Weaponizing Ebola/Marburg

[rakuenso]

However, ebola is a highly capricious virus. The reston strain is proof of this, as its virulence can be easily modified. Does anyone know what strain that test was conducted upon?

[jdurg]

One thing that is also a unknown at this point is how the immune system of people in developed countries differs from the immune system of people in these African Villages. Generally speaking, people in the USA, UK, Japan, etc. have a much greater immune system due to modern medicine than many of the people in these remote African villiages do. All of the effects of Ebola have been seen happening to the people in these African villiages. We still don't know exactly how it will pan out in a more 'modern' civilization.

[claytonm]

I hate to bring up a dead thread but since I am new to the forum and extremely interested in filoviruses and filovirus vaccines I thought I would post something. I find it very hard to believe that scientists have developed vaccines with 100% efficacy in primates. Vaccine developers have been pipetting around in their labs with filoviruses since the 60's trying to find safe, effective vaccines with little success. About a year ago I had to write a research proposal for an immunology class I was taking and chose vaccine development of filoviruses. All of the research I did included the most recent publications found on PubMed. There were several reoccurring themes I noticed in all of the research that was done. First, different animal models acquire different levels of immunity when given the same antigens. No single vaccine concoction was able to infer 100% immunity to a give animal, nor did it even come close. Long term immunity may only be possible with extended booster immunizations. There is enough variation within the viral proteins of the different subtypes of the virus that one single vaccine will not constitute immunity to all subtypes. I would be very interested to see the publication of the research that claims 100% immunity. Nothing is impossible, but from my understanding of the bug it is highly unlikely.

[claytonm]

For anyone who wants more background info on what I found for my paper a year ago, here it is with references: NOTE: THIS IS NOT DIRECTLY RELATED TO THE NY TIMES REPORT

 

Ebola Virus Protein-based Vaccine Development Utilizing NP, VP24, VP30, VP35 and VP40 Packaged inVirus-like Replicon Particles (VRPs)

 

Abstract

 

Ebola viruses are members of the virus family, Filoviridae, and have been implicated as the causative agents of severe hemorrhagic fever outbreaks in human and nonhuman primates. The highly pathogenic virus has a morality rate in excess of 80% with no antiviral therapies or vaccine available. The virus poses a major public health threat to rural African countries that lack proper medical facilities, doctors and scientists researching the virus, attempting elucidating its mechanisms of pathogenesis and the general population in the event of a bioterrorism attack These alarming facts warrant studies to develop safe, efficacious vaccines. Currently, Ebola and other filoviruses are placed at the highest biosafety level (4), due to their extremely virulent nature. This placement limits research to only a few highly specialized labs around the world. An effective vaccine would allow more laboratories the opportunity to study the virus and in turn expand our understanding of the virus. Furthermore, our knowledge that the former Soviet Union was evaluating Ebola virus as a bioterrorism weapon, and recent terrorist attacks on September, 11th 2001, have increased commercial interest in producing a vaccine.

Live virus and attenuated virus vaccine approaches have many safety risks involved, including but not limited to reactogenicity, incomplete inactivation and possible mutations leading to virulent virions, which would never receive approval from the medical community. Furthermore, studies have shown that whole inactivated virion vaccines do no provide effective immunity when challenged with lethal infection. Vaccine efforts have shifted towards the development of subunit based vaccines containing a limited number of viral proteins packaged in Virus-like Replicon Particles (VRPs) Studies have shown that both humoral and cell mediated responses are required for optimal immunity to Ebola virus in humans. It is hypothesized that a vaccine containing both the extracelluarly expressed NP and intracellularly expressed VP proteins (VP24, VP30, VP35 and VP40) packaged in VRPs will induce a strong humoral and cell-mediated response in murine and nonhuman primate animal models to confer robust immunity to Ebola virus, and will serve as predictors for similar immune responses in humans.

 

Introduction

 

Studies have shown that both humoral and cell mediated responses, specifically cytotoxic T-cell responses (CTL), are required for optimal immunity to Ebola virus in humans (1). By understanding the immune mechanisms involved in lethal and nonlethal cases of Ebola, we can better develop vaccines that initiate and activate the protective effector functions of the immune system. Fatal cases of Ebola give us excellent indicators as to what parts of the immune response fail, and in turn demonstrate what is necessary for effective immunity. When determining the importance of cell mediated response, incomplete and unsustained T-cell responses, as noted by the release of exorbitant amounts of the inflammatory cytokines (lead to clotting cascade, microvascular damage, and capillary damage) IL-2, IL-10, IL-1RA, IFN-γ, TNF-α , and IFN-α, are directly associated with fatal cases (2 and 7). Cytokine release is catalyzed by the direct infection of monocytes and macrophages. Of these cytokines, IL-10 acts as a TH1 cytokine inhibitor which suppresses the cell-mediated response (7) These T-cell cytokine levels, as measured through T-cell cytokine RNA, are mostly indicative of failed adaptive immunity (3). Humoral response in fatal cases is also an excellent tool for understanding protective immune response. Decreased and sometimes undetectable level of IL-6, which aids in proliferation of antibody-producing cells, is a common feature associated with fatality (2). The overwhelming majority of cases resulting in death have high Ebola antigens and viral titers in blood serum, but lack detectable antibodies to these anitgens (2).

In contrast, nonfatal cases of Ebola demonstrate cell-mediated and humoral responses that provide protective immunity and illustrate many of the necessary mechanisms needed for this. T-cell cytokine levels observed in nonfatal cases are high throughout infection and convalescence. Survivors are also noted for high levels of IL-6 expression in conjunction with high levels of detectable IgM, specific to Ebola viral antigens (2). This data suggests that an effective vaccine would have to stimulate both humoral and cell mediated immune responses to confer long lasting protection.

The Ebola virus genome contains negative-sense RNA, which codes for seven structural proteins; type I transmember Glycoprotein (GP), Nucleoprotein (NP), four virion structural proteins (VP35), (VP40),(VP30), (VP24) and RNA-dependent RNA polymerase (L) (3). Due to the great diversity of class I-MHC in humans and the limited number of CTL epitopes on a given viral protein, it is most likely necessary to include several Ebola virus proteins in a vaccine (1).

Recent studies have demonstrated immunosuppresive activities of the GP which most likely explains the inability of whole inactivated virus vaccines to confer immunity (4) The GP gene encodes two functionally distinct glycoproteins. The major product of the GP gene is a small nonstructural, ectodomain, secreted glycoprotein (sGP), which is a disulfide linked homodimer consisting of the GP1 and GP2Δ (truncated GP2 proteolytic cleaved at aa position D637) subunits. The minor product of the GP gene is a structural, surface glycoprotein comprised of the GP1 (125kDa, 501aa) and GP2 (26kDa, 175aa) subunits (5). A 40 aa sequence located at the N-terminus of the GP1 subunit has been shown to suppress human lymphocyte mitogen-stimulated proliferation in vitro (blastogenesis) (4 and 5). The full length GP2 subunit contains immunosuppresive motif sequence homology with animal retrovirus protein p15E found in the genomes of Murine Leukemia virus (MuLV) and Feline Leukemia virus (FeLV) (5 and 6). The sGP is present in significant amounts in the blood of infected animals. It has also been noted that levels of the sGP increase between days 6 and 9 in animal systems infected in vivo, suggesting its importance in pathogenesis (5). In vitro experiments utilizing the Ebola-specific antibody KZ52 (neutralization of Ebola virus infectivity >60%) have shown the ability of the sGP to fully inhibit its neutralization activity (1). These findings would indicate that an effective vaccine would need to eliminate the GP in both its forms due to its immunosuppresive nature.

Six Ebola proteins, GP, NP, VP24, VP30, VP35, and VP40, have been evaluated in different vaccine platforms and in different animal models to test there efficacies. Vaccine studies using recombinant forms of these proteins in different combinations have been performed with mixed results, however no combination including the NP with all four VP proteins has been performed.

Recombinant Ebola NP expressed in alphavirus Venezuelan equine encephalitis (VEE) replicons have provided protective immunity in murine models (C57BL/6 mice). However, passive transfer of polyclonal NP-specific antiserum was unable to protect naive recipient mice given lethal challenge using 300 LD50 Ebola virus. In contrast, only adoptive transfer of CD8+ CTLs were able to protect naive mice when given lethal challenge (7). Epitopes restricted to major histocompatibility complex class I were identified for the NP. This study illustrates the importance of CTLs and cell-mediated responses in providing effective immunity.

Recombinant VP proteins expressed in the VEE vector and packaged in VRPs have also given mixed results when used to vaccinate two different mouse strains. VRPs containing VP35 were unable to protect BALB/C mice, allowing only 20-26% efficacy. In contrast, 70% of C57BL/6 mice were protected when vaccinated with the same VEE expressed VP35 (1). These mice had detectable VP protein specific antibodies in serum, but protection was not observed with polyclonal antiserum transfer to naive mice followed by lethal challenge using 300 LD50 Ebola virus. This study indicates that protection is mouse strain dependant when using just VP proteins, as no single VP protein was able to achieve protection in both mouse strains (1).

BALB/C mice have been vaccinated with recombinant GP and VP40 coexpressed in pWRG7077 plasmid vector and packaged in Virus-like particles (VLPs) (8). 100% of these mice were protected when challenged with 9,000 LD50 Ebola virus. Mouse bone marrow-derived dendritic cells were assessed to evaluate their responses to the VLPs. Increases in the surface markers CD40, CD80, CD86, and MHC class I and II were observed. Secretions of IL-6, IL-10, macrophage inflammatory protein (MIP)-1α TNF-α were also noted (8). Neutralizing Ebola virus-specific antibodies were detected. This study failed to test the ability of cell-mediated or humoral transfer to demonstrate passive immunity in naive BALB/C mice.

The efficacy of VRPs containing VEE expressed GP and NP have been tested in Cynomologus Macaques (Macaca fascicularis) (9). Three macaques were vaccinated subcutaneously with the GP and NP mixture, followed by booster immunization 28 days after the first and 28 after the second. A lethal challenge of 1,000 PFU Ebola virus was administered 49 days after the third booster immunization, through intramuscular injection of 1,000 PFU Ebola virus (9). At post challenge day 3, all of the macaques became ill and two of the three died on day 6. This study showed that there are major difference between animal models and the outcome of infection. Rodent models are able to demonstrate immunity when vaccinated with GP and NP, but nonhuman primate models do not.

As mentioned previously, no vaccine has been attempted that combines the immunogenic properties of the NP, VP24, VP30, VP35 and VP40 proteins. Individually, the NP and the respective VP proteins have demonstrated the ability to confer some immunity to rodent or nonhuman primate models. The NP is an extracellularly expressed protein, while the VP proteins are intracellularly expressed (7). The NP should induce humoral immunity and the VP proteins should induce cell mediated immunity. This would suggest that a novel vaccine containing the recombinant, extracellular NP and intracellular VP proteins packaged in immunogenic VRPs should stimulate robust humoral and cell mediated responses in rodents and nonhuman primates. Many different vector systems are available for producing recombinant viral proteins. The alphavirus VEE vector has been used with great success in creating immunogenic VRPs and holds great promise in developing effective subunit vaccines. (1, 2, 3, 7, 8, and 9) The cloned Ebola virus proteins are placed in the genome where structural VEE genes have been deleted. The vector has been designed to only allow replication of cloned proteins along with necessary non-structural VEE proteins. The vector also contains two helper RNAs which encode the VEE spike protein needed for target cell binding and infection by the VRP. The Heterologous proteins are produced upon infection of target cells. No further infection is possible after the initial infection since the VEE structural proteins are absent (7). Also, the VEE vector contains a tropism for professional antigen presenting dendritic cells, which helps stimulate both humoral and cell mediated responses (1 and 7).

The types of animal models used to test the efficacies of filovirus vaccines has been shown to be of importance in predicting vaccination in humans. Due to the highly virulent nature of these viruses, animal models serve as the safest methods for testing. Unfortunately, the models are not perfect indicators of how immunity and vaccination work in humans, but they do serve as valuable predictors. Guinea pigs, mice, and nonhuman primates serve as the three main animal models used in vaccine research. Ebola virus does not have to be adapted to nonhuman primates for studying. For testing in rodent and lower species animals, the Ebola Zaire strain has been adapted through serial passage which attenuates the virus. Adaptation of the virus for a particular species raises many issues. The adaptation can result in sequence changes in the virus which can affect its pathogenesis and alter the epitopes of the specific viral proteins that are used by both B and T cells for recognition (7). Zaire Ebola virus that has been adapted to guinea pigs resulted in a single amino acid change in the NP and L polymerase proteins and three amino acid changes in the VP24 protein (7). The mouse-adapted strain results in five amino acid changes with single mutations occurring in the NP, VP24 and VP35, and two mutations in the L polymerase (7). The need for adaptations to mice and guinea pig models makes them less predictable than nonhuman primate models.

The three animal models all share the conserved tropism of the Ebola virus for mononuclear phagocytes (7). Localization studies of Ebola virus in vivo have indicated that mononuclear phagocytes act as main targets for infection (3). Of the three available animal models, this study will use mice and nonhuman primates to test vaccine efficacy. The pathological features of Ebola virus infection seen in nonhuman primates most closely resembles those described in human infection and is the best predictor of immunity in humans (9). Lymphopenia is commonly associated with the human infection and is most likely due to lymphocyte apoptosis since there is no evidence showing that lymphocytes are directly infected (7 and 9). Lymphocyte apoptosis is also a hallmark feature of Ebola virus infection in nonhuman primates, but not in mice or guinea pigs (9). Disseminated intravascular coagulation (DIC) is a prominent feature found in end-stage lesions of human and nonhuman primates. DIC is absent completely in mice infection and is only found in limited amounts in guinea pig infection (9). Mouse and guinea pig models show a lack of fibrin deposits as seen in human and nonhuman primate infection. Also, viremia and widespread tissue damage is much more apparent in nonhuman primates than in mice and guinea pigs (9). The lack of pathological consistency of mice and guinea pigs with humans indicates that nonhuman primates, like the Cynomologus Macaque, serve as the best animal models. Murine models still serve as very good indicators of vaccine efficacy and will be used in preliminary tests to determine if the use of a more complex nonhuman primate model is warranted.

 

Experimental Plan

 

The initial part of this experiment will test the efficacy of VRPs containing the NP, VP24, VP30, VP35 and VP40 proteins in two murine models. BALB/c (H2d) and C57BL/6 (H2d) mice strains will be used since previous studies have shown different immunity results among the two strains due to genetic differences at the MHC locus (1). The second part of this experiment will test the efficacy of the same VRPs in a nonhuman primate model using Cynomologus Macaques (Macaca fascicularis).

The cloning of Ebola viral genes and construction of the VEE vector will involve RT-PCR of purified RNA from the Ebola Zaire (strain Mayinga). The VEE replicon vector contains a unique ClaI cloning site downstream of the 26S promoter where amplified Ebola proteins can be inserted. Previously published primer sequences, by Wilson et.al., for the VP24, VP30, VP35 and VP40 genes are available and already have unique ClaI restriction sites engineered (in bold) (1). The VP24 forward primer is 5'-GGGATCGATCTCCAGACACCA-AGCAAGACC -3', the VP24 reverse primer is 5'-GGGATCGATGAGTCAGCATATATGAG-TTAGCTC-3', the VP30 forward primer is 5'-CCCATCGATCAGATCTGCGAACCGGTAGA-G-3', the VP30 reverse primer is 5'-CCCATCGATGTACCCTCATCAGACCATGAGC-3', the VP35 forward primer is 5'-GGGATCGATAGAAAAGCTGGTCTAACAAGATGA-3', the VP35 reverse primer is 5'-CCCATCGATCTCACAAGTGTATCATTAATGTAACGT-3', the VP40 forward primer is 5'-CCCATCGATCCTACCTCGGCTGAGAGAGTG-3', the VP40 reverse primer is 5'-CCCATCGATATGTTATGCACTATCCCTGAGAAG-3'.

No known primer sequences have been published to date that are designed to amplify the NP while introducing a ClaI restriction site. However, a known primer sequence to amplify the NP for ELISA testing that contains HaeIII and BamHI restriction sites (in bold) near the 5' end and stop codons (in italics) at the 3' end has been published (10). The forward primer sequence (E-Init) is 5'-GGCCGGATCCCGGAATCACAAAATTCCGAGTATG -3', and the reverse primer sequence (E-Ter) is 5'-GGCCGGATCCATCCCATTGTTCCATGCTCATTCA-3'. Forward and reverse primers were designed to fit the VEE cloning application through editing of the E-Init and E-Ter primers respectively. First, the unneeded HaeIII and BamHI restriction sites were deleted and replaced with a ClaI restriction site (in bold) and flanked by a GGG codon at the 5' end. Secondly, the stop codons at the 3' ends were removed since expression of downstream helper RNAs are required in the VEE vector. The NP forward primer is 5'-GGGATCGATCGGAATCACAAAATTCCGAGT-3', and the NP reverse primer is 5'-GGGAT-CGATATCCCATTGTTCCATGCTCAT-3'. These NP primers are the same length as the previously published VP protein primers and show amplification of the 2,262 bp (754aa) NP gene when performing a pair wise alignment with the complete Ebola Zaire (strain Mayinga) genome (GenBank Accession No. AF086833).

To confirm sequence homology of the RT-PCR amplified Ebola genes to that of the wildtype, RT-PCR, amplicons will be purified on 2% agarose gel, and sent for sequencing. Sequencing data will be compared with the known sequences of the respective Ebola protein genes in the Ebola Zaire (strain Mayinga) genome (GenBank Accession No. AF086833).

Production of the recombinant VRPs will be performed according to Wilson et. al. (1). Capped replicon RNAs will be produced in vitro through T7 runoff transcription of the NotI-digested plasmids using the RiboMAX T7 RNA polymerase kit (Promega) (1). Baby hamster kidney (BHK) cells (ATTC CCL 10) will be cotransfected with the vector RNAs, through electroporation, to allow expression of the Ebola proteins along with helper VEE RNAs and VEE nonstructural protein RNAs (1). The cell culture supernatants will be collected 30 hours after transfection and VRPs will be partially purified by centrifugation for 3 hours at 36,000 rpm in a 20% sucrose cushion (1). The packaged VRPs will be suspended in phosphate-buffered saline (PBS) and titers of the VRPs will be determined through immunofluorescence, measuring focus forming units (1).

Homology of the VRP proteins with wild type proteins in native confirmation will be performed through metabolic labeling and radioimmunoprecipitation as described by Wilson et. al. (1). BHK cells that were transfected with VEE vector RNAs will be incubated for 16 hours and then starved for 30 minutes in medium lacking methionine and cysteine. The cells will then be incubated for 4 hours in minimal essential medium (MEM) containing 0.1 mCi/ml 35S-labeled methionine and cysteine (1). Cell monolayers will be lysed in Zwittergent lysis buffer and the proteins will be analyzed by immunoprecipitation and resolved by 11% SDS-polyacrylamide gel electrophoresis to determine sizes.

Animal research will be conducted in compliance with the Animal Welfare Act and other federal statutes relating to animal experiments, and will adhere to principles stated in the Guide for Care and Use of Laboratory Animals, National Research Council, 1996. The laboratory where research will be conducted will be fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Initial vaccination will be performed in BALB/c (H2d) and C57BL/6 (H2d) strain mice (National Cancer Institute, Frederick MD), using five mice of each strain to determine the efficacy of the vaccine. Mice will be 6-8 week old, pathogen-free females as described by Wilson et. al.. Mice will be housed in separate cages equipped with microisoators and will be provide food and water ad libitum. Four of the mice will be vaccinated with the VRPs at a titer of 2 X 106 focus-forming units through subcutaneous injection at the base of the neck. One mouse will be treated as the negative control and will be immunized with a control replicon encoding Lassa virus nucleoprotein, which does not confer any immunity to filoviruses (1 and 11). Two booster immunizations of the same titer will be given at 30 days post vaccination and at 60 days post vaccination (1). Survival data will be recorded in the vaccinated mice. Anesthetized mice will be bled from the retro-orbital sinus one week prior to lethal challenge to determine neutralizing antibody titers to the viral proteins through plaque reduction in a constant virus:serum dilution format. One month after the final booster immunization, lethal challenge through intraperitoneal inoculation will be performed using the predetermined murine lethal dose of 30 LD50 (10 PFU) of mouse adapted Ebola subtype Zaire (1). Infectious viremia and neutralizing antibody titer will be checked on day 4 (BALB/c mice) and day 5 (C57BL/6 mice) after the challenge. Morbidity and mortality results will be recorded. Thirty days after the lethal challenge, a subsequent back challenge of the same titer will be performed in surviving mice. A 30% or less mortality rate (3 out of 4 Ebola VRP vaccinated mice surviving) will be considered efficient protection. If the 30% or less mortality rate among the mice strains is observed in the initial part of the experiment, vaccination using Cynomologus Macaques as a nonhuman primate model will be used. The nonhuman primate model should be a better predicator of vaccine efficacy in humans based on the similar pathological features of illness observed in these animals.

The secondary part of the vaccination experiment will be performed in Cynomologus Macaques. Five monkeys weighing 4-6 kg will be selected for the study. Macaques will be keep in isolated cages and be given food and water. Four of the Macaques will be vaccinated with 107 focus-forming units of Ebola VRPs in a total volume of 0.5 ml at one site, through subcutaneous injection (9). One Macaque will be will be treated as the negative control and will be immunized with a control replicon encoding influenza hemagglutinin, which has no effect on Ebola virus immunity. Two booster immunizations of the same titer will be given 28 days post vaccination and 56 days post vaccination (9) Survival data will be recorded in the vaccinated Macaques. Telazol (Fort Dodge Laboratories, Fort Dodge, IA) anesthetized Macaques will be bled one week prior to lethal challenge to determine neutralizing antibody titers to the viral proteins through plaque reduction in a constant virus:serum dillution format (9). Forty-nine days after the final booster immunization, lethal challenge through intramuscular inoculation will be performed using 1,000 PFU of Ebola subtype Zaire (9). Infectious viremia and neutralizing antibody titer will be checked at 2 day intervals after the challenge until death or convalescence (9). Morbidity and mortality results will be recorded. Forty-nine days after the lethal challenge, a subsequent back challenge of the same titer will be performed in surviving Macaques. A 30% or less mortality rate (4 out of 5 Ebola VRP vaccinated Macaques surviving) will be considered efficient protection.

Plaque reduction assays for both the mice and Macaques will be performed according to Wilson et. al. (1). To evaluate the presence of Ebola virus neutralizing antibodies in mice, serial diluted mouse sera will be mixed with 100 PFU of mouse adapted Ebola Zaire for 1 hour at 37̊ C, and added to confluent Vero E6 cells (ATCC CRL 1586) (1). The plaque reduction test for Macaques will involve serial diluted Macaque sera mixed with 100 PFU of Ebola Zaire (Mayinga strain) under the same conditions as the mice. Reduction of plaques by 80% will be considered Ebola virus neutralizing (1). Neutralizing antibody titer will be determined through enzyme-linked immunosobent assays (ELISAs) on plates coated with irradiated, sucrose-purified Ebola Zaire virions. Absorbency values 0.2 or greater above control samples will be considered positive, and endpoint titers will be calculated (1). Infectious viremia titers will be determined though plaques assays using Vero E6 cells.

Passive immunization will be determined in both animal models. One ml of pooled immune antisera from BALB/c (H2d) and C57BL/6 (H2d) mice strains and Cynomologus Macaques will be passively administered to one of each of the unvaccinated, disease free animal models. The mice strains will be challenged with 300 LD50 of mouse adapted Ebola subtype Zaire 24 hours post adaptive transfer (1). The nonhuman primates will be Challenged with 1,000 PFU Ebola subtype Zaire 24 hours post adaptive transfer (9). In the Murine models, infectious viremia and neutralizing antibody titer will be checked on day 4 (BALB/c mice) and day 5 (C57BL/6 mice) after the challenge. Morbidity and mortality results will be recorded. In Cynomologus Macaques, infectious viremia and neutralizing antibody titer will be checked at 2 day intervals after the challenge until death or convalescence (9). Morbidity and mortality results will be recorded.

Adoptive transfer will also be determined in the animal models. Effector cells will be separated from cell cultures using the protocols of Wilson et. al. (11). The cells will be separated into three groups containing CD4+, CD8+, and CD4+ and CD8+ effector cells. Phenotype of the effector cells will be determined using fluorescein isothiocyanate-conjugated rat anti-mouse CD4, and rat anti-mouse CD8, and fluorescein isothiocyanate-conjugated anti-Macaques CD4 and anti-Macaques CD8 (11). The three cell groupings will be concentrated individually to 8 X 106 effector cells. The concentrated effector cells will be transferred to three unvaccinated, disease free BALB/c (H2d) mice, three C57BL/6 (H2d) mice, and three Cynomologus Macaques. The mice strains will be challenged with 300 LD50 of mouse adapted Ebola subtype Zaire 4 hours post adoptive transfer (11). The Cynomologus Macaques will be challenged with 1,000 PFU Ebola subtype Zaire 4 hours post adoptive transfer. In the Murine models, infectious viremia and neutralizing antibody titer will be checked on day 4 (BALB/c mice) and day 5 (C57BL/6 mice) after the challenge. Morbidity and mortality results will be recorded. In Cynomologus Macaques, infectious viremia and neutralizing antibody titer will be checked at 2 day intervals after the challenge until death or convalescence (9). Morbidity and mortality results will be recorded.

If the hypothesis that this experiment proposes is correct, both humoral and cell-mediated immunity should be observed in the murine and nonhuman primate models vaccinated with VRPs containing the NP, VP24, VP30, VP35 and VP40 proteins. A mortality rate of less than 30% should be observed in all animal models after lethal challenge and subsequent back challenge. All of the vaccinated animals (excluding negative controls) should show low to undetectable infectious viremia titers following lethal challenge. Humoral immunity should be indicated by reduction of plaques by approximately 80% and ELISA absorbency values 0.2 or greater above control samples in all vaccinated animals, to indicate the presence of neutralizing antibodies to the extracelluarly expressed NP protein. If this is the case, passive immunization in some disease free, unvaccinated animals should also be seen. One hundred percent efficacy is not expected in passive immunization since complete immunity requires both cell-mediated and humoral responses. Cell-mediated immunity should be indicated by CD8+ and CD4+ and CD8+ effector cells, specific to the intracelluarly expressed VP proteins, conferring immunity to the adoptively transferred, disease free, unvaccinated animals, with slightly higher survival rates in the animals receiving just CD8+ cells. Again, one hundred percent efficacy is not expected in adoptive immunization since complete immunity requires both types of immune responses.

This study addresses the immunosuppressive nature of the GP but fails to address the immunosupressive nature of the VP35 protein. VP35 inhibits activation of the interferon regulatory factor 3 which in turn prevents transcriptional activation of the INF-β promoter and the IFN-stimulated ISG54 promoter (12). This VP35 protein immunosuppressive activity could result in delayed immune response, altering the efficacy of a vaccines containing it. Further vaccine studies eliminating both the GP and VP35 protein should be attempted to determine there outcomes on immunity.

 

Literature Cited

 

1. Wilson Julie, Bray Mike, Bakken Russell, Hart Mary. 2001. Vaccine Potential of Ebola VP24, VP30, VP35, and VP40 Proteins. Virology, 286: 384-390.

 

2. Nyamathi Adeline, Fahey John, Sands Heather, Casillas Adrian. 2003. Ebola Virus : Immune Mechanisms of Protection and Vaccine Development. Biological Research for Nursing, 4: (4) 276-281.

 

3. Sullivan Nancy, Yang Zhi-Yong, Nabel Gary. 2003. Ebola Virus Pathogenesis: Implications for Vaccines and Therapies. Journal of Virology, 77: (18) 9733-9737.

 

4. Chepurnov Alexander, Tuzova Marina, Ternovoy Vladimir, Chernukin Igor. 1999. Suppressive Effect of Ebola Virus on T cell Proliferation in vitro is Provided by a 125-kDa GP Viral Protein. Immunology Letters, 68: 257-261.

 

5. Dolnik Olga, Volchkova Valentina, Garten Wolfgang, Carbonnelle Caroline, Becker Stephan, Kahnt Jorg, Stroher Ute, Klenk Hans-Dieter, Volchkov Vikto. 2004. Ecotodomain Shedding of the Glycoprotein GP of Ebola Virus. The EMBO Journal, 23: 2175-2184.

 

6. Peters C.J., Sanchez A., Feldmann H., Rollin P.E., Nichol S., Ksiazek T.G. 1994. Filoviruses as Emerging Pathogens. Seminars in Virology, 4: 147-154.

 

7. Hart Mary. 2003. Vaccine Research Efforts for Filoviruses. International Journal of Parasitology, 33: (5-6) 583-595.

 

8. Warfield Kelly, Bosio Catharine, Welcher Brent, Deal Emily, Mohamadzadeh Mansour, Schmaljohn Alan, Aman M., Bavari Sina. 2003 Ebola Virus-like Particles Protect from Lethal Ebola Virus Infection. PNAS, 100: (26) 15889-15894.

 

9. Geisbert Thomas, Pushko Peter, Anderson Kevin, Smith Johnathan, Davis Kelly, Jahrling Peter. 2002. Evaluation in Nonhuman Primates of Vaccines Against Ebola Virus. Emerging Infectious Diseases, 8: (5)

 

10. Prehaud C., Hellebrand E., Coudrier D., Volchkov V., Volchkova V., Feldmann H., Le

Guenno B., Bouloy M. 1998. Recombinant Ebola Virus Nucleoprotein and Glycoprotein (Gabon 94 Strain) Provide New Tools for the Detection of Human Infections. Journal of General Virology, 79: 2565-2572.

 

11. Wilson Julie, Hart Mary. 2001. Protection from Ebola Virus Mediated by Cytotoxic T Lymphocytes Specific for Viral Nucleoprotein. Journal of Virology, 75: (6) 2660-2664.

 

12. Basler Christopher, Mikulasova Andrea, Martinez-Sorbrido Luis, Paragas Jason, Muhlberger Elke, Bray Mike, Klenk Hnas-Dieter, Palese Peter, Garcia-Sastre Adolfo. 2003. The Ebola Virus VP35 Protein Inhibits Activation of the Interferon Regulatory Factor 3. Journal of Virology, 77: (14) 7945-7956.