In contrast, vaccination with either a single clade B′-derived vaccine or the mixture of multiple vaccines was unable to stimulate a substantial T cell immune response against those fully conservative epitopes; instead, these strategies activated predominant T cell immunity against less conservative or non-conservative epitopes, such as epitopes residing in p24 amino acids 65–75 and 365–384, gp160 amino acids 9–19 and 816–826. One epitope, consensus C Env 93, does not follow this rule. Though this epitope is non-conservative in its amino acid sequences, heterologous vaccination primed higher T cell responses than conventional strategies. We speculate that either this epitope mainly presents in C vaccine (boosting vaccine) or there exist conservative immune epitopes (amino acids sequences may vary but contain conservative antigen specificity) at these sites, just as observed in previous report .
Strategies to raise immune responses preferentially targeting conservative immune epitopes remain to be explored though it has been recognized that an effective HIV-1 vaccine should be capable of eliciting significant cross-reactive immune responses. Early studies using peptides derived from variable region of gp120 have demonstrated that CD8 T cells primed by one peptide are able to recognize or be boosted by homologous determinants containing variant sequence from distinct isolates [7,8]. These studies suggested that it is feasible to prime with one vaccine and to boost with another for raising cross-reactive T cells. Indeed, a recent report on HIV-1 superinfection showed that only one out of nine conserved epitopes developed significant CD8 T cell response (> 1000 SFCs/106 peripheral blood mononuclear cells) during HIV-1 virus A infection whereas an additional six conserved epitopes developed significant CD8 T cells only after infection by the second virus (virus B) . This finding emphasizes the importance of the immune system ‘seeing’ different sequences to develop cross-reactive T cells against conserved epitopes. However, the sequential HIV-1 infection by virus A and B may have severely damaged the immune system as the developed cross-reactive T cell immune responses finally failed to contain HIV-1 replication. Cross-clade HIV-specific T cell immune responses have been observed in Nairobi sex workers who remained seronegative despite frequent exposure to HIV .
We have shown that the heterologous vaccination strategy preferentially enhances immune responses against the most conserved epitopes among various HIV clades, less to epitopes that are less conserved and much less to non-conservative epitopes (Fig. 4). The strategy differs from that tested in mice or primate models in which multiple envelopes from different clades were used in combination for vaccination [17–18]. In these models, more immunogenic epitopes but not more conservative epitopes mounted higher T cell immune responses. Actually, our data show that vaccination with the mixture of multiple vaccines stimulated broader T cell immunity than that with a single vaccine, indicating that this strategy may be more effective in areas where there are multiple HIV-1 clades circulating. However, it did fail to mount significant T cell immunity targeting at conserved epitopes.
To determine whether T cell epitopes raising predominant T cell immune responses in heterologous vaccination are also conserved among other primary isolates, the conservation of one epitope, SPRTLNAW at p24 amino acids 16–23, was examined among different HIV-1 clades in the sequences from Los Alamos database. All primary isolate sequences for CRF07_BC and CRF08_BC were selected and randomly selected sequences for B and C clades (approximately every 10 sequences) and for A/E recombinants (approximately every four sequences). Sequence alignments showed that the epitope SPRTLNAW is highly conserved, appearing in 229 out of 249 sequences (92%) in different clades: 81 out of 93 for B clade (87%), 72 out of 73 for B/C recombinants (98.6%), 61 out of 67 for C clade (91%) and 15 out of 16 for A/E recombinant (93.7%). The biological relevancy of this conservative epitope was examined using the Los Alamos Immune Database, which showed that it is a B*57-restricted epitope. B*57 is an allele closely linked to slow progression during HIV natural infection [19–21]. Importantly, a previous report  demonstrated that the capacity to restrain HIV replication for B*57-positive individuals relied on whether the T cell immunity focused on the conservative epitopes; long-term non-progressors mounted highly focused immune responses against conserved epitopes whereas progressors were less focused. This may illustrate the importance of stimulating immune responses directing at conserved immune determinant.
Interestingly, our data showed that the order for conservative epitopes presenting in heterologous vaccination does influence the magnitude of its specific T cell immunity. The conservative epitope presenting in both DNA priming and vaccinia boosting, such as the epitope residing in gp160 amino acids 545–553, elicited much stronger T cell immunity than those presenting twice in DNA vaccinations, such as epitopes locating at p24 amino acids 65–75 and gp160 amino acids 816–826. These data indicate that a strong boosting does but not the second DNA immunization is critical for effective enhancement of the T cell immunity raised in the previous vaccination. Therefore, we speculate that we will observe a different pattern of dominant T cell epitopes, though all are conserved, by changing the order of vaccines for heterologous vaccination. Furthermore, by introducing a strong boosting (such as administrating recombinant adenoviral vectored vaccine) at the second inoculation, we may overcome the drawback of our current vaccination modality and further increase the T cell immunity against the most conservative epitopes. These hypotheses should be explored further in future experiments with larger groups of mice. In addition, this modality should be tested in a primate model with strong boosts to increase the immunogenicity.
The limited availability of peptide sets gave rise to several limitations in this study. The first is that immune responses were assessed using a consensus clade B and clade C peptide set rather than peptide sets corresponding to the vaccine sequences used. In the light of the sequence differences between B′ and C sequences used for vaccination and the clade B and C consensus sequences (the pairwise similarities are 95% between B′ and consensus B Gag, 91% between B′ and consensus B Env, 91% between our C and consensus C Gag, and 87% between our C and consensus C Env), the consensus peptide set will preferentially identify T cell responses that are cross-reactive between the sequences used for vaccination and the peptide sequences, thereby biasing the detection of responses towards conserved areas of HIV-1; In other words, we may have underestimated the differences in immunity primed by different immunization strategies. The second limitation is that clade E-derived peptides were not included for assessing immune responses yet clade E-derived vaccine was included both in heterologous vaccination and in vaccination with the mixture of multiple vaccines; consequently, we might also have underestimated the immune responses specific for clade E sequences. Overall, our current results may have significantly underestimated the immune responses raised by different immunization strategies, particularly by the use of a mixture of multiple vaccines; this would emphasize the breadth and magnitude of immunity primed by this strategy.
We would like to acknowledge Danli Duan, Yanming Wan, Lianxin Liu and Hong Peng for their kind technical support.
1. Gaschen B, Taylor J, Yusim K, Foley B, Gao F, Lang D, et al
. Diversity considerations in HIV-1 vaccine selection. Science 2002; 296:2354–2360.
2. Brehm M, Pinto A, Daniels K, Schneck J, Welsh R, Selin L. T cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens. Nat Immunol 2002; 3:627–634.
3. Letvin N, Huang Y, Chakrabarti B, Xu L, Seaman M, Beaudry K, et al
. Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys. J Virol 2004; 78:7490–7497.
4. Su L, Graf M, Zhang Y, von Briesen H, Xing H, Kostler J, et al
. Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B′) recombinant strain in China. J Virol 2000; 74:11367–11376.
5. Graf M, Shao Y, Zhao Q, Seidl T, Kostler J, Wolf H, et al
. Cloning and characterization of a virtually full-length HIV type 1genome from a subtype B′-Thai strain representing the most prevalent B-clade isolate in China. AIDS Res Hum Retroviruses 1998; 14:285–288.
6. Piyasirisilp S, McCutchan F, Carr J, Sanders-Buell E, Liu W, Chen J, et al
. A recent outbreak of human immunodeficiency virus type 1 infection in southern China was initiated by two highly homogeneous, geographically separated strains, circulating recombinant form AE and a novel BC recombinant. J Virol 2000; 74:11286–11295.
7. Takahashi H, Nakagawa Y, Pendleton C, Houghten R, Yokomuro K, Germain R, et al
. Induction of broadly cross-reactive cytotoxic T cells recognizing an HIV-1 envelope determinant. Science 1992; 255:333–336.
8. Casement K, Nehete P, Arlinghaus R, Sastry K. Cross-reactive cytotoxic T lymphocytes induced by V3 loop synthetic peptides from different strains of human immunodeficiency virus type 1. Virology 1995; 211:261–267.
9. Altfeld M, Allen T, Yu X, Johnston M, Agrawal D, Korber B, et al
. HIV-1 superinfection despite broad CD8 T-cell responses containing replication of the primary virus. Nature 2002; 420:434–439.
10. Rowland-Jones S, Dong T, Fowke K, Kimani J, Krausa P, Newell H, et al
. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J Clin Invest 1998; 102:1758–1765.
11. Barouch D, Santra S, Schmitz J, Kuroda M, Fu T, Wagner W, et al
. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science 2000; 290:486–492.
12. Amara R, Villinger F, Altman J, Lydy S, O'Neil S, Staprans S, et al
. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 2001; 292:69–74.
13. Edwards B, Bansal A, Sabbaj S, Bakari J, Mulligan M, Goepfert P. Magnitude of functional CD8+ T-cell responses to the Gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma. J Virol 2002; 76:2298–2305.
14. Amara R, Smith J, Staprans S, Montefiori D, Villinger F, Altman J, et al
. Critical role for Env as well as Gag–Pol in control of a simian–human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine. J Virol 2002; 76:6138–6146.
15. Horton H, Vogel T, Carter D, Vielhuber K, Fuller D, Shipley T, et al
. Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239
. J Virol 2002; 76:7187–7202.
16. Verrier B, Le Grand R, Ataman-Onal Y, Terrat C, Guillon C, Durand P, et al
. Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08. DNA Cell Biol 2002; 21:653–658.
17. Kong W, Huang Y, Yang Z, Chakrabarti B, Moodie Z, Nabel G. Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines. J Virol 2003; 77:12764–12772.
18. Seaman M, Xu L, Beaudry K, Martin K, Beddall M, Miura A, et al
. Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys. J Virol 2005; 79:2956–2963.
19. Kaslow R, Carrington M, Apple R, Park L, Munoz A, Saah A, et al
. Influence of combinations of human major histocompatibility complex genes on the course of HIV-1 infection. Nat Med 1996; 2:405–411.
20. Goulder P, Bunce M, Krausa P, McIntyre K, Crowley S, Morgan B, et al
. Novel, cross-restricted, conserved, and immunodominant cytotoxic T lymphocyte epitopes in slow progressors in HIV type 1 infection. AIDS Res Hum Retroviruses 1996; 12:1691–1698.
21. Trachtenberg E, Korber B, Sollars C, Kepler T, Hraber P, Hayes E, et al
. Advantage of a rare HLA supertype in HIV disease progression. Nat Med 2003; 9:928–935.
22. Migueles S, Sabbaghian M, Shupert W, Bettinotti M, Marincola F, Martino L, et al
. HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors. Proc Natl Acad Sci USA 2000; 97:2709–2714.