To date, 14 optimal epitopes within p17 Gag and 23 optimal epitopes within p24 Gag have been defined; however, no optimal CTL epitopes have been defined within p15 Gag . We therefore focused on the three immunodominant regions within p15 Gag and determined the optimal epitopes and their HLA class I restriction within these regions.
The responses to the p15-24 peptide in individuals AC-05, AC-39, AC-55 and 013572j were found to be restricted by HLA-B60 (HLA-B*4001) (Fig. 2a), an allele that is expressed in about 20% of the Caucasian population and is even more prevalent in Asian populations [26,27]. The optimal epitope was identified as KELYPLTSL (amino acid position p15 118–126) and exactly fits the published peptide-binding motif for HLA-B60 , with a glutamic acid at position 2 and a leucine at the C terminus. Four additional individuals recognized the p15-24 peptide but did not express HLA-B60, suggesting that additional epitopes restricted by other HLA class I alleles exist within this region. The precise epitopes targeted by these responses have not yet been defined.
A second CTL epitope appeared to be located in the overlapping region of peptides p15-8 and p15-9. The 9-mer peptide CRAPRKKGC (amino acid position p15 42–50) was defined as the optimal epitope, using serial dilutions of peptide truncations (Fig. 2b). This epitope was shown to be restricted by HLA-B14, which was expressed by both persons who recognized this epitope. The published peptide binding motif for B14 contains an arginine at both positions 2 and 5, and a leucine at the C terminus, and thus the novel epitope within p15 Gag matched sufficiently well with this motif. Overall, 33.3% (2/6) of individuals expressing HLA-B14 tested in the study recognized this novel epitope. This epitope is located within the structurally important linker region between the two zinc fingers in NCp7.
The most frequently recognized response among all subjects was found to be to the p15-14 peptide, which includes the highly conserved protease Gag cleavage site between p7 and p1 . Overall 25% (14/57) of studied individuals had detectable CD8 T-cell responses directed against this peptide (Fig. 1). An HLA-A*0201-restricted epitope (FLGKIWPSYK, amino acid position 70–79) was defined within this peptide (Fig. 2c). This epitope was recognized in 13 out of 24 (54%) individuals expressing the corresponding HLA class I allele.
Taken together, three novel CTL epitopes within immunodominant regions of p15 Gag were characterized in this study. All epitopes map to important functional sites within p15 Gag and were recognized frequently (33–100%) by individuals expressing the restricting HLA class I alleles, suggesting a strong immunogenecity of these epitopes.
Many CTL responses within p17 Gag and p24 Gag have been described so far, but little is known about the contribution of cellular immune responses directed against p15 Gag to the total Gag-specific responses. We therefore screened CD8 T-cell responses against HIV-1 p15 Gag in 57 HIV-1-infected individuals by using overlapping peptides spanning the entire p15 Gag sequence of HIV-1. The data showed that CD8 T-cell responses directed against p15 Gag were clustered within three immunodominant regions and contributed importantly to the CD8 T-cell responses against HIV-1 Gag. In addition, to our knowledge the first three optimal CTL epitopes within HIV-1 p15 Gag are reported in this study.
HIV-1-specific CTL responses directed against Gag have been reported to play an important role in control of HIV-1 infection. CD8 T-cell responses against an HLA-A2-restricted epitope in p17 Gag have been inversely correlated to viral load , as were p24 Gag-specific lymph-proliferative T-helper cell responses . However, p15 Gag, that constitutes 27% of the total length of the HIV-1 Gag protein, has been neglected so far in the analysis of HIV-1-specific immune responses. In this study we show that p15 Gag is frequently targeted by CD8 T cells during HIV-1 infection, with 46% of tested individuals having p15 Gag-specific responses. Adapted to length, p15 Gag was targeted even more frequently than p24 Gag. P15 Gag-specific responses were lower in magnitude than p17 Gag and p24 Gag-specific responses, but contributed up to 17% of total Gag-specific CD8 T-cell responses in infected individuals. In addition, four individuals (9%) had responses only against p15 Gag, but not against p17 Gag and p24 Gag. Taken together, Gag-specific CD8 T-cell responses would have been underestimated without assessment of p15 Gag-specific responses in the study individuals. This is of significance, as HIV-1 Gag is a component of several HIV-1 vaccines and immunogens, and p15 Gag-specific immune responses should be included in the assessment of immune responses induced by these.
In order to determine the HLA class I allele restricting the individual CD8 T-cell responses and to assess the impact of CTL-mediated immune pressure on sequence variation within the virus, the minimal epitopes inducing the CTL responses must be defined. We therefore fine-mapped the optimal CTL epitopes located within the three preferentially targeted regions of p15 Gag and determined their restricting HLA class I alleles. All three novel epitopes mapped to important functional sites within p15 Gag. The HLA-B14-restricted CTL epitope (CRAPRKKGC) covered the entire spatial structure between the two zinc fingers in NCp7, which folds into a globular structure and brings the two zinc fingers into close proximity . This globular structure has been shown to be crucial for viral infectivity and virion morphogenesis, as a single mutation in the linker region, which abolishes the globular structure, yields viruses that are non-infectious and have an immature morphology [29,30]. Not surprisingly, this region is highly conserved within the 64 clade B strains reported in the Los Alamos Database . However, CKAPRKKGC (R42K), CRAPRKR GC (K48R) and CKAPRKRGC (R42K/K48R) variants of this epitope are observed in 8%, 19% and 5% of reported clade B strains, respectively . We therefore tested the corresponding variant peptides to determine if these variants lead to the loss of CD8 T-cell recognition, using a CTL clone specific to the CRAPRKKGC peptide. All three variant peptides induced comparable lysis of labeled target cells by CTL, and required similar peptide concentrations for half-maximal lysis (data not shown) suggesting that CTL directed against this epitope demonstrate a large degree of cross-recognition of epitope variants, as described previously for other HLA-B14-restricted epitopes .
The HLA-B60-restricted CTL epitope KELYPLTSL overlaps another important functional region, in which it covers five amino acids of the eight amino acid-long second Vpr binding site in p6 Gag. Interestingly, all four individuals expressing HLA-B60 alleles included in this study recognized this epitope, demonstrating a high immunogenecity of this epitope in the context of B60. This epitope matched exactly the peptide binding motif described for HLA-B60 . In contrast, the most frequently recognized HLA-A*0201-restricted CTL epitope FLGKIWPSYK does not fit the described binding motif for HLA-A*0201 , as it has a lysine at the C-terminal end of the epitope instead of a leucin, isoleucin or valine. It was therefore not predicted as an HLA-A2-restricted epitope within HIV-1 using the HLA-A2 supertype motif . HLA-A*0201-restricted CTL epitopes that do not fit the described binding motif have also been described for other viral infections such as hepatitis C virus . These findings emphasize the importance of a comprehensive approach using overlapping peptides to characterize HIV-1-specific immune responses and to identify novel CTL epitopes, as these epitopes may otherwise be missed by approaches using epitope prediction based on peptide-binding motifs.
In conclusion, p15 Gag is as frequently targeted by T cells during HIV-1 infection as p17 Gag and p24 Gag, with 46% of individuals having p15-specific CD8 T-cell responses. Overall, p15 Gag-specific responses are lower in magnitude than p17 Gag- and p24 Gag-specific responses, but contributed an average of 17% of total Gag-specific CD8 T-cell responses in infected individuals. This report also includes the description of the first three optimal CTL epitopes defined within p15 Gag, including an HLA-A2-restricted epitope. HIV-1 p15 Gag should be included in the assessment of HIV-1-specific cellular immune responses and may represent an important target for future multicomponent HIV-1 vaccines.
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The HIV Study Collaboration
N. Basgoz, G. K. Robbins, B. Davis and S. A. Kalams, Partners AIDS Research Center and Infectious Disease Division, Massachusetts General Hospital and Harvard Medical School, Boston, USA. P. E. Sax, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. S. Boswell and D. S. Cohen, Fenway Community Health Center, Boston, Massachusetts, USA.