Generally, the results of clinical trials aimed at evaluating conventional DNA vaccines have been disappointing. Even when administered at mg doses, DNA vaccine constructs have usually induced only modest immune responses in humans (2
). To enhance the immunogenicity of DNA vaccines, a new generation of nucleic acid vaccines has been developed, where expression of the encoded antigen involves the alphavirus RNA replication machinery, one of the most efficient replication systems in nature. We and others have previously demonstrated that these alphavirus-based vaccines are considerably more immunogenic than conventional DNA vaccines (19
). In this study, we extend these observations by showing that vaccination with low doses of a single Sindbis virus-based TB DNA vaccine encoding the highly immunogenic TB protein, antigen 85B, provides substantial protection after a virulent M. tuberculosis
challenge. Importantly, the postchallenge survival period for mice immunized with only 5 μg of the single Sin85B plasmid was similar to the mean survival period of BCG-vaccinated mice. These long-term protection results indicate that Sindbis virus-based TB DNA vaccines are attractive human vaccine candidates. Their enhanced immunogenicity suggests that considerably lower doses of the Sindbis TB constructs may yield, in humans, more robust immune responses than conventional DNA vaccines. Additionally, while the effectiveness of other promising gene-based TB vaccines, the modified vaccinia virus- and adenovirus-vectored constructs, may be limited by either preexisting or postimmunization antiviral neutralizing antibodies, humoral responses to the Sindbis virus plasmid vectors have apparently not limited protective responses in mice and likely will not in humans (20
). Clearly, a prime-boost protocol where vaccination with a Sindbis virus-based vector is followed by immunization with a single dose of a viral vector expressing the same TB antigen would be a potentially powerful new vaccination strategy that should be considered for testing in humans.
One mechanism that has been proposed to explain the enhanced immunogenicity of alphaviral vectors involves increased antigen production (25
). While alphaviral-replicase-based vectors were originally designed to generate increased levels of antigen, recent studies have suggested that their enhanced efficacy may not be due to elevated antigen expression. For instance, Leitner and colleagues demonstrated in a tumor model that the enhanced immunogenicity of a Sindbis virus-based construct did not correlate with antigen production (22
). Our results are consistent with this finding. Although the Sin85B plasmid was relatively more protective than the conventional pVax-85B construct, the levels of antigen 85B mRNA detected in cells transfected with these vectors were similar. Furthermore, the eightfold-higher antigen 85B antibody titers detected in mice vaccinated with the pVax-85B plasmid suggests that in vivo expression of antigen 85B from the conventional vector was similar or even slightly elevated compared to expression from the Sin85B plasmid.
The activation of antiviral innate immunity is an alternative immune mechanism that may explain the enhanced efficiency of alphaviral vectors. Alphavirus replicon-based vaccines, like RNA viruses, induce the production of dsRNA molecules which stimulate antiviral pathways. A key protein activated by dsRNA is RNase L, an enzyme responsible for the degradation of mRNA and inhibition of protein translation, which ultimately lead to apoptotic cell death (23
). In support of this mechanism, a recent study demonstrated that activation of the RNase L pathway was required for the induction of immunity by an alphaviral vector (22
). Our results are consistent with the activation of antiviral innate immunity contributing to the immunogenicity of the Sin85B plasmid. We observed an abrogation of protection when RNase L-deficient mice were vaccinated with the Sin85B construct. In contrast, the induction of protective immunity by vaccination with the conventional pVax-85B DNA vaccine and BCG was not significantly impacted in the RNase L−/−
model, indicating that the efficacy of these vaccines is not dependent on stimulating this innate immune pathway for activity. Based on in vitro and in vivo studies, it has been recently reported that apoptosis resulting from the stimulation of antiviral innate immunity is essential for the increased immunogenicity of alphavirus-based vaccines (21
). Similar studies are ongoing to determine whether the Sin85B construct induces increased apoptosis.
Our studies using CD4−/−
, and IFN-γ-depleted mice strongly suggest that CD8 cells and IFN-γ are also important mediators of the Sin85B-induced protective responses. Surprisingly, vaccination-challenge studies using CD4−/−
mice demonstrated that the Sin85B plasmid evoked significantly better protective immunity than the pVax-85B construct. This result strongly suggests that the stimulation of CD8 cells by Sin85B vaccination contributes to the protection seen in CD4−/−
mice. Previously, we had shown that the protection mediated by a TB DNA vaccine cocktail in this same model is dependent on CD8 T-cell activation (8
). Interestingly, while we have demonstrated that CD4-deficient mice can be protected against a tuberculous challenge by a DNA vaccine expressing antigen 85B, D'Souza et al. reported that immunization with a 85A-expressing DNA vaccine induced immune responses that protected β2-microglobulin deficient mice (which lack functional CD8 cells) but not CD4−/−
). Although the specific reasons for these disparate results are unclear, the use of different mouse strains, TB antigens, immunization schedules, and modes of challenge (intravenous versus aerosol) may have contributed to the differing outcomes. Additionally, a recent report indicating that CD4−/−
mice retain major histocompatibility complex class II restricted CD8 cells suggests that data from these CD4-deficient mice should be interpreted with caution (32
In summary, we have shown that vaccination with a highly immunogenic Sindbis virus-based vector expressing the M. tuberculosis protein antigen 85B induces extended antituberculosis protective responses after challenge. Our studies suggest that the Sin85B-induced protective immunity may result partially from the activation of innate immune pathways and may be mediated by CD8 T cells and IFN-γ. These findings suggest that targeting innate immune pathways with specific immunomodulators may allow the induction of powerful innate responses to complement the acquired immune responses induced by more conventional TB vaccines.