needed. For precise development of antibodies and vaccines, the key is to understand which part of SARS-CoV-2 Spike protein is highly immunogenic on a systematic way. We generate a linear epitope landscape of Spike protein by analyzing serum IgG response of 1,051 COVID-19 patients with a peptide microarray. We reveal two regions that rich of linear epitopes, i.e., CTD and a region close to the S2' cleavage site and fusion peptide. Unexpectedly, we find RBD is lack of linear epitope. Besides 3 moderate immunogenic peptides from RBD, 16 highly immunogenic peptides from other regions of Spike protein are determined. These peptides could serve as the base for precise development of antibodies and vaccines for COVID-19 on a systematic level.

negative: SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).

negative: Development of potential COVID-19 vaccines continues to accelerate

negative: Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNFα, CXCL8. We also identified NF-κB and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.

negative: Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector Recombinant vesicular stomatitis virus (rVSV) is currently under evaluation as a human immunodeficiency virus (HIV)-1 vaccine vector. The most compelling reasons to develop rVSV as a vaccine vector include a very low seroprevalence in humans, the ability to infect and robustly express foreign antigens in a broad range of cells, and vigorous growth in continuous cell lines used for vaccine manufacture. Numerous preclinical studies with rVSV vectors expressing antigens from a variety of human pathogens have demonstrated the versatility, flexibility, and potential efficacy of the rVSV vaccine platform. When administered to nonhuman primates (NHPs), rVSV vectors expressing HIV-1 Gag and Env elicited robust HIV-1-specific cellular and humoral immune responses, and animals immunized with rVSV vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env were protected from AIDS after challenge with a pathogenic SIV/HIV recombinant. However, results from an exploratory neurovirulence study in NHPs indicated that these prototypic rVSV vectors might not be adequately attenuated for widespread use in human populations. To address this safety concern, a variety of different attenuation strategies, designed to produce a range of further attenuated rVSV vectors, are currently under investigation. Additional modifications of further attenuated rVSV vectors to upregulate expression of HIV-1 antigens and coexpress molecular adjuvants are also being developed in an effort to balance immunogenicity and attenuation.

negative: Age-related expression of SARS-CoV-2 priming protease TMPRSS2 in the developing lung The emergence of the SARS-CoV-2 novel coronavirus has led to a global pandemic (COVID-19), with more than 5 million cases as of May 2020(1). Available data suggest that severe illness and death from COVID-19 are rare in the pediatric population(2). Integrating single-cell RNA sequencing of the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) in mouse and human lung tissue, we found expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1) and increased with aging in mice and humans. SARS-CoV-2 RNA colocalized with TMPRSS2 mRNA in lung cells from a patient who died of SARS-CoV-2. Together, these data suggest developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.

negative: TMPRSS2, a SARS-CoV-2 internalization protease is downregulated in head and neck cancer patients Objectives Two of the main target tissues of SARS-coronavirus 2 are the oral cavity pharynx-larynx epithelium, the main virus entry site, and the lung epithelium. The virus enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. Herein we aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity-pharynx-larynx and lung tissues as well as neoplastic tissues from the same histological areas. The information provided in this study may contribute to better understanding of SARS-coronavirus 2 ability to interact with different biological systems and contributes to cumulative knowledge on potential mechanisms to inhibit its diffusion. Materials and Methods The study has been conducted using The Cancer Genome Atlas (TCGA) and the Regina Elena Institute (IRE) databases and validated by experimental model in HNSCC and Lung cancer cells. Data from one COVID19 positive patient who was operated on for HNSCC was also included. We have analyzed 478 tumor samples and 44 normal samples from TCGA HNSCC cohort for whom both miRNA and mRNA sequencing was available. The dataset included 391 HPV- and 85 HPV+ cases, with 331 P53 mutated and 147 P53 wild type cases respectively. 352 out of 478 samples were male and 126 female. In IRE cohort we analyzed 66 tumor samples with matched normal sample for miRNA profiling and 23 tumor\normal matched samples for mRNA profiling. 45 out of 66 tumors from IRE cohort were male and 21 female, 38 were P53 mutated and 27 wild type. Most patients (63 of 66) in IRE cohort were HPV negative. Normalized TCGA HNSCC gene expression and miRNA expression data were obtained from Broad Institute TCGA Genome Data Analysis Center (http://gdac.broadinstitute.org/). mRNA expression data from IRE cohort used in this study has been deposited to NCBI’s Gene Expression Omnibus and is accessible through GEO series accession number GSE107591. In order to inference about potential molecular modulation of TMPRSS2, we also included miRNAs expression for the 66 IRE cohort matched tumor and normal samples from Agilent platform. DNA methylation data for TCGA tumors were obtained from Wanderer (http://maplab.imppc.org/wanderer/). We used miRWalk and miRNet web tools for miRNA-target interaction prediction and pathway enrichment analysis. The correlation and regression analyses as well as the miRNA and gene modulation and the survival analysis were conducted using Matlab R2019. Results TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues and had a prognostic value in HNSCC patients. Reduction of TMPRSS2 expression was more evident in women than in men, in TP53 mutated versus wild TP53 tumors as well as in HPV negative patients compared to HPV positive counterparts. Functionally, we assessed the multivariate effect on TMPRSS2 in a single regression model. We observed that all variables had an independent effect on TMPRSS2 in HNSCC patients with HPV negative, TP53 mutated status and with elevated TP53-dependent Myc-target genes associated with low TMPRSS2 expression. Investigation of the molecular modulation of TMPRSS2 in both HNSCC and lung cancers revealed that expression of microRNAs targeting TMPRSS2 anti-correlated in both TCGA and IRE HNSCC datasets, while there was not evidence of TMPRSS2 promoter methylation in both tumor cohorts. Interestingly, the anti-correlation between microRNAs and TMPRSS2 expression was corroborated by testing this association in a SARS-CoV-2 positive HNSCC patient. Conclusions Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. The protective mechanism might occur, at least partially, through the aberrant activation of TMPRSS2 targeting microRNAs; thereby providing strong evidence on the role of non-coding RNA molecule in host viral infection. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.

negative: Designing of a next generation multiepitope based vaccine (MEV) against SARS-COV-2: Immunoinformatics and in silico approaches Coronavirus disease 2019 (COVID-19) associated pneumonia caused by severe acute respiratory coronavirus 2 (SARS-COV-2) was first reported in Wuhan, China in December 2019. Till date, no vaccine or completely effective drug is available for the cure of COVID-19. Therefore, an effective vaccine against SARS-COV-2 is needed to be design. This study was conducted to design an effective multi-epitope vaccine (MEV) against SARS-COV-2. Seven antigenic proteins were taken as a target and epitopes (B cell, IFN-γ and T cell) were predicted. Highly antigenic and overlapping epitopes were shortlisted. Selected T cell epitopes indicated significant interactions with the HLA-binding alleles and 99.29% coverage of the world’s population. Finally, 505 amino acids long MEV was designed by connecting sixteen MHC class I and twelve MHC class II epitopes with suitable linkers and adjuvant. Linkers and adjuvant were added to enhance the immunogenicity response of the vaccine. The allergenicity, physiochemical properties, antigenicity and structural details of MEV were analyzed in order to ensure safety and immunogenicity. MEV construct was non-allergenic and antigenic. Molecular docking demonstrated a stable and strong binding affinity of MEV with TLR3 and TLR8. Codon optimization and in silico cloning ensured increased expression in the Escherichia coli K-12 system. However, to ensure its safety and immunogenic profile, the proposed vaccine needs to be experimentally validated.

negative: Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the 120 available SARS-CoV-2 sequences (as of 21 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

negative: Fighting COVID-19: A quick review of diagnoses, therapies, and vaccines The coronavirus disease 2019 (COVID-19) pandemic caused by a novel coronavirus, SARS-CoV-2, has infected more than 4.9 million individuals and resulted in over 300,000 deaths globally. The rapid spread of the virus and the precipitously increasing numbers of cases necessitate the urgent development of accurate diagnostic methods, effective treatments, and vaccines. Here, we review the progress of developing diagnostic methods, therapies, and vaccines for SARS-CoV-2 with a focus on current clinical trials and their challenges. For diagnosis, nucleic acid amplification tests remain the mainstay diagnostics for laboratory confirmation of SARS-CoV-2 infection, while serological antibody tests are used to aid contact tracing, epidemiological, and vaccine evaluation studies. Viral isolation is not recommended for routine diagnostic procedures due to safety concerns. Currently, no single effective drug or specific vaccine is available against SARS-CoV-2. Some candidate drugs targeting different levels and stages of human responses against COVID-19 such as cell membrane fusion, RNA-dependent RNA polymerase, viral protease inhibitor, interleukin 6 blocker, and convalescent plasma may improve the clinical outcomes of critical COVID-19 patients. Other supportive care measures for critical patients are still necessary. Advances in genetic sequencing and other technological developments have sped up the establishment of a variety of vaccine platforms. Accordingly, numerous vaccines are under development. Vaccine candidates against SARS-CoV-2 are mainly based upon the viral spike protein due to its vital role in viral infectivity, and most of these candidates have recently moved into clinical trials. Before the efficacy of such vaccines in humans is demonstrated, strong international coordination and collaboration among studies, pharmaceutical companies, regulators, and governments are needed to limit further damage due the emerging SARS-CoV-2 virus.

negative: SARS-CoV-2 isolation and propagation from Turkish COVID-19 patients The novel coronavirus pneumonia, which was named later as coronavirus disease 2019 (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2, namely SARS-CoV-2. It is a positive-strand RNA virus that is the seventh coronavirus known to infect humans. The COVID-19 outbreak presents enormous challenges for global health behind the pandemic outbreak. The first diagnosed patient in Turkey has been reported by the Republic of Turkey Ministry of Health on March 11, 2020. In May, over 150,000 cases in Turkey, and 5.5 million cases around the world have been declared. Due to the urgent need for a vaccine and antiviral drug, isolation of the virus is crucial. Here, we report 1 of the first isolation and characterization studies of SARS-CoV-2 from nasopharyngeal and oropharyngeal specimens of diagnosed patients in Turkey. This study provides an isolation and replication methodology,and cell culture tropism of the virus that will be available to the research communities.

negative: Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates Abstract The COVID-19 outbreak has become a global pandemic responsible for over 2,000,000 confirmed cases and over 126,000 deaths worldwide. In this study, we examined the immunogenicity of CHO-expressed recombinant SARS-CoV-2 S1-Fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a COVID-19 vaccine. We demonstrate that the S1-Fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day 7. Strong virus neutralizing activity was observed on day 14 in rabbits immunized with the S1-Fc fusion protein using a pseudovirus neutralization assay. Most importantly, in less than 20 days and three injections of the S1-Fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered COVID-19 patient in a live SARS-CoV-2 infection assay. Our data strongly suggests that the CHO-expressed SARS-CoV-2 S1-Fc recombinant protein could be a strong candidate for vaccine development against COVID-19.

negative: Human and novel coronavirus infections in children: a review Coronaviruses, seven of which are known to infect humans, can cause a spectrum of clinical presentations ranging from asymptomatic infection to severe illness and death. Four human coronaviruses (hCoVs)-229E, HKU1, NL63 and OC43-circulate globally, commonly infect children and typically cause mild upper respiratory tract infections. Three novel coronaviruses of zoonotic origin have emerged during the past two decades: severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV) and the recently discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. These novel coronaviruses are known to cause severe illness and death predominantly in older adults and those with underlying comorbidities. Consistent with what has been observed during the outbreaks of SARS and MERS, children with COVID-19 are more likely to be asymptomatic or to have mild-to-moderate illness, with few deaths reported in children globally thus far. Clinical symptoms and laboratory and radiological abnormalities in children have been similar to those reported in adults but are generally less severe. A rare multisystem inflammatory syndrome in children (MIS-C) which has resulted in critical illness and some deaths has recently been described. Clinical trials for therapeutics and vaccine development should include paediatric considerations. Children may play an important role in the transmission of infection and outbreak dynamics and could be a key target population for effective measures to control outbreaks. The unintended consequences of the unprecedented scale and duration of pandemic control measures for children and families around the world should be carefully examined. ABBREVIATIONS: 2019-nCoV, 2019 novel coronavirus; ADEM, acute demyelinating encephalomyelitis; AAP, American Academy of Pediatrics; ACE-2, angiotensin-converting enzyme 2; ARDS, acute respiratory distress syndrome; BCG, bacillus Calmette-Guérin; BNP, brain natriuretic peptide; CDC, Centers for Disease Control and Prevention; CRP, C-reactive protein; CSF, cerebrospinal fluid; COVID-19, coronavirus disease 2019; CT, computed tomography; CXR, chest X-ray; DOL, day of life; hCoV, human coronavirus; ICU, intensive care unit; IL, interleukin; IVIG, intravenous immunoglobulin; KD, Kawasaki disease; LDH, lactate dehydrogenase; MERS, Middle East respiratory syndrome; MERS-CoV, Middle East respiratory syndrome coronavirus; MEURI, monitored emergency use of unregistered and experimental interventions; MIS-C, multi-system inflammatory syndrome in children; PCR, polymerase chain reaction; PICU, paediatric intensive care unit; RNA, ribonucleic acid; RCT, randomised-controlled trial; RSV, respiratory syncytial virus; SARS, severe acute respiratory syndrome; SARS-CoV-1, severe acute respiratory syndrome coronavirus 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-alpha, tumour necrosis factor alpha; UK United Kingdom; UNICEF, United Nations Children's Fund; USA, United States of America; WHO, World Health Organization.

negative: A Novel Vaccine Employing Non-Replicating Rabies Virus Expressing Chimeric SARS-CoV-2 Spike Protein Domains: Functional Inhibition of Viral/Nicotinic Acetylcholine Receptor Complexes The emergence of the novel ß-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic of coronavirus disease 2019 (COVID-19). Clinical studies have documented that potentially severe neurological symptoms are associated with SARS-CoV-2 infection, thereby suggesting direct CNS penetration by the virus. Prior studies have demonstrated that the destructive neurological effects of rabies virus (RABV) infections are mediated by CNS transport of the virus tightly bound to the nicotinic acetylcholine receptor (nAChR). By comparison, it has been hypothesized that a similar mechanism exists to explain the multiple neurological effects of SARS-CoV-2 via binding to peripheral nAChRs followed by orthograde or retrograde transport into the CNS. Genetic engineering of the RABV has been employed to generate novel vaccines consisting of non-replicating RABV particles expressing chimeric capsid proteins containing human immunodeficiency virus 1 (HIV-1), Middle East respiratory syndrome (MERS-CoV), Ebolavirus, and hepatitis C virus (HCV) sequences. Accordingly, we present a critical discussion that integrates lessons learned from prior RABV research and vaccine development into a working model of a SARS-CoV-2 vaccine that selectively targets and neutralizes CNS penetration of a tightly bound viral nAChR complex.

negative: mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

negative: Cold Exposure-Induced Up-Regulation of Hsp70 Positively Regulates PEDV mRNA Synthesis and Protein Expression In Vitro Porcine epidemic diarrhea (PED) is a highly contagious, intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV as an emerging and re-emerging epizootic virus of swine causes substantial economic losses to the pig industry in China and other countries. In China, the occurrence of PED shows significant seasonal variations, usually outbreak during the winter season. The epidemic characteristics of PED may be highly correlated with the changes of ambient temperature. However, molecular mechanism on the seasonal occurrence of PED still remains unclear. It has been widely observed that low ambient temperature up-regulates the expression of host heat shock protein 70 (Hsp70). Here, we showed that nucleotide and protein levels of Hsp70 were up-regulated in the intestinal of cold exposed pig and cold exposed Vero E6 cells. We found that overexpression of Hsp70 could increase PEDV mRNA synthesis and protein expression in Vero E6 and IPEC-J2 cells, while the siRNAs mediated knockdown of Hsp70 and VER155008 mediated inhibition of Hsp70 resulted in inhibition of viral mRNA synthesis and protein expression in Vero E6 cells. These data suggested that Hsp70 positively regulated PEDV mRNA synthesis and protein expression, which being helpful for understanding the seasonality of PED epidemics and development of novel antiviral therapies in the future.

negative: Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. We describe a model of severe acute respiratory syndrome-coronavirus (SARS-CoV) infection in C57BL/6 mice. A clinical isolate of the virus introduced intranasally replicated transiently to high levels in the lungs of these mice, with a peak on day 3 and clearance by day 9 postinfection. Viral RNA localized to bronchial and bronchiolar epithelium. Expression of mRNA for angiotensin converting enzyme 2, the SARS-CoV receptor, was detected in the lung following infection. The virus induced production in the lung of the proinflammatory chemokines CCL2, CCL3, CCL5, CXCL9, and CXCL10 with differential kinetics. The receptors for these chemokines were also detected. Most impressively, mRNA for CXCR3, the receptor for CXCL9 and CXCL10, was massively up-regulated in the lungs of SARS-CoV-infected mice. Surprisingly Th1 (and Th2) cytokines were not detectable, and there was little local accumulation of leukocytes and no obvious clinical signs of pulmonary dysfunction. Moreover, beige, CD1-/-, and RAG1-/- mice cleared the virus normally. Infection spread to the brain as it was cleared from the lung, again without leukocyte accumulation. Infected mice had a relative failure to thrive, gaining weight significantly more slowly than uninfected mice. These data indicate that C57BL/6 mice support transient nonfatal systemic infection with SARS-CoV in the lung, which is able to disseminate to brain. In this species, proinflammatory chemokines may coordinate a rapid and highly effective innate antiviral response in the lung, but NK cells and adaptive cellular immunity are not required for viral clearance.

negative: Debate on Bacille Calmette-Guérin vaccination against COVID-19: Is it worth performing clinical trials? The non-specific beneficial effects of Bacille Calmette-Guérin (BCG) vaccination suggest that this vaccine might play a role in protecting individuals against severe coronavirus disease (COVID-19).Several studies propose that BCG vaccination may increase the body's immunity, thereby preventing respiratory infections caused by other respiratory pathogens. As the number of deaths due to COVID-19 is increasing rapidly and there is no specific treatment available to date, scientists are evaluating the effectiveness of already approved drugs as therapies against COVID-19, and the results were found to vary widely: from no significant effect being observed to a reduction in the time taken for clinical improvement. This study thus aims to evaluate whether it is worth performing clinical trials to examine the effects of the BCG vaccine on COVID-19. We herein emphasize the need to conduct phase III randomized controlled trials with an adequate sample size and quality to investigate the effects of the BCG vaccine on COVID-19. In the event that BCG vaccination provides non-specific protection against COVID-19, administering it could be helpful in controlling the transmission of COVID-19 and other infectious diseases during future pandemics.

negative: Epitope-based peptide vaccines predicted against novel coronavirus disease caused by SARS-CoV-2 The outbreak of the 2019 novel coronavirus (SARS-CoV-2) has infected millions of people with a large number of deaths across the globe. The existing therapies are limited in dealing with SARS-CoV-2 due to the sudden appearance of the virus. Therefore, vaccines and antiviral medicines are in desperate need. We took immune-informatics approaches to identify B- and T-cell epitopes for surface glycoprotein (S), membrane glycoprotein (M) and nucleocapsid protein (N) of SARS-CoV-2, followed by estimating their antigenicity and interactions with the human leukocyte antigen (HLA) alleles. Allergenicity, toxicity, physiochemical properties analysis and stability were examined to confirm the specificity and selectivity of the epitope candidates. We identified a total of five B cell epitopes in RBD of S protein, seven MHC class-I, and 18 MHC class-II binding T-cell epitopes from S, M and N protein which showed non-allergenic, non-toxic and highly antigenic features and non-mutated in 55,179 SARS-CoV-2 virus strains until June 25, 2020. The epitopes identified here can be a potentially good candidate repertoire for vaccine development.

negative: Rational Vaccine Design in the Time of COVID-19 As scientists consider SARS-CoV-2 vaccine design, we discuss problems that may be encountered and how to tackle them by what we term “rational vaccine design.” We further discuss approaches to pan-coronavirus vaccines. We draw on experiences from recent research on several viruses including HIV and influenza, as well as coronaviruses.

negative: Use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-nCoV: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target A novel coronavirus recently identified in Wuhan, China (SARS-CoV-2) has expanded the number of highly pathogenic coronaviruses affecting humans. The SARS-CoV-2 represents a potential epidemic or pandemic threat, which requires a quick response for preparedness against this infection. The present report uses the informational spectrum methodology to identify the possible origin and natural host of the new virus, as well as putative therapeutic and vaccine targets. The performed in silico analysis indicates that the newly emerging SARS-CoV-2 is closely related to severe acute respiratory syndrome (SARS)-CoV and, to a lesser degree, Middle East respiratory syndrome (MERS)-CoV. Moreover, the well-known SARS-CoV receptor (ACE2) might be a putative receptor for the novel virus as well. Actin protein was also suggested as a host factor that participates in cell entry and pathogenesis of SARS-CoV-2; therefore, drugs modulating biological activity of this protein (e.g. ibuprofen) were suggested as potential candidates for treatment of this viral infection. Additional results indicated that civets and poultry are potential candidates for the natural reservoir of the SARS-CoV-2, and that domain 288-330 of S1 protein from the SARS-CoV-2 represents promising therapeutic and/or vaccine target.

negative: Chapter Four Lipid Nanoparticles for Short Interfering RNA Delivery Abstract The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeutics based on the reversible silencing of specific disease-causing genes. This therapeutic potential depends on the ability to deliver inducers of RNAi, such as short-interfering RNA (siRNA) and micro-RNA (miRNA), to cells of target tissues. This chapter reviews various challenges and delivery strategies for siRNA, with a particular focus on the development of lipid nanoparticle (LNP) delivery technologies. Currently, LNP delivery systems are the most advanced technology for systemic delivery of siRNA, with numerous formulations under various stages of clinical trials. We also discuss methods to improve gene silencing potency of LNP-siRNA, as well as application of LNP technologies beyond siRNA to the encapsulation of other nucleic acids such as mRNA and clustered regularly interspaced short palindromic repeats (CRISPR).

negative: Biovacc-19: A Candidate Vaccine for Covid-19 (SARS-CoV-2) Developed from Analysis of its General Method of Action for Infectivity

negative: Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza Summary The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

negative: Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription. We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The identification of an upstream cis-acting element for MHV subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.

negative: The need for fast-track, high-quality and low-cost studies about the role of the BCG vaccine in the fight against COVID-19 Bacillus Calmette-Guérin (BCG) vaccination is routine and near-universal in many low- and middle-income countries (LMIC). It has been suggested that BCG can have a protective effect on COVID-19 morbidity and mortality. This commentary discusses the limitations of the evidence around BCG and COVID-19. We argue that higher-quality evidence is necessary to understand the protective effect of the BCG vaccine from existing, secondary data, while we await results from clinical trials currently conducted in different settings.

negative: BCG vaccination strategy implemented to reduce the impact of COVID-19: Hype or Hope? Abstract The Bacillus Calmette-Guerin vaccine (BCG vaccine) designed to prevent tuberculosis in children has been shown to induce a adaptive immune response in the body to fight against bacteria as well as other parasites and viruses. This knowledge has been reciprocated to generate the idea that this vaccine can also offer protection against severe acute respiratory syndrome coronavirus-2 (SARS-COV-2). Some recent pre-print articles have highlighted that countries with mass BCG immunizations seems to have a lower incidence of coronavirus disease 2019 (COVID-19) compared to those without BCG immunization. There are yet no experimental proof of any such association and the world health organisation (WHO) is currently testing the theory with clinical trials on selected cohorts. Epidemiologists and other scientific experts has expressed both their hope and concern simultaneously regarding the success theory of BCG vaccination to prevent COVID-19. Though its still not verified in any way whether the BCG vaccination can actually prevent COVID-19 or not but we believe a thorough analytical research in this regard is indeed worth a shot.

negative: Advantages of mRNA display selections over other selection techniques for investigation of protein–protein interactions mRNA display is a genotype–phenotype conjugation method that allows for amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. mRNA display can be used to display both long natural protein and short synthetic peptide libraries with unusually high diversities for the investigation of protein–protein interactions. Here, we summarize the advantages of mRNA display by comparing it with other widely used peptide or protein-selection techniques, and discuss various applications of this technique in studying protein–protein interactions.

negative: The starting line for COVID-19 vaccine development

negative: Current global vaccine and drug efforts against COVID-19: Pros and cons of bypassing animal trials COVID-19 has become one of the biggest health concern, along with huge economic burden. With no clear remedies to treat the disease, doctors are repurposing drugs like chloroquine and remdesivir to treat COVID-19 patients. In parallel, research institutes in collaboration with biotech companies have identified strategies to use viral proteins as vaccine candidates for COVID-19. Although this looks promising, they still need to pass the test of challenge studies in animal models. As various models for SARS-CoV-2 are under testing phase, biotech companies have bypassed animal studies and moved to Phase I clinical trials. In view of the present outbreak, this looks a justified approach, but the problem is that in the absence of animal studies, we can never predict the outcomes in humans. Since animal models are critical for vaccine development and SARS-CoV-2 has different transmission dynamics, in this review we compare different animal models of SARS-CoV-2 with humans for their pathogenic, immune response and transmission dynamics that make them ideal models for vaccine testing for COVID-19. Another issue of using animal model is the ethics of using animals for research; thus, we also discuss the pros and cons of using animals for vaccine development studies.

negative: Increased expression of ACE2, the SARS-CoV-2 entry receptor, in alveolar and bronchial epithelium of smokers and COPD subjects Rationale: Smokers and patients with chronic obstructive pulmonary disease (COPD) are at increased risk for severe Coronavirus Disease 2019 (COVID-19). Objectives: We investigated the expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptor ACE2 and the protease TMPRSS2 in lung tissue from never smokers and smokers with and without COPD. Methods: In a cross-sectional, observational study we measured mRNA expression of ACE2 and TMPRSS2 by RT-PCR in lung tissue samples from 120 well phenotyped subjects. Next, protein levels of ACE2 were visualized by immunohistochemistry on paraffin sections from 87 subjects and quantified in alveolar and bronchial epithelium. Finally, primary human bronchial epithelial cells (HBECs) were cultured at air liquid interface and exposed to air or cigarette smoke. Results: ACE2 mRNA expression was significantly higher in lung tissue from current smokers and subjects with moderate to very severe COPD and correlated with physiological parameters of airway obstruction and emphysema. Pulmonary expression levels of TMPRSS2 were significantly higher in patients with (very) severe COPD and correlated significantly with ACE2 expression. Importantly, protein levels of ACE2 were elevated in both alveolar and bronchial epithelium of current smokers and subjects with moderate to very severe COPD. Finally, TMPRSS2 mRNA expression increased in in vitro cultured HBECs upon acute exposure to cigarette smoke. Conclusions: We demonstrate increased expression of ACE2 in lungs of smokers and COPD subjects, which might facilitate host cell entry of SARS-CoV-2. These findings help identifying populations at risk for severe COVID-19.

negative: Understand Research Hotspots Surrounding COVID-19 and Other Coronavirus Infections Using Topic Modeling Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that causes severe respiratory illness in humans, which eventually results in the current outbreak of novel coronavirus disease (COVID-19) around the world. The research community is interested to know what are the hotspots in coronavirus (CoV) research and how much is known about COVID-19. This study aimed to evaluate the characteristics of publications involving coronaviruses as well as COVID-19 by using a topic modeling analysis. Methods: We extracted all abstracts and retained the most informative words from the COVID-19 Open Research Dataset, which contains all the 35,092 pieces of coronavirus related literature published up to March 20, 2020. Using Latent Dirichlet Allocation modeling, we trained an eight-topic model from the corpus. We then analyzed the semantic relationships between topics and compared the topic distribution between COVID-19 and other CoV infections. Results: Eight topics emerged overall: clinical characterization, pathogenesis research, therapeutics research, epidemiological study, virus transmission, vaccines research, virus diagnostics, and viral genomics. It was observed that COVID-19 research puts more emphasis on clinical characterization, epidemiological study, and virus transmission at present. In contrast, topics about diagnostics, therapeutics, vaccines, genomics and pathogenesis only accounted for less than 10% or even 4% of all the COVID-19 publications, much lower than those of other CoV infections. Conclusions: These results identified knowledge gaps in the area of COVID-19 and offered directions for future research. Keywords: COVID-19, coronavirus, topic modeling, hotspots, text mining

negative: The current understanding and potential therapeutic options to combat COVID-19 Abstract The ongoing wreaking global outbreak of the novel human beta coronavirus (CoV) pathogen was presumed to be from a seafood wholesale market in Wuhan, China, belongs to the Coronaviridae family in the Nidovirales order. The virus is highly contagious with potential human-human transmission which was named as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread across six continents and emerged as a global pandemic in short span with alarming levels of spread and severity. This virus associated symptoms and infectious respiratory illness is designated as coronavirus disease 19 (COVID-19). The SARS-CoV-2 possesses enveloped club-like spike protein projections with positive-sense large RNA genome and has a unique replication strategy. This virus was believed to have zoonotic origin with genetical identity to bat and pangolin CoV. In the current review, we introduce a general overview about the human CoVs and the associated diseases, the origin, structure, replication and key clinical events that occur in the COVID-19 pathogenicity. Furthermore, we focused on possible therapeutic options such as repurposing drugs including antimalarials, antivirals, antiparasitic drugs, and anti-HIV drugs, as well as monoclonal antibodies, vaccines as potential treatment options. Also we have summarized the latest research progress on the usage of stem cell therapy, human convalescent serum, interferon's, in the treatment of COVID-19.

negative: The early landscape of COVID‐19 vaccine development in the UK and rest of the world Since the first WHO notification on 31st December 2019, COVID‐19, the respiratory disease caused by the coronavirus SARS‐CoV‐2, has been responsible for over 4 million confirmed infections, and almost 300,000 deaths worldwide. The pandemic has led to over half of the world's population living under lockdown conditions. To allow normal life to resume, public health interventions will be needed to prevent further waves of infections as lockdown measures are lifted. As one of the most effective countermeasures against infectious diseases, an efficacious vaccine is considered crucial to containing the COVID‐19 pandemic. Following the publication of the genome sequence of SARS‐CoV‐2, vaccine development has accelerated at an unprecedented pace across the world. Here we review the different platforms employed to develop vaccines, the standard timelines of development and how they can be condensed in a pandemic situation. We focus on vaccine development in the UK and vaccines which have entered clinical trials around the world.

negative: A novel two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression, including type I interferon production, by promoting host mRNA degradation and inhibiting host translation, in infected cells. We present evidence that nsp1 uses a novel, two-pronged strategy to inhibit host translation/gene expression. Nsp1 bound to the 40S ribosomal subunit and inactivated the translational activity of the 40S subunits. Furthermore, the nsp1-40S ribosome complex induced the modification of the 5'-region of capped mRNA template and rendered the template RNA translationally incompetent. Nsp1 also induced RNA cleavage in templates carrying the internal ribosome entry site (IRES) from encephalomyocarditis virus, but not in those carrying IRESs from hepatitis C and cricket paralysis viruses, demonstrating that the nsp1-induced RNA modification was template-dependent. We speculate that the mRNAs that underwent the nsp1-mediated modification are marked for rapid turnover by the host RNA degradation machinery.

negative: Within host dynamics of SARS-CoV-2 in humans: Modeling immune responses and antiviral treatments In December 2019, a newly discovered SARS-CoV-2 virus was emerged from China and propagated worldwide as a pandemic. In the absence of preventive medicine or a ready to use vaccine, mathematical models can provide useful scientific insights about transmission patterns and targets for drug development. In this study, we propose a within-host mathematical model of SARS-CoV-2 infection considering innate and adaptive immune responses. We analyze the equilibrium points of the proposed model and obtain an expression of the basic reproduction number. We then numerically show the existence of a transcritical bifurcation. The proposed model is calibrated to real viral load data of two COVID-19 patients. Using the estimated parameters, we perform global sensitivity analysis with respect to the peak of viral load. Finally, we study the efficacy of antiviral drugs and vaccination on the dynamics of SARS-CoV-2 infection. Our results suggest that blocking the production of the virus by infected cells decreases the viral load more than reducing the infection rate of healthy cells. Vaccination is also found useful but during the vaccine development phase, blocking virus production from infected cells can be targeted for antiviral drug development.

negative: Understanding novel COVID-19: its impact on organ failure and risk assessment for diabetic and cancer patients Abstract The current pandemic outbreak of COVID-19 originated from Wuhan, China. It is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with significant mortality and morbidity rate. The severe risk factors are commonly detected in patients of older age and with medical comorbidities like cancer and diabetes. Scientists and doctors have scrambled to gain knowledge about the novel virus and its pathophysiology in order to discover possible therapeutic regimens and vaccines for COVID-19. The therapeutic strategies like targeting the viral genome emphasize the promising approach to target COVID-19. Additionally, blocking the receptor, ACE2 via the neutralizing antibodies for viral escape that prevents it from entering into the cells provides another therapeutic regimen. In this review article, we have presented the effect of SARS-CoV-2 infection in comorbid patients and discussed organ failure caused by this virus. Based on the data available from the scientific literature and ongoing clinical trials, we have focused on therapeutic strategies. We hope that we would fill the gaps that puzzled the researchers and clinicians with the best of our knowledge collected for the betterment of the patients for the coming future.

negative: Can adenoviral vectors overcome a checkered past to win the COVID-19 vaccine race?

negative: Nonsense-Mediated mRNA Decay: Pathologies and the Potential for Novel Therapeutics Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a surveillance pathway used by cells to control the quality mRNAs and to fine-tune transcript abundance. NMD plays an important role in cell cycle regulation, cell viability, DNA damage response, while also serving as a barrier to virus infection. Disturbance of this control mechanism caused by genetic mutations or dys-regulation of the NMD pathway can lead to pathologies, including neurological disorders, immune diseases and cancers. The role of NMD in cancer development is complex, acting as both a promoter and a barrier to tumour progression. Cancer cells can exploit NMD for the downregulation of key tumour suppressor genes, or tumours adjust NMD activity to adapt to an aggressive immune microenvironment. The latter case might provide an avenue for therapeutic intervention as NMD inhibition has been shown to lead to the production of neoantigens that stimulate an immune system attack on tumours. For this reason, understanding the biology and co-option pathways of NMD is important for the development of novel therapeutic agents. Inhibitors, whose design can make use of the many structures available for NMD study, will play a crucial role in characterizing and providing diverse therapeutic options for this pathway in cancer and other diseases.

negative: Use of viral vectors for vaccine production in plants The small size of plant viral genomes, the ease with which they can be manipulated, and the simplicity of the infection process is making the viral vectors an attractive alternative to the transgenic systems for the expression of foreign proteins in plants. One use of these virus expression systems is for vaccine production. There are two basic types of viral system that have been developed for the production of immunogenic peptides and proteins in plants: epitope presentation and polypeptide expression systems. In this review, we discuss advances made in this field.

negative: Emergence of deadly severe acute respiratory syndrome coronavirus-2 during 2019–2020 Wuhan, the city in Hubei province in China is in the focus of global community due to the outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), formerly known as 2019-nCoV. The virus emerged in humans from Wuhan seafood market probably via zoonotic transmission. Within a few days the virus spread its tentacles rapidly to neighboring cities in China and to different geographical regions through travelers and to some extent by human to human transmission leading to significant disease burden globally. More than 2,00,000 people (including more than 8000 deaths) have been infected with this respiratory illness across 167 countries and territories worldwide leading to a pandemic. The present review provides an outline about emergence and spread of SARS-CoV-2 from Wuhan, China in 2019–2020. We have also provided information about the classification, genome, proteins, clinical presentation of COVID-19, type of clinical specimens to be collected and diagnostic methods adopted to identify the respiratory illness. In addition we have also provided information about transmission dynamics, prevention measures and treatment options that are available at the present. Subsequently, we have given a comprehensive overview of the spread of this infection from China to the other parts of the globe. Management of the ongoing outbreak of SARS-CoV-2 encompassing surveillance, clinical, immunological, genetic and evolutionary investigations are likely to provide the desired results. Joint efforts of global scientific community are needed at this hour in terms of enhancement of research on development of accurate diagnostics, antiviral therapeutics and finally into formation of an effective vaccine against the emerging novel coronavirus.

negative: Integration of mRNP formation and export Expression of protein-coding genes in eukaryotes relies on the coordinated action of many sophisticated molecular machineries. Transcription produces precursor mRNAs (pre-mRNAs) and the active gene provides an environment in which the pre-mRNAs are processed, folded, and assembled into RNA–protein (RNP) complexes. The dynamic pre-mRNPs incorporate the growing transcript, proteins, and the processing machineries, as well as the specific protein marks left after processing that are essential for export and the cytoplasmic fate of the mRNPs. After release from the gene, the mRNPs move by diffusion within the interchromatin compartment, making up pools of mRNPs. Here, splicing and polyadenylation can be completed and the mRNPs recruit the major export receptor NXF1. Export competent mRNPs interact with the nuclear pore complex, leading to export, concomitant with compositional and conformational changes of the mRNPs. We summarize the integrated nuclear processes involved in the formation and export of mRNPs.

negative: Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.

negative: In Silico Trial to test COVID-19 candidate vaccines: a case study with UISS platform SARS-CoV-2 is a severe respiratory infection that infects humans. Its outburst entitled it as a pandemic emergence. To get a grip on this outbreak, specific preventive and therapeutic interventions are urgently needed. It must be said that, until now, there are no existing vaccines for coronaviruses. To promptly and rapidly respond to pandemic events, the application of in silico trials can be used for designing and testing medicines against SARS-CoV-2 and speed-up the vaccine discovery pipeline, predicting any therapeutic failure and minimizing undesired effects. Here, we present an in silico platform that showed to be in very good agreement with the latest literature in predicting SARS-CoV-2 dynamics and related immune system host response. Moreover, it has been used to predict the outcome of one of the latest suggested approach to design an effective vaccine, based on monoclonal antibody. Universal Immune System Simulator (UISS) in silico platform is potentially ready to be used as an in silico trial platform to predict the outcome of vaccination strategy against SARS-CoV-2.

negative: Identification of SARS-CoV-2 Vaccine Epitopes Predicted to Induce Long-term Population-Scale Immunity Summary Here we propose a SARS-CoV-2 vaccine design concept based on identification of highly conserved regions of the viral genome and newly acquired adaptations, both predicted to generate epitopes presented on MHC class I and II across the vast majority of the population. We further prioritize genomic regions that generate highly dissimilar peptides from the human proteome, and are also predicted to produce B cell epitopes. We propose sixty-five 33mer peptide sequences, a subset of which can be tested using DNA or mRNA delivery strategies. These include peptides that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor and within a newly evolved furin cleavage site thought to increase membrane fusion. Validation and implementation of this vaccine concept could specifically target specific vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the population.

negative: Implications of SARS-CoV-2 mutations for genomic RNA structure and host microRNA targeting The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2 permissive host cells, we identified twelve separate target sequences in the SARS-CoV-2 genome; eight of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host could be constrained by host miRNA defence. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineered viral attenuation and antigen presentation.

negative: What is the potential function of microRNAs as biomarkers and therapeutic targets in COVID-19? Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19, a pandemic associated with substantial morbidity and mortality. Despite of this, no vaccine or approved drug is available to eradicate the virus. In this manuscript, we present an alternative study area that may contribute to development of diagnostic biomarkers and therapeutic targets for COVID-19. We analyzed sixty SARS-CoV-2 genomes to identify regions that could work as virus-encoded miRNA seed sponges and potentially bind to human miRNA seed sites and prevent interaction with their native targets thereby relieving native miRNA suppression. MicroRNAs (miRNAs) are evolutionally conserved single-stranded RNAs that regulate gene expression at the posttranscriptional level by disrupting translation. MiRNAs are key players in variety of biological processes that regulate differentiation, development and activation of immune cells in both innate and adaptive immunity. We find 34 miRNAs for positive-sense viral RNA and 45 miRNAs for negative-sense that can strongly bind to certain key SARS-CoV-2 genes. The disruption and dysfunction of miRNAs may perturb the immune response and stimulate the release of inflammatory cytokines altering the cellular response to viral infection. Previous studies demonstrate that miRNAs have the potential to be used as diagnostic and therapeutic biomarkers. Therefore, its discovery and validation are essential for improving the diagnosis of infection and clinical monitoring in COVID-19.

negative: Oligonucleotide antiviral therapeutics: Antisense and RNA interference for highly pathogenic RNA viruses Abstract RNA viruses are a significant source of morbidity and mortality in humans every year. Additionally, the potential use of these viruses in acts of bioterrorism poses a threat to national security. Given the paucity of vaccines or postexposure therapeutics for many highly pathogenic RNA viruses, novel treatments are badly needed. Sequence-based drug design, under development for almost 20 years, is proving effective in animal models and has moved into clinical trials. Important advances in the field include the characterization of RNA interference in mammalian cells and chemical modifications that can dramatically increase the in vivo stability of therapeutic oligonucleotides. Antisense strategies utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating the catalytic degradation of target mRNA, or by binding to sites on mRNA essential for translation. Double-stranded RNA oligonucleotides, known as short-interfering RNAs (siRNAs), also mediate the catalytic degradation of complementary mRNAs. As RNA virus infection is predicated on the delivery, replication, and translation of viral RNA, these pathogens present an obvious target for the rapidly advancing field of sequence-specific therapeutics. Antisense oligonucleotides or siRNAs can be designed to target the viral RNA genome or viral transcripts. This article reviews current knowledge on therapeutic applications of antisense and RNA interference for highly pathogenic RNA viral infections.

negative: Viral-Mediated mRNA Degradation for Pathogenesis Cellular RNA decay machinery plays a vital role in regulating gene expression by altering the stability of mRNAs in response to external stresses, including viral infection. In the primary infection, viruses often conquer the host cell’s antiviral immune response by controlling the inherently cellular mRNA degradation machinery to facilitate viral gene expression and establish a successful infection. This review summarizes the current knowledge about the diverse strategies of viral-mediated regulatory RNA shutoff for pathogenesis, and particularly sheds a light on the mechanisms that viruses evolve to elude immune surveillance during infection.

negative: Rapid development and deployment of high‐volume vaccines for pandemic response Overcoming pandemics, such as the current Covid‐19 outbreak, requires the manufacture of several billion doses of vaccines within months. This is an extremely challenging task given the constraints in small‐scale manufacturing for clinical trials, clinical testing timelines involving multiple phases and large‐scale drug substance and drug product manufacturing. To tackle these challenges, regulatory processes are fast‐tracked, and rapid‐response manufacturing platform technologies are used. Here, we evaluate the current progress, challenges ahead and potential solutions for providing vaccines for pandemic response at an unprecedented scale and rate. Emerging rapid‐response vaccine platform technologies, especially RNA platforms, offer a high productivity estimated at over 1 billion doses per year with a small manufacturing footprint and low capital cost facilities. The self‐amplifying RNA (saRNA) drug product cost is estimated at below 1 USD/dose. These manufacturing processes and facilities can be decentralized to facilitate production, distribution, but also raw material supply. The RNA platform technology can be complemented by an a priori Quality by Design analysis aided by computational modeling in order to assure product quality and further speed up the regulatory approval processes when these platforms are used for epidemic or pandemic response in the future.

negative: Withanone and Withaferin-A are predicted to interact with transmembrane protease serine 2 (TMPRSS2) and block entry of SARS-CoV-2 into cells Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use.Communicated by Ramaswamy H. Sarma.

negative: [Progress and challenge of vaccine development against 2019 novel coronavirus (2019-nCoV)]. The outbreak of 2019 novel coronavirus (2019-nCoV) infection poses a serious threat to global public health. Vaccination is an effective way to prevent the epidemic of the virus. 2019-nCoV along with severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) belong to the same β-genus of coronavirus family. Base on the previous experience and the technical platform of developing SARS-CoV and MERS-CoV vaccines, scientists from all over the world are working hard and quickly on the related fields. There are substantial progress in these fields including the characterizing the 2019-nCoV virus, identification of candidate antigens and epitopes, establishment of animal models, characterizing the immune responses, and the design of vaccines. The development of 2019-nCoV vaccines cover all types: inactivated virus vaccine, recombinant protein vaccine, viral vector-based vaccine, mRNA vaccine, and DNA vaccine, et al. As of March 2020, two 2019-nCoV vaccines have entered phase I clinical trials. One is named as Ad5-nCoV developed by the Chinese Institute of Biotechnology of the Academy of Military Medical Sciences and Tianjin Cansino Biotechnology Inc. Ad5-nCoV is based on the replication-defective adenovirus type 5 as the vector to express 2019-nCoV spike protein. The another vaccine is mRNA-1273 developed by the National Institute of Allergy and Infectious Diseases and Moderna, Inc.. RNA-1273 is an mRNA vaccine expressing 2019-nCoV spike protein. Although the rapid development of 2019-nCoV vaccine, it still faces many challenges with unknown knowledge, including the antigenic characteristics of the 2019-nCoV, the influence of antigenic variation, the protective immune response of host, the protection of the elderly population, and the downstream manufacturing process of the new vaccine. The safety and efficacy of vaccines are the first priority for vaccine development and should be carefully evaluated.

negative: Prospective vaccination of COVID-19 using shRNA-plasmid-LDH nanoconjugate COVID-19 is the pandemic outbreak that is caused by SARS-CoV-2 virus from December, 2019. Human race do not know the curative measure of this devastating disease. In today’s era of nanotechnology, it may use its knowledge to develop molecular vaccine to combat this disease. In this article we are intended to propose a hypothesis on the development of a vaccine that is molecular in nature to work against COVID-19. The nanoconjugate may comprise with the inorganic nanoparticle layered double hydroxide intercalated with shRNA-plasmid that have a sequence targeting towards the viral genome or viral mRNA. This nanoconjugate may be used as a nasal spray to deliver the shRNA-plasmid to the target site. The nanoconjugate will have several advantages such as they are biocompatible, they forms as stable knockdown to the target cells and they are stable in the nasal mucosa.

negative: A vector expressing feline mature IL-18 fused to IL-1β antagonist protein signal sequence is an effective adjuvant to a DNA vaccine for feline leukaemia virus DNA vaccination using vectors expressing the gag/pol and env genes of feline leukaemia virus (FeLV) and plasmids encoding feline interleukin-12 (IL-12) and IL-18 completely protected cats from viraemia following challenge [Hanlon L, Argyle D, Bain D, Nicolson L, Dunham S, Golder MC, et al. Feline leukaemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors. J Virol 2001;75:8424–33]. However, the relative contribution of each cytokine gene towards protection is unknown. This study aimed to resolve this issue. IL-12 and IL-18 constructs were modified to ensure effective expression, and bioactivity was demonstrated using specific assays. Kittens were immunised intramuscularly with FeLV DNA and various cytokine constructs. Together with control kittens, these were challenged oronasally with FeLV and monitored for 15 weeks. All six kittens given FeLV, IL-12 and IL-18 were protected from the establishment of persistent viraemia and four from latent infection. Of six kittens immunised with FeLV DNA and IL-18, all were protected from viraemia and five from latent infection. In contrast, three of five kittens given FeLV DNA and IL-12 became persistently viraemic. Therefore, the adjuvant effect on the FeLV DNA vaccine appears to reside in the expression of IL-18.

negative: Data mining and analysis of scientific research data records on covid 19 mortality, immunity, and vaccine development - In the first wave of the Covid-19 pandemic BACKGROUND AND AIMS: Covid-19 is a global pandemic that requires a global and integrated response of all national medical and healthcare systems. Covid-19 exposed the need for timely response and data sharing on fast spreading global pandemics. In this study, we investigate the scientific research response from the early stages of the pandemic, and we review key findings on how the early warning systems developed in previous epidemics responded to contain the virus. METHODS: We conducted data mining of scientific literature records from the Web of Science Core Collection, using the topics Covid-19, mortality, immunity, and vaccine. The individual records are analysed in isolation, and the analysis is compared with records on all Covid-19 research topics combined. The data records are analysed with commutable statistical methods, including R Studio, Bibliometrix package, and the Web of Science data mining tool. RESULTS: From historical analysis of scientific data records on viruses, pandemics and mortality, we identified that Chinese universities have not been leading on these topics historically. However, during the early stages of the Covid-19 pandemic, the Chinese universities are strongly dominating the research on these topics. Despite the current political and trade disputes, we found strong collaboration in Covid-19 research between the US and China. From the analysis on Covid-19 and immunity, we wanted to identify the relationship between different risk factors discussed in the news media. We identified few different clusters, containing references to exercise, inflammation, smoking, obesity and many additional factors. From the analysis on Covid-19 and vaccine, we discovered that although the USA is leading in volume of scientific research on Covid-19 vaccine, the leading 3 research institutions (Fudan, Melbourne, Oxford) are not based in the USA. Hence, it is difficult to predict which country would be first to produce a Covid-19 vaccine. CONCLUSIONS: We analysed the conceptual structure maps with factorial analysis and multiple correspondence analysis (MCA), and identified multiple relationships between keywords, synonyms and concepts, related to Covid-19 mortality, immunity, and vaccine development. We present integrated and corelated knowledge from 276 records on Covid-19 and mortality, 71 records on Covid-19 and immunity, and 189 records on Covid-19 vaccine.

negative: COVID-19: A Review of Emerging Preventative Vaccines and Treatment Strategies COVID-19, which was first detected in the Hubei province of China, has become a global phenomenon. The effects and devastation on both health and economy have been global. At present, there is a substantial amount of research being done to discover suitable treatment modalities. Efforts have been made on the development of potential efficacious vaccines. The development of a vaccine can be complex, expensive as well as time-consuming. Currently, various ongoing clinical trials are in progress that are investigating either pharmacologic therapies or vaccines against this virus. We, in this brief review have tried to address the process and current development efforts of vaccine in progress.

negative: Current status of potential therapeutic candidates for the COVID-19 crisis Abstract As of April 15, 2020, the ongoing coronavirus disease 2019 (COVID-2019) pandemic has swept through 213 countries and infected more than 1,870,000 individuals, posing an unprecedented threat to international health and the economy. There is currently no specific treatment available for patients with COVID-19 infection. The lessons learned from past management of respiratory viral infections have provided insights into treating COVID-19. Numerous potential therapies, including supportive intervention, immunomodulatory agents, antiviral therapy, and convalescent plasma transfusion, have been tentatively applied in clinical settings. A number of these therapies have provided substantially curative benefits in treating patients with COVID-19 infection. Furthermore, intensive research and clinical trials are underway to assess the efficacy of existing drugs and identify potential therapeutic targets to develop new drugs for treating COVID-19. Herein, we summarize the current potential therapeutic approaches for diseases related to COVID-19 infection and introduce their mechanisms of action, safety, and effectiveness.

negative: Need for Speed: From Human SARS-CoV-2 Samples to Protective and Efficacious Antibodies in Weeks The emergence of SARS-CoV-2 has driven a global research effort to identify medical countermeasures at an unprecedented pace. In this issue of Cell, Cao et al. identify thousands of SARS-CoV-2 neutralizing antibodies from convalescent donors. The authors improve our understanding of immunity against the coronavirus spike glycoprotein and detail novel pathways to rapidly identify and characterize protective monoclonal antibodies.

negative: Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines With the increase in international human and material exchanges, contagious and infectious epidemics are occurring. One of the effective methods of epidemic inhibition is the rapid development and supply of vaccines. Considering the safety of the brain during vaccine development is very important. However, manuals for brain safety assays for new vaccines are not uniform or effective globally. Therefore, the aim of this study is to establish a positive-control protocol for an effective brain safety test to enhance rapid vaccine development. The blood-brain barrier's tight junctions provide selective defense of the brain; however, it is possible to destroy these important microstructures by administering lipopolysaccharides (LPSs), thereby artificially increasing the permeability of brain parenchyma. In this study, test conditions are established so that the degree of brain penetration or brain destruction of newly developed vaccines can be quantitatively identified. The most effective conditions were suggested by measuring time-dependent expressions of tight junction biomarkers (zonula occludens-1 [ZO-1] and occludin) in two types of mice (C57BL/6 and ICR) following exposure to two types of LPS (Salmonella and Escherichia). In the future, we hope that use of the developed positive-control protocol will help speed up the determination of brain safety of novel vaccines.

negative: Novel vaccine technologies for the 21st century Novel approaches to vaccine development include structure-based immunogen design, gene-based vaccine platforms and formulation of recombinant antigens with potent adjuvants. These technologies are producing encouraging results in the development of vaccines for globally important diseases such as tuberculosis, influenza and respiratory syncytial virus. Here we highlight the most important developments in these areas over the past 18 months.

negative: Demystifying the myths about COVID-19 infection and its societal importance

negative: Changed in translation: mRNA recoding by −1 programmed ribosomal frameshifting Programmed −1 ribosomal frameshifting (−1PRF) is an mRNA recoding event commonly utilized by viruses and bacteria to increase the information content of their genomes. Recent results have implicated −1PRF in quality control of mRNA and DNA stability in eukaryotes. Biophysical experiments demonstrated that the ribosome changes the reading frame while attempting to move over a slippery sequence of the mRNA – when a roadblock formed by a folded downstream segment in the mRNA stalls the ribosome in a metastable conformational state. The efficiency of −1PRF is modulated not only by cis-regulatory elements in the mRNA but also by trans-acting factors such as proteins, miRNAs, and antibiotics. These recent results suggest a molecular mechanism and new important cellular roles for −1PRF.

negative: SARS vaccine to receive funding in the US

negative: Personalized workflow to identify optimal T-cell epitopes for peptide-based vaccines against COVID-19 Traditional vaccines against viruses are designed to target their surface proteins, i.e., antigens, which can trigger the immune system to produce specific antibodies to capture and neutralize the viruses. However, viruses often evolve quickly, and their antigens are prone to mutations to avoid recognition by the antibodies (antigenic drift). This limitation of the antibody-mediated immunity could be addressed by the T-cell mediated immunity, which is able to recognize conserved viral HLA peptides presented on virus-infected cells. Thus, by targeting conserved regions on the genome of a virus, T-cell epitope-based vaccines are less subjected to mutations and may work effectively on different strains of the virus. Here we propose a personalized workflow to identify an optimal set of T-cell epitopes based on the HLA alleles and the immunopeptidome of an individual person. Specifically, our workflow trains a machine learning model on the immunopeptidome and then predicts HLA peptides from conserved regions of a virus that are most likely to trigger responses from the person T cells. We applied the workflow to identify T-cell epitopes for the SARS-COV-2 virus, which has caused the recent COVID-19 pandemic in more than 100 countries across the globe.

negative: Development of an inactivated vaccine candidate for SARS-CoV-2 The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale production of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6 micrograms per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support the clinical development and testing of PiCoVacc for use in humans.

negative: Two Middle East respiratory syndrome vaccines: first step for other coronavirus vaccines?

negative: Attenuated Subcomponent Vaccine Design Targeting the SARS-CoV-2 Nucleocapsid Phosphoprotein RNA Binding Domain: In silico analysis The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has previously never been identified with humans, thereby creating devastation in public health. The need for an effective vaccine to curb this pandemic cannot be overemphasized. In view of this, we, therefore, designed a subcomponent antigenic peptide vaccine targeting the N-terminal (NT) and C-terminal (CT) RNA binding domains of nucleocapsid protein that aid in viral replication. Promising antigenic B-cells and T cell epitopes were predicted using computational pipelines. The peptides “RIRGGDGKMKDL” and “AFGRRGPEQTQGNFG” were the B cell linear epitopes with good antigenic index and non-allergenic property. Two CD8+ and Three CD4+ T-cell epitopes were also selected considering their safe immunogenic profiling such as allergenicity, antigen level conservancy, antigenicity, peptide toxicity, and putative restrictions to a number of MHC-I and II alleles. With these selected epitopes, a non-allergenic chimeric peptide vaccine incapable of inducing a Type II hypersensitivity reaction was constructed. The molecular interaction between the toll-like receptor-5 (TLR5) which was triggered by the vaccine was analyzed by molecular docking and scrutinized using dynamics simulation. Finally, in silico cloning was performed to ensure the expression and translation efficiency of the vaccine, utilizing pET-28a vector. This research, therefore, provides a guide for experimental investigation and validation.

negative: Challenges in evaluating SARS-CoV-2 vaccines during the COVID-19 pandemic.

negative: SARS-CoV-2: Virology, epidemiology, immunology and vaccine development This first International Alliance for Biological Standardization Covid-19 webinar brought together a broad range of international stakeholders, including academia, regulators, funders and industry, with a considerable delegation from low- and middle-income countries, to discuss the virology, epidemiology and immunology of, and the vaccine development for SARS-CoV-2.

negative: Identification of a small rna containing the trypanosome spliced leader: A donor of shared 5′ sequences of trypanosomatid mRNAs? Abstract The 35 nucleotide spliced leader (SL) sequence is found on the 5′ end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5′-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5′ terminus of many trypanosomatid mRNAs.

negative: Coronavirus disease 2019 (SARS-CoV-2) and colonization of ocular tissues and secretions: a systematic review Coronavirus disease 19 (COVID-19) has been described to potentially be complicated by ocular involvement. However, scant information is available regarding severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and ocular structures tropism. We conducted a systematic review of articles referenced in PubMed, Cochrane Library, Web of Science and Chinese Clinical Trial Register (ChiCTR) from December 20, 2019 to April 6, 2020, providing information on the presence of SARS-CoV-2 in cornea, conjunctiva, lacrimal sac, and tears. We excluded ongoing clinical studies as for unobtainable conclusive results. Of 2422 articles, 11 met the inclusion criteria for analysis and were included in the study. None of the studies were multinational. Among the 11 selected papers there were three original articles, one review, four letters, two editorials, and one correspondence letter. Globally, 252 SARS-CoV-2 infected patients were included in our review. The prevalence of ocular conjunctivitis complicating the course of COVID-19 was demonstrated to be as high as 32% in one study only. Globally, three patients had conjunctivitis with a positive tear-PCR, 8 patients had positive tear-PCR in the absence of conjunctivitis, and 14 had conjunctivitis with negative tear-PCR. The majority of the available data regarding SARS-CoV-2 colonization of ocular and periocular tissues and secretions have to be considered controversial. However, it cannot be excluded that SARS-CoV-2 could both infect the eye and the surrounding structures. SARS-CoV-2 may use ocular structure as an additional transmission route, as demonstrated by the COVID-19 patients’ conjunctival secretion and tears positivity to reverse transcriptase-PCR SARS-CoV-2-RNA assay.

negative: Current situation in the development of a preventive HIV vaccine The uncontrolled progression of the aids epidemic has made the development of an efficacious human immunodeficiency virus (HIV) vaccine a major objective of scientific research. No effective preventive vaccine against HIV is currently available and sterilizing immunity has not yet been achieved in animal models. This review analyses the major challenges in developing an aids vaccine, in particular the mechanisms involved in viral escape from the immune response, and summarizes the results obtained with the different prototypes of therapeutic and preventive vaccines. Finally, social, economic and healthcare aspects of research into HIV vaccines and current controversies regarding the development of clinical trials are discussed.

negative: Human and novel coronavirus infections in children: a review. Coronaviruses, seven of which are known to infect humans, can cause a spectrum of clinical presentations ranging from asymptomatic infection to severe illness and death. Four human coronaviruses (hCoVs)-229E, HKU1, NL63 and OC43-circulate globally, commonly infect children and typically cause mild upper respiratory tract infections. Three novel coronaviruses of zoonotic origin have emerged during the past two decades: severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV) and the recently discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. These novel coronaviruses are known to cause severe illness and death predominantly in older adults and those with underlying comorbidities. Consistent with what has been observed during the outbreaks of SARS and MERS, children with COVID-19 are more likely to be asymptomatic or to have mild-to-moderate illness, with few deaths reported in children globally thus far. Clinical symptoms and laboratory and radiological abnormalities in children have been similar to those reported in adults but are generally less severe. A rare multisystem inflammatory syndrome in children (MIS-C) which has resulted in critical illness and some deaths has recently been described. Clinical trials for therapeutics and vaccine development should include paediatric considerations. Children may play an important role in the transmission of infection and outbreak dynamics and could be a key target population for effective measures to control outbreaks. The unintended consequences of the unprecedented scale and duration of pandemic control measures for children and families around the world should be carefully examined. ABBREVIATIONS 2019-nCoV, 2019 novel coronavirus; ADEM, acute demyelinating encephalomyelitis; AAP, American Academy of Pediatrics; ACE-2, angiotensin-converting enzyme 2; ARDS, acute respiratory distress syndrome; BCG, bacillus Calmette-Guérin; BNP, brain natriuretic peptide; CDC, Centers for Disease Control and Prevention; CRP, C-reactive protein; CSF, cerebrospinal fluid; COVID-19, coronavirus disease 2019; CT, computed tomography; CXR, chest X-ray; DOL, day of life; hCoV, human coronavirus; ICU, intensive care unit; IL, interleukin; IVIG, intravenous immunoglobulin; KD, Kawasaki disease; LDH, lactate dehydrogenase; MERS, Middle East respiratory syndrome; MERS-CoV, Middle East respiratory syndrome coronavirus; MEURI, monitored emergency use of unregistered and experimental interventions; MIS-C, multi-system inflammatory syndrome in children; PCR, polymerase chain reaction; PICU, paediatric intensive care unit; RNA, ribonucleic acid; RCT, randomised-controlled trial; RSV, respiratory syncytial virus; SARS, severe acute respiratory syndrome; SARS-CoV-1, severe acute respiratory syndrome coronavirus 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-alpha, tumour necrosis factor alpha; UK United Kingdom; UNICEF, United Nations Children's Fund; USA, United States of America; WHO, World Health Organization.

negative: The role of passive immunization in the age of SARS-CoV-2: an update The rapid spread of the corona virus pandemic is an existential problem for many people in numerous countries. So far, there is no effective vaccine protection or proven therapy available against the SARS-CoV-2 virus. In this review, we describe the role of passive immunization in times of the corona virus. Passive immunization could be a bridging technology to improve the immune defense of critically ill patients until better approaches with effective medications are available.

negative: Structural and functional conservation of the programmed −1 ribosomal frameshift signal of SARS-CoV-2 17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called −1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of −1 PRF, and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of −1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated −1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.

negative: Putative Roles for Peptidylarginine Deiminases in COVID-19. Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.

negative: Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition involves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases translation of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mRNAs resuming translation after stress and of viral mRNAs suggesting a role of Alu RNPs in adapting the translational output in response to stress and viral infection.

negative: Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model Development of effective vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is still a priority in prevention of re-emergence of SARS. Our previous studies have shown that the receptor-binding domain (RBD) of SARS-CoV spike (S) protein elicits highly potent neutralizing antibody responses in the immunized animals. But it is unknown whether RBD can also induce protective immunity in an animal model, a key aspect for vaccine development. In this study, BALB/c mice were vaccinated intramuscularly (i.m.) with 10 μg of RBD-Fc (RBD fused with human IgG1 Fc) and boosted twice at 3-week intervals and one more time at 12th month. Humoral immune responses of vaccinated mice were investigated for up to 12 months at a 1-month interval and the neutralizing titers of produced antibodies were reported at months 0, 3, 6 and 12 post-vaccination. Mice were challenged with the homologous strain of SARS-CoV 5 days after the last boost, and sacrificed 5 days after the challenge. Mouse lung tissues were collected for detection of viral load, virus replication and histopathological effects. Our results showed that RBD-Fc vaccination induced high titer of S-specific antibodies with long-term and potent SARS-CoV neutralizing activity. Four of five vaccinated mice were protected from subsequent SARS-CoV challenge because no significant virus replication, and no obvious histopathological changes were found in the lung tissues of the vaccinated mice challenged with SARS-CoV. Only one vaccinated mouse had mild alveolar damage in the lung tissues. In contrast, high copies of SARS-CoV RNA and virus replication were detected, and pathological changes were observed in the lung tissues of the control mice. In conclusion, our findings suggest that RBD, which can induce protective antibodies to SARS-CoV, may be further developed as a safe and effective SARS subunit vaccine.

negative: ACE2 expression in human dorsal root ganglion sensory neurons: implications for SARS-CoV-2 virus-induced neurological effects SARS-CoV-2 has created a global crisis. COVID-19, the disease caused by the virus, is characterized by pneumonia, respiratory distress and hypercoagulation and is often fatal1. An early sign of infection is loss of smell, taste and chemesthesis - loss of chemical sensation2. Other neurological effects of the disease have been described, but not explained3,4. We show that human dorsal root ganglion (DRG) neurons express the SARS-CoV-2 receptor5,6, ACE2. ACE2 mRNA is expressed by a subset of nociceptors that express MRGPRD mRNA suggesting that SARS-CoV-2 may gain access to the nervous system through entry into neurons that form free-nerve endings at the outer-most layers of skin and luminal organs. Therefore, sensory neurons are a potential target for SARS-CoV-2 invasion of the nervous system.

negative: Strategies to Prevent SARS-CoV-2-Mediated Eosinophilic Disease in Association with COVID-19 Vaccination and Infection A vaccine to protect against COVID-19 is urgently needed. Such a vaccine should efficiently induce high-affinity neutralizing antibodies which neutralize SARS-CoV-2, the cause of COVID-19. However, there is a concern regarding both vaccine-induced eosinophilic lung disease and eosinophil-associated Th2 immunopotentiation following infection after vaccination. Here, we review the anticipated characteristics of a COVID-19 vaccine to avoid vaccine-associated eosinophil immunopathology.

negative: COVID-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics The novel coronavirus infection (COVID-19 or Coronavirus disease 2019) that emerged from Wuhan, Hubei province of China has spread to many countries worldwide. Efforts have been made to develop vaccines against human coronavirus (CoV) infections such as MERS and SARS in the past decades. However, to date, no licensed antiviral treatment or vaccine exists for MERS and SARS. Most of the efforts for developing CoV vaccines and drugs target the spike glycoprotein or S protein, the major inducer of neutralizing antibodies. Although a few candidates have shown efficacy in in vitro studies, not many have progressed to randomized animal or human trials, hence may have limited use to counter COVID-19 infection. This article highlights ongoing advances in designing vaccines and therapeutics to counter COVID-19 while also focusing on such experiences and advances as made with earlier SARS- and MERS-CoVs, which together could enable efforts to halt this emerging virus infection.

negative: Proteome Organization of COVID-19: Illustrating Targets for Vaccine Development 'COVID-19' the recent virulent viral infection had influenced the lives of millions globally leading to both loss of life, economic and financial crisis Coronavirus belongs to family coronaviridae with four genus viz alpha/beta and gamma-coronavirus, infecting both aves and mammals The SARS-Cov-2 emerged in Wuhan, China in Dec, 2019 and since then had spread to 213 countries Its origin is debatable with both natural origin and conspiracy theory providing no conclusive evidences Coronavirus have '+'ive RNA and encodes for 29 proteins, which carries out its life cycle including infection and disease progression The study of its proteome organization could illustrate the proteins which act as the key molecular players in the infection cycle of the virus These proteins can also act as important drug targets in combating COVID-19 infection Majority of the drugs have been formulated in order to act as agonist to spike proteins inhibiting infection by binding to ACE2 receptors Proteome analysis has also revealed the critical mutated proteins that are responsible for COVID-19 pathogenesis and virulence mRNA based vaccines (mRNA-1273, BNT162) also targets these spike proteins Although DNA vaccine has also been attempted using RDT, but the high rate of mutation associated with COVID-19 have made such vaccines ineffective even before use Thus evolutionarily conserved proteins have been the best candidature for vaccine development Similarly phylogenetic analysis of its proteins could help us to understand the evolutionary pattern of COVID-19 It could be used to develop a predictable model for such pathogenic infections, preparing ourselves to take preventive action against its reoccurrence

negative: Coronavirus replication and pathogenesis: Implications for the recent outbreak of severe acute respiratory syndrome (SARS), and the challenge for vaccine development A novel coronavirus has been recently identified as the causative agent of the severe acute respiratory syndrome (SARS) outbreak that has accounted for more than 8000 infected people worldwide. This review will discuss current knowledge on coronavirus replication, pathogenesis, evolution, and vaccine strategies, as well as the most recent findings on SARS coronavirus.

negative: Biomedical applications of mRNA nanomedicine As an attractive alternative to plasmid DNA, messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapeutics for biomedical applications. Advances in addressing the inherent shortcomings of mRNA and in the development of nanoparticle-based delivery systems have prompted the development and clinical translation of mRNA-based medicines. In this review, we discuss the chemical modification strategies of mRNA to improve its stability, minimize immune responses, and enhance translational efficacy. We also highlight recent progress in nanoparticle-based mRNA delivery. Considerable attention is given to the increasingly widespread applications of mRNA nanomedicine in the biomedical fields of vaccination, protein-replacement therapy, gene editing, and cellular reprogramming and engineering.

negative: Cytokine Storm in COVID-19 patients transforms to a Cytokine Super Cyclone in patients with risk factors The seventh human coronavirus SARS-CoV2 belongs to the cluster of extremely pathogenic coronaviruses like SARS-CoV and MERS-CoV, which are established to cause lower respiratory tract infection. The viral infection can be fatal as the disease advances to pneumonia followed by acute respiratory distress syndrome (ARDS). Increasingly higher cytokine concentration on account of over-stimulated immune response against the virus, or the ‘cytokine storm’, is the reason behind the manifestation of lethal clinical symptoms. In this article, we discuss the immune pathogenesis of cytokine storm and its relation with SARS-CoV2/COVID-19 risk factors. People with underlying risk factors are more susceptible to fatal complications of COVID-19 infection leading to poor clinical outcome. The increased pro-inflammatory immune status in patients with risk factors (diabetes, hypertension, cardiovascular disease, COPD) exacerbates the Cytokine-storm of COVID-19 to a Cytokine Super Cyclone. We also overviewed antiviral immune response provided by BCG vaccine involving the IL-1β, IL-6 and TNF-α secretion via ‘trained monocytes’, which confers early protection against SARS-CoV2.

negative: Recent progress and challenges in drug development against COVID-19 coronavirus (SARS-CoV-2) - an update on the status Abstract Coronaviruses are a large group of viruses known to cause illnesses that vary between the common cold and more severe diseases to include severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). A novel coronavirus was identified in December 2019 in Wuhan city, Hubei province, China. This virus represents a new strain that has not been previously identified in humans. The virus is now known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting disease is called coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) declared the novel coronavirus outbreak a global pandemic in March 2020. Despite rigorous global containment and quarantine efforts, the incidence of COVID-19 continues to rise, with more than 1,948,617 laboratory-confirmed cases and over 121,846 deaths worldwide. Currently, no specific medication is recommended to treat COVID-19 patients. However, governments and pharmaceutical companies are struggling to quickly find an effective drug to defeat the coronavirus. In the current review, we summarize the existing state of knowledge about COVID-19, available medications, and treatment options. Favilavir is an antiviral drug that is approved in Japan for common influenza treatment and is now approved to treat symptoms of COVID-19 in China. Moreover, Chloroquine and hydroxychloroquine, drugs used to treat malaria and arthritis, respectively, were recommended by the National Health Commission of the People's Republic of China for treatment of COVID-19. Presently, chloroquine and hydroxychloroquine are under investigation by the US Food and Drug Administration (FDA) as a treatment for COVID-19. The first COVID-19 vaccine is not expected to be ready for clinical trials before the end of the year.

negative: Overcoming nonstructural protein 15-nidoviral uridylate-specific endoribonuclease (nsp15/NendoU) activity of SARS-CoV-2 COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a ‘double-hit’ whereby two CoV-unique protein elements of the replicase–transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the ‘double-hit hypothesis’.

negative: Merck KGaA helps scale COVID-19 vaccine The University of Oxford’s Jenner Institute and Merck KGaA have developed a manufacturing process for the institute’s COVID-19 vaccine candidate, ChAdOx1 nCoV-19 Using technology developed by Merck, the partners have reduced the process development phase from 12 months to 2 A trial of the vaccine, which is based on an adenovirus vaccine vector and the SARS-Cov-2 spike protein, will start soon and the vaccine could be ready to roll out by the fall, Jenner says

negative: Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle.

negative: Different longitudinal patterns of nucleic acid and serology testing results based on disease severity of COVID-19 patients Effective strategy to mitigate the ongoing pandemic of 2019 novel coronavirus (COVID-19) require a comprehensive understanding of humoral responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the emerging virus causing COVID-19. The dynamic profile of viral replication and shedding along with viral antigen specific antibody responses among COVID-19 patients started to be reported but there is no consensus on their patterns. Here, we conducted a serial investigation on 21 individuals infected with SARS-CoV-2 in two medical centres from Jiangsu Province, including 11 non-severe COVID-19 patients, and 5 severe COVID-19 patients and 5 asymptomatic carriers based on nucleic acid test and clinical symptoms. The longitudinal swab samples and sera were collected from these people for viral RNA testing and antibody responses, respectively. Our data revealed different pattern of seroconversion among these groups. All 11 non-severe COVID-19 patients and 5 severe COVID-19 patients were seroconverted during hospitalization or follow-up period, suggesting that serological testing is a complementary assay to nucleic acid test for those symptomatic COVID-19 patients. Of note, immediate antibody responses were identified among severe cases, compared to non-severe cases. On the other hand, only one were seroconverted for asymptomatic carriers. The SARS-CoV-2 specific antibody responses were well-maintained during the observation period. Such information is of immediate relevance and would assist COVID-19 clinical diagnosis, prognosis and vaccine design.

negative: Severe acute respiratory syndrome (SARS) S protein production in plants: development of recombinant vaccine. In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.

negative: Putative Roles for Peptidylarginine Deiminases in COVID-19 Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.

negative: Epitope-Based Peptide Vaccine Against Severe Acute Respiratory Syndrome-Coronavirus-2 Nucleocapsid Protein: An in silico Approach With an increasing fatality rate, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a promising threat to human health worldwide. SARS-CoV-2 is a member of the Coronaviridae family, which is transmitted from animal to human and because of being contagious, further it transmitted human to human. Recently, the World Health Organization (WHO) has announced the infectious disease caused by SARS-CoV-2, which is known as coronavirus disease-2019 (COVID-2019) as a global pandemic. But, no specific medications are available for the treatment of COVID-19 so far. As a corollary, there is a need for a potential vaccine to impede the progression of the disease. Lately, it has been documented that the nucleocapsid (N) protein of SARS-CoV-2 is responsible for viral replication as well as interferes with host immune responses. We have comparatively analyzed the sequences of N protein of SARS-CoV-2 for the identification of core attributes and analyzed the ancestry through phylogenetic analysis. Subsequently, we have predicted the most immunogenic epitope for T-cell as well as B-cell. Importantly, our investigation mainly focused on major histocompatibility complex (MHC) class I potential peptides and NTASWFTAL interacted with most human leukocyte antigen (HLA) that are encoded by MHC class I molecules. Further, molecular docking analysis unveiled that NTASWFTAL possessed a greater affinity towards HLA and also available in a greater range of the population. Our study provides a consolidated base for vaccine design and we hope that this computational analysis will pave the way for designing novel vaccine candidates.

negative: Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

negative: Improving an Escherichia coli-based biocatalyst for terpenol glycosylation by variation of the expression system Glycosides are becoming increasingly more relevant for various industries as low-cost whole-cell-biocatalysts are now available for the manufacture of glycosides. However, there is still a need to optimize the biocatalysts. The aim of this work was to increase the titre of terpenyl glucosides in biotransformation assays with E. coli expressing VvGT14ao, a glycosyltransferase gene from grape (Vitis vinifera). Seven expression plasmids differing in the resistance gene, origin of replication, promoter sequence, and fusion protein tag were generated and transformed into four different E. coli expression strains, resulting in 18 strains that were tested for glycosylation efficiency with terpenols and a phenol. E. coli BL21(DE3)/pET-SUMO_VvGT14ao yielded the highest titres. The product concentration was improved 8.6-fold compared with E. coli BL21(DE3)pLysS/pET29a_VvGT14ao. The selection of a small solubility-enhancing protein tag and exploitation of the T7 polymerase-induction system allowed the formation of increased levels of functional recombinant protein, thereby improving the performance of the whole-cell biocatalyst. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10295-019-02184-4) contains supplementary material, which is available to authorized users.

negative: Augmentation of immune responses to SARS coronavirus by a combination of DNA and whole killed virus vaccines We studied the immunogenicity of a DNA SARS-vaccine, a whole killed virus, or a whole killed and DNA vaccine combination. The DNA vaccine contained a plasmid encoding the SARS coronavirus (SARS-CoV) S protein under the control of the human CMV promoter and intron A. The whole killed virus vaccine was comprised of SARS-CoV, propagated in Vero-E6 cells, with subsequent β-propilactone inactivation and formulated with aluminum hydroxide adjuvant. Mice immunized twice with the DNA vaccine and once with the whole killed virus elicited higher antibody responses than mice immunized three times with the DNA vaccine or once with the whole killed virus vaccine. Mice immunized twice with the whole killed virus vaccine elicited higher antibody responses than mice immunized three times with the DNA vaccine or once with the whole killed virus vaccine. However, a combination of the vaccines induced T-helper type 1 (Th1) immune responses while the whole killed virus vaccine induced T helper type 2 (Th2) immune response. These results demonstrate that combination of the DNA vaccine and the whole killed virus vaccine can be used to enhance the magnitude and change the bias of the immune responses to SARS-CoV.

negative: Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered COVID-19 patients The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle as a vaccine and demonstrate induction of robust neutralization of a pseudo-virus, proportional to quantity of specific IgG and of higher quantities than recovered COVID-19 patients. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.

negative: Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated Mycobacterium bovis. Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research.

negative: Innovations in HIV-1 Vaccine Design Abstract Purpose The field of HIV-1 vaccinology has evolved during the last 30 years from the first viral vector HIV gene insert constructs to vaccination regimens using a myriad of strategies. These strategies now include germline-targeting, lineage-based, and structure-guided immunogen design. This narrative review outlines the historical context of HIV vaccinology and subsequently highlights the scientific discoveries during the last 6 years that promise to propel the field forward. Methods We conducted a search of 2 electronic databases, PubMed and EMBASE, for experimental studies that involved new HIV immunogen designs between 2013 and 2019. During the title and abstract reviews, publications were excluded if they were written in language other than English and/or were a letter to the editor, a commentary, or a conference-only presentation. We then used ClinicalTrials.gov to identify completed and ongoing clinical trials using these strategies. Findings The HIV vaccinology field has undergone periods of significant growth during the last 3 decades. Findings elucidated in preclinical studies have revealed the importance of the interaction between the cellular and humoral immune system. As a result, several new rationally designed vaccine strategies have been developed and explored in the last 6 years, including native-like envelope trimers, nanoparticle, and mRNA vaccine design strategies among others. Several of these strategies have shown enough promise in animal models to progress toward first-in-human Phase I clinical trials. Implications Rapid developments in preclinical and early-phase clinical studies suggest that a tolerable and effective HIV vaccine may be on the horizon.

negative: Synergistic antiviral effects against SARS-CoV-2 by plant-based molecules The exponential spread of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emphasizes the immediate need for effective antiviral drugs and vaccines that could control and prevent the spread of this pandemic. Several new and repurposed drugs are being tested for their effectiveness in the treatment regime, and the development of vaccines is underway. The availability of genome sequence information of the virus and the identification of potential targets to neutralize and eradicate the infection have enabled the search for novel as well as existing molecules to perform the desired function. However, the application of plants in the development of potential biomolecules, such as antibiotics and vaccines, is limited. Traditional medicines involving plant-based formulations have proven successful in boosting immunity and providing tolerance to virus infections. Still, in-depth studies are not available to explore the bioactive compounds of plant origin and their mechanism of action. Given this, the current opinion article conveys our thoughts and perspectives on the promising usage of plant-based biomolecules in circumventing SARS-CoV-2, and how these molecules can work synergistically with other potential drugs for treating SARS-CoV-2.

negative: In Silico Trial to test COVID-19 candidate vaccines: a case study with UISS platform SARS-CoV-2 is a severe respiratory infection that infects humans. Its outburst entitled it as a pandemic emergence. To get a grip on this, outbreak specific preventive and therapeutic interventions are urgently needed. It must be said that, until now, there are no existing vaccines for coronaviruses. To promptly and rapidly respond to pandemic events, the application of in silico trials can be used for designing and testing medicines against SARS-CoV-2 and speed-up the vaccine discovery pipeline, predicting any therapeutic failure and minimizing undesired effects. Here, we present an in silico platform that showed to be in very good agreement with the latest literature in predicting SARS- CoV-2 dynamics and related immune system host response. Moreover, it has been used to predict the outcome of one of the latest suggested approach to design an effective vaccine, based on monoclonal antibody. UISS is then potentially ready to be used as an in silico trial platform to predict the outcome of vaccination strategy against SARS-CoV-2.

negative: A SARS-CoV-2 vaccine candidate would likely match all currently circulating strains The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Here we analyzed SARS-CoV-2 sequence diversity across 5,700 sequences sampled since December 2019. The Spike protein, which is the target immunogen of most vaccine candidates, showed 93 sites with shared polymorphisms; only one of these mutations was found in more than 1% of currently circulating sequences. The minimal diversity found among SARS-CoV-2 sequences can be explained by drift and bottleneck events as the virus spread away from its original epicenter in Wuhan, China. Importantly, there is little evidence that the virus has adapted to its human host since December 2019. Our findings suggest that a single vaccine should be efficacious against current global strains. One Sentence Summary The limited diversification of SARS-CoV-2 reflects drift and bottleneck events rather than adaptation to humans as the virus spread.

negative: Cytokine regulation in SARS coronavirus infection compared to other respiratory virus infections The pathogenesis of severe acute respiratory syndrome (SARS) is poorly understood and cytokine dysregulation has been suggested as one relevant mechanism to be explored. We compared the cytokine profile in Caco2 cells after infection of SARS coronavirus (SARS‐CoV) with other respiratory viruses including respiratory syncytial virus (RSV), influenza A virus (FluAV), and human parainfluenza virus type 2 (hPIV2). Interferon (IFN) system (production and response) was not suppressed by SARS‐CoV infection. Therefore, SARS‐CoV replication was suppressed by pretreatment with IFN. SARS‐CoV and RSV induced high levels of IL‐6 and RANTES compared with FluAV and hPIV2. Induction level of suppressor of cytokine signaling‐3 (SOCS3) by SARS‐CoV was significantly lower than that by RSV in spite of the significant production of IL‐6. Toll‐like receptors 4 and 9, which correlate with the induction of inflammatory response, were upregulated by SARS‐CoV infection. Collectively, overinduction of inflammatory cytokine and dysregulation of cytokine signaling may contribute to the immunopathology associated with “severe” inflammation in SARS. J. Med. Virol. 78:417–424, 2006. © 2006 Wiley‐Liss, Inc.

negative: In vivo cytokine response to experimental feline infectious peritonitis virus infection Abstract Feline infectious peritonitis virus (FIPV) is a coronavirus that causes sporadic fatal disease in cats characterized by vasculitis, granulomatous inflammation and effusive pleuritis/peritonitis. Histologic changes in lymphoid tissues include lymphoid hyperplasia, lymphoid depletion, histiocytosis, and granuloma formation. Although viremia occurs, histologic lesions are not found uniformly throughout lymphoid tissues. We used experimental infection of cats with a highly pathogenic FIPV isolate, UCD8, to study histologic lesions, virus replication, and cytokine expression in multiple lymphoid tissues during the effusive phase of disease. Viral RNA was found in 76% of central tissues (mediastinal lymph node, spleen, mesenteric lymph node) examined, as compared to 27% of peripheral tissues (popliteal lymph node, cervical lymph node, femoral bone marrow). All tissues positive for virus replication also demonstrated lymphoid depletion. Generally, affected tissues had lower levels of IL-4 and IL-12–p40 mRNA and higher levels of IL-10 mRNA. Although no differences in IFN-γ or TNF-α mRNA were measured, TNF-α protein expression was greater in affected tissues and demonstrated a shift in the source of TNF-α from macrophages to lymphocytes. Together, these results colocalize FIPV replication, lymphocyte depletion in tissues, and alterations in cytokine transcription and translation. A possible role for TNF-α in the previously described FIPV-induced lymphocyte apoptosis is also suggested.

negative: Defining Protective Epitopes for COVID‐19 Vaccination Models Recent papers in the Journal provide tangible avenues for COVID‐19 vaccine production as immunoreactive epitopes are brought to the forefront in these and many other emerging studies.(1,2) The development of a consistent predictable animal model of COVID‐19 infection is evidently also a welcome event for preliminary antiviral and vaccine assessments and surely brings us to another level of progression.(3) This article is protected by copyright. All rights reserved.

negative: The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model BACKGROUND: Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention. METHODS: Recombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay. RESULTS: After the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups. CONCLUSION: Recombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.

negative: Comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains Abstract Infectious bronchitis virus (IBV) produces six subgenomic (sg) mRNAs, each containing a 64 nucleotide (nt) leader sequence, derived from the 5′ end of the genome by a discontinuous process. Several putative functional domains such as a papain-like proteinase (PLpro), main protease (Mpro), RNA-dependent RNA polymerase (RdRp), and RNA helicase encoded by the replicase gene are important for virus replication. We have sequenced four regions of the replicase genes corresponding to the 5′-terminal sequence, PLpro, Mpro, and RdRp domains from 20 heterologous IBV strains, and compared them with previously published coronavirus sequences. All the coronavirus 5′-termini and PLpro domains were divergent, unlike the Mpro and the RdRp domains that were highly conserved with 28% and 48% conserved residues, respectively. Among IBV strains, the 5′ untranslated region including the leader sequence was highly conserved (>94% identical); whereas, the N-terminal coding region and the PLpro domains were highly variable ranging from 84.6% to 100%, and 77.6% to 100% identity, respectively. The IBV Mpro and RdRp domains were highly conserved with 82.7% and 92.7% conserved residues, respectively. The BJ strain was the most different from other IBVs in all four regions of the replicase. Phylogeny-based clustering based on replicase genes was identical to the antigen-based classification of coronaviruses into three groups. However, the IBV strain classification based on replicase gene domains did not correlate with that of the type-specific antigenic groups. The replicase gene sequences of many IBVs recovered from infected chickens were identical to those of vaccine viruses irrespective of serotype, suggesting that either there has been an exchange of genetic material among vaccine and field isolates or that there is a convergent evolution to a specific replicase genotype. There was no correlation between the genotype of any region of the replicase gene and pathotype, suggesting that the replicase is not the sole determinant of IBV pathogenicity.

negative: Convergent antibody responses to SARS-CoV-2 in convalescent individuals During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000. Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

negative: Mapping the Immunodominance Landscape of SARS-CoV-2 Spike Protein for the Design of Vaccines against COVID-19 The ongoing coronavirus disease 2019 (COVID-19) pandemic is a serious threat to global public health, and imposes severe burdens on the entire human society. The severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can cause severe respiratory illness and death. Currently, there are no specific antiviral drugs that can treat COVID-19. Several vaccines against SARS-CoV-2 are being actively developed by research groups around the world. The surface S (spike) protein and the highly expressed internal N (nucleocapsid) protein of SARS-CoV-2 are widely considered as promising candidates for vaccines. In order to guide the design of an effective vaccine, we need experimental data on these potential epitope candidates. In this study, we mapped the immunodominant (ID) sites of S protein using sera samples collected from recently discharged COVID-19 patients. The SARS-CoV-2 S protein-specific antibody levels in the sera of recovered COVID-19 patients were strongly correlated with the neutralising antibody titres. We used epitope mapping to determine the landscape of ID sites of S protein, which identified nine linearized B cell ID sites. Four out of the nine ID sites were found in the receptor-binding domain (RBD). Further analysis showed that these ID sites are potential high-affinity SARS-CoV-2 antibody binding sites. Peptides containing two out of the nine sites were tested as vaccine candidates against SARS-CoV-2 in a mouse model. We detected epitope-specific antibodies and SARS-CoV-2-neutralising activity in the immunised mice. This study for the first time provides human serological data for the design of vaccines against COVID-19.

negative: Hypercytokinemia and Pathogen–Host Interaction in COVID-19 Severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 (SARS-CoV-2) is a novel coronavirus identified as the cause of coronavirus disease-2019 (COVID-19) that began in Wuhan, China in late 2019 and spread now in 210 countries and territories around the world. Many people are asymptomatic or with mild symptoms. However, in some cases (usually the elderly and those with comorbidities) the disease may progress to pneumonia, acute respiratory distress syndrome and multi-organ dysfunction that can lead to death. Such wide interindividual differences in response to SARS-CoV-2 infection may relate to several pathogen- and host-related factors. These include the different levels of the ubiquitously present human angiotensin I converting enzyme 2 (ACE2) receptors gene expression and its variant alleles, the different binding affinities of ACE2 to the virus spike (S) protein given its L- and S-subtypes and the subsequent extent of innate immunity-related hypercytokinemia. The extensive synthesis of cytokines and chemokines in coronavirus diseases was suggested as a major factor in exacerbating lung damage and other fatal complications. The polymorphisms in genes coding for pro-inflammatory cytokines and chemokines have been associated with mediating the response and susceptibility to a wide range of infections and their severe outcomes. Understanding the nature of pathogen–host interaction in COVID-19 symptomatology together with the role of hypercytokinemia in disease severity may permit developing new avenues of approach for prevention and treatment and can delineate public health measures to control the spread of the disease.

negative: Comparison of commercial RT-PCR diagnostic kits for COVID-19 Abstract The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n = 13) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n = 6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

negative: Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS Abstract The SARS-CoV spike glycoprotein (S) is the main target of the protective immune response in humans and animal models of SARS. Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). The presence of both splice sites and WPRE markedly improved the immunogenicity of S-based DNA vaccines against SARS. Upon immunization of mice with low doses (2 μg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. Our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic RNA viruses.

negative: In Silico design and characterization of multi-epitopes vaccine for SARS-CoV2 from its spike proteins COVID 19 is disease caused by novel corona virus, SARS-CoV2 originated in China most probably of Bat origin. Till date, no specific vaccine or drug has been discovered to tackle the infections caused by SARS-CoV2. In response to this pandemic, we utilized bioinformatics knowledge to develop efficient vaccine candidate against SARS-CoV2. Designed vaccine was rich in effective BCR and TCR epitopes screened from the sequence of S-protein of SARS-CoV2. Predicted BCR and TCR epitopes were antigenic in nature non-toxic and probably non-allergen. Modelled and refined tertiary structure was predicted as valid for further use. Protein-Protein interaction prediction of TLR2/4 and designed vaccine indicates promising binding. Designed multiepitope vaccine has induced cell mediated and humoral immunity along with increased interferon gamma response. Macrophages and dendritic cells were also found increased over the vaccine exposure. In silico codon optimization and cloning in expression vector indicates that vaccine can be efficiently expressed in E. coli. In conclusion, predicted vaccine is a good antigen, probable no allergen and has potential to induce cellular and humoral immunity.

negative: Coronavirus mRNA synthesis: Identification of novel transcription initiation signals which are differentially regulated by different leader sequences Abstract The mRNA synthesis of mouse hepatitis virus (MHV) has been proposed to be the result of interaction between the leader RNA and the intergenic sites. Previously, we have identified a transcription initiation site (for mRNA 2-1), which is more efficiently transcribed by viruses containing two copies of UCUAA sequence in the leader RNA than by those with three copies. In this study, we have identified several sites which are regulated in the opposite way, namely, they are efficiently transcribed by the leader RNA with three UCUAA copies but not by those with two copies. These sites were characterized by primer extension and amplification by polymerase chain reaction. One of these sites is in the gene 3 region of a recombinant virus between A59 and JHM strains of MHV. Another is in the gene 2 region of MHV-1 strain. Both of these sites have a sequence similar to but different from the consensus transcription initiation signal (UCUAAUCUAUC and UUUAAUCUU, as opposed to UCUAAAC). These two novel intergenic sequences are not present in the genome of the JHM strain, consistent with the absence of these mRNAs in the JHM-infected cells. The discovery of this type of transcription initiation site provides additional evidence for the importance of the leader RNA in the transcription initiation of MHV mRNAs.

negative: Evidence of SARS-CoV-2 mRNA in endomyocardial biopsies of patients with clinically suspected myocarditis tested negative for COVID-19 in nasopharyngeal swab

negative: Preliminary identification of potential vaccine targets for the COVID-19 coronavirus (SARS-CoV-2) based on SARS-CoV immunological studies The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the available SARS-CoV-2 sequences (as of 9 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

negative: The SARS-CoV-2 Vaccine Pipeline: an Overview Purpose of Review: The goal of this review is to provide a timely overview on efforts to develop a vaccine for the 2019 novel coronavirus SARS-CoV-2, the causative agent of coronavirus disease (COVID-19). Recent Findings: Previous research efforts to develop a severe acute respiratory syndrome coronavirus (SARS-CoV) vaccine in the years following the 2003 pandemic have opened the door for investigators to design vaccine concepts and approaches for the COVID-19 epidemic in China. Both SARS-CoV and SARS-CoV-2 exhibit a high degree of genetic similarity and bind to the same host cell ACE2 receptor. Based on previous experience with SARS-CoV vaccines, it is expected that all COVID-19 vaccines will require careful safety evaluations for immunopotentiation that could lead to increased infectivity or eosinophilic infiltration. Besides this, a COVID-19 vaccine target product profile must address vaccinating at-risk human populations including frontline healthcare workers, individuals over the age of 60, and those with underlying and debilitating chronic conditions. Among the vaccine technologies under evaluation are whole virus vaccines, recombinant protein subunit vaccines, and nucleic acid vaccines. Summary: Each current vaccine strategy has distinct advantages and disadvantages. Therefore, it is paramount that multiple strategies be advanced quickly and then evaluated for safety and efficacy. Ultimately, the safety studies to minimize undesired immunopotentiation will become the most significant bottleneck in terms of time.

negative: Liposomes used as a vaccine adjuvant-delivery system: From basics to clinical immunization Abstract Liposomes are widely utilized as a carrier to improve therapeutic efficacy of agents thanks to their merits of high loading capacity, targeting delivery, reliable protection of agents, good biocompatibility, versatile structure modification and adjustable characteristics, such as size, surface charge, membrane flexibility and the agent loading mode. In particular, in recent years, through modification with immunopotentiators and targeting molecules, and in combination with innovative immunization devices, liposomes are rapidly developed as a multifunctional vaccine adjuvant-delivery system (VADS) that has a high capability in inducing desired immunoresponses, as they can target immune cells and even cellular organelles, engender lysosome escape, and promote Ag cross-presentation, thus enormously enhancing vaccination efficacy. Moreover, after decades of development, several products developed on liposome VADS have already been authorized for clinical immunization and are showing great advantages over conventional vaccines. This article describes in depth some critical issues relevant to the development of liposomes as a VADS, including principles underlying immunization, physicochemical properties of liposomes as the immunity-influencing factors, functional material modification to enhance immunostimulatory functions, the state-of-the-art liposome VADSs, as well as the marketed vaccines based on a liposome VADS. Therefore, this article provides a comprehensive reference to the development of novel liposome vaccines.

negative: Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood Newly emerging human viruses such as Ebola virus, severe acute respiratory syndrome (SARS) virus, and HIV likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. Although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. We describe here mycobacteriophage Patience, a newly isolated phage recovered using Mycobacterium smegmatis mc(2)155 as a host. Patience has genomic features distinct from its M. smegmatis host, including a much lower GC content (50.3% versus 67.4%) and an abundance of codons that are rarely used in M. smegmatis. Nonetheless, it propagates well in M. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over 75% of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. We propose that Patience evolved primarily among lower-GC hosts and that the disparities between its genomic profile and that of M. smegmatis presented only a minimal barrier to host expansion. Rapid adaptions to its new host include recent acquisition of higher-GC genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host.

negative: The coronavirus proofreading exoribonuclease mediates extensive viral recombination Coronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and generates defective viral genomes (DVGs) of unknown function. However, the determinants and patterns of CoV recombination are unknown. Here, we show that divergent β-CoVs SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV) perform extensive RNA recombination in culture, generating similar patterns of recombination junctions and diverse populations of DVGs and sgmRNAs. We demonstrate that the CoV proofreading nonstructural protein (nsp14) 3’-to-5’ exoribonuclease (nsp14-ExoN) is required for normal CoV recombination and that its genetic inactivation causes significantly decreased frequency and altered patterns of recombination in both infected cells and released virions. Thus, nsp14-ExoN is a key determinant of both high fidelity CoV replication and recombination, and thereby represents a highly-conserved and vulnerable target for virus inhibition and attenuation.

negative: A SARS-CoV-2 protein interaction map reveals targets for drug repurposing A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

negative: Polarization of immunity induced by direct injection of naked sequence-stabilized mRNA vaccines In the context of developing a safe genetic vaccination strategy we tested and studied globin-stabilized mRNA-based vaccination in mice. This vaccination strategy has the advantages of genetic vaccination (easy production, adaptability to any disease and inexpensive storage when lyophilized), but not the drawbacks of DNA vaccination (long-term uncontrolled expression of a transgene, possibility of integration into the host genome and possible induction of anti-DNA antibodies). We report here that injection of naked β-globin untranslated region (UTR)-stabilized mRNA coding for β-galactosidase is followed by detectable translation in vivo. In addition, we show that such a vaccination strategy primes a T helper 2 (Th2) type of response which can be enhanced and shifted to a Th1-type immune response by application of recombinant granulocyte/macrophage colony-stimulating factor 1 day after mRNA injection. Our data demonstrate that the administration of globin UTR-stabilized mRNA is a versatile vaccination strategy that can be manipulated to fit the requirement of antiviral, antibacterial or antitumor immunity.

negative: Potential Applications of Plant Biotechnology against SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for an ongoing human pandemic (COVID-19). There is a massive international effort underway to develop diagnostic reagents, vaccines, and antiviral drugs in a bid to slow down the spread of the disease and save lives. One part of that international effort involves the research community working with plants, bringing researchers from all over the world together with commercial enterprises to achieve the rapid supply of protein antigens and antibodies for diagnostic kits, and scalable production systems for the emergency manufacturing of vaccines and antiviral drugs. Here, we look at some of the ways in which plants can and are being used in the fight against COVID-19.

negative: Coronavirus leader‐RNA‐primed transcription: An alternative mechanism to RNA splicing Many viral and cellular mRNA species contain a leader sequence derived from a distant upstream site on the same gene by a process of RNA splicing. This process usually involves either nuclear functions or self‐splicing of RNA molecules. Coronavirus, a cytoplasmic RNA virus, unfolds yet another mechanism of joining RNA, which involves the use of a free leader RNA molecule. This molecule is synthesized and dissociates from the template RNA, and subsequently reassociates with the template RNA at down‐stream initiation sites of subgenomic mRNAs to serve as the primer for transcription. This leader‐primed transcriptional process thus generates viral mRNAs with a fused leader sequence. A similar mechanism might also operate in the mRNA transcription of African trypanosomes.

negative: Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) remains a threat to cause epidemics as evidenced by recent sporadic cases in China. In this communication, we evaluated the efficacy and safety of two SARS vaccine candidates based on the recombinant modified vaccinia Ankara (MVA) expressing SARS-CoV spike or nucleocapsid proteins in ferrets. No clinical signs were observed in all the ferrets challenged with SARS-CoV. On the other hand, vaccination did not prevent SARS-CoV infection in ferrets. In contrast, immunized ferrets (particularly those immunized with rMVA-spike) exhibited significantly stronger inflammatory responses and focal necrosis in liver tissue after SARS-CoV challenge than control animals. Thus, our data suggest that enhanced hepatitis is linked to vaccination with rMVA expressing SARS-CoV antigens.

negative: COVID-19: Risk of increase in smoking rates among England's 6 million smokers and relapse among England's 11 million ex-smokers

negative: Structural basis to design multi-epitope vaccines against Novel Coronavirus 19 (COVID19) infection, the ongoing pandemic emergency: an in silico approach The 2019 novel coronavirus (COVID19 / Wuhan coronavirus), officially named as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a positive-sense single-stranded RNA coronavirus. SARS-CoV-2 causes the contagious COVID19 disease also known as 2019-nCoV acute respiratory disease and has led to the ongoing 2019–20 pandemic COVID19 outbreak. The effective counter measures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidate. In the present study, we have screened and shortlisted 38 CTL, 33 HTL and 12 B cell epitopes from the eleven Protein sequences of SARS-CoV-2 by utilizing different in silico tools. The screened epitopes were further validated for their binding with their respective HLA allele binders and TAP (Transporter associated with antigen processing) molecule by molecular docking. The shortlisted screened epitopes were further utilized to design novel two multi-epitope vaccines (MEVs) composed of CTL, HTL and B cell epitopes overlaps with potential to elicit humoral as well as cellular immune response against SARS-CoV-2. To enhance the immune response for our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has been utilized as an adjuvant at N terminal of both the MEVs. Further molecular models for both the MEVs were prepared and validated for their stable molecular interactions with Toll-Like Receptor 3 (TLR 3). The codon-optimized cDNA of both the MEVs were further analyzed for their potential of high level of expression in a human cell line. The present study is very significant in terms of molecular designing of prospective CTL and HTL vaccine against SARS-CoV-2 infection with the potential to elicit cellular as well as humoral immune response. (SARS-CoV-2), Coronavirus, Human Transporter associated with antigen processing (TAP), Toll-Like Receptor (TLR), Epitope, Immunoinformatics, Molecular Docking, Molecular dynamics simulation, Multi-epitope Vaccine Graphical abstract The designed CTL (Cytotoxic T lymphocyte) and HTL (Helper T lymphocyte) multi-epitope vaccines (MEV) against COVID19 infection. Both the CTL and HTL MEV models show a very stable and well fit conformational complex formation tendency with the Toll like receptor 3. CTL and HTL MEVs: ribbon; Toll like receptor 3: gray cartoon; Adjuvant [truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1]: orange ribbon regions; Epitopes: cyan ribbons regions; 6xHis Tag: magenta ribbon regions.

negative: Characterizations of Coronaviruscis-Acting RNA Elements and the Transcription Step Affecting Its Transcription Efficiency Abstract Seven to eight species of viral subgenomic mRNAs are produced in coronavirus-infected cells. These mRNAs are produced in different quantities, and their molar ratios remain constant during viral replication. We studied RNA elements that affect coronavirus transcription efficiency by characterizing a series of cloned coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNAs containing an inserted intergenic sequence, from which subgenomic DI RNA is transcribed in MHV-infected cells. Certain combinations of upstream and downstream flanking sequences of the intergenic sequence suppressed subgenomic DI RNA transcription, yet changing one of the flanking sequences to a different sequence eliminated transcription suppression. The suppressive effect of certain combinations of flanking sequences, but not all combinations, could be counteracted by altering the intergenic sequence. Thus, the combination of intergenic sequence and flanking sequence affected transcription efficiency. We also characterized another set of DI RNAs designed to clarify which transcription step determines the relative molar ratios of coronavirus mRNAs. Our study indicated that if subgenomic mRNAs were exclusively synthesized from negative-strand genomic RNA, then the relative molar ratios of coronavirus mRNAs were most likely determined after synthesis of the genomic-sized template RNA. If negative-strand subgenomic RNAs were templates for subgenomic mRNAs, then the relative molar ratios of coronavirus mRNAs probably were determined after synthesis of the genomic-sized template RNA used for subgenomic-sized RNA transcription but prior to the completion of the synthesis of subgenomic-sized RNAs containing the leader sequence. The relative molar ratios of coronavirus mRNAs, therefore, seem to have been established prior to a putative replicon-type amplification of subgenomic mRNAs.

negative: Nanotechnology for COVID-19: Therapeutics and Vaccine Research [Image: see text] The current global health threat by the novel coronavirus disease 2019 (COVID-19) requires an urgent deployment of advanced therapeutic options available. The role of nanotechnology is highly relevant to counter this “virus” nano enemy. Nano intervention is discussed in terms of designing effective nanocarriers to counter the conventional limitations of antiviral and biological therapeutics. This strategy directs the safe and effective delivery of available therapeutic options using engineered nanocarriers, blocking the initial interactions of viral spike glycoprotein with host cell surface receptors, and disruption of virion construction. Controlling and eliminating the spread and reoccurrence of this pandemic demands a safe and effective vaccine strategy. Nanocarriers have potential to design risk-free and effective immunization strategies for severe acute respiratory syndrome coronavirus 2 vaccine candidates such as protein constructs and nucleic acids. We discuss recent as well as ongoing nanotechnology-based therapeutic and prophylactic strategies to fight against this pandemic, outlining the key areas for nanoscientists to step in.

negative: Hydroxychloroquine and chloroquine in COVID-19: should they be used as standard therapy? The pandemic of the new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has urged the nations to an unprecedented world-wide reaction, including an accelerated exploration of therapeutic options. In the absence of a vaccine and specifically designed antivirals, the medical community has proposed the use of various previously available medications in order to reduce the number of patients requiring prolonged hospitalizations, oxygen therapy, and mechanical ventilation and to decrease mortality from coronavirus disease 2019 (COVID-19). Hydroxychloroquine and chloroquine are among the proposed drugs and are the most widely used so far, despite the lack of robust evidence on their usefulness. The objective of this article is to review and discuss the possible role of these drugs in the therapy of COVID-19.

negative: Chapter 36 SARS Abstract Five years after the first severe acute respiratory syndrome (SARS) outbreak, several candidate SARS-coronavirus (CoV) vaccines are at various stages of preclinical and clinical development. Based on the observation that SARSCoV infection is efficiently controlled upon passive transfer of antibodies directed against the spike (S) protein of SARS-CoV, vaccines containing the S protein have been formulated. Animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the SARS-CoV S protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type 3, adenovirus, or attenuated vaccinia virus MVA as a vector), as well as mice immunized with DNA vaccines expressing the S protein gene all developed neutralizing antibodies to SARS-CoV and were protected against SARS-CoV challenge. Although much effort has been focused on developing a SARS vaccine, the commercial viability of such a vaccine for SARS-CoV will ultimately depend on whether the virus re-emerges in the near future. This vaccine should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to SARS-CoV and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. Given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals.

negative: Evaluation of nine commercial SARS-CoV-2 immunoassays Due to urgency and demand, numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. Thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of SARS-CoV-2 infection, confirmation of molecular results, contact tracing, and epidemiological studies. This study evaluated the sensitivity and specificity of nine commercially available serological tests. These included three enzyme-linked immunosorbent assays (ELISAs) and six point-of-care (POC) lateral flow tests. The assays were validated using serum samples from: i) SARS-CoV-2 PCR-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China; iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or non-coronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and Epstein Barr virus. The results showed 100% specificity for the Wantai SARS-CoV-2 Total Antibody ELISA, 93% for the Euroimmun IgA ELISA, and 96% for the Euroimmun IgG ELISA with sensitivities of 90%, 90%, and 65%, respectively. The overall performance of the POC tests according to manufacturer were in the rank order of AutoBio Diagnostics > Dynamiker Biotechnology = CTK Biotech > Artron Laboratories > Acro Biotech ≥ Hangzhou Alltest Biotech. Overall, these findings will facilitate selection of serological assays for the detection SARS-CoV-2-specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies.

negative: Knowledge of novel coronavirus (SARS-COV-2) among a Georgian population Introduction Georgia confirmed its first case of SARS-COV-2 infection on February 26, 2020 and by March 31, a total of 110 cases have been reported. Limited understanding about epidemics can lead to panic and disrupt public health efforts to contain transmission. It is important to understand perceptions of the population regarding the disease. This study reports results of a survey designed to understand attitudes and knowledge regarding SARS-COV-2 virus among Georgian population, including health care workers (HCWs). Materials and methods The online survey was conducted using a Facebook advertisement. The target was the whole country and the language used was Georgian. We collected information on demographic data, knowledge of the disease, including perceived differences between coronavirus and influenza. We also included questions to capture the populations perceptions about government preparedness to combat the new coronavirus. Results The survey was open for three days (March 2-4, 2020). 5228 participants completed the survey. Of these, 40.3% were 25-45 years old, 58.2% were female, 20.7% had university degree and 10.3% were HCWs. For 25.8% of respondents, COVID-19 and influenza are the same diseases; 10.9% did not know if they are different. The majority correctly identified the transmission route and symptoms (96.9% and 98.0%, respectively). 19.1% think that Georgia is ready for COVID 19 epidemic, while according to 55% the county is not ready, but HCWs are trying hard to respond properly. For 18% response is inadequate. There was no difference in knowledge between HCWs, non-HCWs and unemployed. 20% of HCWs as well as other study subjects believe that SARS-COV-2 vaccine and medications do exist but are simply not available in Georgia. Conclusion Given that information regarding coronavirus is changing very rapidly, the need to reach people with time-sensitive educational messages as well as prevention strategies is vital.

negative: COVID-19 and smoking: A systematic review of the evidence COVID-19 is a coronavirus outbreak that initially appeared in Wuhan, Hubei Province, China, in December 2019, but it has already evolved into a pandemic spreading rapidly worldwide(1,2). As of 18 March 2020, a total number of 194909 cases of COVID-19 have been reported, including 7876 deaths, the majority of which have been reported in China (3242) and Italy (2505)(3). However, as the pandemic is still unfortunately under progression, there are limited data with regard to the clinical characteristics of the patients as well as to their prognostic factors(4). Smoking, to date, has been assumed to be possibly associated with adverse disease prognosis, as extensive evidence has highlighted the negative impact of tobacco use on lung health and its causal association with a plethora of respiratory diseases(5). Smoking is also detrimental to the immune system and its responsiveness to infections, making smokers more vulnerable to infectious diseases(6). Previous studies have shown that smokers are twice more likely than non-smokers to contract influenza and have more severe symptoms, while smokers were also noted to have higher mortality in the previous MERS-CoV outbreak(7,8). Given the gap in the evidence, we conducted a systematic review of studies on COVID-19 that included information on patients’ smoking status to evaluate the association between smoking and COVID-19 outcomes including the severity of the disease, the need for mechanical ventilation, the need for intensive care unit (ICU) hospitalization and death. The literature search was conducted on 17 March 2020, using two databases (PubMed, ScienceDirect), with the search terms: [‘smoking’ OR ‘tobacco’ OR ‘risk factors’ OR ‘smoker*’] AND [‘COVID-19’ OR ‘COVID 19’ OR ‘novel coronavirus’ OR ‘sars cov-2’ OR ‘sars cov 2’] and included studies published in 2019 and 2020. Further inclusion criteria were that the studies were in English and referred to humans. We also searched the reference lists of the studies included. A total of 71 studies were retrieved through the search, of which 66 were excluded after full-text screening, leaving five studies that were included. All of the studies were conducted in China, four in Wuhan and one across provinces in mainland China. The populations in all studies were patients with COVID-19, and the sample size ranged from 41 to 1099 patients. With regard to the study design, retrospective and prospective methods were used, and the timeframe of all five studies covered the first two months of the COVID-19 pandemic (December 2019, January 2020). Specifically, Zhou et al.(9) studied the epidemiological characteristics of 191 individuals infected with COVID-19, without, however, reporting in more detail the mortality risk factors and the clinical outcomes of the disease. Among the 191 patients, there were 54 deaths, while 137 survived. Among those that died, 9% were current smokers compared to 4% among those that survived, with no statistically significant difference between the smoking rates of survivors and non-survivors (p=0.21) with regard to mortality from COVID-19. Similarly, Zhang et al.(10) presented clinical characteristics of 140 patients with COVID-19. The results showed that among severe patients (n=58), 3.4% were current smokers and 6.9% were former smokers, in contrast to non-severe patients (n=82) among which 0% were current smokers and 3.7% were former smokers , leading to an OR of 2.23; (95% CI: 0.65–7.63; p=0.2). Huang et al.(11) studied the epidemiological characteristics of COVID-19 among 41 patients. In this study, none of those who needed to be admitted to an ICU (n=13) was a current smoker. In contrast, three patients from the non-ICU group were current smokers, with no statistically significant difference between the two groups of patients (p=0.31), albeit the small sample size of the study. The largest study population of 1099 patients with COVID-19 was provided by Guan et al.(12) from multiple regions of mainland China. Descriptive results on the smoking status of patients were provided for the 1099 patients, of which 173 had severe symptoms, and 926 had non-severe symptoms. Among the patients with severe symptoms, 16.9% were current smokers and 5.2% were former smokers, in contrast to patients with non-severe symptoms where 11.8% were current smokers and 1.3% were former smokers. Additionally, in the group of patients that either needed mechanical ventilation, admission to an ICU or died, 25.5% were current smokers and 7.6% were former smokers. In contrast, in the group of patients that did not have these adverse outcomes, only 11.8% were current smokers and 1.6% were former smokers. No statistical analysis for evaluating the association between the severity of the disease outcome and smoking status was conducted in that study. Finally, Liu et al.(13) found among their population of 78 patients with COVID-19 that the adverse outcome group had a significantly higher proportion of patients with a history of smoking (27.3%) than the group that showed improvement or stabilization (3.0%), with this difference statistically significant at the p=0.018 level. In their multivariate logistic regression analysis, the history of smoking was a risk factor of disease progression (OR=14.28; 95% CI: 1.58–25.00; p= 0.018). We identified five studies that reported data on the smoking status of patients infected with COVID-19. Notably, in the largest study that assessed severity, there were higher percentages of current and former smokers among patients that needed ICU support, mechanical ventilation or who had died, and a higher percentage of smokers among the severe cases(12). However, from their published data we can calculate that the smokers were 1.4 times more likely (RR=1.4, 95% CI: 0.98–2.00) to have severe symptoms of COVID-19 and approximately 2.4 times more likely to be admitted to an ICU, need mechanical ventilation or die compared to non-smokers (RR=2.4, 95% CI: 1.43–4.04). In conclusion, although further research is warranted as the weight of the evidence increases, with the limited available data, and although the above results are unadjusted for other factors that may impact disease progression, smoking is most likely associated with the negative progression and adverse outcomes of COVID-19.

negative: Genetic immunization with Hantavirus vaccine combining expression of G2 glycoprotein and fused interleukin-2 In this research, we developed a novel chimeric HTNV-IL-2-G2 DNA vaccine plasmid by genetically linking IL-2 gene to the G2 segment DNA and tested whether it could be a candidate vaccine. Chimeric gene was first expressed in eukaryotic expression system pcDNA3.1 (+). The HTNV-IL-2-G2 expressed a 72 kDa fusion protein in COS-7 cells. Meanwhile, the fusion protein kept the activity of its parental proteins. Furthermore, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response.- The results showed that the chimeric gene could simultaneously evoke specific antibody against G2 glycoprotein and IL-2. And the immunized mice of every group elicited neutralizing antibodies with different titers. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to G2 and IL-2 were significantly higher than that of other groups. Our results suggest that IL-2-based HTNV G2 DNA can induce both humoral and cellular immune response specific for HTNV G2 and can be a candidate DNA vaccine for HTNV infection.

negative: Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2 Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

negative: Nanomaterials and Nanotechnology-Associated Innovations against Viral Infections with a Focus on Coronaviruses Viral infections have recently emerged not only as a health threat to people but rapidly became the cause of universal fatality on a large scale. Nanomaterials comprising functionalized nanoparticles (NPs) and quantum dots and nanotechnology-associated innovative detection methods, vaccine design, and nanodrug production have shown immense promise for interfacing with pathogenic viruses and restricting their entrance into cells. These viruses have been scrutinized using rapid diagnostic detection and therapeutic interventional options against the caused infections including vaccine development for prevention and control. Coronaviruses, namely SARS-CoV, MERS-CoV, and SARS-CoV-2, have endangered human life, and the COVID-19 (caused by SARS-CoV-2) outbreak has become a perilous challenge to public health globally with huge accompanying morbidity rates. Thus, it is imperative to expedite the drug and vaccine development efforts that would help mitigate this pandemic. In this regard, smart and innovative nano-based technologies and approaches encompassing applications of green nanomedicine, bio-inspired methods, multifunctional bioengineered nanomaterials, and biomimetic drug delivery systems/carriers can help resolve the critical issues regarding detection, prevention, and treatment of viral infections. This perspective review expounds recent nanoscience advancements for the detection and treatment of viral infections with focus on coronaviruses and encompasses nano-based formulations and delivery platforms, nanovaccines, and promising methods for clinical diagnosis, especially regarding SARS-CoV-2.

negative: Battling COVID-19 Pandemic: Sphingosine-1-Phosphate Analogs as an Adjunctive Therapy? With the sudden outbreak of COVID-19 patient worldwide and associated mortality, it is critical to come up with an effective treatment against SARS-CoV-2. Studies suggest that mortality due to COVID 19 is mainly attributed to the hyper inflammatory response leading to cytokine storm and ARDS in infected patients. Sphingosine-1-phosphate receptor 1 (S1PR1) analogs, AAL-R and RP-002, have earlier provided in-vivo protection from the pathophysiological response during H1N1 influenza infection and improved mortality. Recently, it was shown that the treatment with sphingosine-1-phosphate receptor 1 analog, CYM5442, resulted in the significant dampening of the immune response upon H1N1 challenge in mice and improved survival of H1N1 infected mice in combination with an antiviral drug, oseltamivir. Hence, here we suggest to investigate the possible utility of using S1P analogs to treat COVID-19.

negative: Design of multi-epitope vaccine candidate against SARS-CoV-2: a in-silico study The best therapeutic strategy to find an effective vaccine against SARS-CoV-2 is to explore the target structural protein. In the present study, a novel multi-epitope vaccine is designed using in silico tools that potentially trigger both CD4 and CD8 T-cell immune responses against the novel Coronavirus. The vaccine candidate was designed using B and T-cell epitopes that can act as an immunogen and elicits immune response in the host system. NCBI was used for the retrieval of surface spike glycoprotein, of novel corona virus (SARS-CoV-2) strains. VaxiJen server screens the most important immunogen of all the proteins and IEDB server gives the prediction and analysis of B and T cell epitopes. Final vaccine construct was designed in silico composed of 425 amino acids including the 50S ribosomal protein adjuvant and the construct was computationally validated in terms of antigenicity, allergenicity and stability on considering all critical parameters into consideration. The results subjected to the modeling and docking studies of vaccine were validated. Molecular docking study revealed the protein-protein binding interactions between the vaccine construct and TLR-3 immune receptor. The MD simulations confirmed stability of the binding pose. The immune simulation results showed significant response for immune cells. The findings of the study confirmed that the final vaccine construct of chimeric peptide could able to enhance the immune response against nCoV-19.

negative: Gene expression, virulence and vaccine development in coronaviruses

negative: Novel 2019 coronavirus: Genome structure, clinical trials, and outstanding questions Novel 2019 coronavirus has created havoc across the globe since its emergence in December 2019 in Wuhan, China, and fast spreading potential. While we were able to identify the causative agent within a few days of the disease outbreak, several questions still remain unanswered. In this review, we discuss the extent of virus spread, current statistics, SARS-CoV-2 genome organization, comparison between the novel coronavirus and causative agents involved in previous outbreaks, ongoing clinical trials and myths associated with the virus. Lastly, we provide insights into the future perspectives which could prove useful for the scientific community as they work on finding the cure against the disease. IMPACT STATEMENT: Early availability of the sequence, the genetic material of SARS-CoV-2 (the virus that causes COVID-19), has prompted efforts towards identifying a safe and effective vaccine in the current public health emergency. To that end, understanding the pathophysiology of disease is crucial for scientists around the world. Since conventional vaccine development and manufacturing may take several years, it is important to think about alternative strategies that we could use to mitigate imminent catastrophe. We hope that this article will open up new avenues and provide insights that could potentially save hundreds of lives affected by COVID-19.

negative: SARS-CoV-2 Productively Infects Human Gut Enterocytes COVID-19, caused by SARS-CoV-2, is an influenza-like disease with a respiratory route of transmission, yet clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids, enterocytes were readily infected by SARS-CoV and SARS-CoV-2 as demonstrated by confocal- and electron-microscopy. Consequently, significant titers of infectious viral particles were measured. mRNA expression analysis revealed strong induction of a generic viral response program. We conclude that intestinal epithelium supports SARS-CoV-2 replication. One Sentence Summary SARS-CoV-2 infection of enterocytes in human small intestinal organoids

negative: SARS-CoV-2/COVID-19: a primer for cardiologists In the late autumn of 2019, a new potentially lethal human coronavirus designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. The pandemic spread of this zoonotic virus has created a global health emergency and an unprecedented socioeconomic crisis. The severity of coronavirus disease 2019 (COVID-19), the illness caused by SARS-CoV‑2, is highly variable. Most patients (~85%) develop no or mild symptoms, while others become seriously ill, some succumbing to disease-related complications. In this review, the SARS-CoV‑2 life cycle, its transmission and the clinical and immunological features of COVID-19 are described. In addition, an overview is presented of the virological assays for detecting ongoing SARS-CoV‑2 infections and the serological tests for SARS-CoV-2-specific antibody detection. Also discussed are the different approaches to developing a COVID-19 vaccine and the perspectives of treating COVID-19 with antiviral drugs, immunomodulatory agents and anticoagulants/antithrombotics. Finally, the cardiovascular manifestations of COVID-19 are briefly touched upon. While there is still much to learn about SARS-CoV‑2, the tremendous recent advances in biomedical technology and knowledge and the huge amount of research into COVID-19 raise the hope that a remedy for this disease will soon be found. COVID-19 will nonetheless have a lasting impact on human society. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12471-020-01475-1) contains supplementary material, which is available to authorized users.

negative: Toward Nanotechnology-Enabled Approaches against the COVID-19 Pandemic The COVID-19 outbreak has fueled a global demand for effective diagnosis and treatment as well as mitigation of the spread of infection, all through large-scale approaches such as specific alternative antiviral methods and classical disinfection protocols. Based on an abundance of engineered materials identifiable by their useful physicochemical properties through versatile chemical functionalization, nanotechnology offers a number of approaches to cope with this emergency. Here, through a multidisciplinary Perspective encompassing diverse fields such as virology, biology, medicine, engineering, chemistry, materials science, and computational science, we outline how nanotechnology-based strategies can support the fight against COVID-19, as well as infectious diseases in general, including future pandemics. Considering what we know so far about the life cycle of the virus, we envision key steps where nanotechnology could counter the disease. First, nanoparticles (NPs) can offer alternative methods to classical disinfection protocols used in healthcare settings, thanks to their intrinsic antipathogenic properties and/or their ability to inactivate viruses, bacteria, fungi, or yeasts either photothermally or via photocatalysis-induced reactive oxygen species (ROS) generation. Nanotechnology tools to inactivate SARS-CoV-2 in patients could also be explored. In this case, nanomaterials could be used to deliver drugs to the pulmonary system to inhibit interaction between angiotensin-converting enzyme 2 (ACE2) receptors and viral S protein. Moreover, the concept of "nanoimmunity by design" can help us to design materials for immune modulation, either stimulating or suppressing the immune response, which would find applications in the context of vaccine development for SARS-CoV-2 or in counteracting the cytokine storm, respectively. In addition to disease prevention and therapeutic potential, nanotechnology has important roles in diagnostics, with potential to support the development of simple, fast, and cost-effective nanotechnology-based assays to monitor the presence of SARS-CoV-2 and related biomarkers. In summary, nanotechnology is critical in counteracting COVID-19 and will be vital when preparing for future pandemics.

negative: Intranasally Administered Antigen 85B Gene Vaccine in Non-Replicating Human Parainfluenza Type 2 Virus Vector Ameliorates Mouse Atopic Dermatitis Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.

negative: COVIDep platform for real-time reporting of vaccine target recommendations for SARS-CoV-2: Description and connections with COVID-19 immune responses and preclinical vaccine trials We introduce COVIDep (https://COVIDep.ust.hk), a web-based platform that provides immune target recommendations for guiding SARS-CoV-2 vaccine development. COVIDep implements a protocol that pools together publicly-available genetic data for SARS-CoV-2 and epitope data for SARS-CoV to identify B cell and T cell epitopes that present potential immune targets for SARS-CoV-2. Correspondences between outputs of COVIDep and immune responses recorded in COVID-19 patients and preclinical vaccine trials are also indicated. The platform is user-friendly, flexible, and based on up-to-date data. It may help guide vaccine designs and associated experimental studies for SARS-CoV-2.

negative: COVID-19 pandemic predictions using the modified Bateman SIZ model and observational data for Heidelberg, Germany: Effect of vaccination with a SARS-CoV-2 vaccine, coronavirus testing and application of the Corona-Warn-App. AIMS OF THE STUDY Published data show that the current progression of the COVID-19 pandemic in Heidelberg, Germany, despite the current lockdown, could continue into 2021 and become more severe. We have used the modified Bateman SIZ algorithm to predict the effects of interventional measures to control the COVID-19 pandemic. MATERIALS AND METHODS Model parameters, e.g., doubling time and rate of decrease in the number of infectious persons were obtained from published reports. Predictions were made for the status quo on June 1, 2020, and for interventional measures obtained for 4 scenarios. These included vaccination of the whole population using a SARS-CoV-2 vaccine having an efficacy of 50% and 100%, mass-testing for COVID-19 coronavirus and application of the Corona-Warn-App. RESULTS The principle findings were 1) without new measures to control the pandemic, the daily number of infectious persons could reach a peak of > 4,500 daily within 18 months when > 67,000 persons would have been infected. This could be prevented by using a vaccine with 50% efficacy which was almost equally effective as a vaccine with 100% efficacy. Application of the Corona-Warn-App was the most effective method and more effective than testing for COVID-19. The methodology used has been described in detail to enable other researchers to apply the modified Bateman SIZ model to obtain predictions for COVID-19 outbreaks in other regions. Application of the model has been verified by independent investigators using different commercial software packages. CONCLUSION The modified Bateman SIZ model has been verified and used to predict the course of the COVID-19 pandemic in Heidelberg. Lockdown measures alone are insufficient to control the pandemic during 2021. Vaccination, diagnostic tests, and use of the Corona-Warn-App with quarantine could successfully control the spread of the coronavirus infection in the community. The Corona-Warn-App applied correctly may be the most effective. The model showed that vaccination with 50% efficacy is almost as effective as vaccination with 100% efficacy.

negative: Anticipating SARS-CoV-2 Vaccine Testing, Licensure, and Recommendations for Use

negative: A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2 Effective countermeasures against the recent emergence and rapid expansion of the 2019 novel coronavirus (SARS-CoV-2) require the development of data and tools to understand and monitor its spread and immune responses to it. However, little information is available about the targets of immune responses to SARS-CoV-2. We used the Immune Epitope Database and Analysis Resource (IEDB) to catalog available data related to other coronaviruses. This includes SARS-CoV, which has high sequence similarity to SARS-CoV-2 and is the best-characterized coronavirus in terms of epitope responses. We identified multiple specific regions in SARS-CoV-2 that have high homology to the SARS-CoV virus. Parallel bioinformatic predictions identified a priori potential B and T cell epitopes for SARS-CoV-2. The independent identification of the same regions using two approaches reflects the high probability that these regions are promising targets for immune recognition of SARS-CoV-2. These predictions can facilitate effective vaccine design against this virus of high priority.

negative: The Importance of Advancing SARS-CoV-2 Vaccines in Children While the role of children in the chain of transmission of SARS-CoV-2 remains to be fully defined, they likely play an important role based on our knowledge of other respiratory viruses. Children are more likely to be asymptomatic or have milder symptoms and less likely to present for healthcare and be tested for SARS-CoV-2; thus, our current estimates are likely under-representative of the true burden of SARS-CoV-2 in children. Given the potential direct benefit of a SARS-CoV-2 vaccine in children and the substantial indirect benefit through community protection or ‘herd immunity’, we argue that planning and implementation of SARS-CoV-2 vaccines should include children. Furthermore, community protection occurred after widespread implementation of prior childhood vaccines against Streptococcus pneumoniae, rubella and rotavirus. We detail considerations for vaccine clinical trials, potential barriers to the implementation of widespread vaccination and argue why children would be an ideal target population for vaccination.

negative: Do COVID-19 and SARS Gene Complexities and Variations Help Overcome the Knowledge Gap? A whole new pathogen, to which humans have virtually no pre-existing immunity, has caused fear all over the world. Severe acute respiratory syndrome coronavirus (SARS CoV-2) is one of the types of human novel-coronavirus of the family coronavirus. The nature of transmission of the virus makes it one of the most infectious pathogenic diseases that has ever existed. Though the human coronaviruses have existed since the discovery of the human coronavirus 229E (HCoV-229E) and human coronavirus OC43 (HCoV-OC43) in 1960, it has been a challenge to develop an effective cure as well as vaccine for the diseases associated with coronaviruses. Commonly, human coronaviruses cause illnesses such as intestinal and respiratory tract illnesses. Nevertheless, the symptoms reflected after infection from the coronaviruses take some time before being identified. Thus, viruses can replicate and cause more harm to the human body before being detected. Moreover, research continues to explain why some gene variations in some individuals increase the risk of some infectious diseases, while others are not affected. Looking at gene variations in people infected with Coronavirus Disease 2019 (COVID-19) and studying how genes influence people's response to infection will help to develop a vaccine that will help strengthen the immune system. Knowing how the human genes respond to the virus COVID-19 will help to cure people more effectively.

negative: Replicative virus shedding in the respiratory tract of patients with Middle East respiratory syndrome coronavirus infection Abstract Background Information on the duration of replicative Middle East respiratory syndrome coronavirus (MERS-CoV) shedding is important for infection control. The detection of MERS-CoV sub-genomic mRNAs indicates that the virus is replicative. This study examined the duration for detecting MERS-CoV sub-genomic mRNA compared with genomic RNA in diverse respiratory specimens. Methods Upper and lower respiratory samples were obtained from 17 MERS-CoV-infected patients. MERS-CoV sub-genomic mRNA was detected by reverse transcription PCR (RT-PCR) and MERS-CoV genomic RNA by real-time RT-PCR. Results In sputum and transtracheal aspirate, sub-genomic mRNA was detected for up to 4 weeks after symptoms developed, which correlated with the detection of genomic RNA. In oropharyngeal and nasopharyngeal swab specimens, the detection of sub-genomic mRNA and genomic RNA did not correlate. Conclusions These findings suggest that MERS-CoV does not replicate well in the upper respiratory tract.

negative: SARS-CoV-2 productively infects human gut enterocytes Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.

negative: Expression cloning of functional receptor used by SARS coronavirus Abstract We have expressed a series of truncated spike (S) glycoproteins of SARS-CoV and found that the N-terminus 14–502 residuals were sufficient to bind to SARS-CoV susceptible Vero E6 cells. With this soluble S protein fragment as an affinity ligand, we screened HeLa cells transduced with retroviral cDNA library from Vero E6 cells and obtained a HeLa cell clone which could bind with the S protein. This cell clone was susceptible to HIV/SARS pseudovirus infection and the presence of a functional receptor for S protein in this cell clone was confirmed by the cell–cell fusion assay. Further studies showed the susceptibility of this cell was due to the expression of endogenous angiotensin-converting enzyme 2 (ACE2) which was activated by inserted LTR from retroviral vector used for expression cloning. When human ACE2 cDNA was transduced into NIH3T3 cells, the ACE2 expressing NIH3T3 cells could be infected with HIV/SARS pseudovirus. These data clearly demonstrated that ACE2 was the functional receptor for SARS-CoV.

negative: A DNA prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge Abstract The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750μg DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200μg 4F/4R S fragment. One dose of 100μg DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750μg DNA vaccine and one dose of 200μg 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100μg DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV.

negative: Comparative studies of the capsid precursor polypeptide P1 and the capsid protein VP1 cDNA vectors for DNA vaccination against foot‐and‐mouth disease virus BACKGROUND: Foot‐and‐mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies. MATERIALS AND METHODS: Here we evaluated comparatively three different viral antigen‐encoding DNA sequences, delivered via two physical means (i.e., gene gun delivery into skin and electroporation delivery into muscle), for naked DNA‐mediated vaccination in a mouse system. RESULTS: Both methods gave similar results, demonstrating commonality of the observed DNA vaccine effects. Immunization with a cDNA vector expressing the major viral antigen (VP1) alone routinely failed to induce the production of anti‐VP1 or neutralizing antibodies in test mice. As a second approach, the plasmid L‐VP1 that produces a transgenic membrane‐anchored VP1 protein elicited a strong antibody response, but all test mice failed in the FMDV challenge experiment. In contrast, for mice immunized with the viral capsid precursor protein (P1) cDNA expression vector, both neutralizing antibodies and 80–100% protection in test mice were detected. CONCLUSIONS: This strategy of using the whole capsid precursor protein P1 cDNA for vaccination, intentionally without the use of virus‐specific protease or other encoding genes for safety reasons, may thus be employed as a relevant experimental system for induction or upgrading of effective neutralizing antibody response, and as a convenient surrogate test system for DNA vaccination studies of FMDV and presumably other viral diseases. Copyright © 2005 John Wiley & Sons, Ltd.

negative: The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19 The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

negative: Stop playing with data: there is no sound evidence that Bacille Calmette-Guérin may avoid SARS-CoV-2 infection (for now) Since the beginning of the COVID-19 epidemic, a possible explanation for the high heterogeneity of infection/mortality rates across involved countries was hinted in the prevalence of tuberculosis vaccination with Bacille Calmette-Guérin (BCG). A systematic review was therefore performed on May 2, 2020. A total of 13 articles were ultimately retrieved, 12 of them as preprint papers. All articles were ecological studies of low quality. Most of them did not include main confounding factors (i.e. demographic of the assessed countries, share of peo- ple residing in urban settings, etc.), and simply assessed the differences among incidence/mortality of COVID-19 with vaccination rates or by having vs. having not any vaccination policy for BCG. Even though all studies shared the very same information sources (i.e. international registries for BCG vaccination rates and open source data for COVID-19 epidemics), results were conflicting, with later studies apparently denying any true correlation between COVID-19 occurrence and BCG vaccination rates and/or policies. As a consequence, there is no sound evidence to recommend BCG vaccination for the prevention of COVID-19.

negative: Bacillus Calmette–Guérin vaccine, age and gender relation to COVID-19 spread and mortality Abstract COVID-19 is affecting different countries all over the world with great variation in infection rate and death ratio. Some reports suggested a relation between the Bacillus Calmette-Guérin (BCG) vaccine and the malaria treatment to the prevention of SARS-CoV-2 infection. Some reports related infant's lower susceptibility to the BCG vaccine. Some other reports a higher risk in males compared to females in such COVID-19 pandemic. Also, some other reports claimed the possible use of chloroquine and hydroxychloroquine as prophylactic in such a pandemic. The present commentary is to discuss the possible relation between those factors and SARS-CoV-2 infection.

negative: SARS‐CoV‐2: What do we know so far? We are living in times where a viral disease has brought normal life in much of the world to a halt. Named after its causative agent, coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus recently renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 manifests as dry cough, frequent fevers and in severe cases pneumonia. Older patients and patients with underlying comorbidities are at a higher risk of death. We are living in times where a viral disease has brought normal life in much of the world to a halt. Named after its causative agent, coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus recently renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 manifests as dry cough, frequent fevers and in severe cases pneumonia.

negative: Immunologic aspects of characteristics, diagnosis, and treatment of coronavirus disease 2019 (COVID-19) On March 11, 2020, the World Health Organization declared the worldwide spread of the infectious disease COVID-19, caused by a new strain of coronavirus, SARS-CoV-2, as a pandemic. Like in all other infectious diseases, the host immune system plays a key role in our defense against SARS-CoV-2 infection. However, viruses are able to evade the immune attack and proliferate and, in susceptible individuals, cause severe inflammatory response known as cytokine storm, particularly in the lungs. The advancement in our understanding of the mechanisms underlying the host immune responses promises to facilitate the development of approaches for prevention or treatment of diseases. Components of immune system, such as antibodies, can also be used to develop sensitive and specific diagnostic methods as well as novel therapeutic agents. In this review, we summarize our knowledge about how the host mounts immune responses to infection by SARS-CoV-2. We also describe the diagnostic methods being used for COVID-19 identification and summarize the current status of various therapeutic strategies, including vaccination, being considered for treatment of the disease.

negative: Emergence of deadly severe acute respiratory syndrome coronavirus-2 during 2019-2020 Wuhan, the city in Hubei province in China is in the focus of global community due to the outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), formerly known as 2019-nCoV. The virus emerged in humans from Wuhan seafood market probably via zoonotic transmission. Within a few days the virus spread its tentacles rapidly to neighboring cities in China and to different geographical regions through travelers and to some extent by human to human transmission leading to significant disease burden globally. More than 2,00,000 people (including more than 8000 deaths) have been infected with this respiratory illness across 167 countries and territories worldwide leading to a pandemic. The present review provides an outline about emergence and spread of SARS-CoV-2 from Wuhan, China in 2019-2020. We have also provided information about the classification, genome, proteins, clinical presentation of COVID-19, type of clinical specimens to be collected and diagnostic methods adopted to identify the respiratory illness. In addition we have also provided information about transmission dynamics, prevention measures and treatment options that are available at the present. Subsequently, we have given a comprehensive overview of the spread of this infection from China to the other parts of the globe. Management of the ongoing outbreak of SARS-CoV-2 encompassing surveillance, clinical, immunological, genetic and evolutionary investigations are likely to provide the desired results. Joint efforts of global scientific community are needed at this hour in terms of enhancement of research on development of accurate diagnostics, antiviral therapeutics and finally into formation of an effective vaccine against the emerging novel coronavirus.

negative: Addressing COVID‐19 Drug Development with Artificial Intelligence The emergence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the virus that led to the COVID‐19 (Coronavirus Disease 2019) pandemic, has resulted in substantial overburdening of healthcare systems as well as an economic crisis on a global scale. This has in turn resulted in widespread efforts to identify suitable therapies to address this aggressive pathogen. Therapeutic antibody and vaccine development are being actively explored, and a phase I clinical trial of mRNA‐1273 which is developed in collaboration between the National Institute of Allergy and Infectious Diseases and Moderna, Inc. is currently underway. Timelines for the broad deployment of a vaccine and antibody therapies have been estimated to be 12–18 months or longer. These are promising approaches that may lead to sustained efficacy in treating COVID‐19. However, its emergence has also led to a large number of clinical trials evaluating drug combinations composed of repurposed therapies. As study results of these combinations continue to be evaluated, there is a need to move beyond traditional drug screening and repurposing by harnessing artificial intelligence (AI) to optimize combination therapy design. This may lead to the rapid identification of regimens that mediate unexpected and markedly enhanced treatment outcomes.

negative: Revisiting potential druggable targets against SARS-CoV-2 and repurposing therapeutics under preclinical study and clinical trials: A comprehensive review Coronavirus disease-19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is one of the most contagious diseases in human history that has already affected millions of lives worldwide. To date, no vaccines or effective therapeutics have been discovered yet that may successfully treat COVID-19 patients or contain the transmission of the virus. Scientific communities across the globe responded rapidly and have been working relentlessly to develop drugs and vaccines, which may require considerable time. In this uncertainty, repurposing the existing antiviral drugs could be the best strategy to speed up the discovery of effective therapeutics against SARS-CoV-2. Moreover, drug repurposing may leave some vital information on druggable targets that could be capitalized in target-based drug discovery. Information on possible drug targets and the progress on therapeutic and vaccine development also needs to be updated. In this review, we revisited the druggable targets that may hold promise in the development of the anti-SARS-CoV-2 agent. Progresses on the development of potential therapeutics and vaccines that are under the preclinical studies and clinical trials have been highlighted. We anticipate that this review will provide valuable information that would help to accelerate the development of therapeutics and vaccines against SARS-CoV-2 infection.

negative: RNA interference inhibits hepatitis E virus mRNA accumulation and protein synthesis in vitro Abstract Hepatitis E virus (HEV) is a zoonotic pathogen to which several species, including human beings, pigs and rodents, are reported to be susceptible. To date, vaccines developed against HEV still need to be improved and a structural gene (ORF2), which encodes a capsid protein with high sequence conservation found across HEV genotypes, is a potential candidate. To exploit the possibility of using RNA interference (RNAi) as a strategy against HEV infection, four small interference RNA (siRNA) duplex targeting ORF2 gene were constructed. A challenge against HEV infection by RNAi was performed in A549 cells. Real-Time quantitative polymerase chain reaction (Real-Time qPCR) and Western blot assay demonstrated that four HEV specific siRNAs (si-ORF2-1, si-ORF2-2, si-ORF2-3 and si-ORF2-4) were capable of protecting cells against HEV infection with very high specificity and efficiency. The results suggest that RNAi is a potent anti-HEV infection prophylaxis strategy.

negative: A Three-Stemmed mRNA Pseudoknot in the SARS Coronavirus Frameshift Signal A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.

negative: Self-replicating alphavirus RNA vaccines. Recombinant nucleic acids are considered as promising next-generation vaccines. These vaccines express the native antigen upon delivery into tissue, thus mimicking live attenuated vaccines without having the risk of reversion to pathogenicity. They also stimulate the innate immune system, thus potentiating responses. Nucleic acid vaccines are easy to produce at reasonable cost and are stable. During the past years, focus has been on the use of plasmid DNA for vaccination. Now mRNA and replicon vaccines have come into focus as promising technology platforms for vaccine development. This review discusses self-replicating RNA vaccines developed from alphavirus expression vectors. These replicon vaccines can be delivered as RNA, DNA or as recombinant virus particles. All three platforms have been pre-clinically evaluated as vaccines against a number of infectious diseases and cancer. Results have been very encouraging and propelled the first human clinical trials, the results of which have been promising.

negative: Treating Influenza and SARS-CoV-2 via mRNA-encoded Cas13a Here, Cas13a has been used to target and mitigate influenza virus A (IAV) and SARS-CoV-2 using a synthetic mRNA-based platform. CRISPR RNAs (crRNA) against PB1 and highly conserved regions of PB2 were screened in conjunction with mRNA-encoded Cas13a. Screens were designed such that only guides that decreased influenza RNA levels in a Cas13-mediated fashion, were valid. Cas13a mRNA and validated guides, delivered post-infection, simulating treatment, were tested in combination and across multiplicities of infection. Their function was also characterized over time. Similar screens were performed for guides against SARS-CoV-2, yielding multiple guides that significantly impacted cytopathic effect. Last, the approach was utilized in vivo, demonstrating the ability to degrade influenza RNA in a mouse model of infection, using polymer-formulated, nebulizer-based mRNA delivery. Our findings demonstrate the applicability of Cas13a in mitigating respiratory infections both in vitro and in a mouse model, paving the way for future therapeutic use.

negative: Application prospect of polysaccharides in the development of anti-novel coronavirus drugs and vaccines Since the outbreak of the novel coronavirus disease COVID-19, caused by the SARS-CoV-2 virus, it has spread rapidly worldwide and poses a great threat to public health. This is the third serious coronavirus outbreak in <20 years, following SARS in 2002–2003 and MERS in 2012. So far, there are almost no specific clinically effective drugs and vaccines available for COVID-19. Polysaccharides with good safety, immune regulation and antiviral activity have broad application prospects in anti-virus, especially in anti-coronavirus applications. Here, we reviewed the antiviral mechanisms of some polysaccharides, such as glycosaminoglycans, marine polysaccharides, traditional Chinese medicine polysaccharides, and their application progress in anti-coronavirus. In particular, the application prospects of polysaccharide-based vaccine adjuvants, nanomaterials and drug delivery systems in the fight against novel coronavirus were also analyzed and summarized. Additionally, we speculate the possible mechanisms of polysaccharides anti-SARS-CoV-2, and propose the strategy of loading S or N protein from coronavirus onto polysaccharide capped gold nanoparticles vaccine for COVID-19 treatment. This review may provide a new approach for the development of COVID-19 therapeutic agents and vaccines.

negative: A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies. Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.

negative: The New Coronavirus, the Current King of China

negative: Could anti-CD20 therapy jeopardise the efficacy of a SARS-CoV-2 vaccine? A vaccine against SARS-CoV-2 might represent the most promising approach to halt durably the current COVID-19 pandemic. We believe that anti-CD20 therapy may jeopardise the efficacy of such a vaccine. This is regrettable because patients receiving anti-CD20 therapy (i.e. those with haematologic malignancies or autoimmune disorders) are particularly at risk of severe COVID-19 and, as such, are the most in need of a vaccine. Here, we review the reasons why anti-CD20 therapy may abrogate or diminish the efficacy of a vaccine against SARS-CoV-2 and we draw physicians' attention towards this potential risk so that it can be considered when evaluating the risk/benefit ratio of anti-CD20 therapy during the current pandemic.

negative: Immunogenicity of a recombinant coronavirus spike glycoprotein expressed in transgenic plants Recently, it has been demonstrated that plants offer the possibility of producing low cost subunit vaccines that can be either parenterally or orally administered. Here we review data we obtained on the immunological response elicited by two recombinant versions of the glycoprotein S from the swine transmissible gastroenteritis coronavirus (TGEV) expressed in transgenic plants. Arabidopsis or potato plants were genetically transformed with cDNAs constructs encoding the N-terminal domain (aa residues 1–750) or the full-length glycoprotein S of TGEV, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Genomic DNA and mRNA analysis of leave extracts from transformed plants demonstrated the incorporation of the foreign cDNA into the plant genomes as well as their transcription. Expression of recombinant polypeptides was observed in most transgenic plants by ELISA using specific antibodies. Mice immunized either parenterally with leave extracts from transgenic arabidopsis plants or, more interestingly, fed with potato tubers, developed antibodies that specifically reacted with TGEV in ELISA, immunoprecipitated the glycoprotein S and in some cases neutralized the virus infectivity. From the above results, we conclude that transgenic plants expressing glycoprotein S polypeptides may be potentially used as a source of recombinant antigen for vaccine production.

negative: A universal design of betacoronavirus vaccines against COVID-19, MERS and SARS Summary Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously-emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10-100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

negative: Recent progress and challenges in drug development against COVID-19 coronavirus (SARS-CoV-2) - an update on the status Coronaviruses are a large group of viruses known to cause illnesses that vary between the common cold and more severe diseases to include severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). A novel coronavirus was identified in December 2019 in Wuhan city, Hubei province, China. This virus represents a new strain that has not been previously identified in humans. The virus is now known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting disease is called coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) declared the novel coronavirus outbreak a global pandemic in March 2020. Despite rigorous global containment and quarantine efforts, the incidence of COVID-19 continues to rise, with more than 1,948,617 laboratory-confirmed cases and over 121,846 deaths worldwide. Currently, no specific medication is recommended to treat COVID-19 patients. However, governments and pharmaceutical companies are struggling to quickly find an effective drug to defeat the coronavirus. In the current review, we summarize the existing state of knowledge about COVID-19, available medications, and treatment options. Favilavir is an antiviral drug that is approved in Japan for common influenza treatment and is now approved to treat symptoms of COVID-19 in China. Moreover, Chloroquine and hydroxychloroquine, drugs used to treat malaria and arthritis, respectively, were recommended by the National Health Commission of the People's Republic of China for treatment of COVID-19. Presently, chloroquine and hydroxychloroquine are under investigation by the US Food and Drug Administration (FDA) as a treatment for COVID-19. The first COVID-19 vaccine is not expected to be ready for clinical trials before the end of the year.

negative: Novel Stem Cells and Nucleic Acid-Based Vaccine Trials Against Viral Outbreak: A Systematic Evaluation During COVID-2019 Pandemic The current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak, the cause of coronavirus disease (COVID-19), has influenced health globally. So far, there are no established management options and prophylaxis for those who have been exposed to SARS-CoV-2, and those who develop COVID-19. Documented scientific evidences in similar viral outbreaks in past suggested few therapy regimens. These rather have not shown promising results in management of current pandemic. So, in the current review, we are exploring novel treatment strategies and therapies that are being explored and are in clinical and preclinical stages of research. To explore more about the same, we directed our search towards stem cell based, DNA based, or RNA based vaccines against COVID-19 under development by various universities, institutes or pharmaceutical companies. The current scientific literature and database search were performed by exploring various Trials registry (NIH: https://clinicaltrials.gov/ and https://www.coronavirus.gov) and Chinese clinical trial registry http://www.chictr.org.cn/) and for preclinical trials various University, Institutions, Pharmaceutical companies websites and news bulletins along with google search were checked routinely from 3rd March 2020 to 16 May 2020. The term “Stem Cell therapy and COVID-19”, “Mesenchymal stem cell and corona 2019 virus”, “DNA Vaccines and COVID-19, RNA Vaccines and COVID-19” and “Cell-based therapy with SARS-CoV-2, University/Institutions and COVID-19 research” were used. The vaccine trials (Stem Cells/DNA/RNA) which were cancelled were not included in this review. Similarly, few others like repurposing of drugs, Nano Vaccines, other miscellaneous trials of Herbs, Music therapy etc., were also excluded. In the present review, we have included the various novel therapies like stem cell therapy, DNA or RNA vaccines which are under development and if proven successful may have a lasting impact on the health industry.

negative: Pandemic Preparedness: Developing Vaccines and Therapeutic Antibodies For COVID-19 Abstract The SARS-CoV-2 pandemic that causes COVID-19 respiratory syndrome has caused global public health and economic crises necessitating rapid development of vaccines and therapeutic countermeasures. The world-wide response to the COVID-19 pandemic has been unprecedented with government, academic and private partnerships working together to rapidly develop vaccine and antibody countermeasures. Many of the technologies being used are derived from prior government-academic partnerships for response to other emerging infections.

negative: Two RNA-binding sites in plant fibrillarin provide interactions with various RNA substrates Fibrillarin, one of the major proteins of the nucleolus, plays several essential roles in ribosome biogenesis including pre-rRNA processing and 2′-O-ribose methylation of rRNA and snRNAs. Recently, it has been shown that fibrillarin plays a role in virus infections and is associated with viral RNPs. Here, we demonstrate the ability of recombinant fibrillarin 2 from Arabidopsis thaliana (AtFib2) to interact with RNAs of different lengths and types including rRNA, snoRNA, snRNA, siRNA and viral RNAs in vitro. Our data also indicate that AtFib2 possesses two RNA-binding sites in the central (138–179 amino acids) and C-terminal (225–281 amino acids) parts of the protein, respectively. The conserved GCVYAVEF octamer does not bind RNA directly as suggested earlier, but may assist with the proper folding of the central RNA-binding site.

negative: Coronavirus (COVID-19): A Review of Clinical Features, Diagnosis, and Treatment Coronavirus (COVID-19) is an enveloped RNA virus that is diversely found in humans and wildlife. A total of six species have been identified to cause disease in humans. They are known to infect the neurological, respiratory, enteric, and hepatic systems. The past few decades have seen endemic outbreaks in the form of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome related coronavirus (SARS-CoV). Yet again, we see the emergence of another outbreak due to a new strain called the SARS-CoV-2 virus. The most recent outbreak initially presented as pneumonia of unknown etiology in a cluster of patients in Wuhan, China. The epicenter of infection was linked to seafood and exotic animal wholesale markets in the city. SARS-CoV-2 is highly contagious and has resulted in a rapid pandemic of COVID-19. As the number of cases continues to rise, it is clear that these viruses pose a threat to public health. This review will introduce a general overview of coronavirus and describe the clinical features, evaluation, and treatment of COVID-19 patients. It will also provide a means to raise awareness among primary and secondary healthcare providers during the current pandemic. Furthermore, our review focuses on the most up-to-date clinical information for the effective management, prevention, and counseling of patients worldwide.

negative: RNA secondary structure packages ranked and improved by high-throughput experiments The computer-aided study and design of RNA molecules is increasingly prevalent across a range of disciplines, yet little is known about the accuracy of commonly used structure prediction packages in real-world tasks. Here, we evaluate the performance of current packages using EternaBench, a dataset comprising 23 in vitro structure mapping and 11 riboswitch activity datasets involving 18,509 synthetic sequences from the crowdsourced RNA design project Eterna. We find that CONTRAfold and RNAsoft, packages with parameters derived through statistical learning, achieve consistently higher accuracy than more widely used packages like the ViennaRNA software, which derive parameters primarily from thermodynamic experiments. Motivated by these results, we develop a multitask-learning-based model, EternaFold, which demonstrates improved performance that generalizes to diverse external datasets, including complete viral genomes probed in vivo and synthetic designs modeling mRNA vaccines.

negative: Repurposed prophylaxis strategies for COVID-19: a systematic review Introduction Efficient therapeutic strategies are needed to counter the COVID-19 pandemic, caused by the SARS- CoV-2 virus. In a context where specific vaccines are not yet available, the containment of the pandemic would be facilitated with efficient prophylaxis. Methods We screened several clinical trials repositories and platforms in search of the prophylactic strategies that are investigated against COVID-19 in late April 2020. Results Up to April 27, 2020, we found 68 clinical trials targeting medical workers (n=43, 63%), patients relatives (n=16, 24%) or individuals at risk of severe COVID-19 (n=5, 7%). (Hydroxy)chloroquine was the most frequently evaluated treatment (n=46, 68%), before BCG vaccine (n=5, 7%). Sixty-one (90%) clinical trials were randomized with a median of planned inclusions of 600 (IQR 255-1515). Conclusion The investigated prophylaxis strategies cover both pre- and post-exposure prophylaxis and study numerous immune enhancers and antivirals, although most research efforts are focused on (hydroxy)chloroquine.

negative: Early Pandemic Evaluation and Enhanced Surveillance of COVID-19 (EAVE II): protocol for an observational study using linked Scottish national data INTRODUCTION: Following the emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 and the ensuing COVID-19 pandemic, population-level surveillance and rapid assessment of the effectiveness of existing or new therapeutic or preventive interventions are required to ensure that interventions are targeted to those at highest risk of serious illness or death from COVID-19. We aim to repurpose and expand an existing pandemic reporting platform to determine the attack rate of SARS-CoV-2, the uptake and effectiveness of any new pandemic vaccine (once available) and any protective effect conferred by existing or new antimicrobial drugs and other therapies. METHODS AND ANALYSIS: A prospective observational cohort will be used to monitor daily/weekly the progress of the COVID-19 epidemic and to evaluate the effectiveness of therapeutic interventions in approximately 5.4 million individuals registered in general practices across Scotland. A national linked dataset of patient-level primary care data, out-of-hours, hospitalisation, mortality and laboratory data will be assembled. The primary outcomes will measure association between: (A) laboratory confirmed SARS-CoV-2 infection, morbidity and mortality, and demographic, socioeconomic and clinical population characteristics; and (B) healthcare burden of COVID-19 and demographic, socioeconomic and clinical population characteristics. The secondary outcomes will estimate: (A) the uptake (for vaccines only); (B) effectiveness; and (C) safety of new or existing therapies, vaccines and antimicrobials against SARS-CoV-2 infection. The association between population characteristics and primary outcomes will be assessed via multivariate logistic regression models. The effectiveness of therapies, vaccines and antimicrobials will be assessed from time-dependent Cox models or Poisson regression models. Self-controlled study designs will be explored to estimate the risk of therapeutic and prophylactic-related adverse events. ETHICS AND DISSEMINATION: We obtained approval from the National Research Ethics Service Committee, Southeast Scotland 02. The study findings will be presented at international conferences and published in peer-reviewed journals.

negative: No evidence that androgen regulation of pulmonary TMPRSS2 explains sex-discordant COVID-19 outcomes The recent emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic have posed a public health crisis. Higher morbidity and mortality of men with COVID-19 may be explained by androgen-driven mechanisms. One such proposed mechanism is androgen regulation of pulmonary TMPRSS2, the host co-receptor for SARS-CoV-2. We find no evidence for increased TMPRSS2 mRNA expression in the lungs of males compared to females in humans or mice. Furthermore, in male mice, treatment with the androgen receptor antagonist enzalutamide does not decrease pulmonary TMPRSS2 expression. Nevertheless, regardless of sex, smoking significantly increases the expression of TMPRSS2, which reverts back to never-smoker levels in former smokers. Finally, we show that in mouse models, despite equivalent AR transcript levels, males express markedly higher amounts of AR protein. If a similar sex-specific regulation of AR protein occurs in human lung, androgens could play important roles in clinical outcome of COVID-19 through mechanisms other than TMPRSS2 regulation.

negative: A method of identifying the blood contributor in mixture stains through detecting blood specific mRNA polymorphism In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identifying individuals in mixture samples composed of two body fluids. In this study, we selected 5 blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension reaction (SBE) followed by capillary electrophoresis. The amplicon size was between 90bp-294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in home-made mixtures which are composed of different body fluids with 1ng peripheral blood mRNA added. This system showed a super sensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method of identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood-specific SNP typing system showed high sensitivity to the typing of blood-source specific markers regardless of other body fluids in the mixture. This article is protected by copyright. All rights reserved.

negative: Human Leukocyte Antigen Susceptibility Map for Severe Acute Respiratory Syndrome Coronavirus 2 Genetic variability across the three major histocompatibility complex (MHC) class I genes (human leukocyte antigen A [HLA-A], -B, and -C genes) may affect susceptibility to and severity of the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). We performed a comprehensive in silico analysis of viral peptide-MHC class I binding affinity across 145 HLA-A, -B, and -C genotypes for all SARS-CoV-2 peptides. We further explored the potential for cross-protective immunity conferred by prior exposure to four common human coronaviruses. The SARS-CoV-2 proteome was successfully sampled and was represented by a diversity of HLA alleles. However, we found that HLA-B*46:01 had the fewest predicted binding peptides for SARS-CoV-2, suggesting that individuals with this allele may be particularly vulnerable to COVID-19, as they were previously shown to be for SARS (M. Lin, H.-T. Tseng, J. A. Trejaut, H.-L. Lee, et al., BMC Med Genet 4:9, 2003, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-4-9). Conversely, we found that HLA-B*15:03 showed the greatest capacity to present highly conserved SARS-CoV-2 peptides that are shared among common human coronaviruses, suggesting that it could enable cross-protective T-cell-based immunity. Finally, we reported global distributions of HLA types with potential epidemiological ramifications in the setting of the current pandemic. IMPORTANCE Individual genetic variation may help to explain different immune responses to a virus across a population. In particular, understanding how variation in HLA may affect the course of COVID-19 could help identify individuals at higher risk from the disease. HLA typing can be fast and inexpensive. Pairing HLA typing with COVID-19 testing where feasible could improve assessment of severity of viral disease in the population. Following the development of a vaccine against SARS-CoV-2, the virus that causes COVID-19, individuals with high-risk HLA types could be prioritized for vaccination.

negative: Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples The ongoing spread of pandemic coronavirus disease (COVID-19) is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). In the lack of specific drugs or vaccines for SARS-CoV-2, it demands rapid diagnosis and management for controlling the outbreak in the community. Here we report the development of the first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 minutes. We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and changes in gold-colloid color from pink to blue in visible range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19 (n=7). The colloid retained pink color when the test includes samples from COVID-19 negative subjects (n=6) or human papillomavirus (HPV) infected women (n=2). The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. Overall, the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

negative: Emergence of Drift Variants That May Affect COVID-19 Vaccine Development and Antibody Treatment New coronavirus (SARS-CoV-2) treatments and vaccines are under development to combat COVID-19. Several approaches are being used by scientists for investigation, including (1) various small molecule approaches targeting RNA polymerase, 3C-like protease, and RNA endonuclease; and (2) exploration of antibodies obtained from convalescent plasma from patients who have recovered from COVID-19. The coronavirus genome is highly prone to mutations that lead to genetic drift and escape from immune recognition; thus, it is imperative that sub-strains with different mutations are also accounted for during vaccine development. As the disease has grown to become a pandemic, B-cell and T-cell epitopes predicted from SARS coronavirus have been reported. Using the epitope information along with variants of the virus, we have found several variants which might cause drifts. Among such variants, 23403A>G variant (p.D614G) in spike protein B-cell epitope is observed frequently in European countries, such as the Netherlands, Switzerland, and France, but seldom observed in China.

negative: Platelet Gene Expression and Function in COVID-19 Patients There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis with the major difference being increased risk of thrombosis rather than bleeding. However, whether SARS-CoV-2 infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene expression changes in pathways associated with protein ubiquitination, antigen presentation and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, ACE2, was not detected by mRNA or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2/25 COVID-19 patients, suggesting platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared to healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity which may contribute to COVID-19 pathophysiology.

negative: Clinical performance of SARS-CoV-2 IgG antibody tests and potential protective immunity As the current SARS-CoV-2 pandemic continues, serological assays are urgently needed for rapid diagnosis, contact tracing and for epidemiological studies. So far, there is little data on how commercially available tests perform with real patient samples and if detected IgG antibodies provide protective immunity. Focusing on IgG antibodies, we demonstrate the performance of two ELISA assays (Euroimmun SARS-CoV-2 IgG & Vircell COVID-19 ELISA IgG) in comparison to one lateral flow assay ((LFA) FaStep COVID-19 IgG/IgM Rapid Test Device) and two in-house developed assays (immunofluorescence assay (IFA) and plaque reduction neutralization test (PRNT)). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. Most of the SARS-CoV-2 samples were from individuals with moderate to severe clinical course, who required an in-patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5-9) and from 93.8 to 100% for the later period (days 10-18) after PCR-diagnosed with COVID-19. With exception of one sample, all positive tested samples in the analysed cohort, using the commercially available assays examined (including the in-house developed IFA), demonstrated neutralizing (protective) properties in the PRNT, indicating a potential protective immunity to SARS-CoV-2. Regarding specificity, there was evidence that samples of endemic coronavirus (HCoV-OC43, HCoV-229E) and Epstein Barr virus (EBV) infected individuals cross-reacted in the ELISA assays and IFA, in one case generating a false positive result (may giving a false sense of security). This need to be further investigated.

negative: Recent Advances in the Vaccine Development Against Middle East Respiratory Syndrome-Coronavirus Middle East respiratory syndrome (MERS) is a deadly viral respiratory disease caused by MERS-coronavirus (MERS-CoV) infection. To date, there is no specific treatment proven effective against this viral disease. In addition, no vaccine has been licensed to prevent MERS-CoV infection thus far. Therefore, our current review focuses on the most recent studies in search of an effective MERS vaccine. Overall, vaccine candidates against MERS-CoV are mainly based upon the viral spike (S) protein, due to its vital role in the viral infectivity, although several studies focused on other viral proteins such as the nucleocapsid (N) protein, envelope (E) protein, and non-structural protein 16 (NSP16) have also been reported. In general, the potential vaccine candidates can be classified into six types: viral vector-based vaccine, DNA vaccine, subunit vaccine, nanoparticle-based vaccine, inactivated-whole virus vaccine and live-attenuated vaccine, which are discussed in detail. Besides, the immune responses and potential antibody dependent enhancement of MERS-CoV infection are extensively reviewed. In addition, animal models used to study MERS-CoV and evaluate the vaccine candidates are discussed intensively.

negative: Protective Adaptive Immunity Against Severe Acute Respiratory Syndrome Coronaviruses 2 (SARS-CoV-2) and Implications for Vaccines Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging human coronavirus responsible for coronavirus disease 2019 (COVID-19), a predominantly respiratory disease that has become a global pandemic. Millions of people worldwide are suffering from COVID-19, and hundreds of thousands of those infected have died. Nevertheless, many more people who have been infected with SARS-CoV-2 are asymptomatic or suffer a mild disease characterized by dry cough and mild fever. This new pandemic poses a threat to public health on a global scale, and an intervention to prevent continued spread of SARS-CoV-2 virus is of the utmost importance. To assess preventive and therapeutic strategies, it is imperative to understand the pathogenesis and immune response against SARS-CoV-2. In this review, we concentrate on the protective adaptive immune response elicited by this novel coronavirus as well as requirements for a successful vaccine inducing optimal protection.

negative: Neuraxial procedures in COVID-19 positive parturients: a review of current reports

negative: Molecular Pathogenesis of Middle East Respiratory Syndrome (MERS) Coronavirus PURPOSE OF REVIEW: Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and is listed in the World Health Organization’s blueprint of priority diseases that need immediate research. Camels are reservoirs of this virus, and the virus spills over into humans through direct contact with camels. Human-to-human transmission and travel-associated cases have been identified as well. Limited studies have characterized the molecular pathogenesis of MERS-CoV. Most studies have used ectopic expression of viral proteins to characterize MERS-CoV and its ability to modulate antiviral responses in human cells. Studies with live virus are limited, largely due to the requirement of high containment laboratories. In this review, we have summarized current studies on MERS-CoV molecular pathogenesis and have mentioned some recent strategies that are being developed to control MERS-CoV infection. RECENT FINDINGS: Multiple antiviral molecules with the potential to inhibit MERS-CoV infection by disrupting virus-receptor interactions are being developed and tested. Although human vaccine candidates are still being developed, a candidate camel vaccine is being tested for efficacy. Combination of supportive treatment with interferon and antivirals is also being explored. SUMMARY: New antiviral molecules that inhibit MERS-CoV and host cell receptor interaction may become available in the future. Additional studies are required to identify and characterize the pathogenesis of MERS-CoV EMC/2012 and other circulating strains. An effective MERS-CoV vaccine, for humans and/or camels, along with an efficient combination antiviral therapy may help us prevent future MERS cases.

negative: Medication for COVID-19-an Overview of Approaches Currently Under Study BACKGROUND: With the worldwide spread of SARS-CoV-2 infection, it is becoming increasingly urgent to develop a vaccine to prevent COVID-19, as well as effective drugs to treat it. METHODS: This article is based on a selective literature search in PubMed and ClinicalTrials.gov, followed by an assessment of the ongoing clinical trials that were revealed by the search. RESULTS: A number of substances have been found to prevent the reproduction of SARS-CoV-2 in vitro. These include virustatic agents that have already been approved for the treatment of other types of viral infection, as well as drugs that are currently used for entirely different purposes. High in vitro activity has been found for the nucleotide analogue remdesivir, for the antimalarial drug chloroquine, and for nitazoxanide, a drug used to treat protozoan infections. Because the virus enters human cells by way of the membrane-associated angiotensin converting enzyme 2 (ACE2), keeping the virus from docking to this receptor is a conceivable treatment approach. Transmembrane protease serine 2 (TMPRSS2) plays a role in the fusion of the virus with cells; inhibitors of this enzyme are known as well. The potential therapeutic efficacy and tolerability of these and other active substances remain to be investigated in clinical trials. At present, more than 80 trials on COVID-10 have already been registered with Clinical- Trials.gov. Some initial findings should already be available in late April 2020. CONCLUSION: Clinical trials are now indispensable in order to determine the true clinical benefits and risks of the substances that have been found to be active against SARSCoV- 2 in vitro. There is not yet any recommendation for the therapeutic use of any particular agent beyond standard supportive treatment.

negative: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development Highly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses. Here we developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses. In addition, the result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses. Instead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity.

negative: Development of SARS-CoV-2 vaccines: should we focus on mucosal immunity?

negative: Immune Response in COVID-19: A Review The immune system protects us from viruses and diseases. It produces an antibody to kill pathogen. This review shows a brief picture about the immune system to protect us from COVID-19. It illustrates the process of the immune system, how it works, and mechanism of the immune system to fight virus. It also provides information on recent COVID-19 treatment and experimental data. Various types of potential challenges are also discussed for the immunes system. At the end, some foods have been suggested and some are discouraged. Physical exercise is also encouraged. This article can be used as a state of the art at this critical moment to the globe for a promising alternative solutions related to the survival of people from coronavirus.

negative: Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA. Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.

negative: A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage.

negative: Potential Therapeutic Targets and Promising Drugs for Combating SARS‐CoV‐2 As of April 9, 2020, a novel coronavirus (SARS‐CoV‐2) had caused 89,931 deaths and 1,503,900 confirmed cases worldwide, which indicates an increasingly severe and uncontrollable situation. Initially, little was known about the virus. As research continues, we have learned the genome structure, epidemiological and clinical characteristics and pathogenic mechanisms of SARS‐CoV‐2. Based on these discoveries, identifying potential targets involved in the processes of virus pathogenesis is urgently needed, and discovering or developing promising drugs based on potential targets is the most pressing need. Therefore, we summarize the potential therapeutic targets involved in virus pathogenesis and discuss the advancements, possibilities and significance of promising drugs based on these targets for treating SARS‐CoV‐2. This review will facilitate the identification of potential targets and provide promising clues for drug development that can be translated into clinical applications for combating SARS‐CoV‐2.

negative: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response There is a great need for the development of vaccines for preventing SARS-CoV-2 infection and mitigating the COVID-19 pandemic. Here, we developed two modified vaccinia Ankara (MVA) based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the S1 region of the spike (MVA/S1) which forms trimers and is secreted. Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization. Following immunizations with MVA/S or MVA/S1, both spike protein recombinants induced strong IgG antibodies to purified full-length SARS-CoV-2 spike protein. The MVA/S induced a robust antibody response to purified RBD, S1 and S2 whereas MVA/S1 induced an antibody response to the S1 region outside of the RBD region. Both vaccines induced an antibody response in the lung and that was associated with induction of bronchus-associated lymphoid tissue. MVA/S but not MVA/S1 vaccinated mice generated robust neutralizing antibody responses against SARS-CoV-2 that strongly correlated with RBD antibody binding titers. Mechanistically, S1 binding to ACE-2 was strong but reduced following prolonged pre-incubation at room temperature suggesting confirmation changes in RBD with time. These results demonstrate MVA/S is a potential vaccine candidate against SARS-CoV-2 infection.

negative: Molecular characterization of attenuated vaccine strains of transmissible gastroenteritis virus. Previous studies in our laboratory demonstrated that 2 attenuated strains of transmissible gastroenteritis virus (TGEV) contain deletions affecting messenger (m) RNAs 2, 3, or 4. In this report, we have compared mRNAs of four modified-live virus vaccines for TGEV with the virulent Miller PP3 isolate to determine whether any transcriptional patterns are shared among attenuated strains. Using northern blot analysis, all vaccine viruses expressed mRNAs indistinguishable in size from those of Miller PP3. However, using S1 nuclease protection experiments, alterations in the regions of the genome from which mRNAs 2 and 3 are transcribed were detected in 2 of the vaccine strains. When genomic cDNA fragments derived from the coding region for mRNA 2 were sequenced, a 6-nucleotide deletion, also found in the attenuated strain Purdue-115, was discovered. The product of mRNA 2, a spike glycoprotein, was visualized by western blotting for each vaccine strain, and no profound differences in mobility were detected relative to Miller PP3. Alterations in the region of the genome from which mRNA 3 is transcribed appear to be identical or very similar to sequence alterations already described in this region for Purdue-115, one of which is likely to alter the polypeptide product of mRNA 3. Insertions or deletions in mRNAs 2 or 3 may contribute to attenuation but are not a prerequisite for this phenotype. The S1 nuclease protection analysis is a sensitive tool for differentiating particular strains of TGEV.

negative: Fated for decay: RNA elements targeted by viral endonucleases For over a decade, studies of messenger RNA regulation have revealed an unprecedented level of connectivity between the RNA pool and global gene expression. These connections are underpinned by a vast array of RNA elements that coordinate RNA-protein and RNA-RNA interactions, each directing mRNA fate from transcription to translation. Consequently, viruses have evolved an arsenal of strategies to target these RNA features and ultimately take control of the pathways they influence, and these strategies contribute to the global shutdown of the host gene expression machinery known as “Host Shutoff”. This takeover of the host cell is mechanistically orchestrated by a number of non-homologous virally encoded endoribonucleases. Recent large-scale screens estimate that over 70 % of the host transcriptome is decimated by the expression of these viral nucleases. While this takeover strategy seems extraordinarily well conserved, each viral endonuclease has evolved to target distinct mRNA elements. Herein, we will explore each of these RNA structures/sequence features that render messenger RNA susceptible or resistant to viral endonuclease cleavage. By further understanding these targeting and escape mechanisms we will continue to unravel untold depths of cellular RNA regulation that further underscores the integral relationship between RNA fate and the fate of the cell.

negative: High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

negative: Synthetic antibodies neutralize SARS-CoV-2 infection of mammalian cells Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

negative: Nucleotide sequence of the human coronavirus HCV 229E mRNA 4 and mRNA 5 unique regions.

negative: ACE-inibitori, sartani e sindrome respiratoria acuta da coronavirus 2./ [ACE-inhibitors, angiotensin receptor blockers and severe acute respiratory syndrome caused by coronavirus] Some Authors recently suggested that angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) should be discontinued, even temporarily, given the current pandemic of SARS-CoV-2 virus. The suggestion is based on the hypothesis that ACE-inhibitors and ARBs may favor the entry and diffusion of SARS-CoV-2 virus into the human cells. ACE-inhibitors and ARBs may increase the expression of ACE2 receptors, which are the sites of viral entry into the human organism. ACE2 receptors are ubiquitous, although they are extremely abundant on the cell surface of type 2 pneumocytes. Type 2 pneumocytes are small cylindrical alveolar cells located in close vicinity to pulmonary capillaries and responsible for the synthesis of alveolar surfactant, which is known to facilitate gas exchanges. The increased expression of ACE2 for effect of ACE-inhibitors and ARBs can be detected by increased production of angiotensin1-7 and mRNA related to ACE2. There is the fear that the increased expression of ACE2 induced by ACE-inhibitors and ARBs may ultimately facilitate the entry and diffusion of the SARS-CoV-2 virus. However, there is no clinical evidence to support this hypothesis. Furthermore, available data are conflicting and some counter-intuitive findings suggest that ARBs may be beneficial, not harmful. Indeed, studies conducted in different laboratories demonstrated that ACE2 receptors show a down-regulation (i.e. the opposite of what would happen with ACE-inhibitors and ARBs) for effect of their interaction with the virus. In animal studies, down-regulation of ACE2 has been found as prevalent in the pulmonary areas infected by virus, but not in the surrounding areas. In these studies, virus-induced ACE2 down-regulation would lead to a reduced formation of angiotensin1-7 (because ACE2 degrades angiotensin II into angiotensin1-7) with consequent accumulation of angiotensin II. The excess angiotensin II would favor pulmonary edema and inflammation, a phenomenon directly associated with angiotensin II levels, along with worsening in pulmonary function. Such detrimental effects have been blocked by ARBs in experimental models. In the light of the above considerations, it is reasonable to conclude that the suggestion to discontinue ACE-inhibitors or ARBs in all patients with the aim of preventing or limiting the diffusion of SARS-CoV-2 virus is not based on clinical evidence. Conversely, experimental studies suggest that ARBs might be useful in these patients to limit pulmonary damage through the inhibition of type 1 angiotensin II receptors. Controlled clinical studies in this area are eagerly awaited. This review discusses facts and theories on the potential impact of ACE-inhibitors and ARBs in the setting of the SARS-CoV-2 pandemic.

negative: Artificial intelligence predicts the immunogenic landscape of SARS-CoV-2: toward universal blueprints for vaccine designs The global population is at present suffering from a pandemic of Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The goals of this study were to use artificial intelligence (AI) to predict blueprints for designing universal vaccines against SARS-CoV-2, that contain a sufficiently broad repertoire of T-cell epitopes capable of providing coverage and protection across the global population. To help achieve these aims, we profiled the entire SARS-CoV-2 proteome across the most frequent 100 HLA-A, HLA-B and HLA-DR alleles in the human population, using host-infected cell surface antigen presentation and immunogenicity predictors from the NEC Immune Profiler suite of tools, and generated comprehensive epitope maps. We then used these epitope maps as input for a Monte Carlo simulation designed to identify statistically significant “epitope hotspot” regions in the virus that are most likely to be immunogenic across a broad spectrum of HLA types. We then removed epitope hotspots that shared significant homology with proteins in the human proteome to reduce the chance of inducing off-target autoimmune responses. We also analyzed the antigen presentation and immunogenic landscape of all the nonsynonymous mutations across 3400 different sequences of the virus, to identify a trend whereby SARS-COV-2 mutations are predicted to have reduced potential to be presented by host-infected cells, and consequently detected by the host immune system. A sequence conservation analysis then removed epitope hotspots that occurred in less-conserved regions of the viral proteome. Finally, we used a database of the HLA genotypes of approximately 22 000 individuals to develop a “digital twin” type simulation to model how effective different combinations of hotspots would work in a diverse human population, and used the approach to identify an optimal constellation of epitopes hotspots that could provide maximum coverage in the global population. By combining the antigen presentation to the infected-host cell surface and immunogenicity predictions of the NEC Immune Profiler with a robust Monte Carlo and digital twin simulation, we have managed to profile the entire SARS-CoV-2 proteome and identify a subset of epitope hotspots that could be harnessed in a vaccine formulation to provide a broad coverage across the global population.

negative: Interferon-Induced Transmembrane Protein (IFITM3) Is Upregulated Explicitly in SARS-CoV-2 Infected Lung Epithelial Cells Current guidelines for COVID-19 management recommend the utilization of various repurposed drugs. Despite ongoing research toward the development of a vaccine against SARS-CoV-2, such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. Therefore, there is an urgent need to develop a framework for the rapid identification of novel targets for diagnostic and therapeutic interventions. We analyzed publicly available transcriptomic datasets of SARS-CoV infected humans and mammals to identify consistent differentially expressed genes then validated in SARS-CoV-2 infected epithelial cells transcriptomic datasets. Comprehensive toxicogenomic analysis of the identified genes to identify possible interactions with clinically proven drugs was carried out. We identified IFITM3 as an early upregulated gene, and valproic acid was found to enhance its mRNA expression as well as induce its antiviral action. These findings indicate that analysis of publicly available transcriptomic and toxicogenomic data represents a rapid approach for the identification of novel targets and molecules that can modify the action of such targets during the early phases of emerging infections like COVID-19.

negative: Vectored vaccines to protect against PRRSV Abstract PRRSV is the causative agent of the most important infectious disease affecting swine herds worldwide, producing great economic losses. Commercially available vaccines are only partially effective in protection against PRRSV. Moreover, modified live vaccines may allow virus shedding, and could revert generating virulent phenotypes. Therefore, new efficient vaccines are required. Vaccines based on recombinant virus genomes (virus vectored vaccines) against PRRSV could represent a safe alternative for the generation of modified live vaccines. In this paper, current vectored vaccines to protect against PRRSV are revised, including those based on pseudorabies virus, poxvirus, adenovirus, and virus replicons. Special attention has been provided to the use of transmissible gastroenteritis virus (TGEV) as vector for the expression of PRRSV antigens. This vector has the capability of expressing high levels of heterologous genes, is a potent interferon-α inducer, and presents antigens in mucosal surfaces, eliciting both secretory and systemic immunity. A TGEV derived vector (rTGEV) was generated, expressing PRRSV wild type or modified GP5 and M proteins, described as the main inducers of neutralizing antibodies and cellular immune response, respectively. Protection experiments showed that vaccinated animals developed a faster and stronger humoral immune response than the non-vaccinated ones. Partial protection in challenged animals was observed, as vaccinated pigs showed decreased lung damage when compared with the non-vaccinated ones. Nevertheless, the level of neutralizing antibodies was low, what may explain the limited protection observed. Several strategies are proposed to improve current rTGEV vectors expressing PRRSV antigens.

negative: The promise of mRNA vaccines: a biotech and industrial perspective mRNA technologies have the potential to transform areas of medicine, including the prophylaxis of infectious diseases. The advantages for vaccines range from the acceleration of immunogen discovery to rapid response and multiple disease target manufacturing. A greater understanding of quality attributes that dictate translation efficiency, as well as a comprehensive appreciation of the importance of mRNA delivery, are influencing a new era of investment in development activities. The application of translational sciences and growing early-phase clinical experience continue to inform candidate vaccine selection. Here we review the state of the art for the prevention of infectious diseases by using mRNA and pertinent topics to the biotechnology and pharmaceutical industries.

negative: Vector-based genetically modified vaccines: Exploiting Jenner’s legacy Abstract The global vaccine market is diverse while facing a plethora of novel developments. Genetic modification (GM) techniques facilitate the design of ’smarter’ vaccines. For many of the major infectious diseases of humans, like AIDS and malaria, but also for most human neoplastic disorders, still no vaccines are available. It may be speculated that novel GM technologies will significantly contribute to their development. While a promising number of studies is conducted on GM vaccines and GM vaccine technologies, the contribution of GM technology to newly introduced vaccines on the market is disappointingly limited. In this study, the field of vector-based GM vaccines is explored. Data on currently available, actually applied, and newly developed vectors is retrieved from various sources, synthesised and analysed, in order to provide an overview on the use of vector-based technology in the field of GM vaccine development. While still there are only two vector-based vaccines on the human vaccine market, there is ample activity in the fields of patenting, preclinical research, and different stages of clinical research. Results of this study revealed that vector-based vaccines comprise a significant part of all GM vaccines in the pipeline. This study further highlights that poxviruses and adenoviruses are among the most prominent vectors in GM vaccine development. After the approval of the first vectored human vaccine, based on a flavivirus vector, vaccine vector technology, especially based on poxviruses and adenoviruses, holds great promise for future vaccine development. It may lead to cheaper methods for the production of safe vaccines against diseases for which no or less perfect vaccines exist today, thus catering for an unmet medical need. After the introduction of Jenner’s vaccinia virus as the first vaccine more than two centuries ago, which eventually led to the recent eradication of smallpox, this and other viruses may now be the basis for constructing vectors that may help us control other major scourges of mankind.

negative: A single dose of ChAdOx1 MERS provides protective immunity in rhesus macaques Developing a vaccine to protect against the lethal effects of the many strains of coronavirus is critical given the current global pandemic. For Middle East respiratory syndrome coronavirus (MERS-CoV), we show that rhesus macaques seroconverted rapidly after a single intramuscular vaccination with ChAdOx1 MERS. The vaccine protected against respiratory injury and pneumonia and reduced viral load in lung tissue by several orders of magnitude. MERS-CoV replication in type I and II pneumocytes of ChAdOx1 MERS–vaccinated animals was absent. A prime-boost regimen of ChAdOx1 MERS boosted antibody titers, and viral replication was completely absent from the respiratory tract tissue of these rhesus macaques. We also found that antibodies elicited by ChAdOx1 MERS in rhesus macaques neutralized six different MERS-CoV strains. Transgenic human dipeptidyl peptidase 4 mice vaccinated with ChAdOx1 MERS were completely protected against disease and lethality for all different MERS-CoV strains. The data support further clinical development of ChAdOx1 MERS.

negative: Competing endogenous RNA network profiling reveals novel host dependency factors required for MERS-CoV propagation Circular RNAs (circRNAs) are an integral component of the host competitive endogenous RNA (ceRNA) network. These noncoding RNAs are characterized by their unique splicing reactions to form covalently closed loop structures and play important RNA regulatory roles in cells. Recent studies showed that circRNA expressions were perturbed in viral infections and circRNAs might serve as potential antiviral targets. We investigated the host ceRNA network changes and biological relevance of circRNAs in human lung adenocarcinoma epithelial (Calu-3) cells infected with the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV). A total of ≥49337 putative circRNAs were predicted. Among the 7845 genes which generated putative circRNAs, 147 (1.9%) of them each generated ≥30 putative circRNAs and were involved in various biological, cellular, and metabolic processes, including viral infections. Differential expression (DE) analysis showed that the proportion of DE circRNAs significantly (P < 0.001) increased at 24 h-post infection. These DE circRNAs were clustered into 4 groups according to their time-course expression patterns and demonstrated inter-cluster and intra-cluster variations in the predicted functions of their host genes. Our comprehensive circRNA-miRNA-mRNA network identified 7 key DE circRNAs involved in various biological processes upon MERS-CoV infection. Specific siRNA knockdown of two selected DE circRNAs (circFNDC3B and circCNOT1) significantly reduced MERS-CoV load and their target mRNA expression which modulates various biological pathways, including the mitogen-activated protein kinase (MAPK) and ubiquitination pathways. These results provided novel insights into the ceRNA network perturbations, biological relevance of circRNAs, and potential host-targeting antiviral strategies for MERS-CoV infection.

negative: Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.

negative: Triaging of Respiratory Protective Equipment on the assumed risk of SARS-CoV-2 aerosol exposure in patient-facing healthcare workers delivering secondary care: a rapid review ABSTRACT BACKGROUND Objectives: "In patient-facing healthcare workers delivering secondary care, what is the evidence behind UK Government PPE Guidance on surgical masks versus respirators for SARS-CoV-2 protection?" METHODS Two independent reviewers searched MEDLINE, Google Scholar and grey literature 11th-30th April 2020. Studies published on any date containing primary data comparing surgical facemasks and respirators specific to SARS-CoV-2, and studies underpinning government PPE guidance, were included. Appraisal was performed using CASP checklists. Results were synthesised by comparison of findings and appraisals. RESULTS In all three laboratory studies of 14 different respirators and 12 surgical facemasks, respirators were significantly more effective than facemasks in protection factors, reduction factors, filter penetrations, and total inspiratory leakages at differing particle sizes, mean inspiratory flows, and breathing rates. Tests included live viruses and inert particles on dummies and humans. In six clinical studies, 6,502 participants, there was no consistent definition of "exposure" to determine the efficacy of RPE. It is difficult to define "safe". The only statistically significant result found continuous use of respirators more effective in clinical respiratory illness compared to targeted use or surgical facemask. CONCLUSIONS There is a paucity of evidence on the comparison of FRSMs and respirators specific to SARS-CoV-2, and poor-quality evidence in other contexts. Indirectness results in extrapolation of non-SARS-CoV-2 specific data to guide UK Government PPE guidance. The appropriateness of this is unknown given the uncertainty over the transmission of SARS-CoV-2. 1. The evidence base for UK Government PPE guidelines is not based on SARS-CoV-2 and requires generalisation from low-quality evidence of other pathogens/particles. 2. There is a paucity of high-quality evidence regarding the efficacy of RPE specific to SARS-CoV-2. 3. HMG's PPE guidelines are underpinned by the assumption of droplet transmission of SARS-CoV-2. Triaging the use of FFP3 respirators might increase the risk of COVID-19 faced by some. FUNDING This review was unfunded and unsponsored.

negative: COVID-19 Drug Discovery Using Intensive Approaches Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

negative: Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13567-019-0631-5) contains supplementary material, which is available to authorized users.

negative: Updated Approaches against SARS-CoV-2 Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lies behind the ongoing outbreak of coronavirus disease 2019 (COVID-19). There is a growing understanding of SARS-CoV-2 in virology, epidemiology, and clinical management strategies. However, no anti-SARS-CoV-2 drug or vaccine has been officially approved due to the absence of adequate evidence. Scientists are racing to develop a treatment for COVID-19. Recent studies have revealed many attractive therapeutic options, even if some of them remain to be further confirmed in rigorous preclinical models and clinical trials. In this minireview, we aim to summarize the updated potential approaches against SARS-CoV-2. We emphasize that further efforts are warranted to develop the safest and most effective approach.

negative: Middle East Respiratory Syndrome Vaccine Candidates: Cautious Optimism Efforts towards developing a vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV) have yielded promising results. Utilizing a variety of platforms, several vaccine approaches have shown efficacy in animal models and begun to enter clinical trials. In this review, we summarize the current progress towards a MERS-CoV vaccine and highlight potential roadblocks identified from previous attempts to generate coronavirus vaccines.

negative: Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial BACKGROUND: A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. METHODS: We did a dose-escalation, single-centre, open-label, non-randomised, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults aged between 18 and 60 years were sequentially enrolled and allocated to one of three dose groups (5â×â1010, 1â×â1011, and 1·5â×â1011 viral particles) to receive an intramuscular injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralising antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralisation and pseudovirus neutralisation tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127. FINDINGS: Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36·3 years) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the analysis. At least one adverse reaction within the first 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]. Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralising antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination. INTERPRETATION: The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation. FUNDING: National Key R&D Program of China, National Science and Technology Major Project, and CanSino Biologics.

negative: Native Tertiary Structure and Nucleoside Modifications Suppress tRNA’s Intrinsic Ability to Activate the Innate Immune Sensor PKR Interferon inducible protein kinase PKR is an essential component of innate immunity. It is activated by long stretches of dsRNA and provides the first line of host defense against pathogens by inhibiting translation initiation in the infected cell. Many cellular and viral transcripts contain nucleoside modifications and/or tertiary structure that could affect PKR activation. We have previously demonstrated that a 5′-end triphosphate–a signature of certain viral and bacterial transcripts–confers the ability of relatively unstructured model RNA transcripts to activate PKR to inhibit translation, and that this activation is abrogated by certain modifications present in cellular RNAs. In order to understand the biological implications of native RNA tertiary structure and nucleoside modifications on PKR activation, we study here the heavily modified cellular tRNAs and the unmodified or the lightly modified mitochondrial tRNAs (mt-tRNA). We find that both a T7 transcript of yeast tRNA(Phe) and natively extracted total bovine liver mt-tRNA activate PKR in vitro, whereas native E. coli, bovine liver, yeast, and wheat tRNA(Phe) do not, nor do a variety of base- or sugar-modified T7 transcripts. These results are further supported by activation of PKR by a natively folded T7 transcript of tRNA(Phe) in vivo supporting the importance of tRNA modification in suppressing PKR activation in cells. We also examine PKR activation by a T7 transcript of the A14G pathogenic mutant of mt-tRNA(Leu), which is known to dimerize, and find that the misfolded dimeric form activates PKR in vitro while the monomeric form does not. Overall, the in vitro and in vivo findings herein indicate that tRNAs have an intrinsic ability to activate PKR and that nucleoside modifications and native RNA tertiary folding may function, at least in part, to suppress such activation, thus serving to distinguish self and non-self tRNA in innate immunity.

negative: SARS‐CoV‐2 and human milk: What is the evidence? The novel coronavirus SARS‐CoV‐2 has emerged as one of the most compelling and concerning public health challenges of our time. To address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical and public health communities has rapidly engaged to collectively find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS‐CoV‐2 transmission. Although respiratory droplets are a known mechanism of transmission, other mechanisms are likely. Of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. However, there is limited published literature related to vertical transmission of any human coronaviruses (including SARS‐CoV‐2) via human milk and/or breastfeeding. Results of the literature search reported here (finalized on 17 April 2020) revealed a single study providing some evidence of vertical transmission of human coronavirus 229E; a single study evaluating presence of SARS‐CoV in human milk (it was negative); and no published data on MERS‐CoV and human milk. We identified 13 studies reporting human milk tested for SARS‐CoV‐2; one study (a non‐peer‐reviewed preprint) detected the virus in one milk sample, and another study detected SARS‐CoV‐2 specific IgG in milk. Importantly, none of the studies on coronaviruses and human milk report validation of their collection and analytical methods for use in human milk. These reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, SARS‐CoV‐2) during breastfeeding are discussed.

negative: Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site, generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We propose that natural selection has favored a “Don’t burn down the house” strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.

negative: Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2 Abstract Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

negative: siRNA silencing of angiotensin-converting enzyme 2 reduced severe acute respiratory syndrome-associated coronavirus replications in Vero E6 cells The outbreak of severe acute respiratory syndrome (SARS) in 2002–2003 has had a significant impact worldwide. No effective prophylaxis or treatment for SARS is available up to now. Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for SARS-associated coronavirus (SARS-CoV). By expressing a U6 promoter-driven small interfering RNA containing sequences homologous to part of ACE2 mRNA, we successfully silenced ACE2 expression in Vero E6 cells. By detecting negative strand SARS-CoV RNA and measuring RNA copy numbers of SARS-CoV by real-time reverse transcription polymerase chain reaction (RT-PCR), we demonstrated that SARS-CoV infection was reduced in the ACE2-silenced cell lines. These findings support the involvement of ACE2 in SARS-CoV infections and provide a basis for further studies on potential use of siRNA targeting ACE2 as a preventive or therapeutic strategy for SARS.

negative: Assay Techniques and Test Development for COVID-19 Diagnosis An ongoing theme of the COVID-19 pandemic is the need for widespread availability of accurate and eﬃcient diagnostic testing for detection of SARS-CoV-2 and antiviral antibodies in infected individuals. This report describes various assay techniques and tests for COVID-19 diagnosis. Most tests for early detection of SARS-CoV-2 RNA rely on the reverse transcription-polymerase chain reaction, but isothermal nucleic acid ampliﬁcation assays, including transcription-mediated ampliﬁcation and CRISPR-based methodologies, are promising alternatives. Identiﬁcation of individuals who have developed antibodies to the SARS-CoV-2 virus requires serological tests, including enzyme-linked immunosorbent assay (ELISA) and lateral ﬂow immunoassay. This report also provides an overview of current development in COVID-19 diagnostic techniques and products to facilitate future improvement and innovation.

negative: Infectious Bronchitis Virus as a Vector for the Expression of Heterologous Genes The avian coronavirus infectious bronchitis virus (IBV) is the causative agent of the respiratory disease infectious bronchitis of domestic fowl, and is controlled by routine vaccination. To explore the potential use of IBV as a vaccine vector a reverse genetics system was utilised to generate infectious recombinant IBVs (rIBVs) expressing the reporter genes enhanced green fluorescent protein (eGFP) or humanised Renilla luciferase (hRluc). Infectious rIBVs were obtained following the replacement of Gene 5 or the intergenic region (IR) with eGFP or hRluc, or the replacement of ORFs 3a and 3b with hRluc. The replacement of Gene 5 with an IBV codon-optimised version of the hRluc gene also resulted in successful rescue of infectious rIBV. Reporter gene expression was confirmed by fluorescence microscopy, or luciferase activity assays, for all successfully rescued rIBVs following infection of primary chick kidney (CK) cells. The genetic stability of rIBVs was analysed by serial passage on CK cells. Recombinant IBV stability varied depending on the genome region being replaced, with the reporter genes maintained up to at least passage 8 (P8) following replacement of Gene 5, P7 for replacement of the IR and P5 for replacement of ORFs 3a and 3b. Codon-optimisation of the hRluc gene, when replacing Gene 5, resulted in an increase in genome stability, with hRluc expression stable up to P10 compared to P8 for standard hRluc. Repeated passaging of rIBVs expressing hRluc at an MOI of 0.01 demonstrated an increase in stability, with hRluc expression stable up to at least P12 following the replacement of Gene 5. This study has demonstrated that heterologous genes can be incorporated into, and expressed from a range of IBV genome locations and that replacement of accessory Gene 5 offers a promising target for realising the potential of IBV as a vaccine vector for other avian pathogens.

negative: Influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of SARS-CoV and its implication for subunit vaccine development The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated S protein of the TW1 strain, S(TR2) (88 kDa), carrying three S fragments (S74–253, S294–739, and S1129–1255) was investigated to study the influences of intron and exon splicing enhancers to improve S(TR2) protein expression in mammalian cells. Our results showed that S(TR2) protein expression with the use of an 138 base-pair intron addition increased by 1.9-, 2.5-, and 4.1-fold in Vero E6, QBI-293A cells, and CHO/dhFr− cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. Using the exon splicing enhancers, including a bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), were also found to increase S(TR2) protein expression in CHO/dhFr− cells by 1.7- and 2.6-fold. Nevertheless, combination of the intron and the exon splicing enhancers resulted in suppressing the intron-enhancing e S(TR2) protein expression in in CHO/dhFr− cells. Our studies also demonstrated the S(TR2) protein was mainly as the Endo H-sensitive glycoprotein (115 kDa) expressed in Vero E6, QBI-293A, and CHO/dhFr− cells. However, only a minor form of the Endo H-resistant glycoproteins (∼130 kDa) was detected in CHO/dhFr− cells. Taken together, our results indicated that intron had a better enhancing effect on S(TR2) protein expression than exon splicing enhancers, and the expression of ∼130 kDa S(TR2) glycoprotein was enhanced by the intron addition into the expression vector construct. Results of the present study can provide an optimal strategy to enhance SARS-CoV S protein expression in mammalian cells and may contribute to the development of SARS-CoV subunit vaccine.

negative: Molecular targets for COVID-19 drug development: Enlightening Nigerians about the pandemic and future treatment There is little or no research initiated on enlightening Nigerians about the pathogenesis, targets for drug development, and drug repositioning for SARS-CoV-2 infection. COVID-19 is a viral infection causing symptoms like dry cough, sore throat, nasal congestion, tiredness, fever, loss of taste and smell etc. Th disease was first reported in Wuhan, China, in December 2019. The infection is caused by SARS-CoV-2, which is the third introduction of a highly pathogenic coronavirus into the human population. Coronaviruses are viruses with a positive RNA envelope assigned to α, β, γ, and δ genera. Moreover, SARS-CoV-2 belongs to the β genus. The four structural proteins of β coronavirus are membrane (M), envelope (E), spike (S), and nucleocapsid (N) protein, mediation of coronavirus host infection is established by spike (S) protein. Therefore, the search for drug development targets and repositioning of existing therapeutics is essential for fighting the present pandemic. It was reviewed that therapeutics targeting SARS-CoV-2 binding to ACE2 receptor, viral RNA synthesis and replication, 3CLpro, RdRp, and helicase will play a crucial role in the development of treatment for SARS-CoV-2 infection. Furthermore, the RdRp and spike protein of SARS-CoV-2 are the most promising targets for drug development and repositioning and vaccine development. Remdesivir combination with chloroquine/hydroxychloroquine are promising drug repositioning for the treatment of COVID-19, and mRNA-1273 targeting spike protein is the promising vaccine. However, as patient management and drug repositioning are taking place, it is imperative to identify other promising targets used by SARS-CoV-2 to establish infection, to develop novel therapeutics.

negative: Kunjin replicon-based simian immunodeficiency virus gag vaccines Abstract An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines encoded the wild type gag gene, an RNA-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. The vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the SIV gag-pol vaccine providing the optimal performance. These results illustrate that for an RNA-based vector the RNA sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour.

negative: Presence of Subgenomic mRNAs in Virions of Coronavirus IBV Abstract The presence of subgenomic mRNAs in virions of IBV was examined by probing Northern blots of RNA extracted from virions using as a probe a cDNA of the 3′-terminal nucleocapsid protein (N) gene. This detects all five mRNAs because of the 3′-coterminal, nested-set arrangement of coronavirus mRNAs. The mRNAs were readily detected even after extensive purification of virions and after RNase A treatment of virions. In sucrose gradients the peaks of virus particles, genomic RNA (gRNA), and mRNAs were coincident. Cellular mRNA was not detected in virions. The molar ratio of gRNA to each mRNA ranged from about 10 to 30 for IBV-Beaudette and 25 to 800 for IBV-M41. The molar ratio of genomic to intracellular viral mRNAs was also determined. From this it was estimated that the efficiency of incorporation of gRNA into virions was at least 20- to 100-fold greater, depending on the mRNA species, for IBV-Beaudette and 100- to 500-fold for IBV-M41. It is concluded that most virions contain only gRNA or contain only one species of mRNA on average in addition to gRNA.

negative: Bioinformatic prediction of potential T cell epitopes for SARS-Cov-2 To control and prevent the current COVID-19 pandemic, the development of novel vaccines is an emergent issue. In addition, we need to develop tools that can measure/monitor T-cell and B-cell responses to know how our immune system is responding to this deleterious virus. However, little information is currently available about the immune target epitopes of novel coronavirus (SARS-CoV-2) to induce host immune responses. Through a comprehensive bioinformatic screening of potential epitopes derived from the SARS-CoV-2 sequences for HLAs commonly present in the Japanese population, we identified 2013 and 1399 possible peptide epitopes that are likely to have the high affinity (<0.5%- and 2%-rank, respectively) to HLA class I and II molecules, respectively, that may induce CD8(+) and CD4(+) T-cell responses. These epitopes distributed across the structural (spike, envelope, membrane, and nucleocapsid proteins) and the nonstructural proteins (proteins corresponding to six open reading frames); however, we found several regions where high-affinity epitopes were significantly enriched. By comparing the sequences of these predicted T cell epitopes to the other coronaviruses, we identified 781 HLA-class I and 418 HLA-class II epitopes that have high homologies to SARS-CoV. To further select commonly-available epitopes that would be applicable to larger populations, we calculated population coverages based on the allele frequencies of HLA molecules, and found 2 HLA-class I epitopes covering 83.8% of the Japanese population. The findings in the current study provide us valuable information to design widely-available vaccine epitopes against SARS-CoV-2 and also provide the useful information for monitoring T-cell responses.

negative: The potential for antibody-dependent enhancement of SARS-CoV-2 infection: Translational implications for vaccine development There is an urgent need for vaccines to the 2019 coronavirus (COVID19; SARS-CoV-2). Vaccine development may not be straightforward, due to antibody-dependent enhancement (ADE). Antibodies against viral surface proteins can, in some cases, increase infection severity by ADE. This phenomenon occurs in SARS-CoV-1, MERS, HIV, Zika, and dengue virus infection and vaccination. Lack of high-affinity anti-SARS-CoV-2 IgG in children may explain the decreased severity of infection in these groups. Here, we discuss the evidence for ADE in the context of SARS-CoV-2 infection and how to address this potential translational barrier to vaccine development, convalescent plasma, and targeted monoclonal antibody therapies.

negative: Emerging roles for RNA degradation in viral replication and antiviral defense Abstract Viral replication significantly alters the gene expression landscape of infected cells. Many of these changes are driven by viral manipulation of host transcription or translation machinery. Several mammalian viruses encode factors that broadly dampen gene expression by directly targeting messenger RNA (mRNA). Here, we highlight how these factors promote mRNA degradation to globally regulate both host and viral gene expression. Although these viral factors are not homologous and use distinct mechanisms to target mRNA, many of them display striking parallels in their strategies for executing RNA degradation and invoke key features of cellular RNA quality control pathways. In some cases, there is a lack of selectivity for degradation of host versus viral mRNA, indicating that the purposes of virus-induced mRNA degradation extend beyond redirecting cellular resources towards viral gene expression. In addition, several antiviral pathways use RNA degradation as a viral restriction mechanism, and we will summarize new findings related to how these host-encoded ribonucleases target and destroy viral RNA.

negative: COVID‐19 treatment by repurposing drugs until the vaccine is in sight Corona virus disease (COVID‐19) has created pandemic in the world as declared by WHO on March 12, 2020. It is a viral disease caused by SARS‐CoV 2 virus and has affected large populations in over 120 countries. There is no specific treatment available and management is empirical. Until such time that an effective vaccine is available for COVID‐19 viral infection, one can repurpose known therapeutic drug molecules such as angiotensin receptor 2 blocker, a commonly used antihypertensive drug, to control COVID‐19 virus from gaining entry into the host cell by blocking the angiotensin receptor. Clinical trials should also be undertaken to use statins, which are lipid‐lowering drugs but have anti‐inflammatory and immunomodulatory properties to prevent acute lung injury in COVID‐19 infection.

negative: A COVID-19 pandemic guideline in evidence-based medicine Data sources This review article scrutinised 16 clinical studies (clinical trials and observational studies) concerning coronavirus disease of 2019 (COVID-19). Additionally, 18 guidelines about the COVID-19 were reviewed and the key points were represented in this study. Study selection The review included human trials, in-vitro studies, review articles, and credible news reports about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 complications, treatment guidelines, management strategies, and epidemiological features. There were no exclusion criteria reported in this review and the included information was in English and Chinese languages. Data extraction and synthesis A descriptive review of the literature was conducted, taking a comprehensive approach. The paper consisted of three main parts: introduction, presentation, and management. The introduction part presented basic information about the SARS-CoV-2, its evolution and transmission, and the course of disease. The presentation section introduced the signs and symptoms, diagnosis, high risk groups, and complications of COVID-19. Eventually, some evidence was presented about the prevention, medical management, and measuring responses to the treatments in the management section. Results Based on the results of this study, non-pharmaceutical interventions, including strict social isolation and distancing measures, might reduce the spread of the SARS-CoV-2 by nearly 99.3 percent (reproduction number mitigating from 406 to 2.5 in 30 days). In the supportive management section, monitoring vital signs and neonatal feeding were stated as the most important factors to consider. For symptomatic neonates, medical management and intervention were mentioned as essential. It was claimed that for adults with mild infection, the best option would be home quarantine with further medical monitoring or hospitalisation if required. The following sequence was also suggested as early supportive therapy and monitoring: intravenous fluid administration, oxygen therapy, and application of corticosteroids. Management of critical patients with critical COVID-19 included admission to intensive care unit, use of continuous positive airway pressure and bi-level positive airway pressure in certain circumstances, endotracheal intubation, invasive mechanical ventilation, extracorporeal membrane oxygenation, and fluid resuscitation and vasopressors. Additionally, this study suggested oseltamivir, iopinavir, remdesivir, chloroquine, baricitinib, ruxolitinib, and fedratinib as possible drugs to help manage COVID-19. A soaring c-reactive protein level and decreased albumin content in the blood were reported to be associated with a deteriorating status in COVID-19 patients. To keep the number of exposures to a minimum, two separate viral clearance tests taken at least 24 hours apart, were stated as necessary laboratory results before the discharge of patients with COVID-19. Conclusions The study warns about possible exponential spread of COVID-19 and proposes to adhering to tighter restrictions of social distancing. Besides the clinical guidelines presented within the study, it also encourages further up-to-date and evidence-based management guidelines for patients with COVID-19.

negative: Comparison of commercial RT-PCR diagnostic kits for COVID-19 The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

negative: A versatile platform technology for recombinant vaccines using non-propagative human parainfluenza virus type 2 vector Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.

negative: One-Health: a Safe, Efficient, Dual-Use Vaccine for Humans and Animals against Middle East Respiratory Syndrome Coronavirus and Rabies Virus. Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and is a highly pathogenic respiratory virus. There are no treatment options against MERS-CoV for humans or animals, and there are no large-scale clinical trials for therapies against MERS-CoV. To address this need, we developed an inactivated rabies virus (RABV) that contains the MERS-CoV spike (S) protein expressed on its surface. Our initial recombinant vaccine, BNSP333-S, expresses a full-length wild-type MERS-CoV S protein; however, it showed significantly reduced viral titers compared to those of the parental RABV strain and only low-level incorporation of full-length MERS-CoV S into RABV particles. Therefore, we developed a RABV-MERS vector that contained the MERS-CoV S1 domain of the MERS-CoV S protein fused to the RABV G protein C terminus (BNSP333-S1). BNSP333-S1 grew to titers similar to those of the parental vaccine vector BNSP333, and the RABV G-MERS-CoV S1 fusion protein was efficiently expressed and incorporated into RABV particles. When we vaccinated mice, chemically inactivated BNSP333-S1 induced high-titer neutralizing antibodies. Next, we challenged both vaccinated mice and control mice with MERS-CoV after adenovirus transduction of the human dipeptidyl peptidase 4 (hDPP4) receptor and then analyzed the ability of mice to control MERS-CoV infection. Our results demonstrated that vaccinated mice were fully protected from the MERS-CoV challenge, as indicated by the significantly lower MERS-CoV titers and MERS-CoV and mRNA levels in challenged mice than those in unvaccinated controls. These data establish that an inactivated RABV-MERS S-based vaccine may be effective for use in animals and humans in areas where MERS-CoV is endemic. IMPORTANCE Rabies virus-based vectors have been proven to be efficient dual vaccines against rabies and emergent infectious diseases such as Ebola virus. Here we show that inactivated rabies virus particles containing the MERS-CoV S1 protein induce potent immune responses against MERS-CoV and RABV. This novel vaccine is easy to produce and may be useful to protect target animals, such as camels, as well as humans from deadly MERS-CoV and RABV infections. Our results indicate that this vaccine approach can prevent disease, and the RABV-based vaccine platform may be a valuable tool for timely vaccine development against emerging infectious diseases.

negative: A SARS-CoV-2 vaccine candidate: In silico cloning and validation SARS-CoV-2 is spreading globally at a rapid pace. To contain its spread and prevent further fatalities, the development of a vaccine against SARS-CoV-2 is an urgent prerequisite. Thus, in this article, by utilizing the in silico approach, a vaccine candidate for SARS-CoV-2 has been proposed. Moreover, the effectiveness and safety measures of our proposed epitopic vaccine candidate have been evaluated by in silico tools and servers (AllerTOP and AllergenFP servers). We observed that the vaccine candidate has no allergenicity and successfully combined with Toll-like receptor (TLR) protein to elicit an inflammatory immune response. Stable, functional mobility of the vaccine-TLR protein binding interface was confirmed by the Normal Mode Analysis. The in silico cloning model demonstrated the efficacy of the construct vaccine along with the identified epitopes against SARS-CoV-2. Taken together, our proposed in silico vaccine candidate has potent efficacy against COVID-19 infection, and successive research work might validate its effectiveness in in vitro and in vivo models.

negative: SARS-CoV-2 productively infects human gut enterocytes The virus severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission via the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2 as demonstrated by confocal- and electron-microscopy. Consequently, significant titers of infectious viral particles were detected. mRNA expression analysis revealed strong induction of a generic viral response program. Hence, intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology

negative: Virus-induced translational arrest through 4EBP1/2-dependent decay of 5'-TOP mRNAs restricts viral infection. The mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV), is a highly successful pathogen for which there are no vaccines or therapeutics. Translational arrest is a common antiviral strategy used by hosts. In response, RVFV inhibits two well-known antiviral pathways that attenuate translation during infection, PKR and type I IFN signaling. Despite this, translational arrest occurs during RVFV infection by unknown mechanisms. Here, we find that RVFV infection triggers the decay of core translation machinery mRNAs that possess a 5'-terminal oligopyrimidine (5'-TOP) motif in their 5'-UTR, including mRNAs encoding ribosomal proteins, which leads to a decrease in overall ribosomal protein levels. We find that the RNA decapping enzyme NUDT16 selectively degrades 5'-TOP mRNAs during RVFV infection and this decay is triggered in response to mTOR attenuation via the translational repressor 4EBP1/2 axis. Translational arrest of 5'-TOPs via 4EBP1/2 restricts RVFV replication, and this increased RNA decay results in the loss of visible RNA granules, including P bodies and stress granules. Because RVFV cap-snatches in RNA granules, the increased level of 5'-TOP mRNAs in this compartment leads to snatching of these targets, which are translationally suppressed during infection. Therefore, translation of RVFV mRNAs is compromised by multiple mechanisms during infection. Together, these data present a previously unknown mechanism for translational shutdown in response to viral infection and identify mTOR attenuation as a potential therapeutic avenue against bunyaviral infection.

negative: Epitope based peptide vaccine against SARS-COV2: an immune-informatics approach World is witnessing exponential growth of SARS-CoV2 and fatal outcomes of COVID 19 has proved its pandemic potential already by claiming more than 3 lakhs deaths globally. If not controlled, this ongoing pandemic can cause irreparable socio-economic and psychological impact worldwide. Therefore a safe and effective vaccine against COVID 19 is exigent. Recent advances in immunoinformatics approaches could potentially decline the attrition rate and accelerate the process of vaccine development in these unprecedented times. In the present study, a multivalent subunit vaccine targeting S2 subunit of the SARS-CoV2 S glycoprotein has been designed using open source, immunoinformatics tools. Designed construct comprises of epitopes capable of inducing T cell, B cell (Linear and discontinuous) and Interferon Î³. physiologically, vaccine construct is predicted to be thermostable, antigenic, immunogenic, non allergen and non toxic in nature. According to population coverage analysis, designed multiepitope vaccine covers 99.26% population globally. 3D structure of vaccine construct was designed, validated and refined to obtain high quality structure. Refined structure was docked against Toll like receptors to confirm the interactions between them. Vaccine peptide sequence was reverse transcribed, codon optimized and cloned in pET vector. Our in-silico study suggests that proposed vaccine against fusion domain of virus has the potential to elicit an innate as well as humoral immune response in human and restrict the entry of virus inside the cell. Results of the study offer a framework for in-vivo analysis that may hasten the process of development of therapeutic tools against COVID 19.Communicated by Ramaswamy H. Sarma.

negative: Expression of the COVID-19 receptor ACE2 in the human conjunctiva SARS-CoV-2 is assumed to use angiotensin-converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and there has been speculation that SARS-CoV-2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high-throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS-CoV-2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS-CoV-2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median: 0.0 transcripts per million [TPM], min: 0.0 TPM, max: 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS-CoV-2 via these mediators unlikely.

negative: Coronavirus Vaccine: Light at the End of the Tunnel The world is currently facing an unprecedented global pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Predicting the next source of the pandemic can be very challenging. As vaccination is the best way to prevent an infectious disease, the development of an effective vaccine against SARS-CoV-2 can not only reduce the morbidity and mortality associated with it, but can also lessen the economic impact. As the traditional method of vaccine development takes many years for a vaccine to be available to the society, the vaccine development for SARS-CoV-2 should be speeded up using a pandemic approach with fast-track approvals from the regulatory authorities. Various challenges associated with developing a vaccine during the pandemic such as technological hurdles, clinical development pathways, regulatory issues, and support from global funding agencies are expressed here.

negative: In situ expression of tumor antigens by messenger RNA-electroporated dendritic cells in lymph nodes of melanoma patients. Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for cancer immunotherapy has been proved efficient and clinically safe. It obviates prior knowledge of CTL and Th epitopes in the antigen and leads to the presentation of multiple epitopes for several HLA alleles. Here we studied the migration capacity and the antigen expression of mRNA-electroporated DC (mRNA-DC) in lymph nodes after vaccination in melanoma patients. DC were electroporated with mRNA encoding gp100 or tyrosinase, labeled with indium-111 and superparamagnetic iron oxide particles, and injected intranodally in melanoma patients 24 to 48 hours before scheduled dissection of regional lymph nodes. Immunohistochemical analysis of the lymph nodes after surgery revealed that mRNA-DC migrated from the injection site into the T-cell areas of the same and subsequent lymph nodes, where they expressed the antigen encoded by the electroporated mRNA. Furthermore, vaccine-related CD8(+) T-cell responses could be detected in 7 of 11 patients vaccinated with mRNA-DC. Together these data show that mature DC electroporated with mRNA encoding TAA migrate and express antigens in the lymph nodes and induce specific immune responses.

negative: Target selectivity in mRNA silencing Despite the excitement and promise of RNA interference in treating neurodegenerative disease, disease gene mRNA might resist mRNA silencing. Conventional siRNA design does not uniformly distinguish a mutant from a wild-type allele. CAG expansions in trinucleotide repeat diseases are unselective targets for small siRNAs. This review will consider recent discoveries in mechanisms of RNA interference and siRNA modifications that improve siRNA selectivity, delivery and performance.

negative: Epitope-based peptide vaccines predicted against novel coronavirus disease caused by SARS-CoV-2 The outbreak of the 2019 novel coronavirus (SARS-CoV-2) has infected thousands of people with a large number of deaths across 26 countries. The sudden appearance of the virus leads to the limited existing therapies for SARS-CoV-2. Therefore, vaccines and antiviral medicines are in desperate need. This study took immune-informatics approaches to identify B- and T-cell epitopes for surface glycoprotein (S) of SARS-CoV-2, followed by estimating their antigenicity and interactions with the human leukocyte antigen (HLA) alleles. We identified four B cell epitopes, two MHC class-I and nine MHC class-II binding T-cell epitopes, which showed highly antigenic features. Allergenicity, toxicity and physiochemical properties analysis confirmed the specificity and selectivity of epitopes. The stability and safety of epitopes were confirmed by digestion analysis. No mutations were observed in all the selected B- and T-cell epitopes across all isolates from different locations worldwide. Epitopes were thus identified and some of them can be potential candidates for vaccine development.

negative: Specific Nucleotide Changes in the Subgenomic Promoter Region Influence Infectivity of the Sindbis Virus Transcription of the subgenomic mRNA of Sindbis virus (SINV) is initiated at a subgenomic promoter (SP). Alignment of SINV sequences identified a 68-nucleotide conserved domain spanning −19 to +49 relative to the subgenomic mRNA start site. Nucleotide T or C is present at −18 or +49 in all known SINVs while a Sindbis-like virus XJ-160 has an A or T at a corresponding position. Our results indicate that deletion or substitution of the T at +49 decreased the activity of SP, while substituting T for A at −18 did not decrease the activity of SP or genetic stability of recombinant SINV.

negative: Data analytics for novel coronavirus disease This paper describes different aspects of novel coronavirus disease (COVID-19), presents visualization of the spread of the infection, and discusses the potential applications of data analytics on this viral infection. Firstly, a literature survey is done on COVID-19 highlighting a number of factors including its origin, its similarity with previous coronaviruses, its transmission capacity, its symptoms, etc. Secondly, data analytics is applied on a dataset of Johns Hopkins University to find out the spread of the viral infection. It is shown here that although the disease started in China in December 2019, the highest number of confirmed cases up to June 04, 2020 is in the USA. Thirdly, the worldwide increase in the number of confirmed cases over time is modelled here using a polynomial regression algorithm with degree 2. Fourthly, classification algorithms are applied on a dataset of 5644 samples provided by Hospital Israelita Albert Einstein of Brazil in order to diagnose COVID-19. It is shown here that multilayer perceptron (MLP), XGBoost and logistic regression can classify COVID-19 patients at an accuracy above 91%. Finally, a discussion is presented on the potential applications of data analytics in several important factors of COVID-19.

negative: Consensus Summary Report for CEPI/BC March 12-13, 2020 Meeting: Assessment of Risk of Disease Enhancement with COVID-19 Vaccines A novel coronavirus (CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China and has since spread as a global pandemic. Safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of Coronavirus Disease 2019 (COVID-19) disease and ease the major economic impact. There has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. However, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. A syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. Animal models allowed scientists to determine the underlying mechanism for the former in the case of Respiratory Syncytial virus (RSV) vaccine and have been utilized to design and screen new RSV vaccine candidates. Because some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for SARS-CoV-2 vaccines. To address this challenge, the Coalition for Epidemic Preparedness Innovations (CEPI) and the Brighton Collaboration (BC) Safety Platform for Emergency vACcines (SPEAC) convened a scientific working meeting on March 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. This report summarizes the evidence presented and provides considerations for safety assessment of COVID-19 vaccine candidates in accelerated vaccine development.

negative: The First 75 Days of Novel Coronavirus (SARS-CoV-2) Outbreak: Recent Advances, Prevention, and Treatment The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously known as 2019-nCoV) outbreak has engulfed an unprepared world amidst a festive season. The zoonotic SARS-CoV-2, believed to have originated from infected bats, is the seventh member of enveloped RNA coronavirus. Specifically, the overall genome sequence of the SARS-CoV-2 is 96.2% identical to that of bat coronavirus termed BatCoV RaTG13. Although the current mortality rate of 2% is significantly lower than that of SARS (9.6%) and Middle East respiratory syndrome (MERS) (35%), SARS-CoV-2 is highly contagious and transmissible from human to human with an incubation period of up to 24 days. Some statistical studies have shown that, on average, one infected patient may lead to a subsequent 5.7 confirmed cases. Since the first reported case of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 on December 1, 2019, in Wuhan, China, there has been a total of 60,412 confirmed cases with 1370 fatalities reported in 25 different countries as of February 13, 2020. The outbreak has led to severe impacts on social health and the economy at various levels. This paper is a review of the significant, continuous global effort that was made to respond to the outbreak in the first 75 days. Although no vaccines have been discovered yet, a series of containment measures have been implemented by various governments, especially in China, in the effort to prevent further outbreak, whilst various medical treatment approaches have been used to successfully treat infected patients. On the basis of current studies, it would appear that the combined antiviral treatment has shown the highest success rate. This review aims to critically summarize the most recent advances in understanding the coronavirus, as well as the strategies in prevention and treatment.

negative: ACE-2 Expression in the Small Airway Epithelia of Smokers and COPD Patients: Implications for COVID-19 Introduction: Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by the severe acute respiratory syndrome coronavirus2 (SARSCoV-2). This virus uses the angiotensin converting enzyme II (ACE2) as the cellular entry receptor to infect the lower respiratory tract. Because individuals with chronic obstructive pulmonary disease (COPD) are at increased risk of severe COVID19, we determined whether ACE2 expression in the lower airways was related to COPD and cigarette smoking. Methods: Using RNAseq, we determined gene expression levels in bronchial epithelia obtained from cytologic brushings of 6th to 8th generation airways in individuals with and without COPD. We externally validated these results from two additional independent cohorts, which used microarray technologies to measure gene expression levels from 6th to 12th generation airways. Results: In the discovery cohort (n=42 participants), we found that ACE2 expression levels were increased by 48% in the airways of COPD compared with non-COPD subjects (COPD=2.52 (0.66) log2 counts per million reads (CPM) versus non-COPD= 1.70 (0.51) CPM , p=.000762). There was a significant inverse relationship between ACE2 gene expression and FEV1% of predicted (r=negative 0.24; p=0.035). Current smoking also significantly increased ACE2 expression levels compared with never smokers (never current smokers=2.77 (90.91) CPM versus smokers=1.78 (0.39) CPM, p=0.024). These findings were replicated in the two external cohorts. Conclusions: ACE2 expression in lower airways is increased in patients with COPD and with current smoking. These data suggest that these two subgroups are at increased risk of serious COVID19 infection and highlight the importance of smoking cessation in reducing the risk.

negative: Towards the design of multiepitope-based peptide vaccine candidate against SARS-CoV-2 Coronavirus disease 2019 is a current pandemic health threat especially for elderly patients with comorbidities. This respiratory disease is caused by a beta coronavirus known as severe acute respiratory syndrome coronavirus 2. The disease can progress into acute respiratory distress syndrome that can be fatal. Currently, no specific drug or vaccine are available to combat this pandemic outbreak. Social distancing and lockdown have been enforced in many places worldwide. The spike protein of coronavirus 2 is essential for viral entry into host target cells via interaction with angiotensin converting enzyme 2. This viral protein is considered a potential target for design and development of a drug or vaccine. Previously, we have reported several potential epitopes on coronavirus 2 spike protein with high antigenicity, low allergenicity and good stability against specified proteases. In the current study, we have constructed and evaluated a peptide vaccine from these potential epitopes by using in silico approach. This construct is predicted to have a protective immunogenicity, low allergenicity and good stability with minor structural flaws in model build. The population coverage of the used T-cells epitopes is believed to be high according to the employed restricted alleles. The vaccine construct can elicit efficient and long-lasting immune response as appeared through simulation analysis. This multiepitope-based peptide vaccine may represent a potential candidate against coronavirus 2. However, further in vitro and in vivo verification are required.

negative: A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

negative: Cell-mediated immune responses in the head-associated lymphoid tissues induced to a live attenuated avian coronavirus vaccine Humoral immunity is important for controlling viral diseases of poultry, but recent studies have indicated that cytotoxic T cells also play an important role in the immune response to infectious bronchitis virus (IBV). To better understand the cell mediated immune responses to IBV in the mucosal and systemic immune compartments chickens were ocularly vaccinated with IBV. This induced a lymphocyte expansion in head-associated lymphoid tissues (HALT) and to a lesser extent in the spleen, followed by a rapid decline, probably due to homing of lymphocytes out of these organs and contraction of the lymphocyte population. This interpretation was supported by observations that changes in mononuclear cells were mirrored by that in CD3(+)CD44(+) T cell abundance, which presumably represent T effector cells. Increased interferon gamma (IFN-γ) expression was observed in the mucosal immune compartment, i.e., HALT, after primary vaccination, but shifted to the systemic immune compartment after boosting. In contrast, the expression of cytotoxicity-associated genes, i.e., granzyme A (GZMA) and perforin mRNA, remained associated with the HALT after boosting. Thus, an Ark-type IBV ocular vaccine induces a central memory IFN-γ response in the spleen while the cytotoxic effector memory response, as measured by GZMA and perforin mRNA expression, remains associated with CALT after boosting.

negative: Relationship between mRNA stability and intron presence Abstract Introns were found to enhance almost every steps of gene expression except increasing mRNA stability. By analyzing the genome-wide data of mRNA stability published by someone previously, we found that human intron-containing genes have more stable mRNAs than intronless genes, and the Arabidopsis thaliana genes with the most unstable mRNAs have fewer introns than other genes in the genome. After controlling for mRNA length, we found mRNA stability is still positively correlated with intron number in human intron-containing genes. But in yeast Saccharomyces cerevisiae, two different datasets on mRNA half-life gave conflicting results. The components of messenger ribonucleoprotein particles recruited during intron splicing may be retained in cytoplasmic mRNPs and act as signals of mRNA stability or simply insulators to avoid mRNA degradation.

negative: Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

negative: Recent advances in influenza vaccines Seasonal influenza remains a major public health problem, responsible for hundreds of thousands of deaths every year, mostly of elderly people. Despite the wide availability of vaccines, there are multiple problems decreasing the effectiveness of vaccination programs. These include viral variability and hence the requirement to match strains by estimating which will become prevalent each season, problems associated with vaccine and adjuvant production, and the route of administration as well as the perceived lower vaccine efficiency in older adults. Clinical protection is still suboptimal for all of these reasons, and vaccine uptake remains too low in most countries. Efforts to improve the effectiveness of influenza vaccines include developing universal vaccines independent of the circulating strains in any particular season and stimulating cellular as well as humoral responses, especially in the elderly. This commentary assesses progress over the last 3 years towards achieving these aims. Since the beginning of 2020, an unprecedented international academic and industrial effort to develop effective vaccines against the new coronavirus SARS-CoV-2 has diverted attention away from influenza, but many of the lessons learned for the one will synergize with the other to mutual advantage. And, unlike the SARS-1 epidemic and, we hope, the SARS-CoV-2 pandemic, influenza will not be eliminated and thus efforts to improve influenza vaccines will remain of crucial importance.

negative: A novel delivery platform based on Bacteriophage MS2 virus-like particles Abstract Our objective here is to review the novel delivery platform based on Bacteriophage MS2 virus-like particles (VLPs), including introduction to their structure, their potential as a delivery platform, and their expected use in medicine and other fields. Bacteriophage MS2 VLPs are nanoparticles devoid of viral genetic material and can self-assemble from the coat protein into an icosahedral capsid. As a novel delivery platform, they possess numerous features that make them suitable and attractive for targeted delivery of RNAs or DNAs, epitope peptides, and drugs within the protein capsid. In short, as a novel delivery platform, MS2 VLPs are suitable for delivery of targeted agents and hold promise for use in diagnostics, vaccines, and therapeutic modalities.

negative: Dogs immunized with LBSap vaccine displayed high levels of IL-12 and IL-10 cytokines and CCL4, CCL5 and CXCL8 chemokines in the dermis Abstract The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-β and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-β expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.

negative: Tuberculosis vaccine development: from classic to clinical candidates Bacillus Calmette-Guérin (BCG) has been in use for nearly 100 years and is the only licensed TB vaccine. While BCG provides protection against disseminated TB in infants, its protection against adult pulmonary tuberculosis (PTB) is variable. To achieve the ambitious goal of eradicating TB worldwide by 2050, there is an urgent need to develop novel TB vaccines. Currently, there are more than a dozen novel TB vaccines including prophylactic and therapeutic at different stages of clinical research. This literature review provides an overview of the clinical status of candidate TB vaccines and discusses the challenges and future development trends of novel TB vaccine research in combination with the efficacy of evaluation of TB vaccines, provides insight for the development of safer and more efficient vaccines, and may inspire new ideas for the prevention of TB.

negative: Coronaviruses: Molecular Biology☆ Abstract Coronaviruses (CoVs) are enveloped, positive-strand RNA viruses with characteristic spike glycoproteins that project outward like the rays of the sun (corona – Latin for ‘crown’), when visualized by electron microscopy. CoV are classified, together with the toroviruses, in the family Coronaviridae and the order Nidovirales. All nidoviruses have a common genome organization and generate a nested set (nido – Latin for ‘nest’) of 3′ co-terminal mRNAs. CoVs have been isolated from a variety of species, including birds, livestock, domestic animals, and humans. CoV infections can cause respiratory, gastrointestinal, and neurologic disease, depending on the strain of the virus and the site of infection. Importantly, CoVs have been shown to cross species barriers and have emerged from animal reservoirs to infect humans and cause severe disease. The CoV responsible for an outbreak of severe acute respiratory disease (SARS-CoV) in 2002–03 likely originated as a bat coronavirus which, during replication in an intermediate host (such as the palm civet), evolved to be able to infect humans efficiently. SARS-CoV infected over 8000 people with approximately 10% mortality rate before it was controlled by public health measures of isolation of infected individuals and contacts. Middle East Respiratory Syndrome CoV (MERS-CoV), first reported in 2012, is likely transmitted from camels to humans with potentially fatal consequences. To date, there are no approved vaccines or direct acting antiviral drugs to combat coronavirus infections in humans. The emergence or re-emergence of CoVs from animal reservoirs is a potential concern for public health.

negative: Differential expression of lncRNAs during the HIV replication cycle: an underestimated layer in the HIV-host interplay Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell’s molecular network. Here, we performed transcriptome profiling throughout a primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Many lncRNAs were suggested to play a role in mechanisms relying on proteasomal and ubiquitination pathways, apoptosis, DNA damage responses and cell cycle regulation. Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile as compared to protein coding mRNAs, suggesting that mRNAs and lncRNAs are independently modulated. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function. Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and should be further investigated as they may represent targets for controlling HIV replication.

negative: Transmissible gastroenteritis coronavirus packaging signal is located at the 5' end of the virus genome. To locate the transmissible gastroenteritis coronavirus (TGEV) packaging signal, the incorporation of TGEV subgenomic mRNAs (sgmRNAs) into virions was first addressed. TGEV virions were purified by three different techniques, including an immunopurification using an M protein-specific monoclonal antibody. Detection of sgmRNAs in virions by specific reverse transcription-PCRs (RT-PCRs) was related to the purity of virus preparations. Interestingly, virus mRNAs were detected in partially purified virus but not in virus immunopurified using stringent conditions. Analyses by quantitative RT-PCR confirmed that virus mRNAs were not present in highly purified preparations. Lack of sgmRNA encapsidation was probably due to the absence of a packaging signal (Psi) within these mRNAs. This information plus that from the encapsidation of a collection of TGEV-derived minigenomes suggested that Psi is located at the 5' end of the genome. To confirm that this was the case, a set of minigenomes was expressed that included an expression cassette for an mRNA including the beta-glucuronidase gene (GUS) plus variable sequence fragments from the 5' end of the virus genome potentially including Psi. Insertion of the first 649 nucleotides (nt) of the TGEV genome led to the specific encapsidation of the mRNA, indicating that a Psi was located within this region which was absent from all of the other virus mRNAs. The presence of this packaging signal was further confirmed by showing the expression and rescue of the mRNA including the first 649 nt of the TGEV genome under control of the cytomegalovirus promoter in TGEV-infected cells. This mRNA was successfully amplified and encapsidated, indicating that the first 649 nt of TGEV genome also contained the 5' cis-acting replication signals. The encapsidation efficiency of this mRNA was about 30-fold higher than the genome encapsidation efficiency, as estimated by quantitative RT-PCR. In contrast, viral mRNAs presented significantly lower encapsidation efficiencies (about 100-fold) than those of the virus genome, strongly suggesting that TGEV mRNAs in fact lacked an alternative TGEV Psi.

negative: Current Clinical Trials and Vaccine Development Strategies for Corona Virus Disease (COVID-19) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached global epidemic status claiming more than 319K lives and affecting more than 4 81M people and counting worldwide Considering the severity of the situation and low recovery rate many research institutions and pharmaceutical industries are rushing to learn more about this new virus and the morbid physiology of this disease with effective diagnostic methods, therapeutic agents and vaccines Various approaches are highlighted for comparing the possible treatment methods available for COVID-19 some of which are BCG vaccination on COVID-19 and Non-pharmaceutical interventions, drug based clinical trials of Hydroxychloroquine-Azithromycin, chloroquine, lopinavir/ritonavir, ChAdOx1 nCoV-19, Remdesivir, Stem Cell therapy and mesenchymal stromal cell therapy, etc

negative: Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice. Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.

negative: A spike with which to beat COVID-19? This month’s Under the Lens discusses how structural studies of the SARS-CoV-2 spike glycoprotein might guide a path towards a vaccine.

negative: Development of an inactivated vaccine candidate for SARS-CoV-2 The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. There are currently no SARS-CoV-2-specific treatments or vaccines available due to the novelty of the virus. Hence, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against SARS-CoV-2 strains. Three immunizations using two different doses (3 μg or 6 μg per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support clinical development of SARS-CoV-2 vaccines for humans.

negative: COVID-19 treatment by repurposing drugs until the vaccine is in sight Corona virus disease (COVID-19) has created pandemic in the world as declared by WHO on March 12, 2020. It is a viral disease caused by SARS-CoV 2 virus and has affected large populations in over 120 countries. There is no specific treatment available and management is empirical. Until such time that an effective vaccine is available for COVID-19 viral infection, one can repurpose known therapeutic drug molecules such as angiotensin receptor 2 blocker, a commonly used antihypertensive drug, to control COVID-19 virus from gaining entry into the host cell by blocking the angiotensin receptor. Clinical trials should also be undertaken to use statins, which are lipid-lowering drugs but have anti-inflammatory and immunomodulatory properties to prevent acute lung injury in COVID-19 infection.

negative: Immunogenicity of a DNA vaccine candidate for COVID-19 The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

negative: Monte Carlo simulation of SARS-CoV-2 radiation-induced inactivation for vaccine development Immunization with an inactivated virus is one of the strategies currently being tested towards developing a SARS-CoV-2 vaccine. One of the methods used to inactivate viruses is exposure to high doses of ionizing radiation to damage their nucleic acids. Although gamma-rays effectively induce lesions in the RNA, envelope proteins are also highly damaged in the process. This in turn may alter their antigenic properties, affecting their capacity to induce an adaptive immune response able to confer effective protection. Here, we modelled the impact of sparsely and densely ionizing radiation on SARS-CoV-2 using the Monte Carlo toolkit Geant4-DNA. With a realistic 3D target virus model, we calculated the expected number of lesions in the spike and membrane proteins, as well as in the viral RNA. We show that gamma-rays produce significant spike protein damage, but densely ionizing charged particles induce less membrane damage for the same level of RNA lesions, because a single ion traversal through the nuclear envelope is sufficient to inactivate the virus. We propose that accelerated charged particles produce inactivated viruses with little structural damage to envelope proteins, thereby representing a new and effective tool for developing vaccines against SARS-CoV-2 and other enveloped viruses.

negative: COVID-19 vaccine development: Time to consider SARS-CoV-2 challenge studies? While a human challenge study holds the prospect of accelerating the development of a vaccine for the coronavirus SARS-CoV-2, it may be opposed due to risks of harm to participants and researchers. Given the increasing number of human deaths and severe disruption to lives worldwide, we argue that a SARS-CoV-2 challenge study is ethically justifiable as its social value substantially outweighs the risks. Such a study should therefore be seriously considered as part of the global research response towards the COVID-19 pandemic. In this paper, we contribute to the debate by addressing the misperception that a challenge study for the coronavirus would lower scientific and ethical standards for vaccine research and development, and examine how it could be ethically conducted. We also set out information that needs to be disclosed to prospective participants to obtain their consent.

negative: Adding the missing sugars to coronavirus protein structures Protein structures solved by cryo-electron microscopy (cryo-EM) or X-ray crystallography often miss the sugar molecules known as glycans that cover large swaths of the proteins’ surfaces The recently reported structures of the spike protein on SARS-CoV-2, the novel coronavirus that causes COVID-19, are no different Researchers are now working to fill in some of those sugary blanks to better understand the virus’s biology and to help drugmakers develop vaccines and treatments “People usually go for what they can analyze” and focus mostly on the protein components of a structure, says Andrea Thorn, a structural biologist at the Julius Maximilian University of Würzburg She is a member of the Coronavirus Structural Task Force, a group of scientists who specialize in modeling and processing crystallographic and cryo-EM data Because structural biology measurements don’t reveal much about glycans on proteins, “we often fail to recognize what an important part of the structure they

negative: Sanofi adds new virus to vaccine pact Sanofi's vaccine business Translate Bio will work together to develop an mRNA vaccine for the novel coronavirus The collaboration will build on a 2018 pact in which Sanofi paid Translate $45 million to develop up to five mRNA vaccines for infectious diseases For Sanofi, one of the world’s leading vaccine makers, the agreement complements a collaboration it formed in February with the US Biomedical Advanced Research and Development Authority to develop a cell-based vaccine against the virus

negative: Timely development of vaccines against SARS-CoV-2

negative: An mRNA Vaccine Protects Mice against Multiple Tick-Transmitted Flavivirus Infections Powassan virus (POWV) is an emerging tick-transmitted flavivirus that circulates in North America and Russia. Up to 5% of deer ticks now test positive for POWV in certain regions of the northern United States. Although POWV infections cause life-threatening encephalitis, there is no vaccine or counter-measure available for prevention or treatment. Here, we developed a lipid nanoparticle (LNP)-encapsulated modified mRNA vaccine encoding the POWV prM and E genes and demonstrated its immunogenicity and efficacy in mice following immunization with one or two doses. The POWV mRNA vaccine induced high titers of neutralizing antibody and sterilizing immunity against lethal challenge with different POWV strains. The mRNA vaccine also induced cross-neutralizing antibodies against multiple other tick-borne flaviviruses and protected mice against the distantly related Langat virus. These data demonstrate the utility of the LNP-mRNA vaccine platform for the development of vaccines with protective activity against multiple flaviviruses.

negative: Towards effective COVID19 vaccines: Updates, perspectives and challenges (Review) In the current context of the pandemic triggered by SARS-COV-2, the immunization of the population through vaccination is recognized as a public health priority. In the case of SARSCOV2, the genetic sequencing was done quickly, in one month. Since then, worldwide research has focused on obtaining a vaccine. This has a major economic impact because new technological platforms and advanced genetic engineering procedures are required to obtain a COVID19 vaccine. The most difficult scientific challenge for this future vaccine obtained in the laboratory is the proof of clinical safety and efficacy. The biggest challenge of manufacturing is the construction and validation of production platforms capable of making the vaccine on a large scale.

negative: Verification with the utility of an established rapid assessment of brain safety for newly developed vaccines In the twenty-first century, high contagious infectious diseases such as SARS (Severe Acute Respiratory Syndrome), MERS (Middle East Respiratory Syndrome), FMD (Foot-and-Mouth Disease) and AI (Avian Influenza) have become very prevalent, causing treat harm to humans and animals in aspect of public health, and economical issues. The critical problem is that newly-reported infectious diseases that humans firstly experience are expected to continue to emerge, and these diseases will be spreading out rapidly. Therefore, rapid and safe supplies of effective vaccines are most pivotal to prevent the rapid prevalent of new infection, but international standards or assessing protocol the safety of urgent vaccines are not established well. In our previous study, since we established a module to assess the brain safety of urgent vaccines, therefore, it is necessary to verify that this established module for assessing brain safety could work effectively in commercially available two vaccines (one killed- and on live-vaccines). We compared the results of Evans blue (EB) assay and qPCR analysis by injection of two kinds of vaccines, PBS and Lipopolysaccharide (LPS) under the condition of the module previously reported. We confirmed that the brain safety test module for urgent vaccine we established is very reproducible. Therefore, it is believed that this vaccine safety testing method can be used to validate brain safety when prompt supply of a newly developed vaccines is needed.

negative: Silver Lining of COVID-19: heightened global interest in pneumococcal and influenza vaccines, an infodemiology study BACKGROUND: Health-seeking behaviors change during pandemics and may increase with regard to illnesses with symptoms similar to the pandemic. The global reaction to COVID-19 may drive interest in vaccines for other diseases. OBJECTIVES: Our study investigated the correlation between global online interest in COVID-19 and interest in CDC-recommended routine vaccines. DESIGN, SETTINGS, MEASUREMENTS: This infodemiology study used Google Trends data to quantify worldwide interest in COVID-19 and CDC-recommended vaccines using the unit search volume index (SVI), which estimates volume of online search activity relative to highest volume of searches within a specified period. SVIs from December 30, 2019 to March 30, 2020 were collected for “coronavirus (Virus)” and compared with SVIs of search terms related to CDC-recommended adult vaccines. To account for seasonal variation, we compared SVIs from December 30, 2019 to March 30, 2020 with SVIs from the same months in 2015 to 2019. We performed country-level analyses in ten COVID-19 hotspots and ten countries with low disease burden. RESULTS: There were significant positive correlations between SVIs for “coronavirus (Virus)” and search terms for pneumococcal (R=0.89, p<0.0001) and influenza vaccines (R=0.93, p<0.0001) in 2020, which were greater than SVIs for the same terms in 2015-2019 (p= 0.005, p<0.0001, respectively). Eight in ten COVID-19 hotspots demonstrated significant positive correlations between SVIs for coronavirus and search terms for pneumococcal and influenza vaccines. LIMITATIONS: SVIs estimate relative changes in online interest and do not represent for people with no Internet access. CONCLUSION: A peak in worldwide interest in pneumococcal and influenza vaccines coincided with the COVID-19 pandemic in February and March 2020. Trends are likely not seasonal in origin and may be driven by Covid-19 hotspots. Global events may change public perception about the importance of vaccines. Our findings may herald higher demand for pneumonia and influenza vaccines in the upcoming season.

negative: B-Cell Engineering: A promising approach towards vaccine development for COVID-19 With the number of cases crossing three million (and more than two hundred and fifty thousand deaths) worldwide, there is a dire need of a vaccine (and repurposing of drugs) for SARS-CoV-2 disease (COVID-19). It can be argued that a vaccine may be the most efficient way to contain the spread of this disease and prevent its future onset. While many attempts are being made to design and develop a vaccine for SARS-CoV-2, pertinent technological hitches do exist. That is perhaps one of the reasons that we don’t have vaccine for coronaviruses (including SARS-CoV-1 and MERS). Recently developed CRISPR-mediated genome editing approach can be repurposed into a cell modification endeavor rather than correcting defective parts of genome. With this premise, B-cells can be engineered into universal donor, antigen specific, perpetually viable, long lasting, non-oncogenic, relatively benign, antibody producing cells which may serve as an effective vaccine for SARS-CoV-2 and, by the same rationale, other viruses and pathogens.

negative: Development of improved therapeutic mesothelin-based vaccines for pancreatic cancer Pancreatic cancer is the 5(th) leading cause of cancer deaths, and there are no effective treatments. We developed a poxvirus platform vaccine with improved immunogenicity and inserted the mesothelin gene to create an anti-mesothelin cancer vaccine. Mesothelin expression is mostly restricted to tumors in adult mammals and thus may be a good target for cancer treatment. We show here that the modified vaccinia virus Ankara (MVA) virus expressing mesothelin and the enhanced MVA virus missing the immunosuppressive A35 gene and expressing mesothelin were both safe in mice and were able to induce IFN-gamma secreting T cells in response to mesothelin expressing tumor cells. In addition, the MVA virus has oncolytic properties in vitro as it can replicate in and kill Panc02 pancreatic adenocarcinoma cell line tumor cells, even though it is unable to replicate in most mammalian cells. Deletion of the A35 gene in MVA improved T cell responses as expected. However, we were unable to demonstrate inhibition of Panc02 tumor growth in immunocompetent mice with pre-vaccination of mice, boosts, or even intratumoral injections of the recombinant viruses. Vaccine efficacy may be limited by shedding of mesothelin from tumor cells thus creating a protective screen from the immune system.

negative: Temporal regulation of bovine coronavirus RNA synthesis Abstract The structure and synthesis of bovine coronavirus (BCV)-specific intracellular RNA were studied. A genome-size RNA and seven subgenomic RNAs with molecular weights of approximately 3.3 × 106, 3.1 × 106, 2.6 × 106, 1.1 × 106, 1.0 × 106, 0.8 × 106 and 0.6 × 106 were detected. Comparisons of BCV intracellular RNAs with those of mouse hepatitis virus (MHV) demonstrated the presence of an additional RNA for BCV, species 2a, of 3.1 × 106 daltons. BCV RNAs contain a nested-set structure similar to that of other coronaviruses. This nested-set structure would suggest that the new RNA has a capacity to encode a protein of approximately 430 amino acids. Kinetic studies demonstrated that the synthesis of subgenomic mRNAs and genomic RNA are differentially regulated. At 4 to 8 h post-infection (p.i.), subgenomic RNAs are synthesized at a maximal rate and represent greater than 90% of the total viral RNA synthesized, whereas genome-size RNA accounts for only 7%. Later in infection, at 70 to 72 h p.i., genome-size RNA is much more abundant and accounts for 88% of total RNA synthesized. Immunoprecipitations of [35S]methionine-pulse-labeled viral proteins demonstrated that viral protein synthesis occurs early in the infection, concurrent with the peak of viral subgenomic RNA synthesis. Western blot analysis suggests that these proteins are stable since the proteins are present at high level as late as 70 to 72 h p.i. The kinetics of production of virus particles coincides with the synthesis of genomic RNA. These studies thus indicate that there is a differential temporal regulation of the synthesis of genomic RNA and subgenomic mRNAs, and that the synthesis of genomic RNA is the rate-limiting step regulating the production of virus particles.

negative: Current development of COVID-19 diagnostics, vaccines and therapeutics Abstract A novel coronavirus, designated as SARS-CoV-2, first emerged in Wuhan City, Hubei Province, China, in late December 2019. The rapidly increasing number of cases has caused worldwide panic. In this review, we describe some currently applied diagnostic approaches, as well as therapeutics and vaccines, to prevent, treat and control further outbreaks of SARS-CoV-2 infection.

negative: Prediction and Evolution of B Cell Epitopes of Surface Protein in SARS-CoV-2 The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. The sequences of the surface protein of SARS-CoV-2 and its proximal sequences were obtained by BLAST, the sequences of the whole genome of SARS-CoV-2 were obtained from the GenBank. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed. 7 epitopes were predicted, including 5 linear epitopes and 2 conformational epitopes, one of the linear and one of the conformational were coincide. The epitope D mutated easily, but the other epitopes were very conservative and the epitope C was the most conservative. It is worth mentioning that all of the 6 dominated epitopes were absolutely conservative in nearly 1000 SARS-CoV-2 genomes, and they deserved further study. The findings would facilitate the vaccine development, had the potential to be directly applied on the treatment in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.

negative: Possible method for the production of a Covid-19 vaccine.

negative: The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates entry of SARS-CoV-2 into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. Antibodies to the RBD domain of SARS-CoV (SARS-CoV-1), a closely related coronavirus which emerged in 2002-2003, have been shown to potently neutralize SARS-CoV-1 S-protein-mediated entry, and the presence of anti-RBD antibodies correlates with neutralization in SARS-CoV-2 convalescent sera. Here we show that immunization with the SARS-CoV-2 RBD elicits a robust neutralizing antibody response in rodents, comparable to 100 µg/ml of ACE2-Ig, a potent SARS-CoV-2 entry inhibitor. Importantly, anti-sera from immunized animals did not mediate antibody-dependent enhancement (ADE) of S-protein-mediated entry under conditions in which Zika virus ADE was readily observed. These data suggest that an RBD-based vaccine for SARS-CoV-2 could be safe and effective.

negative: Reverse genetic systems: Rational design of coronavirus live attenuated vaccines with immune sequelae Since the end of 2019, the global COVID-19 outbreak has once again made coronaviruses a hot topic. Vaccines are hoped to be an effective way to stop the spread of the virus. However, there are no clinically approved vaccines available for coronavirus infections. Reverse genetics technology can realize the operation of RNA virus genomes at the DNA level and provide new ideas and strategies for the development of new vaccines. In this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. An efficient reverse genetics platform is useful for obtaining the ideal attenuated strain to prepare an attenuated live vaccine.

negative: Establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain H120 Abstract Infectious bronchitis virus (IBV) strain H120 was successfully rescued as infectious clone by reverse genetics. Thirteen 1.5–2.8kb fragments contiguously spanning the virus genome were amplified and cloned into pMD19-T. Transcription grade complete length cDNA was acquired by a modified “No See’m” ligation strategy, which employed restriction enzyme Bsa I and BsmB I and ligated more than two fragments in one T4 ligase reaction. The full-length genomic cDNA was transcribed and its transcript was transfected by electroporation into BHK-21 together with the transcript of nucleocapsid gene. At 48h post transfection, the medium to culture the transfected BHK-21 cells was harvested and inoculated into 10-days old SPF embryonated chicken eggs (ECE) to replicate the rescued virus. After passage of the virus in ECE five times, the rescued H120 virus (R-H120) was successfully recovered. R-H120 was subsequently identified to possess the introduced silent mutation site in its genome. Some biological characteristics of R-H120 such as growth curve, EID50 and HA titers, were tested and all of them were very similar to its parent strain H120. In addition, both R-H120 and H120 induced a comparable titer of HA inhibition (HI) antibody in immunized chickens and also provided up to 85% of immune protection to the chickens that were challenged with Mass41 IBV strain. The present study demonstrated that construction of infectious clone from IBV vaccine strain H120 is possible and IBV-H120 can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach.

negative: Renin-Angiotensin System Blockers and the COVID-19 Pandemic: At Present There Is No Evidence to Abandon Renin-Angiotensin System Blockers During the spread of the severe acute respiratory syndrome coronavirus-2, some reports of data still emerging and in need of full analysis indicate that certain groups of patients are at risk of COVID-19. This includes patients with hypertension, heart disease, diabetes mellitus, and clearly the elderly. Many of those patients are treated with renin-angiotensin system blockers. Because the ACE2 (angiotensin-converting enzyme 2) protein is the receptor that facilitates coronavirus entry into cells, the notion has been popularized that treatment with renin-angiotensin system blockers might increase the risk of developing a severe and fatal severe acute respiratory syndrome coronavirus-2 infection. The present article discusses this concept. ACE2 in its full-length form is a membrane-bound enzyme, whereas its shorter (soluble) form circulates in blood at very low levels. As a mono-carboxypeptidase, ACE2 contributes to the degradation of several substrates including angiotensins I and II. ACE (angiotensin-converting enzyme) inhibitors do not inhibit ACE2 because ACE and ACE2 are different enzymes. Although angiotensin II type 1 receptor blockers have been shown to upregulate ACE2 in experimental animals, the evidence is not always consistent and differs among the diverse angiotensin II type 1 receptor blockers and differing organs. Moreover, there are no data to support the notion that ACE inhibitor or angiotensin II type 1 receptor blocker administration facilitates coronavirus entry by increasing ACE2 expression in either animals or humans. Indeed, animal data support elevated ACE2 expression as conferring potential protective pulmonary and cardiovascular effects. In summary, based on the currently available evidence, treatment with renin-angiotensin system blockers should not be discontinued because of concerns with coronavirus infection.

negative: Development of epitope-based peptide vaccine against novel coronavirus 2019 (SARS-COV-2): Immunoinformatics approach Recently, a novel coronavirus (SARS-COV-2) emerged which is responsible for the recent outbreak in Wuhan, China. Genetically, it is closely related to SARS-CoV and MERS-CoV. The situation is getting worse and worse, therefore, there is an urgent need for designing a suitable peptide vaccine component against the SARS-COV-2. Here, we characterized spike glycoprotein to obtain immunogenic epitopes. Next, we chose 13 Major Histocompatibility Complex-(MHC) I and 3 MHC-II epitopes, having antigenic properties. These epitopes are usually linked to specific linkers to build vaccine components and molecularly dock on toll-like receptor-5 to get binding affinity. Therefore, to provide a fast immunogenic profile of these epitopes, we performed immunoinformatics analysis so that the rapid development of the vaccine might bring this disastrous situation to the end earlier.

negative: Interaction of the HIV-1 frameshift signal with the ribosome Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.

negative: Immunogenic profile of SARS-CoV-2 spike in individuals recovered from COVID-19 The rapid global spread of SARS-CoV-2 and resultant mortality and social disruption have highlighted the need to better understand coronavirus immunity to expedite vaccine development efforts. Multiple candidate vaccines, designed to elicit protective neutralising antibodies targeting the viral spike glycoprotein, are rapidly advancing to clinical trial. However, the immunogenic properties of the spike protein in humans are unresolved. To address this, we undertook an in-depth characterisation of humoral and cellular immunity against SARS-CoV-2 spike in humans following mild to moderate SARS-CoV-2 infection. We find serological antibody responses against spike are routinely elicited by infection and correlate with plasma neutralising activity and capacity to block ACE2/RBD interaction. Expanded populations of spike-specific memory B cells and circulating T follicular helper cells (cTFH) were detected within convalescent donors, while responses to the receptor binding domain (RBD) constitute a minor fraction. Using regression analysis, we find high plasma neutralisation activity was associated with increased spike-specific antibody, but notably also with the relative distribution of spike-specific cTFH subsets. Thus both qualitative and quantitative features of B and T cell immunity to spike constitute informative biomarkers of the protective potential of novel SARS-CoV-2 vaccines.

negative: Molecular Targets for the Testing of COVID-19 The pandemic outbreaks of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread all over the world in a short period of time. Efficient identification of the infection by SARS-CoV-2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID-19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid-February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID-19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS-CoV-2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID-19 virus.

negative: Non-specific Primers Reveal False-negative Risk in Detection of COVID-19 Infections Background: A novel coronavirus disease 2019 (COVID-19) broke out in Wuhan of Hubei province and had spread throughout the world since December 2019. Because the clinically diagnosed cases in Hubei province were reported for the first time on February 13, 2020, a very high peak of new cases in China was observed. The reason why so many clinically diagnosed cases appeared was not clear. Methods: All data of new cases in China were acquired from WHO situation reports. Linear fitting was used to infer the ability to detect COVID-19 infections. Primer-BLAST and nucleotide blast were applied to check the specificity of primers. Expression data of human mRNA in different tissues was obtained from Human Protein Atlas. Finding: Based on the data and analysis of changes of new laboratory-confirmed cases and new clinically diagnosed cases, it was inferred that there were many false-negative results in all clinically diagnosed cases in Hubei province. There were eight non-specific primers in dozens of primers used in clinical or research detection of COVID-19. Among them, a pair of primer for the ORF1ab regions of SARS-CoV-2 genome, which widely applied to detect SARS-CoV-2 virus in China, well matched some human mRNAs such as Cathepsin C transcripts. Compared to other transcripts, Cathepsin C mRNA had a high abundance in tonsil, lung and small intestine. Interpretation: Some non-specific RT-PCR primers could cause the serious interference during RT-PCR amplification so as to increase the risk of false-negative diagnoses for COVID-19 infections. Funding Key Research Project of the Higher Education of Henan Province

negative: Consensus summary report for CEPI/BC March 12-13, 2020 meeting: Assessment of risk of disease enhancement with COVID-19 vaccines A novel coronavirus (CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China and has since spread as a global pandemic. Safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of Coronavirus Disease 2019 (COVID-19) disease and ease the major economic impact. There has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. However, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. A syndrome of "disease enhancement" has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. Animal models allowed scientists to determine the underlying mechanism for the former in the case of Respiratory syncytial virus (RSV) vaccine and have been utilized to design and screen new RSV vaccine candidates. Because some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for SARS-CoV-2 vaccines. To address this challenge, the Coalition for Epidemic Preparedness Innovations (CEPI) and the Brighton Collaboration (BC) Safety Platform for Emergency vACcines (SPEAC) convened a scientific working meeting on March 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. This report summarizes the evidence presented and provides considerations for safety assessment of COVID-19 vaccine candidates in accelerated vaccine development.

negative: mRNA transcript therapy. mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery.

negative: [Comment] Treatment strategies to fight the new coronavirus SARS-CoV-2: A challenge for a Rubik's Cube solver SARS-coronavirus-2 (SARS-CoV-2), the etiologic agent of the new lung disease COVID-19 is closely related to SARS-CoV, and together with MERS-CoV are three new human coronaviruses that emerged in the last 20 years. The COVID-19 outbreak is a rapidly evolving situation with higher transmissibility and infectivity compared with SARS and MERS. Clinical presentations range from asymptomatic or mild symptoms to severe illness. The prevalent cause of mortality is pneumonia that progresses to ARDS. The ongoing pandemic has already resulted in more than 135,000 deaths and an unprecedented burden on national health systems worldwide. Pending the availability of a vaccine, there is a critical need to identify effective treatments and a number of clinical trials have been implemented worldwide. Trials are based on repurposed drugs that are already approved for other infections, have acceptable safety profiles or have performed well in animal studies against the other two deadly coronaviruses. Supportive care remains the mainstay of therapy at present, as it is still unclear how well these data can be extrapolated to SARS-CoV-2. Most of those emerging re-introduced drugs are administered to patients in the context of clinical trials. In this review, we summarize the strategies currently employed in the treatment of COVID-19.

negative: 3CL hydrolase-based multiepitope peptide vaccine against SARS-CoV-2 using immunoinformatics The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-Î³-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.

negative: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development BACKGROUND: Highly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses. METHODS: We developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses. RESULTS: The result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses. CONCLUSION: Instead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity.

negative: Convergent antibody responses to SARS-CoV-2 in convalescent individuals. During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000. Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

negative: The SARS-CoV-2 N protein is a good component in a vaccine. Dutta and co-workers suggest in a recent letter (1) that the SARS-CoV-2 nucleoprotein (N) might be a good vaccine target.….

negative: Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose and prevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several aspects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguards that can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to prevent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that in general has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidates described has to be performed in humans.

negative: A Call for Vaccine Against COVID-19: Implications for Cardiovascular Morbidity and Healthcare Utilization

negative: A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide. They include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), many of which seriously endanger human health and well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host responses. The role of the C terminus of betacoronavirus nsp1 in virulence has been characterized, but the location of the alphacoronavirus nsp1 region that is important for virulence remains unclear. Here, using TGEV nsp1 as a model to explore the function of this protein in alphacoronaviruses, we demonstrate that alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal structure of full-length TGEV at 1.85-Å resolution and conducting several biochemical analyses, we observed that a specific motif (amino acids 91–95) of alphacoronavirus nsp1 is a conserved region that inhibits host protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9 technology to construct a recombinant TGEV in which this specific nsp1 motif was altered, we found that this mutation does not affect virus replication in cell culture but significantly reduces TGEV pathogenicity in pigs. Taken together, our findings suggest that alphacoronavirus nsp1 is an essential virulence determinant, providing a potential paradigm for the development of a new attenuated vaccine based on modified nsp1.

negative: Multigene RNA vector based on coronavirus transcription. Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5' and 3' ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells.

negative: Unusual heterogeneity of leader-mRNA fusion in a murine coronavirus: implications for the mechanism of RNA transcription and recombination. Coronavirus mRNA transcription was thought to be regulated by the interaction between the leader RNA and the intergenic sequence (IS), probably involving direct RNA-RNA interactions between complementary sequences. In this study, we found that a particular strain of mouse hepatitis virus, JHM2c, which has a deletion of a 9-nucleotide (nt) sequence (UUUAUAAAC) immediately downstream of the leader RNA, transcribed subgenomic mRNA species containing a whole array of heterogeneous leader fusion sites. Using a transfected defective interfering RNA which contains an IS and a reporter (chloramphenicol acetyltransferase) gene and JHM2c as a helper virus, we demonstrated that subgenomic mRNAs transcribed from the defective interfering RNAs were extremely heterogeneous. The leader-mRNA fusion sites in this virus can be grouped into five types. In type I, the leader is fused with the consensus IS of the template RNA at a site within the UCUAA repeats, consistent with the classical model of discontinuous transcription. In type II, the leader is fused with the consensus IS as in type I, but the leader of mRNA contains some nucleotide substitutions within the UCUAA repeats. In type III, the leader is fused with mRNAs at a site either upstream or downstream of the consensus IS. The sequences around the fusion sites bear little or no homology to the leader. As a result, mRNAs contain sequences complementary to the template sequences upstream of the IS or have sequence deletions downstream of the IS. In type IV, the leader is fused to the IS at the 9-nt sequence immediately downstream of the UCUAA repeats. In type V, the leader-mRNA fusion site contains a duplication of a portion of the leader sequence or an insertion of nontemplated sequences which are not present in either leader or template RNA. These patterns of leader-mRNA fusion resemble the aberrant homologous recombination frequently seen in other RNA viruses. The degree of heterogeneity of leader fusion sites is dependent on the sequences of both the leader RNA and IS. These results suggest that leader-mRNA fusion in coronavirus transcription does not require direct RNA-RNA interaction between complementary sequences. A modified model of RNA transcription and recombination based on protein-RNA and protein-protein interactions is proposed. This study also provides a paradigm for aberrant homologous recombination.

negative: Expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis. Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.

negative: COVID-19 vaccines: Knowing the unknown Vaccine development against SARS-CoV-2 has drawn attention around the globe due to the exploding pandemic. Although COVID-19 is caused by a new coronavirus, SARS-CoV-2, previous research on other coronavirus vaccines, such as FIPV, SARS, and MERS, has provided valuable information for the rapid development of COVID-19 vaccine. However, important knowledge gaps remain - some are specific to SARS-CoV-2, others are fundamental to immunology and vaccinology. Here, we discuss areas that need to be addressed for COVID-19 vaccine development, and what can be learned from examples of vaccine development in the past. Since the beginning of the outbreak, the research progress on COVID-19 has been remarkable. We are therefore optimistic about the rapid development of COVID-19 vaccine.

negative: Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses Seven coronaviruses (CoVs) have been isolated from humans so far. Among them, three emerging pathogenic CoVs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and a newly identified CoV (2019-nCoV), once caused or continue to cause severe infections in humans, posing significant threats to global public health. SARS-CoV infection in humans (with about 10% case fatality rate) was first reported from China in 2002, while MERS-CoV infection in humans (with about 34.4% case fatality rate) was first reported from Saudi Arabia in June 2012. 2019-nCoV was first reported from China in December 2019, and is currently infecting more than 70000 people (with about 2.7% case fatality rate). Both SARS-CoV and MERS-CoV are zoonotic viruses, using bats as their natural reservoirs, and then transmitting through intermediate hosts, leading to human infections. Nevertheless, the intermediate host for 2019-nCoV is still under investigation and the vaccines against this new CoV have not been available. Although a variety of vaccines have been developed against infections of SARS-CoV and MERS-CoV, none of them has been approved for use in humans. In this review, we have described the structure and function of key proteins of emerging human CoVs, overviewed the current vaccine types to be developed against SARS-CoV and MERS-CoV, and summarized recent advances in subunit vaccines against these two pathogenic human CoVs. These subunit vaccines are introduced on the basis of full-length spike (S) protein, receptor-binding domain (RBD), non-RBD S protein fragments, and non-S structural proteins, and the potential factors affecting these subunit vaccines are also illustrated. Overall, this review will be helpful for rapid design and development of vaccines against the new 2019-nCoV and any future CoVs with pandemic potential. This review was written for the topic of Antivirals for Emerging Viruses: Vaccines and Therapeutics in the Virology section of Frontiers in Microbiology.

negative: Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses BACKGROUND: Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA. RESULTS: In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220(+ )cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-A(d)) were increased on CD11c(+ )dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice. CONCLUSION: These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

negative: Assessing and Managing the Risks of Potential Pandemic Pathogen Research

negative: An overview of the safety, clinical application and antiviral research of the COVID-19 therapeutics Since a novel coronavirus pneumonia outbreak in late December 2019, coronavirus disease -19 (COVID-19) epidemic has gradually spread worldwide, becoming a major public health event. No specific antivirals are currently available for COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The treatments for COVID-19 are mainly based on the experiences of similar virus such SARS-CoV, MERS-CoV, HIV and influenza viruses. Scientists have taken great efforts to investigate the effective methods for the treatment of COVID-19. Up to now, there are over 1000 clinical studies for COVID-19 all over the world. In this article, we reviewed the current options for COVID-19 therapy including small molecules such as Remdesivir, Favipiravir, Lopinavir/Ritonavir etc, peptide inhibitors of ACE2, Traditional Chinese Medicines and Biologics such as SARS-CoV-2-specific neutralizing antibodies, mesenchymal stem cells and vaccines etc. Meanwhile, we systematically reviewed their clinical safety, clinical applications and progress of antiviral researches. The therapeutic effect of these antiviral drugs is summarized and compared, hoping to provide some ideas for clinical options of COVID-19 treatment and also provide experiences for the life-threatening virus diseases in the future.

negative: Exercising caution in correlating COVID-19 incidence and mortality rates with BCG vaccination policies due to variable rates of SARS CoV-2 testing TThe Bacillus Calmette-Guerin (BCG) vaccine provides protection against tuberculosis (TB), and is proposed to provide protection to non-TB infectious diseases. The COVID-19 outbreak results from infection with the novel coronavirus SARS-CoV-2 (CoV-2) and was declared a pandemic on March 11th, 2020. We queried whether the BCG vaccine offers protection against CoV-2 infection. We observed that countries with a current universal BCG vaccination policy have a significantly lower COVID-19 incidence than countries which never had a universal BCG policy or had one in the past. However, population density, median age, TB incidence, urban population, and, most significantly, CoV-2 testing rate, were also connected with BCG policy and could potentially confound the analysis. By limiting the analysis to countries with high CoV-2 testing rates, defined as greater than 2,500 tests per million inhabitants, these parameters were no longer statistically associated with BCG policy. When analyzing only countries with high testing rates, there was no longer a significant association between the number of COVID-19 cases per million inhabitants and the BCG vaccination policy. Although preliminary, our analyses indicate that the BCG vaccination may not offer protection against CoV-2 infection. While reporting biases may confound our observations, our findings support exercising caution in determining potential correlation between BCG vaccination and COVID-19 incidence, in part due significantly lower rates of CoV-2 testing per million inhabitants in countries with current universal BCG vaccination policy.

negative: Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs, there are examples of miRNA like sequences from RNA viruses with regulatory functions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with viral replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA-based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Overall, 950 hairpin structured sequences were extracted from the virus genome and based on the prediction results, 29 of them could be precursor miRNAs. Targeting analysis showed that 30 viral mature miRNA-like sequences could target 1,367 different human genes. PANTHER gene function analysis results indicated that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Protein class-based grouping of targeted human genes showed that host transcription might be one of the main targets of the virus since 96 genes involved in transcriptional processes were potential targets of predicted viral miRNAs. For instance, basal transcription machinery elements including several components of human mediator complex (MED1, MED9, MED12L, MED19), basal transcription factors such as TAF4, TAF5, TAF7L and site-specific transcription factors such as STAT1 were found to be targeted. In addition, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF1ab, ORF3a, ORF8, ORF7a and ORF10. Considering the fact that miRNA-based therapies have been paid attention, based on the findings of this study, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.

negative: Vaccines for emerging infectious diseases: Lessons from MERS coronavirus and Zika virus The past decade and a half has been characterized by numerous emerging infectious diseases. With each new threat, there has been a call for rapid vaccine development. Pathogens such as the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Zika virus represent either new viral entities or viruses emergent in new geographic locales and characterized by novel complications. Both serve as paradigms for the global spread that can accompany new pathogens. In this paper, we review the epidemiology and pathogenesis of MERS-CoV and Zika virus with respect to vaccine development. The challenges in vaccine development and the approach to clinical trial design to test vaccine candidates for disease entities with a changing epidemiology are discussed.

negative: Detection of SARS-CoV-2 neutralizing antibodies with a cell-free PCR assay Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 4 million confirmed infections worldwide and over 300,000 deaths. While Remdesivir has recently received FDA emergency use authorization for treatment of SARS-CoV-2 infection, convalescent plasma (CP) with high titers of SARS-CoV-2 neutralizing antibodies (NAbs) from recovered donors remains a promising and widely accessible method to mitigate severe disease symptoms. Here, we describe the development and validation of a cell-free neutralization PCR assay using SARS-CoV-2 spike protein S1 and human ACE2 receptor-DNA conjugates. By comparing with samples collected prior to the outbreak, we confirmed that NAbs were specifically detected in COVID-19 cases. Using our unique assay, the NAb signals are detectable as early as 10 days after onset of symptoms and continue to rise, plateauing after 18 days. Notably, we showed that the use of a licensed pathogen reduction technology to inactivate potentially contaminating infectious pathogens in CP did not alter NAb signals, paving a path to safely administer effective CP therapies. The described neutralization PCR assay can serve as a qualification tool to easily identify suitable CP donors of a potentially lifesaving therapy. In addition, this assay tool is readily deployable in standard laboratories with biosafety level 2 capability, and can yield results within 2–3 hrs. This advancement can facilitate research on factors driving diverse COVID-19 disease manifestations, help evaluate the impact of various CP processing protocols on CP therapeutic efficacy and assist in accelerating vaccine efficacy assessment.

negative: [Recent developments in SARS vaccine studies]. Severe acute respiratory syndrome (SARS) caused thousands of human infections worldwide and hundreds of deaths in just a few months. Evidence indicates that SARS coronavirus (SARS-CoV) has been circulating from animals to humans since before the 2002-2003 outbreak, suggesting that another pandemic may occur. This possibility has focused continuous action on SARS vaccine research. Inactivated vaccines, viral and bacterial vector vaccines, recombinant protein vaccines, subunit vaccines, DNA vaccines, and live-attenuated virus vaccines have been studied in different animal models. Although different animal models are used in vaccine studies, the most appropriate model for studying SARS is ferret since it develops the typical clinical signs, viral replication patterns and lung pathology compatible with that of SARS pathogenesis in humans. While there is much evidence that various vaccine strategies against SARS are safe and immunogenic, vaccinated animals still display significant disease upon challenge. Moreover, potential vaccine enhancement of SARS have also been shown in some studies. Data from the studies give an important information of the demand for further vaccine development research, especially focusing on mucosal immunization, T-cell immunity and combinations of heterologous vaccines in prime-boost regimens. In this review article developments on SARS vaccines have been discussed under the light of recent literature.

negative: Characterization of the African Swine Fever Virus Decapping Enzyme during Infection African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro. Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts. IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development.

negative: The Improbability of the Rapid Development of a Vaccine for SARS-CoV-2

negative: Gene delivery for immunoengineering A growing number of gene delivery strategies are being employed for immunoengineering in applications ranging from infectious disease prevention to cancer therapy. Viral vectors tend to have high gene transfer capability but may be hampered by complications related to their intrinsic immunogenicity. Non-viral methods of gene delivery, including polymeric, lipid-based, and inorganic nanoparticles as well as physical delivery techniques, have also been widely investigated. By using either ex vivo engineering of immune cells that are subsequently adoptively transferred or in vivo transfection of cells for in situ genetic programming, researchers have developed different approaches to precisely modulate immune responses. In addition to expressing a gene of interest through intracellular delivery of plasmid DNA and mRNA, researchers are also delivering oligonucleotides to knock down gene expression and immunostimulatory nucleic acids to tune immune activity. Many of these biotechnologies are now in clinical trials and have high potential to impact medicine.

negative: Global imperative to combat stigma associated with the coronavirus disease 2019 pandemic

negative: E-cigarette use frequency by smoking status among youth in the United States, 2014-2019 Aims: To report annual 2014-2019 youth estimates of past 30-day e-cigarette use frequency by smoking status in the United States (US). Design: Weighted prevalence estimates of students e-cigarette use using the 2014-2019 National Youth Tobacco Surveys (NYTS). For each year, t-tests for significance were used to compare estimates with those from the preceding year; t-tests were not performed on data for 2019 due to the change in survey format from paper to electronic. Setting: The NYTS is an annual school-based cross-sectional survey of US middle school (MS) and high school (HS) students. Participants: 117,472 students. Measurements: Self-report of past 30 day e-cigarette use based on students smoking status. Smoking status is assessed by asking if students have ever tried smoking, even one or two puffs, with never smokers responding no. Former smokers respond yes but have not smoked at all in the past 30 days. Current smokers used cigarettes at least once in the past 30 days. Frequent e-cigarette use is defined as use on 20 or more days in the past month. Findings: Past 30-day and frequent e-cigarette use increased among never, former, and current smoker youth from 2014-2019. In 2019, a greater proportion of current smokers used e-cigarettes frequently (HS = 46.1%, 95% CI: 39.1, 53.2; MS = 27.4%, 95% CI: 21.1, 33.6) compared to former smokers (HS = 23.2%, 95% CI: 18.1, 28.2; MS = 10.9%, 95% CI: 6.1, 15.7) and never smokers (HS = 3.7%, 95% CI: 3.0, 4.3; MS = 0.7%, 95% CI: 0.4, 0.9). From 2018 to 2019, the total number of youth using e-cigarettes frequently who were never smokers (2018: 180,000; 2019: 490,000) or former smokers (2018: 260,000; 2019: 640,000) surpassed that of current smokers (2018: 420,000; 2019: 460,000). Conclusions: The proportion and number of never smoker youth using e-cigarettes frequently increased greatly since 2014.

negative: Therapeutic Strategy for Patients with Coronavirus Disease 2019 During Left Ventricular Assist Device Supports

negative: Coronaviruses hijack the complement system

negative: Comparative review of respiratory diseases caused by coronaviruses and influenza A viruses during epidemic season Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to sweep the world, causing infection of millions and death of hundreds of thousands. The respiratory disease that it caused, COVID-19 (stands for coronavirus disease in 2019), has similar clinical symptoms with other two CoV diseases, severe acute respiratory syndrome and Middle East respiratory syndrome (SARS and MERS), of which causative viruses are SARS-CoV and MERS-CoV, respectively. These three CoVs resulting diseases also share many clinical symptoms with other respiratory diseases caused by influenza A viruses (IAVs). Since both CoVs and IAVs are general pathogens responsible for seasonal cold, in the next few months, during the changing of seasons, clinicians and public heath may have to distinguish COVID-19 pneumonia from other kinds of viral pneumonia. This is a discussion and comparison of the virus structures, transmission characteristics, clinical symptoms, diagnosis, pathological changes, treatment and prevention of the two kinds of viruses, CoVs and IAVs. It hopes to provide information for practitioners in the medical field during the epidemic season.

negative: Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals Summary Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide ‘megapools’, circulating SARS-CoV-2−specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike and N proteins each accounted for 11-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2−reactive CD4+ T cells in ∼40-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating ‘common cold’ coronaviruses and SARS-CoV-2.

negative: In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes. A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3.9 kb (M39). Expression of the reporter gene beta-glucuronidase (GUS) (2-8 microg per 10(6) cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1-2 microg per 10(6) cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development.

negative: Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery [Image: see text] Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.

negative: SARS-CoV-2 vaccines: 'Warp Speed' needs mind melds not warped minds In this review, we address issues that relate to the rapid "Warp Speed" development of vaccines to counter the COVID-19 pandemic. We review the antibody response that is triggered by SARS-CoV-2 infection of humans, and how it may inform vaccine research. The isolation and properties of neutralizing monoclonal antibodies from COVID-19 patients provide additional information on what vaccines should try to elicit. The nature and longevity of the antibody response to coronaviruses are relevant to the potency and duration of vaccine-induced immunity. We summarize the immunogenicity of leading vaccine candidates tested to date in animals and humans, and discuss the outcome and interpretation of virus-challenge experiments in animals. By far the most immunogenic vaccine candidates for antibody responses are recombinant proteins, which are not included in the initial wave of "Warp Speed" immunogens. A substantial concern for SARS-CoV-2 vaccines is adverse events, which we review by considering what was seen in studies of SARS-CoV-1 and MERS-CoV vaccines. We conclude by outlining the possible outcomes of the "Warp Speed" vaccine program, which range from the hoped-for rapid success to a catastrophic adverse influence on vaccine uptake generally.

negative: How did we get here? Short history of COVID-19 and other coronavirus-related epidemics The COVID-19 epidemic was not the first coronavirus epidemic of this century and represents one of the increasing number of zoonoses from wildlife to impact global health. SARS CoV-2, the virus causing the COVID-19 epidemic is distinct from, but closely resembles SARS CoV-1, which was responsible for the severe acute respiratory syndrome (SARS) outbreak in 2002. SARS CoV-1 and 2 share almost 80% of genetic sequences and use the same host cell receptor to initiate viral infection. However, SARS predominantly affected individuals in close contact with infected animals and health care workers. In contrast, CoV-2 exhibits robust person to person spread, most likely by means of asymptomatic carriers, which has resulted in greater spread of disease, overall morbidity and mortality, despite its lesser virulence. We review recent coronavirus-related epidemics and distinguish clinical and molecular features of CoV-2, the causative agent for COVID-19, and review the current status of vaccine trials.

negative: A method of identifying the blood contributor in mixture stains through detecting blood specific mRNA polymorphism. In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identifying individuals in mixture samples composed of two body fluids. In this study, we selected 5 blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension reaction (SBE) followed by capillary electrophoresis. The amplicon size was between 90bp-294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in home-made mixtures which are composed of different body fluids with 1ng peripheral blood mRNA added. This system showed a super sensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method of identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood-specific SNP typing system showed high sensitivity to the typing of blood-source specific markers regardless of other body fluids in the mixture. This article is protected by copyright. All rights reserved.

negative: Potential T-cell and B-cell Epitopes of 2019-nCoV As of early March, 2019-nCoV has infected more than one hundred thousand people and claimed thousands of lives. 2019-nCoV is a novel form of coronavirus that causes COVID-19 and has high similarity with SARS-CoV. No approved vaccine yet exists for any form of coronavirus. Here we use computational tools from structural biology and machine learning to identify 2019-nCoV T-cell and B-cell epitopes based on viral protein antigen presentation and antibody binding properties. These epitopes can be used to develop more effective vaccines and identify neutralizing antibodies. We identified 405 viral peptides with good antigen presentation scores for both human MHC-I and MHC-II alleles, and two potential neutralizing B-cell epitopes near the 2019-nCoV spike protein receptor binding domain (440-460 and 494-506). Analyzing mutation profiles of 68 viral genomes from four continents, we identified 96 coding-change mutations. These mutations are more likely to occur in regions with good MHC-I presentation scores (p=0.02). No mutations are present near the spike protein receptor binding domain. Based on these findings, the spike protein is likely immunogenic and a potential vaccine candidate. We validated our computational pipeline with SARS-CoV experimental data. The novel coronavirus 2019-nCoV has affected more than 100 countries and continues to spread. There is an immediate need for effective vaccines that contain antigens which trigger responses from human T-cells and B-cells (known as epitopes). Here we identify potential T-cell epitopes through an analysis of human antigen presentation, as well as B-cell epitopes through an analysis of protein structure. We identify a list of top candidates, including an epitope located on 2019-nCoV spike protein that potentially triggers both T-cell and B-cell responses. Analyzing 68 samples, we observe that viral mutations are more likely to happen in regions with strong antigen presentation, a potential form of immune evasion. Our computational pipeline is validated with experimental data from SARS-CoV.

negative: Vectors for gene expression in mammalian cells Publisher Summary The achievement of robust and regulated protein production in mammalian cells is a complex process that requires careful consideration of many factors, including transcriptional and translational control elements, RNA processing, gene copy number, mRNA stability, the chromosomal site of gene integration, potential toxicity of recombinant proteins to the host cell, and the genetic properties of the host. Gene transfer into mammalian cells may be effected either by infection with virus that carries the recombinant gene of interest, or by direct transfer of plasmid DNA. This chapter discusses the molecular architecture of non-viral vectors for high-level protein production. Virus-based vectors for gene therapy, protein production, vaccine development and other applications are summarized in a table and described.

negative: Recombinant adenoviral vaccine encoding the spike 1 subunit of the Middle East Respiratory Syndrome Coronavirus elicits strong humoral and cellular immune responses in mice BACKGROUND AND AIM: Middle East respiratory syndrome coronavirus (MERS-CoV) has rapidly spread throughout the Middle East since its discovery in 2012. The virus poses a significant global public health threat with potentially devastating effects. In this study, a recombinant adenoviral-based vaccine encoding the spike 1 (S1) subunit of the MERS-CoV genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. MATERIALS AND METHODS: Mice were immunized initially by intramuscular injection and boosted 3 weeks later by intranasal application. Expression of the S1 protein in the lungs and kidneys was detected using conventional polymerase chain reaction (PCR) and immunohistochemistry (IHC) targeting specific regions within the S1 subunit at weeks 3, 4, 5, and 6 after the first vaccination. Antigen-specific humoral and cellular immune responses were evaluated in serum and in cell culture following in vitro stimulation with a specific 9-mer epitope within the S1 protein (CYSSLILDY). RESULTS: S1 protein expression was only detected by IHC in the kidneys of the Ad-MERS-S1 group at week 6 from first immunization, and in both lungs and kidneys of Ad-MERS-S1 group by conventional PCR at weeks 3 and 5 post-prime. The vaccine elicited a specific S1-immunoglobulin G antibody response, which was detected in the sera of the vaccinated mice at weeks 4 and 6 from the onset of the first immunization. There was a significant increase in the amount of Th1-related cytokines (interferon-γ and interleukin [IL] 12), and a significant decrease in the Th2-related cytokine IL-4 in splenocyte cell culture of the vaccinated group compared with the control groups. CONCLUSION: The results of this study suggest that this recombinant adenovirus vaccine encoding the S1 subunit of MERS-CoV elicits potentially protective antigen-specific humoral and cellular immune responses in mice. This study demonstrates a promising vaccine for the control and/or prevention of MERS-CoV infection in humans.

negative: Smoking and the risk of COVID-19 infection in the UK Biobank Prospective Study Several studies suggest a lower prevalence of smoking than expected among adults with coronavirus disease (COVID-19). We conducted logistic regression analyses of the UK Biobank prospective study of 0.5 million adults followed for an average of 11 years. Compared to women, men were more likely to be tested and to test positive. In sex-stratified analyses, current smokers had higher adjusted Odds Ratios (OR) for being tested (male OR 1.60, 95%CI 1.32-1.95 and female OR 1.50, 1.21-.1.86). Current smokers were more slightly more likely than never smokers to test positive for COVID-19. Further examination of smoking as a risk factor for COVID-19 is required. These must take into account reverse causality, where smokers quit to avoid disease as well as prior diseases.

negative: Olfactory and gustatory dysfunctions due to the coronavirus disease (COVID-19): a review of current evidence INTRODUCTION: It is reported that coronavirus disease (COVID-19) can affect the sense of smell and taste of infected people. The pathobiology of this virus is still incompletely known, and it is therefore important to explore the impact of COVID-19 infections on olfactory and gustatory functions. We aimed to review current evidence on olfactory and gustatory dysfunctions caused by COVID-19. METHODS: This study was a narrative review performed in 2020 to investigate the olfactory and gustatory dysfunctions of the COVID-19. We searched eight keywords in six databases to determine the related documents on the main objective of the study. To discover studies meeting the inclusion criteria, the authors screened the titles and abstracts of the identified articles. The appropriate studies were included and their results were discussed to make the final selection. RESULTS: We have studied 24 current articles on the olfactory and gustatory dysfunctions due to COVID-19. A review of current studies has shown that we have a surge in the spread of olfactory and gustatory dysfunctions that happened during the epidemic of COVID-19 infection. Most studies (95.8%) have confirmed the symptoms of anosmia in patients with SARS-CoV-2 infection. A review of current studies showed that, in addition to anosmia, evidence of ageusia and dysgeusia (parageusia) was also seen in patients with COVID-19. CONCLUSION: The results of our study support recent reports that SARS-CoV-2 may infect oral and nasal tissues and cause olfactory and gustatory dysfunctions. These findings may aid future research on the diagnosis, prevention, and treatment of COVID-19 consequences.

negative: CORONAVIRUS VIRULENCE GENES WITH MAIN FOCUS ON SARS-CoV ENVELOPE GENE Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and 7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM) is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients.

negative: COVID-19: Risk of increase in smoking rates among England’s 6 million smokers and relapse among England’s 11 million ex-smokers

negative: Challenges for Experimentation on Living Beings at the Dawn of the 21(st) Century “We can talk endlessly about moral progress, about social progress, about poetic progress, about progress made in happiness; nevertheless, there is a type of progress that defies any discussion, and that is scientific progress, as soon as we judge it within the hierarchy of knowledge, from a specifically intellectual point of view.”

negative: COVID-19 and Bacillus Calmette-Guérin: What is the Link? The debate around the role of vaccination with Bacillus Calmette-Guérin has revived right in the time of the Coronavirus disease 19 pandemic. Since Bacillus Calmette-Guérin is one of the most commonly delivered therapies in urology, in this editorial we discuss some points that we think will be of interest and guidance to practicing urologists during this public health emergency.

negative: SARS-CoV-2 (COVID-19) Vaccine Development and Production: An Ethical Way Forward The world awaits a SARS-CoV-2 virus (i.e., COVID-19 disease) vaccine to keep the populace healthy, fully reopen their economies, and return their social and healthcare systems to "normal." Vaccine safety and efficacy requires meticulous testing and oversight; this paper describes how despite grandiose public statements, the current vaccine development, testing, and production methods may prove to be ethically dubious, medically dangerous, and socially volatile. The basic moral concern is the potential danger to the health of human test subjects and, eventually, many vaccine recipients. This is further complicated by economic and political pressures to reduce government oversight on rushed vaccine testing and production, nationalistic distribution goals, and failure to plan for the widespread immunization needed to produce global herd immunity. As this paper asserts, the public must be better informed to assess promises about the novel vaccines being produced and to tolerate delays and uncertainty.

negative: Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. Positive-stranded genomic RNA of coronavirus MHV and its six subgenomic mRNAs are synthesized in the cytoplasm of the host cell. The mRNAs are composed of leader and body sequences which are non-contiguous on the genome and are fused together in the cytoplasm by a mechanism which appears to involve an unusual and specific 'polymerase jumping' event.

negative: Preconditioning Vaccine Sites for mRNA-Transfected Dendritic Cell Therapy and Antitumor Efficacy. Messenger RNA (mRNA)-transfected dendritic cell (DC) vaccines have been shown to be a powerful modality for eliciting antitumor immune responses in mice and humans; however, their application has not been fully optimized since many of the factors that contribute to their efficacy remain poorly understood. Work stemming from our laboratory has recently demonstrated that preconditioning the vaccine site with a recall antigen prior to the administration of a dendritic cell vaccine creates systemic recall responses and resultantly enhances dendritic cell migration to the lymph nodes with improved antitumor efficacy. This chapter describes the generation of murine mRNA-transfected DC vaccines, as well as a method for vaccine site preconditioning with protein antigen formulations that create potent recall responses.

negative: An International Review of Tobacco Use and the COVID-19 Pandemic: Examining Hospitalization, Asymptomatic Cases, and Severity Background and objectives: Since the World Health Organization (WHO) declared a public health emergency of imminent concern in March 2020, the novel coronavirus (SARS-CoV-2) and its related disease (COVID-19) has become a topic of much-needed research. This study primarily focused on what effect smoking had on hospitalization; however, asymptomaticity and severity were discussed. Data: Data was collected through searches on databases including PubMed and Google Scholar. Eligibility criteria included being RT-PCR verified and including smoking data. Discussion: This study found that smokers were significantly underrepresented in COVID-19 hospitalization on a purely epidemiological level in some areas, including China and Manhattan, but not others: Seattle, Greater New York City Area, and Italy. Furthermore, smokers were equally represented in asymptomatic populations, but smokers will likely experience a more severe manifestation of the disease if they are symptomatic. Further inquiry into possible mechanisms by which the observed epidemiological effect is necessary, as it has implications for recommendations on loosening restrictions on social distancing measures. Conclusions and Recommendations: This study recommends that smokers consider themselves to be at higher risk for COVID-19, as they may experience a more severe manifestation of the disease. Health care providers and policymakers should consider smokers at higher risk as well, as there is evidence to claim that smokers may contract a more severe form of COVID-19.

negative: Comparative analysis of coronavirus genomic RNA structure reveals conservation in SARS-like coronaviruses Coronaviruses, including SARS-CoV-2 the etiological agent of COVID-19 disease, have caused multiple epidemic and pandemic outbreaks in the past 20 years(1–3). With no vaccines, and only recently developed antiviral therapeutics, we are ill equipped to handle coronavirus outbreaks(4). A better understanding of the molecular mechanisms that regulate coronavirus replication and pathogenesis is needed to guide the development of new antiviral therapeutics and vaccines. RNA secondary structures play critical roles in multiple aspects of coronavirus replication, but the extent and conservation of RNA secondary structure across coronavirus genomes is unknown(5). Here, we define highly structured RNA regions throughout the MERS-CoV, SARS-CoV, and SARS-CoV-2 genomes. We find that highly stable RNA structures are pervasive throughout coronavirus genomes, and are conserved between the SARS-like CoV. Our data suggests that selective pressure helps preserve RNA secondary structure in coronavirus genomes, suggesting that these structures may play important roles in virus replication and pathogenesis. Thus, disruption of conserved RNA secondary structures could be a novel strategy for the generation of attenuated SARS-CoV-2 vaccines for use against the current COVID-19 pandemic.

negative: Towards the first multi-epitope recombinant vaccine against Crimean-Congo hemorrhagic fever virus: A computer-aided vaccine design approach Abstract Crimean-Congo hemorrhagic fever (CCHF) is considered one of the major public health concerns with case fatality rates of up to 80%. Currently, there is no effective approved vaccine for CCHF. In this study, we used a computer-aided vaccine design approach to develop the first multi-epitope recombinant vaccine for CCHF. For this purpose, linear B-cell and T-cell binding epitopes from two structural glycoproteins of CCHF virus including Gc and Gn were predicted. The epitopes were further studied regarding their antigenicity, allergenicity, hydrophobicity, stability, toxicity and population coverage. A total number of seven epitopes including five T-cell and two B-cell epitopes were screened for the final vaccine construct. Final vaccine construct composed of 382 amino acid residues which were organized in four domains including linear B-cell, T-cell epitopes and cholera toxin B-subunit (CTxB) along with heat labile enterotoxin IIc B subunit (LT-IIc) as adjuvants. All the segments were joined using appropriate linkers. The physicochemical properties as well as the presence of IFN-γ inducing epitopes in the proposed vaccine, was also checked to determining the vaccine stability, solubility and its ability to induce cell-mediated immune responses. The 3D structure of proposed vaccine was subjected to the prediction of computational B-cell epitopes and molecular docking studies with MHC-I and II molecules. Furthermore, molecular dynamics stimulations were performed to study the vaccine-MHCs complexes stability during stimulation time. The results suggest that our proposed vaccine was stable, well soluble in water and potentially antigenic. Results also demonstrated that the vaccine can induce both humoral and cell-mediated immune responses and could serve as a promising anti-CCHF vaccine candidate.

negative: mRNA as novel technology for passive immunotherapy While active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. In either case, administration or expression of recombinant proteins plays a fundamental role. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. With its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mRNA combines several advantages. For example, compared to transfected DNA, mRNA harbors inherent safety features. It is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. Compared to the administration of recombinant proteins produced in bioreactors, mRNA allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. Based on great progress in understanding and manipulating mRNA properties, efficacy data in various models have now demonstrated that IVT mRNA constitutes a potent and flexible platform technology. Starting with an introduction into passive immunotherapy, this review summarizes the current status of IVT mRNA technology and its application to such immunological interventions.

negative: A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-ΔG-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-ΔG-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-ΔG-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters’ lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-ΔG-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

negative: The Importance Of Naturally Attenuated Sars‐Cov‐2 In The Fight Against Covid‐19 The current SARS‐CoV‐2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID‐19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS‐CoV‐2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS‐CoV‐2 would offer significant relief, although vaccine developers have warned that design, testing, and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (1), but the earliest vaccines were based on live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS‐CoV‐2 will mutate and may naturally attenuate over time (2). What makes the current pandemic unique is that, thanks to state‐of‐the‐art nucleic acid sequencing technologies, we can follow in detail how SARS‐CoV‐2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS‐CoV‐2 variants across the globe should be of key interest in our fight against the pandemic. This article is protected by copyright. All rights reserved.

negative: Vaccinia Virus as a Master of Host Shutoff Induction: Targeting Processes of the Central Dogma and Beyond The synthesis of host cell proteins is adversely inhibited in many virus infections, whereas viral proteins are efficiently synthesized. This phenomenon leads to the accumulation of viral proteins concurrently with a profound decline in global host protein synthesis, a phenomenon often termed “host shutoff”. To induce host shutoff, a virus may target various steps of gene expression, as well as pre- and post-gene expression processes. During infection, vaccinia virus (VACV), the prototype poxvirus, targets all major processes of the central dogma of genetics, as well as pre-transcription and post-translation steps to hinder host cell protein production. In this article, we review the strategies used by VACV to induce host shutoff in the context of strategies employed by other viruses. We elaborate on how VACV induces host shutoff by targeting host cell DNA synthesis, RNA production and processing, mRNA translation, and protein degradation. We emphasize the topics on VACV’s approaches toward modulating mRNA processing, stability, and translation during infection. Finally, we propose avenues for future investigations, which will facilitate our understanding of poxvirus biology, as well as fundamental cellular gene expression and regulation mechanisms.

negative: An exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies. Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.

negative: COVID-19: An updated review/ COVID-19: ОБНОВЛЕННЫЙ ВЗГЛЯД COVID-19 is a zoonotic disease that showed higher levels of transmissibility in humans. Coronavirus has the largest recognized genome (28–33 kb) of a positive-sense single stranded RNA. The genome composed of 5′-end, the translationable mRNA sequences for the key proteins; replicase, spike, envelop membrane, and nucleocapsid and 3′-end (polyA tail). This highly contagious virus may impair the immune system in the early phase of the disease, hence the symptoms of the disease appear very rapidly. Importantly until now, there is no efficient strategy for containing the disease. So, all the world scientists today are in a race against time to find a vaccine or treatment to COVID-19, which requires a deeper understanding.

negative: Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs. Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free leader RNA species. As a result, each virus-specific mRNA contains an identical leader RNA derived from the 5', end of the genomic RNA. In this study, we demonstrate by primer extension studies that the leader-fusion sites on a given species of coronavirus subgenomic mRNA are heterogeneous. The heterogeneity was due to variation in the number of pentanucleotide (UCUAA) repeats present at the leader fusion site. This pentanucleotide repeat region was complementary between the free leader RNA and the transcription start sites on the template RNA. This result suggests that the discontinuous transcription of coronavirus mRNAs occurs within the complementary sequences localized in two different RNA segments and that RNA joining occurs at variable sites.

negative: CEPI commits $384 million to Novavax’s COVID-19 vaccine The Coalition for Epidemic Preparedness Innovations (CEPI) will commit up to $384 million for manufacturing and clinical testing of Novavax’s nanoparticle-based COVID-19 vaccine Novavax was one of the first companies to disclose that it was working on a COVID-19 vaccine in January, and a Phase I clinical trial on the safety of that experimental vaccine will begin in Australia this month That trial will make it one of the first 10 vaccines to enter clinical testing since the pandemic began CEPI, a nonprofit organization that funds infectious disease vaccine development, counts at least 115 vaccine programs underway for COVID-19 CEPI has so far provided funding to nine of them Before now, the coalition had invested $58 million across all its COVID-19 vaccine efforts, including an initial $4 million for Novavax The new, $384 million commitment to Novavax is the largest single investment CEPI has ever made in a vaccine

negative: New Insights of Emerging SARS-CoV-2: Epidemiology, Etiology, Clinical Features, Clinical Treatment, and Prevention Since the first reports that the novel coronavirus was showing human-to-human transmission characteristics and asymptomatic cases, the number of patients with associated pneumonia has continued to rise and the epidemic has grown. It now threatens the health and lives of people across the world. The governments of many countries have attached great importance to the prevention of SARS-CoV-2, via research into the etiology and epidemiology of this newly emerged disease. Clinical signs, treatment, and prevention characteristics of the novel coronavirus pneumonia have been receiving attention worldwide, especially from medical personnel. However, owing to the different experimental methods, sample sizes, sample sources, and research perspectives of various studies, results have been inconsistent, or relate to an isolated aspect of the virus or the disease it causes. Currently, systematic summary data on the novel coronavirus are limited. This review combines experimental and clinical evidence into a systematic analysis and summary of the current progress of research into SARS-CoV-2, from multiple perspectives, with the aim of gaining a better overall understanding of the disease. Our report provides important information for current clinicians, for the prevention and treatment of COVID-19 pneumonia.

negative: COVID-19, mobile health and serious mental illness

negative: SARS-CoV-2 Isolation and Propagation from Turkish COVID-19 patients The novel coronavirus pneumonia, which was named later as Coronavirus Disease 2019 (COVID-19), is caused by the Severe Acute Respiratory Syndrome Coronavirus 2, namely SARS-CoV-2. It is a positive-strand RNA virus that is the seventh coronavirus known to infect humans. The COVID-19 outbreak presents enormous challenges for global health behind the pandemic outbreak. The first diagnosed patient in Turkey has been reported by the Republic of Turkey Ministry of Health on March 11, 2020. Today, over ninety thousand cases in Turkey, and two million cases around the world have been declared. Due to the urgent need for vaccine and anti-viral drug, isolation of the virus is crucial. Here, we report one of the first isolation and characterization studies of SARS-CoV-2 from nasopharyngeal and oropharyngeal specimens of diagnosed patients in Turkey. This study provides an isolation and replication methodology, and cell culture tropism of the virus that will be available to the research communities. Article Summary Scientists have isolated virus from Turkish COVID-19 patients. The isolation, propagation, and plaque and immune response assays of the virus described here will serve in following drug discovery and vaccine testing.

negative: Chapter Eight Molecular Basis of Coronavirus Virulence and Vaccine Development Abstract Virus vaccines have to be immunogenic, sufficiently stable, safe, and suitable to induce long-lasting immunity. To meet these requirements, vaccine studies need to provide a comprehensive understanding of (i) the protective roles of antiviral B and T-cell-mediated immune responses, (ii) the complexity and plasticity of major viral antigens, and (iii) virus molecular biology and pathogenesis. There are many types of vaccines including subunit vaccines, whole-inactivated virus, vectored, and live-attenuated virus vaccines, each of which featuring specific advantages and limitations. While nonliving virus vaccines have clear advantages in being safe and stable, they may cause side effects and be less efficacious compared to live-attenuated virus vaccines. In most cases, the latter induce long-lasting immunity but they may require special safety measures to prevent reversion to highly virulent viruses following vaccination. The chapter summarizes the recent progress in the development of coronavirus (CoV) vaccines, focusing on two zoonotic CoVs, the severe acute respiratory syndrome CoV (SARS-CoV), and the Middle East respiratory syndrome CoV, both of which cause deadly disease and epidemics in humans. The development of attenuated virus vaccines to combat infections caused by highly pathogenic CoVs was largely based on the identification and characterization of viral virulence proteins that, for example, interfere with the innate and adaptive immune response or are involved in interactions with specific cell types, such as macrophages, dendritic and epithelial cells, and T lymphocytes, thereby modulating antiviral host responses and viral pathogenesis and potentially resulting in deleterious side effects following vaccination.

negative: Toward Nanotechnology-Enabled Approaches against the COVID-19 Pandemic [Image: see text] The COVID-19 outbreak has fueled a global demand for effective diagnosis and treatment as well as mitigation of the spread of infection, all through large-scale approaches such as specific alternative antiviral methods and classical disinfection protocols. Based on an abundance of engineered materials identifiable by their useful physicochemical properties through versatile chemical functionalization, nanotechnology offers a number of approaches to cope with this emergency. Here, through a multidisciplinary Perspective encompassing diverse fields such as virology, biology, medicine, engineering, chemistry, materials science, and computational science, we outline how nanotechnology-based strategies can support the fight against COVID-19, as well as infectious diseases in general, including future pandemics. Considering what we know so far about the life cycle of the virus, we envision key steps where nanotechnology could counter the disease. First, nanoparticles (NPs) can offer alternative methods to classical disinfection protocols used in healthcare settings, thanks to their intrinsic antipathogenic properties and/or their ability to inactivate viruses, bacteria, fungi, or yeasts either photothermally or via photocatalysis-induced reactive oxygen species (ROS) generation. Nanotechnology tools to inactivate SARS-CoV-2 in patients could also be explored. In this case, nanomaterials could be used to deliver drugs to the pulmonary system to inhibit interaction between angiotensin-converting enzyme 2 (ACE2) receptors and viral S protein. Moreover, the concept of “nanoimmunity by design” can help us to design materials for immune modulation, either stimulating or suppressing the immune response, which would find applications in the context of vaccine development for SARS-CoV-2 or in counteracting the cytokine storm, respectively. In addition to disease prevention and therapeutic potential, nanotechnology has important roles in diagnostics, with potential to support the development of simple, fast, and cost-effective nanotechnology-based assays to monitor the presence of SARS-CoV-2 and related biomarkers. In summary, nanotechnology is critical in counteracting COVID-19 and will be vital when preparing for future pandemics.

negative: Avian infectious bronchitis virus genomic RNA contains sequence homologies at the intergenic boundaries Abstract cDNAs derived from the 3'-terminus of IBV Beaudette genomic RNA by oligo(dT)-primed reverse transcription were cloned in PAT153/HB101. 25 clones were characterised and two of these were found to cover 3.3 kb extending from the poly(A) tail. The relationships of the two smallest IBV mRNAs, A and B, to the cloned sequences were established by restriction fragment hybridisation to Northern blots. The 5'-termini of mRNAs A and B were located on the clones by S1 mapping. DNA sequencing of cloned sequences corresponding to the intergenic boundaries of mRNAs A and B was carried out. Homologies between the sequences at these boundaries were observed.

negative: Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication. Stress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. The MERS-CoV 4a accessory protein is known to inhibit SG formation in cells in which it was expressed by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-Δp4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-Δp4-induced SG formation. MERS-CoV-Δp4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells, and inhibition of SG formation by small interfering RNA-mediated depletion of the SG components promoted MERS-CoV-Δp4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-Δp4 replication was not due to either the induction of type I and type III interferons or the accumulation of viral mRNAs in the SGs. Rather, it was due to the inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2α phosphorylation and likely by the confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of the 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that the MERS-CoV 4a accessory protein, which was reported to block SG formation in cells in which it was expressed, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this report is the first to show the biological significance of SG in CoV replication and provides insight into the interplay between MERS-CoV and antiviral stress responses.

negative: On facing the SARS-cov-2 (COVID-19) with combination of nanomaterials and medicine: Possible strategies and first challenges Global health is facing the most dangerous situation regarding the novel severe acute respiratory syndrome called coronavirus 2 (SARS-CoV-2), which is widely known as the abbreviated COVID-19 pandemic. This is due to the highly infectious nature of the disease and its possibility to cause pneumonia induced death in approximately 6.89% of infected individuals (data until 27 April 2020). The pathogen causing COVID-19 is called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is believed to be originated from the Wuhan Province in China. Unfortunately, an effective and approved vaccine for SARS-CoV-2 virus is still not available, making the situation more dangerous and currently available medical care futile. This unmet medical need thus requires significant and very urgent research attention to develop an effective vaccine to address the SARS-CoV-2 virus. In this review, the state-of-the-art drug design strategies against the virus are critically summarized including exploitations of novel drugs and potentials of repurposed drugs. The applications of nanochemistry and general nanotechnology was also discussed to give the status of nanodiagnostic systems for COVID-19.

negative: Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

negative: A Targeted Vaccine against COVID-19: S1-Fc Vaccine Targeting the Antigen-Presenting Cell Compartment Elicits Protection against SARS-CoV-2 Infection Vaccination efficacy is enhanced by targeting the antigen-presenting cell compartment. Here, we show that S1-Fc antigen delivery targeting the FcγR+ antigen-presenting cell compartment elicits anti-SARS-CoV-2 S1-antigen specific IgG production in vivo exerting biologically functional and protective activity against live virus infection, assessed in a stringent experimental virus challenge assay in vitro. The S1-domain of the SARS-CoV-2 spike protein was genetically fused to a human immunoglobulin Fc moiety, which contributes to mediate S1-Fc cellular internalization by FcγR+ antigen-presenting cells. Immediately upon administration intramuscularly, our novel vaccine candidate recombinant rS1-Fc homes to lymph nodes in vivo where FcγR+ antigen-presenting cells reside. Seroconversion is achieved as early as day 7, mounting considerably increased levels of anti-S1 IgGs in vivo. Interestingly, immunization at elevated doses with non-expiring S1-Fc encoding dsDNA favors the education of a desired antigen-specific adaptive T cell response. However, low-dose immunization, safeguarding patient safety, using recombinant rS1-Fc, elicits a considerably elevated protection amplitude against live SARS-CoV-2 infection. Our promising findings on rS1-Fc protein immunization prompted us to further develop an affordable and safe product for delivery to our communities in need for COVID-19 vaccinations.

negative: Reversible ADP-ribosylation of RNA ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD(+)) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.

negative: An in-silico approach to develop of a multi-epitope vaccine candidate against SARS-CoV-2 envelope (E) protein Since the first appearance of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) in China on December 2019, the world has now witnessed the emergence of the SARS- CoV-2 outbreak. Therefore, due to the high transmissibility rate of virus, there is an urgent need to design and develop vaccines against SARS-CoV-2 to prevent more cases affected by the virus. In this study, a computational approach is proposed for vaccine design against the envelope (E) protein of SARS-CoV-2, which contains a conserved sequence feature. First, we sought to gain potential B-cell and T-cell epitopes for vaccine designing against SARS-CoV-2. Second, we attempted to develop a multi-epitope vaccine. Immune targeting of such epitopes could theoretically provide defense against SARS-CoV-2. Finally, we evaluated the affinity of the vaccine to major histocompatibility complex (MHC) molecules to stimulate the immune system response to this vaccine. We also identified a collection of B-cell and T-cell epitopes derived from E proteins that correspond identically to SARS-CoV-2 E proteins. The in-silico design of our potential vaccine against E protein of SARS-CoV-2 demonstrated a high affinity to MHC molecules, and it can be a candidate to make a protection against this pandemic event.

negative: Dynamics of translation by single ribosomes through mRNA secondary structures During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream stem-loops containing 100% G-C base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit (E) site. Downstream stem-loops or pseudoknots containing both G-C and A-U pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat surprisingly, unfolding of mRNA 2° structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

negative: Malaria vaccines: high-throughput tools for antigens discovery with potential for their development Malaria is a disease induced by parasites of the Plasmodium genus, which are transmitted by Anopheles mosquitoes and represents a great socio-economic burden Worldwide. Plasmodium vivax is the second species of malaria Worldwide, but it is the most prevalent in Latin America and other regions of the planet. It is currently considered that vaccines represent a cost-effective strategy for controlling transmissible diseases and could complement other malaria control measures; however, the chemical and immunological complexity of the parasite has hindered development of effective vaccines. Recent availability of several genomes of Plasmodium species, as well as bioinformatic tools are allowing the selection of large numbers of proteins and analysis of their immune potential. Herein, we review recently developed strategies for discovery of novel antigens with potential for malaria vaccine development.

negative: SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques CD4 T follicular helper (Tfh) cells are important for the generation of long-lasting and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that, following infection with SARS-CoV-2, adult rhesus macaques exhibited transient accumulation of activated, proliferating Tfh cells in their peripheral blood on a transitory basis. The CD4 helper cell responses were skewed predominantly toward a Th1 response in blood, lung, and lymph nodes, reflective of the interferon-rich cytokine environment following infection. We also observed the generation of germinal center Tfh cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies but delayed or absent IgA antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.

negative: Preliminary Virtual Screening Studies to Identify GRP78 Inhibitors Which May Interfere with SARS-CoV-2 Infection SARS-CoV-2 Spike protein was predicted by molecular docking to bind the host cell surface GRP78, which was suggested as a putative good molecular target to inhibit Covid-19. We aimed to confirm that GRP78 gene expression was increased in blood of SARS-CoV-2 (+) versus SARS-CoV-2 (−) pneumonia patients. In addition, we aimed to identify drugs that could be repurposed to inhibit GRP78, thus with potential anti-SARS-CoV-2 activity. Gene expression studies were performed in 10 SARS-CoV-2 (−) and 24 SARS-CoV-2 (+) pneumonia patients. A structure-based virtual screen was performed with 10,761 small molecules retrieved from DrugBank, using the GRP78 nucleotide binding domain and substrate binding domain as molecular targets. Results indicated that GRP78 mRNA levels were approximately four times higher in the blood of SARS-CoV-2 (+) versus SARS-CoV-2 (−) pneumonia patients, further suggesting that GRP78 might be a good molecular target to treat Covid-19. In addition, a total of 409 compounds were identified with potential as GRP78 inhibitors. In conclusion, we found preliminary evidence that further proposes GRP78 as a possible molecular target to treat Covid-19 and that many clinically approved drugs bind GRP78 as an off-target effect. We suggest that further work should be urgently carried out to confirm if GRP78 is indeed a good molecular target and if some of those drugs have potential to be repurposed for SARS-CoV-2 antiviral activity.

negative: Ten challenging questions about SARS-CoV-2 and COVID-19. INTRODUCTION Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently introduced as a global public health problem by the World Health Organization (WHO). The virus outbreak has been documented around the world. Updating data in different aspects of the virus could force us to revise our idea about the main questions concerning coronavirus disease-19 (COVID-19). AREAS COVERED Although our knowledge about the SARS-CoV-2 and COVID-19 is largely based on the very limited data, the information is growing rapidly. The renewed answers to the specific research questions concerning updating data not only reveal gaps for future research but also re-categorized our information. Here, we attempt to briefly discuss ten important questions about SARS-CoV-2 and COVID-19. EXPERT OPINION Since our knowledge about different aspects of SARS-CoV-2 appears to be in its infancy and is rapidly changing, the provision of the right data is more difficult in this regard. However, we try to rely on results from more extensive research to answer the main questions about this new virus. Therefore, further studies, particularly in the context of the virus pathogenesis, diagnosis, treatment, and vaccine development, are warranted.

negative: Safety and immunogenicity of a potential checkpoint blockade vaccine for canine melanoma Human immunotherapy with checkpoint blockades has achieved significant breakthroughs in recent years. In this study, a checkpoint blockade vaccine for canine melanoma was tested for safety and immunogenicity. Five healthy adult dogs received a mixture of three replication-defective chimpanzee-derived adenoviral vectors, one expressing mouse fibroblast-associated protein (mFAP) and the others expressing canine melanoma-associated antigens Trp-1 or Trp-2 fused into Herpes Simplex-1 glycoprotein D, a checkpoint inhibitor of herpes virus entry mediator (HVEM) pathways. The vaccine mixture was shown to be well tolerated and increased frequencies of canineTrp-1-specific activated CD8(+) and CD4(+) T cells secreting interferon-(IFN)-γ, tumor necrosis factor (TNF)-α, or interleukin (IL)-2 alone or in combinations in four and five out of five dogs, respectively. To avoid excessive bleeds, responses to cTrp-2 were not analyzed. All dogs responded with increased frequencies of mFAP-specific activated CD8(+) and CD4(+) T cells. The results of this safety/immunogenicity trial invite further testing of this checkpoint blockade vaccine combination in dogs with melanoma. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00262-018-2201-5) contains supplementary material, which is available to authorized users.

negative: Epitranscriptomic marks: Emerging modulators of RNA virus gene expression Epitranscriptomics, the study of posttranscriptional chemical moieties placed on RNA, has blossomed in recent years. This is due in part to the emergence of high‐throughput detection methods as well as the burst of discoveries showing biological function of select chemical marks. RNA modifications have been shown to affect RNA structure, localization, and functions such as alternative splicing, stabilizing transcripts, nuclear export, cap‐dependent and cap‐independent translation, microRNA biogenesis and binding, RNA degradation, and immune regulation. As such, the deposition of chemical marks on RNA has the unique capability to spatially and temporally regulate gene expression. The goal of this article is to present the exciting convergence of the epitranscriptomic and virology fields, specifically the deposition and biological impact of N7‐methylguanosine, ribose 2′‐O‐methylation, pseudouridine, inosine, N6‐methyladenosine, and 5‐methylcytosine epitranscriptomic marks on gene expression of RNA viruses. This article is categorized under: RNA in Disease and Development > RNA in Disease. RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications.

negative: Smoking and the risk of COVID-19 in a large observational population study BACKGROUND Smokers are generally more susceptible to infectious respiratory diseases and are at higher risk of developing severe complications from these infections. Conflicting reports exist regarding the impact of smoking on the risk of Coronavirus disease 2019 (COVID-19) infection. METHODS We carried out a population-based study among over 3,000,000 adult members of Clalit Health Services, the largest health provider in Israel. Since the beginning of the disease outbreak, 114,545 individuals underwent RT-PCR testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and 4.0% had positive results. We performed a case-control study among patients who underwent SARS-CoV-2 testing, to assess the impact of smoking on infection incidence and severity. Individuals with positive tests were matched in a 1:5 ratio to individuals tested negative, of the same sex, age, and ethnicity/religion. Conditional logistic regressions were performed to evaluate odds ratios for current and previous smoking on the risk of testing positive. Multivariable logistic regressions were performed among patients infected with COVID-19 to estimate the association between smoking and fatal or severe disease requiring ventilation. Regressions were performed with and without adjustment for preexisting medical conditions. RESULTS In the matched cohort, current smokers (9.8%) were significantly less prevalent among members tested positive compared to the general population, and to matched members tested negative (19.4%, P<0.001). Current smoking was associated with significantly reduced odds ratio (OR) for testing positive OR=0.457 (95% confidence interval (CI) 0.407-0.514). Among patients tested positive, there was no evidence of significantly increased risk of developing severe or fatal disease. CONCLUSION The risk of infection by COVID-19 appears to be reduced by half among current smokers. This intriguing finding may reveal unique infection mechanisms present for COVID-19 which may be targeted to combat the disease and reduce its infection rate.

negative: In Vivo Delivery of Nucleic Acid-Encoded Monoclonal Antibodies Antibody immunotherapy is revolutionizing modern medicine. The field has advanced dramatically over the past 40 years, driven in part by major advances in isolation and manufacturing technologies that have brought these important biologics to the forefront of modern medicine. However, the global uptake of monoclonal antibody (mAb) biologics is impeded by biophysical and biochemical liabilities, production limitations, the need for cold-chain storage and transport, as well as high costs of manufacturing and distribution. Some of these hurdles may be overcome through transient in vivo gene delivery platforms, such as non-viral synthetic plasmid DNA and messenger RNA vectors that are engineered to encode optimized mAb genes. These approaches turn the body into a biological factory for antibody production, eliminating many of the steps involved in bioprocesses and providing several other significant advantages, and differ from traditional gene therapy (permanent delivery) approaches. In this review, we focus on nucleic acid delivery of antibody employing synthetic plasmid DNA vector platforms, and RNA delivery, these being important approaches that are advancing simple, rapid, in vivo expression and having an impact in animal models of infectious diseases and cancer, among others.

negative: COVID-19 coronavirus vaccine design using reverse vaccinology and machine learning To ultimately combat the emerging COVID-19 pandemic, it is desired to develop an effective and safe vaccine against this highly contagious disease caused by the SARS-CoV-2 coronavirus. Our literature and clinical trial survey showed that the whole virus, as well as the spike (S) protein, nucleocapsid (N) protein, and membrane protein, have been tested for vaccine development against SARS and MERS. We further used the Vaxign reverse vaccinology tool and the newly developed Vaxign-ML machine learning tool to predict COVID-19 vaccine candidates. The N protein was found to be conserved in the more pathogenic strains (SARS/MERS/COVID-19), but not in the other human coronaviruses that mostly cause mild symptoms. By investigating the entire proteome of SARS-CoV-2, six proteins, including the S protein and five non-structural proteins (nsp3, 3CL-pro, and nsp8–10) were predicted to be adhesins, which are crucial to the viral adhering and host invasion. The S, nsp3, and nsp8 proteins were also predicted by Vaxign-ML to induce high protective antigenicity. Besides the commonly used S protein, the nsp3 protein has not been tested in any coronavirus vaccine studies and was selected for further investigation. The nsp3 was found to be more conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV than among 15 coronaviruses infecting human and other animals. The protein was also predicted to contain promiscuous MHC-I and MHC-II T-cell epitopes, and linear B-cell epitopes localized in specific locations and functional domains of the protein. Our predicted vaccine targets provide new strategies for effective and safe COVID-19 vaccine development.

negative: LC16m8: An attenuated smallpox vaccine The frequency of moderate to severe adverse reactions associated with smallpox vaccines currently stockpiled in the US, and the continued threat of bioterrorism have prompted the development of effective vaccines with improved safety profiles. LC16m8, an attenuated, replicating smallpox vaccine derived from the Lister strain of vaccinia, is currently licensed in Japan where it was safely used in over 50,000 children in the 1970s. It has been shown to have markedly less neurotoxicity than unattenuated vaccines in nonclinical studies. LC16m8 is immunogenic after a single dose, and recent studies in two different animal models have demonstrated protective efficacy equivalent to that of the only FDA-licensed smallpox vaccine. This article reviews the history and available scientific literature regarding LC16m8 and provides comparisons to other smallpox vaccines.

negative: Identification of conserved epitopes in SARS-CoV-2 spike and nucleocapsid protein Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first occurred in Wuhan (China) in December 2019, is a novel virus that causes a severe acute respiratory disease. The virus spike glycoproteins and nucleocapsid proteins are the main targets for the development of vaccines and antiviral drugs, to control the disease spread. We herein study the structural order-disorder propensity and the rates of evolution of these two proteins to characterize their B cell and T cell epitopes, previously suggested to contribute to immune response caused by SARS-CoV-2 infections. We first analyzed the rates of evolution along the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes. For this purpose, we compared orthologs from seven human coronaviruses: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. We then focus on the local, structural order-disorder propensities of the protein regions where the SARS-CoV-2 epitopes are located. We show that the vast majority of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains and some of them are characterized by low rates of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well-conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by high rates of evolution, probably to overcome host immunity. In conclusion, our results provide evidence for conserved epitopes that may help to develop long-lasting, broad-spectrum SARS-CoV-2 vaccines.

negative: The incidence of the novel coronavirus SARS-CoV-2 among asymptomatic patients: a systematic review BACKGROUND: the recent outbreak of the coronavirus disease 2019 (COVID‐19) has quickly spread globally since its discovery in Wuhan, China, in December 2019. A comprehensive strategy, including surveillance, diagnostics, research, and clinical treatment is urgently needed to win the battle against COVID-19. Recently, numerous studies reported the incidence of SARS-CoV-2 in asymptomatic patients. Yet, the incidence and viral transmission from the asymptomatic cases are not apparent yet. AIM: this study aims to systematically review the published literature on SARS-CoV-2 in the asymptomatic patients to estimate the incidence of COVID-19 among asymptomatic cases, as well as describe its epidemiological and clinical significance. METHOD: the literature was searched through four scientific databases: PubMed, Web of Science, Scopus, and Science Direct. RESULTS: a total of 63 studies satisfied the inclusion criteria where the majority of the reported studies were from China. However, there was a lack of SARS-CoV-2 epidemiological studies from several countries worldwide, tracing the actual incidence of COVID-19, especially in asymptomatic patients. Studies with a large sample size (n>1000) estimated that percentage of people contracting SARS-CoV-2 and are likely to be asymptomatic ranges from 1.2-12.9%. However, the other studies with a smaller sample size reported a much higher incidence and indicated that up to 87.9% of COVID-19 infected individuals could be asymptomatic. Most of these studies indicated that asymptopatics are a potential source of infection to the community. CONCLUSION: this review highlighted the need for more robust and well-designed studies to better estimate COVID-19 incidence among asymptomatic patients worldwide. The early identification of the asymptomatic cases, as well as monitoring and tracing close contact, could help in mitigating the spread of COVID-19.

negative: COVID-19: Therapeutics and Their Toxicities SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is causing the COVID-19 pandemic. There is no current standard of care. Clinicians need to be mindful of the toxicity of a wide variety of possibly unfamiliar substances being tested or repurposed to treat COVID-19. The United States Food and Drug Administration (FDA) has provided emergency authorization for the use of chloroquine and hydroxychloroquine. These two medications may precipitate ventricular dysrhythmias, necessitating cardiac and electrolyte monitoring, and in severe cases, treatment with epinephrine and high-doses of diazepam. Recombinant protein therapeutics may cause serum sickness or immune complex deposition. Nucleic acid vaccines may introduce mutations into the human genome. ACE inhibitors and ibuprofen have been suggested to exacerbate the pathogenesis of COVID-19. Here, we review the use, mechanism of action, and toxicity of proposed COVID-19 therapeutics.

negative: A PCR-enhanced method for determining the 5' end sequence of mRNAs. A method was developed to amplify ligated cDNA copies of mRNA 5' ends before cloning and sequencing. The method facilitates the study of the 5' end sequence on many molecules of an individual mRNA species. It can also be used to study the 5' end sequence on other kinds of single-stranded RNA molecules.

negative: Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: Implication for developing SARS vaccines Abstract Development of an effective vaccine for severe acute respiratory syndrome (SARS) remains to be a priority to prevent possible re-emergence of SARS coronavirus (SARS-CoV). We previously demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies. This suggests that the RBD may serve as an ideal vaccine candidate. Recombinant adeno-associated virus (rAAV) has been proven to be an effective system for gene delivery and vaccine development. In this study, a novel vaccine against SARS-CoV was developed based on the rAAV delivery system. The gene encoding RBD was cloned into a pAAV-IRES-hrGFP plasmid. The immunogenicity induced by the resulting recombinant RBD-rAAV was evaluated in BALB/c mice. The results demonstrated that (1) a single dose of RBD-rAAV vaccination could induce sufficient neutralizing antibody against SARS-CoV infection; (2) two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and (3) the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. These results suggested that RBD-rAAV is a promising SARS candidate vaccine.

negative: New Twists in Detecting mRNA Modification Dynamics Modified nucleotides in mRNA are an essential addition to the standard genetic code of four nucleotides in animals, plants, and their viruses. The emerging field of epitranscriptomics examines nucleotide modifications in mRNA and their impact on gene expression. The low abundance of nucleotide modifications and technical limitations, however, have hampered systematic analysis of their occurrence and functions. Selective chemical and immunological identification of modified nucleotides has revealed global candidate topology maps for many modifications in mRNA, but further technical advances to increase confidence will be necessary. Single-molecule sequencing introduced by Oxford Nanopore now promises to overcome such limitations, and we summarize current progress with a particular focus on the bioinformatic challenges of this novel sequencing technology.

negative: Coronavirus SARS-CoV-2: Analysis of subgenomic mRNA transcription, 3CLpro and PL2pro protease cleavage sites and protein synthesis Coronaviruses have recently caused world-wide severe outbreaks: SARS (Severe Acute Respiratory Syndrome) in 2002 and MERS (Middle-East Respiratory Syndrome) in 2012. At the end of 2019, a new coronavirus outbreak appeared in Wuhan (China) seafood market as first focus of infection, becoming a pandemics in 2020, spreading mainly into Europe and Asia. Although the virus family is well-known, this specific virus presents considerable differences, as higher transmission rates, being a challenge for diagnostic methods, treatments and vaccines. Coronavirus.pro is a C++ application which simulates Coronavirus replication cycle. This software has identified virus type in short times and provided FASTA files of virus proteins, a list of mRNA sequences and secondary structures. Furthermore, the software has identified a list of structural, non-structural and accessory proteins in 2019-nCoV virus genome more similar to SARS than to MERS, as several fusion proteins characteristics of this virus type. These results are useful as a first step in order to develop diagnostic methods, new vaccines or antiviral drugs, which could avoid virus replication in any stage: fusion inhibitors, RdRp inhibitors and PL2pro/3CLpro protease inhibitors.

negative: Scrutinizing the SARS-CoV-2 protein information for the designing an effective vaccine encompassing both the T-cell and B-cell epitopes Novel SARS coronavirus (SARS-CoV-2) has caused a pandemic condition world-wide and has been declared as public health emergency of International concern by WHO in a very short span of time. The community transmission of this highly infectious virus has severely affected various parts of China, Italy, Spain and USA among others. The prophylactic solution against SARS-CoV-2 infection is challenging due to the high mutation rate of its RNA genome. Herein, we exploited a next generation vaccinology approach to construct a multi-epitope vaccine candidate against SARS-CoV-2 with high antigenicity, safety and efficacy to combat this deadly infectious agent. The whole proteome was scrutinized for the screening of highly conserved, antigenic, non-allergen and non-toxic epitopes having high population coverage that can elicit both humoral and cellular mediated immune response against COVID-19 infection. These epitopes along with four different adjuvants were utilized to construct a multi-epitope vaccine candidate that can generate strong immunological memory response having high efficacy in humans. Various physiochemical analyses revealed the formation of a stable vaccine product having a high propensity to form a protective solution against the detrimental SARS-CoV-2 strain with high efficacy. The vaccine candidate interacted with immunological receptor TLR3 with high affinity depicting the generation of innate immunity. Further, the codon optimization and in silico expression show the plausibility of the high expression and easy purification of the vaccine product. Thus, this present study provides an initial platform of the rapid generation of an efficacious protective vaccine for combating COVID-19.

negative: New insights about the regulation of Nidovirus subgenomic mRNA synthesis Abstract The members of the Order Nidovirales share a similar genome organization with two overlapping nonstructural polyproteins encoded in the 5′ two-thirds and the structural proteins encoded in the 3′ third. They also express their 3′ region proteins from a nested set of 3′ co-terminal subgenomic messenger RNAs (sg mRNAs). Some but not all of the Nidovirus sg mRNAs also have a common 5′ leader sequence that is acquired by a discontinuous RNA synthesis mechanism regulated by multiple 3′ body transcription regulating sequences (TRSs) and the 5′ leader TRS. Initial studies detected a single major body TRS for each 3′ sg mRNA with a few alternative functional TRSs reported. The recent application of advanced techniques, such as next generation sequencing and ribosomal profiling, in studies of arteriviruses and coronaviruses has revealed an expanded sg mRNA transcriptome and coding capacity.

negative: Epitope based vaccine prediction for SARS-COV-2 by deploying immuno-informatics approach Abstract A new virus termed SARS-COV-2 (causing COVID-19 disease) can exhibit a progressive, fatal impact on individuals. The World Health Organization (WHO) has declared the spread of the virus to be a global pandemic. Currently, there are over 1 million cases and over 100,000 confirmed deaths due to the virus. Hence, prophylactic and therapeutic strategies are promptly needed. In this study we report an epitope, ITLCFTLKR, which is biochemically fit to HLA allelic proteins. We propose that this could be used as a potential vaccine candidate against SARS-COV-2. A selected putative epitope and HLA-allelic complexes show not only better binding scores, but also RMSD values in the range of 0–1 Å. This epitope was found to have a 99.8% structural favorability as per Ramachandran-plot analysis. Similarly, a suitable range of IC50 values and population coverage was obtained to represent greater validation of T-cell epitope analysis. Stability analysis using MDWeb and half-life analysis using the ProtParam tool has confirmed that this epitope is well-selected. This new methodology of epitope-based vaccine prediction is fundamental and fast in application, ad can be economically beneficial and viable.

negative: Use of personal protective equipment against coronavirus disease 2019 by healthcare professionals in Wuhan, China: cross sectional study OBJECTIVE: To examine the protective effects of appropriate personal protective equipment for frontline healthcare professionals who provided care for patients with coronavirus disease 2019 (covid-19). DESIGN: Cross sectional study. SETTING: Four hospitals in Wuhan, China. PARTICIPANTS: 420 healthcare professionals (116 doctors and 304 nurses) who were deployed to Wuhan by two affiliated hospitals of Sun Yat-sen University and Nanfang Hospital of Southern Medical University for 6-8 weeks from 24 January to 7 April 2020. These study participants were provided with appropriate personal protective equipment to deliver healthcare to patients admitted to hospital with covid-19 and were involved in aerosol generating procedures. 77 healthcare professionals with no exposure history to covid-19 and 80 patients who had recovered from covid-19 were recruited to verify the accuracy of antibody testing. MAIN OUTCOME MEASURES: Covid-19 related symptoms (fever, cough, and dyspnoea) and evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, defined as a positive test for virus specific nucleic acids in nasopharyngeal swabs, or a positive test for IgM or IgG antibodies in the serum samples. RESULTS: The average age of study participants was 35.8 years and 68.1% (286/420) were women. These study participants worked 4-6 hour shifts for an average of 5.4 days a week; they worked an average of 16.2 hours each week in intensive care units. All 420 study participants had direct contact with patients with covid-19 and performed at least one aerosol generating procedure. During the deployment period in Wuhan, none of the study participants reported covid-19 related symptoms. When the participants returned home, they all tested negative for SARS-CoV-2 specific nucleic acids and IgM or IgG antibodies (95% confidence interval 0.0 to 0.7%). CONCLUSION: Before a safe and effective vaccine becomes available, healthcare professionals remain susceptible to covid-19. Despite being at high risk of exposure, study participants were appropriately protected and did not contract infection or develop protective immunity against SARS-CoV-2. Healthcare systems must give priority to the procurement and distribution of personal protective equipment, and provide adequate training to healthcare professionals in its use.

negative: COVID-19 Diagnostics, Tools, and Prevention The Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), outbreak from Wuhan City, Hubei province, China in 2019 has become an ongoing global health emergency. The emerging virus, SARS-CoV-2, causes coughing, fever, muscle ache, and shortness of breath or dyspnea in symptomatic patients. The pathogenic particles that are generated by coughing and sneezing remain suspended in the air or attach to a surface to facilitate transmission in an aerosol form. This review focuses on the recent trends in pandemic biology, diagnostics methods, prevention tools, and policies for COVID-19 management. To meet the growing demand for medical supplies during the COVID-19 era, a variety of personal protective equipment (PPE) and ventilators have been developed using do-it-yourself (DIY) manufacturing. COVID-19 diagnosis and the prediction of virus transmission are analyzed by machine learning algorithms, simulations, and digital monitoring. Until the discovery of a clinically approved vaccine for COVID-19, pandemics remain a public concern. Therefore, technological developments, biomedical research, and policy development are needed to decipher the coronavirus mechanism and epidemiological characteristics, prevent transmission, and develop therapeutic drugs.

negative: Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed. Previous studies suggested that coronavirus RNA transcription involves interaction between leader RNA and the intergenic (IG) sequences, probably via protein-RNA interactions (X. M. Zhang, C.-L. Liao, and M. M. C. Lai, J. Virol., 68:4738-4746, 1994; X. M. Zhang and M. M. C. Lai, J. Virol., 68:6626-6633, 1994). To determine whether cellular proteins are involved in this process, we performed UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG (promoter) sequence between genes 6 and 7 (IG7) and the 5' untranslational region of mouse hepatitis virus genomic RNA. We demonstrated that three different cellular proteins (p70, p48, and p35/38) bound to the promoter sequence of the template RNA. Deletion analyses of the template RNA mapped the binding site of p35/38 at the consensus transcription initiation signal. In contrast, the binding of p70 and p48 was less specific. p35/38 is the same protein as the one previously identified to bind to the complementary strand of the leader RNA; its binding affinity to the leader was approximately 15 times stronger than that to IG7. Site-directed mutagenesis of the IG sequence revealed that mutations in the consensus sequence of IG7 (UCUAAUCUAAAC to UCGAAAC and GCUAAAG), which resulted in reduced subgenomic mRNA transcription, also caused correspondingly reduced levels of p35/38 binding. These results demonstrated that the extent of protein binding to the IG sequences correlated with the amounts of subgenomic mRNAs transcribed from the IG site. These studies suggest that these RNA-binding proteins are involved in coronavirus RNA transcription and may represent transcription factors.

negative: SARS-CoV-2 pandemic and research gaps: Understanding SARS-CoV-2 interaction with the ACE2 receptor and implications for therapy The COVID-19 pandemic is an emerging threat to global public health. While our current understanding of COVID-19 pathogenesis is limited, a better understanding will help us develop efficacious treatment and prevention strategies for COVID-19. One potential therapeutic target is angiotensin converting enzyme 2 (ACE2). ACE2 primarily catalyzes the conversion of angiotensin I (Ang I) to a nonapeptide angiotensin or the conversion of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and has direct effects on cardiac function and multiple organs via counter-regulation of the renin-angiotensin system (RAS). Significant to COVID-19, ACE2 is postulated to serve as a major entry receptor for SARS-CoV-2 in human cells, as it does for SARS-CoV. Many infected individuals develop COVID-19 with fever, cough, and shortness of breath that can progress to pneumonia. Disease progression promotes the activation of immune cells, platelets, and coagulation pathways that can lead to multiple organ failure and death. ACE2 is expressed by epithelial cells of the lungs at high level, a major target of the disease, as seen in post-mortem lung tissue of patients who died with COVID-19, which reveals diffuse alveolar damage with cellular fibromyxoid exudates bilaterally. Comparatively, ACE2 is expressed at low level by vascular endothelial cells of the heart and kidney but may also be targeted by the virus in severe COVID-19 cases. Interestingly, SARS-CoV-2 infection downregulates ACE2 expression, which may also play a critical pathogenic role in COVID-19. Importantly, targeting ACE2/Ang 1-7 axis and blocking ACE2 interaction with the S protein of SARS-CoV-2 to curtail SARS-CoV-2 infection are becoming very attractive therapeutics potential for treatment and prevention of COVID-19. Here, we will discuss the following subtopics: 1) ACE2 as a receptor of SARS-CoV-2; 2) clinical and pathological features of COVID-19; 3) role of ACE2 in the infection and pathogenesis of SARS; 4) potential pathogenic role of ACE2 in COVID-19; 5) animal models for pathological studies and therapeutics; and 6) therapeutics development for COVID-19.

negative: Rapid development of an inactivated vaccine for SARS-CoV-2 The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3μg or 6 μg per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans. One Sentence Summary A purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology

negative: Development of epitope‐based peptide vaccine against novel coronavirus 2019 (SARS‐COV‐2): Immunoinformatics approach Recently, a novel coronavirus (SARS‐COV‐2) emerged which is responsible for the recent outbreak in Wuhan, China. Genetically, it is closely related to SARS‐CoV and MERS‐CoV. The situation is getting worse and worse, therefore, there is an urgent need for designing a suitable peptide vaccine component against the SARS‐COV‐2. Here, we characterized spike glycoprotein to obtain immunogenic epitopes. Next, we chose 13 Major Histocompatibility Complex‐(MHC) I and 3 MHC‐II epitopes, having antigenic properties. These epitopes are usually linked to specific linkers to build vaccine components and molecularly dock on toll‐like receptor‐5 to get binding affinity. Therefore, to provide a fast immunogenic profile of these epitopes, we performed immunoinformatics analysis so that the rapid development of the vaccine might bring this disastrous situation to the end earlier.

negative: A rapidly adaptable biomaterial vaccine for SARS-CoV-2 The global COVID-19 pandemic motivates accelerated research to develop safe and efficacious vaccines. To address this need, we leveraged a biomaterial vaccine technology that consists of mesoporous silica rods (MSRs) that provide a sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and adjuvants to concentrate and mature antigen-presenting cells at the vaccine site. Here we explored the humoral responses resulting from the use of monophosphoryl lipid A (MPLA) as the adjuvant and SARS-CoV-2 spike proteins S1, S2, the nucleocapsid (N) protein, and receptor binding domain (RBD) as the target antigens. The dose of antigen and impact of pre-manufacturing of vaccines as versus loading antigen just-in-time was explored in these studies. Single shot MSR vaccines induced rapid and robust antibody titers to the presented antigens, even without the use of a boost, and sera from vaccinated animals demonstrated neutralizing activity against a SARS-CoV-2 pseudovirus. Overall, these results suggest the MSR vaccine system may provide potent protective immunity when utilized to present SARS-CoV-2 antigens.

negative: Progress and Challenges in the Development of COVID-19 Vaccines and Current Understanding of SARS-CoV-2- Specific Immune Responses. The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally, and the WHO has declared this outbreak a pandemic. Vaccines are an effective way to prevent the rapid spread of COVID-19. Furthermore, the immune response against SARS-CoV-2 infection needs to be understood for the development of an efficient and safe vaccine. Here, we review the current understanding of vaccine targets and the status of vaccine development for COVID-19. We also describe host immune responses to highly pathogenic human coronaviruses in terms of innate and adaptive immunities.

negative: The outbreak of SARS-CoV-2 pneumonia calls for viral vaccines The outbreak of 2019-novel coronavirus disease (COVID-19) that is caused by SARS-CoV-2 has spread rapidly in China, and has developed to be a Public Health Emergency of International Concern However, no specific antiviral treatments or vaccines are available yet This work aims to share strategies and candidate antigens to develop safe and effective vaccines against SARS-CoV-2 An outbreak of 2019-novel coronavirus (SARS-CoV-2) that causes atypical pneumonia (COVID-19) has raged in China since mid-December 2019 and has spread to 26 countries (February 20, 2020) The epidemic was identified by the first four cases confirmed on December 29, 2019 and was traced to the Huanan Seafood Wholesale Market, Wuhan city, Hubei Province, China1 A total of 75,465 cases with SARS-CoV-2 infections have been confirmed up to date (February 20, 2020), and 2,236 people have died in China2 COVID-19 spreads rapidly by human-to-human transmission with a median incubation period of 3 0 days (range, 0 to 24 0), and the time from symptom onset to developing pneumonia is 4 0 days (range, 2 0 to 7 0)3 Respiratory droplets and direct contact are conventional transmission routes for SARS-CoV-2, and fecal-to-oral transmission might also have a role3 Fever, dry cough, and fatigue are common symptoms at onset of COVID-194 Most patients have lymphopenia and bilateral ground-glass opacity changes on chest CT scans4,5 No specific antiviral treatments or vaccines are available because it is a new emerging viral disease Development of SARS-CoV-2-based vaccines is urgently required The entire virus particle-based preparation of vaccines, including inactivated and attenuated virus vaccines is advisable, because it is based on previous studies about the prevention and control of seasonal influenza vaccines6 The first SARS-CoV-2 (Wuhan-Hu-1) was successfully sequenced and its genomic sequence submitted to GenBank on January 5, 2020 (Accession no MN908947 3)7 Subsequently large-scale culture of SARS-CoV-2 was quickly performed, and an inactivated virus vaccine could be prepared through the employment of established physical and chemical methods such as UV light, formaldehyde, and β-propiolactone8 The development of attenuated-virus vaccines is also possible by carefully screening the serially propagated SARS-CoV-2 with reduced pathogenesis such as induced minimal lung injury, diminished limited neutrophil influx, and increased anti-inflammatory cytokine expressions compared with the wild-type virus9 Both inactivated and attenuated virus vaccines have their own disadvantages and side effects (Table 1) Alternatively, new vaccine designs based on the putative protective antigen/peptides derived from SARS-CoV-2 should be considered

negative: Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

negative: [ACE-inhibitors, angiotensin receptor blockers and severe acute respiratory syndrome caused by coronavirus]. Some Authors recently suggested that angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) should be discontinued, even temporarily, given the current pandemic of SARS-CoV-2 virus. The suggestion is based on the hypothesis that ACE-inhibitors and ARBs may favor the entry and diffusion of SARS-CoV-2 virus into the human cells. ACE-inhibitors and ARBs may increase the expression of ACE2 receptors, which are the sites of viral entry into the human organism. ACE2 receptors are ubiquitous, although they are extremely abundant on the cell surface of type 2 pneumocytes. Type 2 pneumocytes are small cylindrical alveolar cells located in close vicinity to pulmonary capillaries and responsible for the synthesis of alveolar surfactant, which is known to facilitate gas exchanges. The increased expression of ACE2 for effect of ACE-inhibitors and ARBs can be detected by increased production of angiotensin1-7 and mRNA related to ACE2. There is the fear that the increased expression of ACE2 induced by ACE-inhibitors and ARBs may ultimately facilitate the entry and diffusion of the SARS-CoV-2 virus. However, there is no clinical evidence to support this hypothesis. Furthermore, available data are conflicting and some counter-intuitive findings suggest that ARBs may be beneficial, not harmful. Indeed, studies conducted in different laboratories demonstrated that ACE2 receptors show a down-regulation (i.e. the opposite of what would happen with ACE-inhibitors and ARBs) for effect of their interaction with the virus. In animal studies, down-regulation of ACE2 has been found as prevalent in the pulmonary areas infected by virus, but not in the surrounding areas. In these studies, virus-induced ACE2 down-regulation would lead to a reduced formation of angiotensin1-7 (because ACE2 degrades angiotensin II into angiotensin1-7) with consequent accumulation of angiotensin II. The excess angiotensin II would favor pulmonary edema and inflammation, a phenomenon directly associated with angiotensin II levels, along with worsening in pulmonary function. Such detrimental effects have been blocked by ARBs in experimental models. In the light of the above considerations, it is reasonable to conclude that the suggestion to discontinue ACE-inhibitors or ARBs in all patients with the aim of preventing or limiting the diffusion of SARS-CoV-2 virus is not based on clinical evidence. Conversely, experimental studies suggest that ARBs might be useful in these patients to limit pulmonary damage through the inhibition of type 1 angiotensin II receptors. Controlled clinical studies in this area are eagerly awaited. This review discusses facts and theories on the potential impact of ACE-inhibitors and ARBs in the setting of the SARS-CoV-2 pandemic.

negative: Epidemiological impact of SARS-CoV-2 vaccination: mathematical modeling analyses Background: Several SARS-CoV-2 vaccine candidates are currently in the pipeline. This study aims to inform SARS-CoV-2 vaccine development, licensure, decision-making, and implementation by determining key preferred vaccine product characteristics and associated population-level impact. Methods: Vaccination impact was assessed at various efficacies using an age-structured mathematical model describing SARS-CoV-2 transmission and disease progression, with application for China. Results: A prophylactic vaccine with efficacy against acquisition (VEs) of ≥70% is needed to eliminate this infection. A vaccine with VEs <70% will still have a major impact, and may control the infection if it reduces infectiousness or infection duration among those vaccinated who acquire the infection, or alternatively if supplemented with a moderate social-distancing intervention (<20% reduction in contact rate), or complemented with herd immunity. Vaccination is cost-effective. For a vaccine with VEs of 50%, number of vaccinations needed to avert one infection is only 2.4, one severe disease case is 25.5, one critical disease case is 33.2, and one death is 65.1. Gains in effectiveness are achieved by initially prioritizing those ≥60 years. Probability of a major outbreak is virtually zero with a vaccine with VEs ≥70%, regardless of number of virus introductions. Yet, an increase in social contact rate among those vaccinated (behavior compensation) can undermine vaccine impact. Conclusions: Even a partially-efficacious vaccine can offer a fundamental solution to control SARS-CoV-2 infection and at high cost-effectiveness. In addition to the primary endpoint on infection acquisition, developers should assess natural history and disease progression outcomes and/or proxy biomarkers, since such secondary endpoints may prove critical in licensure, decision-making, and vaccine impact.

negative: Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription. Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus-specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis.

negative: Coronavirus mRNA transcription: UV light transcriptional mapping studies suggest an early requirement for a genomic-length template. Mouse hepatitis virus (MHV) synthesizes seven to eight mRNAs, each of which contains a leader RNA derived from the 5' end of the genome. To understand the mechanism of synthesis of these mRNAs, we studied how the synthesis of each mRNA was affected by UV irradiation at different time points after infection. When MHV-infected cells were UV irradiated at a late time in infection (5 h postinfection), the syntheses of the various mRNAs were inhibited to different extents in proportion to the sizes of the mRNAs. Analysis of the UV inactivation kinetics revealed that the UV target size of each mRNA was equivalent to its own physical size. In contrast, when cells were irradiated at 2.5 or 3 h postinfection, there appeared to be two different kinetics of inhibition of mRNA synthesis: the synthesis of every mRNA was inhibited to the same extent by a small UV dose, but the remaining mRNA synthesis was inhibited by additional UV doses at different rates for different mRNAs in proportion to RNA size. The analysis of the UV inactivation kinetics indicated that the UV target sizes for the majority of mRNAs were equivalent to that of the genomic-size RNA early in the infection. These results suggest that MHV mRNA synthesis requires the presence of a genomic-length RNA template at least early in the infection. In contrast, later in the infection, the sizes of the templates used for mRNA synthesis were equivalent to the physical sizes of each mRNA. The possibility that the genomic-length RNA required early in the infection was used only for the synthesis of a polymerase rather than as a template for mRNA synthesis was ruled out by examining the UV sensitivity of a defective interfering (DI) RNA. We found that the UV target size for the DI RNA early in infection was much smaller than that for mRNAs 6 and 7, which are approximately equal to or smaller in size than the DI RNA. This result indicates that even though DI RNA and viral mRNAs are synthesized by the same polymerase, mRNAs are synthesized from a larger (genomic-length) template. We conclude that a genomic-length RNA template is required for MHV subgenomic mRNA synthesis at least early in infection. Several transcription models are proposed.

negative: Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2) About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

negative: A human monoclonal antibody blocking SARS-CoV-2 infection The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV). This cross-neutralizing antibody targets a communal epitope on these viruses and offers potential for prevention and treatment of COVID-19.

negative: Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5′-to-3′ direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the K(m) of ATP for M-nsp13 is inversely proportional to the length of the 5′ loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5′ loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target.

negative: N-glycosylation network construction and analysis to modify glycans on the spike S glycoprotein of SARS-CoV-2 Background The spike S-protein of SARS-CoV-2 is N-glycosylated. The N-glycan structure and composition of this glycoprotein influence how the virus interacts with host cells. Objective To identify a putative N-glycan biosynthesis pathway of SARS-CoV-2 (HEK293 cell recombinant) from previously published mass spectrometric studies, and to identify what effect blocking some enzymes has on the overall glycoprotein profile. Finally, our goal was to provide the biosynthesis network, and glycans in easy-to-use format for further glycoinformatics work. Methods We reconstructed the glycosylation network based on previously published empirical data using GNAT, a glycosylation network analysis tool. Our compilation of the network tool had 23 glycosyltransferase and glucosidase enzymes, and could infer the pathway of glycosylation machinery based on glycans identified in the virus spike protein. Once the glycan biosynthesis pathway was generated, we simulated the effect of blocking specific enzymes - Mannosidase-II and alpha-1,6-fucosyltransferase to see how they would affect the biosynthesis network. Results Of the 23 enzymes, a total of 12 were involved in glycosylation of SARS-CoV-2 - Man-Ia, MGAT1, MGAT2, MGAT4, MGAT5, B4GalT, B4GalT, Man II, SiaT, ST3GalI, ST3GalVI and FucT8. Blocking enzymes resulted in a substantially modified glycan profile of the protein. Conclusions A network analysis of N-glycan biosynthesis of SARS-CoV-2 spike protein shows an elaborate enzymatic pathway with several intermediate glycans, along with the ones identified by mass spectrometric studies. Variations in the final N-glycan profile of the virus, given its site-specific microheterogeneity, could be a factor in the host response to the infection and response to antibodies. Here we provide all the resources generated - the glycans derived from mass spectrometry and intermediate glycans in glycoCT xml format, and the biosynthesis network for future drug and vaccine development work.

negative: Does BCG protect against SARS-CoV-2 infection ?: elements of proof. Background : There are several factors explaining the difference in the spread of SARS-CoV-2 infection including the BCG vaccination. This fact is supported by the concept of beneficial non specific effect of this live vaccine associated to its interaction with the immune system. Our study aims to identify the correlation between the universal BCG vaccination policy and the mortality attributed to COVID-19. Methods : We conducted an epidemiological study in which we collected COVID-19 pandemic data of April 11th, 2020 from the web site worldometers.info . The exclusion criteria for our study were a number of inhabitants less than one million, low-income countries according to the World Bank classification, a total number of infection cases less than 500 and countries that have performed less than one hundred tests per million inhabitants. Results : Countries that never had universal BCG vaccination policy have a higher mortality (correlated to performed diagnostic tests) attributed to SARS-CoV-2 infection (p<0.001). We found that the year of introduction of vaccination influenced significantly the mortality. Countries that started immunization policy before 1960 had more favorable results (p=0.049). For countries that started the BCG vaccination after 1960, countries with current policies have lower mortality attributed to SARS-CoV-2 infection than countries that have stopped immunization (p=0.047). Conclusions : Countries that have a BCG vaccination policy have a lower mortality attributed to SARS-CoV-2 infection. The populations of countries that applied this immunization before 1960 are more protected even if this universal policy has been interrupted.

negative: Coronavirus Pandemic—Therapy and Vaccines The current coronavirus COVID-19 pandemic, which originated in Wuhan, China, has raised significant social, psychological and economic concerns in addition to direct medical issues. The rapid spread of severe acute respiratory syndrome-coronavirus (SARS-CoV)-2 to almost every country on the globe and the failure to contain the infections have contributed to fear and panic worldwide. The lack of available and efficient antiviral drugs or vaccines has further worsened the situation. For these reasons, it cannot be overstated that an accelerated effort for the development of novel drugs and vaccines is needed. In this context, novel approaches in both gene therapy and vaccine development are essential. Previous experience from SARS- and MERS-coronavirus vaccine and drug development projects have targeted glycoprotein epitopes, monoclonal antibodies, angiotensin receptor blockers and gene silencing technologies, which may be useful for COVID-19 too. Moreover, existing antivirals used for other types of viral infections have been considered as urgent action is necessary. This review aims at providing a background of coronavirus genetics and biology, examples of therapeutic and vaccine strategies taken and potential innovative novel approaches in progress.

negative: MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA Abstract MERS-CoV is the only lethal human CoV still endemic in the Arabian Peninsula and neither vaccine nor therapeutics against MERS-CoV infection is available. The nsp1 of CoV is thought to be a major virulence factor because it suppresses protein synthesis through the degradation of host mRNA. In contrast, viral RNA circumvents the nsp1-mediated translational shutoff for an efficient propagation. In this study, we identified amino acid residue in MERS-CoV nsp1 that differ from those of SARS-CoV nsp1, and that appear to be crucial for circumventing the translational shutoff. In addition, reverse genetics analysis suggested the presence of a cis-acting element at the 5′-terminus of the nsp1-coding region, which contributes to the specific recognition of viral RNA that is required for an efficient viral replication. Our results suggest the CoVs share a common mechanism for circumventing the nsp1-mediated translational shutoff.

negative: Inactivated trivalent influenza vaccine is associated with lower mortality among Covid-19 patients in Brazil We analyzed data from 92,664 clinically and molecularly confirmed Covid-19 cases in Brazil to understand the potential associations between influenza vaccination and Covid-19 outcomes. Controlling for health facility of treatment, comorbidities as well as an extensive range of sociodemographic factors, we show that patients who received a recent influenza vaccine experienced on average 8% lower odds of needing intensive care treatment (95% CIs [0.86, 0.99]), 18% lower odds of requiring invasive respiratory support (0.74, 0.88) and 17% lower odds of death (0.75, 0.89). Large scale promotion of influenza vaccines seems advisable, especially in populations at high risk of severe SARS-CoV-2 infection.

negative: Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice Abstract A new type of coronavirus has been identified as the causative agent underlying Middle East Respiratory Syndrome (MERS). The MERS coronavirus (MERS-CoV) has spread in the Middle East, but cases originating in the Middle East have also occurred in the European Union and the USA. Eight hundred and thirty-seven cases of MERS-CoV infection have been confirmed to date, including 291 deaths. MERS-CoV has infected dromedary camel populations in the Middle East at high rates, representing an immediate source of human infection. The MERS-CoV spike (S) protein, a characteristic structural component of the viral envelope, is considered as a key target of vaccines against coronavirus infection. In an initial attempt to develop a MERS-CoV vaccine to ultimately target dromedary camels, we constructed two recombinant adenoviral vectors encoding the full-length MERS-CoV S protein (Ad5.MERS-S) and the S1 extracellular domain of S protein (Ad5.MERS-S1). BALB/c mice were immunized with both candidate vaccines intramuscularly and boosted three weeks later intranasally. All the vaccinated animals had antibody responses against spike protein, which neutralized MERS-CoV in vitro. These results show that an adenoviral-based vaccine can induce MERS-CoV-specific immune responses in mice and hold promise for the development of a preventive vaccine that targets the animal reservoir, which might be an effective measure to eliminate transmission of MERS-CoV to humans.

negative: SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in 'lasso' for the multi-faced virus During the current COVID-19 pandemic, the global ratio between the dead and the survivors is approximately 1 to 10, which has put humanity on high alert and provided strong motivation for the intensive search for vaccines and drugs. It is already clear that if we follow the most likely scenario, which is similar to that used to create seasonal influenza vaccines, then we will need to develop improved vaccine formulas every year to control the spread of the new, highly mutable coronavirus SARS-CoV-2. In this article, using well-known RNA viruses (HIV, influenza viruses, HCV) as examples, we consider the main successes and failures in creating primarily highly effective vaccines. The experience accumulated dealing with the biology of zoonotic RNA viruses suggests that the fight against COVID-19 will be difficult and lengthy. The most effective vaccines against SARS-CoV-2 will be those able to form highly effective memory cells for both humoral (memory B cells) and cellular (cross-reactive antiviral memory T cells) immunity. Unfortunately, RNA viruses constantly sweep their tracks and perhaps one of the most promising solutions in the fight against the COVID-19 pandemic is the creation of 'universal' vaccines based on conservative SARS-CoV-2 genome sequences (antigen-presenting) and unmethylated CpG dinucleotides (adjuvant) in the composition of the phosphorothioate backbone of single-stranded DNA oligonucleotides (ODN), which can be effective for long periods of use. Here, we propose a SARS-CoV-2 vaccine based on a lasso-like phosphorothioate oligonucleotide construction containing CpG motifs and the antigen-presenting unique ACG-containing genome sequence of SARS-CoV-2. We found that CpG dinucleotides are the least rare dinucleotides in the genomes of SARS-CoV-2 and other known human coronaviruses, and hypothesized that their higher frequency could be responsible for the unwanted increased lethality to the host, causing a 'cytokine storm' in people who overexpress cytokines through the activation of specific Toll-like receptors in a manner similar to TLR9-CpG ODN interactions. Interestingly, the virus strains sequenced in China (Wuhan) in February 2020 contained on average one CpG dinucleotide more in their genome than the later strains from the USA (New York) sequenced in May 2020. Obviously, during the first steps of the microevolution of SARS-CoV-2 in the human population, natural selection tends to select viral genomes containing fewer CpG motifs that do not trigger a strong innate immune response, so the infected person has moderate symptoms and spreads SARS-CoV-2 more readily. However, in our opinion, unmethylated CpG dinucleotides are also capable of preparing the host immune system for the coronavirus infection and should be present in SARS-CoV-2 vaccines as strong adjuvants.

negative: Translation initiation of viral mRNAs Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell‐type‐specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs. Copyright © 2010 John Wiley & Sons, Ltd.

negative: Development of an inactivated vaccine candidate, BBIBP-CorV, with potent protection against SARS-CoV-2 Summary The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 μg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.

negative: siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.

negative: Adenovirus-based vaccine prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates robust immune responses in macaques A ferret model of severe acute respiratory syndrome (SARS)-CoV infection was used to evaluate the efficacy of an adenovirus vaccine. Animals were subjected to heterologous prime-boost using vectors from human serotype 5 and chimpanzee derived adenoviruses (human AdHu5 and chimpanzee AdC7) expressing spike protein followed by intranasal challenge with SARS-CoV. Vaccination led to a substantial reduction in viral load and prevented the severe pneumonia seen in unvaccinated animals. The same prime-boost strategy was effective in rhesus macaques in eliciting SARS-CoV specific immune responses. These data indicate that a heterologous adenovirus-based prime-boost vaccine strategy could safely stimulate strong immunity that may be needed for complete protection against SARS-CoV infection.

negative: Scores of coronavirus vaccines are in competition - how will scientists choose the best?

negative: SARS-CoV-2 codon usage bias downregulates host expressed genes with similar codon usage Severe acute respiratory syndrome is quickly spreading throughout the world and was declared as a pandemic by the World Health Organisation (WHO). The pathogenic agent is a new coronavirus (SARS-CoV-2) that infects pulmonary cells with great effectiveness. In this study we focus on the codon composition for the viral proteins synthesis and its relationship with the proteins synthesis of the host. Our analysis reveals that SARS-CoV-2 preferred codons have poor representation of G or C nucleotides in the third position, a characteristic which could conduct to an unbalance in the tRNAs pools of the infected cells with serious implications in host protein synthesis. By integrating this observation with proteomic data from infected cells, we observe a reduced translation rate of host proteins associated with highly expressed genes, and that they share the codon usage bias of the virus. The functional analysis of these genes suggests that this mechanism of epistasis contributes to understand some deleterious collateral effect as result of the viral replication. In this manner, our finding contribute to the understanding of the SARS-CoV-2 pathogeny and could be useful for the design of a vaccine based on the live attenuated strategy.

negative: Immune responses in Balb/c mice induced by a candidate SARS-CoV inactivated vaccine prepared from F69 strain The immunogenicity of a candidate-inactivated vaccine prepared from SARS-CoV F69 strain was evaluated in Balb/c mice. Potent humoral immune responses were induced under the elicitation of three times of immunizations at 2-week intervals with this vaccine, combined with three types of adjuvants (Freund's adjuvant, Al(OH)(3) adjuvant and CpG adjuvant). Titers of specific IgG antibodies in three test groups all peaked in the sixth week after first vaccination, but significant differences existed in the kinetics of specific IgG antibody levels. The strong neutralizing capacity exhibited in micro-cytopathic effect neutralization tests indicated the specific antibodies are protective. Western blot assay further demonstrated the specificity of the induced serum antibodies.

negative: Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2(1–5). Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC(50)s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

negative: Applying lessons from the Ebola vaccine experience for SARS-CoV-2 and other epidemic pathogens The world is experiencing an unprecedented global pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus, Severe Acute Respiratory Syndrome-coronavirus-2 (SARS-CoV-2). Development of new vaccines and therapeutics are important to achieve long-term prevention and control of the virus. Experience gained in the development of vaccines for Ebola virus disease provide important lessons in the regulatory, clinical, and manufacturing process that can be applied to SARS-CoV-2 and other epidemic pathogens. This report outlines the main lessons learned by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD) during development of an Ebola Zaire vaccine (ERVEBO®) and looks ahead to critical lessons beyond vaccine development. It highlights focus areas for public-private partnership and regulatory harmonization that can be directly applied to current vaccine development efforts for SARS-CoV-2, while drawing attention to the need for parallel consideration of issues beyond development that are equally important to achieve global preparedness and response goals.

negative: Implications of the virus-encoded miRNA and host miRNA in the pathogenicity of SARS-CoV-2 The outbreak of COVID-19 caused by SARS-CoV-2 has rapidly spread worldwide and has caused over 1,400,000 infections and 80,000 deaths. There are currently no drugs or vaccines with proven efficacy for its prevention and little knowledge was known about the pathogenicity mechanism of SARS-CoV-2 infection. Previous studies showed both virus and host-derived MicroRNAs (miRNAs) played crucial roles in the pathology of virus infection. In this study, we use computational approaches to scan the SARS-CoV-2 genome for putative miRNAs and predict the virus miRNA targets on virus and human genome as well as the host miRNAs targets on virus genome. Furthermore, we explore miRNAs involved dysregulation caused by the virus infection. Our results implicated that the immune response and cytoskeleton organization are two of the most notable biological processes regulated by the infection-modulated miRNAs. Impressively, we found hsa-miR-4661-3p was predicted to target the S gene of SARS-CoV-2, and a virus-encoded miRNA MR147-3p could enhance the expression of TMPRSS2 with the function of strengthening SARS-CoV-2 infection in the gut. The study may provide important clues for the mechisms of pathogenesis of SARS-CoV-2.

negative: Immunoinformatic Analysis of T- and B-Cell Epitopes for SARS-CoV-2 Vaccine Design Currently, there is limited knowledge about the immunological profiles of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) We used computer-based immunoinformatic analysis and the newly resolved 3-dimensional (3D) structures of the SARS-CoV-2 S trimeric protein, together with analyses of the immunogenic profiles of SARS-CoV, to anticipate potential B-cell and T-cell epitopes of the SARS-CoV-2 S protein for vaccine design, particularly for peptide-driven vaccine design and serological diagnosis Nine conserved linear B-cell epitopes and multiple discontinuous B-cell epitopes composed of 69 residues on the surface of the SARS-CoV-2 trimeric S protein were predicted to be highly antigenic We found that the SARS-CoV-2 S protein has a different antigenic profile than that of the SARS-CoV S protein due to the variations in their primary and 3D structures Importantly, SARS-CoV-2 may exploit an immune evasion mechanism through two point mutations in the critical and conserved linear neutralization epitope (overlap with fusion peptide) around a sparsely glycosylated area These mutations lead to a significant decrease in the antigenicity of this epitope in the SARS-CoV-2 S protein In addition, 62 T-cell epitopes in the SARS-CoV-2 S protein were predicted in our study The structure-based immunoinformatic analysis for the SARS-CoV-2 S protein in this study may improve vaccine design, diagnosis, and immunotherapy against the pandemic of COVID-19

negative: New Vaccine Design and Delivery Technologies Technological advances in immunology, protein design, and genetic delivery have unlocked new possibilities for vaccine concepts and delivery technologies that were previously inaccessible. These next-generation vaccine design efforts are particularly promising in their potential to provide solutions to challenging targets for which conventional approaches have proven ineffective—for example, a universal influenza vaccine. In this perspective, we discuss emerging approaches to vaccine design and engineering based on recent insights into immunology, structural biology, computational biology, and immunoengineering. We anticipate that these cutting-edge, interdisciplinary approaches will lead to breakthrough vaccine concepts for ever-evolving and (re)emerging influenza viruses, with important ramifications for global public health.

negative: SARS-CoV-2 and human milk: what is the evidence? The novel coronavirus SARS-CoV-2 has emerged as one of the most compelling public health challenges of our time. To address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical, and public health communities have rapidly engaged to find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS-CoV-2 transmission. While respiratory droplets are a known mechanism of transmission, other mechanisms are possible. Of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. However, there is limited published literature related to vertical transmission of any human coronavirus (including SARS-CoV-2) via human milk and/or breastfeeding. There is a single study providing some evidence of vertical transmission of human coronavirus 229E, a single study evaluating presence of SARS-CoV in human milk (it was negative), and no published data on MERS-CoV and human milk. There are 9 case studies of human milk tested for SARS-CoV-2; none detected the virus. Importantly, none of the published studies on coronaviruses and human milk report validation of their analytical methods for use in human milk. These reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, SARS-CoV-2) during breastfeeding are discussed.

negative: Known SARS‐CoV‐2 infections: The tip of an important iceberg

negative: Potential adjuvants for the development of a SARS-CoV-2 vaccine based on experimental results from similar coronaviruses The extensive efforts around the globe are being made to develop a suitable vaccine against COVID-19 (Coronavirus Disease-19) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2). An effective vaccine should be able to induce high titers of neutralizing antibodies to prevent the virus from attaching to the host cell receptors. However, to elicit the protective levels of antibodies, a vaccine may require multiple doses or assistance from other immunostimulatory molecules. Further, the vaccine should be able to induce protective levels of antibodies rapidly with the least amount of antigen used. This not only decreases the cost of a vaccine and make it affordable, but also reduces the quantity of the antigen used. As the pandemic has hit most countries across the globe, there will be an overwhelming demand for the vaccine in a quick time. Incorporating a suitable adjuvant in a SARS-CoV-2 vaccine may address these requirements. This review paper will discuss the experimental results of the adjuvanted vaccine studies with similar coronaviruses (CoVs) which might be useful to select an appropriate adjuvant for a vaccine against rapidly emerging SARS-CoV-2. We also discuss the current progress in the development of adjuvanted vaccines against the disease.

negative: Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine. Some of the structural proteins of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) carry major epitopes involved in virus neutralization and are essential for the induction of protective humoral responses and the development of an effective vaccine. Rabbit antisera were prepared using full-length N and M proteins and eight expressed fragments covering the S protein. Antisera to S and M proteins were found to have different neutralizing titres towards SARS-CoV infection in vivo, ranging from 1:35 to 1:128. Antiserum to the N protein did not contain neutralizing antibodies. Epitopes inducing protective humoral responses to virus infection were located mainly in the M protein and a region spanning residues 13-877 of the S protein. The neutralizing ability of antisera directed against the expressed structural proteins was greater than that of convalescent patient antisera, confirming that, as immunogens, the former induce strong, SARS-CoV-specific neutralizing antibody responses. The in vitro neutralization assay has important implications for the design of an effective, protein-based vaccine preventing SARS-CoV infection.

negative: COVID-19 vaccine design: the Janus face of immune enhancement Previous work on severe acute respiratory syndrome coronavirus (SARS-CoV) vaccines identified cellular immunopathology and antibody-dependent enhancement as potential safety issues. We discuss the implications of these findings for COVID-19 vaccine development and our approach to optimizing for safety and efficacy.

negative: Excavating SARS-coronavirus 2 genome for epitope-based subunit vaccine synthesis using immunoinformatics approach Since the outbreak of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in December 2019 in China, there has been an upsurge in the number of deaths and infected individuals throughout the world, thereby leading to the World Health Organization declaration of a pandemic. Since no specific therapy is currently available for the same, the present study was aimed to explore the SARS-CoV-2 genome for the identification of immunogenic regions using immunoinformatics approach. A series of computational tools were applied in a systematic way to identify the epitopes that could be utilized in vaccine development. The screened-out epitopes were passed through several immune filters, such as promiscuousity, conservancy, antigenicity, nonallergenicity, population coverage, nonhomologous to human proteins, and affinity with human leukocyte antigen alleles, to screen out the best possible ones. Further, a construct comprising 11 CD4, 12 CD8, 3 B cell, and 3 interferon-Î³ epitopes, along with an adjuvant ß-defensin, was designed in silico, resulting in the formation of a multiepitope vaccine. The in silico immune simulation and population coverage analysis of the vaccine sequence showed its capacity to elicit cellular, humoral, and innate immune cells and to cover up a worldwide population of more than 97%. Further, the interaction analysis of the vaccine construct with Toll-like receptor 3 (immune receptor) was carried out by docking and dynamics simulations, revealing high affinity, constancy, and pliability between the two. The overall findings suggest that the vaccine may be highly effective, and is therefore required to be tested in the lab settings to evaluate its efficacy.

negative: The Potential of Various Nanotechnologies for Coronavirus Diagnosis/Treatment Highlighted through a Literature Analysis With the current COVID-19 outbreak, it has become essential to develop efficient methods for the treatment and detection of this virus. Among the new approaches that could be tested, that relying on nanotechnology finds one of its main grounds in the similarity between nanoparticle (NP) and coronavirus (COV) sizes, which promotes NP-COV interactions. Since COVID-19 is very recent, most studies in this field have focused on other types of coronavirus than COVID-19, such as those involved in MERS or SARS diseases. Although their number is limited, they have led to promising results on various COV using a wide range of different types of nanosystems, e.g., nanoparticles, quantum dos, or nanoassemblies of polymers/proteins. Additional efforts deserve to be spent in this field to consolidate these findings. Here, I first summarize the different nanotechnology-based methods used for COV detection, i.e., optical, electrical, or PCR ones, whose sensitivity was improved by the presence of nanoparticles. Furthermore, I present vaccination methods, which comprise nanoparticles used either as adjuvants or as active principles. They often yield a better-controlled immune response, possibly due to an improved antigen presentation/processing than in non-nanoformulated vaccines. Certain antiviral approaches also took advantage of nanoparticle uses, leading to specific mechanisms such as the blocking of virus replication at the cellular level or the reduction of a COV induced apoptotic cellular death.

negative: Single-cell RNA expression profiling of ACE2 and AXL in the human maternal-Fetal interface 2019 novel coronavirus disease has resulted in thousands of critically ill patients in China, which is a serious threat to people's life and health. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) was reported to share the same receptor, angiotensin-converting enzyme 2 (ACE2), with SARS-CoV. Here, based on the public single-cell RNA-sequencing database, we analyzed the mRNA expression profile of putative receptor ACE2 and AXL receptor tyrosine kinase (AXL) in the early maternal-fetal interface. The result indicates that the ACE2 has very low expression in the different cell types of early maternal-fetal interface, except slightly high in decidual perivascular cells cluster 1 (PV1). Interestingly, we found that the Zika virus (ZIKV) receptor AXL expression is concentrated in perivascular cells and stromal cells, indicating that there are relatively more AXL-expressing cells in the early maternal-fetal interface. This study provides a possible infection route and mechanism for the SARS-CoV-2- or ZIKV-infected mother-to-fetus transmission disease, which could be informative for future therapeutic strategy development. Zheng Qing-Liang 1 Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204 Duan Tao 2 Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204 Jin Li-Ping 3 Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204 Wang W, Tang J, Wei F. Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan, China. J Med Virol 2020. [Ahead of print]. doi: 10.1002/jmv.25689. Chen H, Guo J, Wang C, Luo F, Yu X, Zhang W, et al. Clinical characteristics and intrauterine vertical transmission potential of COVID-19 infection in nine pregnant women: A retrospective review of medical records. Lancet 2020. [Ahead of print]. doi: 10.1016/S0140-6736(20) 30360-3. Xu X, Chen P, Wang J, Feng J, Zhou H, Li X, et al. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci China Life Sci 2020. [Ahead of print]. doi: 10.1007/s11427-020-1637-5. Li W, Sui J, Huang IC, Kuhn JH, Radoshitzky SR, Marasco WA, et al. The S proteins of human coronavirus NL63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ACE2. Virology 2007;367:367-74. doi: 10.1016/j.virol.2007.04.035. Ferreira LM, Meissner TB, Tilburgs T, Strominger JL. HLA-G: At the interface of maternal-fetal tolerance. Trends Immunol 2017;38:272-86. doi: 10.1016/j.it.2017.01.009. Picelli S, Faridani OR, Björklund AK, Winberg G, Sagasser S, Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 2014;9:171-81. doi: 10.1038/nprot.2014.006. Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature 2018;563:347-53. doi: 10.1038/s41586-018-0698-6. Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. BioRxiv 2020. [Ahead of print]. doi: 10.1101/2020.01.26.919985. Lazear HM, Diamond MS. Zika virus: New clinical syndromes and its emergence in the western hemisphere. J Virol 2016;90:4864-75. doi: 10.1128/JVI.00252-16. Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika virus. N Engl J Med 2016;374:1552-63. doi: 10.1056/NEJMra1602113. Rasmussen SA, Jamieson DJ, Honein MA, Petersen LR. Zika virus and birth defects - Reviewing the evidence for causality. N Engl J Med 2016;374:1981-7. doi: 10.1056/NEJMsr1604338. Brasil P, Pereira JP Jr., Moreira ME, Ribeiro Nogueira RM, Damasceno L, Wakimoto M, et al. Zika virus infection in pregnant women in Rio de Janeiro. N Engl J Med 2016;375:2321-34. doi: 10.1056/NEJMoa1602412. Tabata T, Petitt M, Puerta-Guardo H, Michlmayr D, Wang C, Fang-Hoover J, et al. Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission. Cell Host Microbe 2016;20:155-66. doi: 10.1016/j.chom.2016.07.002. Quicke KM, Bowen JR, Johnson EL, McDonald CE, Ma H, O'Neal JT, et al. Zika virus Infects Human Placental Macrophages. Cell Host Microbe 2016;20:83-90. doi: 10.1016/j.chom.2016.05.015.

negative: An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. No differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. There were also no significant differences in virulence in mice following intranasal challenge. After immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. Both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. In addition, the synthetic approach turned out to be very accurate, since the rescued WNV genome contained no undesired mutations. Thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. This demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research.

negative: Coronavirus disease (COVID-19): a scoping review BACKGROUND: In December 2019, a pneumonia caused by a novel coronavirus (SARS-CoV-2) emerged in Wuhan, China and has rapidly spread around the world since then. AIM: This study aims to understand the research gaps related to COVID-19 and propose recommendations for future research. METHODS: We undertook a scoping review of COVID-19, comprehensively searching databases and other sources to identify literature on COVID-19 between 1 December 2019 and 6 February 2020. We analysed the sources, publication date, type and topic of the retrieved articles/studies. RESULTS: We included 249 articles in this scoping review. More than half (59.0%) were conducted in China. Guidance/guidelines and consensuses statements (n = 56; 22.5%) were the most common. Most (n = 192; 77.1%) articles were published in peer-reviewed journals, 35 (14.1%) on preprint servers and 22 (8.8%) posted online. Ten genetic studies (4.0%) focused on the origin of SARS-CoV-2 while the topics of molecular studies varied. Nine of 22 epidemiological studies focused on estimating the basic reproduction number of COVID-19 infection (R(0)). Of all identified guidance/guidelines (n = 35), only ten fulfilled the strict principles of evidence-based practice. The number of articles published per day increased rapidly until the end of January. CONCLUSION: The number of articles on COVID-19 steadily increased before 6 February 2020. However, they lack diversity and are almost non-existent in some study fields, such as clinical research. The findings suggest that evidence for the development of clinical practice guidelines and public health policies will be improved when more results from clinical research becomes available.

negative: Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2 Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.

negative: Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Although CoV mRNAs are of cytoplasmic origin, the presence of several NMD-inducing features (including multiple ORFs with internal termination codons that create a long 3′ untranslated region) in CoV mRNAs led us to explore the interplay between the NMD pathway and CoVs. Our study using murine hepatitis virus as a model CoV showed that CoV mRNAs are recognized by the NMD pathway as a substrate, resulting in their degradation. Furthermore, CoV replication induced the inhibition of the NMD pathway, and N protein (a viral structural protein) had an NMD inhibitory function that protected viral mRNAs from rapid decay. Our data further suggest that the NMD pathway interferes with optimal viral replication by degrading viral mRNAs early in infection, before sufficient accumulation of N protein. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus.

negative: In vivo structural characterization of the whole SARS-CoV-2 RNA genome identifies host cell target proteins vulnerable to re-purposed drugs SARS-CoV-2 is an RNA virus of the Coronaviridae family that is the causal pathogen of the ongoing Coronavirus Disease 2019 pandemic. There are currently no antiviral drugs or vaccines to treat COVID-19, and the failure to identify effective interventions can be blamed on our incomplete understanding of the nature of this virus and its host cell infection process. Here, we experimentally determined structural maps of the SARS-CoV-2 RNA genome in infected human cells and also characterized in vitro refolded RNA structures for SARS-CoV-2 and 6 other coronaviruses. Our in vivo data confirms several structural elements predicted from theoretical analysis and goes much further in revealing many previously unknown structural features that functionally impact viral translation and discontinuous transcription in cells. Importantly, we harnessed our in vivo structure data alongside a deep-learning tool and accurately predicted several dozen functionally related host cell proteins that bind to the SARS-CoV-2 RNA genome, none of which were known previously. Thus, our in vivo structural study lays a foundation for coronavirus RNA biology and indicates promising directions for the rapid development of therapeutics to treat COVID-19. HIGHLIGHTS We mapped the in vivo structure and built secondary structural models of the SARS-CoV-2 RNA genome We discovered functionally impactful structural features in the RNA genomes of multiple coronaviruses We predicted and validated host cell proteins that bind to the SARS-CoV-2 RNA genome based on our in vivo RNA structural data using a deep-learning tool

negative: Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79 μg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-γ and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice.

negative: Serodiagnostics for Severe Acute Respiratory Syndrome–Related Coronavirus-2: A Narrative Review Accurate serologic tests to detect host antibodies to severe acute respiratory syndrome–related coronavirus-2 (SARS-CoV-2) will be critical for the public health response to the coronavirus disease 2019 pandemic. Many use cases are envisaged, including complementing molecular methods for diagnosis of active disease and estimating immunity for individuals. At the population level, carefully designed seroepidemiologic studies will aid in the characterization of transmission dynamics and refinement of disease burden estimates and will provide insight into the kinetics of humoral immunity. Yet, despite an explosion in the number and availability of serologic assays to test for antibodies against SARS-CoV-2, most have undergone minimal external validation to date. This hinders assay selection and implementation, as well as interpretation of study results. In addition, critical knowledge gaps remain regarding serologic correlates of protection from infection or disease, and the degree to which these assays cross-react with antibodies against related coronaviruses. This article discusses key use cases for SARS-CoV-2 antibody detection tests and their application to serologic studies, reviews currently available assays, highlights key areas of ongoing research, and proposes potential strategies for test implementation.

negative: SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.

negative: Platelet Gene Expression and Function in COVID-19 Patients. There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis with the major difference being increased risk of thrombosis rather than bleeding. However, whether SARS-CoV-2 infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene expression changes in pathways associated with protein ubiquitination, antigen presentation and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, ACE2, was not detected by mRNA or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2/25 COVID-19 patients, suggesting platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared to healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity which may contribute to COVID-19 pathophysiology.

negative: Could individuals from countries using bcg vaccination be resistant to sars-cov-2 induced infections?/ Verem aşısı uygulayan ülkelerin bireyleri sars-cov-2 virüsünün yol açtığı enfeksiyona karşı daha dirençli olabilir mi? The lower than expected number of SARS-CoV-2 cases in countries with fragile health systems is puzzling. Herein, we hypothesize that BCG vaccination policies and vaccine strain preferences adopted by different countries might influence the SARS-CoV-2 transmission patterns and/or COVID-19 associated morbidity and mortality. We also postulate that until a specific vaccine is developed, SARS-CoV-2 vulnerable populations could be immunized with BCG vaccines to attain heterologous nonspecific protection from the new coronavirus. In the lights of our investigations the most resistant countries appear to be the ones using Group I BCG strain. Within these countries, however, those who employs Russian strain is even more protected against COVID-19 infection.

negative: SARS-CoV-2 transcriptome analysis and molecular cataloguing of immunodominant epitopes for multi-epitope based vaccine design SARS-CoV-2 is a single-stranded RNA virus that has caused more than 0.29 million deaths worldwide as of May 2020, and influence of COVID-19 pandemic is increasing continuously in the absence of approved vaccine and drug. Moreover, very limited information is available about SARS-CoV-2 expressed regions and immune responses. In this paper an effort has been made, to facilitate vaccine development by proposing multiple epitopes as potential vaccine candidates by utilising SARS-CoV-2 transcriptome data. Here, publicly available RNA-seq data of SARS-CoV-2 infection in NHBE and A549 human cell lines were used to construct SARS-CoV-2 transcriptome to understand disease pathogenesis and immune responses. In the first step, epitope prediction, MHC class I and II gene identification for epitopes, population coverage, antigenicity, immunogenicity, conservation and crossreactivity analysis with host antigens were performed by using SARS-CoV-2 transcriptome, and in the second step, structural compatibility of identified T-and B-cell epitopes were evaluated with MHC molecules and B-cell receptors through molecular docking studies. Quantification of MHC gene expression was also performed that indicated high variation in allele types and expression level of MHC genes with respect to cell lines. In A549 cell line, HLA-A*30:01:01:01 and HLA-B*44:03:01:01 were highly expressed, whereas 92 variants of HLA-A*24 genes such as HLA-A*24:02:01:01, HLA-A*24:286, HLA-A*24:479Q, HLA-A*24:02:134 and HLA-A*24:02:116 were highly expressed in NHBE cell lines. Prevalence of HLA-A*24 alleles was suggested as risk factors for H1N1 infection, and associated with type-1 diabetes. HLA-C*03:03, linked with male infertility factors was also highly expressed in SARS-CoV-2 infected NHBE cell lines. Finally, three potential T-cell and five B-cell epitopes were selected for molecular docking studies with twenty-two MHC molecules and two B-cell receptors respectively. The results of in silico analysis indicated that proposed epitopes have high potential to recognize immune response of SARS-CoV-2 infection. This study will facilitate in vitro and in vivo vaccine related research studies.

negative: Construction and immunogenicity of a recombinant fowlpox vaccine coexpressing S1 glycoprotein of infectious bronchitis virus and chicken IL-18 Infectious bronchitis virus (IBV) poses a major threat to the chicken industry worldwide. In this study, we developed a recombinant fowlpox virus (rFPV) vaccine expressing the IBV S1 gene and chicken interleukin-18 gene (IL-18), rFPV-S1/IL18. Recombinant plasmid pSY-S1/IL18 was constructed by cloning chicken IL-18 into fowlpox virus transfer plasmid containing S1 gene and transfected into the chicken embryo fibroblasts cell pre-infected with S-FPV-017 to generate rFPV-S1/IL18. Expression of the recombinant proteins was confirmed by RT-PCR and IFA. We also constructed the recombinant fowlpox virus rFPV-S1 without IL-18. One-day-old chickens were vaccinated by wing-web puncture with the two rFPVs, and the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels (P < 0.05) elicited by either rFPV-S1 or rFPV-S1/IL18. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-S1/IL18 were significantly higher (P < 0.05) than in those immunized with rFPV-S1. All chickens immunized with rFPV-S1/IL18 were completely protected (20/20) after challenge with the virulent IBV HN99 strain 43 days after immunization, while only 15 out of 20 of the chickens immunized with the rFPV-S1 were protected. Our results show that the protective efficacy of the rFPV-S1 vaccine could be enhanced significantly by simultaneous expression of IL-18.

negative: Coronavirus in Continuous Flux: From SARS‐CoV to SARS‐CoV‐2 The world is currently experiencing a global pandemic caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which causes severe respiratory disease similar to SARS. Previous studies have suggested that SARS‐CoV‐2 shares 79% and 96% sequence identity to SARS‐CoV and to bat coronavirus RaTG13, respectively at the whole‐genome level. Furthermore, a series of studies have shown that SARS‐CoV‐2 induces clusters of severe respiratory illnesses (i.e., pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS)) resembling SARS‐CoV. Moreover, the pathological syndrome may, in part, be caused by cytokine storms and dysregulated immune responses. Thus, in this work the recent literature surrounding the biology, clinical manifestations, and immunology of SARS‐CoV‐2 is summarized, with the aim of aiding prevention, diagnosis, and treatment for SARS‐CoV‐2 infection. This article is protected by copyright. All rights reserved

negative: Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic As the world is witnessing the epidemic of COVID-19, a disease caused by a novel coronavirus, SARS-CoV-2, emerging genetics and clinical evidences suggest a similar path to those of SARS and MERS. The rapid genomic sequencing and open access data, together with advanced vaccine technology, are expected to give us more knowledge on the pathogen itself, including the host immune response as well as the plan for therapeutic vaccines in the near future. This review aims to provide a comparative view among SARS-CoV, MERS-CoV and the newly epidemic SARS-CoV-2, in the hope to gain a better understanding of the host-pathogen interaction, host immune responses, and the pathogen immune evasion strategies. This predictive view may help in designing an immune intervention or preventive vaccine for COVID-19 in the near future.

negative: Making the Mark: The Role of Adenosine Modifications in the Lifecycle of RNA Viruses Viral epitranscriptomics is a newly emerging field that has identified unique roles for RNA modifications in modulating lifecycles of RNA viruses. Despite the observation of a handful of modified viral RNAs five decades ago, very little was known about how these modifications regulate viral lifecycles, until recently. Here we review the pro- and anti-viral effects of methyl-6-adenosine in distinct viral life cycles, the role of 2′ O-methyl modifications in RNA stability and innate immune sensing, and functions of adenosine to inosine modifications in retroviral life cycles. With roles for over 100 modifications in RNA still unknown, this is a rapidly emerging field that is destined to suggest novel antiviral therapies.

negative: Saliva: a diagnostic option and a transmission route for 2019-nCoV Data sources This review included 13 clinical studies (observational or clinical trial) which reported results of studies of the 2019 novel coronavirus (2019-nCoV). The other 62 referenced papers were of different types (eg, reviews, WHO protocols, letter to editor etc). Study selection The study selected trials, reviews, and in-vitro research assessing the critical aspects of saliva as an easily accessible and early-stage diagnostic source, and also an entry route for 2019-nCoV. Most of the clinical studies were descriptive case series of patients who had contracted 2019-nCoV in China. These were mainly studies designed to compare saliva samples with throat swabs, with regard to the presence of 2019-nCoV RNA. Another aspect of the included studies was the susceptibility of oral tissues to 2019-nCoV due to the expression of angiotensin-converting enzyme II (ACE2) as a receptor for the 2019-nCoV. Some review studies and clinical infection control protocols were also included to discuss the transmission patterns of 2019-nCoV from the oral cavity. Studies were not restricted to English language and they were not all full-text papers. Data extraction and synthesis A narrative synthesis of the results was conducted using distinct headings and subheadings, defined by the authors based on relevancy to the consensus about the importance of saliva with reference to 2019-nCoV. Results There was an inherent heterogeneity among the included clinical studies concerning their designs, sampling techniques, and the results about the diagnostic value of saliva. The percentage of coronavirus disease of 2019 (COVID-19) patients with positive 2019-nCoV RNA varied from 12.9% to 91.67% among these studies. Regarding the possibility of direct virus invasion into the oral tissues, the results suggested that ACE2+ cells in salivary glands could possibly be the target cells of 2019-nCoV and theoretically could generate infectious saliva in a sustained way. Furin was suggested as another protein which makes the tongue more vulnerable to 2019-nCoV, especially in conditions inducing its upregulation (for example, squamous cell carcinoma). According to the comparisons between 2019-nCoV and SARS-CoV, saliva could be considered of diagnostic value via the early detection of viral RNA for both of the viruses. Whilst the viral peak was shown to be at onset of symptoms for 2019-nCoV, it can linger up to the tenth day after the appearance of symptoms for SARS-CoV. Finally, this paper warns about airborne transmission, particularly for close contacts. Conclusions Saliva can be proposed as an easily accessible diagnostic source although further clinical studies are required. Given the presence of viral RNA in saliva in the early stages of COVID-19, the recommendations to wear masks to prevent the rapid transmission of infectious droplets into the air, and keep a safe distance from other people are clearly based in evidence.

negative: Elevated expression of ACE2 in tumor-adjacent normal tissues of cancer patients The rapidly developing COVID-19 pandemic has raised a concern that cancer patients may have increased susceptibility to SARS-CoV-2 infection. This discussion has mostly focused on therapy-induced immune suppression. Here, we examined the expression patterns of ACE2, the receptor through which SARX-CoV2 enters human cells, and found that ACE2 mRNA levels are elevated in tumor-adjacent normal tissues of cancer patients, including in normal-adjacent lung tissues of lung cancer patients. These observations raise the possibility that the elevated COVID-19 risk of cancer patients may not be limited to those undergoing immune-suppressing treatment.

negative: The quest to find an effective vaccine for COVID-19. Professor Alan Glasper, from the University of Southampton, delves into history and discusses the international quest to develop an effective vaccine to tackle COVID-19.

negative: Construction of Epitope-Based Peptide Vaccine Against SARS-CoV-2: Immunoinformatics Study Recently, a novel coronavirus (SARS-CoV-2) appeared which is conscientious for the current outbreak in China and rapidly spread worldwide Unluckily, there is no approved vaccine found against SARS-CoV-2 Therefore, there is an urgent need for designing a suitable peptide vaccine constituent against the SARS-CoV-2 In this study, we characterized the spike glycoprotein of SARS-CoV-2 to obtain immunogenic epitopes In addition, we used 58 SARS-CoV-2 isolates were retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) and National Center for Biotechnology Information (NCBI), then aligned to obtain the conserved region of SARS-CoV-2 spike glycoprotein The interaction between the conserved region with ACE2 receptor, a SARS-CoV-2 receptor on the host cell, has been evaluated through molecular docking approach The B-cell epitope was identified using the immune epitope database (IEDB) web server Interestingly, we recommend Pep_4 ADHQPQTFVNTELH as a epitope-based peptide vaccine candidate to deal with the SARS-CoV-2 outbreak Pep_4 has a high level of immunogenicity and does not trigger autoimmune mechanisms Pep_4 is capable of forming BCR/Fab molecular complexes with the lowest binding energy for activation of transduction signal the direct B-cell immune response However, further study is suggested for confirmation (in vitro and in vivo)

negative: Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19 This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

negative: An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review The present state of diagnostic and therapeutic developmental race for vaccines against the SARS CoV-2 (nCOVID-19) focuses on prevention and control of this global pandemic which also represents a critical challenge to the global health community. Although development of novel vaccines can prevent the SARS CoV-2 infections, it is still impeded by several other factors and therefore novel approaches towards treatment and management of this disease is the urgent need. Passive immunotherapy plays a vital role as a possible alternative to meet this challenge and among various antibody sources, chicken egg yolk antibodies (IgY) can be used as an alternative to mammalian antibodies which have been previously studied against SARS CoV outbreak in China. In this review, we discuss the strategies for the use of chicken egg yolk (IgY) antibodies in the development of rapid diagnosis and immunotherapy against SARS CoV-2. Also, IgY antibodies have previously been used against various respiratory bacterial and viral infections in humans and animals. Compared to mammalian antibodies (IgG), chicken egg yolk antibodies (IgY) have greater binding affinity to specific antigens, ease of extraction and lower production costs, hence possessing remarkable pathogen-neutralizing activity of pathogens in respiratory and lungs. We provide an overall importance for the use of monoclonal chicken egg yolk antibodies (IgY) using phage display method describing their potential passive immunotherapeutic application for the treatment and prevention of SARS CoV-2 infection which is simple, fast and safe way of approach for treating patients effectively.

negative: Progress and Challenges in the Development of COVID-19 Vaccines and Current Understanding of SARS-CoV-2- Specific Immune Responses The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally, and the WHO has declared this outbreak a pandemic. Vaccines are an effective way to prevent the rapid spread of COVID-19. Furthermore, the immune response against SARS-CoV-2 infection needs to be understood for the development of an efficient and safe vaccine. Here, we review the current understanding of vaccine targets and the status of vaccine development for COVID-19. We also describe host immune responses to highly pathogenic human coronaviruses in terms of innate and adaptive immunities.

negative: Coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression The recent emergence of two highly pathogenic human coronaviruses (CoVs), severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, has ignited a strong interest in the identification of viral factors that determine the virulence and pathogenesis of CoVs. The nonstructural protein 1 (nsp1) of CoVs has attracted considerable attention in this regard as a potential virulence factor and a target for CoV vaccine development because of accumulating evidence that point to its role in the downregulation of host innate immune responses to CoV infection. Studies have revealed both functional conservation and mechanistic divergence among the nsp1 of different mammalian CoVs in perturbing host gene expression and antiviral responses. This review summarizes the current knowledge about the biological functions of CoV nsp1 that provides an insight into the novel strategies utilized by this viral protein to modulate host and viral gene expression during CoV infection.

negative: DNA vaccine protection against SARS-CoV-2 in rhesus macaques The global COVID-19 pandemic caused by the SARS-CoV-2 virus has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 Spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. Following vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

negative: Effects of Renin-Angiotensin Inhibition on ACE2 and TMPRSS2 Expression: Insights into COVID-19 Angiotensin-converting enzyme 2 (ACE2), a component of the renin-angiotensin system, is a receptor for SARS-CoV-2, the virus that causes COVID-19. To determine whether the renin-angiotensin inhibition regulates ACE2 expression, either enalapril (an angiotensin-converting enzyme inhibitor) or losartan (an AT1 receptor blocker) was infused subcutaneously to male C57BL/6J mice for two weeks. Neither enalapril nor losartan changed abundance of ACE2 mRNA in lung, ileum, kidney, and heart. Viral entry also depends on transmembrane protease serine 2 (TMPRSS2) to prime the S protein. TMPRSS2 mRNA was abundant in lungs and ileum, modest in kidney, but barely detectable in heart. TMPRSS2 mRNA abundance was not altered by either enalapril or losartan in any of the 4 tissues. Next, we determined whether depletion of angiotensinogen (AGT), the unique substrate of the renin-angiotensin system, changes ACE2 and TMPRSS2 mRNA abundance. AGT antisense oligonucleotides (ASO) were injected subcutaneously to male C57BL/6J mice for 3 weeks. Abundance of ACE2 mRNA was unchanged in any of the 4 tissues, but TMPRSS2 mRNA was significantly decreased in lungs. Our data support that the renin-angiotensin inhibition does not regulate ACE2 and hence are not likely to increase risk for COVID-19.

negative: Immunization with SARS-CoV S DNA vaccine generates memory CD4(+) and CD8(+) T cell immune responses An effective vaccine for severe acute respiratory syndrome (SARS) will probably require the generation and maintenance of both humoral and cellular immune responses. It has been reported that after natural infection in humans and immunization in animals with SARS-CoV vaccine, antibody is produced and persistent for a long period of time. In the present study, mice were immunized i.m. with SARS-CoV S DNA vaccine, and three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses when the cells were stimulated in vitro with a pool of peptides overlapping entire SARS spike protein. The results show that prime-immunization with SARS-CoV S DNA vaccine can induce both CD4(+) and CD8(+) T cell responses. Boosting with the same vaccine enhances CD4(+) and CD8(+) T cell responses in both lymphoid and nonlymphoid organs and were persistent over two months. The SARS-CoV S-specific CD4(+) and CD8(+) T cells were CD62L(−), a marker for memory cells, and −30 to 50% of the cells expressed IL-7Rα (CD127), a marker for the capacity of effector cells to develop into memory cells. In addition, immunization with the DNA vaccine elicited high levels of antibody production. Taken together, these data demonstrate that immunization with SARS-CoV S DNA vaccine can generate antigen-specific humoral and cellular immune responses that may contribute to long-term protection.

negative: Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases Since the outbreak of the novel coronavirus disease COVID-19, caused by the SARS-CoV-2 virus, this disease has spread rapidly around the globe. Considering the potential threat of a pandemic, scientists and physicians have been racing to understand this new virus and the pathophysiology of this disease to uncover possible treatment regimens and discover effective therapeutic agents and vaccines. To support the current research and development, CAS has produced a special report to provide an overview of published scientific information with an emphasis on patents in the CAS content collection. It highlights antiviral strategies involving small molecules and biologics targeting complex molecular interactions involved in coronavirus infection and replication. The drug-repurposing effort documented herein focuses primarily on agents known to be effective against other RNA viruses including SARS-CoV and MERS-CoV. The patent analysis of coronavirusrelated biologics includes therapeutic antibodies, cytokines, and nucleic acid-based therapies targeting virus gene expression as well as various types of vaccines. More than 500 patents disclose methodologies of these four biologics with the potential for treating and preventing coronavirus infections, which may be applicable to COVID-19. The information included in this report provides a strong intellectual groundwork for the ongoing development of therapeutic agents and vaccines.

negative: DPP4, the Middle East Respiratory Syndrome Coronavirus Receptor, is Upregulated in Lungs of Smokers and Chronic Obstructive Pulmonary Disease Patients BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) causes pneumonia with a relatively high case fatality rate in humans. Smokers and chronic obstructive pulmonary disease (COPD) patients have been reported to be more susceptible to MERS-CoV infection. Here, we determined the expression of MERS-CoV receptor, dipeptidyl peptidase IV (DPP4), in lung tissues of smokers without airflow limitation and COPD patients in comparison to nonsmoking individuals (never-smokers). METHODS: DPP4 expression was measured in lung tissue of lung resection specimens of never-smokers, smokers without airflow limitation, COPD GOLD stage II patients and in lung explants of end-stage COPD patients. Both control subjects and COPD patients were well phenotyped and age-matched. The mRNA expression was determined using qRT-PCR and protein expression was quantified using immunohistochemistry. RESULTS: In smokers and subjects with COPD, both DPP4 mRNA and protein expression were significantly higher compared to never-smokers. Additionally, we found that both DPP4 mRNA and protein expression were inversely correlated with lung function and diffusing capacity parameters. CONCLUSIONS: We provide evidence that DPP4 is upregulated in the lungs of smokers and COPD patients, which could partially explain why these individuals are more susceptible to MERS-CoV infection. These data also highlight a possible role of DPP4 in COPD pathogenesis.

negative: Prevalence, Severity and Mortality associated with COPD and Smoking in patients with COVID-19: A Rapid Systematic Review and Meta-Analysis Background: Coronavirus disease 2019 (COVID-19) is an evolving infectious disease that dramatically spread all over the world in the early part of 2020. No studies have yet summarised the potential severity and mortality risks caused by COVID-19 in patients with chronic obstructive pulmonary disease (COPD), and we update information in smokers. Methods: We systematically searched electronic databases from inception to March 24, 2020. Data were extracted by two independent authors in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Study quality was assessed using a modified version of the Newcastle Ottawa Scale. We synthesised a narrative from eligible studies and conducted a meta-analysis using a random-effects model to calculate pooled prevalence rates and 95% confidence intervals (95%CI). Results: In total, 123 abstracts were screened and 61 full-text manuscripts were reviewed. A total of 15 studies met the inclusion criteria, which included a total of 2473 confirmed COVID-19 patients. All studies were included in the meta-analysis. The crude case fatality rate of COVID-19 was 6.4%. The pooled prevalence rates of COPD patients and smokers in COVID-19 cases were 2% (95% CI, 1%-3%) and 9% (95% CI, 4%-14%) respectively. COPD patients were at a higher risk of more severe disease (risk of severity = 63%, (22/35) compared to patients without COPD 33.4% (409/1224) [calculated RR, 1.88 (95% CI, 1.4-2.4)]. This was associated with higher mortality (60%). Our results showed that 22% (31/139) of current smokers and 46% (13/28) of ex-smokers had severe complications. The calculated RR showed that current smokers were 1.45 times more likely [95% CI: 1.03-2.04] to have severe complications compared to former and never smokers. Current smokers also had a higher mortality rate of 38.5%. Conclusion: Although COPD prevalence in COVID-19 cases was low in current reports, COVID-19 infection was associated with substantial severity and mortality rates in COPD. Compared to former and never smokers, current smokers were at greater risk of severe complications and higher mortality rate. Effective preventive measures are required to reduce COVID-19 risk in COPD patients and current smokers.

negative: Repurpose Open Data to Discover Therapeutics for COVID-19 using Deep Learning There have been more than 850,000 confirmed cases and over 48,000 deaths from the human coronavirus disease 2019 (COVID-19) pandemic, caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), in the United States alone. However, there are currently no proven effective medications against COVID-19. Drug repurposing offers a promising way for the development of prevention and treatment strategies for COVID-19. This study reports an integrative, network-based deep learning methodology to identify repurposable drugs for COVID-19 (termed CoV-KGE). Specifically, we built a comprehensive knowledge graph that includes 15 million edges across 39 types of relationships connecting drugs, diseases, genes, pathways, and expressions, from a large scientific corpus of 24 million PubMed publications. Using Amazon AWS computing resources, we identified 41 repurposable drugs (including indomethacin, toremifene and niclosamide) whose therapeutic association with COVID-19 were validated by transcriptomic and proteomic data in SARS-CoV-2 infected human cells and data from ongoing clinical trials. While this study, by no means recommends specific drugs, it demonstrates a powerful deep learning methodology to prioritize existing drugs for further investigation, which holds the potential of accelerating therapeutic development for COVID-19.

negative: Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine Abstract Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200–1:6400, average titre is 1:4580) and high CTL activity (67.4±8.4% (E:T=25:1), 69.6±6.7% (E:T=50:1) and 71.8±6.2% (E:T=100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6±12.7% (E:T=25:1), 57.4±11.7% (E:T=50:1) and 63.0±6.3% (E:T=100:1)). However, antibody titres were much lower (1:200–1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.

negative: Engineering DNA vaccines against infectious diseases Abstract Engineering vaccine-based therapeutics for infectious diseases is highly challenging, as trial formulations are often found to be nonspecific, ineffective, thermally or hydrolytically unstable, and/or toxic. Vaccines have greatly improved the therapeutic landscape for treating infectious diseases and have significantly reduced the threat by therapeutic and preventative approaches. Furthermore, the advent of recombinant technologies has greatly facilitated growth within the vaccine realm by mitigating risks such as virulence reversion despite making the production processes more cumbersome. In addition, seroconversion can also be enhanced by recombinant technology through kinetic and nonkinetic approaches, which are discussed herein. Recombinant technologies have greatly improved both amino acid-based vaccines and DNA-based vaccines. A plateau of interest has been reached between 2001 and 2010 for the scientific community with regard to DNA vaccine endeavors. The decrease in interest may likely be attributed to difficulties in improving immunogenic properties associated with DNA vaccines, although there has been research demonstrating improvement and optimization to this end. Despite improvement, to the extent of our knowledge, there are currently no regulatory body-approved DNA vaccines for human use (four vaccines approved for animal use). This article discusses engineering DNA vaccines against infectious diseases while discussing advantages and disadvantages of each, with an emphasis on applications of these DNA vaccines. Statement of Significance This review paper summarizes the state of the engineered/recombinant DNA vaccine field, with a scope entailing “Engineering DNA vaccines against infectious diseases”. We endeavor to emphasize recent advances, recapitulating the current state of the field. In addition to discussing DNA therapeutics that have already been clinically translated, this review also examines current research developments, and the challenges thwarting further progression. Our review covers: recombinant DNA-based subunit vaccines; internalization and processing; enhancing immune protection via adjuvants; manufacturing and engineering DNA; the safety, stability and delivery of DNA vaccines or plasmids; controlling gene expression using plasmid engineering and gene circuits; overcoming immunogenic issues; and commercial successes. We hope that this review will inspire further research in DNA vaccine development.

negative: Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M(pro)). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC(50) values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC(50) values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic M(pro) inhibitors. A complex crystal structure of SARS-CoV-2 M(pro) with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M(pro). Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

negative: Modern mRNA Proofreading and Repair: Clues that the Last Universal Common Ancestor Possessed an RNA Genome? RNA repair has now been demonstrated to be a genuine biological process and appears to be present in all three domains of life. In this article, we consider what this might mean for the transition from an early RNA-dominated world to modern cells possessing genetically encoded proteins and DNA. There are significant gaps in our understanding of how the modern protein-DNA world could have evolved from a simpler system, and it is currently uncertain whether DNA genomes evolved once or twice. Against this backdrop, the discovery of RNA repair in modern cells is timely food for thought and brings us conceptually one step closer to understanding how RNA genomes were replaced by DNA genomes. We have examined the available literature on multisubunit RNA polymerase structure and function and conclude that a strong case can be made that the Last Universal Common Ancestor (LUCA) possessed a repair-competent RNA polymerase, which would have been capable of acting on an RNA genome. However, while this lends credibility to the proposal that the LUCA had an RNA genome, the alternative, that LUCA had a DNA genome, cannot be completely ruled out.

negative: mRNA Metabolism in Cardiac Development and Disease: Life After Transcription The central dogma of molecular biology illustrates the importance of mRNAs as critical mediators between genetic information encoded at the DNA level and proteomes/metabolomes that determine the diverse functional outcome at the cellular and organ levels. Although the total number of protein-producing (coding) genes in the mammalian genome is ~20,000, it is evident that the intricate processes of cardiac development and the highly regulated physiological regulation in the normal heart, as well as the complex manifestation of pathological remodeling in a diseased heart, would require a much higher degree of complexity at the transcriptome level and beyond. Indeed, in addition to an extensive regulatory scheme implemented at the level of transcription, the complexity of transcript processing following transcription is dramatically increased. RNA processing includes post-transcriptional modification, alternative splicing, editing and transportation, ribosomal loading, and degradation. While transcriptional control of cardiac genes has been a major focus of investigation in recent decades, a great deal of progress has recently been made in our understanding of how post-transcriptional regulation of mRNA contributes to transcriptome complexity. In this review, we highlight some of the key molecular processes and major players in RNA maturation and post-transcriptional regulation. In addition, we provide an update to the recent progress made in the discovery of RNA processing regulators implicated in cardiac development and disease. While post-transcriptional modulation is a complex and challenging problem to study, recent technological advancements are paving the way for a new era of exciting discoveries and potential clinical translation in the context of cardiac biology and heart disease.

negative: Hepatitis C virus vaccine design: focus on the humoral immune response Despite the recent development of safe and highly effective direct-acting antivirals, hepatitis C virus (HCV) infection remains a significant health problem. In 2016, the World Health Organization set out to reduce the rate of new HCV infections by 90% by 2030. Still, global control of the virus does not seem to be achievable in the absence of an effective vaccine. Current approaches to the development of a vaccine against HCV include the production of recombinant proteins, synthetic peptides, DNA vaccines, virus-like particles, and viral vectors expressing various antigens. In this review, we focus on the development of vaccines targeting the humoral immune response against HCV based on the cumulative evidence supporting the important role of neutralizing antibodies in protection against HCV infection. The main targets of HCV-specific neutralizing antibodies are the glycoproteins E1 and E2. Recent advances in the knowledge of HCV glycoprotein structure and their epitopes, as well as the possibility of getting detailed information on the human antibody repertoire generated by the infection, will allow rational structure-based antigen design to target specific germline antibodies. Although obtaining a vaccine capable of inducing sterilizing immunity will be a difficult task, a vaccine that prevents chronic hepatitis C infections, a more realistic goal in the short term, would have a considerable health impact.

negative: SARS virus: The beginning of the unraveling of a new coronavirus Severe acute respiratory syndrome (SARS) virus caused a severe outbreak in several regions of the world in 2003. The virus is a novel coronavirus, which may have an origin in wild animals such as civet cats in southern China. Its genome structure, gene expression pattern and protein profiles are similar to those of other coronaviruses. However, distinct patterns of several open reading frames in the SARS virus genome may contribute to its severe virulence. The potential mutability of the coronavirus genome may pose problems in the control of future SARS outbreaks. The mechanism of SARS pathogenesis may involve both direct viral cytocidal effects on the target cells and immune-mediated mechanisms. The life cycle of the SARS virus is largely unknown; however, based on the analogy with other coronaviruses, several potential targets for antiviral development are identified. Vaccines offer an important preventive measure for possible future recurrences of SARS, but the prospect for their development is still unknown because of the uncertainty regarding the role of immune responses in SARS virus pathogenesis. The comparative studies of other coronaviruses offer insights into the understanding of SARS virus.

negative: Influenza and SARS-Coronavirus Activating Proteases TMPRSS2 and HAT Are Expressed at Multiple Sites in Human Respiratory and Gastrointestinal Tracts The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.

negative: Clinical Use of DNA Vaccines Owing to their unique advantages in simplicity, safety, scalability, and possibility of repeated administrations, DNA vaccines represent an appealing and competitive immunization approach for a wide array of conditions, including but not limited to infectious diseases and cancer immunotherapy. Despite the exciting efficacy observed in preclinical studies, DNA vaccines have faced challenges in inducing strong immune responses in humans. This unexpected poor immunogenicity has severely hampered the translation of DNA vaccines from investigational medications to licensed products. To overcome this obstacle, tremendous efforts have been made to improve antigen expression and enhance immunogenicity. Among these endeavors, in vivo DNA electroporation (EP) has proved to be a breakthrough technology capable of mediating efficient DNA uptake and resulting in enhanced antigen expression and vaccine immunogenicity. EP-mediated DNA delivery has become one of the major platforms used in clinical trials to evaluate DNA vaccines in humans. In this chapter, in addition to EP delivery, other progress made in DNA vaccine development including plasmid optimization, antigen design, and immunologic adjuvants is also reviewed. Finally, the use of DNA vaccines in the context of clinical trials for infectious diseases and cancer immunotherapy is summarized. Specifically, the strategies that allow DNA vaccines to overcome antigenic diversity for viral infection and break immune tolerance for cancer therapy are explored. Based on the advantages of DNA vaccines and the immense progress, led by the electroporation-mediated vaccine delivery, DNA vaccines appear to have the potential to fundamentally transform the vaccine field, providing important benefits for preventing and curing diseases.

negative: The potential of drug repositioning as a short-term strategy for the control and treatment of COVID-19 (SARS-CoV-2): a systematic review The novel human coronavirus (SARS-CoV-2), the causative agent of COVID-19, has quickly become a threat to the public health and economy worldwide. Despite the severity of some cases, there are no current pathogen-specific antivirals available to treat the disease. Therefore, many studies have focused on the evaluation of the anti-SARS-CoV-2 activity of clinically available drugs. Here, we conducted a systematic review to describe the drug repositioning strategy against SARS-CoV-2 and to discuss the clinical impact of this approach in the current pandemic context. The systematic review was performed on March 23, 2020, using PubMed/MEDLINE, Scopus, Cochrane Library, and Biblioteca Virtual de Saúde (BVS). The data were summarized in tables and critically analyzed. After the database search, 12 relevant studies were identified as eligible for the review. Among the drugs reported in these studies, 57 showed some evidence of antiviral activity. Antivirals, especially antiretrovirals, are the main class of therapeutic agents evaluated against COVID-19. Moreover, studies have reported the anti-SARS-CoV-2 activity of antitumor (16%; 9/57), antimalarial (7%, 4/57), and antibacterial (5%; 3/57) agents. Additionally, seven pharmacological agents (chloroquine, tetrandrine, umifenovir (arbidol), carrimycin, damageprevir, lopinavir/ritonavir) are in phase IV of clinical trials. Due to the evidence of the anti-SARS-CoV-2 activity of various clinically available agents, drug repositioning stands out as a promising strategy for a short-term response in the fight against the novel coronavirus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00705-020-04693-5) contains supplementary material, which is available to authorized users.

negative: A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting. The molecular determinants of stimulation of -1 programmed ribosomal frameshifting (-1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes -1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3' nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2'-OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1-2'-OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2-S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV/BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of -1 PRF (15 +/- 2%) relative to the BWYV pseudoknot (6 +/- 1%), a difference traced largely to the identity of the 3' nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by approximately 1.5 kcal x mol(-1) (pH 7.0, 37 degrees C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs.

negative: Multi-epitope vaccine design using an immunoinformatics approach for 2019 novel coronavirus (SARS-CoV-2) A new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. An in silico approach based on the available virus genome was applied to identify 19 high immunogenic B-cell epitopes and 499 human-leukocyte-antigen (HLA) restricted T-cell epitopes. Thirty multi-epitope peptide vaccines were designed by iNeo Suite, and manufactured by solid-phase synthesis. Docking analysis showed stable hydrogen bonds of epitopes with their corresponding HLA alleles. When four vaccine peptide candidates from the spike protein of SARS-CoV-2 were selected to immunize mice, a significantly larger amount of IgG in serum as well as an increase of CD19+ cells in ILNs was observed in peptide-immunized mice compared to the control mice. The ratio of IFN-γ-secreting lymphocytes in CD4+ or CD8+ cells in the peptides-immunized mice were higher than that in the control mice. There were also a larger number of IFN-γ-secreting T cells in spleen in the peptides-immunized mice. This study screened antigenic B-cell and T-cell epitopes in all encoded proteins of SARS-CoV-2, and further designed multi-epitope based peptide vaccine against viral structural proteins. The obtained vaccine peptides successfully elicited specific humoral and cellular immune responses in mice. Primate experiments and clinical trial are urgently required to validate the efficacy and safety of these vaccine peptides. Importance So far, a new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. Different from the development approaches for traditional vaccines, the development of our peptide vaccine is faster and simpler. In this study, we performed an in silico approach to identify the antigenic B-cell epitopes and human-leukocyte-antigen (HLA) restricted T-cell epitopes, and designed a panel of multi-epitope peptide vaccines. The resulting SARS-CoV-2 multi-epitope peptide vaccine could elicit specific humoral and cellular immune responses in mice efficiently, displaying its great potential in our fight of COVID-19.

negative: SARS-CoV-2 / COVID-19 and its Transmission, Prevention, Treatment and Control - An Update Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2), pandemic has caused huge panic, havoc and global threats worldwide The origin of this virus has been linked to animals, intermediate host is still to be identified, and studies are being carried out that how it got transmitted to humans and acquired rapid human-to-human transmission Within a short time period of only 05 months, SARS-CoV-2 has spread to 213 countries, and till 28th May, 2020, nearly 5 8 million confirmed cases have been reported while taking lives of 0 36 million persons Seeing the current situation of rapid increase in COVID-19 cases daily in many countries, this seems to be the deadliest pandemic after the 1918 Spanish Flu There is currently no specific effective treatment for COVID-19 and also in absence of vaccine the radical cure of the disease is far away Researchers are pacing high to design and develop effective vaccines, drugs and therapeutics to counter COVID-19, however such efforts, clinical trials, necessary approvals and then to reach the level of bulk production of many millions of doses may still take much time Prevention and control of COVID-19 outbreaks requires an evidence-based, multi-factorial and effective mitigation strategy to be adopted The current review discusses on the research advancements, challenges and opportunities in COVID 19 management with a focus on its transmission, prevention, treatment and control

negative: Genome structure and transcriptional regulation of human coronavirus NL63 BACKGROUND: Two human coronaviruses are known since the 1960s: HCoV-229E and HCoV-OC43. SARS-CoV was discovered in the early spring of 2003, followed by the identification of HCoV-NL63, the fourth member of the coronaviridae family that infects humans. In this study, we describe the genome structure and the transcription strategy of HCoV-NL63 by experimental analysis of the viral subgenomic mRNAs. RESULTS: The genome of HCoV-NL63 has the following gene order: 1a-1b-S-ORF3-E-M-N. The GC content of the HCoV-NL63 genome is extremely low (34%) compared to other coronaviruses, and we therefore performed additional analysis of the nucleotide composition. Overall, the RNA genome is very low in C and high in U, and this is also reflected in the codon usage. Inspection of the nucleotide composition along the genome indicates that the C-count increases significantly in the last one-third of the genome at the expense of U and G. We document the production of subgenomic (sg) mRNAs coding for the S, ORF3, E, M and N proteins. We did not detect any additional sg mRNA. Furthermore, we sequenced the 5' end of all sg mRNAs, confirming the presence of an identical leader sequence in each sg mRNA. Northern blot analysis indicated that the expression level among the sg mRNAs differs significantly, with the sg mRNA encoding nucleocapsid (N) being the most abundant. CONCLUSIONS: The presented data give insight into the viral evolution and mutational patterns in coronaviral genome. Furthermore our data show that HCoV-NL63 employs the discontinuous replication strategy with generation of subgenomic mRNAs during the (-) strand synthesis. Because HCoV-NL63 has a low pathogenicity and is able to grow easily in cell culture, this virus can be a powerful tool to study SARS coronavirus pathogenesis.

negative: Landscape and Selection of Vaccine Epitopes in SARS-CoV-2 There is an urgent need for a vaccine with efficacy against SARS-CoV-2. We hypothesize that peptide vaccines containing epitope regions optimized for concurrent B cell, CD4(+) T cell, and CD8(+) T cell stimulation would drive both humoral and cellular immunity with high specificity, potentially avoiding undesired effects such as antibody-dependent enhancement (ADE). Additionally, such vaccines can be rapidly manufactured in a distributed manner. In this study, we combine computational prediction of T cell epitopes, recently published B cell epitope mapping studies, and epitope accessibility to select candidate peptide vaccines for SARS-CoV-2. We begin with an exploration of the space of possible T cell epitopes in SARS-CoV-2 with interrogation of predicted HLA-I and HLA-II ligands, overlap between predicted ligands, protein source, as well as concurrent human/murine coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, viral source protein abundance, sequence conservation, coverage of high frequency HLA alleles and co-localization of CD4(+) and CD8(+) T cell epitopes. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering to select regions with surface accessibility, high sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. From 58 initial candidates, three B cell epitope regions were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we propose a set of SARS-CoV-2 vaccine peptides for use in subsequent murine studies and clinical trials.

negative: Coronavirus genomes carry the signatures of their habitats Coronaviruses such as SARS-CoV-2 regularly infect host tissues that express antiviral proteins (AVPs) in abundance. Understanding how they evolve to adapt or evade host immune responses is important in the effort to control the spread of COVID-19. Two AVPs that may shape viral genomes are the zinc finger antiviral protein (ZAP) and the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 protein (APOBEC3). The former binds to CpG dinucleotides to facilitate the degradation of viral transcripts while the latter deaminates C into U residues leading to dysfunctional transcripts. We tested the hypothesis that both APOBEC3 and ZAP may act as primary selective pressures that shape the genome of an infecting coronavirus by considering a comprehensive number of publicly available genomes for seven coronaviruses (SARS-CoV-2, SARS-CoV, MERS, Bovine CoV, Murine MHV, Porcine HEV, and Canine CoV). We show that coronaviruses that regularly infect tissues with abundant AVPs have CpG-deficient and U-rich genomes; whereas viruses that do not infect tissues with abundant AVPs do not share these sequence hallmarks. In SARS-CoV-2, CpG is most deficient in the S protein region to evaded ZAP-mediated antiviral defense during cell entry. Furthermore, over four months of SARS-CoV-2 evolutionary history, we observed a marked increase in C to U substitutions in the 5’ UTR and ORF1ab regions. This suggests that the two regions could be under constant C to U deamination by APOBEC3. The evolutionary pressures exerted by host immune systems onto viral genomes may motivate novel strategies for SARS-CoV-2 vaccine development.

negative: En route to Peptide Therapeutics for COVID 19: Harnessing Potential Antigenic Mimicry Between Viral and Human Proteins Molecular mimicry is a general strategy used by pathogens to infect the host cells. The emergence of SARS-CoV-2 virus has resulted in more than 6,700,000 infections and 390,000 deaths worldwide. Coronavirus disease (COVID-19) is an infectious disease caused by this virus. In this project concept, we aim to focus on the peptide-protein interaction analysis using two important drug targets in SARS-CoV-2 such as spike (S) protein and nucleocapsid (N) protein. These proteins play an important role in the virus entry and encapsidation of the viral particles. Motifs or functional regions in these two proteins must be sharing sequence homology with human protein (ACE2) which may be involved in the binding mechanism. The results will show a set of motif regions which can disrupt the viral infection. Once we identify these sets of antigenic determinant regions, antibody binding activity studies can be performed by in vitro methods. Our results from this study may suggest the existence of antigenic mimicry between SARS-CoV-2 and host proteins. The hit peptide components will have therapeutic applications to be developed into a wide variety of medicinal formulations against SARS-CoV-2 such as vaccine, intranasal and inhalation formulations. Also, the choice of conserved regions will lead to development of cross protective therapeutics against wide range of coronaviruses.

negative: Zinc Iodide in combination with Dimethyl Sulfoxide for treatment of SARS-CoV-2 and other viral infections Zinc Iodide and Dimethyl Sulfoxide composition is proposed as therapeutic agents to treat and prevent chronic and acute viral infections including SARS-CoV-2 infected patients. The therapeutic combinations have a wide range of virucidal effect on DNA and RNA containing viruses. The combination also exhibits anti-inflammatory, immunomodulating, antifibrotic, antibacterial, antifungal and antioxidative effects. Given the fact that Zinc Iodide has been used as an oral antiseptic agent and DMSO have already proven as a safe pharmaceutical solvent and therapeutic agent, we hypothesize that the combination of these two agents can be applied as an effective, safe and inexpensive treatment for SARS-CoV-2 and other viral infection. The therapeutic compound can be applied as both etiological and pathogenesis therapy and used as an effective and safe antiseptic (disinfectant) for human and animals as well.

negative: Coronavirus Vaccines: Light at the End of the Tunnel The world is currently facing an unprecedented global pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Predicting the next source of the pandemic can be very challenging As vaccination is the best way to prevent an infectious disease, the development of an effective vaccine against SARS-CoV-2 can not only reduce the morbidity and mortality associated with it, but can also lessen the economic impact As the traditional method of vaccine development takes many years for a vaccine to be available to the society, the vaccine development for SARS-CoV-2 should be speeded up using a pandemic approach with fast-track approvals from the regulatory authorities Various challenges associated with developing a vaccine during the pandemic such as technological hurdles, clinical development pathways, regulatory issues, and support from global funding agencies are expressed here

negative: Viral replicase gene products suffice for coronavirus discontinuous transcription. We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5' and 3' ends of the human coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of coronavirus-mediated transcription. These data show that the coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.

negative: RNA-cleaving properties of human apurinic/apyrimidinic endonuclease 1 (APE1). We have recently identified apurinic/apyrimidinic endonuclease 1 (APE1) as an endoribonuclease that cleaves c-myc mRNA in vitro and regulates c-myc mRNA levels and half-life in cells. This study was undertaken to further unravel the RNA-cleaving properties of APE1. Here, we show that APE1 cleaves RNA in the absence of divalent metal ions and, at 2 mM, Zn(2+), Ni(2+), Cu(2+), or Co(2+) inhibited the endoribonuclease activity of APE1. APE1 is able to cleave CD44 mRNA, microRNAs (miR-21, miR-10b), and three RNA components of SARS-corona virus (orf1b, orf3, spike) suggesting that, when challenged, it can cleave any RNAs in vitro. APE1 does not cleave strong doublestranded regions of RNA and it has a strong preference for 3' of pyrimidine, especially towards UA, CA, and UG sites at single-stranded or weakly paired regions. It also cleaves RNA weakly at UC, CU, AC, and AU sites in single-stranded or weakly paired regions. Finally, we found that APE1 can reduce the ability of the Dicer enzyme to process premiRNAs in vitro. Overall, this study has revealed some previously unknown biochemical properties of APE1 which has implications for its role in vivo.

negative: Repurposing Nimesulide, a Potent Inhibitor of the B0AT1 Subunit of the SARS-CoV-2 Receptor, as a Therapeutic Adjuvant of COVID-19 The global pandemic caused by the SARS-CoV-2 infection is a health emergency that needs to be addressed immediately. The international scientific community, following World Health Organization (WHO) indications, launched different trials for testing drugs putatively able to block the SARS-CoV-2 infection or treat the COVID-19 disease symptoms. In parallel, studies devoted to a better understanding of SARS-CoV-2 biology are in the course for designing an effective vaccine. One of the human membrane proteins known to be docked by the virus is angiotensin-converting enzyme 2 (ACE2), proposed to be responsible for viral entry in target cells. Recently, the 3D structure of ACE2 has been obtained, showing its physical interaction with B0AT1 (SLC6A19), a plasma membrane transporter involved in the trafficking of amino acids in cells. The receptor targeted by SARS-CoV-2 is a supercomplex formed by a dimer of ACE2-B0AT1, in which ACE2 binds the viral protein and B0AT1 stabilizes the heterodimer. As a serendipity occurrence, nimesulide was shown to abolish the transport function of B0AT1. Here we suggest including nimesulide in the list of drugs to be tested for the identification of co-adjuvants in the treatment of COVID-19.

negative: Could anti-CD20 therapy jeopardize the efficacy of a SARS-CoV-2 vaccine? A vaccine against SARS-CoV-2 might represent the most promising approach to halt durably the current COVID-19 pandemic. We believe that anti-CD20 therapy may jeopardize the efficacy of such a vaccine. This is regrettable because patients receiving anti-CD20 therapy (i.e. those with hematologic malignancies or autoimmune disorders) are particularly at risk of severe COVID-19 and, as such, are the most in need of a vaccine. Here, we review the reasons why anti-CD20 therapy may abrogate or diminish the efficacy of a vaccine against SARS-CoV-2 and we draw physicians’ attention towards this potential risk so that it can be considered when evaluating the risk/benefit ratio of anti-CD20 therapy during the current pandemic.

negative: A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype, IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x10(4.7) EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV.

negative: The Possible of Immunotherapy for COVID-19: a Systematic Review The novel coronavirus (2019-nCoV) is an emerging pathogen that was first described in late December 2019 and causes a severe respiratory infection in humans. Since the outbreak of COVID-19, international attention has raised to develop treatment and control options such as types of immunotherapies. The immunotherapy is an effective method for fighting against similar viral infections such as SARS-CoV, and MERS-CoV. These methods include several types of vaccines, monoclonal antibody candidates, and etc. This systematic review article was designed to evaluate the existing evidence and experience related to immunotherapy for 2019-nCoV. Web of Science (ISI), PubMed, and Scopus databases were used to search for suitable keywords such as 2019-nCoV, novel coronavirus, Immunotherapy, interleukin, vaccine and the related words for relevant publications up to 24.3.2020. The present systematic review was performed based on PRISMA protocol. Data extraction and quality valuation of articles were performed by two reviewers. 51 articles were the results of the search and based on the inclusions and exclusions criteria, 7 articles were included in the final review. As a conclusion of these studies demonstrated that although no serious research has been done on this subject at the time of writing this article, similar studies on the related viruses showed notable results. So immunotherapy for this virus can also be a suitable option.

negative: Scientific Response to the Coronavirus Crisis in Spain: Collaboration and Multidisciplinarity

negative: Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation. Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

negative: Determination of host RNA helicases activity in viral replication RNA helicases are encoded by all eukaryotic and prokaryotic cells and a minority of viruses. Activity of RNA helicases is necessary for all steps in the expression of cells and viruses and the host innate response to virus infection. Their vast functional repertoire is attributable to the core ATPase-dependent helicase domain in conjunction with flanking domains that are interchangeable and engage viral and cellular cofactors. Here, we address the important issue of host RNA helicases that are necessary for replication of a virus. The chapter covers approaches to identification and characterization of candidate helicases and methods to define the biochemical and biophysical parameters of specificity and functional activity of the enzymes. We discuss the context of cellular RNA helicase activity and virion-associated RNA helicases. The methodology and choice of controls fosters the assessment of the virologic scope of RNA helicases across divergent cell lineages and viral replication cycles.

negative: Immune responses of a designed HIV-1 DNA vaccine on rhesus monkeys An effective HIV-1 vaccine will be the ultimate solution for the prevention of HIV/AIDS, though HAART plays important roles in treating the disease. In this study, a large-scale recombinant DNA plasmid containing a designed HIV-1 multi-epitope-p24 chimeric gene was prepared and purified. Rhesus monkeys were then inoculated muscularly with the plasmid for four times in week 0, 4, 8 and 18. Whole blood was collected two weeks after the third and fourth inoculation, followed by serum and peripheral blood mononuclear cell (PBMC) separation. The CTL activity and proliferation of PBMCs stimulated by macaque MHC-I-restricted HIV-1 CTL epitope peptide were analyzed by MTT and LDH release assay, respectively. Th1 cytokines in supernatant of cultured PBMC stimulated by HIV-1 CTL epitope peptide and anti-HIV-1 antibody in serum were assayed by ELISA. The results showed that increased CTL target-killing activity, higher secretion of Th1 cytokines (IFN-γ and IL-2) and promoted proliferative reaction of monkey PBMCs stimulated by HIV-1 CTL epitope peptide were detected in the immunization group inoculated by the recombinant DNA vaccine for three times, which were further enhanced by the fourth inoculation. At the same time, HIV-1 specific antibody in serum of immunized monkeys was higher than that in controls. We concluded that the designed HIV-1 DNA vaccine may induce HIV-1 specific cellular and humoral immunity on monkeys.

negative: Advancing a multivalent ‘Pan-anthelmintic’ vaccine against soil-transmitted nematode infections ASCARIS LUMBRICOIDES: The Sabin Vaccine Institute Product Development Partnership is developing a Pan-anthelmintic vaccine that simultaneously targets the major soil-transmitted nematode infections, in other words, ascariasis, trichuriasis and hookworm infection. The approach builds off the current bivalent Human Hookworm Vaccine now in clinical development and would ultimately add both a larval Ascaris lumbricoides antigen and an adult-stage Trichuris trichiura antigen from the parasite stichosome. Each selected antigen would partially reproduce the protective immunity afforded by UV-attenuated Ascaris eggs and Trichuris stichosome extracts, respectively. Final antigen selection will apply a ranking system that includes the evaluation of expression yields and solubility, feasibility of process development and the absence of circulating antigen-specific IgE among populations living in helminth-endemic regions. Here we describe a five year roadmap for the antigen discovery, feasibility and antigen selection, which will ultimately lead to the scale-up expression, process development, manufacture, good laboratory practices toxicology and preclinical evaluation, ultimately leading to Phase 1 clinical testing.

negative: Is SARS-CoV-2 Vertically Transmitted? At the end of 2019, in Wuhan (China), the onset of a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was observed. The disease, named COVID-19, has a wide spectrum of clinical presentations, ranging from asymptomatic or mild to critical, and for some patients the disease is even fatal. Apparently, being a child or being pregnant does not represent an additional risk for adverse outcomes. The purpose of this mini-review was to investigate what is in the scientific literature, so far, in regard to vertical transmission of SARS-CoV-2. Data were obtained independently by the two authors, who carried out a systematic search in the PubMed, Embase, LILACS, Cochrane, Scopus and SciELO databases using the Medical Subject Heading terms “coronavirus,” “COVID-19,” and “vertical transmission.” Few studies about the vertical transmission of SARS-CoV-2 are found in the literature. In all case reports and case series, the mothers' infection occurred in the third trimester of pregnancy, there were no maternal deaths, and most neonates had a favorable clinical course. The virus was not detected in the neonate nasopharyngeal swab samples at birth, in the placenta, in the umbilical cord, in the amniotic fluid, in the breast milk or in the maternal vaginal swab samples in any of these articles. Only three papers reported neonatal SARS-CoV-2 infection, but there is a bias that positive pharyngeal swab samples were collected at 36 h and on the 2nd, 4th, and 17th days of life. The possibility of intrauterine infection has been based mainly on the detection of IgM and IL-6 in the neonates' serum. In conclusion, to date, no convincing evidence has been found for vertical transmission of SARS-CoV-2.

negative: Bovine coronavirus mRNA replication continues throughout persistent infection in cell culture. The existence of viral mRNA replicons was demonstrated in cells infected with the bovine coronavirus by showing a minus-strand counterpart and a corresponding replicative intermediate for each subgenomic mRNA species. mRNA replication is thus a universal property of coronaviruses, since this is now the third coronavirus for which it has been demonstrated. During the acute phase of infection (first 48 h), minus and plus strands accumulated at the same rate initially, but maximal accumulation of minus strands peaked earlier than that for plus strands, indicating that minus- and plus-strand levels are differentially regulated. In addition, packaged (input) mRNAs appeared to serve as templates for their own early replication. mRNA replication continued throughout establishment and maintenance of persistent infection (studied for 120 days), which is consistent with our hypothesis that mRNA replication contributes mechanistically to virus persistence. A replication-defective (potentially interfering) species of RNA existed transiently (beginning at day 2 and ending before day 76 postinfection), but because of its transient nature it cannot be considered essential to the long-term maintenance of virus persistence.

negative: [Strategies for vaccine development of COVID-19] An epidemic of acute respiratory syndrome in humans, which appeared in Wuhan, China in December 2019, was caused by a novel coronavirus (SARS-CoV-2). This disease was named as "Coronavirus Disease 2019" (COVID-19). SARS-CoV-2 was first identified as an etiological pathogen of COVID-19, belonging to the species of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV). The speed of both the geographical transmission and the sudden increase in numbers of cases is much faster than SARS and Middle East respiratory syndrome (MERS). COVID-19 is the first global pandemic caused by a coronavirus, which outbreaks in 211 countries/territories/areas. The vaccine against COVID-19, regarded as an effective prophylactic strategy for control and prevention, is being developed in about 90 institutions worldwide. The experiences and lessons encountered in the previous SARS and MERS vaccine research can be used for reference in the development of COVID-19 vaccine. The present paper hopes to provide some insights for COVID-19 vaccines researchers.

negative: Expression of Immunogenic Glycoprotein S Polypeptides from Transmissible Gastroenteritis Coronavirus in Transgenic Plants Abstract The use of transgenic plants as vaccine production systems was described recently. We report on the immunological response elicited by two recombinant versions of the glycoprotein S from the swine-transmissible gastroenteritis coronavirus (TGEV) expressed in transgenic plants. Arabidoposis plants were genetically transformed with cDNAs constructs encoding either the N-terminal domain (amino acid residues 1–750) or the full-length glycoprotein S of TGEV, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Genomic DNA and mRNA analyses of leaf extracts from transformed plants demonstrated the incorporation of the foreign cDNA into the arabidopsis genome, as well as their transcription. Expression of recombinant polypeptides were observed in most transgenic plants by ELISA using specific antibodies. Mice immunized with leaf extracts from transgenic plants developed antibodies that reacted specifically with TGEV in ELISA, immunoprecipitated the virus-induced protein, and neutralized the virus infectivity. From these results, we conclude that transgenic plants expressing glycoprotein S polypeptides may possibly be used as a source of recombinant antigen for vaccine production.

negative: A review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination Abstract Feline infectious peritontis (FIP) has been an elusive and frustrating problem for veterinary practitioners and cat breeders for many years. Over the last several years, reports have begun to elucidate aspects of the molecular biology of the causal virus (FIPV). These papers complement a rapidly growing base of knowledge concerning the molecular organization and replication of coronaviruses in general. The fascinating immunopathogenesis of FIPV infection and the virus' interaction with macrophages has also been the subject of several recent papers. It is now clear that FIPV may be of interest to scientists other than veterinary virologists since its pathogenesis may provide a useful model system for other viruses whose infectivity is enhanced in the presence of virus-specific antibody. With these advances and the recent release of the first commercially-available FIPV vaccine, it is appropriate to review what is known about the organization and replication of coronaviruses and the pathogenesis of FIPV infection.

negative: SEROPREVALENCE AND CLINICAL SPECTRUM OF SARS-CoV-2 INFECTION IN THE FIRST VERSUS THIRD TRIMESTER OF PREGNANCY Introduction: Case registries of pregnant women diagnosed with coronavirus disease (COVID-19) by polymerase chain reaction (PCR) have reported that the majority experienced mild infection, but up to 9% may require critical care. Most COVID-19 cases published were in the third trimester of pregnancy, which could reflect reporting bias, higher risk of infection or increased disease severity in late pregnancy. Seroprevalence studies may allow reliable estimates of the susceptibility to infection and clinical spectrum since they include asymptomatic and mild infections not tested for PCR. We evaluated the seroprevalence and clinical presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pregnant women in the first and third trimester. Methods: The study was approved by the Institutional Review Board at each institution and informed consent was obtained. We recruited 874 consecutive pregnancies attending for first trimester screening (10-16 weeks of gestation, n=372) or delivery (n=502) from April 14 to May 5. All women were interviewed with a structured questionnaire for COVID-19 symptoms two months prior to sampling. SARS-CoV-2 IgG and IgM/IgA antibodies were tested (COVID-19 VIRCLIA Monotest, Vircell Microbiologist, Spain; reported sensitivity 70% IgG and 89% IgM/IgA, and specificity 89% and 99% respectively). Indeterminate results were re-tested (VITROS Immunodiagnostic Products Anti-SARS-CoV2 Total Tests, Ortho Clinical Diagnostics, USA; 100% sensitivity and specificity) and re-classified as positive or negative. Women with COVID-19 were diagnosed and managed according to standard protocols and guidelines3,4. Statistical differences were tested using the {chi}2 test or Student t-test as appropriate (p<0.05). Results: A total of 125 of 874 women (14.3%) were positive for either IgG or IgM/IgA SARS-CoV-2 antibodies, 54/372 (14.5%) in the first and 71/502 (14.1%) in the third trimester. A total of 75/125 (60%) reported no symptoms of COVID-19 in the past 2 months, whereas 44 (35.2%) reported one or more symptoms, of which 31 (24.8%) had at least 3 symptoms or anosmia and 8 (6.4%) dyspnea. Overall, 7 women (5.6%) were admitted for persistent fever despite paracetamol and dyspnea, of which 3 had signs of pneumonia on chest radiography. All 3 had criteria for severity (bilateral chest condensation, respiratory rate>30 and leukopenia) and required oxygen support but not critical care or mechanical ventilation, and they were all discharged well. The rates of symptomatic infection, hospital admission or dyspnea were significantly higher in third trimester women (Table and Figure). Discussion: The 14.3% seroprevalence of SARS-COV-2 in pregnant women in this study was substantially larger than the contemporary rates of PCR positive cases (0.78%) reported for women 20-40y in Barcelona. The data confirm that COVID-19 is asymptomatic in the majority of pregnant women6 and illustrate the value of seroprevalence studies to capture the high proportion of asymptomatic or mild infections. In this study, none of the 125 pregnant women with SARS-CoV-2 infection required critical care as compared to 9% reported in cases diagnosed with PCR. However, the proportion of infections with symptoms or dyspnea was remarkably higher in the third trimester, and these results are in line with COVID-19 registries, reporting that 81% of hospitalized women were in late pregnancy or peripartum. These results provide reassuring information that, even in settings with a high prevalence, SARS-CoV-2 infection in pregnancy mostly presents with asymptomatic or mild clinical forms. The susceptibility to infection seemed to be the same in the first and the third trimesters of gestation. The data further suggest that, as with other respiratory viruses, COVID-19 could be more severe and require increased surveillance in late pregnancy. These findings should be confirmed and extended with larger consecutive prevalence studies in pregnancy.

negative: Severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology. UNLABELLED Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses. IMPORTANCE Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

negative: Natural History of COVID-19 and Current Knowledge on Treatment Therapeutic Options Despite intense research there is currently no effective vaccine available against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in the later 2019 and responsible for the COVID-19 pandemic. This infectious and communicable disease has become one of the major public health challenges in the world. The clinical management of COVID-19 has been limited to infection prevention and control measures associated with supportive care such as supplemental oxygen and mechanical ventilation. Meanwhile efforts to find an effective treatment to inhibit virus replication, mitigate the symptoms, increase survival and decrease mortality rate are ongoing. Several classes of drugs, many of them already in use for other diseases, are being evaluated based on the body of clinical knowledge obtained from infected patients regarding to the natural history and evolution of the infection. Herein we will provide an updated overview of the natural history and current knowledge on drugs and therapeutic agents being tested for the prevention and treatment of COVID-19. These include different classes of drugs such as antiviral agents (chloroquine, ivermectin, nitazoxanide, hydroxychloroquine, lopinavir, remdesivir, tocilizumab), supporting agents (Vitamin C, Vitamin D, azithromycin, corticosteroids) and promising investigational vaccines. Considering the controversies and excessive number of compounds being tested and reported in the literature we hope that this review can provide useful and updated consolidated information on potential drugs used to prevent, control and treat COVID-19 patients worldwide.

negative: WHO informal consultation on the guidelines for evaluation of the quality, safety, and efficacy of DNA vaccines, Geneva, Switzerland, December 2019 Consultations have been held to promote the revision of the WHO guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines adopted by the Expert Committee on Biological Standardization (ECBS) in 2005. The drivers for this revision are described, including the need for regulatory convergence highlighted by the WHO R&D Blueprint. These consultations have driven the revision to its current form, where a new guideline that includes quality, nonclinical, and clinical evaluation of plasmid DNA vaccines is being prepared for public consultation with a view to present to an upcoming ECBS. Major changes to the guidelines include streamlining the existing quality (part A) and nonclinical (part B) sections to reflect the two decades of experience, with manufacturing and control, nonclinical evaluation, and clinical testing of plasmid DNA vaccines, as a platform technology. The urgency for gaining regulatory convergence on this topic is that development of such a platform technology as DNA vaccines for routine use immunizations will prepare manufacturers and regulators across the globe in dealing with rapid development of medical countermeasures against emerging infectious diseases even in the face of an emergency setting. Two examples are described of Zika candidate vaccines that have rapidly advanced in development based on preexisting nonclinical and clinical data that precluded the need to repeat nonclinical toxicology. This report describes the progress stemming from the most recent consultation on the guidelines, including topics discussed and consensus reached.

negative: Age-dependent assessment of genes involved in cellular senescence, telomere and mitochondrial pathways in human lung tissue of smokers, COPD and IPF: Associations with SARS-CoV-2 COVID-19 ACE2-TMPRSS2-Furin-DPP4 axis Aging is one of the key contributing factors for chronic obstructive pulmonary diseases (COPD) and other chronic inflammatory lung diseases. Cigarette smoke is a major etiological risk factor that has been shown to alter cellular processes involving mitochondrial function, cellular senescence and telomeric length. Here we determined how aging contribute to the alteration in the gene expression of above mentioned cellular processes that play an important role in the progression of COPD and IPF. We hypothesized that aging may differentially alter the expression of mitochondrial, cellular senescence and telomere genes in smokers and patients with COPD and IPF compared to non-smokers. Total RNA from human lung tissues from non-smokers, smokers, and patients with COPD and IPF were processed and analyzed based on their ages (younger: <55 yrs and older: >55 yrs). NanoString nCounter panel was used to analyze the gene expression profiles using a custom designed codeset containing 112 genes including 6 housekeeping controls (mitochondrial biogenesis and function, cellular senescence, telomere replication and maintenance). mRNA counts were normalized, log2 transformed for differential expression analysis using linear models in the limma package (R/Bioconductor). Data from non-smokers, smokers and patients with COPD and IPF were analyzed based on the age groups (pairwise comparisons between younger vs. older groups). Several genes were differentially expressed in younger and older smokers, and patients with COPD and IPF compared to non-smokers which were part of the mitochondrial biogenesis/function (HSPD1, FEN1, COX18, COX10, UCP2 & 3), cellular senescence (PCNA, PTEN, KLOTHO, CDKN1C, TNKS2, NFATC1 & 2, GADD45A) and telomere replication/maintenance (PARP1, SIRT6, NBN, TERT, RAD17, SLX4, HAT1) target genes. Interestingly, NOX4 and TNKS2 were increased in the young IPF as compared to the young COPD patients. Genes in the mitochondrial dynamics and other quality control mechanisms like FIS1 and RHOT2 were decreased in young IPF compared to their age matched COPD subjects. ERCC1 (Excision Repair Cross-Complementation Group 1) and GADD45B were higher in young COPD as compared to IPF. Aging plays an important role in various infectious diseases. Elderly patients with chronic lung disease and smokers were found to have high incidence and mortality rates in the current pandemic of SARS-CoV-2 infection. Immunoblot analysis in the lung homogenates of smokers, COPD and IPF subjects revealed increased protein abundance of important proteases and spike proteins like TMPRSS2, furin and DPP4 in association with a slight increase in SARS-CoV-2 receptor ACE2 levels. This may further strengthen the observation that smokers, COPD and IPF subjects are more prone to COVID-19 infection. Overall, these findings suggest that altered transcription of target genes that regulate mitochondrial function, cellular senescence, and telomere attrition add to the pathobiology of lung aging in COPD and IPF and other smoking-related chronic lung disease in associated with alterations in SARS-CoV-2 ACE2-TMPRSS2-Furin-DPP4 axis for COVID-19 infection.

negative: Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine Abstract It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1–422); N2 (residues: 1–109); N3 (residues: 110–422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1–1269 nt), N2 (1–327 nt) and N3 (328–1269 nt)—expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4+:CD8+ assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4+:CD8+ assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.

negative: Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens. AIMS To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results. METHODS SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control subjects. The relation between SARS positivity and GAPDH mRNA concentration was investigated using the chi2 test. Increasing the sensitivity for SARS-CoV and GAPDH mRNA detection was investigated in follow up specimens in which SARS-CoV and GAPDH mRNA were not detected initially. RESULTS Varying amounts of SARS-CoV were found in the 26 SARS-CoV positive specimens and SARS-CoV was not detected in the 40 follow up specimens and controls. In addition, concentrations of GAPDH mRNA were significantly different between the patients with SARS, follow up specimens, and healthy controls (Kruskal-Wallis test, p<0.05). Moreover, GAPDH mRNA concentrations were highly correlated with SARS-CoV positivity (chi2 = 5.43; p<0.05). Finally, SARS-CoV and GAPDH mRNA were both detected in three follow up urine specimens that were initially negative when the amount of cDNA used was increased from 5 microl to 10 and 15 microl. CONCLUSIONS This Q-RT-PCR assay can be used to detect SARS-CoV. Moreover, GAPDH mRNA may be useful to rule out false negative results in SARS-CoV detection, and the current extraction method for urine may not be sensitive enough to detect low titres of SARS-CoV.

negative: Lessons for COVID-19 immunity from other coronavirus infections Abstract A key goal to controlling COVID-19 is developing an effective vaccine. Development of a vaccine requires knowledge of what constitutes a protective immune response and also features that might be pathogenic. Protective and pathogenic aspects of the response to SARS-CoV-2 are not well understood, partly because the virus has infected humans for only 6 months. However, insight into coronavirus immunity can be informed by previous studies of immune responses to non-human coronaviruses, to common cold coronaviruses, and to SARS-CoV and MERS-CoV. Here we review the literature describing these responses and discuss their relevance to the SARS-CoV-2 immune response.

negative: COVIEdb: A database for potential immune epitopes of coronaviruses 2019 novel coronavirus (2019-nCoV) has caused large-scale pandemic COVID-19 all over the world. It’s essential to find out which parts of the 2019-nCoV sequence are recognized by human immune system for vaccine development. And for the prevention of the potential outbreak of similar coronaviruses in the future, vaccines against immunogenic epitopes shared by different human coronaviruses are essential. Here we predict all the potential B/T-cell epitopes for SARS-CoV, MERS-CoV, 2019-nCoV and RaTG13-CoV based on the protein sequences. We found YFKYWDQTY in ORF1ab protein, VYDPLQPEL and TVYDPLQPEL in spike (S) protein might be pan-coronavirus targets for vaccine development. All the predicted results are stored in a database COVIEdb (http://biopharm.zju.edu.cn/coviedb/).

negative: Immunoinformatics-guided designing of epitope-based subunit vaccine against the SARS Coronavirus-2 (SARS-CoV-2) SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus which has caused deaths of numerous infected people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. This study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against SARS-CoV-2. Thereafter, molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive treatment for this lethal disease.

negative: Stop playing with data: there is no sound evidence that Bacille Calmette-Guérin may avoid SARS-CoV-2 infection (for now). Since the beginning of the COVID-19 epidemic, a possible explanation for the high heterogeneity of infection/mortality rates across involved countries was hinted in the prevalence of tuberculosis vaccination with Bacille Calmette-Guérin (BCG). A systematic review was therefore performed on May 2, 2020. A total of 13 articles were ultimately retrieved, 12 of them as preprint papers. All articles were ecological studies of low quality. Most of them did not include main confounding factors (i.e. demographic of the assessed countries, share of peo- ple residing in urban settings, etc.), and simply assessed the differences among incidence/mortality of COVID-19 with vaccination rates or by having vs. having not any vaccination policy for BCG. Even though all studies shared the very same information sources (i.e. international registries for BCG vaccination rates and open source data for COVID-19 epidemics), results were conflicting, with later studies apparently denying any true correlation between COVID-19 occurrence and BCG vaccination rates and/or policies. As a consequence, there is no sound evidence to recommend BCG vaccination for the prevention of COVID-19.

negative: Identification of potential vaccine candidates against SARS-CoV-2, A step forward to fight novel coronavirus 2019-nCoV: A Reverse Vaccinology Approach The recent Coronavirus Disease 2019 (COVID-19) causes an immense health crisis to global public health. The World Health Organization (WHO) declared the COVID-19 as a pandemic. The COVID-19 is the etiologic agent of a recently arose disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Presently, there is no vaccine available against this emerged viral disease. Therefore, it is indeed a need of the hour to develop an effectual and safe vaccine against this decidedly pandemic disease. In the current study, we collected SARS-CoV-2 genome from Indian geographical origin against human host, further more using reverse vaccinology and immunoinformatics tools here we claim effective vaccine candidates that can be mile stone in battle against COVID19. This novel study divulged two promising antigenic peptide GVYFASTEK and NFRVQPTESIV from surface glycoproteins (protein accession no. – QIA98583.1 and QHS34546.1) of SARS-CoV-2, which were predicated to be interacted with class I and class II MHC alleles and showed up to 90% conservancy and high value of antigenicity. Subsequently, the molecular docking studies were verified molecular interaction of these prime antigenic peptides with the residues of HLA-A*11-01 allele for MHC Class I and HLA DRB1*04-01 allele for MHC class II. After vigorous analysis, these peptides were predicted to be suitable epitopes which are capable to elicit the strong cell-mediated immune response against the SARS-CoV-2. Consequences from the present study could facilitate selecting SARS-CoV-2 epitopes for vaccine production pipelines in the immediate future. This novel research will certainly pave the way for a fast, reliable and virtuous platform to provide timely countermeasure of this dangerous pandemic disease, COVID-19.

negative: The ACE2 expression in human heart indicates new potential mechanism of heart injury among patients infected with SARS-CoV-2 A new type of pneumonia caused by a novel coronavirus SARS-CoV-2 outbreaks recently in China and spreads into many other countries. This disease, named as COVID-19, is similar to patients infected by SARS-CoV and MERS-CoV, and nearly 20% of patients developed severe condition. Cardiac injury is a prevalent complication of severe patients, exacerbating the disease severity in coronavirus disease 2019 (COVID-19) patients. Angiotensin-converting enzyme 2 (ACE2), the key host cellular receptor of SARS-CoV-2, has been identified in multiple organs, but its cellular distribution in human heart is not illuminated clearly. This study performed the first state-of-art single cell atlas of adult human heart, and revealed that pericytes with high expression of ACE2 might act as the target cardiac cell of SARS-CoV-2. The pericytes injury due to virus infection may result in capillary endothelial cells dysfunction, inducing microvascular dysfunction. And patients with basic heart failure disease showed increased ACE2 expression at both mRNA and protein levels, meaning that if infected by the virus these patients may have higher risk of heart attack and critically ill condition. The finding of this study explains the high rate of severe cases among COVID-19 patients with basic cardiovascular disease; and these results also perhaps provide important reference to clinical treatment of cardiac injury among severe patients infected by SARS-CoV-2.

negative: Closing in on the causes of host shutoff Influenza A virus suppresses the translation of host mRNA by selectively remodeling and dominating the pool of mRNA in infected cells.

negative: siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA Abstract SARS-associated coronavirus (SCoV) M protein plays a key role in viral assembly and budding. Recent studies revealed that M protein could interact with N protein in the Golgi complex. In this study, we showed that SCoV M protein co-localized in the Golgi apparatus with a Golgi vector marker. To study M protein function, three candidate small interfering RNAs (siRNAs) corresponding to M gene sequences were designed, transcribed in vitro, and then tested for their ability to silence M protein expression. The plasmid, pEGFP-M, encoding SCoV M protein as a fusion protein with EGFP, was used for silencing and for reporter gene detection in HEK 293T cells transfected with siRNA constructs. The results showed that the mean green fluorescence intensity and M RNA transcripts were significantly reduced, and that the expression of M glycoprotein was strongly inhibited in those cells co-transfected with M-specific siRNAs. These findings demonstrated that the three M-specific siRNAs were able to specifically and effectively inhibit M glycoprotein expression in cultured cells by blocking the accumulation of mRNA, which provides an approach for studies on the functions of M protein and for the development of novel prophylactic or therapeutic agents for SCoV infection.

negative: Clinical characteristics of severe acute respiratory syndrome coronavirus 2 reactivation Summary Objectives Previous studies on the pneumonia outbreak caused by the 2019 novel coronavirus disease (COVID-19) were based on information from the general population. However, limited data was available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reactivation. This study aimed to evaluate the clinical characteristics of the SARS-CoV-2 reactivation. Methods Clinical records, laboratory results, and chest CT scans were retrospectively reviewed for 55 patients with laboratory-confirmed COVID-19 pneumonia (i.e., with throat swab samples that were positive for SARS-CoV-2) who were admitted to Zhongnan Hospital of Wuhan University, Wuhan, China, from Jan. 8 to Feb. 10, 2020. Results All 55 patients had a history of epidemiological exposure to COVID-19, and 5 (9%) patients who discharged from hospital presented with SARS-CoV-2 reactivation. Among the 5 reactivated patients, other symptoms were also observed, including fever, cough, sore throat, and fatigue. One of the 5 patients had progressive lymphopenia (from 1.3 to 0.56 × 109 cells per L) and progressive neutrophilia (from 4.5 to 18.28 × 109 cells per L). All 5 reactivated patients presented normal aminotransferase levels. Throat swab samples from the 5 reactivated patients were tested for SARS-CoV-2, indicating all positive for the virus. Conclusions Findings from this small group of cases suggested that there was currently evidence for reactivation of SARS-CoV-2 and there might be no specific clinical characteristics to distinguish them.

negative: CHAPTER 28 Coronaviridae Publisher Summary Coronaviruses are ssRNA viruses that infect a wide range of mammalian and avian species; they are important causes of respiratory and enteric disease, encephalomyelitis, hepatitis, serositis, and vasculitis in domestic animals. In humans, coronaviruses are one of several groups of viruses that cause the common cold. The prototype of the family, avian infectious brochitis virus, is one of the most infectious of all viruses and causes an acute respiratory disease, which in young chicks can cause very high mortality. Outbreaks can be explosive, involving nearly every bird in the flock at about the same time, because of respiratory transmission and a very short incubation period. In many ways, transmissible gastroenteritis virus of swine and mouse hepatitis virus behave similarly, affecting young animals most severely, spreading very quickly to all animals at risk, and causing major economic losses before control strategies can be put into place. The strategy of expression of the coronavirus genome is unique. The input virion RNA molecule is translated directly, one of the products being an RNA polymerase that then transcribes a full-length (—) sense copy RNA, from which in turn is transcribed a 3′-coterminal nested set of subgenomic mRNAs. The nested set comprises six overlapping species of mRNAs that extend for different lengths from a common 3′ terminus. Only the unique sequence toward the 5′ end, which is not shared with the next smallest mRNA in the nested set, is translated, each product, therefore, being a unique protein.

negative: Coronaviruses: General Features Coronaviruses have the largest known RNA genomes (∼30kb), which are of positive sense. Together with toroviruses, they are classified in the family Coronaviridae, order Nidovirales. All coronaviruses have four common proteins, three in the envelope and one associated with the genome. Assembly of virus particles occurs at internal membranes. The genes for the structural proteins are at the 3′ end of the genome. Most of the genome (∼20kb) is gene 1, which encodes 15–16 proteins associated with RNA replication and transcription. Translation of gene 1 involves ribosomal frameshifting. Transcription is by a discontinuous process which results in a 3′ co-terminal nested set of mRNAs, each of which has a common leader sequence transcribed from the 5′ terminus of the genome. Only the most 5′-proximal gene of each mRNA is translated. Recombination is a feature of coronavirus evolution. The outbreak of severe acute respiratory syndrome (SARS) has resulted in the discovery of more coronaviruses in humans, other mammals, and avian species, and the realization that the host range of coronaviruses is wider than previously acknowledged. Coronaviruses are associated with a wide range of diseases, including the respiratory and enteric systems, though not necessarily restricted to these, for example, some coronaviruses affect the central nervous system, kidneys, and gonads. The most widely used coronavirus vaccine (billions of doses annually) is against infectious bronchitis virus, which affects chickens.

negative: Immune and bioinformatics identification of T cell and B cell epitopes in the protein structure of SARS-CoV-2: A Systematic Review The beginning of 2020 was marked as the emergence of a COVID-19 outbreak caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, there is no vaccine or approved treatment for this infectious virus so the invention of an efficient vaccine is certainly a high priority. Some studies have employed several techniques to facilitate the combination of the immunoinformatics approach and comparative genomic approach in order to determine the potential peptides for designing the T-cell epitope-based peptide vaccine using the 2019-nCoV envelope protein as a target. Via screening the bioimmunoinformatic SARS-CoV2 derived B-cell and T-cell epitopes within the basic immunogenic of SARS-CoV2 proteins, we presented a set of inferred B-cell and T-cell epitopes from the spike (S) and nucleocapsid (N) proteins with high antigenicity and without allergenic property or toxic effects. Our findings provide a screened set of epitopes that can be introduced as potential targets for developing peptide vaccines against the SARS-CoV-2 virus.

negative: A phase trial of the oral Lactobacillus casei vaccine polarizes Th2 cell immunity against transmissible gastroenteritis coronavirus infection Transmissible gastroenteritis coronavirus (TGEV) is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. TGEV is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. An oral Lactobacillus casei (L. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. In this L. casei vaccine, repetitive peptides expressed by L. casei (specifically the MDP and tuftsin fusion protein (MT)) were repeated 20 times and the D antigenic site of the TGEV spike (S) protein was repeated 6 times. Immunization with recombinant Lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. The first immunization is more important than the last immunization in the series. The recombinant Lactobacillus elicited specific systemic and mucosal immune responses. Recombinant L. casei had a strong potentiating effect on the cellular immunity induced by the oral L. casei vaccine. However, during TGEV infection, the systemic and local immune responses switched from Th1 to Th2-based immune responses. The systemic humoral immune response was stronger than the cellular immune response after TGEV infection. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Furthermore, the Lactobacillus vaccine stimulated an anti-TGEV infection Th17 pathway. The histopathological examination showed tremendous potential for recombinant Lactobacillus to enable rapid and effective treatment for TGEV with an intestinal tropism in piglets. The TGEV immune protection was primarily dependent on mucosal immunity.

negative: Environmental concern regarding the effect of humidity and temperature on 2019-nCoV survival: fact or fiction The new coronavirus, called 2019-nCoV, is a new type of virus that was first identified in Wuhan, China, in December 2019. Environmental conditions necessary for survival and spread of 2019-nCoV are somewhat transparent but unlike animal coronaviruses. We are poorly aware of their survival in environment and precise factors of their transmission. Countries located in east and west of globe did not have a significant impact on prevalence of disease among communities, and on the other hand, north and south have provided a model for relative prediction of disease outbreaks. The 2019-nCoV can survive for up to 9 days at 25 °C, and if this temperature rises to 30 °C, its lifespan will be shorter. The 2019-nCoV is sensitive to humidity, and lifespan of viruses in 50% humidity is longer than that of 30%. Also, temperature and humidity are important factors influencing the COVID-19 mortality rate and may facilitate 2019-nCoV transmission. Thus, considering the available and recent evidence, it seems that low temperatures, as well as dry and unventilated air, may affect stability and transmissibility of 2019-nCoV.

negative: Outbreak of COVID-19: An emerging global pandemic threat Since the first outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Wuhan, Hubei, China in December 2019, it is now recognized as a pandemic by the World Health Organization (WHO) as more than 200 countries and territories worldwide are affected with an increasing incidence. The SARS-CoV-2 infection results in a spectrum of non-specific signs and symptoms, ranging from asymptomatic infection, to flu-like illness such as fever, cough, dry cough and fatigue, to pneumonia, acute respiratory distress syndrome, and even multi-organ failures with high morbidity and mortality. SARS-CoV-2 is mainly transmitted through respiratory droplets that infected people exhale during incubation and onset period. By 12 June 2020, over 7.5 million confirmed cases of Coronavirus disease 2019 (COVID-19) with more than 421,000 deaths in the world have been reported to the WHO. No specific medication is approved to treat COVID-19, raising the urgent need for antiviral drug development. By 12 June 2020, there are over 1000 clinical trials registered in clinicaltrials.gov for treatment of COVID-19. This review summarizes the epidemiology, virology, clinical presentation, pathophysiology, diagnosis, and particularly the antiviral drugs currently under clinical trials for treatment of SARS-CoV-2 infection, together with the challenges and perspectives of this disease are also discussed.

negative: SARS Coronavirus nsp1 Protein Induces Template-Dependent Endonucleolytic Cleavage of mRNAs: Viral mRNAs Are Resistant to nsp1-Induced RNA Cleavage SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

negative: The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause zoonotic infections and serious disease, primarily in the Arabian Peninsula, due to repeated spill-over from dromedary camels and subsequent nosocomial transmission. Approved MERS vaccines for use in animals or humans are not currently available. MERS-CoV replication requires the virus-encoded papain-like protease (PLpro) to cleave multiple sites in the viral replicase polyproteins, thereby releasing functional non-structural proteins. Additionally, PLpro is a deubiquitinating enzyme (DUB) that can remove ubiquitin(-like) moieties from substrates, presumably to counteract host antiviral responses. In previous work, we determined the crystal structure of MERS-CoV PLpro in complex with ubiquitin, facilitating the design of PLpro mutations that impair DUB activity without affecting viral polyprotein cleavage. Here, we introduced these DUB-inactivating mutations into the viral genome and examined their impact on MERS-CoV infection both in cell culture and in a lethal mouse model. Although overall replication of DUB-negative and wild-type (wt) recombinant MERS-CoV was comparable in multiple cell lines, infection with DUB-negative virus markedly increased mRNA levels for interferon (IFN)-β and IFN-stimulated genes. Moreover, compared to a wt virus infection, the survival rate was significantly increased when DUB-negative MERS-CoV was used to infect transgenic mice expressing a human MERS-CoV receptor. Interestingly, DUB-negative and wt MERS-CoV replicated to the same titers in lungs of infected mice, but the DUB-negative virus was cleared faster, likely due to the observed accelerated and better-regulated innate immune responses, in contrast to delayed and subsequently excessive responses in wt virus-infected mice. This study provides the first direct evidence that the DUB activity of a coronaviral protease contributes to innate immune evasion and can profoundly enhance virulence in an animal model. Thus, reduction or removal of the innate immune-suppressive DUB activity of PLpros is a promising strategy for coronavirus attenuation in the context of rational vaccine development. Although zoonotic coronaviruses such as Middle East respiratory coronavirus (MERS-CoV) have pandemic potential, therapeutics and vaccines that counteract this public health threat are not currently available. Coronaviruses typically employ multiple strategies to evade the host’s innate immune response, which may enhance clinical disease and/or reduce the efficacy of modified live vaccines. The MERS-CoV-encoded papain-like protease (PLpro) is not only crucial for the expression of functional replicase proteins, but has also been postulated to antagonize ubiquitination-dependent steps during the activation of the innate immune response. Here, we report the generation of engineered MERS-CoVs mutants in which PLpro’s deubiquitinating (DUB) activity was specifically disrupted without affecting virus viability. In this manner, we could demonstrate that the DUB activity of PLpro suppresses the interferon response in MERS-CoV-infected cells. Strikingly, in the lungs of mice infected with DUB-negative MERS-CoV, innate immune responses were induced at an earlier stage of infection than in wt virus-infected mice. This group also showed a clearly increased survival, indicating that the DUB activity is an important MERS-CoV virulence factor. This proof-of-concept study establishes that the engineering of DUB-negative coronaviruses, which elicit a more effective immune response in the host, is a viable strategy for vaccine development.

negative: A Novel Vaccine Employing Non-Replicating Rabies Virus Expressing Chimeric SARS-CoV-2 Spike Protein Domains: Functional Inhibition of Viral/Nicotinic Acetylcholine Receptor Complexes The emergence of the novel β-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic of coronavirus disease 2019 (COVID-19). Clinical studies have documented that potentially severe neurological symptoms are associated with SARS-CoV-2 infection, thereby suggesting direct CNS penetration by the virus. Prior studies have demonstrated that the destructive neurological effects of rabies virus (RABV) infections are mediated by CNS transport of the virus tightly bound to the nicotinic acetylcholine receptor (nAChR). By comparison, it has been hypothesized that a similar mechanism exists to explain the multiple neurological effects of SARS-CoV-2 via binding to peripheral nAChRs followed by orthograde or retrograde transport into the CNS. Genetic engineering of the RABV has been employed to generate novel vaccines consisting of non-replicating RABV particles expressing chimeric capsid proteins containing human immunodeficiency virus 1 (HIV-1), Middle East respiratory syndrome (MERS-CoV), Ebolavirus, and hepatitis C virus (HCV) sequences. Accordingly, we present a critical discussion that integrates lessons learned from prior RABV research and vaccine development into a working model of a SARS-CoV-2 vaccine that selectively targets and neutralizes CNS penetration of a tightly bound viral nAChR complex.

negative: Immunogenicity of next-generation HPV vaccines in non-human primates: Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. HPVs are oncogenic small double-stranded DNA viruses that are the primary causal agent of cervical cancer and other types of cancers, including in the anus, oropharynx, vagina, vulva, and penis. Prophylactic vaccination against HPV is an attractive strategy for preventing cervical cancer and some other types of cancers. However, there are few safe and effective vaccines against HPV infections. Current first-generation commercial HPV vaccines are expensive to produce and deliver. The goal of this study was to develop an alternate potent HPV recombinant L1-based vaccines by producing HPV virus-like particles into a vaccine that is currently used worldwide. Live attenuated measles virus (MV) vaccines have a well-established safety and efficacy record, and recombinant MV (rMV) produced by reverse genetics may be useful for generating candidate HPV vaccines to meet the needs of the developing world. We studied in non-human primate rMV-vectored HPV vaccine in parallel with a classical alum adjuvant recombinant HPV16L1 and 18L1 protein vaccine produced in Pichia pastoris. A combined prime-boost approach using both vaccines was evaluated, as well as immune interference due to pre-existing immunity against the MV. The humoral immune response induced by the MV, Pichia-expressed vaccine, and their combination as priming and boosting approaches was found to elicit HPV16L1 and 18L1 specific total IgG and neutralizing antibody titres. Pre-existing antibodies against measles did not prevent the immune response against HPV16L1 and 18L1.

negative: Sequencing of coronavirus IBV genomic RNA: a 195-base open reading frame encoded by mRNA B Abstract DNA sequencing of genomic cDNA clones of avian infectious bronchitis virus (IBV) has been carried out. 770 bases have been determined which include genomic sequences spanning the 5' termini of the two smallest mRNAs of the 3'-coterminal “nested” set: mRNA A and mRNA B. This region contains the complete coding sequences for mRNA B which are additional to those present in mRNA A. Two open reading frames are present, predicting proteins of M rs 7500 and 9500.

negative: Genome based Evolutionary study of SARS-CoV-2 towards the Prediction of Epitope Based Chimeric Vaccine SARS-CoV-2 is known to infect the neurological, respiratory, enteric, and hepatic systems of human and has already become an unprecedented threat to global healthcare system. COVID-19, the most serious public condition caused by SARS-CoV-2 leads the world to an uncertainty alongside thousands of regular death scenes. Unavailability of specific therapeutics or approved vaccine has made the recovery of COVI-19 more troublesome and challenging. The present in silico study aimed to predict a novel chimeric vaccines by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein (S), membrane protein (M), envelope protein and nucleocapsid protein (N) were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins (S, E, M and N) reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and Normal Mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. The development of preventive measures to combat COVID-19 infections might be aided the present study. However, the in vivo and in vitro validation might be ensured with wet lab trials using model animals for the implementation of the presented data.

negative: Combating the Coronavirus Pandemic: Early Detection, Medical Treatment, and a Concerted Effort by the Global Community The World Health Organization (WHO) has declared the outbreak of 2019 novel coronavirus, known as 2019-nCoV, a pandemic, as the coronavirus has now infected over 2.6 million people globally and caused more than 185,000 fatalities as of April 23, 2020. Coronavirus disease 2019 (COVID-19) causes a respiratory illness with symptoms such as dry cough, fever, sudden loss of smell, and, in more severe cases, difficulty breathing. To date, there is no specific vaccine or treatment proven effective against this viral disease. Early and accurate diagnosis of COVID-19 is thus critical to curbing its spread and improving health outcomes. Reverse transcription-polymerase chain reaction (RT-PCR) is commonly used to detect the presence of COVID-19. Other techniques, such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), clustered regularly interspaced short palindromic repeats (CRISPR), and microfluidics, have allowed better disease diagnosis. Here, as part of the effort to expand screening capacity, we review advances and challenges in the rapid detection of COVID-19 by targeting nucleic acids, antigens, or antibodies. We also summarize potential treatments and vaccines against COVID-19 and discuss ongoing clinical trials of interventions to reduce viral progression.

negative: Human H-ferritin presenting RBM of spike glycoprotein as potential vaccine of SARS-CoV-2 The outbreak of COVID-19 has so far inflicted millions of people all around the world and will have a long lasting effect on every aspect of everyone’s life. Yet there is no effective approved treatment for the disease. In an effort of utilizing human ferritin as nanoplatform for drug delivery, we engineered a fusion protein by presenting receptor-binding motif (RBM) of SARS-CoV-2 virus spike glycoprotein on the N-terminus of ferritin subunits. The designed fusion protein with a cage-like structure, similar to that of corona virus, is a potential anti-SARS-CoV-2 vaccine. We hereby show the construction, preparation, and characterization of the fusion protein RBM-HFtn. Our initial affinity study confirmed its biological activity towards ACE2 receptor which suggests its mode of action against SARS-CoV-2 could be either through vaccine therapy or blocking the cellular entry of virus as antagonist of ACE2 receptor.

negative: Epidemiological and clinical characteristics of 26 asymptomatic SARS-CoV-2 carriers BACKGROUND: We retrospectively analysed 26 persistently asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carriers. METHODS: Epidemiological and clinical characteristics from the 26 asymptomatic patients with positive results for SARS-CoV-2 RNA testing were obtained. RESULTS: Twenty-two patients (84.6%) correlated with clustering occurrence. The median period from contact to diagnosis and the last positive nucleic acid test was 19 (8–24 days) and 21.5 days (10–36 days), respectively. The median period from diagnosis to negative nucleic acid test was significantly different between patients with normal or atypical chest computed tomography (CT) findings (n=16, 61.5%; 7.5 days [2–20 days]) and patients with typical ground-glass or patchy opacities on CT(n=10, 38.5%; 12.5 days[8–22 days]; P<0.01). Seven patients (70.0%) with initial positive nucleic acid test results had a negative result simultaneously with improved CT findings. Obvious improvement in CT findings was observed in three patients (30.0%) despite positive nucleic acid test results. CONCLUSION: In asymptomatic patients, changes in biochemical and inflammatory variables are small and changes on chest CT can occur. It is worth noting the long existence of SARS-CoV-2 in some asymptomatic patients and false-negative results need to be considered in SARS-CoV-2nucleic acid test.

negative: Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection HIV infection of CD4(+) T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs. To survey these dynamic changes, we applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq). These time points afforded a view of the transcriptomic changes occurring both before and during viral replication. In the resulting small RNA-Seq data set, we detected a phased pattern of microRNA expression. Largely distinct sets of microRNAs were found to be suppressed at 5 and 12 h postinfection, and both sets of changes rebounded later in infection. A larger set of microRNA changes was observed at 24 h postinfection. When integrated with mRNA expression data, the small RNA-Seq data indicated a role for microRNAs in transcriptional regulation, T cell activation, and cell cycle during HIV infection. As a unique benefit of next-generation sequencing, we also detected candidate novel host microRNAs differentially expressed during infection, including one whose downregulation at 24 h postinfection may allow full replication of HIV to proceed. Collectively, our data provide a uniquely comprehensive view of the changes in host microRNAs induced by HIV during cellular infection.

negative: COVID‐19 vaccines: knowing the unknown Vaccine development against SARS‐CoV‐2 has drawn attention around the globe due to the exploding pandemic. Although COVID‐19 is caused by a new coronavirus, SARS‐CoV‐2, previous research on other coronavirus vaccines, such as FIPV, SARS and MERS, has provided valuable information for the rapid development of COVID‐19 vaccine. However, important knowledge gaps remain – some are specific to SARS‐CoV‐2, others are fundamental to immunology and vaccinology. Here we discuss areas that need to be addressed for COVID‐19 vaccine development, and what can be learned from examples of vaccine development in the past. Since the beginning of the outbreak, the research progress on COVID‐19 has been remarkable. We are therefore optimistic about the rapid development of COVID‐19 vaccine. This article is protected by copyright. All rights reserved

negative: Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.

negative: Companion vaccine Bioinformatic design tool reveals limited functional genomic variability of SARS-Cov-2 Spike Receptor Binding Domain Tracking the genetic variability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for several reasons, such as to identify target sequences to generate robust vaccines and neutralizing monoclonal antibodies; to track viral genetic temporal and geographic evolution; and to mine for viral variants associated with reduced or increased severity. Several online tools and bioinformatic analyses have been released for this purpose; four main viral clades have been described via phylogenetic analyses. Here, we present an open-source bioinformatic protocol focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Results on the whole GISAID sequence dataset at the time of the writing (April–June 2020) confirms no high variability pressure on the viral genome in terms of quality and quantity of mutations. We then focused our analysis on the receptor-binding domain region of the spike protein and only found a few variants associated with particular geographic locations that should be tracked over time. Finally, additional immunogenomic analyses revealed some variation in mutated epitope MHC compatibility and T-cell recognition for most frequent human HLAs.

negative: Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/gt.2010.149) contains supplementary material, which is available to authorized users.

negative: Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections Several viruses cause pulmonary infections due to their shared tropism with cells of the respiratory tract. These respiratory problems due to viral infection become a public health concern due to rapid transmission through air/aerosols or via direct-indirect contact with infected persons. In addition, the cross-species transmission causes alterations to viral genetic makeup thereby increasing the risk of emergence of pathogens with new and more potent infectivity. With the introduction of effective nucleic acid-based technologies, post translational gene silencing (PTGS) is being increasingly used to silence viral gene targets and has shown promising approach towards management of many viral infections. Since several host factors are also utilized by these viruses during various stages of infection, silencing these host factors can also serve as promising therapeutic tool. Several nucleic acid-based technologies such as short interfering RNAs (siRNA), antisense oligonucleotides, aptamers, deoxyribozymes (DNAzymes), and ribozymes have been studied and used against management of respiratory viruses. These therapeutic nucleic acids can be efficiently delivered through the airways. Studies have also shown efficacy of gene therapy in clinical trials against respiratory syncytial virus (RSV) as well as models of respiratory diseases including severe acute respiratory syndrome (SARS), measles and influenza. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and highlighted the emerging roles of nucleic acids in the management of some of the severe respiratory viral infections. We have also focused on the methods of their delivery and associated challenges.

negative: Going Virtual to Support Anatomy Education: A STOPGAP in the Midst of the Covid-19 Pandemic

negative: Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics The objective of this Personal View is to compare transmissibility, hospitalisation, and mortality rates for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with those of other epidemic coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and pandemic influenza viruses. The basic reproductive rate (R(0)) for SARS-CoV-2 is estimated to be 2·5 (range 1·8–3·6) compared with 2·0–3·0 for SARS-CoV and the 1918 influenza pandemic, 0·9 for MERS-CoV, and 1·5 for the 2009 influenza pandemic. SARS-CoV-2 causes mild or asymptomatic disease in most cases; however, severe to critical illness occurs in a small proportion of infected individuals, with the highest rate seen in people older than 70 years. The measured case fatality rate varies between countries, probably because of differences in testing strategies. Population-based mortality estimates vary widely across Europe, ranging from zero to high. Numbers from the first affected region in Italy, Lombardy, show an all age mortality rate of 154 per 100 000 population. Differences are most likely due to varying demographic structures, among other factors. However, this new virus has a focal dissemination; therefore, some areas have a higher disease burden and are affected more than others for reasons that are still not understood. Nevertheless, early introduction of strict physical distancing and hygiene measures have proven effective in sharply reducing R(0) and associated mortality and could in part explain the geographical differences.

negative: Ten challenging questions about SARS-CoV-2 and COVID-19 INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently introduced as a global public health problem by the World Health Organization (WHO). The virus outbreak has been documented around the world. Updating data in different aspects of the virus could force us to revise our idea about the main questions concerning coronavirus disease-19 (COVID-19). AREAS COVERED: Although our knowledge about the SARS-CoV-2 and COVID-19 is largely based on the very limited data, the information is growing rapidly. The renewed answers to the specific research questions concerning updating data not only reveal gaps for future research but also re-categorized our information. Here, we attempt to briefly discuss 10 important questions about SARS-CoV-2 and COVID-19. EXPERT OPINION: Since our knowledge about different aspects of SARS-CoV-2 appears to be in its infancy and is rapidly changing, the provision of the right data is more difficult in this regard. However, we try to rely on results from more extensive research to answer the main questions about this new virus. Therefore, further studies, particularly in the context of the virus pathogenesis, diagnosis, treatment, and vaccine development, are warranted.

negative: Potential adjuvants for the development of a SARS-CoV-2 vaccine based on experimental results from similar coronaviruses The extensive efforts around the globe are being made to develop a suitable vaccine against COVID-19 (Coronavirus Disease-19) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2). An effective vaccine should be able to induce high titers of neutralizing antibodies to prevent the virus from attaching to the host cell receptors. However, to elicit the protective levels of antibodies, a vaccine may require multiple doses or assistance from other immunostimulatory molecules. Further, the vaccine should be able to induce protective levels of antibodies rapidly with the least amount of antigen used. This decreases the cost of a vaccine and makes it affordable. As the pandemic has hit most countries across the globe, there will be an overwhelming demand for the vaccine in a quick time. Incorporating a suitable adjuvant in a SARS-CoV-2 vaccine may address these requirements. This review paper will discuss the experimental results of the adjuvanted vaccine studies with similar coronaviruses (CoVs) which might be useful to select an appropriate adjuvant for a vaccine against rapidly emergingSARS-CoV-2. We also discuss the current progress in the development of adjuvanted vaccines against the disease.

negative: Chapter 3 Replication and Expression Strategies of Viruses Summary Regardless of their genetic constitution, viral genomes are replicated, expressed, and assembled in association with living host cells. These entities do not undergo division, but rather generate new particles through the assembly of preformed components. Some viruses go further by modifying cellular metabolism to create a more favorable environment for viral gene expression. In a few instances, these clever strategies also facilitate escape from the host’s defense responses. The process of replication is typically divided into the phases of attachment, entry, uncoating, genome replication and expression, assembly, maturation, and finally, egress or release from the host cell. In this chapter, we address genome replication and expression, including the multiple strategies that serve to control gene expression and ensure preferential propagation of the virus.

negative: Protective immunity by an engineered DNA vaccine for Mayaro virus Mayaro virus (MAYV) of the genus alphavirus is a mosquito-transmitted emerging infectious disease that causes an acute febrile illness, rash, headaches, and nausea that may turn into incapacitating, persistent arthralgias in some victims. Since its discovery in Trinidad in 1954, cases of MAYV infection have largely been confined there and to the northern countries of South America, but recently, MAYV cases have been reported in some island nations in the Caribbean Sea. Accompanying these reports is evidence that new vectors, including Aedes spp. mosquitos, recently implicated in the global spread of Zika and chikungunya viruses, are competent for MAYV transmission, which, if true, could facilitate the spread of MAYV beyond its current range. Despite its status as an emerging virus, there are no licensed vaccines to prevent MAYV infection nor therapeutics to treat it. Here, we describe the development and testing of a novel DNA vaccine, scMAYV-E, that encodes a synthetically-designed consensus MAYV envelope sequence. In vivo electroporation-enhanced immunization of mice with this vaccine induced potent humoral responses including neutralizing antibodies as well as robust T-cell responses to multiple epitopes in the MAYV envelope. Importantly, these scMAYV-E-induced immune responses protected susceptible mice from morbidity and mortality following a MAYV challenge.

negative: Current Therapies Under Investigation for COVID-19. In response to the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), researchers are expeditiously searching for antiviral treatments able to alleviate the symptoms of infection, which can be life-threatening. Here, we provide a general overview of what is currently known about the structure and characteristic features of SARS-CoV-2, some of which could potentially be exploited for the purposes of antiviral therapy and vaccine development. This mini-review also covers selected and noteworthy antiviral agents/supportive therapy out of hundreds of drugs that are being repurposed or tested as potential treatments for COVID-19, the disease caused by SARS-CoV.

negative: Decoding SARS-CoV-2 transmission, evolution and ramification on COVID-19 diagnosis, vaccine, and medicine Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 6156 genome samples collected up to April 24, 2020, we report that SARS-CoV-2 has had 4459 alarmingly mutations which can be clustered into five subtypes. We introduce mutation ratio and mutation $h$-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively non-conservative. In particular, the nucleocapsid protein has more than half its genes changed in the past few months, signaling devastating impacts on the ongoing development of COVID-19 diagnosis, vaccines, and drugs.

negative: Bioinformatic prediction of potential T cell epitopes for SARS-Cov-2 To control and prevent the current COVID-19 pandemic, the development of novel vaccines is an emergent issue. In addition, we need to develop tools that can measure/monitor T-cell and B-cell responses to know how our immune system is responding to this deleterious virus. However, little information is currently available about the immune target epitopes of novel coronavirus (SARS-CoV-2) to induce host immune responses. Through a comprehensive bioinformatic screening of potential epitopes derived from the SARS-CoV-2 sequences for HLAs commonly present in the Japanese population, we identified 2013 and 1399 possible peptide epitopes that are likely to have the high affinity (<0.5%- and 2%-rank, respectively) to HLA class I and II molecules, respectively, that may induce CD8+ and CD4+ T-cell responses. These epitopes distributed across the structural (spike, envelope, membrane, and nucleocapsid proteins) and the nonstructural proteins (proteins corresponding to six open reading frames); however, we found several regions where high-affinity epitopes were significantly enriched. By comparing the sequences of these predicted T cell epitopes to the other coronaviruses, we identified 781 HLA-class I and 418 HLA-class II epitopes that have high homologies to SARS-CoV. To further select commonly-available epitopes that would be applicable to larger populations, we calculated population coverages based on the allele frequencies of HLA molecules, and found 2 HLA-class I epitopes covering 83.8% of the Japanese population. The findings in the current study provide us valuable information to design widely-available vaccine epitopes against SARS-CoV-2 and also provide the useful information for monitoring T-cell responses.

negative: The epidemiology, diagnosis and treatment of COVID-19 Abstract In December 2019, the outbreak of the novel coronavirus disease (COVID-19) in China spread worldwide, becoming an emergency of major international concern. SARS-CoV-2 infection causes clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus. Human-to-human transmission via droplets, contaminated hands or surfaces has been described, with incubation times of 2-14 days. Early diagnosis, quarantine, and supportive treatments are essential to cure patients. This paper reviews the literature on all available information about the epidemiology, diagnosis, isolation and treatments of COVID-19. Treatments, including antiviral agents, chloroquine and hydroxychloroquine, corticosteroids, antibodies, convalescent plasma transfusion and vaccines, are discussed in this article. In addition, registered trials investigating treatment options for COVID-19 infection are listed.

negative: Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia BACKGROUND: The role of human bocavirus (HBoV) in respiratory illness is uncertain. HBoV genomic DNA is frequently detected in both ill and healthy children. We hypothesized that spliced viral capsid messenger RNA (mRNA) produced during active replication might be a better marker for acute infection. METHODS: As part of the Etiology of Pneumonia in the Community (EPIC) study, children aged <18 years who were hospitalized with community-acquired pneumonia (CAP) and children asymptomatic at the time of elective outpatient surgery (controls) were enrolled. Nasopharyngeal/oropharyngeal specimens were tested for HBoV mRNA and genomic DNA by quantitative polymerase chain reaction. RESULTS: HBoV DNA was detected in 10.4% of 1295 patients with CAP and 7.5% of 721 controls (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.0–2.0]); HBoV mRNA was detected in 2.1% and 0.4%, respectively (OR, 5.1 [95% CI, 1.6–26]). When adjusted for age, enrollment month, and detection of other respiratory viruses, HBoV mRNA detection (adjusted OR, 7.6 [95% CI, 1.5–38.4]) but not DNA (adjusted OR, 1.2 [95% CI, .6–2.4]) was associated with CAP. Among children with no other pathogens detected, HBoV mRNA (OR, 9.6 [95% CI, 1.9–82]) was strongly associated with CAP. CONCLUSIONS: Detection of HBoV mRNA but not DNA was associated with CAP, supporting a pathogenic role for HBoV in CAP. HBoV mRNA could be a useful target for diagnostic testing.

negative: Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry Abstract The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by β-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.

negative: ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 20191,2 and is responsible for the COVID-19 pandemic3. Vaccines are an essential countermeasure urgently needed to control the pandemic4. Here, we show that the adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, is immunogenic in mice, eliciting a robust humoral and cell-mediated response. This response was not Th2 dominated, as demonstrated by IgG subclass and cytokine expression profiling. A single vaccination with ChAdOx1 nCoV-19 induced a humoral and cellular immune response in rhesus macaques. We observed a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated rhesus macaques. Importantly, no evidence of immune-enhanced disease following viral challenge in vaccinated animals was observed. ChAdOx1 nCoV-19 is currently under investigation in a phase I clinical trial. Safety, immunogenicity and efficacy against symptomatic PCR-positive COVID-19 disease will now be assessed in randomised controlled human clinical trials.

negative: Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications Background The COVID-19 pandemic caused by SARS-CoV-2 coronavirus threatens global public health. Currently, neutralizing antibodies (NAbs) versus this virus are expected to correlate with recovery and protection of this disease. However, the characteristics of these antibodies have not been well studied in association with the clinical manifestations in patients. Methods Plasma collected from 175 COVID-19 recovered patients with mild symptoms were screened using a safe and sensitive pseudotyped-lentiviral-vector-based neutralization assay. Spike-binding antibody in plasma were determined by ELISA using RBD, S1, and S2 proteins of SARS-CoV-2. The levels and the time course of SARS-CoV-2-specific NAbs and the spike-binding antibodies were monitored at the same time. Findings SARS-CoV-2 NAbs were unable to cross-reactive with SARS-CoV virus. SARS-CoV-2-specific NAbs were detected in patients from day 10-15 after the onset of the disease and remained thereafter. The titers of NAb among these patients correlated with the spike-binding antibodies targeting S1, RBD, and S2 regions. The titers of NAbs were variable in different patients. Elderly and middle-age patients had significantly higher plasma NAb titers (P<0.0001) and spike-binding antibodies (P=0.0003) than young patients. Notably, among these patients, there were ten patients whose NAb titers were under the detectable level of our assay (ID50: < 40); while in contrast, two patients, showed very high titers of NAb, with ID50 :15989 and 21567 respectively. The NAb titers were positive correlated with plasma CRP levels but negative correlated with the lymphocyte counts of patients at the time of admission, indicating an association between humoral response and cellular immune response. Interpretation The variations of SARS-CoV-2 specific NAbs in recovered COVID-19 patients may raise the concern about the role of NAbs on disease progression. The correlation of NAb titers with age, lymphocyte counts, and blood CRP levels suggested that the interplay between virus and host immune response in coronavirus infections should be further explored for the development of effective vaccine against SARS-CoV-2 virus. Furthermore, titration of NAb is helpful prior to the use of convalescent plasma for prevention or treatment. Funding Ministry of Science and Technology of China, National Natural Science Foundation of China, Shanghai Municipal Health Commission, and Chinese Academy of Medical Sciences

negative: Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial BACKGROUND: A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. METHODS: We did a dose-escalation, single-centre, open-label, non-randomised, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults aged between 18 and 60 years were sequentially enrolled and allocated to one of three dose groups (5 × 10(10), 1 × 10(11), and 1·5 × 10(11) viral particles) to receive an intramuscular injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralising antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralisation and pseudovirus neutralisation tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127. FINDINGS: Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36·3 years) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the analysis. At least one adverse reaction within the first 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]. Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralising antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination. INTERPRETATION: The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation. FUNDING: National Key R&D Program of China, National Science and Technology Major Project, and CanSino Biologics.

negative: Overview of lethal human coronaviruses Coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. Seven coronaviruses that infect humans have been identified: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Among them, SARS-CoV and MERS-CoV caused outbreaks in 2002 and 2012, respectively. SARS-CoV-2 (COVID-19) is the most recently discovered. It has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. Viruses are genetically simple, yet highly diverse. However, the recent outbreaks of SARS-CoV and MERS-CoV, and the ongoing outbreak of SARS-CoV-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. Only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. This paper mainly focuses on SARS-CoV, MERS-CoV, and SARS-CoV-2. Here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment.

negative: Passive immunotherapy with convalescent plasma against COVID-19? What about the evidence base and clinical trials?

negative: Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development BACKGROUND: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. METHODS: We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. FINDINGS: Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. INTERPRETATION: MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.

negative: Altered microRNA expression and pre-mRNA splicing events reveal new mechanisms associated with early stage Mycobacterium avium subspecies paratuberculosis infection The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was “proliferation of endothelial cells”, indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as “proliferation of endothelial cells” (bta-miR-196 b), “bacteria recognition” (bta-miR-146 b), and “regulation of the inflammatory response” (bta-miR-146 b).

negative: Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.

negative: Early experience with convalescent plasma as immunotherapy for COVID‐19 in China: Knowns and unknowns BACKGROUND AND OBJECTIVES: In the absence of a vaccine or specific drug treatment options for coronavirus disease (COVID‐19), attention has been shifted in China to the possible therapeutic use of convalescent plasma. COVID‐19 convalescent plasma (CCP) is currently under investigation. We summarized clinical studies and other research data available as of May 5, 2020, on CCP therapy according to the Clinical Treatment Guideline of COVID‐19 Convalescent Plasma in China, as well as clinical experience at the First Affiliated Hospital of Zhejiang University, as part of a comprehensive anti‐epidemic strategy. MATERIALS AND METHODS: As of May 5, 2020, when the epidemic was well‐controlled in China, healthcare databases and sources of English literature relating to convalescent plasma were searched and reviewed. Sources of clinical and methodological heterogeneity were identified. RESULTS: As of May 5, 2020, up to 2,000 samples of CCP had been collected across China and administered to 700 COVID‐19 patients. From donors, 200–400 mL of plasma were collected at each donation, with antibody titers >1:160. We identified three clinical studies for COVID‐19 in China. Analyses showed a statistically significant improvement in clinical outcomes compared with untreated cases (P<0.001). No adverse effects were reported. CONCLUSION: From initial studies, convalescent plasma therapy appears effective and safe for COVID‐19. However, there is clearly a need for well‐designed RCTs (randomized controlled trials) or other formal studies to further evaluate the efficacy and any potential adverse effects of CCP.

negative: Evaluation of sensitivities and specificities of SARS-CoV detection by real-time quantitative reverse transcription-PCR assays The etiological agent of severe acute respiratory syndrome (SARS) was identified as a new coronavirus, termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study, we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreen™ dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities, 13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection, but because of its sequence variability in the different viral strains, primers and a probe based on the N gene were suitable substitutions. Meanwhile, we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

negative: Olfactory and taste disorders in COVID-19: a systematic review()() INTRODUCTION: The SARS-CoV-2 virus causes COVID-19, and it is responsible for the largest pandemic since the 1918 H1N1 influenza outbreak. The classic symptoms of the disease have been well defined by the World Health Organization; however, olfactory/gustatory disorders have been reported in some studies, but there are still several missing points in the understanding and in the consensus about the clinical management of these cases. OBJECTIVE: To identify evidence in the scientific literature about olfactory/gustatory disorders, their clinical presentation, prevalence and possible specific treatments associated with COVID-19. METHODS: A systematic review of articles published up to April 25, 2020 was performed in Medline, Cochrane Clinical Trials, ScienceDirect, Lilacs, Scopus and Google Schoolar, OpenGrey.eu, DissOnline, The New York Academy of Medicine and Reasearch Gate. Inclusion criteria: (1) Studies on patients with COVID-19; (2) Records of COVID-19 signs/symptoms, and olfactory/gustatory functions. Exclusion criteria: (1) Studies on non-human coronavirus; (2) Review articles; (3) Experimental studies (in animals or in vitro); (4) Olfactory/gustatory disorders initiated prior to SARS-CoV-2 infection. The risk assessment of bias of the selected studies was performed using the Newcastle-Ottawa scale. RESULTS: Six articles from the 1788 records met the inclusion criteria and were analyzed. A total of 1457 patients of different ethnicities were assessed; of them, 885 (60.7%) and 822 (56.4%) had smell and taste disorders, respectively, with women being most often affected. There were olfactory/gustatory disorders even without nasal obstruction/rhinorrhea and beginning even before the signs/symptoms of COVID-19; the recovery of smell/taste, when it occurs, usually happened in the first two weeks after COVID-19 resolution. There is evidence that olfactory/gustatory disorders are strong predictors of infection by SARS-CoV-2, and it is possible to recommend patient isolation, as early as of the medical consultation, preventing the spread of the virus. No scientific evidence has been identified for effective treatments for any of the disorders. CONCLUSION: Olfactory/gustatory disorders may occur at varying intensities and prior to the general symptoms of COVID-19 and should be considered as part of the clinical features of COVID-19, even in mild cases. There is still no scientific evidence of specific treatments for such disorders in COVID-19 disease.

negative: The Human Coronavirus Disease COVID-19: Its Origin, Characteristics, and Insights into Potential Drugs and Its Mechanisms The emerging coronavirus disease (COVID-19) swept across the world, affecting more than 200 countries and territories. Genomic analysis suggests that the COVID-19 virus originated in bats and transmitted to humans through unknown intermediate hosts in the Wuhan seafood market, China, in December of 2019. This virus belongs to the Betacoronavirus group, the same group of the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV), and for the similarity, it was named SARS-CoV-2. Given the lack of registered clinical therapies or vaccines, many physicians and scientists are investigating previously used clinical drugs for COVID-19 treatment. In this review, we aim to provide an overview of the CoVs origin, pathogenicity, and genomic structure, with a focus on SARS-CoV-2. Besides, we summarize the recently investigated drugs that constitute an option for COVID-19 treatment.

negative: COVID-19 vaccine development: Time to consider SARS-CoV-2 challenge studies? Abstract While a human challenge study holds the prospect of accelerating the development of a vaccine for the coronavirus SARS-CoV-2, it may be opposed due to risks of harm to participants and researchers. Given the increasing number of human deaths and severe disruption to lives worldwide, we argue that a SARS-CoV-2 challenge study is ethically justifiable as its social value substantially outweighs the risks. Such a study should therefore be seriously considered as part of the global research response towards the COVID-19 pandemic. In this paper, we contribute to the debate by addressing the misperception that a challenge study for the coronavirus would lower scientific and ethical standards for vaccine research and development, and examine how it could be ethically conducted. We also set out information that needs to be disclosed to prospective participants to obtain their consent.

negative: Mesenchymal Stem Cell Infusion Shows Promise for Combating Coronavirus (COVID-19)- Induced Pneumonia A new study published by the journal Aging & Disease reported that intravenous administration of clinical-grade human mesenchymal stem cells (MSCs) into patients with coronavirus disease 2019 (COVID-19) resulted in improved functional outcomes (Leng et al., Aging Dis, 11:216-228, 2020). This study demonstrated that intravenous infusion of MSCs is a safe and effective approach for treating patients with COVID-19 pneumonia, including elderly patients displaying severe pneumonia. COVID-19 is a severe acute respiratory illness caused by a new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, treating COVID-19 patients, particularly those afflicted with severe pneumonia, is challenging as no specific drugs or vaccines against SARS-CoV-2 are available. Therefore, MSC therapy inhibiting the overactivation of the immune system and promoting endogenous repair by improving the lung microenvironment after the SARS-CoV-2 infection found in this study is striking. Additional studies in a larger cohort of patients are needed to validate this therapeutic intervention further, however.

negative: A Scalable Topical Vectored Vaccine Candidate Against SARS-CoV-2 The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) caused an ongoing unprecedented global public health crises of coronavirus disease in 2019 (CoVID-19). The precipitously increased death rates, its impact on livelihood and trembling economies warrant the urgent development of SARS-CoV-2 vaccine which would be safe, efficacious and scalable. Owing to unavailability of the vaccine, we propose a de novo synthesised avian orthoavulavirus 1 (AOaV-1)-based topical respiratory vaccine candidate against CoVID-19. Avirulent strain of Newcastle disease virus, proto-type virus of AOaV-1, was engineered to express full length spike (S) glycoprotein which is highly neutralizing and major protective antigen of the SARS-CoV-2. Broad-scale in vitro characterization of recombinant vaccine candidate demonstrated efficient co-expression of the hemagglutinin-neuraminidase (HN) of AOaV-1 and S protein of SARS-CoV-2, and comparable replication kinetics were observed in cell culture model. The recombinant vaccine candidate virus actively replicated and spread within cells independently of exogenous trypsin. Interestingly, incorporation of S protein of SARS-CoV-2 into the recombinant AOaV-1 particles attributed the sensitivity to anti-SARS-CoV-2 antiserum and more prominently to anti-AOaV-1 antiserum. Finally, our results demonstrated that the recombinant vaccine vector stably expressed S protein after multiple propagation in chicken embryonated eggs, and this expression did not significantly impact the in vitro growth characteristics of the recombinant. Taken together, the presented respiratory vaccine candidate is highly attenuated in primates per se, safe and lacking pre-existing immunity in human, and carries the potential for accelerated vaccine development against CoVID-19 for clinical studies.

negative: Naturally Derived Membrane Lipids Impact Nanoparticle-Based Messenger RNA Delivery INTRODUCTION: Lipid based nanoparticles (LNPs) are clinically successful vectors for hepatic delivery of nucleic acids. These systems are being developed for non-hepatic delivery of mRNA for the treatment of diseases like cystic fibrosis or retinal degeneration as well as infectious diseases. Localized delivery to the lungs requires aerosolization. We hypothesized that structural lipids within LNPs would provide features of integrity which can be tuned for attributes required for efficient hepatic and non-hepatic gene delivery. Herein, we explored whether naturally occurring lipids that originate from the cell membrane of plants and microorganisms enhance mRNA-based gene transfection in vitro and in vivo and whether they assist in maintaining mRNA activity after nebulization. METHODS: We substituted DSPC, a structural lipid used in a conventional LNP formulation, to a series of naturally occurring membrane lipids. We measured the effect of these membrane lipids on size, encapsulation efficiency and their impact on transfection efficiency. We further characterized LNPs after nebulization and measured whether they retained their transfection efficiency. RESULTS: One plant-derived structural lipid, DGTS, led to a significant improvement in liver transfection of mRNA. DGTS LNPs had similar transfection ability when administered in the nasal cavity to conventional LNPs. In contrast, we found that DGTS LNPs had reduced transfection efficiency in cells pre-and post-nebulization while maintaining size and encapsulation similar to DSPC LNPs. CONCLUSIONS: We found that structural lipids provide differential mRNA-based activities in vitro and in vivo which also depend on the mode of administration. Understanding influence of structural lipids on nanoparticle morphology and structure can lead to engineering potent materials for mRNA-based gene therapy applications.

negative: Preliminary Results of Seroprevalence of SARS-CoV-2 at Community Clinics in Tokyo Serological evaluation with SARS-CoV-2 specific IgG antibody will be an alternative way to know the pandemic of novel coronavirus disease (COVID-19) if the capacity for diagnostic PCR test is limited. The point-of-care test to detect SARS-CoV-2 specific IgG antibody in peripheral blood (n =202) was performed in two community clinics in Tokyo, Japan. The overall positive rate of SRAS-CoV-2 IgG antibody was 5.9% (95% confidence interval[CI]: 3.1-10.1). Higher rate was observed for healthcare workers (n =55, 9.1 [3.0-20.0]). The limitation on antibody tests includes low sensitivity and potent cross-reactivity with the previous coronavirus. Robust healthcare policy to efficiently monitor COVID-19 spread is warranted in Tokyo.

negative: Effects of a SARS-associated coronavirus vaccine in monkeys The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus. Here, we have investigated the ability of adenoviral delivery of codon-optimised SARS-CoV strain Urbani structural antigens spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce virus-specific broad immunity in rhesus macaques. We immunised rhesus macaques intramuscularly with a combination of the three Ad5-SARS-CoV vectors or a control vector and gave a booster vaccination on day 28. The vaccinated animals all had antibody responses against spike protein S1 fragment and T-cell responses against the nucleocapsid protein. All vaccinated animals showed strong neutralising antibody responses to SARS-CoV infection in vitro. These results show that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS causal agent.

negative: Selection of proteins with desired properties from natural proteome libraries using mRNA display. mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca(2+)-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display-based selection can be accomplished in ~7 d.

negative: Potential for elimination of SAR-CoV-2 through vaccination as inspired by elimination of multiple influenza viruses through natural pandemics or mass vaccination The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed many lives worldwide. To combat the pandemic, multiple types of vaccines are under development with unprecedented rapidity. Theoretically, future vaccination against COVID-19 may fall into long-term costly guerrilla warfare between SARS-CoV-2 and humans. Elimination of SARS-CoV-2 through vaccination to avoid the potential long-term costly guerrilla warfare, if possible, is highly desired and worth intensive consideration. Human influenza pandemics emerging in 1957, 1968, and 2009 established strong global herd immunity and led to the elimination of three human influenza viruses, which circulated worldwide for years before the pandemics. Moreover, both clade 7.2 of subtype H5 highly pathogenic avian influenza virus and subtype H7N9 avian influenza virus circulated in poultry in China for years, and they have been virtually eliminated through mass vaccination in recent years. These facts suggest that the rapid establishment of global herd immunity through mass vaccination using an appropriate vaccine could eliminate SARS-CoV-2. The coming 2 years are a golden time for elimination through vaccination, which requires tremendous national and international collaboration. This review also prioritizes the efficacy of vaccines for COVID-19 and elucidates the importance of the development of more live vaccines for COVID-19.

negative: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 10(4) PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERS(MA)6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 10(6) PFU UV-inactivated MERS(MA)6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.

negative: Epitope based vaccine prediction for SARS-COV-2 by deploying immuno-informatics approach A new virus termed SARS-COV-2 (causing COVID-19 disease) can exhibit a progressive, fatal impact on individuals. The World Health Organization (WHO) has declared the spread of the virus to be a global pandemic. Currently, there are over 1 million cases and over 100,000 confirmed deaths due to the virus. Hence, prophylactic and therapeutic strategies are promptly needed. In this study we report an epitope, ITLCFTLKR, which is biochemically fit to HLA allelic proteins. We propose that this could be used as a potential vaccine candidate against SARS-COV-2. A selected putative epitope and HLA-allelic complexes show not only better binding scores, but also RMSD values in the range of 0-1â¯Å. This epitope was found to have a 99.8% structural favorability as per Ramachandran-plot analysis. Similarly, a suitable range of IC50 values and population coverage was obtained to represent greater validation of T-cell epitope analysis. Stability analysis using MDWeb and half-life analysis using the ProtParam tool has confirmed that this epitope is well-selected. This new methodology of epitope-based vaccine prediction is fundamental and fast in application, ad can be economically beneficial and viable.

negative: Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2 Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.

negative: Severe acute respiratory syndrome coronaviruses with mutations in the E protein are attenuated and promising vaccine candidates. UNLABELLED Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4(+) and CD8(+) T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates. IMPORTANCE Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo. Here, we show that small deletions and modifications within the E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4(+) and CD8(+) T cell counts in vivo, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development.

negative: COVID-19 and the Risk to Health Care Workers: A Case Report

negative: SARS-CoV-2 (COVID-19) Vaccine Development and Production: An Ethical Way Forward The world awaits a SARS-CoV-2 virus (i.e., COVID-19 disease) vaccine to keep the populace healthy, fully reopen their economies, and return their social and healthcare systems to “normal.” Vaccine safety and efficacy requires meticulous testing and oversight; this paper describes how despite grandiose public statements, the current vaccine development, testing, and production methods may prove to be ethically dubious, medically dangerous, and socially volatile. The basic moral concern is the potential danger to the health of human test subjects and, eventually, many vaccine recipients. This is further complicated by economic and political pressures to reduce government oversight on rushed vaccine testing and production, nationalistic distribution goals, and failure to plan for the widespread immunization needed to produce global herd immunity. As this paper asserts, the public must be better informed to assess promises about the novel vaccines being produced and to tolerate delays and uncertainty.

negative: Dynamic RNA Modifications in Posttranscriptional Regulation Cellular RNAs can be chemically modified over a hundred different ways. These modifications were once thought to be static, discrete, and utilized to fine-tune RNA structure and function. However, recent studies have revealed that some modifications, like mRNA methylation, can be reversed, and these reversible modifications may play active roles in regulating diverse biological processes. In this perspective, we summarize examples of dynamic RNA modifications that affect biological functions. We further propose that reversible modifications might occur on tRNA, rRNA, and other noncoding RNAs to regulate gene expression analogous to the reversible mRNA methylation.

negative: Development of Gene-Based Vectors for Immunization

negative: Dynamic profiles, biodistribution and integration evaluation after intramuscular/intravenous delivery of a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in vaccinated normal rodent BACKGROUND: The persistence, biodistribution, and risk of integration into the host genome of any new therapeutic DNA vaccine must be established in preclinical studies. We previously developed the DNA vaccine pcDNA-CCOL2A1 encoding chicken type II collagen (CCII) for the treatment of rheumatoid arthritis (RA). In the present study, we characterized its dynamic profile, biodistribution, and potential for genomic DNA integration in normal vaccinated rodent. RESULTS: A real-time quantitative PCR analysis (RT-qPCR) of animals administered a single dose of pcDNA-CCOL2A1 (300 μg/kg by intramuscular injection) showed that CCOL2A1 mRNA level in the blood peaked between 2 and 6 h post-immunization and then rapidly declined, and was undetectable between day 1–42. CCOL2A1 transcript was detected at the muscle injection site on days 3–14 post-immunization. Starting from day 14, the transcript was detected in the heart, liver, lung, and kidney but not in the spleen or thymus, and was expressed only in the lung on day 28. There was no CCOL2A1 mRNA present in the testes or ovaries at any time point. Non-invasive in vivo fluorescence imaging revealed CCII protein expression from 2 h up to day 10 and from 2 h up to day 35 after administration of pcDNA-CCOL2A1 via the intravenous and intramuscular routes, respectively; the protein had disappeared by day 42. Importantly, CCOL2A1 was not integrated into the host genome. CONCLUSIONS: These results indicate that pcDNA-CCOL2A1 vaccine is rapidly cleared within a short period of time and is therefore safe, and merits further development as a therapeutic vaccine for RA treatment.

negative: Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination Abstract The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

negative: The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 The present outbreak of a coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) is the third documented spillover of an animal coronavirus to humans in only two decades that has resulted in a major epidemic. The Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the classification of viruses and taxon nomenclature of the family Coronaviridae, has assessed the placement of the human pathogen, tentatively named 2019-nCoV, within the Coronaviridae. Based on phylogeny, taxonomy and established practice, the CSG recognizes this virus as forming a sister clade to the prototype human and bat severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and designates it as SARS-CoV-2. In order to facilitate communication, the CSG proposes to use the following naming convention for individual isolates: SARS-CoV-2/host/location/isolate/date. While the full spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined, the independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying viruses at the species level to complement research focused on individual pathogenic viruses of immediate significance. This will improve our understanding of virus–host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

negative: Potential SARS-CoV-2 Preimmune IgM Epitopes While studying the human public IgM igome as represented by a library of 224,087 linear mimotopes, three exact matches to peptides in the proteins of SARS-CoV-2 were found: two in the open reading frame 1ab and one in the spike protein. Joining the efforts to fast track SARS-CoV-2 vaccine development, here we describe briefly these potential epitopes in comparison to mimotopes representing peptides of SARS-CoV, HCoV 229E and OC43.

negative: COVID-19: Emergence, Spread, Possible Treatments, and Global Burden The Coronavirus (CoV) is a large family of viruses known to cause illnesses ranging from the common cold to acute respiratory tract infection. The severity of the infection may be visible as pneumonia, acute respiratory syndrome, and even death. Until the outbreak of SARS, this group of viruses was greatly overlooked. However, since the SARS and MERS outbreaks, these viruses have been studied in greater detail, propelling the vaccine research. On December 31, 2019, mysterious cases of pneumonia were detected in the city of Wuhan in China's Hubei Province. On January 7, 2020, the causative agent was identified as a new coronavirus (2019-nCoV), and the disease was later named as COVID-19 by the WHO. The virus spread extensively in the Wuhan region of China and has gained entry to over 210 countries and territories. Though experts suspected that the virus is transmitted from animals to humans, there are mixed reports on the origin of the virus. There are no treatment options available for the virus as such, limited to the use of anti-HIV drugs and/or other antivirals such as Remdesivir and Galidesivir. For the containment of the virus, it is recommended to quarantine the infected and to follow good hygiene practices. The virus has had a significant socio-economic impact globally. Economically, China is likely to experience a greater setback than other countries from the pandemic due to added trade war pressure, which have been discussed in this paper.

negative: Elucidation of Cellular Targets and Exploitation of the Receptor Binding Domain of SARS-CoV-2 for vaccine and monoclonal antibody synthesis The pandemic caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has resulted in over 452,822 deaths in the first twenty days of June 2020 due to the coronavirus virus disease 2019 (COVID-19). The SARS-CoV-2 uses the host angiotensin-converting enzyme 2 (ACE2) receptor to gain entry inside the human cells where it replicates by using the cell protein synthesis mechanisms. The knowledge of the tissue distribution of ACE2 in human organs is therefore important to predict the clinical course of the COVID-19. Also important is the understanding of the viral receptor-binding domain (RBD) a region within the spike (S) proteins that enable the entry of the virus into the host cells to synthesize vaccine and monoclonal antibodies (mAbs). We performed an exhaustive search of human protein databases to establish the tissues that express ACE2 and performed an in-depth analysis like sequence alignments and homology modeling of the spike protein (S) of the SARS-CoV-2 to identify antigenic regions in the RBD that can be exploited to synthesize vaccine and mAbs. Our results show that ACE2 is widely expressed in human organs that may explain the pulmonary, systemic, and neurological deficits seen in COVID-19 patients. We show that though the S protein of the SARS-CoV-2 is a homolog of S protein of SARS-CoV-1, it has regions of dissimilarities in the RBD and transmembrane segments. We show peptide sequences in the RBD of SARS-CoV-2 that can bind to the MHC alleles and serve as effective epitopes for vaccine and mAbs synthesis. This article is protected by copyright. All rights reserved.

negative: Computers and viral diseases. Preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the SARS-CoV-2 (2019-nCoV, COVID-19) coronavirus This paper concerns study of the genome of the Wuhan Seafood Market isolate believed to represent the causative agent of the disease COVID-19. This is to find a short section or sections of viral protein sequence suitable for preliminary design proposal for a peptide synthetic vaccine and a peptidomimetic therapeutic, and to explore some design possibilities. The project was originally directed towards a use case for the Q-UEL language and its implementation in a knowledge management and automated inference system for medicine called the BioIngine, but focus here remains mostly on the virus itself. However, using Q-UEL systems to access relevant and emerging literature, and to interact with standard publically available bioinformatics tools on the Internet, did help quickly identify sequences of amino acids that are well conserved across many coronaviruses including 2019-nCoV. KRSFIEDLLFNKV was found to be particularly well conserved in this study and corresponds to the region around one of the known cleavage sites of the SARS virus that are believed to be required for virus activation for cell entry. This sequence motif and surrounding variations formed the basis for proposing a specific synthetic vaccine epitope and peptidomimetic agent. The work can, nonetheless, be described in traditional bioinformatics terms, and readily reproduced by others, albeit with the caveat that new data and research into 2019-nCoV is emerging and evolving at an explosive pace. Preliminary studies using molecular modeling and docking, and in that context the potential value of certain known herbal extracts, are also described.

negative: Medication for COVID-19—an Overview of Approaches Currently Under Study BACKGROUND: With the worldwide spread of SARS-CoV-2 infection, it is becoming increasingly urgent to develop a vaccine to prevent COVID-19, as well as effective drugs to treat it. METHODS: This article is based on a selective literature search in PubMed and ClinicalTrials.gov, followed by an assessment of the ongoing clinical trials that were revealed by the search. RESULTS: A number of substances have been found to prevent the reproduction of SARS-CoV-2 in vitro. These include virustatic agents that have already been approved for the treatment of other types of viral infection, as well as drugs that are currently used for entirely different purposes. High in vitro activity has been found for the nucleotide analogue remdesivir, for the antimalarial drug chloroquine, and for nitazoxanide, a drug used to treat protozoan infections. Because the virus enters human cells by way of the membrane-associated angiotensin converting enzyme 2 (ACE2), keeping the virus from docking to this receptor is a conceivable treatment approach. Transmembrane protease serine 2 (TMPRSS2) plays a role in the fusion of the virus with cells; inhibitors of this enzyme are known as well. The potential therapeutic efficacy and tolerability of these and other active substances remain to be investigated in clinical trials. At present, more than 80 trials on COVID-10 have already been registered with ClinicalTrials.gov. Some initial findings should already be available in late April 2020. CONCLUSION: Clinical trials are now indispensable in order to determine the true clinical benefits and risks of the substances that have been found to be active against SARS-CoV-2 in vitro. There is not yet any recommendation for the therapeutic use of any particular agent beyond standard supportive treatment.

negative: A recombinant Lactobacillus plantarum strain expressing the spike protein of SARS-CoV-2 Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic in the past four months and causes respiratory disease in humans of almost all ages. Although several drugs have been announced to be partially effective treatments for this disease, no approved vaccine is available. Here, we described the construction of a recombinant Lactobacillus plantarum strain expressing the SARS-CoV-2 spike protein. The results showed that the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity. The highest protein yield was obtained under the following conditions: cells were induced with 50 ng/mL SppIP at 37 °C for 6-10 h. The recombinant spike (S) protein was stable under normal conditions and at 50 °C, pH = 1.5, or a high salt concentration. Recombinant L. plantarum may provide a promising food-grade oral vaccine candidate against SARS-CoV-2 infection.

negative: Food Restriction Differentially Affects mRNAs Encoding the Major Anterior Pituitary Tropic Hormones Chronic food restriction (FR) leads to adaptive cellular changes, some of which retard aging. Moreover, some of these changes occur within weeks after onset of FR. Because neuroendocrine mechanisms may mediate these effects, we measured the effect of FR on the messenger ribonucleicacids (mRNAs) encoding all of the tropic hormones of the anterior pituitary (AP). Slot blot and solution hybridization were conducted on AP ribonucleicacid (RNA) samples obtained at 0500 h (AM) and 1500 h (PM) from 3-month-old male Fischer 344 rats fed ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. Poly A RNA/μg total RNA was similar in AL and FR rats, indicating that there was no overall effect of FR on mRNA levels. The level of proopiomelanocortin (POMC) mRNA was not reduced by FR when expressed per fig of RNA or as total AP content. By contrast, the total AP content of the mRNAs encoding LHβ, FSHβ TSHβ, GH, and PRL was markedly reduced by FR. When expressed per fig of RNA, however, only GH (AM and PM), FSHβ (AM), TSHbeta; (PM), and PRL (PM) were reduced by FR. These results reveal that FR differentially affects pituitary tropic hormone mRNA levels within weeks after onset of FR, and are consistent with a role for neuroendocrine alterations in the initiation of adaptive cellular responses to FR.

negative: SARS-CoV-2 Infection and the Liver A novel strain of coronoviridae (SARS-CoV-2) was reported in Wuhan China in December 2019. Initially, infection presented with a broad spectrum of symptoms which typically included muscle aches, fever, dry cough, and shortness of breath. SARS-CoV-2 enters cells via ACE2 receptors which are abundant throughout the respiratory tract. However, there is evidence that these receptors are abundant throughout the body, and just as abundant in cholangiocytes as alveolar cells, posing the question of possible direct liver injury. While liver enzymes and function tests do seem to be deranged in some patients, it is questionable if the injury is due to direct viral damage, drug-induced liver injury, hypoxia, or microthromboses. Likely, the injury is multifactoral, and management of infected patients with pre-existing liver disease should be taken into consideration. Ultimately, a vaccine is needed to aid in reducing cases of SARS-CoV-2 and providing immunity to the general population. However, while considering the types of vaccines available, safety concerns, particularly of RNA- or DNA-based vaccines, need to be addressed.

negative: Epitope based peptide vaccine against SARS-COV2: an immune-informatics approach. World is witnessing exponential growth of SARS-CoV2 and fatal outcomes of COVID 19 has proved its pandemic potential already by claiming more than 3 lakhs deaths globally. If not controlled, this ongoing pandemic can cause irreparable socio-economic and psychological impact worldwide. Therefore a safe and effective vaccine against COVID 19 is exigent. Recent advances in immunoinformatics approaches could potentially decline the attrition rate and accelerate the process of vaccine development in these unprecedented times. In the present study, a multivalent subunit vaccine targeting S2 subunit of the SARS-CoV2 S glycoprotein has been designed using open source, immunoinformatics tools. Designed construct comprises of epitopes capable of inducing T cell, B cell (Linear and discontinuous) and Interferon γ. physiologically, vaccine construct is predicted to be thermostable, antigenic, immunogenic, non allergen and non toxic in nature. According to population coverage analysis, designed multiepitope vaccine covers 99.26% population globally. 3D structure of vaccine construct was designed, validated and refined to obtain high quality structure. Refined structure was docked against Toll like receptors to confirm the interactions between them. Vaccine peptide sequence was reverse transcribed, codon optimized and cloned in pET vector. Our in-silico study suggests that proposed vaccine against fusion domain of virus has the potential to elicit an innate as well as humoral immune response in human and restrict the entry of virus inside the cell. Results of the study offer a framework for in-vivo analysis that may hasten the process of development of therapeutic tools against COVID 19.Communicated by Ramaswamy H. Sarma.

negative: Nascent Synthesis of Leader Sequence-Containing Subgenomic mRNAs in Coronavirus Genome-Length Replicative Intermediate RNA Abstract Infection with coronavirus results in the accumulation of genomic-sized mRNA and six to eight subgenomic mRNAs that make up a 3′ coterminal nested-set structure. Genome-length negative-strand RNA and subgenomic-length negative-strand RNAs, each of which corresponds to each of the subgenomic mRNAs, also accumulate in infected cells. The present study examined whether the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis. Genome-length replicative intermediate (RI) RNA was purified by two-dimensional gel electrophoresis of intracellular RNAs from cells infected with mouse hepatitis virus. RNase A treatment of the purified genome-length RI resulted in the production of the genome-length replicative form RNA, indicating that the genome-length RI included genome-length template RNA. RNase protection assays using the purified genome-length RI and two probes, which corresponded to the 5′ 300-nt region of mRNA 6 and to the same region of mRNA 7, showed the presence of nascent leader sequence-containing subgenomic mRNAs in the genome-length RI. These data demonstrated that the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis.

negative: DNA vaccine protection against SARS-CoV-2 in rhesus macaques The global COVID-19 pandemic caused by the SARS-CoV-2 virus has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 Spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. Following vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log(10) reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

negative: A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread world-wide with millions of cases and hundreds of thousands of deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8+ and CD4+ T cell responses with S protein-specific killing activity were detected. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines. Author Contributions CH performed research, analyzed data, and wrote the paper; CS performed research and analyzed data; AA performed research and analyzed data; AE performed research and analyzed data; SM performed research, analyzed data, and wrote the paper; MH developed the bioinformatics pipeline and analyzed data; BS contributed new reagents and concepts; MDM designed and supervised research, analyzed data and wrote the paper; all authors read, corrected and approved the final manuscript. Significance Statement The COVID-19 pandemic has caused hundreds of thousands of deaths, yet. Therefore, effective vaccine concepts are urgently needed. In search for such a concept, we have analysed a measles virus-based vaccine candidate targeting SARS-CoV-2. Using this well known, safe vaccine backbone, we demonstrate here induction of functional immune responses in both arms of adaptive immunity with the desired immune bias. Therefore, occurrence of immunopathologies such as antibody-dependent enhancement or enhanced respiratory disease is rather unlikely. Moreover, the candidate still induces immunity against the measles, recognized as a looming second menace, when countries are entrapped to stop routine vaccination campaigns in the face of COVID-19. Thus, a bivalent measles-based COVID-19 vaccine could be the solution for two significant public health threats.

negative: Management of hypertension in COVID-19 The ACE2 receptor plays a central role in severe acute respiratory syndrome coronavirus 2 host cell entry and propagation. It has therefore been postulated that angiotensin converting enzyme inhibitors and angiotensin receptor blockers may upregulate ACE2 expression and thus increase susceptibility to infection. We suggest that alternative anti-hypertensive agents should be preferred among individuals who may be exposed to this increasingly common and potentially lethal virus.

negative: [Detection of the mRNA expression of human angiotensin-converting enzyme 2 as a SARS coronavirus functional receptor in human femoral head]. OBJECTIVE To investigate the mRNA expression of severe acute respiratory syndrome-associated coronavirus (SARS-COV) functional receptor, angiotensin-converting enzyme 2 (ACE2), in human femoral head and conjunctiva, and explore the possible entry route of SARS-COV in human femoral head. METHODS ACE2 mRNA in human femoral head was detected by nested RT-PCR with human beta actin gene as the positive control. RESULTS The mRNA of human beta actin gene could be amplified efficiently in all the tissue samples. The mRNA of human ACE2 was expressed efficiently in the normal lung tissue, but not in the cartilage and cancellous bone under the weight-bearing area of the femoral head. CONCLUSION SARS-COV can not infect the femoral head tissue and lead to avascular necrosis of the femoral head directly by the spike glycoprotein, and mechanism of the virus for causing avascular necrosis needs further investigation.

negative: Quantification of individual subgenomic mRNA species during replication of the coronavirus transmissible gastroenteritis virus Abstract A biotinylated-oligonucleotide-based method was used to isolate the subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) to investigate the amounts of the mRNAs produced at early, middle and late times in the replication cycle. TGEV mRNA 6, which encodes the N protein, was observed to be the most abundant species throughout the replication cycle. The ratios of mRNA 6 to the other mRNAs were 1:0.11 (mRNA 2), 1:0.16 (mRNAs 3 and 4) and 1:0.37 (mRNA 5) at 12 h post-infection. All the mRNA species were differentially regulated throughout the replication cycle, although the rate of accumulation of mRNAs 4, 5, and 6, but not mRNA 3, increased markedly towards the end of the replication cycle. mRNA 7 was not detected in the system used. There was no observable correlation between the amounts of each mRNA synthesised and the potential degree of base pairing between the 3′ end of the leader sequence and the transcription associated sequences on the genomic RNA at any time during the replication cycle. This indicates that the extent of base pairing was not the only factor involved in the control of subgenomic mRNA synthesis.

negative: Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-stranded RNA Accumulation and Anti-Viral Responses By accelerating global mRNA decay, many viruses impair host protein synthesis, limiting host defenses and stimulating virus mRNA translation. Vaccinia virus (VacV) encodes two decapping enzymes (D9, D10) that remove protective 5′ caps on mRNAs, presumably generating substrates for degradation by the host exonuclease Xrn1. Surprisingly, we find VacV infection of Xrn1-depleted cells inhibits protein synthesis, compromising virus growth. These effects are aggravated by D9-deficiency and dependent upon a virus transcription factor required for intermediate and late mRNA biogenesis. Considerable double-stranded RNA (dsRNA) accumulation in Xrn1-depleted cells is accompanied by activation of host dsRNA-responsive defenses controlled by PKR and 2′-5′-oligoadenylate synthetase (OAS), which respectively inactivate the translation initiation factor eIF2 and stimulate RNA cleavage by RNase L. This proceeds despite VacV-encoded PKR and RNase L antagonists being present. Moreover, Xrn1-depletion sensitizes uninfected cells to dsRNA treatment. Thus, Xrn1 is a cellular factor regulating dsRNA accumulation and dsRNA-responsive innate immune effectors.

negative: Risk of Virus Contamination Through Surgical Smoke During Minimally Invasive Surgery: A Systematic Review of Literature on a Neglected Issue Revived in the COVID-19 Pandemic Era CONTEXT: The coronavirus disease 2019 (COVID-19) pandemic raised concerns about the safety of laparoscopy due to the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diffusion in surgical smoke. Although no case of SARS-CoV-2 contagion related to surgical smoke has been reported, several international surgical societies recommended caution or even discouraged the use of a laparoscopic approach. OBJECTIVE: To evaluate the risk of virus spread due to surgical smoke during surgical procedures. EVIDENCE ACQUISITION: We searched PubMed and Scopus for eligible studies, including clinical and preclinical studies assessing the presence of any virus in the surgical smoke from any surgical procedure or experimental model. EVIDENCE SYNTHESIS: We identified 24 studies. No study was found investigating SARS-CoV-2 or any other coronavirus. About other viruses, hepatitis B virus was identified in the surgical smoke collected during different laparoscopic surgeries (colorectal resections, gastrectomies, and hepatic wedge resections). Other clinical studies suggested a consistent risk of transmission for human papillomavirus (HPV) in the surgical treatments of HPV-related disease (mainly genital warts, laryngeal papillomas, or cutaneous lesions). Preclinical studies showed conflicting results, but HPV was shown to have a high risk of transmission. CONCLUSIONS: Although all the available data come from different viruses, considering that the SARS-CoV-2 virus has been shown in blood and stools, the theoretical risk of virus diffusion through surgical smoke cannot be excluded. Specific clinical studies are needed to understand the effective presence of the virus in the surgical smoke of different surgical procedures and its concentration. Meanwhile, adoption of all the required protective strategies, including preoperative patient nasopharyngeal swab for COVID-19, seems mandatory. PATIENT SUMMARY: In this systematic review, we looked at the risk of virus spread from surgical smoke exposure during surgery. Although no study was found investigating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or any other coronavirus, we found that the theoretical risk of virus diffusion through surgical smoke cannot be excluded.

negative: Evaluation of Serologic and Antigenic Relationships Between Middle Eastern Respiratory Syndrome Coronavirus and Other Coronaviruses to Develop Vaccine Platforms for the Rapid Response to Emerging Coronaviruses Background. Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs. Methods. Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs. Results. Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43. Conclusions. Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs.

negative: Smoking is Associated with COVID-19 Progression: A Meta-Analysis Objective: To determine the association between smoking and progression of COVID-19. Design: A meta-analysis of 12 published papers. Data Source: PubMed database was searched on April 6, 2020. Eligibility criteria and data analysis: We included studies reporting smoking behavior of COVID-19 patients and progression of disease. Search terms included smoking, smoker*, characteristics, risk factors, outcomes, and COVID-19, COVID, coronavirus, sar cov-2, sar cov 2. There were no language limitations. One author extracted information for each study, screened the abstract or the full text, with questions resolved through discussion among both authors. A random effects meta-analysis was applied. Main Outcome Measures: The study outcome was progression of COVID-19 among people who already had the disease. Results: We identified 12 papers with a total of 9,025 COVID-19 patients, 878 (9.7%) with severe disease and 495 with a history of smoking (5.5%). The meta-analysis showed a significant association between smoking and progression of COVID-19 (OR 2.25, 95% CI 1.49-3.39, p=0.001). Limitations in the 12 papers suggest that the actual risk of smoking may be higher. Conclusions: Smoking is a risk factor for progression of COVID-19, with smokers having higher odds of COVID-19 progression than never smokers. Physicians and public health professionals should collect data on smoking as part of clinical management and add smoking cessation to the list of practices to blunt the COVID-19 pandemic.

negative: Prevention and Control Strategies for SARS-CoV-2 Infection The population of 168 countries all over the world is struggling with the outbreak of COVID-19. The outbreak is declared as pandemic and public health emergency of international concern declared by WHO. SARS-CoV-2 responsible for the present health emergency exhibited close resemblance with SARS-CoV. Both the viruses are zoonotic and belong to a large family of viruses Coronaviridae. The complete virus particle is made up of four major structural proteins, namely spikes (S), nucleocapsid (N), membrane (M), and envelope (E) encoded by virus genome. The S protein of virus shows similarity to S protein of SARS-CoV. COVID-19 spreads from person to person, and this makes it more vulnerable for causing infection. Several efforts are taken to find prevention strategies for COVID-19. Researchers across the globe are working to find effective vaccination for SARS-CoV-2. There is no vaccine or medication available till date for COVID-19. Preventive measures such as social distancing, awareness, maintenance of hygiene, isolation, and movement restrictions can help in control of COVID-19 spread. Proper sanitization and cleaned and sanitized public transport can be effective in inhibiting the spread of the virus. In the present situation of medical emergency, cooperation and support by following advices from the WHO and government only facilitate everyone to come over.

negative: Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag Abstract Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.

negative: N6-methyladenosine regulates PEDV replication and host gene expression Methylation of the N6 position of adenosine (m(6)A) is a widespread RNA modification that is critical for various physiological and pathological processes. Although this modification was also found in the RNA of several viruses almost 40 years ago, its biological functions during viral infection have been elucidated recently. Here, we investigated the effects of viral and host RNA methylation during porcine epidemic diarrhea virus (PEDV) infection. The results demonstrated that the m(6)A modification was abundant in the PEDV genome and the host methyltransferases METTL3 and METTL14 and demethylase FTO were involved in the regulation of viral replication. The knockdown of the methyltransferases increased PEDV replication while silencing the demethylase decreased PEDV output. Moreover, the proteins of the YTHDF family regulated the PEDV replication by affecting the stability of m(6)A-modified viral RNA. In particular, PEDV infection could trigger an increasement of m(6)A in host RNA and decrease the expression of FTO. The m(6)A modification sites in mRNAs and target genes were also altered during PEDV infection. Additionally, part of the host responses to PEDV infection was controlled by m(6)A modification, which could be reversed by the expression of FTO. Taken together, our results identified the role of m(6)A modification in PEDV replication and interactions with the host.

negative: Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific SARS proteins, 7a/7b, 3a/3b and S Abstract The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.

negative: Vasculopathy and Coagulopathy Associated with SARS-CoV-2 Infection. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has resulted in > 500,000 deaths worldwide, including > 125,000 deaths in the U.S. since its emergence in late December 2019 and June 2020. Neither curative anti-viral drugs nor a protective vaccine is currently available for the treatment and prevention of COVID-19. Recently, new clinical syndromes associated with coagulopathy and vasculopathy have emerged as a cause of sudden death and other serious clinical manifestations in younger patients infected with SARS-CoV-2 infection. Angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 and other coronaviruses, is a transmembrane protein expressed by lung alveolar epithelial cells, enterocytes, and vascular endothelial cells, whose physiologic role is to induce the maturation of angiotensin I to generate angiotensin 1-7, a peptide hormone that controls vasoconstriction and blood pressure. In this review, we provide the general context of the molecular and cellular mechanisms of SARS-CoV-2 infection with a focus on endothelial cells, describe the vasculopathy and coagulopathy syndromes in patients with SARS-CoV-2, and outline current understanding of the underlying mechanistic aspects.

negative: Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host’s immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.

negative: Selenium – associated gene signatures within the SARS-CoV-2 – host genomic interaction interface A recent study by Sies and Parnham expanded on the therapeutic potential of Epselen, an organoselenium compound, as an inhibitor of SARS-CoV-2’s main protease. In this study, using an unbiased, data driven bioinformatic screening of two independently extracted SARS-CoV-2 – induced host gene signatures confirms Selenium as a potential drug candidate. This pathway based approach adds transcriptomic evidence complimenting the protein-centered rationale expanded on by the authors. Given that aside from host – protein interactions, antisense transcript mRNA-mRNA interactions have been demonstrated to occur at selenocysteine related insertions in RNA viruses, transcriptomic-level evidence offer unique insight into viral lifecycles. Furthermore, the pathways identified from these two selenium associated signatures provide further insight into the perturbations of selenium metabolism introduced by SARS-CoV-2 during its lifecycle.

negative: When a COVID-19 vaccine is ready, will we all be ready for it?

negative: Withanone and Withaferin-A are predicted to interact with transmembrane protease serine 2 (TMPRSS2) and block entry of SARS-CoV-2 into cells Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use. Communicated by Ramaswamy H. Sarma

negative: Inhibition of measles virus infections in cell cultures by peptide-conjugated morpholino oligomers. Measles virus (MeV) is a highly contagious human pathogen. Despite the success of measles vaccination programs, measles is still responsible for an estimated 245,000 deaths each year. There are currently no antiviral compounds available for the treatment of measles. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are antisense compounds that enter cells readily and can interfere with mRNA function by steric blocking. A panel of PPMO was designed to target various sequences of MeV RNA that are known to be important for viral replication. Five PPMO, targeting MeV genomic RNA or mRNA, inhibited the replication of MeV, in a dose-responsive and sequence-specific manner in cultured cells. One of the highly active PPMO (PPMO 454), targeting a conserved sequence in the translation start site of the mRNA coding for the nucleocapsid protein, inhibited multiple genotypes of MeV. This report provides evidence that PPMO treatment represents a promising approach for developing antiviral agents against measles and other paramyxoviruses.

negative: From COVID-19 research to vaccine application: why might it take 17 months not 17 years and what are the wider lessons? It is often said that it takes 17 years to move medical research from bench to bedside. In a coronavirus disease (COVID-19) world, such time-lags feel intolerable. In these extraordinary circumstances could years be made into months? If so, could those lessons be used to accelerate medical research when the crisis eases? To measure time-lags in health and biomedical research as well as to identify ways of reducing them, we developed and published (in 2015) a matrix consisting of overlapping tracks (or stages/phases) in the translation from discovery research to developed products, policies and practice. The matrix aids analysis by highlighting the time and actions required to develop research (and its translation) both (1) along each track and (2) from one track to another, e.g. from the discovery track to the research-in-humans track. We noted four main approaches to reducing time-lags, namely increasing resources, working in parallel, starting or working at risk, and improving processes. Examining these approaches alongside the matrix helps interpret the enormous global effort to develop a vaccine for the 2019 novel coronavirus SARS-CoV-2, the causative agent of COVID-19. Rapid progress in the discovery/basic and human research tracks is being made through a combination of large-scale funding, work being conducted in parallel (between different teams globally and through working in overlapping tracks), working at greater (but proportionate) risk to safety than usual, and adopting various new processes. The overlapping work of some of the teams involves continuing animal research whilst entering vaccine candidates into Phase I trials alongside planning their Phase II trials. The additional funding available helps to reduce some of the usual financial risks in moving so quickly. Going forward through the increasingly large human trials for safety, dosage and efficacy, it will be vital to overlap work in parallel in the often challenging public policy and clinical tracks. Thus, regulatory and reimbursement bodies are beginning and preparing rapid action to pull vaccines proving to be safe and effective through to extraordinarily rapid application to the general population. Monitoring the development of a COVID-19 vaccine using the matrix (modified as necessary) could help identify which of the approaches speeding development and deployment could be usefully applied more widely in the future.

negative: The development of Chinese specific human cytomegalovirus polyepitope recombinant vaccine Abstract Human cytomegalovirus (HCMV) infection is a major cause of morbidity in the recipients of organ transplants and in the congenitally infected infants. HCMV vaccine has emerged as an effective approach to prevent HCMV infection particularly for the development of multiple viral antigens vaccination and human leukocyte antigen (HLA)-restricted polyepitope technology. As the Chinese population makes up more than one fifth of the population worldwide, it is important to develop HCMV vaccines more specific for the Chinese population by targeting Chinese-restricted HLA alleles and antigens. In the present study, we designed a novel chimeric polyepitope vaccine based on the replication-deficient adenovirus Ad5F35, which encodes 83 HCMV T cell epitopes from 15 different HCMV antigens, restricted to 14 HLA I and 7 HLA II alleles that cover 92% of the Chinese population. Our results show that the recombinant adenovirus vaccine Ad5F35-CTL·Th can be efficiently transfected and expressed in peripheral blood mononuclear cells (PBMCs) with little cytopathic activity. Ad5F35-CTL·Th can also be endogenously processed and presented by PBMCs. Ad5F35-CTL·Th-stimulated HCMV-specific cytotoxic T lymphocytes (CTLs) showed strong cytolytic activity against HCMV polyepitope-sensitized target cells. The CTL activity was accompanied by high levels of IFN-γ production after Ad5F35-CTL·Th stimulation. The specificity and vigorous response to the recombinant adenovirus vaccine in vitro makes it a potential candidate to be used for transplantation recipients or congenitally infected infants.

negative: The Complex Relationship between HTLV-1 and Nonsense-Mediated mRNA Decay (NMD) Before the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated.

negative: Molecular biology of severe acute respiratory syndrome coronavirus The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. Coronaviruses and their closest relatives possess extremely large plus-strand RNA genomes and employ unique mechanisms and enzymes in RNA synthesis that separate them from all other RNA viruses. The SARS epidemic prompted a variety of studies on multiple aspects of the coronavirus replication cycle, yielding both rapid identification of the entry mechanisms of SARS-CoV into host cells and valuable structural and functional information on SARS-CoV proteins. These recent advances in coronavirus research have important implications for the development of anti-SARS drugs and vaccines.

negative: Identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus RNA genome. We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) synthesize an additional mRNA species (mRNA 2b, previously called mRNA 2a) with a size intermediate between that of mRNAs 2 and 3, suggesting the presence of an optional transcriptional initiation site. This transcriptional start is dependent on the leader sequence of the virus strains. To study the mechanism of coronavirus transcriptional regulation, we have cloned and sequenced the region of the viral genome corresponding to the 5' unique coding region of mRNA 2 of the JHM strain of MHV. In addition to the open reading frame (ORF) predicted to encode the viral nonstructural protein p30, a second complete ORF, with the potential to encode a 439-amino-acid polypeptide, was discovered. The transcriptional initiation sites of both mRNA 2a (formerly called mRNA 2) and mRNA 2b were determined by primer extension studies and RNA sequencing. The data indicated that transcription of mRNA 2a initiated at a site, UCUAUAC, that resembled the consensus intergenic sequence. In contrast, the start signal of the optional mRNA 2b, UAAUAAAC, deviated from the consensus sequence. mRNA 2b is a functional mRNA, as shown by in vitro translation studies of mRNA and ORF 2b and by the detection of an additional viral structural protein, gp65, in the JHM strain that synthesized this mRNA. Although the A59 strain of MHV was found to retain ORF 2b, it lacked the correct transcriptional and translational start signals for this gene. This study has therefore identified an optional gene product for murine coronaviruses and provided insights into the mechanism of regulation of MHV RNA transcription.

negative: Improvement of different vaccine delivery systems for cancer therapy Cancer vaccines are the promising tools in the hands of the clinical oncologist. Many tumor-associated antigens are excellent targets for immune therapy and vaccine design. Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and/or delivery strategies to achieve positive clinical results. Various vaccine delivery systems such as different routes of immunization and physical/chemical delivery methods have been used in cancer therapy with the goal to induce immunity against tumor-associated antigens. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target antigen-presenting cells (APCs) have demonstrated to be effective in animal models. New developments in vaccine delivery systems will improve the efficiency of clinical trials in the near future. Among them, nanoparticles (NPs) such as dendrimers, polymeric NPs, metallic NPs, magnetic NPs and quantum dots have emerged as effective vaccine adjuvants for infectious diseases and cancer therapy. Furthermore, cell-penetrating peptides (CPP) have been known as attractive carrier having applications in drug delivery, gene transfer and DNA vaccination. This review will focus on the utilization of different vaccine delivery systems for prevention or treatment of cancer. We will discuss their clinical applications and the future prospects for cancer vaccine development.

negative: Characterization of an internal ribosome entry site within mRNA 5 of murine hepatitis virus The unique region of mRNA 5 of murine hepatitis virus contains two open reading frames, ORF 5a and ORF 5b. The downstream ORF 5b encodes the envelope (E) protein, an integral membrane protein of the virus. We have shown previously that the expression of ORF 5b is mediated by the internal entry of ribosomes. In the experiments reported here, we have used the in vitro translation of synthetic mRNAs to identify the region of mRNA 5 that mediates internal ribosome entry. Our results show that the 5′ border of the MHV mRNA 5 IRES element is located between nucleotides 227 and 244 in ORF 5a, while the 3′ border is located between nucleotides 140 and 172 in ORF 5b. The MHV mRNA 5 IRES element, therefore, contains not more than 280 nucleotides and encompasses the ORF 5b initiation codon. As evidenced by electrophoretic mobility shift assays, the IRES element of mRNA 5 interacts specifically with protein factors present in an L-cell lysate.

negative: Early detection and visualization of human adenovirus serotype 5-viral vectors carrying foot-and-mouth disease virus or luciferase transgenes in cell lines and bovine tissues. Recombinant replication-defective human adenovirus type 5 (Ad5) vaccines containing capsid-coding regions from foot-and-mouth disease virus (FMDV) have been demonstrated to induce effective immune responses and provide homologous protective immunity against FMDV in cattle. However, basic mechanisms of Ad5-FMDV vaccine function including virus tropism, transgene expression, and antigen presentation, remain incompletely understood. The current study characterized the dynamics of Ad5 viral vector (Ad5-FMDV-A24 and Ad5-luciferase) infection in cell lines and early post-inoculation vector-host interactions in cattle. Adenovirus dissemination was described utilizing novel rPCR, rRT-PCR, luminometry, and immunomicroscopy techniques. In vitro infection of human and bovine cells with both Ad5 vectors resulted in dose-dependent detection of vector DNA, mature mRNA transcripts, and transgene-encoded proteins. Subsequent to intramuscular inoculation of cattle, Ad5 and transgene products were detected at the injection sites of all animals at all time-points examined (6, 24, and 48 hpi). Microscopically, injection sites were characterized by marked infiltrates of interstitium consisting of predominantly large mononuclear cells. Immunomicroscopy indicated these cells infrequently contained adenovirus and/or transgenic proteins and were phenotypically consistent with antigen-presenting cells (macrophages and dendritic cells). Vector DNA and mature mRNA transcripts were first detected at the draining and local lymph nodes as early as 6 hpi and systemically at 24 hpi. These results provide novel insights for understanding Ad5-mediated immunity against FMDV using novel techniques that will contribute to ongoing efforts for the improvement of future Ad-FMDV vaccine platforms.

negative: Leader-mRNA Junction Sequences Are Unique for Each Subgenomic mRNA Species in the Bovine Coronavirus and Remain So Throughout Persistent Infection Abstract The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3′ end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junction region, and in its place was the nonconforming sequence GGTAGAC that occurs just 15 nt downstream in the genome. Leader-mRNA junction sequences found after 296 days of persistent infection were the same as those found during acute infection (<18 hr postinfection). These data indicate that, in contrast to the closely related mouse hepatitis virus, the bovine coronavirus maintains a stable leader-mRNA junction sequence for each mRNA. Interestingly, this stability may be related to the fact that a UCUAA sequence element, postulated by others to be a regulator of the leader-mRNA fusion event, occurs only once within the 3′ flanking sequence of the genomic leader donor and once at intergenic sites in the bovine coronavirus genome, whereas it occurs two to four times at these sites in the mouse hepatitis coronavirus.

negative: The rise and evolution of COVID-19 Abstract Coronaviridae (CoV) is a large family of zoonotic viruses linked to a range of diseases from the common cold to the severe SARS-CoV and MERS-CoV epidemics. In 2019, a novel virus emerged from Wuhan, China characterized by a marked worldwide outbreak of respiratory illness. Prevention and containment became the prioritized intervention against COVID-19, coupled with a continued search for hallmarks of the disease which would allow early detection and provide insight to management and triage. Cutaneous findings associated with COVID include diffuse maculopapular rashes, livedo reticularis, and acro-ischemic ‘COVID toes’. These skin findings occurred anywhere from days before respiratory symptom onset to weeks after recovery, and predominantly in the child and adolescent population. The role of dermatologists can be expanded during this COVID-19 pandemic to help identify disease through cutaneous presentations.

negative: The evolution of pulmonary pathology in fatal COVID-19 disease: an autopsy study with clinical correlation The pandemia of coronavirus disease 2019 (COVID-19) has caused more than 355,000 confirmed deaths worldwide. However, publications on postmortem findings are scarce. We present the pulmonary findings in four cases of fatal COVID-19 with a spectrum of lung pathology reflecting disease course and duration, invasive therapies, and laboratory features. Early disease is characterized by neutrophilic, exudative capillaritis with microthrombosis and high levels of IL-1beta and IL-6. Later stages are associated with diffuse alveolar damage and ongoing intravascular thrombosis in small to medium-sized pulmonary vessels, occasionally with areas of infarction equivalents, accompanied by laboratory features of disseminated intravascular coagulation. In late stages, organizing pneumonia with extensive intra-alveolar proliferation of fibroblasts and marked metaplasia of alveolar epithelium can be observed. Viral RNA is encountered in the lung, with virus particles in endothelial cells and pneumocytes. In many patients, multi-organ failure with severe liver damage sets in finally, possibly as consequence of an early-onset pro-inflammatory cytokine storm and/or thrombotic microangiopathy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00428-020-02881-x) contains supplementary material, which is available to authorized users.

negative: Measles virus gene expression in subacute sclerosing panencephalitis Abstract RNA was extracted from the diseased brain of a case of human subacute sclerosing panencephalitis (SSPE) and analysed for the expression of measles-specific RNA. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho- (P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. RNA obtained from the human brain was also translated in vitro and measles virus nucleocapsid and P protein was produced. However, in marked contrast to control reactions M protein was not detected in the products formed by translation in vitro. These results indicate an impaired measles virus M protein mRNA synthesis in infected brain tissue.

negative: Down-regulated gene expression spectrum and immune responses changed during the disease progression in COVID-19 patients BACKGROUND: WHO characterizes novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a pandemic. Here, we investigated the clinical, cytokine levels, T cell proportion and related gene expression occurring in COVID-19 patients on admission and after intial treatment. METHODS: 11 patients diagnosed as COVID-19 with similar initial treatment regimen were enrolled in the hospital. Plasma cytokines, CyTOF and microfluidic qPCR for gene expression were conducted. RESULTS: 5 mild and 6 severe patients were included. Cough and fever were the top symptoms in the 11 COVID-2019 cases. The elder age, more neutrophils numbers and higher C-reactive protein level were found in severe cases. IL-10 level was significantly varied with disease progression and treatment. The decreased T cell proportions were observed in COVID-19 patients especially in severe cases, and all elevated to normal in mild patiens after initial treatment but only CD4+T cells return to normal in severe cases. The number of DEGs increased with the disease progress, and decreased after initial treatment. All down-regulated DEGs in severe cases mainly involved in Th17 cell differentiation, cytokine-mediated signaling pathway and T cell activation. After initial treatmen in severe cases, MAP2K7 and SOS1 were upregulated relative to that on admission. CONCLUSIONS: Our findings show a decreased T cell proportion with down-regulated gene expression related to T cell activation and differentiation were occurred in COVID-19 severe patients, which may help to provide effective treatment strategies for COVID-19 .

negative: A compendium answering 150 questions on COVID-19 and SARS-CoV-2 In December 2019, China reported the first cases of the coronavirus disease 2019 (COVID-19). This disease, caused by the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), has developed into a pandemic. To date it has resulted in ~6.5 million confirmed cases and caused almost 400,000 related deaths worldwide. Unequivocally, the COVID-19 pandemic is the gravest health and socio-economic crisis of our time. In this context, numerous questions have emerged in demand of basic scientific information and evidence-based medical advice on SARS-CoV-2 and COVID-19. Although the majority of the patients show a very mild, self-limiting viral respiratory disease, many clinical manifestations in severe patients are unique to COVID-19, such as severe lymphopenia and eosinopenia, extensive pneumonia, a "cytokine storm" leading to acute respiratory distress syndrome, endothelitis, thrombo-embolic complications and multiorgan failure. The epidemiologic features of COVID-19 are distinctive and have changed throughout the pandemic. Vaccine and drug development studies and clinical trials are rapidly growing at an unprecedented speed. However, basic and clinical research on COVID-19-related topics should be based on more coordinated high-quality studies. This paper answers pressing questions, formulated by young clinicians and scientists, on SARS-CoV-2, COVID-19 and allergy, focusing on the following topics: virology, immunology, diagnosis, management of patients with allergic disease and asthma, treatment, clinical trials, drug discovery, vaccine development and epidemiology. Over 140 questions were answered by experts in the field providing a comprehensive and practical overview of COVID-19 and allergic disease.

negative: SARS-CoV-2: A New Song Recalls an Old Melody The viruses causing the SARS outbreak of 2002–2003 and current COVID-19 pandemic are related betacoronaviruses. What insights were learned from SARS that can inform SARS-CoV-2 vaccine development? Focusing on important lessons from SARS vaccine development and two SARS vaccines evaluated in humans may guide SARS-CoV-2 vaccine design, testing, and implementation.

negative: Coordinate induction of IFN-α and -γ by SARS-CoV also in the absence of virus replication Abstract Background: Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV, characterized by an over-exuberant immune response with lung lymphomononuclear cells infiltration and proliferation that may account for tissue damage more than the direct effect of viral replication. This study is aimed at investigating the capability of SARS-CoV to activate IFN-α and -γ expression in lymphomonocytes (PBMC) from healthy donors, evaluating whether viral replication is necessary for this activation. Results: SARS-CoV virus is able to induce both IFN-α and -γ mRNA accumulation and protein release in a dose-dependent manner, MOI 10 being the most effective. The time course curve indicated that IFN-α mRNA induction peaked at 24 h.p.i,. whereas IFN-γ mRNA was still increasing at 48 h.p.i. Released IFN (both types) reached a plateau after 24–48 h.p.i. and remained rather stable over a 5-day period. A transient peak of negative strand viral RNA was detected after 1–2 days of infection, but neither infectious virus progeny yield nor newly produced viral genomic RNA could be evidenced in infected cultures, even after prolonged observation time (up to 13 days). Cocultivation of PBMC with fixed SARS-CoV-infected Vero cells was even more efficient than exposure to live virus in eliciting IFN-α and -γ induction. A combination of IFN-α and -γ strongly inhibited SARS-CoV replication in Vero cells, while the single cytokines were much less effective. Conclusions: This study provides evidence that SARS-CoV is able to induce in normal PBMC a coordinate induction of IFN-α and -γ gene expression. Virus replication is not necessary for IFN induction since efficient IFN expression could be obtained also by the cocultivation of normal PBMC with fixed SARS-CoV-infected cells. Concomitant activation of IFN-α and -γ gene expression by SARS-CoV in vivo may be relevant for the pathogenesis of the disease, both for the possible involvement in immunomediated damage of the tissues and for the strong inhibition of SARS-CoV replication as a result of combined cytokine action.

negative: Vasculopathy and Coagulopathy Associated with SARS-CoV-2 Infection The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has resulted in > 500,000 deaths worldwide, including > 125,000 deaths in the U.S. since its emergence in late December 2019 and June 2020. Neither curative anti-viral drugs nor a protective vaccine is currently available for the treatment and prevention of COVID-19. Recently, new clinical syndromes associated with coagulopathy and vasculopathy have emerged as a cause of sudden death and other serious clinical manifestations in younger patients infected with SARS-CoV-2 infection. Angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 and other coronaviruses, is a transmembrane protein expressed by lung alveolar epithelial cells, enterocytes, and vascular endothelial cells, whose physiologic role is to induce the maturation of angiotensin I to generate angiotensin 1-7, a peptide hormone that controls vasoconstriction and blood pressure. In this review, we provide the general context of the molecular and cellular mechanisms of SARS-CoV-2 infection with a focus on endothelial cells, describe the vasculopathy and coagulopathy syndromes in patients with SARS-CoV-2, and outline current understanding of the underlying mechanistic aspects.

negative: Current Insight into the Novel Coronavirus Disease 2019 (COVID-19) SARS-CoV-2 is a novel strain of coronavirus that has not been previously identified in humans. It has been declared a pandemic and has infected at least 1,844,683 individuals and caused 117,021 deaths as of 14th April 2020. Transmission among humans occurs via close contact with an infected individual that produces respiratory droplets. Patients have been shown to undergo acute respiratory distress syndrome, which is defined as cytokine storm. The diagnosis relies on detection of nucleic acid, IgG/IgM antibodies, and a chest radiograph of the suspected individuals. The genome of SARS-CoV-2 is similar to other coronaviruses that comprise of ten open reading frames (ORFs). SARS-CoV-2 spike protein exhibits higher affinity to ACE2 receptor as compared with SARS-CoV. Repurposing drugs like favipiravir, remdesivir, chloroquine, and TMPRSS2 protease inhibitors have been shown to be effective for the treatment of COVID-19. Personal protective measures should be followed to prevent SARS-CoV-2 infection. In addition, a clinical trial of SARS-CoV-2 vaccine, mRNA-1273, has been started. This chapter provides a glimpse of advancements made in the area of SARS-CoV-2 infection by proving recent clinical and research trials in the field.

negative: A single-dose live-attenuated YF17D-vectored SARS-CoV2 vaccine candidate The explosively expanding COVID-19 pandemic urges the development of safe, efficacious and fast-acting vaccines to quench the unrestrained spread of SARS-CoV-2. Several promising vaccine platforms, developed in recent years, are leveraged for a rapid emergency response to COVID-191. We employed the live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express the prefusion form of the SARS-CoV-2 Spike antigen. In mice, the vaccine candidate, tentatively named YF-S0, induces high levels of SARS-CoV-2 neutralizing antibodies and a favorable Th1 cell-mediated immune response. In a stringent hamster SARS-CoV-2 challenge model2, vaccine candidate YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose confers protection from lung disease in most vaccinated animals even within 10 days. These results warrant further development of YF-S0 as a potent SARS-CoV-2 vaccine candidate.