In this study, we demonstrated that the group M consensus env gene (CON6) induced a higher number of cross-reactive and more potent T-cell responses to subtype A, B, and C viruses than any single subtype env immunogen alone. CON6 induced cross-reactive T-cell responses to all three subtypes, while single WT clade A, B, or C immunogens tended to induce subtype-specific T-cell immune responses and induced fewer responses that were cross-reactive with other subtypes. Interestingly, the polyvalent immunogen induced similar numbers of epitope responses compared to CON6. While we recognize that immunogenicity of an immunogen in mice does not necessarily predict immunogenicity in nonhuman primates or humans, these results provide proof of the concept that a consensus immunogen can induce broader and more potent T-cell responses across clades than individual strain-based immunogens. Thus, the theoretical advantages of the consensus approach as a T-cell immunogen have been experimentally validated in an animal model system. Further testing of centralized gene vaccines in nonhuman primates and humans, alone or in combination with subtype consensus or WT immunogens, are thus warranted.
While it was anticipated that intraclade responses would be more intense and more common than interclade responses, the performance of the CON6 immunogen with respect to both the breadth and magnitude of the elicited immune response was not expected. Previous studies have shown that a polyvalent immunogen induces broader immune responses than single immunogens (6
). In two studies, the T-cell responses were analyzed with peptide pools but not individual peptides (22
). In such an analysis, a few predominant T-cell responses might overshadow many weaker T-cell responses and the breadth of T-cell responses could not be evaluated on the basis of individual T-cell epitopes. In the current study, we defined T-cell epitopes using individual peptides from five overlapping Env peptide sets of three subtypes (A, B, and C) and found that the majority of the T-cell epitopes were recognized by responses elicited by the polyvalent immunogen. However, some T-cell epitopes were not detected in mice immunized with the polyvalent immunogen, while the same epitopes were detected in the animals immunized with individual subtype immunogens. The failure to induce some T-cell responses might be due to the lower doses of each immunogen in the polyvalent cocktail. Alternatively, there may have been interference between some of the epitopes among the multiple immunogens. Interference has been reported in some (17
) but not all (18
) studies of multivalent immunogens, thus leaving its potential impact on our results unclear. Still another explanation may be that the ancestral nature of CON6 (group M consensus and ancestor sequences being the same in this case) (13
) may have features that make it more immunogenic, while some of these immune responses have been lost in modern strains that may have accumulated immunorefractive mutations as HIV has evolved in people. While HLA and MHC molecules are very different in humans and mice, the processing of T-cell epitopes by enzymes may not be.
A recent study by Doria-Rose et al. showed that an ancestral subtype B env
immunogen (An1-envB) induced weak neutralizing activity in rabbits (10
). We have observed neutralizing activity induced with CON6 gp120 and gp140 proteins in guinea pigs similar to those induced by An1-envB (15
). The study by Doria-Rose et al. did not perform T-cell immune response analysis. In a more recent study, we have studied a second generation of group M consensus Env immunogen (CON-S) with shorter variable loops for its ability to induce neutralizing antibodies and discovered that a newer generation of group M consensus env
gene (CON-S) with shorter variable loops can induce broader neutralizing antibody responses to subtype B and C viruses than wild-type Env immunogens (27
). This study showed that the consensus gene approach has potential for inducing more broadly reactive neutralizing antibodies than current immunogens and was superior to wild-type subtype A, B, or C primary HIV-1 Envs. Further studies are needed to compare the neutralizing antibody responses induced by the group M consensus env
gene with a large panel of polyvalent Env
Few studies have been performed to map T-cell epitopes with single overlapping Env peptide sets in mice (5
). T-cell epitopes in each subtype WT Env protein used in this study have not been identified before. Our study is the first to systemically map the Env T-cell epitopes induced by consensus and multiple subtype Env immunogens with different subtype overlapping peptide sets. In the Los Alamos HIV Molecular Immunology Database, many CD4 epitopes were registered from the literature but only four CD8 epitopes were reported in mice (23
). In this study, we identified three new CD8 epitopes (C1, C7, and C8) in C57BL/6 mice and four new CD4 epitopes (B9/H3, B10/H4, H7, and C9) in three strains of mice, although we did not detect some CD4 epitopes that have been previously reported (23
The majority of T-cell epitopes identified in this study were in conserved envelope regions (Fig. ). These data differed from results from other studies in which Th epitopes were identified in envelope variable regions (9
). This discrepancy may be partly explained by the use of different immunogens, peptides, and detection methods of Th responses. However, Yusim et al. showed that HIV CTL epitopes were concentrated in relatively conserved regions when heterologous reference peptides were used for detection of CTL activity (39
). They also found that variable regions tend to have higher concentrations of amino acids that never serve as C-terminal anchor residues and to have reduced propensity for epitope cleavage and processing. The analysis suggests that variable regions that more readily accommodate changes in HIV-1 genomes tend to accumulate mutations that make them generally less immunogenic as well as less likely to be cross-recognized either within a subtype or among subtypes. Thus, the sequence analysis is in agreement with our observation.
Based on the crystal structure of gp120 protein (25
), we found most of the Th epitope targets (H1, H2, C3/B3, C4/B4, C5, B5, and B8) were in the nonexposed inner domain and only three Th epitope targets (C6, C7, and B7) were in the outer domain. Other Th epitope targets were in either the signal peptide region (C1) or the C1 region (B1 and B2/C2), for which no structural data were available. Therefore, our data do not support the hypothesis that exposed protein fragments are uniquely susceptible to proteolytic activity and are thus preferentially shuttled through the antigen-processing pathway in mice (5
T-cell responses were sensitive to amino acid substitutions between immunogens and peptides, and nearly all nonrecognized T-cell epitopes had amino acid substitutions in the epitope sequences in the vaccines relative to testing strains. This was also true for the subtype B immunogen (JRFL) when three heterologous subtype B Env overlapping peptide sets were used for epitope mapping. Since vaccinated individuals will not be exposed to, nor infected with, homologous viruses, a successful vaccine must be able to prevent or control heterologous viral infection. Therefore, our study with heterologous immunogens and screening peptides within the same subtype was designed to begin to mimic this viral diversity. The level of cross-reactivity induced by the subtype B immunogen to three heterologous subtype B Env-overlapping peptide sets was similar to what we observed with CON6 immunogen (Table ). Our sequence analysis showed that the differences in amino acid sequences between group M consensus and subtype Env proteins were the same as those between two viruses within the same subtype (Table ) (14
). Therefore, our experimental results were in agreement with our expectations based on sequence analysis and suggest that the group M consensus immunogens may be as efficient as a natural appropriately selected single subtype immunogen for induction of T-cell immune responses against viruses in regions where one subtype is predominant.
The inbred mouse is not an ideal model with which to study T-cell responses, since mice from the same strain have a common genetic background and mouse MHC molecules are different from HLA molecules in humans. The other major difference is that viruses constantly evolve under the immune selection pressure and accumulate many escape mutations along infection history in humans, while they are completely new in mice when used as immunogens. However, if the mechanisms for T-cell responses to HIV-1 immunogens in general are similar between humans and mice, more cross-reactive T-cell responses after immunization with group M consensus immunogens might be anticipated in humans, although the T-cell epitopes recognized may be different. To address this concern, a similar comparison experiment is warranted to determine if cross-subtype T-cell responses can also be induced in nonhuman primates and, if successful, in human clinical trials.