Evaluation in nonhuman primates of vaccines against Ebola virus

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.     

Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

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.     

Recombinant subunit vaccines protect guinea pigs from lethal Ebola virus challenge

Ebola virus (EBOV) is among the deadliest pathogens known to man causing infrequent outbreaks of hemorrhagic disease. In humans, the case fatality rates in the outbreaks can reach 90%. During the West African epidemic almost 30,000 people were infected and of these over 11,000 fatalities were reported. Currently, we are facing an uncontained larger outbreak in the Democratic Republic of the Congo. Even though EBOV was discovered in 1976, extensive efforts to develop countermeasures, particularly therapeutics and vaccines, started late and there is still no FDA-approved product available. Nevertheless, one candidate vaccine, the rVSV-ZEBOV, is being used in clinical trials during the current outbreak with the hope of ending the human transmission chains. However, adverse reactions to administration of some EBOV vaccines have been reported; therefore, we have developed a safe and efficacious formulation of insect-cell derived adjuvanted protein vaccines. Vaccine candidates containing the EBOV glycoprotein with or without matrix proteins VP24 and VP40 formulated with one of three different adjuvants were tested in guinea pigs for immunogenicity and efficacy against lethal EBOV challenge. The results demonstrated that these vaccine candidates engendered high titers of antigen-specific antibodies in immunized animals and two of these vaccine candidates afforded complete or nearly complete protection against lethal challenge. Interestingly, we found a sex bias in partially protected immunized groups with male guinea pigs succumbing to disease and females surviving. In summary, we developed a safe and immunogenic adjuvanted subunit vaccine uniformly protective against EBOV disease in guinea pigs.     

Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice

The development of multiagent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. We report here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease, anthrax; a viral disease, Marburg fever; and against a toxin-mediated disease, botulism. The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)). CBA/J mice inoculated with a mixture of VRP expressing BoNT H(C) serotype C (BoNT/C H(C)) and MAT-PA were 80% protected from a B. anthracis (Sterne strain) challenge and then 100% protected from a sequential BoNT/C challenge. Swiss mice inoculated with individual VRP or with mixtures of VRP vaccines expressing BoNT H(C) serotype A (BoNT/A H(C)), MAT-PA, and MBGV-GP produced antibody responses specific to the corresponding replicon-expressed protein. Combination of the different VRP vaccines did not diminish the antibody responses measured for Swiss mice inoculated with formulations of two or three VRP vaccines as compared to mice that received only one VRP vaccine. Swiss mice inoculated with VRP expressing BoNT/A H(C) alone or in combination with VRP expressing MAT-PA and MBGV GP, were completely protected from a BoNT/A challenge. These studies demonstrate the utility of combining individual VRP vaccines into multiagent formulations for eliciting protective immune responses to various types of diseases.     

Protective efficacy of neutralizing antibodies against Ebola virus infection

Ebola virus causes lethal hemorrhagic fever in humans and nonhuman primates, but no effective antiviral compounds are available for the treatment of this infection. The surface glycoprotein (GP) of Ebola virus is an important target of neutralizing antibodies. Although passive transfer of GP-specific antibodies has been evaluated in mouse and guinea pig models, protection was achieved only by treatment shortly before or after virus challenge. Using these animal models, we evaluated the protective efficacy of two monoclonal antibodies whose epitopes are distinct from those of the antibodies tested by others. Treatment of mice with these antibodies 2 days after challenge completely protected most of the animals; even treatment 3 or 4 days after challenge was partially effective. Although antibody treatment in the guinea pig model was not as effective as in the mouse model, single-dose treatment of guinea pigs 1 day before, or 1 or 2 days after challenge did protect some animals. Interestingly, the protective effects seen in these animal models did not correlate with the in vitro neutralizing activity of the antibodies, suggesting different mechanisms of the neutralization by these antibodies. These results underscore the potential therapeutic utility of monoclonal antibodies for postexposure treatment of Ebola virus infections.     

Marburg virus-like particles protect guinea pigs from lethal Marburg virus infection

Ongoing outbreaks of filoviruses in Africa and concerns about their use in bioterrorism attacks have led to intense efforts to find safe and effective vaccines to prevent the high mortality associated with these viruses. We previously reported the generation of virus-like particles (VLPs) for the filoviruses, Marburg (MARV) and Ebola (EBOV) virus, and that vaccinating mice with Ebola VLPs (eVLPs) results in complete survival from a lethal EBOV challenge. The objective of this study was to determine the efficacy of Marburg VLPs (mVLPs) as a potential vaccine against lethal MARV infection in a guinea pig model. Guinea pigs vaccinated with mVLPs or inactivated MARV developed MARV-specific antibody titers, as tested by ELISA or plaque-reduction and neutralization assays and were completely protected from a MARV challenge over 2000 LD50. While eVLP vaccination induced high EBOV-specific antibody responses, it did not cross-protect against MARV challenge in guinea pigs. Vaccination with mVLP or eVLP induced proliferative responses in vitro only upon re-exposure to the homologous antigen and this recall proliferative response was dependent on the presence of CD4+ T cells. Taken together with our previous work, these findings suggest that VLPs are a promising vaccine candidate for the deadly filovirus infections.     

Virus-like particles exhibit potential as a pan-filovirus vaccine for both Ebola and Marburg viral infections

A safe and effective pan-filovirus vaccine is highly desirable since the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause highly lethal disease typified by unimpeded viral replication and severe hemorrhagic fever. Previously, we showed that expression of the homologous glycoprotein (GP) and matrix protein VP40 from a single filovirus, either EBOV or MARV, resulted in formation of wild-type virus-like particles (VLPs) in mammalian cells. When used as a vaccine, the wild-type VLPs protected from homologous filovirus challenge. The aim of this work was to generate a multi-agent vaccine that would simultaneously protect against multiple and diverse members of the Filoviridae family. Our initial approach was to construct hybrid VLPs containing heterologous viral proteins, of EBOV and MARV, and test the efficacy of the hybrid VLPs in a guinea pig model. Our data indicate that vaccination with GP was required and sufficient to protect against a homologous filovirus challenge, as heterologous wild-type VLPs or hybrid VLPs that did not contain the homologous GP failed to protect. Alternately, we vaccinated guinea pigs with a mixture of wild-type Ebola and Marburg VLPs. Vaccination with a single dose of the multivalent VLP vaccine elicited strong immune responses to both viruses and protected animals against EBOV and MARV challenge. This work provides a critical foundation towards the development of a pan-filovirus vaccine that is safe and effective for use in primates and humans.     

Intranasal vaccination with ebola virus GP amino acids 258–601 protects mice against lethal challenge

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.     

Alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in BALB/c mice and functional serum antibodies in rhesus macaques

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.     

Immunization with vesicular stomatitis virus vaccine expressing the Ebola glycoprotein provides sustained long-term protection in rodents

Ebola virus (EBOV) infections cause lethal hemorrhagic fever in humans, resulting in up to 90% mortality. EBOV outbreaks are sporadic and unpredictable in nature; therefore, a vaccine that is able to provide durable immunity is needed to protect those who are at risk of exposure to the virus. This study assesses the long-term efficacy of the vesicular stomatitis virus (VSV)-based vaccine (VSVΔG/EBOVGP) in two rodent models of EBOV infection. Mice and guinea pigs were first immunized with 2 × 10⁴ or 2 × 10⁵ plaque forming units (PFU) of VSVΔG/EBOVGP, respectively. Challenge of mice with a lethal dose of mouse-adapted EBOV (MA-EBOV) at 6.5 and 9 months after vaccination provided complete protection, and 80% (12 of 15 survivors) protection at 12 months after vaccination. Challenge of guinea pigs with a lethal dose of guinea pig-adapted EBOV (GA-EBOV) at 7, 12 and 18 months after vaccination resulted in 83% (5 of 6 survivors) at 7 months after vaccination, and 100% survival at 12 and 18 months after vaccination. No weight loss or clinical signs were observed in the surviving animals. Antibody responses were analyzed using sera from individual rodents. Levels of EBOV glycoprotein-specific IgG antibody measured immediately before challenge appeared to correlate with protection. These studies confirm that vaccination with VSVΔG/EBOVGP is able to confer long-term protection against Ebola infection in mice and guinea pigs, and support follow-up studies in non-human primates.     

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa.     

Evaluation of Lassa virus vaccine immunogenicity in a CBA/J-ML29 mouse model

Lassa fever (LF) is one of the most prevalent viral hemorrhagic fevers in West Africa responsible for thousands of deaths annually. The BSL-4 containment requirement and lack of small animal model to evaluate Lassa virus (LASV)-specific cell-mediated immunity (CMI) complicate development of effective LF vaccines. Here we have described a CBA/J-ML29 model allowing evaluation of LASV-specific CMI responses in mice. This model is based on Mopeia virus reassortant clone ML29, an attractive immunogenic surrogate for LASV. A single intraperitoneal (i.p.) immunization of CBA/J mice with ML29 protected animals against a lethal homologous intracerebral (i.c.) challenge with 588 LD(50). The ML29-immunized mice displayed negligible levels of LASV-specific antibody titers, but LASV-specific CMI responses were detectable early and peaked on day 8-10 after immunization. A T cell cytotoxicity assay in vivo showed a correlation between LASV-specific cytotoxicity and the timing of protection induced by the ML29 immunization. Notably, CBA/J mice that received CD8+ T cell-depleted splenocytes from ML29-immunized donors all succumbed to a lethal i.c. challenge, demonstrating that CD8+ T cells are critical in protection. The CBA/J-ML29 model can be useful immunological tool for the preliminary evaluation of immunogenicity and efficacy of vaccine candidates against LASV outside of BSL-4 containment facilities.     

Expression, processing, and immunogenicity of the structural proteins of Venezuelan equine encephalitis virus from recombinant baculovirus vectors

Recombinant baculoviruses expressing the structural proteins of Venezuelan equine encephalitis virus (VEE) have been constructed and the intracellular processing, antigenicity, and immunogenicity of the expression products have been assessed. Baculoviruses expressing the entire structural protein region (C-E3-E2-6K-E1), or the complete glycoprotein region (E3-E2-6K-E1), generated products in Sf9 cells that were accurately and completely processed, and resulted in mature proteins that were antigenically and electrophoretically indistinguishable from authentic viral proteins. These products were highly immunogenic in BALB/c mice, induced efficient VEE neutralizing responses, and protected these animals against challenge with virulent VEE. Expression of individual glycoprotein regions (E3-E2 and 6K-E1) generated products that were accurately but incompletely processed, and induced non-neutralizing antibodies that reacted more efficiently with denatured than native VEE proteins. Nonetheless, immunization with the 6K-E1 expression product provided complete protection against VEE challenge.     

Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein

We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection.     

Matrix-M adjuvant enhances antibody,cellular and protective immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine in mice

Ebola virus (EBOV) causes severe hemorrhagic fever for which there is no approved treatment or preventive vaccine. Immunological correlates of protective immunity against EBOV disease are not well understood. However, non-human primate studies have associated protection of experimental vaccines with binding and neutralizing antibodies to the EBOV glycoprotein (GP) as well as EBOV GP-specific CD4⁺ and CD8⁺ T cells. In this report a full length, unmodified Zaire EBOV GP gene from the 2014 EBOV Makona strain (EBOV/Mak) was cloned into a baculovirus vector. Recombinant EBOV/Mak GP was produced in Sf9 insect cells as glycosylated trimers and, when purified, formed spherical 30–40 nm particles. In mice, EBOV/Mak GP co-administered with the saponin adjuvant Matrix-M was significantly more immunogenic, as measured by virus neutralization titers and anti-EBOV/Mak GP IgG as compared to immunization with AlPO₄ adjuvanted or non-adjuvanted EBOV/Mak GP. Similarly, antigen specific T cells secreting IFN-γ were induced most prominently by EBOV/Mak GP with Matrix-M. Matrix-M also enhanced the frequency of antigen-specific germinal center B cells and follicular helper T (T_FH) cells in the spleen in a dose-dependent manner. Immunization with EBOV/Mak GP with Matrix-M was 100% protective in a lethal viral challenge murine model; whereas no protection was observed with the AlPO₄ adjuvant and only 10% (1/10) mice were protected in the EBOV/Mak GP antigen alone group. Matrix-M adjuvanted vaccine induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8⁺ T cells, specific T_FH cells, germinal center B cells, and persistence of EBOV GP-specific plasma B cells in the bone marrow. Taken together, the addition of Matrix-M adjuvant to the EBOV/Mak GP nanoparticles enhanced both B and T-cell immune stimulation which may be critical for an Ebola subunit vaccine with broad and long lasting protective immunity.     

DNA vaccines induce partial protection against Leishmania mexicana

As part of an ongoing effort to develop a vaccine against Leishmania mexicana, we tested DNA vaccines encoding L. mexicana GP63, CPb, and LACK, and L. amazonensis GP46, to evaluate this strategy and define the best antigen candidates. Immune responses and vaccine efficacy were evaluated in BALB/c mice immunized with plasmid DNA encoding the different antigens. All four DNA vaccines induced Leishmania-specific humoral and lympho-proliferative immune responses. However, only mice immunized with VR1012-GP46, VR1012-GP63 and VR1012-CPb were partially protected against infection, as evidenced by reduced lesion size and parasite burden. Interestingly, immunization of mice with a mixture of these three plasmids further increased protection. Thus, plasmids encoding CPb, GP63 and GP46 represent good candidates for further development of DNA vaccines against L. mexicana.     

Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat in swine industry and causes heavy economic losses worldwide. Currently, the available vaccines are the inactivated and attenuated virus vaccines, but the use of PRRSV in their production raises the issue of safety. We developed a chimeric virus-like particles (VLPs) vaccine candidate for PRRSV protection. The chimeric VLPs was composed of M1 protein from H1N1 influenza virus and a fusion protein, denoted as NA/GP5, containing the cytoplasmic and transmembrane domains of H1N1 virus NA protein and PRRSV GP5 protein. Vaccination of BALB/c mice with 10 μg of chimeirc VLPs by intramuscular immunization stimulated antibody responses to GP5 protein, and induced cellular immune response. The data suggested that the chimeric VLP vaccine candidate may provide a new strategy for further development of vaccines against PRRSV infection.     

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.     

Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice

An orally delivered foot-and-mouth disease (FMD) vaccine has not previously been reported. By using a T4 bacteriophage nanoparticle surface gene-protein display system (T4-S-GPDS), we created a foot-and-mouth disease virus (FMDV) entire capsid protein vaccine candidate. On the T4 phage surface SOC site, a full length FMDV capsid precursor polyprotein (P1, 755 aa) and proteinase 3C (213 aa) derived from an infected pig of serotype O strain GD-10 (1999), were separately displayed on different T4 phage particle surfaces through inserting their coding region DNAs into the T4 phage genome, yielding phage strains T4-P1 and T4-3C. We also constructed a series of FMDV sub-full length capsid structural protein (subunit) containing T4 phage recombinant vaccines. Both sucking and young BALB/c mice were used as two kinds of FMDV vaccine potency evaluation models. Many groups of both model mice were vaccinated orally or by subcutaneous injection with varying FMDV-T4 phage recombinant vaccines, with and without addition of adjuvant, then challenged with a lethal dose of cattle source virulent FMDV. In the case of immunization with a mixture of phage T4-P1 and phage T4-3C particles without any adjuvant added, all mice were 100% protected following either oral or injection immunization, whereas 100% of the control, non-immunized mice and mice immunized with only T4 phage vector Z1/Zh(-) or wild-type T4(+)D phage died; in contrast, with FMDV subunit vaccine, less than 75% protection followed the same potency challenge in both mice model groups. In addition, two pigs immunized with a phage T4-P1 and phage T4-3C mix were protected upon housing together with infected pigs. This study represents a clear example of how FMD and other pathogenic disease vaccines can be prepared by a simple and efficient bacteriophage route.     

De novo syntheses of Marburg virus antigens from adenovirus vectors induce potent humoral and cellular immune responses

Marburg virus (MARV) is an African filovirus that causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, there are no MARV vaccines or therapies approved for human use. We hypothesized that developing a vaccine that induces a de novo synthesis of MARV antigens in vivo will lead to strong induction of both a humoral and cell-mediated immune response against MARV. Here, we develop and characterize three novel gene-based vaccine candidates which express the viral glycoprotein (GP) from either the Ci67, Ravn or Musoke strain of MARV. Immunization of mice with complex adenovirus (Ad)-based vaccine candidates (cAdVax vaccines), led to efficient production of both antibodies and cytotoxic T lymphocytes (CTL) specific to Musoke strain GP and Ci67 strain GP, respectively. Antibody responses were also shown to be cross-reactive across the MARV strains, but not cross-reactive to Ebola virus, a related filovirus. Additionally, three 1 x 10(8)pfu doses of vaccine vector were demonstrated to be safe in mice, as this did not lead to any detectable toxicity in liver or spleen. These promising results indicate that a cAdVax-based vaccine could be effective for induction of both humoral and cell-mediated immune responses to multiple strains of the Marburg virus.