Ebola virus outbreaks are associated with high lethality due to the absence of treatment options or a licensed vaccine. Both DNA priming with rAd vector boosting and rAd alone can confer protection to lethal challenge in an animal model that closely parallels human disease [21
]. The rAd vector vaccine conferred protection in an accelerated vaccine regimen in a nonhuman primate species [3
]. Although in vitro cytopathicity has been observed by overexpression of Ebola GP (one of the vaccine components), we have not seen toxicity in animals vaccinated by vectors expressing Ebola GP (unpublished data). However, because this hypothetical complication has been raised, we sought to modify GP to eliminate in vitro cytopathicity yet retain antigenic properties that are necessary for protective immunity. Here, the efficacies of different forms of GP were evaluated using doses at the threshold of protection in the accelerated vaccination model. We have identified a vaccine with decreased in vitro cytopathicity that retained immunogenicity necessary to protect against Ebola infection.
We find that alternative forms of GP confer differential immune protection. Deletion of the GP transmembrane domain abolished cytopathic effects in transfected HEK293 cells, but the corresponding ΔTM(Z) vaccine was less efficacious than wild-type GP(Z) in protecting the cynomolgus macaques against infection. Although cellular and humoral immune responses were indistinguishable between groups receiving the different immunogen forms, the inherent variability in quantitating the responses in outbred macaques may obscure our ability to identify immune responses responsible for higher survival. Alternatively, ΔTM(Z) may differ from wild-type GP(Z) in antigenic qualities that are not captured by measurements of total antigen-specific IgG or intracellular cytokine responses stimulated by a broad peptide pool. For example, the transmembrane-deleted protein is secreted and likely shows conformational differences from the membrane-anchored protein. Subsequent modifications of the glycoprotein to retain membrane attachment and a more native envelope structure yielded a mutant, E71D, with reduced in vitro cytopathicity. Recently, it was suggested that this region of GP contributes to viral receptor binding [22
]. It is noteworthy that the envelope glycoprotein cytopathicity of other viruses such as HIV is linked to receptor binding and fusion [23
], raising the possibility that Ebola GP shares similar properties.
Ongoing outbreaks of both Ebola and Marburg viruses illustrate the importance of developing a filovirus vaccine for human use. This report shows that protective immunity against Ebola infection is achieved in nonhuman primates by the generation of antigen-specific immune responses to a single protein, GP, which has been modified to eliminate in vitro cytopathic effects. Since the vaccine will be licensed initially against one agent, protective immunity was evaluated against challenge with this species, Zaire ebolavirus. Future studies will address whether this vaccine can protect against other Ebola species, such as SEBOV. Preliminary studies suggest that protection can be observed in animals receiving the bivalent vaccine that survive ZEBOV challenge when subsequently exposed to the Gulu strain of SEBOV (unpublished data). However, complete analysis of the question of cross-strain protection will require additional studies that fall beyond the scope of the present study.
In a dose-ranging experiment with rAd expressing GP and NP, immune protection correlated well with GP-specific ELISA IgG titers. This observation is consistent with early Ebola vaccine studies performed using a gene-based GP and NP vaccine in guinea pigs. In the current study there was also a trend toward increased survival in animals with higher CD8+ T cell responses when vaccines containing different antigen combinations were evaluated for protective efficacy. In combination, these findings suggest that the GP-specific humoral immune response provides a correlate of immunity; however, it is likely that the T cell response contributes to protection, and the Ig response may be a reflection of T cell help. Further improvements in the sensitivity of the T cell assay, as well as immune depletion studies, may provide further insight into this question.
Ebola virus protective immunity has also been achieved more recently using replication competent vesicular stomatitis virus vectors [24
], supporting the concept that vaccination for Ebola can successfully protect against disease mortality. While this vaccine merits further investigation, and replication-competent viruses have contributed to many effective vaccines, concerns remain about live viruses because the amount of viral replication cannot be controlled, and their environmental effects are uncertain. Such considerations may pose challenges to development of vesicular stomatitis virus-based vaccines for human use. The recent recommendation that the live-attenuated polio vaccine, a relatively safe vaccine, no longer be used in the United States [25
], and the inactivated virus be used instead, exemplifies these challenges. In contrast, the rAd vector vaccine is nonreplicating and can be manufactured to high yields, and safety data exist for this platform. Here we show that immunity follows a single injection with 1010
rAd particles, a dose that is two orders of magnitude lower than previously reported for this single-modality vaccine. Such doses of rAd vectors have proved to be well tolerated and immunogenic for other recombinant genes in vivo and can be evaluated for the vectors reported here, alone or in DNA prime/rAd boost combinations. One theoretical concern regarding a rAd vaccine is the possibility that preexisting immunity to natural Ad5 infection, occurring in up to 50% of the U.S. population, may affect vaccine efficacy. Recent preliminary results from HIV vaccine trials suggest that antigen-specific immune responses can be readily elicited by rAd5 vectors even in study participants with prior immunity to adenovirus type 5, especially when vaccine doses higher than 109
are used (unpublished data). Given that Ebola GP is more immunogenic than HIV Env, we would expect that significant immune responses can be generated to the present vaccine; future clinical trials will resolve this question. In addition, DNA priming can be used prior to rAd boost to overcome Ad immunity and has also been shown to be effective in preventing Ebola infection [2
]. In case Ad5 immunity should become problematic, several alternative rAd platforms, involving Ad35 and other exotic serotypes, are under development. Preexisting immunity to Ad35 is quite low (~10%–15%) worldwide [26
], and the same principles reported in this paper would apply to the development of such vaccine candidates.
Immunization with 1010 rAd particles of E71D(Z)+E71D(S/G) was effective against infectious challenge with ZEBOV. These data provide evidence that protection does not require the inclusion of the NP gene in the vaccine, and document that protective immunity can be conferred by responses to the GP antigen alone. Indeed, diminished survival was observed only in vaccine groups containing this gene, raising the possibility that under some conditions inclusion of NP may dilute protective immune responses. However, the significance of this result is limited by the relatively small animal numbers used here. It is noteworthy that elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection, and simplify the vaccine for future development in human trials. A first-generation Ebola DNA vaccine is currently under evaluation in phase I clinical studies, and the rAd component is currently being manufactured as a clinical grade preparation that is undergoing safety studies and will be tested in Phase I trials pending regulatory approvals. After the completion of initial studies testing individual DNA and rAd vaccine components, these candidates will be evaluated in prime boost combination. Vaccine safety and immunogenicity relative to the nonhuman primate challenge model will guide assessments regarding its utility as a preventive vaccine in humans.