The studies reported herein were undertaken to examine whether DNA vaccines could be improved to protect against lethal MARV challenge and to study the relative potency of vaccines when administered as recombinant DNA and/or viral vectors for immune protection against MARV Angola. Although methylated CpG in DNA plasmids theoretically serves as a natural adjuvant, stimulation through TLR9 appears to be less effective than activation mechanisms by viral vectors for the induction of innate responses, therefore resulting in lower immunogenicity of DNA vaccines in primates (13
). Approaches to improve the potency of DNA vaccines have included methods to increase antigen expression, alter tissue targeting, and enhance immune responses through the use of adjuvants (12
). In the current work we focused on two approaches to generate efficient antigen expression, promoter modification to enable efficient transcription in mammalian cells (1
) and alteration of the GP gene insert for optimum codon usage (27
). Partial protection against MARV (Musoke strain) was previously observed after DNA immunization at multiple sites by gene gun (17
), and it was suggested that efficacy was due to gene gun targeting of the immunogen to antigen-presenting cells in skin tissue. Therefore, the use of Biojector delivery of the DNA vaccine in our studies may also have contributed to the improved efficacy for protection. Prior to this report, uniform protection from MARV mortality in NHP has been achieved only with vaccines containing viral components, as either vectors or virus-like particles (8
). The results herein demonstrate the potential utility of DNA plasmids for use as vaccine vectors in primates and suggest that improvements in antigen expression and delivery may be a valid approach for enhancing immune responses induced by DNA vaccines.
The results presented here also shed light on the nature of immune responses that are associated with vaccine protection against MARV Angola mortality. We showed previously that a prime-boost vaccine using DNA and rAd vectors protected against lethal infection with the Zaire species of EBOV, but homologous vector vaccines were not tested in that study and so the contribution of each component to protective immunity could not be assessed (23
). Here, the vaccine vectors were compared singly and in combination. It is interesting that the group receiving only DNA vaccines generated levels of antibody against MARV GP similar to those of the rAd-only group and yet exhibited a reduced level of protection against illness. Riemenschneider et al. also observed a lack of correlation between antibody levels and protection after vaccination with a DNA vaccine (17
). Cell-mediated immunity was not reported in that study, but the present work reveals the balance of T-cell responses (CD4/CD8) and functional phenotype to be distinguishing features between groups that became ill and those that were fully protected. Both vaccine modalities that induced skewing toward CD4+
T-cell immunity mediated incomplete protection against symptoms of MARV infection, and for the DNA/rAd vaccine, this was despite the induction of a robust CD8+
T-cell response. This observation suggests that protective immunity against MARV Angola depends on qualitative differences that are not captured by measures of the absolute magnitude of antibody or cell-mediated immune responses, as observed for other pathogens (5
). Vaccine modalities using DNA to prime the immune response generated T cells with a polyfunctional quality: cells that simultaneously produce TNF-α, IFN-γ, and IL-2. Although this phenotype has defined protective immunity for a variety of pathogens (2
), the generation of polyfunctional CD4+
cells did not associate with the rAd-GP vaccine group that provided the most robust protection (absence of symptoms) against MARV Angola. It is noteworthy that many of the previous studies were designed to evaluate long-term memory against persistent chronic infections where it would be important to generate long-lived cells with a triple-positive cytokine phenotype. Filovirus infection kills susceptible hosts within days to weeks, and the unvaccinated NHP in this study were moribund or euthanized between days 8 and 9. Therefore, it is not surprising that the rapid generation of CD8+
T cells lacking the polyfunctional phenotype that includes IL-2 but instead possessing a dominant effector phenotype (TNF-α and IFN-γ), such as that induced by the rAd-GP vaccine, provided robust protection against infection. The relatively small number of animals used in these studies did not reveal statistically significant differences in T-cell polyfunctional quality within each vaccine group between subjects who remained healthy and those exhibiting clinical symptoms, but it is perhaps noteworthy that the single symptom-free subject in the DNA vaccine group had a higher proportion of CD8+
T cells expressing TNF-α and IFN-γ (with or without IL-2) and a lower representation of cells single positive for IL-2, indicating the dominance of T cells with greater effector potential in the healthy subject (data not shown). Similarly, in the DNA/rAd vaccine subjects that remained healthy there were higher proportions of CD8+
T cells expressing the effector cytokines TNF-α and IFN-γ.
The protection from mortality afforded by the DNA vaccine used herein suggests that plasmid vectors, previously thought to be ineffective as vaccines for filoviruses, may hold promise. The inherent safety and stability of this platform make it attractive as a vaccine to be used in areas lacking refrigeration, such as regions of Africa where filovirus outbreaks are endemic. Because of the high magnitude of antigen-specific responses achieved by heterologous prime-boost, it has been proposed that long-term immunity may be optimally achieved by priming rAd with DNA (18
). This is another important consideration for vaccines used in populations where immunization programs are less well developed and subjects may have only a single opportunity to be vaccinated. The studies shown here suggest that the value of DNA priming will depend on the pathogen, antigen, and mechanism of immune protection. For MARV Angola, these studies suggest that it will be important for DNA and rAd vaccines to be optimized to skew immunity toward CD8+
T cells with effector properties, while also maintaining CD4+
and antibody responses.