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|Jun 29, 1998
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|Michael C. Hevey, Diane I. Negley, Peter Pushko, Jonathan F. Smith, Alan L. Schmaljohn
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|The United States Of America As Represented By The Secretary Of The Army
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This application is a divisional application of application Ser. No. 10/267,322, filed Oct. 9, 2002 now U.S. Pat. No. 7,090,852, which is a divisional application of U.S. Utility application Ser. No. 09/336,910, filed Jun. 21, 1999, now U.S. Patent No. 6,517,842, issued Feb. 11, 2003, which claims priority provisional application Ser. No. 60/091,403, filed Jun. 29, 1998.
Marburg virus (MBGV) was first recognized in 1967, when an outbreak of hemorrhagic fever in humans occurred in Germany and Yugoslavia, after the importation of infected monkeys from Uganda (Martini and Siegert, 1971, Marburg Virus Disease. Berlin: Springer-Verlag; Smith et al., 1982, Lancet 1, 816-820). Thirty-one cases of MBGV hemorrhagic fever were identified that resulted in seven deaths. The filamentous morphology of the virus was later recognized to be characteristic, not only of additional MBGV isolates, but also of Ebola virus (EBOV) (Johnson et al., 1977, J. Virol. 71, 3031-3038; Smith et al., 1982, Lancet 1, 816-820; Pattyn et al., 1977, Lancet 1, 573-574). MBGV and EBOV are now known to be distinctly different lineages in the family Filoviridae, within the viral order Mononegavirales (Kiley et al., 1982, Intervirology 18, 24-32; Feldmann and Klenk, 1996, Adv. Virus Res. 47, 1-52).
Few natural outbreaks of MBGV disease have been recognized, and all proved self-limiting, with no more than two cycles of human-to-human transmission. However, the actual risks posed by MBGV to global health cannot be assessed because factors which restrict the virus to its unidentified ecological niche in eastern Africa, and those that limit its transmissibility, remain unknown (Feldmann and Klenk, 1996, supra). Concern about MBGV is further heightened by its known stability and infectivity in aerosol form (Belanov et al., 1996, Vopr. Virusol. 41, 32-34; Frolov and Gusev Iu, 1996, Vopr. Virusol. 41, 275-277). Thus, laboratory research on MBGV is necessarily performed at the highest level of biocontainment. To minimize future risk, our primary interest has been the identification of appropriate antigens and vaccine strategies that can provide immunity to MBGV.
Early efforts to demonstrate the feasibility of vaccination against MBGV were only partially successful, as inoculation with formalin-inactivated viruses only protected about half the experimental animals (guinea pigs or nonhuman primates) from fatal disease (Ignat'ev. et al., 1991, Vopr. Virusol. 36, 421-423; Ignat'ev et al., 1996, J. Biotechnol. 44, 111-118). We recently demonstrated that the MBGV GP, cloned into a baculovirus vector and expressed as a soluble antigen to be administered in adjuvant, was sufficient to protect most but not all guinea pigs from lethal MBGV challenge (Hevey et al., 1997, Virology 239, 206-216). In addition, purified, 60Co-irradiated virus, administered in adjuvant, completely protected guinea pigs from challenge with either of two different strains of MBGV, thus setting a standard for future, more pragmatic, vaccine candidates (Hevey et al., 1997, supra). Experiences with EBOV vaccines have been similar to those with MBGV, reinforcing the difficulties of classical approaches (Lupton et al., 1980, Lancet 2, 1294-1295). Recent efforts to develop EBOV vaccines, using three distinctly different approaches (vaccinia recombinants, VEE replicon, and naked DNA) to achieve viral antigen expression in cells of vaccinated animals, showed that nucleoprotein (NP) as well as GP protected BALB/c mice (VanderZanden et al., 1998, Virology 245), whereas protection of guinea pigs by NP was unsuccessful (Gilligan et al., 1997, In: Brown, F., Burton, D., Doherty, P., Mekalanos, J., Norrby, E. (eds). 1997. Vaccines 97 Cold Spring Harbor Press. Cold Spring Harbor, N.Y.; Pushko et al., 1997, In: Brown, F., Burton, D., Doherty, P., Mekalanos, J., Norrby, E. (eds). 1997. Vaccines 97 Cold Spring Harbor Press. Cold Spring Harbor, N.Y.) or equivocal (Xu et al., 1998, Nat. Med. 4, 37-42).
Irrespective of how encouraging filovirus vaccine results may appear in guinea pigs or mice, protection of nonhuman primates is widely taken as the more definitive test of vaccine potential for humans. Low-passage viral isolates from fatal human cases of MBGV or EBOV tend to have uniform lethality in nonhuman primates, but not in guinea pigs or mice. Small animal models with fatal disease outcomes have been achieved only with a subset of filovirus isolates and only then by multiple serial passages in the desired host (Hevey et al., 1997, supra; Connolly et al., 1999, J. Infect. Dis. 179, suppl. 1, S203 ; Xu et al., 1998, supra; Bray et al., 1998, J. Infect. Dis. 178, 661-665). While highly useful for identification and initial characterization of vaccine candidates, guinea pig and murine models remain somewhat suspect with regard to the possibility that protection in such animals is easier to achieve than in nonhuman primates and, by inference, in humans. For example, with MBGV, peak viremias and viral titers in organs are more than 100 times higher in nonhuman primates than in guinea pigs.
Therefore, there is a need for an efficacious vaccine for MBGV useful for protecting humans against Marburg hemorrhagic fever.
The present invention satisfies the need discussed above. The present invention relates to a method and composition for use in inducing an immune response which is protective against infection with MBGV.
In this study a vaccine delivery system based on a Venezuelan equine encephalitis (VEE) virus replicon was used to identify candidate protective antigens in nonhuman primates. In this vaccine strategy, a gene coding for a protein of interest is cloned in place of the VEE virus structural genes; the result is a self-replicating RNA molecule that encodes its own replicase and transcriptase functions, and in addition makes abundant quantities of mRNA encoding the foreign protein. When replicon RNA is transfected into eukaryotic cells along with two helper RNAs that express the VEE structural proteins (glycoproteins and nucleocapsid), the replicon RNA is packaged into VEE virus-like particles by the VEE virus structural proteins, which are provided in trans. Since the helper RNAs lack packaging signals neccessary for further propagation, the resulting VEE replicon particles (VRPs) which are produced are infectious for one cycle but are defective thereafter. Upon infection of an individual cell with a VRP, an abortive infection occurs in which the infected cell produces the protein of interest in abundance, is ultimately killed by the infection, but does not produce any viral progeny (Pushko et al., 1997, Virology 239, 389-401). The VEE replicon is described in greater detail in U.S. Pat. No. 5,792,462 issued to Johnston et al. on Aug. 11, 1998.
Results shown here demonstrate that the VEE replicon is a potent tool for vaccination with MBGV antigens. Guinea pigs were protected by vaccination with packaged replicons that expressed GP, or by either of two replicons which expressed internal MBGV antigens (NP and VP35). GP expressed from the VEE replicon elicited an even more robust immunity than was achieved previously with a baculovirus-produced soluble GP administered in adjuvant. When results were extended to nonhuman primates, complete protection with GP was demonstrated. The data shown here constitute the most emphatic proof to date that an efficacious vaccine for MBGV is feasible, and define candidate antigens for such a vaccine.
Therefore, it is one object of the present invention to provide a VEE virus replicon vector comprising a VEE virus replicon and a DNA fragment encoding any of the MBGV GP, NP, VP40, VP35, VP30, and VP24, and GPΔTM, a GP deletion mutant from which the C-terminal 39 amino acids encoding the transmembrane region and cytoplasmic tail of MBGV GP were removed.
It is another object of the present invention to provide a self replicating RNA comprising the VEE virus replicon and any of the MBGV GP, GPΔTM, NP, VP40, VP35, VP30, and VP24 described above.
It is another object of the present invention to provide infectious VEE virus replicon particles produced from the VEE virus replicon RNA described above.
It is further an object of the invention to provide an immunological composition for the protection of mammals against MBGV infection comprising VEE virus replicon particles containing nucleic acids encoding any of the MBGV GP, GPΔTM, NP, VP40, VP35, VP30, and VP24 or a combination of different VEE virus replicons each containing nucleic acids encoding a different MBGV protein from any of MBGV GP, GPΔTM, NP, VP40, VP35, VP30, and VP24.
These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims, and accompanying drawings where:
In the description that follows, a number of terms used in recombinant DNA, virology and immunology are extensively utilized. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Filoviruses. The filoviruses (e.g. Marburg virus, MBGV) cause acute hemorrhagic fever characterized by high mortality. Humans can contract filoviruses by infection in endemic regions, by contact with imported primates, and by performing scientific research with the virus. However, there currently are no available vaccines or effective therapeutic treatments for filovirus infection. The virions of filoviruses contain seven proteins which include a surface glycoprotein (GP), a nucleoprotein (NP), an RNA-dependent RNA polymerase (L), and four virion structural proteins (VP24, VP30, VP35, and VP40). Little is known about the biological functions of these proteins and it is not known which antigens significantly contribute to protection and should therefore be used to induce an immune response by an eventual vaccine candidate.
Replicon. A replicon is equivalent to a full length virus from which all of the viral structural proteins have been deleted. A multiple cloning site can be cloned into the site previously occupied by the structural protein genes. Virtually any heterologous gene may be cloned into this cloning site. Transcription of the RNA from the replicon yields an RNA capable of initiating infection of the cell identical to that seen with the full-length infectious virus clone. However, in lieu of the viral structural proteins, the heterologous antigen is expressed. This system does not yield any progeny virus particles because there are no viral structural proteins available to package the RNA into particles.
Particles which appear structurally identical to virus particles can be produced by supplying structural proteins for packaging of the replicon RNA in trans. This is typically done with two helpers also called defective helper RNAs. One helper consists of a full length infectious clone from which the nonstructural protein genes and the glycoprotein genes are deleted. The helper retains only the terminal nucleotide sequences, the promoter for subgenomic mRNA transcription and the sequences for the viral nucleocapsid protein. The second helper is identical to the first except that the nucleocapsid gene is deleted and only the glycoprotein genes are retained. The helper RNA's are transcribed in vitro and co-transfected with replicon RNA. Because the replicon RNA retains the sequences for packaging by the nucleocapsid protein, and because the helpers lack these sequences, only the replicon RNA is packaged by the viral structural proteins and released from the cell. The particles can then by inoculated into animals similar to parent virus. The replicon particles will initiate only a single round of replication because the helpers are absent, they produce no progeny virus particles, and express only the viral nostructural proteins and the product of the heterologous gene cloned in place to the structural proteins.
The VEE virus replicon is a genetically reorganized version of the VEE virus genome in which the structural proteins genes are replaced with a gene from an immunogen of interest, in this invention, the MBGV virion proteins. The result is a self replicating RNA (replicon) that can be packaged into infectious particles using defective helper RNAs that encode the glycoprotein and capsid proteins of the VEE virus.
Subject. Includes both human, animal, e.g., horse, donkey, pig, guinea pig, mouse, hamster, monkey, chicken, bats, birds and insects such as mosquito.
In one embodiment, the present invention relates to a recombinant DNA molecule that includes a VEE replicon and a DNA sequence encoding any of MBGV virion proteins GP, GPΔTM, NP, VP40, VP35, VP30, VP24. The sequences encoding the Marburg proteins GP, GPΔTM, NP, VP40, VP35, VP30, VP24 corresponding to nucleotides 104-11242 of the Genbank sequence is presented in SEQ ID NO:1; the GP DNA fragment extends from nucleotide 5932 to 8033, of which nucleotides 5940-7985 encode the protein identified in SEQ ID NO:2; the GPΔTM DNA fragment, a GP deletion mutant from which the C-terminal 39 amino acids encoding the transmembrane region and cytoplasmic tail of MBGV GP were removed, extends from nucleotides 5933 to 7869, of which nucleotides 5940-7871 encode the protein; NP, identified in SEQ ID NO:3, is encoded by the DNA fragment extending from nucleotides 104 to 2195; VP40 DNA fragment extends from nucleotide 4564 to 5958, of which nucleotides 4567-5416 encode the protein identified in SEQ ID NO:4; VP35 DNA fragment extends from nucleotide 2938 to 4336, of which nucleotides 2944-3933 encode the protein identified in SEQ ID NO:5; VP30 DNA fragment extends from nucleotide 8861 to 9979, of which nucleotides 8864-9697 encode the protein identified in SEQ ID NO:6; VP24 DNA fragment extends from nucleotide 10182 to 11242, of which nucleotides 10200-10961 encode the protein identified in SEQ ID NO:7.
When the DNA sequences described above are in a replicon expression system, such as the VEE replicon described above, the proteins can be expressed in vivo. The DNA sequence for any of the MBGV virion proteins described above can be cloned into the multiple cloning site of a replicon such that transcription of the RNA from the replicon yields an infectious RNA containing the sequence(s) which encodes the MBGV virion protein or proteins of interest. Use of helper RNA containing sequences necessary for encapsulation of the viral transcript will result in the production of viral particles containing replicon RNA which are able to infect a host and initiate a single round of replication resulting in the expression of the MBGV virion proteins. Such replicon constructs include, for example, VP24 cloned into a VEE replicon, pRep Mus VP24, VP30 cloned into a VEE replicon, pRep Mus VP30, VP35 cloned into a VEE replicon, pRep Mus VP35, and VP40 cloned into a VEE replicon, pRep Mus VP40, NP cloned into a VEE replicon, pRep Mus NP, GP cloned into a VEE replicon, pRep Mus GP, GPΔTM cloned into a VEE replicon, pRep Mus GPΔTM. The sequences encoding the MBGV proteins were cloned into the replicon vector by methods known in the art and described below in Materials and Methods. Schematic diagrams of the resulting constructs are shown in the Figures. The VEE constructs containing Marburg proteins can be used as a DNA vaccine, or for the production of RNA molecules as described below.
In another embodiment, the present invention relates to RNA molecules resulting from the transcription of the constructs described above. The RNA molecules can be prepared by in vitro transcription using methods known in the art and described in the Examples below. Alternatively, the RNA molecules can be produced by transcription of the constructs in vivo, and isolating the RNA. These and other methods for obtaining RNA transcripts of the constructs are known in the art. Please see Current Protocols in Molecular Biology. Frederick M. Ausubel et al. (eds.), John Wiley and Sons, Inc. The RNA molecules can be used, for example, as a direct RNA vaccine, or to transfect cells along with RNA from helper plasmids, one of which expresses VEE glycoproteins and the other VEE capsid proteins, as described above, in order to obtain replicon particles.
In a further embodiment, the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs. The host cell can be prokaryotic (for example, bacterial), lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to mouse and human). Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with, prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods. The DNA sequence can be present in the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of MBGV virion proteins, such as glutathione S-transferase. The recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts. Control sequences for yeast vectors are known in the art. Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), such as CHO cells, vero cells, and COS cells to name a few. Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV), and cytomegalovirus (CMV). Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequences are known in the art.
The transformed or transfected host cells can be used as a source of DNA sequences described above. When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein cloned into the VEE replicon, or a source of RNA transcribed from the replicon as described above, or a source of replicon particles.
In a further embodiment, the present invention relates to a method of producing the recombinant or fusion protein which includes culturing the above-described host cells, under conditions such that the DNA fragment is expressed and the recombinant or fusion protein is produced thereby. The recombinant or fusion protein can then be isolated using methodology well known in the art. The recombinant or fusion protein can be used as a vaccine for immunity against infection with MBGV or as a diagnostic tool for detection of MBGV infection. The transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit MBGV virus function, such as host proteins or chemically derived agents or other proteins which may interact with the virus to inhibit its replication or survival.
In another embodiment, the present invention relates to a MBGV vaccine comprising one or more replicon particles derived from one or more replicons encoding one or more MBGV virion proteins. The present invention relates to a method for providing immunity against MBGV virus said method comprising administering one or more replicon particles containing any combination of the MBGV virion proteins to a subject such that a protective immune reaction is generated. Even though the MBGV strain Musoke was used in the examples below, it is expected that protection would be afforded using virion proteins from other MBGV strains, as well as significant cross protection between strains.
Vaccine formulations of the present invention comprise an immunogenic amount of a replicon particle, resulting from one of the replicon constructs described above, or a combination of replicon particles as a multivalent vaccine, in combination with a pharmaceutically acceptable carrier. An "immunogenic amount" is an amount of the replicon particles sufficient to evoke an immune response in the subject to which the vaccine is administered. An amount of from about 105 to 108 or more replicon particles per dose with one to three doses one month apart is suitable, depending upon the age and species of the subject being treated. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution.
Administration of the replicon particles disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), by in ovo injection in birds, orally and by topical application of the virus (typically carried in the pharmaceutical formulation) to an airway surface. Topical application of the virus to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the virus to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the replicon as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.
When the replicon RNA or DNA is used as a vaccine, the replicon RNA or DNA can be administered directly using techniques such as delivery on gold beads (gene gun), delivery by liposomes, or direct injection, among other methods known to people in the art. Any one or more constructs or replicating RNA described above can be use in any combination effective to illicit an immunogenic response in a subject. Generally, the nucleic acid vaccine administered may be in an amount of about 1-5 ug of nucleic acid per dose and will depend on the subject to be treated, capacity-of the subject's immune system to develop the desired immune response, and the degree of protection desired. Precise amounts of the vaccine to be administered may depend on the judgement of the practitioner and may be peculiar to each subject and antigen.
The vaccine may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Examples of suitable immunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired immune responses expected to confer protective immunity, or reduce disease symptoms, or reduce severity of disease.
The following MATERIALS AND METHODS were used in the examples that follow.
Cell Cultures and Viruses
Vero E6 (Vero C1008, ATCC CRL 1586), Vero 76 (ATCC CRL 1587), and BHK (ATCC CCL 10) cells were grown in minimal essential medium with Earle's salts supplemented with 10% fetal bovine serum and gentamicin (50 μg/ml). MBGV (strain Musoke) was isolated from a human case in 1980 in. Kenya (Smith et al., 1982, Lancet 1, 816-820), and a derivative of this virus (six passages in Vero 76 cells) was used to challenge the cynomolgus monkeys. The MBGV (Musoke) that was adapted for guinea pig lethality and plaque-picked three times was described previously (Hevey et al., 1997, Virology 239, 206-210).
Construction of Recombinant VEE Replicons
MBGV gene clones pGem-GP, pGem-NP, pTM1-VP40, pTM1-VP35, pTM1-VP30, and pTM1-VP24 were generously provided by Heinz Feldmann and Anthony Sanchez (Centers for Disease Control and Prevention, Atlanta, Ga.) (Will et al., 1993, J. Virol. 67, 1203-1210; Sanchez et al., 1992, J. Gen. Virol. 73, 347-357; Feldman et al., 1992, Virus Res. 24, 1-19). VEE replicon and shuttle vector as well as the replicons that express Lassa virus NP and Flu HA were previously described (Pushko et al., 1997, Virology 239, 289-401). The MBGV GP gene from pGem-GP was excised with Sal I and subcloned into the Sal I site of the shuttle vector by using standard techniques (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor). A clone with the MBGV GP gene in the correct orientation was excised with Apa I and Not I and this fragment was cloned into the Apa I and Not I sites of the VEE replicon plasmid.
Construction of pBluescript-KS(+)-GPΔTM, a deletion mutant of MBGV from which the C-terminal 39 amino acids (transmembrane region and cytoplasmic tail) of MBGV GP were removed, was previously described (Hevey et al., 1997, supra). Here, the MBGV GPΔTM gene was excised from pBluescript-KS(+) with Hind III, and the resulting fragment ligated into the Hind III site of the shuttle vector. MBGV GPΔTM gene was excised from the shuttle vector using-Cla I, and the resulting fragment ligated into the VEE replicon plasmid.
The MBGV NP gene was amplified by PCR performed with 1 ng of pGem NP as template DNA, 1 μg each of forward (5′-CCG ACC ATG GAT TTA CAC AGT TTG TTG G-3′, SEQ ID NO:8) and reverse primer (5′-CTA GCC ATG GCT GGA CTA CAA GTT CAT CGC-3′ SEQ ID NO:9), and AmpliTaq polymerase (GeneAmp PCR reagent kit, Perkin Elmer, Branchburg, N.J.). The primers contained an NcoI recognition sequence at the 3′ terminus end (5-10 inclusive for both the forward and reverse primers). The reaction conditions were: 40 cycles of 94� C. for 45 sec, 50� C. for 45 sec, and 72� C. for 1 min., followed by a final extension step at 72� C. for 5 min. The product was cloned into the pCR™II (InVitrogen, Carlsbad, Calif.) vector, excized with Eco RI, then subcloned into the shuttle vector using Eco RI sites. The MBGV NP gene was excised with Cla I and ligated into the VEE replicon plasmid.
The MBGV VP40, VP35, VP30, and VP24 genes were excised from pTM1 with Bam HI and ligated into the Bam HI site of the shuttle vector. These MBGV genes were then excised from shuttle vectors using either Cla I (VP35, VP30, and VP24) or Apa I and Not I (VP40) and ligated into the VEE replicon plasmid.
Packaging of Replicons into VEE-Like Particles and Determination of Replicon Titer
Replicon RNAs were packaged into VRPs as described previously (Pushko et al., 1997, Virology 239, 389-401). Briefly, BHK cells were cotransfected with RNA transcribed in vitro from the replicon plasmid and from two helper plasmids, one of which expressed VEE glycoproteins and the other VEE capsid protein. The cell culture supernatant was harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by pelleting through a 20% sucrose cushion (SW28 rotor, 25,000 rpm, 4 h), after which they were resuspending in 1 ml PBS. To assay titers of packaged replicons, Vero cells (105 cells per well in eight-chamber slides, Labtek slides, Nunc Inc.) were infected with serial dilutions of the replicon particles and incubated for 16-18 h at 37� C. to allow for expression of the MBGV genes. After rinsing and fixating with acetone, antigen-positive cells were identified by indirect immunofluorescence assay (IFA) as described previously (Schmaljohn et al, 1995, Virology 206, 963-972). The antibodies used included MAbs specific for MBGV GP (II-7C11), NP (III-5F8), VP40 (III-1H11), VP35 (XBC04-BG06), and VP30 (III-5F11 and 5F12) (Hevey et al., 1997, supra). To detect VP24 antigen, a monkey anti-MBGV serum was used, a monkey anti-Lassa serum was used to detect expression of Lassa NP in cells, and influenza HA was detected with serum from a mouse immunized with a VEE replicon expressing influenza HA (provided by Dr. Mary Kate Hart, USAMRIID).
Immunoprecipitation and Gel Electrophoresis of Proteins Expressed by VEE Replicons
Expressed MBGV antigens were immunoprecipitated and analyzed by gel electrophoresis as described previously (Hevey et al., 1997, supra). Briefly, Vero cells were infected (MOI≧23) with VRP expressing a single MBGV antigen. Complete medium was replaced 16-18 h postinfection by methionine- and cysteine-free medium for 1 h, and monolayers were then labeled with 35S-methionine and cysteine for 4 h. Convalescent guinea pig anti-MBGV (group 1, Table 5, in Hevey et al., 1997, supra) was used to immunoprecipitate MBGV-specific proteins from the resulting cell lysates.
Vaccination of Guinea Pigs with VEE Replicons Expressing MBGV Proteins
Inbred strain 13 guinea pigs (maintained as a colony at USAMRIID) were inoculated subcutaneously with 106 focus-forming units (FFU) of VRP in a total volume of 0.5 ml administered at two dorsal sites. Guinea pigs were anesthetized, bled, and those that received two or three doses of replicon inoculated (as described for the first vaccine dose) 28 days after the primary vaccination. Guinea pigs were anesthetized and bled again 28 days later, and animals that received three doses of replicons were inoculated, as described above. Animals were anesthetized and bled 21 days later, and challenged 7 days after the last bleed with 103.0 plaque forming units (PFU) (ca. 2000 LD50) guinea pig adapted MBGV. Animals were examined daily for signs of illness. Heparinized plasma was obtained from the retroorbital sinus of anesthetized animals 7 days postinfection for assay of viremia. Surviving guinea pigs were observed for at least 30 days after challenge, then anesthetized and exsanguinated. Viremia titers was measured by plaque assay on Vero E6 cells.
Vaccination of Cynomolgus Monkeys with Replicons
Twelve cynomolgus macaques (Macaca fascicularis), 11 females and 1 male, ranging from 2.8 to 4.5 Kg, were inoculated subcutaneously with 107 FFU of VRP in a total volume of 0.5 ml at one site. Monkeys were anesthetized with ketamine, bled, and inoculated (as described for the first vaccine dose) 28 days after the primary injection, and again 28 days after the second. Animals were anesthetized and bled 21 days after the third vaccine dose, then were challenged 14 days later with 103.9 PFU MBGV subcutaneously. Here and in guinea pig experiments, the inoculum was back-titrated to ensure proper dose delivery. Animals were examined daily by the attending veterinarian for signs of illness, and given buprenorphine (Buprenex) at a dosage of 0.01 mg/kg body weight, to provide-analgesia upon signs of distress. Of the unprotected animals, three succumbed abruptly, while one was euthanized in extremis. A detailed clinical evaluation, serum for viremia determination and blood chemistries, as well as EDTA blood was obtained from anesthetized animals 17 days before and 3, 5, 7, 10, 17, and 32 days postinfection. Viremia was measured by plaque assay on Vero E6 cells.
MBGV ELISA and Infectivity Assays
Antibody titers in guinea pig plasmas or monkey sera were determined by an indirect ELISA as described previously (Hevey et al., 1997, supra). Briefly, antigen consisting of purified, irradiated virus was coated directly onto PVC plates and serial dilutions of test serum were added to wells containing antigen. The presence of bound antibody was detected by use of the appropriate horseradish peroxidase conjugated anti-species antibody (HPO-goat-anti-guinea pig IgG H+L; HPO-goat-anti-monkey IgG H+L). Endpoint of reactivity was defined as the dilution at which OD405 was 0.2 as determined by extrapolation of a four parameter curve fit (SOFTmax�, Molecular Devices Corp. Sunnydale, Calif.) of background-subtracted mean OD versus dilution. Results shown in any table or figure are from a single assay to allow more valid comparison of endpoints. Plaque assays were performed on Vero E6 cells with a semi-solid overlay on serial dilutions of samples. Viral plaques were visualized by staining viable cells with neutral red 6-7 days postinfection. To measure plaque reduction neutralization, equal volumes of a virus stock (target plaque dose was 100 PFU) and serum diluted in cell culture medium were mixed and incubated at 37� C. for 1 h. The resulting sample was assayed by plaque assay on Vero E6 cells for more than a 50% reduction in PFU compared to control samples.
Clinical Laboratory Assays
For nonhuman primate studies, hematological results were obtained with a Coulter instrument, and differential counts were performed manually. Clinical chemistry results were obtained with a Piccolo™ analyzer (Abaxis, Inc., Sunnydale, Calif.) using the diagnostic panel General Chemistry 12, which measures alanine aminotransferase (ALT), albumin, alkaline phosphatase (ALP), amylase, aspartate aminotransferase (AST), calcium, cholesterol, creatinine, glucose, total bilirubin, total protein, and urea nitrogen.
Results of indirect immunofluorescence assay (IFA) analyses of Vero cells infected with different recombinant VEE replicons expressing MBGV proteins, are shown in
Expression, antigenicity, and size determination of the MBGV proteins were confirmed by immunoprecipitation and gel electrophoresis. The results obtained from expression of MBGV GP, GPΔTM, NP, and VP40 in Vero cells are shown in
Groups of strain 13 guinea pigs were inoculated with packaged recombinant VEE replicons expressing individual MBGV proteins, and later challenged with 103.3 LD50 guinea pig-adapted MBGV subcutaneously. Results are shown in Table 1. MBGV GP protected guinea pigs from both death and viremia when administered as a three dose regimen. In addition, no reduction in efficacy or potency was observed when a two dose regimen was instituted, and significant efficacy was observed even when a single dose of 106 FFU of VRP expressing MBGV GP was used as an immunogen. The efficacy of either the two or three dose vaccine schedule was further demonstrated by the observation that no boost in postchallenge ELISA titers were observed. This result suggested minimal antigen exposure after challenge with MBGV, and thus robust or even sterile immunity in these animals. MBGV GPΔTM, which was previously shown to be protective as a vaccine when produced from insect cells, also protected guinea pigs from death and viremia when delivered in an VEE virus replicon. Again, there were no increases in postchallenge ELISA titers in the group of animals immunized with GPΔTM, thus no differences were discerned in the vaccine efficacy of membrane-bound versus soluble GP.
Protection of replicon inoculated strain 13 guinea
pigs from lethal challenge with Marburg virus (Musoke
Log 10 ELISA Titer*
# of Doses
Day 64
Lassa NP
*Endpoint titer of equal volumes of serum pooled from animals in each group against MBGV Musoke
aSurvivors/Total (S/T) on day 30 postinfection.
★★indicates p < 0.01,
★indicates p < 0.05.
bViremia (Log10 PFU/ml) day 7 postinfection. Where ≧2 animals were viremic, a GMT was calculated.
cViremic animals/total (V/T) on day 7 postinfection. All animals that died were viremic.
In the experiment shown, MBGV NP protected all vaccinated guinea pigs from both viremia and death, while MBGV VP35 vaccination resulted in five of six animals surviving, but four of the five survivors were viremic seven days postinfection. None of the other MBGV viral proteins cloned into VEE replicons evoked significant protection against a lethal challenge with MBGV. Thus, the proteins that showed the most promise as vaccine candidates in the guinea pig model were MBGV GP and NP. Cumulative results from this and additional experiments (not shown) in strain 13 guinea pigs inoculated three times with VRPs demonstrated complete survival with GP (18/18), and less complete protection with NP (16/18) and VP35 (13/18) as compared with controls (2/24).
Encouraged by the success in vaccinating guinea pigs against MBGV, we evaluated the ability of these same VEE replicons to protect cynomolgus macaques from lethal MBGV infection. The monkeys received 10-fold higher doses of replicons, but on an identical schedule as tested in the guinea pigs. Four groups contained three monkeys each. One group received VRPs which expressed MBGV GP; a second group received VRPs which expressed MBGV NP; a third group received a mixture of MBGV GP and MBGV NP VRPs; and a fourth received VRPs which expressed a control antigen (influenza HA) irrelevant to MBGV immunity. Anti-MBGV ELISA antibody titers were monitored throughout the experiment.
All animals that received VEE replicons expressing MBGV GP, either alone or in combination with MBGV NP, survived challenge with 8000 PFU MBGV without any observed signs of illness (Table 2). Of the three animals vaccinated with MBGV NP, one died 8 days after challenge from MBGV disease. The other two NP recipients displayed signs of illness 7-9 days after challenge, but eventually recovered. One NP-inoculated survivor had a relatively mild disease (slightly reduced activity and responsiveness), while the other had severe disease which included obvious petechiae, loss of weight, reduced activity, and fever. All control animals succumbed, with clinical signs first noted on day 7 or 8, and deaths occurring on days 9 or 10 postchallenge.
Survival of replicon-inoculated cynomolgus monkeys*
Day of Death
Influenza HA
9, 9, 10
*surviving animals remain healty >90 days postchallenge.
aAntigen delivered by VEE replicon.
★Indicates p = 0.05.
The pre- and postchallenge ELISA antibody titers of the cynomolgus macaques are shown in
A plaque reduction neutralization assay was performed on pre- and postchallenge serum samples. No neutralization activity was observed, at 1:20 or higher dilutions, in any sample. It should be noted that it is frequently difficult to demonstrate filovirus neutralizing antibody in vitro; however, antibodies may nonetheless be relevant in vivo (Hevey et al., 1997, Virology 239, 206-216), perhaps via mechanisms other than classical neutralization (Schmaljohn et al., 1982, supra).
The viremia levels in each of the monkeys at several time points after MBGV challenge are shown in
Upon necropsy of the control and the unprotected NP-inoculated monkeys, MBGV titers in their livers were 9.2, 9.7, 9.4, and 9.6 Log10 PFU/gm. Virus was detected in all other organs examined as well, and although abundant, was at least 10-fold lower than in the liver. Not surprisingly, elevated liver enzymes were the most obvious abnormal feature in clinical chemistries. As shown in
To our knowledge, this is the first report of any filovirus vaccine shown to be completely efficacious in nonhuman primates. Before these observations, we were cautiously optimistic about the overall feasibility of an efficacious vaccine for MBGV, but were also concerned that proofs of filovirus vaccine concepts in guinea pigs may not necessarily forecast success in nonhuman primates and, by inference, in humans. Results presented here defined GP, possibly in combination with NP, as candidate antigens for a MBGV vaccine, and demonstrated that nearly complete immunity is achievable in nonhuman primates.
We chose an alphavirus replicon based on VEE virus to deliver the antigens of interest. This method of vaccination has several advantages (Pushko et al., 1997, Virology 239, 389-401), including the ability to produce large quantities of antigen in situ, so that native processing of the antigens might evoke a broad array of immune responses. In addition, all transcription of RNA occurs in the cytoplasm of cells, which avoids RNA splicing problems sometimes observed when proteins of RNA viruses are expressed from the nucleus. Moreover, VEE replicons have proven stable after packaging into VRPs. In addition to robust antibody induction, alphavirus replicons have been demonstrated to elicit cytotoxic T lymphocytes in mice (Caley et al., 1997, J. Virol. 71, 3031-3038; Zhou et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92, 3009-3013). The success reported here using VEE replicons to vaccinate monkeys against lethal MBGV challenge justifies a more detailed analysis of the potential of these vectors for use as human vaccines. These analyses may include such factors as the relevance of host-vector interactions that may affect vaccine potency, overall safety of the vector, and the duration and minimal requirements for immunity to MBGV disease induced by this vector.
Two viral antigens demonstrated unambiguous potential as protective antigens in the guinea pig model: MBGV GP and MBGV NP. Another viral antigen, VP35, provided significant protection from death; however, most (5/6) animals vaccinated with VP35 exhibited viremias 7 days after infection. Consequently, VP35 was not considered a candidate for the initial examination of vaccine efficacy in nonhuman primates. While none of the other viral antigens showed significant promise as protective antigens in the guinea pig model, some were only weakly immunogenic, at least when delivered as VRPs. Thus, we have not formally excluded the possibility that such antigens may prove protective under different circumstances, or in species other than guinea pigs.
As a more definitive test of efficacy, the two most promising guinea pig protective antigens from MBGV were used to inoculate nonhuman primates either alone or in combination. Using recombinant VEE replicons, MBGV GP was clearly shown to be protective. The observation that none of the animals developed overt illness or viremia was conclusive proof that this vaccine approach had protected animals from a substantial challenge dose of MBGV. However, there were some significant differences observed between guinea pigs and cynomolgus macaques. Most notable was the observation that two-thirds of the GP-vaccinated monkeys demonstrated rises in ELISA antibody titers following MBGV challenge, whereas there was apparently sterile immunity (i.e. no further increases in antibody titers) to viral challenge in guinea pigs given a 10-fold lower dose of the same vaccine. This may be attributable to the overall higher prechallenge ELISA antibody titers observed in guinea pigs when compared to those observed in the monkeys (Table 1 vs.
The second antigen examined, MBGV NP, was less effective at protecting nonhuman primates compared to guinea pigs. All the monkeys inoculated with NP displayed signs of illness, with one animal dying in the same time frame as control animals. All animals were viremic, and viremia levels were predictive of outcome. As expected, the two animals that survived illness had large boosts in their ELISA antibody titers against MBGV when pre- and postchallenge sera were examined. Though not statistically significant in a group of only three animals, MBGV NP was apparently able to provide a measure of protection from death, but not from disease in two monkeys. We surmise that the immune response to NP was sufficient to suppress replication of MBGV until augmented by additional host immune responses.
The monkeys that were vaccinated with both MBGV GP and NP demonstrated the same degree of protection as the animals vaccinated with GP alone. No viremias were observed at any time point, and two of three animals demonstrated postchallenge increases in ELISA antibody titers to MBGV. These results demonstrated that the, NP replicon, equivocal by itself as a macaque vaccine, did not interfere with a GP-based vaccine when protective efficacy was used as a measurement.
For these studies, in the interest of expedient vaccine development, protection from viral disease was prioritized over the detailed study of immune mechanisms in two relatively difficult animal species for immunological studies, guinea pigs and cynomolgus macaques. It was already clear from studies done in guinea pigs that ELISA antibody titers to MBGV were not wholly predictive of clinical outcome, but rather one measure of immunogenicity of the vaccine candidate. However, it was also known that administration of polyclonal antisera or a neutralizing MAb could protect some guinea pigs from lethal challenge, indicating that antibodies can play a role in the protective response to MBGV (Hevey et al., 1997, supra). As for immunity to virtually all viruses, T cell responses to MBGV are almost certainly important in their immunoregulatory and effector functions. Indeed, we observed protection in both guinea pigs (NP and VP35) and nonhuman primates (NP) with antigens for which the most logical protective mechanisms involve cellular immunity. However, it also proved emphatically true in the most susceptible animals—nonhuman primates—that protective immunity was elicited by an antigen (GP) that theoretically favored a redundant protective response of both T cells and antibodies.
|Cited Patent
|Filing date
|Publication date
|May 23, 1995
|Aug 11, 1998
|University Of North Carolina At Chapel Hill
|Alphavirus RNA replicon systems
|Feb 19, 1997
|Sep 22, 1998
|The University Of North Carolina At Chapel Hill
|System for the in vivo delivery and expression of heterologous genes in the bone marrow
|Nov 10, 1997
|Apr 1, 2003
|The University Of North Carolina At Chapel Hill
|Alphavirus RNA replicon systems
|Becker et al (Virology 225:145-155, 1996).
|Bray et al., "A mouse model for evaluatio nof prophylaxis and therapy of Ebola hemorrhagive fever", J. Infectious Diseases, 1998, 178:651-61.
|Caley et al, "Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus Type I immunogen expressed by a Venezuelan Equine Encephalitis virus vaccine vector", J. Virology, Apr. 1997, vol. 71, No. 4, p. 3031-3038.
|Connolly et al, "Pathogenesis of experimental Ebola virus infection in guinea pigs", J. Infectious Diseases, 1999: 179(suppl 1): S203-17.
|Dubensky et al, "Sindbis virus DNA-based expression vectors: utility for in vrito and in vivo gene transfer", J. Virology, Jan. 1996, vol. 70, No. 1, pp. 508-519.
|Feldmann and Klenk, "Marburg and Ebola Viruses", Advances in Virus Research, vol. 47, 1996, pp. 1-52.
|Feldmann et al., "Characterization of Filoviruses based on differences in structure and antigenicity of the virion glycoprotein", Virology 199, 469-473 (1994).
|Feldmann et al., "Glycosylation and oligomerization of the spike protein of Marburg virus", Virology 182, 353-356 (1991).
|Feldmann et al., "Marburg virus, a filovirus: messenger RNAs, gene order, and regulatory elements of the replication cycle", Virus Research, 24 (1992) 1-19.
|Gilligan et al., "Assessment of protective immunity conferred by recombinant vaccinia viruses to guinea pigs challenged with Ebola virus", Vaccines 97 (1997), p. 87-92.
|Hevey et al (Virology 239:206-216, 1997).
|Hevey et al., "Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by Baculovirus recombinants", Virology 239:206-216 (1997).
|Hevey et al., "Recombinant Marbug Virus glycoprotein subunit vaccine protects guinea pigs from lethal infection", Vaccines 97, 1997, pp. 93-98.
|Ignat'ev et al., "Comparative analysis of some immunological parameters of inactivated Marbug virus injected into guinea pigs", Voprosy Virusologii, No. 5, pp. 118-120, 1991.
|Ignatyev et al, "Inactivated Marburg virus elicits a nonprotective immune response in Rhesus monkeys", J. Biotechnology 44 (1996) 111-118.
|Johnson et al., "Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus", Int. J. Exp. Path. (1995) 76:227-236.
|Kiley et al, "Filoviridae: a taxonomic home of Marburg and Ebola Viruses?", Taxonomy, Intervirology 18:24-32, (1982).
|Lupton et al., "New Activated Factor IX Product in Haemophilia", Letters to the Editor, The Lancet, Dec. 13, 1980, pp. 1294-1295.
|Marburg Virus Disease, Ed. Martini and Siegert, Springer-Verlag, New York, Heidelberg, Berlin (1971), pp. 1-230.
|Pattyn et al., "Isolation of Marburg-Like Virus from a Case of Haemorrhagic Fever in Zaire," The Lancet, Mar. 1977, p. 573-574.
|Pushko et al (Vaccines 97, p. 253-258, 1997).
|Pushko et al (Virology 239:389-401, 1997).
|Pushko et al., "Replicon-helper systems from attenuated Venezuelan Equine Encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo", Virology 239:389-401 (1997).
|Pushko et al., "Venezuelan Equine Encephalitis virus replicon vector: immunogenicity studies with Ebola NP and GP genes in guinea pigs", Vaccines 97, 1997, p. 253-258.
|Sanchez et al., "Sequence analysis of the Marburg virus nucleoprotein gene: comparison to Ebola virus and other non-segmented negative-strand RNA viruses", J. Gen. Virology (1992) 73:347-357.
|Smith et al., "Fatal Human Disease from Vervet Monkeys", Preliminary Communications, The Lancet, No. 7256, Nov. 25, 1967, pp. 1119 and 1121.
|Vanderzanden et al., "DNA Vaccines Expressing Either the GP or NP Genes of Ebola Virus Ptoect Mice from Lethal Challenge", Virology 245, 000-000 (1998), pp. 1-10.
|Volchkov et al., "Processing of the Ebola virus glycoprotein by the proprotein convertase furin", PNAS USA, vol. 95, pp. 5762-5767 (May 1998).
|Will et al., "Marburg virus gene 4 encosed the virion membrance protein, a Type I transmembrane glycoprotein", J. Virology, Mar. 1993, vol. 67, No. 3, p. 1203-1210.
|Xianzheng, et al. "Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus", PNAS USA, vol. 92, p. 3009-3013, Mar. 1995.
|Xu et al, "Immunization for Ebola Virus infection", Nature Medicine, vol. 4, No. 1, Jan. 1998, pp. 37-42.
|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|Oct 1, 2008
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Mumbai / New Delhi: Merger negotiations between senior managers of India's biggest mobile phone services company Bharti Airtel Ltd and Johannesburg-listed MTN Group Ltd, a phone firm with interests spanning Africa and west Africa, will take a decisive turn next week with the two sides set to decide which funding structure to opt for in a potential amalgamation, a person close to the discussions said.
The funding structure is important because it will decide how many fresh shares the acquiring company will potentially have to issue, thereby diluting its earnings per share and price of shares. Shares of Bharti Airtel, on market expectations that the New Delhi firm will be the buyer, have already lost 6.5% in value since the merger talks were confirmed by the two companies late on Monday.
"It is still blue sky discussions between senior executives, bankers, lawyers and others," the person said, asking not to be identified because the talks were still at an early stage. "But, some direction should emerge next week on whether it is a pure cash offer, cash and stock, cash-stock-debt... The stage where the (MTN and Bharti) managements will recommend a deal is far away." The person did not specify a date next week when this choice would be made.
A deal paid for only through shares, which is highly unlikely, will dilute Bharti Airtel's equity by up to two-fifths. Financial analysts such as Kawaljeet Saluja and Rohit Chordia at Kotak Institutional Equity Research in Mumbai say Bharti Airtel may have to dilute its equity up to 40%, if the company has to raise around $16.1 billion or Rs64,400 crore (at $21.8 a share), if it funds the MTN acquisition on its own. "We believe that viable option is for joint bids with another partner, which can facilitate a complete buyout without stretching the balance sheet of Bharti," the analysts wrote in a note earlier this week.
It is likely then, say analysts, that Bharti Airtel, which has $163 million in cash reserves and a long-term debt-equity ratio of nearly 35%, according to Reuters estimates, will also depend on cash and debt to pay for the acquisition. And, although appetite for acquisition finance is drying up among lenders in the US and Europe, the person quoted earlier, said the Indian phone firm had what are called "letters of comfort" from "more than a dozen" global banks "running into several billion dollars" to finance the transaction.
Earlier this week, the Financial Times, which first reported Bharti Airtel's interest in MTN, had said the Indian company had tied up for $12 billion in debt financing for the deal from lenders including Standard Chartered Bank Plc and Goldman Sachs and Co.
Bharti Airtel's shares closed at Rs842 each on Friday, valuing the company at just under $41.6 billion, while MTN shares ended trades at Rand 15,330, boosting the value of the Johannesburg-based firm at $37.17 billion.
Another person close to the bankers who have committed to fund Bharti Airtel in the MTN bid said the Makati family, a significant MTN shareholder with a nearly 11% stake through a company named M1 Ltd, had given its in-principle approval to the deal. But before the family gives its formal assent to the deal, the MTN board will first have to accept Bharti Airtel's offer. This could not be independently verified by Mint.
What makes MTN attractive to acquirers is its 68.2 million customers across 21 countries, including South Africa, Nigeria, Iran and Cyprus, netting profits of $1.58 billion on revenues of $9.62 billion. The more-profitable Bharti Airtel, in comparison, with some 62 million customers reported net profits of $1.67 billion on $6.76 billion sales.
Meanwhile, rating firm Standard and Poor's (S&P) said it may review Bharti Airtel's rating as its closes in on the MTN acquisition.
"While a prominent overseas investment adds diversity to Bharti's businesses and cash flows, the financing and the debt requirements are of greater concern, especially since Bharti has budgeted capital expenditures of about $3.3 billion per annum," Yasmin Wirjawan and Anshukant Taneja, rating analysts at S&P wrote in a note on Friday. S&P rates Bharti Airtel's debt BBB-, the lowest investment-grade rating, with a stable outlook.
Fitch Ratings, another international rating firm, said it's not taking any rating action because of insufficient information on the possible transaction and the likelihood of other bids for MTN.
Fitch has a BB+ rating on Bharti's debt, its highest non-investment grade rating, with a stable outlook.
Andrea Tan and Harichandan Arakali of Bloomberg contributed to this story.

subset: commoncrawl


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The Camellia Villa is a home away from home, but better. The decor sets the mood for a relaxing southern style vacation. Open the windows and enjoy bay breezes or watch one of our breathless sunsets. There is even an extra bedroom for the children to ensure privacy.
Discovered Our Memorable Hideaway


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Tuesday, January 22, 2013
Math Notes for PH Mathematics Course ~
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We are starting a new Math Course with our youngest. It is
Prentice Hall Math Course 1.
Since the Teachers Editions are now all on CD ROM in PDF version. I took some time to go through it and made some notes for myself so I can quickly skip to where I need to be in the program. You can find these notes
Anyone using this program might really find these notes helpful, which is why I thought to share, when looking at the Teachers Resources on CD ROM.
Note: I am finding the easiest way to work this program is to load the New Paced Lesson plans into the Lesson Book. The Lesson Plans that are pre-loaded shows all the information, objectives, lesson vocabulary, materials needed and th elist of activities and descriptions along with links for the information and resource.
Happy Math Learning!
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The Ministry of Foreign Affairs of Denmark (MFA) provides grants for development research activities as part of Denmark's international development cooperation. Within this framework, the MFA invites applications for individual Danida Mobility Grants to facilitate the creation and development of international networks and research collaboration through research stays in Denmark by researchers from the growth and transition countries included in the "Partnering with Denmark" programme: Bangladesh, Brazil, China, Colombia, Ghana, Indonesia, Kenya, Mexico, Myanmar, South Africa, Turkey, and Vietnam.
Mobility Grants – Call for applications for individual mobility grants for researchers from growth and transition countries (involved in the programme "Partnering with Denmark")
The applicant (Danish host institution) must fill in the electronic application form with:
Appendix B: template for agreement.
CALL OPEN – no application deadline.
Reference is made to the section with Questions and Answers under Calls for Applications.


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Your Home is not an Investment, It's a Lifestyle Choice
In this article, I plan to take a hard look at the true costs of home ownership, its value as an investment, and how it fits into your plans for financial independence (FI).
I've heard it many times over the years from those I assumed were wiser than me, "Purchase a home, it's a great investment." or "Renting is throwing money away." Then there was, "Buy as much home as you can afford." After all, home ownership is the American dream. It's just what you do… Right?
That logic has come under great scrutiny these days, especially since the financial crisis of 2008. I'm not going to tell you that a home purchase is a poor investment. Honestly, it depends on your unique situation and includes factors such as where you would like to live, the cost of purchasing vs. renting in your area, how nomadic you are, how disciplined you are with saving and investing, etc. To make an informed decision, you need to understand all of the costs of home ownership, sit down and do the math, as well as consider the value of your personal desires.
The True Cost of Home Ownership
Many home buyers fail to consider the true cost of home ownership (I use the word "ownership" lightly here because you don't really own a home until you are mortgage free. Even then, you must pay property taxes or risk losing your home). They typically think only in terms of the mortgage payment (principal + interest) and their desire to start building equity. However, there is a myriad of other costs that go beyond the mortgage payment, as well as obstacles that get in the way of building equity. The true cost of home ownership involves Ongoing Costs, Opportunity Costs, as well as Costs to your Freedom.
Ongoing Costs
Very simply stated, the ongoing costs of a home look something like this:
Mortgage + PMI + Property Tax + Insurance + Maintenance + Repairs + Remodels + Association Fees + Utilities = a Big 'ol Hunk of your Income and Time.
In addition to these costs, many home buyers fail to consider the impact of the mortgage amortization schedule. An amortization schedule is a timeline for paying off a loan. The schedule breaks down the payments into interest and principal. As the mortgage is paid down, the ratio of payment between interest and principle changes.
Unfortunately for the home buyer, mortgages are front end loaded. In other words, during the early years of a mortgage payment schedule, the payment is mostly interest and the principle of the loan is reduced by very little. If the loan were payed off as scheduled, it would take approximately 20 years to get half way through a 30 year $200,000 mortgage at 4% interest.
What does this mean for the typical home buyer chasing the American dream of bigger is better? Every time a home buyer upgrades and takes out a new mortgage, the amortization of that loan starts over. As a result, it is very expensive to keep moving and "keep movin' on up." Now, combine that with the average 10% hit you take in fees when selling and buying a home. Well, you can see how this process inhibits your ability to build equity and prolongs the process of becoming debt free.
Opportunity Costs
In regard to home ownership, opportunity cost is lost income due to money that is tied up in a home instead of being invested elsewhere, such as the stock market. For a more in depth description of opportunity cost, check this out: Investopedia – Opportunity Cost Definition.
Although home prices have consistently trended up throughout history (2007-2011 being a recent exception), you can see how ongoing costs can cut into the return on your investment. In addition, the S&P 500 has historically averaged a 7% return when adjusted for inflation. Now, calculate what that down payment and other ongoing costs would have yielded over that 30 year loan. You most likely come out head investing in index funds.
Do the math for yourself. There are plenty of calculators out there to help you tackle the rent vs. own debate. This calculator at Financial Mentor is one of the better ones I have come across: Financial Mentor – Rent vs. Buy Calculator. Not only does it consider opportunity costs, but it comes complete with terms and definitions at the end of the article. Start running the numbers and take it from there.
Costs to your Freedom
Generally speaking, real estate is not a very liquid asset and it can be difficult to sell unless you live in a hot market. Home ownership can tie your money up for long periods of time, making it difficult to move around as you see fit. In contrast, as a renter, you can move on when you are ready without the stress, expenses, or time required to sell a home.
The importance of this depends largely on how nomadic you plan to be. Do you plan on long term travel before and after FI? Are you unsure of your plans to stay put in a certain geographical location for at least five to ten years? Do you plan to regularly explore living in other geographical locations in the near future? If you answered yes to any of these questions, renting would be the better decision in terms of freedom, as well as financially.
The Case for Home Ownership
There are many arguments for the benefits of owning a home including tax benefits, providing an inflation protected asset, and diversification. However, the true benefits of these can, arguably, be obtained through other means. To many, home ownership is not solely a financial decision.
Most of the reasons for owning a home that hold any value for me have more to do with lifestyle choices rather than finances. They include increased options as well as psychological benefits. Consider the following examples:
Home ownership can provide you with more options regarding the type of home and area that you would like to reside. This is especially true if rental options are limited. If you happen to find your utopia, your home base, and plan to stay put, then home ownership becomes more valuable.
If you are the type of person who finds a sense of security in "owning" your home, or you are uncomfortable with a landlord having any degree of control over your life, then home ownership may hold more value for you.
Finally, if you have already gone down the path of home ownership and your mortgage is either paid or almost paid off, home ownership holds value here as well. Debt free and rent free is a good place to be, both psychologically and financially as you approach financial independence and retirement. Just remember to account for the opportunity and freedom costs of home ownership mentioned previously.
All of these reasons may come at a financial cost of varying degrees. Only you can decide how much of your life they are worth. Again, start by running the numbers and take it from there. Knowledge is power.
Don't Buy as Much House as You Can Afford, Buy Only as Much as You Need
The bottom line is to make a choice that enables you to live well below your means, regardless if you rent or buy. In my article, Where I've Been and How I Started on the Path to FI, I revealed how the significant downsizing of my home has made a huge positive impact on our lives. It freed up a large amount of disposable income that we applied to our investment vehicle of choice, low cost index funds. I can't emphasize enough how important this has been in our success toward FI.
I'm not suggesting you live in a shack. Just be realistic and do the math. Purchase or rent a home that doesn't make you feel imprisoned by debt and allows you to maintain a VERY high savings rate.
Honestly, if I could go back with the same knowledge I have now, I would have put off home ownership for a while. Instead, I would have utilized that money to develop a strong financial base through investing early in life. The power of compounding interest would have started working its magic for me much sooner. As they say, hindsight is 20/20.


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The greenback tumbled right after the Federal Reserve declared Wednesday
that it lifted its benchmark desire fee.
The US greenback index was down by
.eight% at a hundred.89 around 2:eighteen p.m. ET.
The Federal Open Marketplaces Committee lifted
the benchmark federal funds fee by 25 foundation details to a vary of
.75%-1%, marking its third raise because the Excellent
Economic downturn. This hike will finally trickle down to
raise borrowing expenses
for small-time period loans and
credit rating playing cards.
Almost every person
experienced been
this final result. In point, the Globe
Desire Amount probability details offered by Bloomberg
traders viewing a a hundred% opportunity of a
The FOMC projected
much more fee hikes
this year, unchanged from its prior
estimate of 3 in 2017. Only a single member of the FOMC —
Minneapolis Fed's Neel Kashkari — voted
in opposition to a fee hike.
Head around right here to get all the facts on the Fed's
As for the rest of the entire world, here's the scoreboard as of eight:47
a.m. ET:
- The British pound is up by .nine% at
1.2260 in opposition to the greenback. Previously,
details launched by the Business office for Countrywide Data confirmed
that the headline unemployment fee fell to four.7%,
a level last noticed in the summer of 1975. On the other hand,
actual wage expansion in the Uk is slowing: regular pay back rose by just
.eight% — the most affordable fee because October 2014.
- The euro is higher by .7% at 1.0675
in opposition to the greenback as Europe gears up for the very first of
a lot of significant elections this year:
the Dutch elections. The country's political landscape is
fragmented, with around 28 parties in these elections, indicating
that a coalition among two or much more parties would be necessary to
sort a vast majority govt.
New polls counsel that Geert Wilder's populist celebration has
dipped in acceptance, but even if his party were to
get the well-liked vote, it is unlikely that he would grow to be the
up coming key minister specified that lots of of his
rivals have ruled out forming a coalition with the
- The South African rand
is more robust by 1.nine% at 12.9089 for each greenback right after poor retail
income, which fell by 2.three% year-around-year in January, in comparison to
forecasts of an uptick of 1.2%. "The slide in retail income in
January is disappointing specified the strengthening photo painted by
[Tuesday's] mining and production figures. Even so, we hope
that expansion will decide on up later in the year as agricultural
output rebounds and inflation eases," John Ashbourne, Africa
economist at Capital Economics, wrote.
- The Russian ruble is up .7% at
fifty eight.7269 for each greenback, when Brent crude oil, the international
benchmark, is higher by 1.five% at $fifty one.68 for each barrel.
- The Japanese yen is up by .7% at
113.ninety four for each greenback.


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Bummerville (GNIS FID: 220146) is a populated place located in Calaveras County at latitude 38.401 and longitude -120.506.
The elevation of Bummerville is 2,956 feet. Bummerville appears on the West Point U.S. Geological Survey Map. Calaveras County is in the Pacific Time Zone (UTC -8 hours).
Calaveras County: Cities & Towns | ZIP Codes | Features (airports, churches, hospitals, etc.)
BIG Map |
Driving Directions |
Data Source - USGS GNIS FID: 220146
BOUNDARY MAPS: Arnold-West Point CCD | Calaveras County
West Point, CA
(1.4 miles W)
Buckhorn, CA (3.6 miles NNW)
Pioneer, CA (4.1 miles WNW)
Rail Road Flat, CA (4.1 miles S)
Red Corral CDP, CA (5.5 miles W)
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Pine Grove, CA (8.3 miles W)
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Independence, CA (3.6 miles S)
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(3.9 miles NNW)
Porter, CA (4.3 miles E)
Fuchs, CA (4.5 miles SE)
Glencoe, CA (5.5 miles SW)
Sandoz, CA (5.5 miles SE)


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Enrollment Problems Increasing, Soviet Communist Party Admits
By BILL KELLER, Special to The New York Times
Published: July 12, 1989
MOSCOW, July 11— The Soviet Communist Party is troubled by dwindling recruitment and a steep decline in prestige, according to an unusually candid collection of articles and letters published today in Moscow's main party newspaper.
The material included an article by a teacher at a top party training school who reported that many of the most active young party candidates are defecting to join informal political organizations, where the activities are ''more relevant and effective.''
Occasional letters expressing anguish about the condition of the party began to creep into the press last year, but the hand-wringing has become more pronounced since March, when many prominent party figures were trounced in the first competitive elections for a new national legislature. Special Concern in Leningrad
In another sign of the party's distress, Mikhail S. Gorbachev, the President and party leader, today went to Leningrad, where voters rejected the six top Communists in the city's party organization. The defeated Leningrad officials have refused to give up their party leadership positions.
At the time of the elections, Mr. Gorbachev said the outcome was not a repudiation of the party. Emphasizing that nearly 90 percent of the winners were party members, he said the elections were a victory of fresher and more progressive Communists over conservative party functionaries.
But the party-controlled press is beginning to admit that the party itself is held in low repute.
The declining prestige of the party is most evident in statistics showing plummeting enrollment of new party members, despite the fact that membership has traditionally brought career advancement and privileges.
According to data published recently in the journal of the party Central Committee, induction of new members began to fall off in 1987, and last year plunged 20 percent. After years of steady growth, the total party membership barely held steady at 19.5 million.
The party does not publish statistics on the number of people who quit, but the letters published today in Moskovskaya Pravda indicated that this, too, has become a growing problem. Doing Their Own Thing
One writer, the party secretary at a Moscow metallurgical factory, reported that in the first six months of this year more workers in that factory had quit the party than had joined.
''The most active Communists often leave to 'do their own thing' - that is, they join more lively and flexible, more relevant and effective voluntary movements,'' wrote the teacher at the party training school.
Several writers said the greatest blow to the party's prestige was its decision to reserve 100 seats in the new 2,250-member Congress for deputies chosen by the party without a vote of the general public.
''No matter which way you look at it, the party still retains Stalin's pattern, a rigid, command-administrative structure,'' the teacher wrote. ''It lags behind the changes in life. It cannot prevent undesirable changes and it lags behind the positive ones.''


text:
If Charlie Parker Was a Gunslinger,
There'd Be a Whole Lot of Dead Copycats
Containing Multitudes Since 2004
To Whom Does the World Belong? #2
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Daniel Riccuito
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Sylvester Stallone: 'Grief is like a virus'
Sage Stallone, 36, was found dead at his home in the Hollywood Hills in July (12), and a Los Angeles County Coroner subsequently determined the actor/producer suffered a heart attack.
The tragedy hit The Expendables star hard, and Stallone admits he is still consumed by grief four months after his son's passing.
He tells Britain's Hello! magazine, "I fall to pieces from time to time. You just do. It's a cycle you just have to deal with, like a virus that keeps coming back.
"You go through this time of thinking, 'It's really horrible', then you come out at the other end and realise there's not much you can do about it and you forgive yourself… Then you go through another cycle where you don't forgive yourself, then you get over it again… It's just an ongoing thing. And I don't think it ever goes away."