Mary Kate Hart

Protection from Ebola Virus Mediated by Cytotoxic T Lymphocytes Specific for the Viral Nucleoprotein

ABSTRACT Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocompatibility complex class I-restricted CTL response to an 11-amino-acid sequence in the amino-terminal portion of the Ebola virus NP. Passive transfer of polyclonal NP-specific antiserum did not protect recipient mice. In contrast, adoptive transfer of CTLs specific for the Ebola virus NP protected unvaccinated mice from lethal Ebola virus challenge. The protective CTLs were CD8+, restricted to the Db class I molecule, and recognized an epitope within amino acids 43 to 53 (VYQVNNLEEIC) in the Ebola virus NP. The demonstration that CTLs can prevent lethal Ebola virus infection affects vaccine development in that protective cellular immune responses may be required for optimal protection from Ebola virus.

Induction of Humoral and CD8+ T Cell Responses Are Required for Protection against Lethal Ebola Virus Infection

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4+ and CD8+ IFN-γ+ T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8+, but not CD4+, T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-γ-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8+ T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.

Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus.

Multiagent DNA vaccines for highly pathogenic organisms offer an attractive approach for preventing naturally occurring or deliberately introduced diseases. Few animal studies have compared the feasibility of combining unrelated gene vaccines. Here, we demonstrate that DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models. In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species. These results demonstrate for the first time the potential of combined DNA vaccines for these agents and point to a possible method of rapid development of multiagent vaccines for disparate pathogens such as those that might be encountered in a biological attack.

Protective Cytotoxic T-Cell Responses Induced by Venezuelan Equine Encephalitis Virus Replicons Expressing Ebola Virus Proteins

ABSTRACT 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.

Vaccine research efforts for filoviruses.

Ebola and Marburg viruses belong to the family Filoviridae, and cause acute, frequently fatal, haemorrhagic fever in humans and non-human primates. No vaccines are available for human use. This review describes the status of research efforts to develop vaccines for these viruses and to identify the immune mechanisms of protection. The vaccine approaches discussed include DNA-based vaccines, and subunit vaccines vectored by adenovirus, alphavirus replicons, and vaccinia virus.

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live- attenuated V3526 vaccine candidate.

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.

Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

ABSTRACT There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice.

Immunization with either a live-attenuated (TC-83) or formalin-inactivated (C-84) vaccine for Venezuelan equine encephalitis (VEE) virus protected BALB/c mice from lethal VEE infection acquired subcutaneously or by aerosol. While vaccinated C3H/HeN mice were also protected from parenteral infection, neither vaccine protected these mice from an aerosol infection. The apparent vaccine failures in C3H/HeN mice could not be attributed to deficiencies in virus-neutralizing antibodies in serum, as these responses were typically of equal or higher titer than those observed in protected BALB/c mice before challenge. IgG subclass analysis offered no facile explanation: profiles of IgG2 alpha dominance were observed in C3H/HeN mice given either vaccine and in BALB/c mice given the live-attenuated vaccine, whereas BALB/c antibody responses shifted toward IgGl dominance after immunization with the killed C-84 vaccine. Data from immunized congenic mice showed that the H-2 genes from the C3H/He mice were not singularly responsible for the inability of these mice to resist aerosol infection with VEE virus. VEE virus-specific IgA responses were detected more frequently in respiratory and vaginal secretions obtained from the protected BALB/c mice.

Development of a cAdVax-Based Bivalent Ebola Virus Vaccine That Induces Immune Responses against both the Sudan and Zaire Species of Ebola Virus

ABSTRACT Ebola virus (EBOV) causes a severe hemorrhagic fever for which there are currently no vaccines or effective treatments. While lethal human outbreaks have so far been restricted to sub-Saharan Africa, the potential exploitation of EBOV as a biological weapon cannot be ignored. Two species of EBOV, Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV), have been responsible for all of the deadly human outbreaks resulting from this virus. Therefore, it is important to develop a vaccine that can prevent infection by both lethal species. Here, we describe the bivalent cAdVaxE(GPs/z) vaccine, which includes the SEBOV glycoprotein (GP) and ZEBOV GP genes together in a single complex adenovirus-based vaccine (cAdVax) vector. Vaccination of mice with the bivalent cAdVaxE(GPs/z) vaccine led to efficient induction of EBOV-specific antibody and cell-mediated immune responses to both species of EBOV. In addition, the cAdVax technology demonstrated induction of a 100% protective immune response in mice, as all vaccinated C57BL/6 and BALB/c mice survived challenge with a lethal dose of ZEBOV (30,000 times the 50% lethal dose). This study demonstrates the potential efficacy of a bivalent EBOV vaccine based on a cAdVax vaccine vector design.

Comparison of the immunological responses and efficacy of gamma-irradiated V3526 vaccine formulations against subcutaneous and aerosol challenge with Venezuelan equine encephalitis virus subtype IAB

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel further augmented the immune response in mice and resulted in 100% protection against aerosol challenge.