The relatively low immunogenicity of DNA vaccines in clinical trials to date has led to the development of various strategies to improve the immunogenicity of these vaccines. Approaches that have proven effective in nonhuman primates include the use of plasmid cytokines, polymer adjuvants, and in vivo electroporation techniques (4
). A disadvantage of these approaches is that they require additional materials or devices that add manufacturing, regulatory, and practical complexities that may limit their large-scale clinical application. In this study, we investigated the potential utility of a strategy aimed at optimizing regulatory elements in the plasmid DNA vaccine. We demonstrated that the addition of the HTLV-1 R element to a CMV expression cassette markedly increased DNA vaccine immunogenicity in both mice and cynomolgus monkeys. Importantly, such a modification does not involve any additional components or devices and thus does not increase vaccine complexity. Optimizing immunogenicity by engineering novel regulatory elements into the plasmid backbone therefore represents a simple, practical, and effective approach that could be advanced into clinical trials.
A previous study demonstrated that adding the HTLV-1 R element downstream of an SV40 early promoter increased transgene expression by approximately 10- to 40-fold in vitro (30
). The present study extends this observation by demonstrating that the R element also potentiates expression from a strong CMV promoter/enhancer. More importantly, we showed that this enhanced expression translates into significantly augmented immune responses in both rodents and nonhuman primates. We speculate that the beneficial effects of the R region likely involve recruiting key cellular transcription factors that enhance transgene transcription (12
), although the precise mechanism of action remains to be determined. The R region may also function as an internal ribosome entry site, suggesting that it may act at a posttranscriptional step as well (2
). Alternatively, it is possible that the combination of CMV enhancer and HTLV-1 R regions stimulate optimal expression in professional antigen-presenting cells, which would best facilitate the antigen-specific T-cell response. Interestingly, the RSV/R and mUB/R DNA vaccines exhibited comparable transgene expression in vitro compared with CMV/R DNA vaccines (Fig. ) but nevertheless failed to result in improved immunogenicity (Fig. ). Thus, enhanced expression in vitro is not the sole determinant of enhanced immunogenicity in vivo.
In both mice and cynomolgus monkeys, CMV/R DNA vaccines expressing HIV-1 antigens elicited higher cellular immune responses than the parental 1012 DNA vaccines expressing the same antigens. However, the magnitude of the observed effects differed substantially between the two species. While the CMV/R DNA vaccines elicited only twofold higher responses in mice (Fig. ), the CMV/R DNA vaccines elicited >10-fold higher cellular immune responses to Gag, Pol, and Nef and seven- to ninefold higher responses to Env after two immunizations in cynomolgus monkeys (Fig. and ). We suspect that this difference may reflect the lower baseline immunogenicity of the parental 1012 DNA vaccines in nonhuman primates and that the beneficial effects of the R element may appear more striking in limiting situations. Consistent with this observation, the R element had the greatest effect at enhancing the weakest responses elicited by the parental 1012 DNA vaccine against Gag and Nef. However, Env- and Pol-specific cellular immune responses were also significantly higher when induced by CMV/R DNA vaccines compared with the parental 1012 DNA vaccines.
We also observed that the six-plasmid mixture of CMV/R DNA vaccines that included Gag, Pol, and Nef on separate plasmids elicited significantly higher cellular immune responses to these antigens compared to the four-plasmid mixture of CMV/R DNA vaccines that included the Gag-Pol-Nef fusion protein. These effects are particularly notable since the separate gag, pol, and nef plasmids were each utilized at one-third the dose of the plasmid encoding the Gag-Pol-Nef fusion protein. We speculate that this may reflect enhanced translation or mRNA stability of the shorter genes compared with the fusion gene, which might potentially affect antigen processing and presentation.
A limitation of the current study is that we were not able to assess the protective efficacy afforded by the CMV/R DNA vaccines. Since HIV-1 does not infect cynomolgus monkeys, we could not perform viral challenges in these animals. However, accumulating data have confirmed the importance of cellular immune responses in controlling HIV-1 replication in humans and simian immunodeficiency virus replication in rhesus monkeys (14
). Moreover, vaccines aimed at eliciting virus-specific cellular immune responses have afforded partial control of simian-human immunodeficiency virus and simian immunodeficiency virus challenges in rhesus monkeys (1
). Thus, we believe that the markedly increased magnitude and breadth of HIV-1-specific cellular immune responses afforded by the CMV/R DNA vaccines in nonhuman primates in the present study will likely prove beneficial in the development of second-generation DNA vaccines for HIV-1 and other pathogens. In particular, incorporating the HTLV-1 R element and utilizing separate genes in place of fusion genes represent simple and practical strategies to improve DNA vaccines. These strategies could be advanced readily into clinical trials.