One requirement of a highly effective AIDS vaccine is the need to induce both neutralizing antibodies and cellular immunity to the many strains of HIV-1 that circulate throughout the world. In this report, we have evaluated the ability of plasmid DNA vaccines to elicit immune responses to multiple gene products of HIV-1 from alternative clades of virus. The goal was to elicit both antibody and T-cell responses against various HIV genes from these different clades. Env, Gag, Pol, and Nef were chosen as targets because they represent the major expressed proteins during viral infection. A mutant Env with deletions in the cleavage site, fusion domain, and a region between the heptad repeats was used for its ability to elicit a more potent humoral immune response while retaining its ability to stimulate Env-specific CTLs (7
A variety of previous studies have shown that CTLs contribute to the control of viremia and protect against the progression of HIV disease (6
). Processed forms of Gag, Pol, Nef, and Env presented on class I major histocompatibility complex antigens can serve as the targets of CTLs that recognize and lyse HIV-1 infected cells, in this way contributing to the efficacy of a preventive vaccine. It is hoped that if the T-cell response is sufficiently robust, these cells will kill HIV-infected cells before the virus can replicate and establish a reservoir of infection in vivo. For a globally effective vaccine, it will be necessary to elicit CTL that react with strains from multiple clades. Though there may be some cross-clade reactivity after immunization with a single clade (e.g., see reference 13
), there is also evidence of disparities in such immune responses (e.g., see reference 9
). It therefore appeared desirable to include representatives of the major classes of virus in a DNA vaccine to induce cross-clade immunity. However, the main concern of such a cocktail is whether it will cause interference between gene-specific immune responses. Interference among immune responses to various viral genes has been seen previously in murine HIV immunization studies (15
). Recently, studies of modifications to HIV DNA vaccines, including different combinations of viral genes, altered RNA structure or codon usage, and/or stimulatory cytokine genes, have shown more encouraging results in mice (41
). More importantly, some approaches have shown promise in challenge studies using nonhuman primates (1
), though complete protection against infection has been difficult to achieve. Additional modifications were therefore incorporated in this study in an attempt to improve efficacy.
When the immune responses to different combinations of Env and Gag-Pol-Nef were compared, there was no decrease in the humoral and cellular response to clade B mutant Env plasmid and Gag-Pol-Nef plasmids when mixed compared with the responses to the two plasmids individually (Fig. ). When the complexity was increased to four components, including gp145ΔCFI from three clades and Gag-Pol-Nef from clade B, there was no interference with the humoral response to B-Env while the immune response to other clades was enhanced.
When the complexity of the vaccine was increased to six components, ABC(×6), containing the same Env-gp145ΔCFI from different clades as in group ABC(×4) plus the Gag-Pol-Nef fusion protein from clades A and C, minor differences in immunogenicity were seen. Analysis of the Gag response showed that ABC(×4) elicited CD4+ and CD8+ responses to clades A and B, while ABC(×6) improved the response to clade C Gag peptides. The lack of CD4+ and CD8+ responses to clade C Gag in ABC(×4) probably is due to the absence of clades A and C Gag; however, ABC(×4) containing only clade B Gag could induce both CD4+ and CD8+ responses to clade A Gag even though it shares only 85% homology in amino acid sequence. This result suggested that clades A and B Gag share some common CD4+ and CD8+ epitopes but differ more substantially from clade C Gag in mice. In contrast, the CD4+ and CD8+ responses against Env between ABC(×4) and ABC(×6) were similar: both groups elicited comparable CD4+ responses against all three clades and generated similar CD8+ responses against clade B Env. ABC(×6) also induced a significant CD8+ response to Env from clade A. Since both ABC(×4) and ABC(×6) contained the same combination of Env from clades A, B, and C, the similar CD4+ and CD8+ responses against Env from the two groups were not unexpected.
For Pol responses, both groups demonstrated CD4+
responses against Pol from clade B. The ABC(×4) group elicited a CD4+
response to both sets of Pol peptides, while ABC(×6) stimulated a CD4+
response only against one of the two Pol peptide pools (Table ). Both groups induced CD8+
responses to the first half of the clade B Pol (Fig. , left panel). For Nef, only the ABC(×4) group elicited a CD4+
response against Nef from clade B (Table ). The poor anti-Nef response also may be due to the inability of BALB/c mice to recognize Nef epitopes, as other groups have reported that Nef is highly immunogenic in other strains of mice (15
In addition, we attempted to determine whether CD4+ and CD8+ T-cell responses against multigenes would affect humoral responses. There was no significant difference among different groups in ELISA titers (summarized in Table ). All the groups showed similar antibody titers to Env protein from clades A, B, and C (Fig. ). These data suggested that there was no interference among different clades of Env in antibody response. Equally importantly, there was no interference among various viral genes between humoral and cellular responses.
In summary, the ABC(×4) vaccine regimen was able to induce substantial and balanced CD4+
T-cell responses to the viral antigens from different clades. Though ABC(×6) induced a comparable response, the lower complexity of ABC(×4) suggests that it is a better candidate for development for clinical production. The more complex combination, ABC(×6), could also be more variable because more epitopes are included, and the larger number of components could complicate production and quality control that might also affect immune responses. These factors suggest that more complexity does not always yield incremental immunity, as with ABC(×6). Though there was no significant loss of immunogenicity with the vaccine studied here, this complexity has been problematic in previous studies (15
). For this reason, immune analyses, such as those performed in this study, are needed to address these issues and facilitate the development of safe and effective vaccines. The results here suggest that a multigene HIV-1 DNA vaccine is feasible because the immune responses to individual genes do not cause interference when combined with one another. Because mouse serum is difficult to evaluate for neutralizing antibody activity due to its high background in neutralization assays, future studies in other animal species will address this issue. As the HIV-1 pandemic continues to grow, virus variability becomes increasingly problematic. Though a few subtypes of HIV-1 predominate in different regions of the world, a rising number of recombinant strains have been reported lately (18
). Such viruses continually mutate and escape (2
) during different stages of infection. A multiepitope and multiclade immune response should help to reduce the likelihood of viral escape. The data presented in this report may therefore help to guide the development of improved vaccines against diverse strains of HIV.