Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease

Abstract

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.

FIG. 1.

Association of HLA class I alleles and sequence variation in HIV-1 PR. (A) Bars indicate the percentages of subjects with viruses carrying mutations at each individual amino acid position within HIV-1 PR. Mutations and polymorphisms were defined as variations from the sequence of HIV-1 HXB2 PR (32). Amino acid substitutions showing correlations with defined HLA alleles are indicated by black bars. Drug-associated mutations are given in bold; major drug mutations are marked by an asterisk, and minor drug mutations are marked by a triangle. Newly defined drug mutations presumably involved in darunavir resistance (for preliminary data, see Johnson et al. [15]) are marked by a diamond. (B and C) HLA alleles associated positively (B) or negatively (C) with mutations at the indicated amino acid positions. (D) Previously defined CTL epitopes (19) are indicated by solid lines; dashed lines indicate predicted epitopes.

FIG. 2.

Cross-reactivity of CD8⁺ T cells recognizing the HLA-B44-restricted CTL epitope EW9 (amino acids 34 to 42). PBMC from HLA-B44-positive patients were stimulated with EEMNLPGRW (EW9; wild-type) and the variant peptides EEINLPGRW (EW9 M/I), EEMDLPGRW (EW9 N/D), EDINLPGRW (EW9 DI), and EDMNLPGRW (EW9 E/D; mutations are indicated in bold italics) and subsequently tested for reactivity by IFN-γ ELISPOT analysis. After stimulation, outgrowing CD8⁺-T-cell lines were analyzed for cross-reactivity with the peptides EW9, EW9 E/D, EW9 DI, EW9 M/I, EW9 N/D, EW9 P/S, and EW9 R/K, which carry the most frequent drug resistance mutations and/or polymorphisms occurring in the epitope. Peptides tested for cross-reactivity are given on the left side of each graph. Dashes indicate consensus with the wild-type peptide sequence; amino acid substitutions are given in capital letters. The numbers of spot-forming units (SFU) triggered by wild-type EW9, corresponding variant peptides, and the no-peptide control in CD8⁺ T cells are shown by differently shaded bars.

FIG. 3.

Cross-reactivity of CD8⁺ T cells recognizing the HLA-A2- and HLA-B62-restricted epitope KI10 (amino acids 45 to 54). PBMC from HLA-A2-positive patients (A to C), an HLA-B62-positive patient (D), and patients carrying both HLA-A2 and B62 (E and F) were stimulated with the peptide KMIGGIGGFI (wild type) and the variant peptides KIIGGIGGFI (KI10-I) and KMIGGIGGFV (KI10-V) comprising the drug mutations M46I and I54V, respectively. Outgrowing cell lines were evaluated for cross recognition of these peptides by IFN-γ ELISPOT analysis. Peptide sequences are given on the left side of each graph. Dashes indicate consensus with the HXB2 sequence; amino acid substitutions are given in capital letters. The numbers of spot-forming units (SFU) triggered by KI10, the corresponding variant peptides, and the negative control (no peptide) are shown by white, gray, and black bars. Restricting HLA alleles are indicated in the upper right corner of each graph.

FIG. 4.

Influence of drug-induced mutations and polymorphisms on recognition by CD8⁺ T cells. PBMC from HLA-matched HIV-1-positive patients were stimulated with the indicated wild-type or mutant peptides and tested for recognition of the peptides by IFN-γ ELISPOT analyses. After peptide stimulation, outgrowing CD8⁺-T-cell lines were analyzed for cross recognition by using the peptides indicated on the left side of each graph. Dashes indicate consensus with the wild-type peptide sequence; amino acid substitutions are given in capital letters. The numbers of spot-forming units (SFU) triggered by wild-type peptides, corresponding variant peptides, and the negative (no-peptide) control are shown by white and black bars. The position(s) of the mutated amino acid(s) is indicated in parentheses in the upper right corner of each graph.