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1.  Protective Cytotoxic T-Cell Responses Induced by Venezuelan Equine Encephalitis Virus Replicons Expressing Ebola Virus Proteins 
Journal of Virology  2005;79(22):14189-14196.
Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8+ T-cell responses to six Ebola virus proteins were examined. CD8+ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8+ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8+ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.
PMCID: PMC1280180  PMID: 16254354
2.  Individual and Bivalent Vaccines Based on Alphavirus Replicons Protect Guinea Pigs against Infection with Lassa and Ebola Viruses 
Journal of Virology  2001;75(23):11677-11685.
Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.
PMCID: PMC114754  PMID: 11689649
3.  Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs 
PLoS Medicine  2006;3(6):e177.
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.
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
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
• 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)
PMCID: PMC1459482  PMID: 16683867
4.  Passive Transfer of Antibodies Protects Immunocompetent and Immunodeficient Mice against Lethal Ebola Virus Infection without Complete Inhibition of Viral Replication 
Journal of Virology  2001;75(10):4649-4654.
Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.
PMCID: PMC114218  PMID: 11312335
5.  Effective Post-Exposure Treatment of Ebola Infection  
PLoS Pathogens  2007;3(1):e2.
Ebola viruses are highly lethal human pathogens that have received considerable attention in recent years due to an increasing re-emergence in Central Africa and a potential for use as a biological weapon. There is no vaccine or treatment licensed for human use. In the past, however, important advances have been made in developing preventive vaccines that are protective in animal models. In this regard, we showed that a single injection of a live-attenuated recombinant vesicular stomatitis virus vector expressing the Ebola virus glycoprotein completely protected rodents and nonhuman primates from lethal Ebola challenge. In contrast, progress in developing therapeutic interventions against Ebola virus infections has been much slower and there is clearly an urgent need to develop effective post-exposure strategies to respond to future outbreaks and acts of bioterrorism, as well as to treat laboratory exposures. Here we tested the efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50% and 100% of the animals, respectively, following treatment as late as 24 h after lethal challenge. More important, four out of eight rhesus macaques were protected if treated 20 to 30 min following an otherwise uniformly lethal infection. Currently, this approach provides the most effective post-exposure treatment strategy for Ebola infections and is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate release.
Author Summary
Being highly pathogenic for humans and monkeys and the subject of former weapons programs makes Ebola virus one of the most feared pathogens worldwide today. Due to a lack of licensed pre- and post-exposure intervention, our current response depends on rapid diagnostics, proper isolation procedures, and supportive care of case patients. Consequently, the development of more specific countermeasures is of high priority for the preparedness of many nations. In this study, we investigated an attenuated vesicular stomatitis virus expressing the Ebola virus surface glycoprotein, which had previously demonstrated convincing efficacy as a vaccine against Ebola infections in rodents and monkeys, for its potential use in the treatment of an Ebola virus infection. Surprisingly, treatment of guinea pigs and mice as late as 24 h after lethal Ebola virus infection resulted in 50% and 100% survival, respectively. More important, 50% of rhesus macaques (4/8) were protected if treated 20 to 30 min after Ebola virus infection. Currently, this approach provides the most effective treatment strategy for Ebola infections and seems particularly suited for the use in accidental exposures and the control of human-to-human transmission during outbreaks.
PMCID: PMC1779298  PMID: 17238284
6.  Protection against lethal challenge by Ebola virus-like particles produced in insect cells 
Virology  2008;383(1):12-21.
Ebola virus-like particles (VLPs) were produced in insect cells using a recombinant baculovirus expression system and their efficacy for protection against Ebola virus infection was investigated. Two immunizations with 50 ug Ebola VLPs (high dose) induced a high level of antibodies against Ebola GP that exhibited strong neutralizing activity against GP-mediated virus infection and conferred complete protection of vaccinated mice against lethal challenge by a high dose of mouse-adapted Ebola virus. In contrast, two immunizations with 10 ug Ebola VLPs (low dose) induced 5-fold lower levels of antibodies against GP and these mice were not protected against lethal Ebola virus challenge, similar to control mice that were immunized with 50 ug SIV Gag VLPs. However, the antibody response against GP were boosted significantly after a third immunization with 10 ug Ebola VLPs to similar levels as those induced by two immunizations with 50 ug Ebola VLPs, and vaccinated mice were also effectively protected against lethal Ebola virus challenge. Furthermore, serum viremia levels in protected mice were either below the level of detection or significantly lower compared to the viremia levels in control mice. These results show that effective protection can be achieved by immunization with Ebola VLPs produced in insect cells, which give high production yields, and lend further support to their development as an effective vaccine strategy against Ebola virus.
PMCID: PMC2657000  PMID: 18986663
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.
PMCID: PMC2582959  PMID: 19043556
8.  Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge. 
Journal of Virology  1994;68(6):3505-3511.
The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.
PMCID: PMC236854  PMID: 7514677
9.  Identification of Protective Epitopes on Ebola Virus Glycoprotein at the Single Amino Acid Level by Using Recombinant Vesicular Stomatitis Viruses 
Journal of Virology  2003;77(2):1069-1074.
Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.
PMCID: PMC140786  PMID: 12502822
10.  Ebola GP-Specific Monoclonal Antibodies Protect Mice and Guinea Pigs from Lethal Ebola Virus Infection 
Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as pools of 3–4 MAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted EBOV. Each of the 8 MAbs (100 µg) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven MAbs were effective 2 days post-infection (dpi), with 1 MAb demonstrating partial protection 3 dpi. In the guinea pigs each MAb showed partial protection at 1 dpi, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3–4 MAbs completely protected the majority of animals, while administration at 2–3 dpi achieved 50–100% protection. This data suggests that the MAbs generated are capable of protecting both animal species against lethal Ebola virus challenge. These results indicate that MAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection.
Author Summary
Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against the Ebola virus surface glycoprotein, in mice and guinea pigs. Various combinations and doses of the neutralizing and non-neutralizing MAbs were tested, and a post-exposure treatment protocol was determined. There was 100% survival when guinea pigs received a mix of 3 neutralizing MAbs two days after a challenge with 1,000 LD50 of guinea pig-adapted EBOV. This data suggests that the MAbs generated are effective as a post-exposure therapeutic for a lethal Ebola virus infection. Development of a post-exposure therapeutic for an Ebola virus infection is vital due to the high lethality of the disease, the relative speed in which it kills, and the fact that no vaccine has been approved for human use. Additionally, is it unlikely that preventative vaccines will be employed, because Ebola virus infections occur primarily in Africa, and to date have only killed approximately 2,300 people making it financially unfeasible for a mass vaccination. Therefore, having an effective therapy in the event of an outbreak would be extremely beneficial.
PMCID: PMC3308939  PMID: 22448295
11.  Mechanisms of Immunity in Post-Exposure Vaccination against Ebola Virus Infection 
PLoS ONE  2015;10(3):e0118434.
Ebolaviruses can cause severe hemorrhagic fever that is characterized by rapid viral replication, coagulopathy, inflammation, and high lethality rates. Although there is no clinically proven vaccine or treatment for Ebola virus infection, a virus-like particle (VLP) vaccine is effective in mice, guinea pigs, and non-human primates when given pre-infection. In this work, we report that VLPs protect Ebola virus-infected mice when given 24 hours post-infection. Analysis of cytokine expression in serum revealed a decrease in pro-inflammatory cytokine and chemokine levels in mice given VLPs post-exposure compared to infected, untreated mice. Using knockout mice, we show that VLP-mediated post-exposure protection requires perforin, B cells, macrophages, conventional dendritic cells (cDCs), and either CD4+ or CD8+ T cells. Protection was Ebola virus-specific, as marburgvirus VLPs did not protect Ebola virus-infected mice. Increased antibody production in VLP-treated mice correlated with protection, and macrophages were required for this increased production. However, NK cells, IFN-gamma, and TNF-alpha were not required for post-exposure-mediated protection. These data suggest that a non-replicating Ebola virus vaccine can provide post-exposure protection and that the mechanisms of immune protection in this setting require both increased antibody production and generation of cytotoxic T cells.
PMCID: PMC4364937  PMID: 25785602
12.  Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine 
PLoS ONE  2009;4(4):e5308.
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.
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.
PMCID: PMC2669164  PMID: 19390586
13.  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.
PMCID: PMC2873919  PMID: 20502688
14.  Role of Natural Killer Cells in Innate Protection against Lethal Ebola Virus Infection 
Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1–3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell–mediated protection clearly depended on perforin, but not interferon-γ secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection.
PMCID: PMC2212007  PMID: 15249592
virus-like particles; filoviruses; immunity; matrix protein; glycoprotein
15.  Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice 
Vaccine  2011;29(16):2968-2977.
Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.
PMCID: PMC3070761  PMID: 21329775
16.  Evaluation in Nonhuman Primates of Vaccines against Ebola Virus 
Emerging Infectious Diseases  2002;8(5):503-507.
Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.
PMCID: PMC3369765  PMID: 11996686
Keywords: Ebola; macaque; vaccine; Vaccinia virus; replicon
17.  Neutralizing Antibody Fails to Impact the Course of Ebola Virus Infection in Monkeys 
PLoS Pathogens  2007;3(1):e9.
Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.
Author Summary
Ebola virus is one of the most feared of human pathogens with a mortality that can approach 90% and an extremely rapid disease course that can lead to death within days of infection. Antibodies able to inhibit viral infection in culture, neutralizing antibodies, can typically prevent viral infection in animals and humans when present prior to infection, at sufficient concentration. Such neutralizing antibodies may be provided through passive administration or induced by vaccination. We have previously shown that a human neutralizing antibody can protect guinea pigs against Ebola virus. However, here we show that this antibody does not protect monkeys against Ebola virus and surprisingly appears to have very little impact upon the rapid course of infection, despite being present at very high levels in the blood of the monkeys. We conclude that administering antibody prior to or immediately following exposure to Ebola virus, for example, after an accident in a research setting or a bioterrorist attack, is unlikely to be effective in preventing disease. Recent successes in protecting monkeys against Ebola virus through vaccination may be independent of antibody, or, more likely, critically dependent on the cooperation of antibody and cellular immunity.
PMCID: PMC1779296  PMID: 17238286
18.  A Single Sublingual Dose of an Adenovirus-Based Vaccine Protects against Lethal Ebola Challenge in Mice and Guinea Pigs 
Molecular Pharmaceutics  2011;9(1):156-167.
Sublingual (SL) delivery, a non-invasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and Guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa. A SL dose of 1 × 108 infectious particles induced Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naïve mice in a manner similar to the same dose given IN. Ex vivo CFSE and in vivo cytotoxic T lymphocyte (CTL) assays confirmed that SL immunization elicits a notable population of effector memory CD8+ T cells and strong CTL responses in spleen and SMLN. SL immunization induced significant ZGP-specific Th1 and Th2 type responses unaffected by pre-existing immunity (PEI) that protected mice and Guinea pigs from lethal challenge. SL delivery protected more mice with PEI to Ad5 than IM injection. SL immunization also reduced systemic anti-Ad5 T and B cell responses in naïve mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.
PMCID: PMC3358355  PMID: 22149096
adenovirus 5; Ebola Zaire; sublingual; vaccine; mouse; Guinea pig; pre-existing immunity; CD4 T cell; memory response; toxicity
19.  Successful Topical Respiratory Tract Immunization of Primates against Ebola Virus▿  
Journal of Virology  2007;81(12):6379-6388.
Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys—in which HPIV3 infection is mild and asymptomatic—and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.
PMCID: PMC1900097  PMID: 17428868
20.  Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs 
PLoS Pathogens  2015;11(6):e1005016.
The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.
Author Summary
The filoviruses have caused multiple outbreaks among humans this decade, including a 90% lethal outbreak of Marburg virus in Angola and a significant, sustained outbreak of Ebola virus in West Africa. The viral surface glycoprotein (GP), which enables filoviruses to infect host cells, is the primary target of the immune system. Antibodies that target filovirus GP have been shown to provide life-saving therapy in nonhuman primates. However, the majority of known antibodies are only reactive against Ebola virus and not other emerging filoviruses. In this study, we present ten antibodies against Marburg virus, elicited by immunization of mice using engineered forms of its GP. Surprisingly, two antibodies exhibit some cross-reactivity to ebolaviruses (including species Ebola, Sudan, Bundibugyo, Reston). Other antibodies in this panel recognize a novel “wing” feature on a portion of GP that is unique to Marburg and does not exist in ebolaviruses, and protect 90%-100% of mice from lethal exposure. These antibodies, and their structural and functional analysis presented here, illuminate directions forward for therapeutics against Marburg virus.
PMCID: PMC4482612  PMID: 26115029
21.  Molecularly engineered vaccine which expresses an immunodominant T-cell epitope induces cytotoxic T lymphocytes that confer protection from lethal virus infection. 
Journal of Virology  1989;63(10):4311-4316.
Identification of a single viral T-cell epitope, associated with greater than 95% of the virus-specific cytotoxic T-lymphocyte (CTL) activity in BALB/c (H-2d) mice (J. L. Whitton, A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. A. Oldstone, J. Virol. 63:4303-4310, 1989), permitted us to design a CTL vaccine and test its ability to protect against a lethal virus challenge. Here we show that a single immunization with a recombinant vaccinia virus-lymphocytic choriomeningitis virus (LCMV) vaccine (VVNPaa1-201) expressing the immunodominant epitope completely protected H-2d mice from lethal infection with LCMV but did not protect H-2b mice. Furthermore, we show that the success or failure of immunization was determined entirely by the host class I major histocompatibility glycoproteins. The difference in outcome between mice of these two haplotypes was consistent with the presence or absence in the immunizing sequences of an epitope for CTL recognition and is correlated with the induction of LCMV-specific H-2-restricted CTL in H-2d mice. Protection is not conferred by a humoral immune response, since LCMV-specific antibodies were not detectable in sera from VVNPaa1-201-immunized mice. In addition, passive transfer of sera from vaccinated mice did not confer protection upon naive recipients challenged with LCMV. Hence, the molecular dissection of viral proteins can uncover immunodominant CTL epitope(s) that can be engineered into vaccines that elicit CTL. A single CTL epitope can protect against a lethal virus infection, but the efficacy of the vaccine varies in a major histocompatibility complex-dependent manner.
PMCID: PMC251047  PMID: 2476571
22.  Replication-Deficient Ebolavirus as a Vaccine Candidate▿  
Journal of Virology  2009;83(8):3810-3815.
Ebolavirus causes severe hemorrhagic fever, with case fatality rates as high as 90%. Currently, no licensed vaccine is available against Ebolavirus. We previously generated a replication-deficient, biologically contained Ebolavirus, EbolaΔVP30, which lacks the essential VP30 gene, grows only in cells stably expressing this gene product, and is genetically stable. Here, we evaluated the vaccine potential of EbolaΔVP30. First, we demonstrated its safety in STAT-1-knockout mice, a susceptible animal model for Ebolavirus infection. We then tested its protective efficacy in two animal models, mice and guinea pigs. Mice immunized twice with EbolaΔVP30 were protected from a lethal infection of mouse-adapted Ebolavirus. Virus titers in the serum of vaccinated mice were significantly lower than those in nonvaccinated mice. Protection of mice immunized with EbolaΔVP30 was associated with a high antibody response to the Ebolavirus glycoprotein and the generation of an Ebolavirus NP-specific CD8+ T-cell response. Guinea pigs immunized twice with EbolaΔVP30 were also protected from a lethal infection of guinea pig-adapted Ebolavirus. Our study demonstrates the potential of the EbolaΔVP30 virus as a new vaccine platform.
PMCID: PMC2663241  PMID: 19211761
23.  Induction of Immune Responses in Mice and Monkeys to Ebola Virus after Immunization with Liposome-Encapsulated Irradiated Ebola Virus: Protection in Mice Requires CD4+ T Cells 
Journal of Virology  2002;76(18):9176-9185.
Ebola Zaire virus (EBO-Z) causes severe hemorrhagic fever in humans, with a high mortality rate. It is thought that a vaccine against EBO-Z may have to induce both humoral and cell-mediated immune responses to successfully confer protection. Because it is known that liposome-encapsulated antigens induce both antibody and cellular responses, we evaluated the protective efficacy of liposome-encapsulated irradiated EBO-Z [L(EV)], which contains all of the native EBO-Z proteins. In a series of experiments, mice immunized intravenously with L(EV) were completely protected (94/94 mice) against illness and death when they were challenged with a uniformly lethal mouse-adapted variant of EBO-Z. In contrast, only 55% of mice immunized intravenously with nonencapsulated irradiated virus (EV) survived challenge, and all became ill. Treatment with anti-CD4 antibodies before or during immunization with L(EV) eliminated protection, while treatment with anti-CD8 antibodies had no effect, thus indicating a requirement for CD4+ T lymphocytes for successful immunization. On the other hand, treatment with either anti-CD4 or anti-CD8 antibodies after immunization did not abolish the protection. After immunization with L(EV), antigen-specific gamma interferon (IFNγ)-secreting CD4+ T lymphocytes were induced as analyzed by enzyme-linked immunospot assay. Anti-CD4 monoclonal antibody treatment abolished IFNγ production (80 to 90% inhibition compared to that for untreated mice). Mice immunized with L(EV), but not EV, developed cytotoxic T lymphocytes specific to two peptides (amino acids [aa] 161 to 169 and aa 231 to 239) present in the amino-terminal end of the EBO-Z surface glycoprotein. Because of the highly successful results in the mouse model, L(EV) was also tested in three cynomolgus monkeys. Although immunization of the monkeys with L(EV)-induced virus-neutralizing antibodies against EBO-Z caused a slight delay in the onset of illness, it did not prevent death.
PMCID: PMC136452  PMID: 12186901
24.  Chemical Modifications of Antisense Morpholino Oligomers Enhance Their Efficacy against Ebola Virus Infection▿  
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5′ or the 3′ terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.
PMCID: PMC2681561  PMID: 19223614
25.  Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species 
PLoS ONE  2012;7(12):e44115.
Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.
PMCID: PMC3516506  PMID: 23236343

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