Human immunodeficiency virus type 1 (HIV-1) is able to evade the host cytotoxic T-lymphocyte (CTL)response through a variety of escape avenues. Epitopes that are presented to CTLs are first processed in the presenting cell in several steps, including proteasomal cleavage, transport to the endoplasmic reticulum, binding by the HLA molecule, and finally presentation to the T-cell receptor. An understanding of the potential of the virus to escape CTL responses can aid in designing an effective vaccine. To investigate such a potential, we analyzed HIV-1 gag from 468 HIV-1-positive Kenyan women by using several bioinformatic approaches that allowed the identification of positively selected amino acids in the HIV-1 gag region and study of the effects that these mutations could have on the various stages of antigen processing. Correlations between positively selected residues and mean CD4 counts also allowed study of the effect of mutation on HIV disease progression. A number of mutations that could create or destroy proteasomal cleavage sites or reduce binding affinity of the transport antigen processing protein, effectively hindering epitope presentation, were identified. Many mutations correlated with the presence of specific HLA alleles and with lower or higher CD4 counts. For instance, the mutation V190I in subtype A1-infected individuals is associated with HLA-B*5802 (P ‫؍‬ 4.73 ؋ 10 ؊4 ), a rapid-progression allele according to other studies, and also to a decreased mean CD4 count (P ‫؍‬ 0.019). Thus, V190I is a possible HLA escape mutant. This method classifies many positively selected mutations across the entire gag region according to their potential for immune escape and their effect on disease progression.

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HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate. While T242N was ubiquitous in clade D HLA-B*57 ؉ subjects, this mutation was rare (15%) in clade A1. Instead, P243T and I247L were selected by clade A1-infected HLA-B*57 subjects but not by HLA-B*5801 ؉ subjects. Our data suggest that clade A1 consensus proline at Gag residue 243 might represent an inherent block to T242N escape in clade A1. We confirmed immunologically that P243T and I247L likely represent escape mutations. HLA-B*57 evolution also differed between clades in the KF11 and IW9 epitopes. A better understanding of clade-specific evolution is important for the development of HIV vaccines in regions with multiple clades.

We examined the effect of HLA class I haplotypes on HIV-1 seroconversion and disease progression in the Pumwani sex worker cohort. This study included 595 HIV-1 positive patients and 176 HIV negative individuals. HLA-A, -B, and -C were typed to 4-digit resolution using sequence-based typing method. HLA class I haplotype frequencies were estimated using PyPop 32-0.6.0. The influence of haplotypes on time to seroconversion and CD4+ T cell decline to <200 cells/mm3 were analyzed by Kaplan-Meier analysis using SPSS 13.0. Before corrections for multiple comparisons, three 2-loci haplotypes were significantly associated with faster seroconversion, including A*23∶01-C*02∶02 (p = 0.014, log rank(LR) = 6.06, false-discovery rate (FDR) = 0.056), B*42∶01-C*17∶01 (p = 0.01, LR = 6.60, FDR = 0.08) and B*07∶02-C*07∶02 (p = 0.013, LR = 6.14, FDR = 0.069). Two A*74∶01 containing haplotypes, A*74∶01-B*15∶03 (p = 0.047, LR = 3.942, FDR = 0.068) and A*74∶01-B*15∶03-C*02∶02 (p = 0.045, LR = 4.01, FDR = 0.072) and B*14∶02-C*08∶02 (p = 0.021, LR = 5.36, FDR = 0.056) were associated with slower disease progression. Five haplotypes, including A*30∶02-B*45∶01 (p = 0.0008, LR = 11.183, FDR = 0.013), A*30∶02-C*16∶01 (p = 0.015, LR = 5.97, FDR = 0.048), B*53∶01-C*04∶01 (p = 0.010, LR = 6.61, FDR = 0.08), B*15∶10-C*03∶04 (p = 0.031, LR = 4.65, FDR = 0.062), and B*58∶01-C*03∶02 (p = 0.037, LR = 4.35, FDR = 0.066) were associated with faster progression to AIDS. After FDR corrections, only the associations of A*30∶02-B*45∶01 and A*30∶02-C*16∶01 with faster disease progression remained significant. Cox regression and deconstructed Kaplan-Meier survival analysis showed that the associations of haplotypes of A*23∶01-C*02∶02, B*07∶02-C*07∶02, A*74∶01-B*15∶03, A*74∶01-B*15∶03-C*02∶02, B*14∶02-C*08∶02 and B*58∶01-C*03∶02 with differential seroconversion or disease progression are due to the dominant effect of a single allele within the haplotypes. The true haplotype effect was observed with A*30∶02-B*45∶01, A*30∶02-C*16∶02, B*53∶01-C*04∶01 B*15∶10-C*03∶04, and B*42∶01-C*17∶01. In these cases, the presence of both alleles accelerated the disease progression or seroconversion than any of the single allele within the haplotypes. Our study showed that the true effects of HLA class I haplotypes on HIV seroconversion and disease progression exist and the associations of HLA class I haplotype can also be due to the dominant effect of a single allele within the haplotype.

Objectives The p1 region of HIV-1 gag contains the frameshift stem-loop, gag–pol transframe and a protease cleavage site that are crucial for viral assembly, replication and infectivity. Identifying and characterizing CD8+ epitopes that are under host immune selection in this region will help in designing effective vaccines for HIV-1. Design An approach combining bioinformatical analysis and interferon gamma enzyme-linked immunosorbent spot (ELISPOT) assays is used to identify and characterize the epitopes. Potential human leukocyte antigen (HLA)-restricted epitopes were identified by correlating the positively selected mutations with host HLA alleles. Methods ELISPOT analysis with overlapping peptides was used to confirm and characterize the epitopes. Results Four positively selected residues were significantly associated with HLA class I alleles, including HLA B*1302 (K4R, P=0.0008 and I5L, P=0.0108), A*7401 (S9N, P=0.0002) and A*30 genotypes (P7S, P=0.009), suggesting epitopes restricted by these alleles are present in this region. ELISPOT analysis with patient peripheral blood mononuclear cells identified 7 novel epitopes restricted by the 3 alleles. Two types of epitopes were observed in this region based on the ELISPOT responses, Type I: the positively selected variation does not affect CD8+ T-cell responses; and Type II: the CD8+ T-cell responses are determined by the epitope variants. Conclusion We identified and characterized seven novel CD8+ epitopes in the p1 spacer protein region. Classifying the effects of positively selected variants on CD8+ T-cell responses will help in designing effective vaccines for HIV-1.

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