Differential Immunodominance Hierarchy of CD8 (cid:2) T-Cell Responses in HLA-B * 27:05- and -B * 27:02-Mediated Control of HIV-1 Infection

The well-characterized association between HLA-B * 27:05 and protection against HIV disease progression has been linked to immunodominant HLA-B * 27:05-restricted CD8 (cid:2) T-cell responses toward the conserved Gag KK10 (residues 263 to 272) and polymerase (Pol) KY9 (residues 901 to 909) epitopes. We studied the impact of the 3 amino acid differences between HLA-B * 27:05 and the closely related HLA-B * 27:02 on the HIV-specific CD8 (cid:2) T-cell response hierarchy and on immune control of HIV. Genetic epidemiological data indicate that both HLA-B * 27:02 and the CD8 (cid:2) T-cell response to a Nef epitope (residues 142 to 150 [VW9]), with Pol KY9 subdominant and Gag KK10 further subdominant. This selection was driven by structural differences in the F pocket, mediated by a polymorphism between these two HLA alleles at position 81. Analysis of autologous virus sequences showed that in HLA-B * 27:02-positive subjects, all three of these CD8 (cid:2) T-cell responses impose selection pressure on the virus, whereas in HLA-B * 27:05-positive subjects, there is no Nef VW9-mediated selection pressure. These studies demonstrate that HLA-B * 27:02 mediates protection against HIV disease progression that is at least as strong as or stronger than that mediated by HLA-B * 27:05. In combination with the protective Gag KK10 and Pol KY9 CD8 (cid:2) T-cell responses that dominate HIV-specific CD8 (cid:2) T-cell activity in HLA-B * 27:05-positive subjects, a Nef VW9-specific response is additionally present and immunodominant in HLA-B * 27:02-positive subjects, mediated through a polymorphism at residue 81 in the F pocket, that contributes to selection pressure against HIV. T cell responses restricted by those alleles. We focus here on HLA-B * 27:05, one of the best-characterized protective HLA molecules, and the closely related HLA-B * 27:02, which differs by only 3 amino acids and which has not been well studied in relation to control of HIV infection. We show that HLA-B * 27:02 is also protective against HIV disease progression, but the CD8 (cid:2) T-cell immunodominance hierarchy of HLA-B * 27:02 differs strikingly from that of HLA-B * 27:05. These findings indicate that the immunodominant HLA-B * 27:02-restricted Nef response adds to protection mediated by the Gag and Pol specificities that dominate anti-HIV CD8 (cid:2) T-cell activity in HLA-B * 27:05-positive subjects. Sequencing was undertaken using the BigDye Ready Reaction Terminator Mix (V3) (Applied Biosystems UK) analyzed using Sequencher v4.8 (Gene Codes Corporation) and manually aligned using Se_Al software. IFN- (cid:3) ELISpot assays. We tested ex vivo peripheral blood mononuclear cells (PBMCs) against a panel of 410 overlapping peptides (OLPs) spanning the entire HIV-1 proteome to screen for IFN- (cid:2) ELISpot responses (14). We additionally tested putative optimal epitopes by ELISpot assay using ex vivo PBMCs from HLA-B * 27:02- and HLA-B * 27:05-positive subjects. Cell staining and flow cytometry. Cell staining from cryopreserved PBMCs was undertaken using anti-CD3-Pacific orange (Invitrogen), anti-CD8-Alexa Fluor 700 (BD Biosciences), and HLA-B * 27 tetramers conjugated to phycoerythrin (PE). Dead cells were gated out using a LIVE/DEAD viability kit (Invitrogen). assay. Peptide affinity to HLA class molecules was determined using a luminescent oxygen channeling immunoassay Briefly, peptides were dissolved in phosphate-buffered saline (PBS)–0.1% Lutrol F68 by sonication for 10 Peptides were titrated in 384-well microplates using a Microlab STAR liquid handling robot (Hamilton Robotics). Recombinant, denatured HLA-C heavy chain (HC) was diluted into PBS–0.1% Lutrol F68/100 mM Tris/maleate (pH 6.6) containing prefolded, recombinant beta-2 microglobulin ( (cid:4) 2 m) on ice. The HC- (cid:4) 2 m mix was added 1:1 to the peptide titrations and incubated for 48 h at 18°C to allow peptide–MHC-I complex folding. After complex folding, samples were transferred to 384-well