Seth J. Zost,Robert H. Carnahan
A budding new approach strengthens an important message
In vaccinology, both mRNA-based delivery of genes encoding antigens as well as nanoparticle-based vaccines have shown great promise in tackling challenging pathogens. In this issue of Cell, Hoffmann et al. combine these two approaches, harnessing the same cellular pathway hijacked by many viruses to boost immune responses to SARS-CoV-2 vaccination.
A budding new approach strengthens an important message

In vaccinology, both mRNA-based delivery of genes encoding antigens as well as nanoparticle-based vaccines have shown great promise in tackling challenging pathogens. In this issue of Cell, Hoffmann et al. combine these two approaches, harnessing the same cellular pathway hijacked by many viruses to boost immune responses to SARS-CoV-2 vaccination.

The emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic have seen mRNA-based vaccine platforms move into routine clinical use with dizzying speed. Approved mRNA vaccines target the spike (S) protein of SARS-CoV-2 and stimulate high levels of neutralizing antibodies, which are known to correlate with protection from infection. Much of the excitement surrounding mRNA vaccines stems from several advantages that could lead to more effective vaccines against tricky pathogens. Since mRNA vaccines result in expression of viral glycoproteins at the cell surface, membrane-bound regions of viral antigens do not need to be altered in order to make the viral proteins soluble, avoiding issues with protein stability or the need to introduce stabilizing domains. As a result, mRNA vaccines largely avoid the lengthy optimization of custom production workflows that can stymie the development of traditional protein vaccines, accelerating vaccine development against emerging pathogens. mRNA vaccines can also engage cytotoxic T cell responses in ways that conventional protein-based vaccines cannot, as translation of viral antigens produces immunogenic peptides that can be presented via endogenous antigen presentation pathways. Finally, the genes encoded by mRNA vaccines can be updated to match circulating strains, an essential feature when the goal is to provide continued protection against rapidly evolving RNA viruses such as influenza virus and SARS-CoV-2. In addition to mRNA-based vaccine platforms, another promising vaccine innovation has been used in the development of an expanding number of approaches to display viral antigens in ordered multimeric arrays, mimicking the natural structure of intact virions. For example, virus-like particles (VLPs), which are covered in viral antigens but are not infectious, have been used successfully in vaccines such as those for human papillomavirus. A related approach, protein nanoparticles (NPs), utilizes self-assembling proteins with antigen attachment sites that result in ordered display of antigens on the NP surface. Through mimicking the repetitive organization of proteins on the viral surface, VLPs and NPs improve the immune response by maximizing B cell activation to induce high titers of protective antibodies. For example, an NP-based vaccine displaying SARS-CoV-2 S has been shown to induce high levels of neutralizing antibodies. In addition, NP-display platforms have been leveraged to display multiple antigenically distinct viral glycoproteins on the same particle. These “mosaic” NPs have been shown to excel at inducing broadly cross-reactive antibody responses, a major goal of vaccine design. , , In this issue of Cell, Hoffmann et al. set out to merge the advantages of both mRNA and nanoparticle-based vaccines. In theory, combining the modular features of mRNA vaccine platforms with the strong immunogenicity of NP-based vaccines would be beneficial. In practice, this is easier said than done. Many protein NP-display platforms involve multiple proteins that self-assemble into the NP and would require co-expression of several different proteins in vivo. Delivering genes encoding all of these to the same cell via mRNA-lipid nanoparticles is a tall order. However, Hoffmann et al. cleverly repurpose the cellular pathway some viruses use to bud from infected cells and create a novel vaccine platform that blends the desirable features of mRNA and NP-based vaccines. The endosomal sorting complex required for transport (ESCRT) is a set of proteins that drive membrane budding in cellular processes such as the formation of multivesicular bodies. To harness the ESCRT machinery, the authors add a short sequence into the cytoplasmic tail of SARS-CoV-2 S that facilitates interactions with TSG101 and ALIX, two proteins that are involved in the recruitment of ESCRT machinery to sites of budding. This sequence, which the authors term an ESCRT- and ALIX-binding region (EABR), recruits host ESCRT proteins to S at the plasma membrane. This ultimately leads to self-assembly and budding of enveloped VLPs (eVLPs) bearing the S glycoprotein (Figure 1 ). After finding an optimal EABR sequence, the authors demonstrate that expression of the S-EABR construct is sufficient to produce eVLPs in vitro. Mice vaccinated with purified eVLPs expressed from the optimized S-EABR construct produced high levels of neutralizing antibodies, verifying that eVLPs have comparable immunogenicity to a protein NP-based vaccine. After showing the potential of the S-EABR construct to generate immunogenic eVLPs in vitro, the authors pivot to assessing the efficacy of an S-EABR mRNA vaccine approach. In a series of experiments, the authors demonstrate that mice vaccinated with an S-EABR mRNA immunization regimen make superior neutralizing antibody responses when compared with mice vaccinated with an S-only mRNA regimen (an analog for approved human mRNA vaccines). In addition to the magnitude of induced antibodies, a wider breadth of the polyclonal antibody response was observed for the S-EABR vaccine, including greater neutralization of the SARS-CoV-2 Omicron variants BA.1 and BA.2. Importantly, while S-EABR-vaccinated mice have considerable reductions in neutralization potency against these antigenically advanced Omicron variants, they retain 8- to 10-fold higher neutralizing titers compared with mice vaccinated with the S-only mRNA vaccine. Given the continued circulation and antigenic evolution of SARS-CoV-2, these improvements in neutralizing antibody breadth would likely translate to meaningful improvements in vaccine effectiveness. A smaller but significant increase in T cell responses by an S-EABR mRNA approach was also observed compared to both direct eVLP immunization and S-only mRNA immunization. The novel vaccine strategy developed by Hoffmann and colleagues combines the flexibility and speed of mRNA-based vaccines with the superior immunogenicity of VLP and NP platforms, with exciting applications to other pathogens. While the authors show that incorporating an EABR into the envelope protein of HIV also leads to robust eVLP production, some viruses, such as influenza virus, have multiple glycoproteins that play roles in attachment (hemagglutinin) and release from infected cells (neuraminidase). It remains to be seen how compatible these proteins are with the EABR-based eVLP platform and whether expression of these proteins with EABR domains would lead to eVLP production. As the authors note, important immunologic questions remain unanswered, such as whether the cross-reactivity observed in S-EABR vaccination is due to more effective priming of antibodies that target conserved sites but are subdominant in the immune response to S-only vaccination. While Hoffmann and colleagues use a single flavor of antigen, the eVLP system could also be applied to mRNA vaccines containing multiple antigens. Recent work has demonstrated that mRNA-based delivery of 20 different influenza virus hemagglutinin proteins induces broad protection against different influenza virus strains. It is possible that combining multivalent mRNA vaccines encoding different antigens with the eVLP approach would further enhance protective immune responses. Thanks to the work of Hoffmann et al., the field of vaccine design has one more way to get the message across.