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1.  Vesicular Stomatitis Virus-Based Vaccines Protect Nonhuman Primates against Bundibugyo ebolavirus 
Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29–36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.
Author Summary
Ebola viruses (EBOV), of which there are five species, are categorized as Category A Priority Pathogens and Tier 1 Select Agents by several US Government agencies as a result of their high mortality rates and potential for use as agents of bioterrorism. Currently, there are no vaccines or therapeutics approved for human use. Replication-competent, recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins (GP), in place of the VSV glycoprotein have shown promise in lethal nonhuman primate (NHP) models of filovirus infection as both single injection preventive vaccines and as post-exposure treatments. The recent outbreak of the fifth recognized EBOV species, Bundibugyo ebolavirus (BEBOV), demonstrates the need for vaccines that can be rapidly deployed to combat an outbreak of a new filovirus species. To date, rVSV-filovirus GP-based vaccines have only been able to protect against challenge with a homologous species of EBOV. Here, we show that the two heterologous rVSV-based filovirus vaccines available at the time of the original BEBOV outbreak can protect NHPs against BEBOV challenge using a short prime-boost vaccination strategy. While the prime-boost strategy was successful, a single injection blended vaccination strategy with the same vaccine vectors failed to provide protection. These data suggest that an abbreviated prime-boost regimen of 36 days may have utility for quickly responding to outbreaks caused by new species of EBOV.
doi:10.1371/journal.pntd.0002600
PMCID: PMC3868506  PMID: 24367715
2.  A Replicating Cytomegalovirus-Based Vaccine Encoding a Single Ebola Virus Nucleoprotein CTL Epitope Confers Protection against Ebola Virus 
Background
Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the ‘bush-meat’ trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.
Methodology/Principal Findings
We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a ‘proof-of-concept’ for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NPCTL). MCMV/ZEBOV-NPCTL induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.
Conclusions/Significance
This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for ‘disseminating’ CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.
Author Summary
Human outbreaks of hemorrhagic disease caused by Ebola virus (EBOV) are a serious health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans. Candidate EBOV vaccines do not spread from the initial vaccinee. In addition to being highly immunogenic, vaccines based on the cytomegalovirus (CMV) platform have the unique potential to re-infect and disseminate through target populations. To explore the utility of CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a region of nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NPCTL). MCMV/ZEBOV-NPCTL induced high levels of long-lasting CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. The absence of ZEBOV neutralizing and only low, sporadic levels of total anti-ZEBOV IgG antibodies in protected animals prior to ZEBOV challenge indicate a role, albeit perhaps not exclusive, for CD8+ T cells in mediating protection. This study demonstrates the ability of a CMV-based vaccine approach to protect against ZEBOV, and provides a ‘proof-of-concept’ for the potential for a ‘disseminating’ CMV-based EBOV vaccine to prevent EBOV transmission in wild animal populations.
doi:10.1371/journal.pntd.0001275
PMCID: PMC3153429  PMID: 21858240
3.  Vesicular Stomatitis Virus–Based Ebola Vaccines With Improved Cross-Protective Efficacy 
The Journal of Infectious Diseases  2011;204(Suppl 3):S1066-S1074.
For Ebola virus (EBOV), 4 different species are known: Zaire, Sudan, Côte d’Ivoire, and Reston ebolavirus. The newly discovered Bundibugyo ebolavirus has been proposed as a 5th species. So far, no cross-neutralization among EBOV species has been described, aggravating progress toward cross-species protective vaccines. With the use of recombinant vesicular stomatitis virus (rVSV)–based vaccines, guinea pigs could be protected against Zaire ebolavirus (ZEBOV) infection only when immunized with a vector expressing the homologous, but not a heterologous, EBOV glycoprotein (GP). However, infection of guinea pigs with nonadapted wild-type strains of the different species resulted in full protection of all animals against subsequent challenge with guinea pig–adapted ZEBOV, showing that cross-species protection is possible. New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP. After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40. Our data demonstrate that cross-protection between the EBOV species can be achieved, although EBOV-GP alone cannot induce the required immune response.
doi:10.1093/infdis/jir348
PMCID: PMC3203393  PMID: 21987743
4.  Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector▿  
Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.
doi:10.1128/CVI.00467-09
PMCID: PMC2849326  PMID: 20181765
5.  Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species 
PLoS Pathogens  2010;6(5):e1000904.
A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.
Author Summary
Ebola virus causes death, fear, and economic disruption during outbreaks. It is a concern worldwide as a natural pathogen and a bioterrorism agent, and has caused death to residents and tourists of Africa where the virus circulates. A vaccine strategy to protect against all circulating Ebola viruses is complicated by the fact that there are five different virus species, and individual vaccines provide protection only against those included in the vaccine. Making broad vaccines that contain multiple components is complicated, expensive, and poses challenges for regulatory approval. Therefore, in the present work, we examined whether a prime-boost immunization strategy with a vaccine targeted to one Ebola virus species could cross protect against a different species. We found that genetic immunization with vectors expressing the Ebola virus glycoprotein from Zaire blocked infection with a newly emerged virus species, Bundibugyo EBOV, not represented in the vaccine. Protection occurred in the absence of antibodies against the second species and was mediated instead by cellular immune responses. Therefore, single-component vaccines may be improved to protect against multiple Ebola viruses if they are designed to generate this type of immunity.
doi:10.1371/journal.ppat.1000904
PMCID: PMC2873919  PMID: 20502688
6.  Cathepsin B & L Are Not Required for Ebola Virus Replication 
Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB−/− and catL−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.
Author Summary
It is currently believed that Ebola virus (EBOV) enters cells via macropinocytosis following which, the cysteine proteases cathepsin B and L (CatB, CatL) cleave the viral glycoprotein (GP) allowing exposure of its core receptor-binding and fusion domain thus facilitating subsequent infection. We studied the effect of CatB and CatL on in vitro and in vivo EBOV replication. Our results demonstrate a reduction of Zaire ebolavirus (ZEBOV) entry upon selective inhibition of CatB, but not CatL in cell culture. Interestingly, all other EBOV species enter the cells efficiently when CatB and/or CatL activity is blocked. Moreover, when wild-type (control), catB−/− and catL−/− mice were infected with a lethal dose of mouse-adapted ZEBOV, all animals were equally susceptible to lethal challenge with no difference in virus replication and time to death. Therefore, we conclude that EBOV replication is dispensable of CatB and CatL, and proteolytic processing of GP can also be mediated by other endosomal proteases.
doi:10.1371/journal.pntd.0001923
PMCID: PMC3516577  PMID: 23236527
7.  Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates 
PLoS Pathogens  2008;4(11):e1000225.
Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.
Author Summary
Ebola virus is among the most lethal microbes known to man, with case fatality rates often exceeding 80%. Since its discovery in 1976, outbreaks have been sporadic and geographically restricted, primarily to areas of Central Africa. However, concern about the natural or unnatural introduction of Ebola outside of the endemic areas has dramatically increased both research interest and public awareness. A number of candidate vaccines have been developed to combat Ebola virus, and these vaccines have shown varying degrees of success in nonhuman primate models. Safety is a significant concern for any vaccine and in particular for vaccines that replicate in the host. Here, we evaluated the safety of our replication-competent vesicular stomatitus virus (VSV)-based Ebola vaccine in SHIV-infected rhesus monkeys. We found that the vaccine caused no evidence of overt illness in any of these immunocompromised animals. We also demonstrated that this vaccine partially protected the SHIV-infected monkeys against a lethal Ebola challenge and that there appears to be an association with levels of CD4+ lymphocytes and survival. Our study suggests that the VSV-based Ebola vaccine will be safe in immunocompromised populations and supports further study and development of this promising vaccine platform for its use in humans.
doi:10.1371/journal.ppat.1000225
PMCID: PMC2582959  PMID: 19043556
8.  Single-Injection Vaccine Protects Nonhuman Primates against Infection with Marburg Virus and Three Species of Ebola Virus▿  
Journal of Virology  2009;83(14):7296-7304.
The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (CIEBOV), and Marburgvirus (MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVΔG/SEBOVGP, VSVΔG/ZEBOVGP, and VSVΔG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVΔG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic filovirus species.
doi:10.1128/JVI.00561-09
PMCID: PMC2704787  PMID: 19386702
9.  Single Immunization With a Monovalent Vesicular Stomatitis Virus–Based Vaccine Protects Nonhuman Primates Against Heterologous Challenge With Bundibugyo ebolavirus 
The Journal of Infectious Diseases  2011;204(Suppl 3):S1082-S1089.
The recombinant vesicular stomatitis virus (rVSV) vector-based monovalent vaccine platform expressing a filovirus glycoprotein has been demonstrated to provide protection from lethal challenge with Ebola (EBOV) and Marburg (MARV) viruses both prophylactically and after exposure. This platform provides protection between heterologous strains within a species; however, protection from lethal challenge between species has been largely unsuccessful. To determine whether the rVSV-EBOV vaccines have the potential to provide protection against a newly emerging, phylogenetically related species, cynomolgus macaques were vaccinated with an rVSV vaccine expressing either the glycoprotein of Zaire ebolavirus (ZEBOV) or Côte d’Ivoire ebolavirus (CIEBOV) and then challenged with Bundibugyo ebolavirus (BEBOV), which was recently proposed as a new EBOV species following an outbreak in Uganda in 2007. A single vaccination with the ZEBOV–specific vaccine provided cross-protection (75% survival) against subsequent BEBOV challenge, whereas vaccination with the CIEBOV–specific vaccine resulted in an outcome similar to mock-immunized animals (33% and 25% survival, respectively). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens.
doi:10.1093/infdis/jir350
PMCID: PMC3189995  PMID: 21987745
10.  Single dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus 
Nature  2015;520(7549):688-691.
The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus1. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal hemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in nearly 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primate (NHPs) against ZEBOV2. In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately ten-fold lower vaccine-associated viremia compared to the first generation vaccine and both provided complete, single dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.
doi:10.1038/nature14428
PMCID: PMC4629916  PMID: 25853476
11.  A Complex Adenovirus-Vectored Vaccine against Rift Valley Fever Virus Protects Mice against Lethal Infection in the Presence of Preexisting Vector Immunity▿  
Clinical and Vaccine Immunology : CVI  2009;16(11):1624-1632.
Rift Valley fever virus (RVFV) has been cited as a potential biological-weapon threat due to the serious and fatal disease it causes in humans and animals and the fact that this mosquito-borne virus can be lethal in an aerosolized form. Current human and veterinary vaccines against RVFV, however, are outdated, inefficient, and unsafe. We have incorporated the RVFV glycoprotein genes into a nonreplicating complex adenovirus (CAdVax) vector platform to develop a novel RVFV vaccine. Mice vaccinated with the CAdVax-based vaccine produced potent humoral immune responses and were protected against lethal RVFV infection. Additionally, protection was elicited in mice despite preexisting immunity to the adenovirus vector.
doi:10.1128/CVI.00182-09
PMCID: PMC2772385  PMID: 19776190
12.  Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine 
PLoS Pathogens  2013;9(5):e1003389.
We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.
Author Summary
Ebola virus (EBOV) has been associated with outbreaks in human and nonhuman primate populations since 1976. With a fatality rate approaching 90%, EBOV is one of the most lethal infectious diseases in humans. The increased frequency of EBOV outbreaks along with its potential to be used as a bioterrorism agent has dramatically strengthened filovirus vaccine research and development. While there are currently no approved vaccines or post exposure treatments available for human use, several vaccine candidates have shown to protect nonhuman primates from lethal EBOV challenge. Our primary focus is to develop vaccine candidates to protect humans and endangered wildlife species at risk of infection in Africa. Here, we evaluated the efficacy and immunogenicity of our dual vaccines against EBOV and rabies virus (RABV) in rhesus macaques. Our live replication-competent vaccine provided 100% protection following EBOV challenge while the replication-deficient and inactivated candidates provided 50% protection. Interestingly, protection is dependent on the quality of the antibodies rather than the quantity. All three RABV-based EBOV vaccines did induce antibody levels necessary for protection from RABV infection. These results encourage the further development of these novel dual vaccines directed against two of the most lethal viral diseases.
doi:10.1371/journal.ppat.1003389
PMCID: PMC3667758  PMID: 23737747
13.  Prediction and identification of mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins 
Virology Journal  2012;9:111.
Background
Ebola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs).
Results
Computer-assisted algorithms were used to predict H-2d-specific T cell epitopes in two species of EBOV (Sudan and Zaire) GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV), GPCAGDFAF and LYDRLASTV (Zaire EBOV) could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma.
Conclusion
Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.
doi:10.1186/1743-422X-9-111
PMCID: PMC3411508  PMID: 22695180
Ebola virus; T cell epitope; Replication-deficient adenovirus; Computer-assisted algorithms
14.  Molecular Determinants of Ebola Virus Virulence in Mice 
PLoS Pathogens  2006;2(7):e73.
Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in humans and nonhuman primates, with fatality rates in humans of up to 90%. The molecular basis for the extreme virulence of ZEBOV remains elusive. While adult mice resist ZEBOV infection, the Mayinga strain of the virus has been adapted to cause lethal infection in these animals. To understand the pathogenesis underlying the extreme virulence of Ebola virus (EBOV), here we identified the mutations responsible for the acquisition of the high virulence of the adapted Mayinga strain in mice, by using reverse genetics. We found that mutations in viral protein 24 and in the nucleoprotein were primarily responsible for the acquisition of high virulence. Moreover, the role of these proteins in virulence correlated with their ability to evade type I interferon-stimulated antiviral responses. These findings suggest a critical role for overcoming the interferon-induced antiviral state in the pathogenicity of EBOV and offer new insights into the pathogenesis of EBOV infection.
Synopsis
Zaire ebolavirus causes severe hemorrhagic fever in humans with up to 90% case-fatality rates. Currently, there are no vaccines or specific therapeutic interventions available for this devastating viral disease due, at least in part, to a lack of knowledge regarding the molecular basis of virulence for this extremely pathogenic agent. While adult mice resist wild-type Zaire ebolavirus infection, the virus has recently been adapted to cause lethal infection in mice. In order to understand the pathogenesis underlying Zaire ebolavirus infection, the authors identified the mutations responsible for the acquisition of virulence in mice, using reverse genetics technology, which allows the generation of genetically altered mutant viruses from cloned cDNA. By testing the virulence of mutant viruses, two viral proteins, viral protein 24 and the nucleoprotein, were found to be primarily responsible for the acquisition of virulence in mice. Moreover, the role of these proteins in virulence correlated with their ability to confer resistance to interferon-stimulated antiviral responses in mouse cells. These findings suggest a critical role of these proteins in overcoming the interferon-induced antiviral state in the pathogenicity of Zaire ebolavirus and offer new insights into the pathogenesis of Zaire ebolavirus infection.
doi:10.1371/journal.ppat.0020073
PMCID: PMC1513261  PMID: 16848640
15.  Inactivated or Live-Attenuated Bivalent Vaccines That Confer Protection against Rabies and Ebola Viruses ▿  
Journal of Virology  2011;85(20):10605-10616.
The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.
doi:10.1128/JVI.00558-11
PMCID: PMC3187516  PMID: 21849459
16.  Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge 
PLoS ONE  2016;11(9):e0162446.
Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.
doi:10.1371/journal.pone.0162446
PMCID: PMC5021345  PMID: 27622456
17.  Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine 
PLoS ONE  2009;4(4):e5308.
Background
The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector.
Methodology/Principal Findings
Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge.
Conclusions/Significance
We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the molecular components of adenovirus-based vaccines can produce potent, optimized product, useful for vaccination and post-exposure therapy.
doi:10.1371/journal.pone.0005308
PMCID: PMC2669164  PMID: 19390586
18.  A Tetravalent Dengue Vaccine Based on a Complex Adenovirus Vector Provides Significant Protection in Rhesus Monkeys against All Four Serotypes of Dengue Virus▿  
Journal of Virology  2008;82(14):6927-6934.
Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.
doi:10.1128/JVI.02724-07
PMCID: PMC2446963  PMID: 18480438
19.  An Ebola whole-virus vaccine is protective in nonhuman primates 
Science (New York, N.Y.)  2015;348(6233):439-442.
Zaire ebolavirus is the causative agent of the current outbreak of hemorrhagic fever disease in West Africa. Previously, we showed that a whole Ebola virus (EBOV) vaccine based on a replication-defective EBOV (EBOVΔVP30) protects immunized mice and guinea pigs against lethal challenge with rodent-adapted EBOV. Here, we demonstrate that EBOVΔVP30 protects nonhuman primates against lethal infection with EBOV. Although EBOVΔVP30 is replication-incompetent, we additionally inactivated the vaccine with hydrogen peroxide; the chemically inactivated vaccine remained antigenic and protective in nonhuman primates. EBOVΔVP30 thus represents a safe, efficacious, whole-EBOV vaccine candidate that differs from other EBOV vaccine platforms in that it presents all viral proteins and the viral RNA to the host immune system, which might contribute to protective immune responses.
doi:10.1126/science.aaa4919
PMCID: PMC4565490  PMID: 25814063
20.  Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs 
PLoS Medicine  2006;3(6):e177.
Background
Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine.
Methods and Findings
To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 1010 particles, two logs lower than that used previously.
Conclusions
Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.
A simplified Ebola vaccine that consists of a modified GP protein (which is well-tolerated by human cells even at high concentrations) in a replication-defective adenoviral vector protects macaques.
Editors' Summary
Background.
Humans who get infected with Ebola virus develop an illness called Ebola hemorrhagic fever (EHV), which is one of the most deadly viral diseases known; 50%–90% of all ill patients die, and there is no available treatment for EHV. Scientists think that the occasional outbreaks of the disease occur because the virus “jumps” from an infected animal to a person (a rare event) and then is transmitted between people by direct contact with infected blood or other body fluids or parts. Several strains or variants of the Ebola virus exist. Most outbreaks have been caused either by the Zaire strain or by the Sudan/Gulu strain (so-called because that is where the particular virus was first isolated). Scientists are working on a vaccine against Ebola that could be given to people before they get infected and then protect them when they come in contact with the virus. A number of candidate vaccines have been developed and tested in animals.
Why Was This Study Done?
The researchers who did this study are working on a vaccine that consists of two particular parts of the virus. One part is called GP (which stands for glycoprotein) and is from the outer coat of the virus; the other, NP (nucleoprotein), is from its inside. Without the rest of the virus, GP and NP cannot cause EBV. However, the hope is that giving these parts of the virus to an individual can educate their immune system to build a response against GP and NP, which would then recognize the virus should the vaccinated person become infected with the whole virus, and destroy it before it can cause disease. To get the GP and NP parts into the body so that they can cause a strong immune response (which is what effective vaccines do), the researchers used a manmade version of another, harmless virus called recombinant adenovirus 5 (or rAd5) to carry the NP and GP. The researchers have shown previously that this strategy for introducing a vaccine works in animals. The vaccine—i.e., the combination of the rAd5 virus and the two Ebola virus parts—can protect animals against subsequent infection with real Ebola virus that would otherwise kill them. However, during these earlier studies, the researchers had noticed that the GP part, when present at high levels, seemed to make human cells sick. They had not seen any similar problems in the experimental animals, but to be on the safe side they decided to see whether they could change the GP part so that it would still be effective as a vaccine but no longer make human cells sick.
What Did the Researchers Do and Find?
They changed the GP part of the vaccine in different ways so that it would no longer make human cells sick and then tested whether the resulting vaccines (combined with the original NP part and the Ad5 virus) could still protect monkeys from EHF after they were infected with Ebola virus. They found that some of the new GP versions made the vaccine less effective, but others did what they had hoped for; namely, they gave the same level of protection as when the original GP part was present. While doing these experiments, the researchers also found that the NP component seemed unnecessary and in some cases even weakened the vaccine's effect.
What Do These Findings Mean?
The researchers have now developed a simplified vaccine against Ebola virus that is effective in monkeys. This vaccine consists of only a modified GP component (which is well tolerated by human cells even at high concentrations) and the rAd5 component. This vaccine is not the only candidate currently being developed against Ebola, but it seems likely that it is one of a few that will be tested in human volunteers in the near future. The initial clinical trials will test whether the vaccine is safe in humans, and whether it can cause the immune system to produce an immune response that is specific for the Ebola virus. Assuming that the outcomes of these trials are positive, the next question is whether the vaccine can protect humans against Ebola disease. Because Ebola is so dangerous and outbreaks are relatively rare, the vaccine will likely be tested only during an actual outbreak. At that time, an experimental vaccine might be given to people at immediate risk of becoming infected, especially health-care workers who, because they take care of infected patients, are themselves at very high risk of becoming infected. In addition to trials in humans, the scientists will also explore whether this vaccine, which was developed based on the GP component of the Zaire strain, can protect monkeys against infections with other strains of the Ebola virus.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030177.
• World Health Organization
• MedlinePlus Medical Encyclopedia
• US Centers for Disease Control and Prevention
• Wikipedia (note: Wikipedia is a free Internet encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030177
PMCID: PMC1459482  PMID: 16683867
21.  A New Approach for Monitoring Ebolavirus in Wild Great Apes 
Background
Central Africa is a “hotspot” for emerging infectious diseases (EIDs) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. Ebolavirus is suspected to have caused recent declines in resident great apes. While ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm Ebola virus disease (EVD) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure.
Methodology/Principal findings
Here we report the first successful noninvasive detection of antibodies against Ebola virus (EBOV) from wild ape feces. Using this method, we have been able to identify gorillas with antibodies to EBOV with an overall prevalence rate reaching 10% on average, demonstrating that EBOV exposure or infection is not uniformly lethal in this species. Furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to EBOV (protected from exposure by rivers as topological barriers of transmission).
Conclusions/Significance
Our new approach will contribute to a strategy to protect apes from future EBOV infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. Finally, since human EVD is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where EBOV causes case-fatality rates of up to 88%.
Author Summary
Ebolavirus causes deadly outbreaks in wild great apes, and has been reported as a significant threat to the survival of wild lowland gorillas in Central Africa. Improved knowledge of basic information regarding geographic distribution of ebolavirus in great ape populations, including the identification of immunologically naïve populations and the determination of whether apes survive virus exposure, will be needed in order for protective interventions such as immunization to be effective. However, monitoring ebolavirus infection in wild gorillas by current methods is challenging because of the difficulty in obtaining diagnostic samples from these elusive primates. Additionally, there are limitations associated with the available laboratory assays used to document ebolavirus infection. Here we report the first successful noninvasive detection of EBOV immunity in wild great apes, demonstrating survival in this species. This tool will be useful in a comprehensive strategy aimed at the protection of this endangered species and improved prevention of EVD outbreaks in human populations.
doi:10.1371/journal.pntd.0003143
PMCID: PMC4169258  PMID: 25232832
22.  Serologic Cross-Reactivity of Human IgM and IgG Antibodies to Five Species of Ebola Virus 
Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30–45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n = 24), Gulu, Uganda (n = 20), Bundibugyo, Uganda (n = 33), and the Philippines (n = 18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV.
Author Summary
Ebola virus (EBOV) is a highly pathogenic virus, capable of causing Ebola hemorrhagic fever in humans and non-human primates. Five species of EBOV have been identified. To examine whether infection with one EBOV species results in antibodies that cross-react with other EBOV species, we selected groups of human diagnostic samples from four outbreaks, which were each due to a different EBOV species, and compared IgM and IgG responses by ELISA to each of the five EBOV species. For samples from an individual outbreak, we found limited IgM reactivity to species of EBOV other than the virus species the individual was infected with. In contrast, for all groups of outbreak samples we observed strong cross-reactive IgG antibodies to all EBOV species. Our study demonstrates that IgG antibody responses tend to be more cross-reactive than IgM antibody responses in people infected with EBOV, a finding that has implications for the development of diagnostic assays and vaccines to EBOV.
doi:10.1371/journal.pntd.0001175
PMCID: PMC3110169  PMID: 21666792
23.  Vaccine To Confer to Nonhuman Primates Complete Protection against Multistrain Ebola and Marburg Virus Infections▿  
Filoviruses (Ebola and Marburg viruses) are among the deadliest viruses known to mankind, with mortality rates nearing 90%. These pathogens are highly infectious through contact with infected body fluids and can be easily aerosolized. Additionally, there are currently no licensed vaccines available to prevent filovirus outbreaks. Their high mortality rates and infectious capabilities when aerosolized and the lack of licensed vaccines available to prevent such infectious make Ebola and Marburg viruses serious bioterrorism threats, placing them both on the category A list of bioterrorism agents. Here we describe a panfilovirus vaccine based on a complex adenovirus (CAdVax) technology that expresses multiple antigens from five different filoviruses de novo. Vaccination of nonhuman primates demonstrated 100% protection against infection by two species of Ebola virus and three Marburg virus subtypes, each administered at 1,000 times the lethal dose. This study indicates the feasibility of vaccination against all current filovirus threats in the event of natural hemorrhagic fever outbreak or biological attack.
doi:10.1128/CVI.00431-07
PMCID: PMC2268273  PMID: 18216185
24.  Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein 
Journal of Virology  2015;90(1):279-291.
ABSTRACT
Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models.
IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.
doi:10.1128/JVI.02172-15
PMCID: PMC4702572  PMID: 26468532
25.  Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus 
The Journal of Infectious Diseases  2015;212(Suppl 2):S443-S451.
Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7–9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines.
doi:10.1093/infdis/jiv316
PMCID: PMC4564554  PMID: 26109675
attenuation; rVSV vector; Ebola vaccine; glycoprotein; challenge; nonhuman primates

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