Nef-specific CD8+ T cell responses contribute to HIV-1 immune control

Abstract

Recent studies in the SIV-macaque model of HIV infection suggest that Nef-specific CD8+ T-cell responses may mediate highly effective immune control of viraemia. In HIV infection Nef recognition dominates in acute infection, but in large cohort studies of chronically infected subjects, breadth of T cell responses to Nef has not been correlated with significant viraemic control. Improved disease outcomes have instead been associated with targeting Gag and, in some cases, Pol. However analyses of the breadth of Nef-specific T cell responses have been confounded by the extreme immunogenicity and multiple epitope overlap within the central regions of Nef, making discrimination of distinct responses impossible via IFN-gamma ELISPOT assays. Thus an alternative approach to assess Nef as an immune target is needed. Here, we show in a cohort of >700 individuals with chronic C-clade infection that >50% of HLA-B-selected polymorphisms within Nef are associated with a predicted fitness cost to the virus, and that HLA-B alleles that successfully drive selection within Nef are those linked with lower viral loads. Furthermore, the specific CD8+ T cell epitopes that are restricted by protective HLA Class I alleles correspond substantially to effective SIV-specific epitopes in Nef. Distinguishing such individual HIV-specific responses within Nef requires specific peptide-MHC I tetramers. Overall, these data suggest that CD8+ T cell targeting of certain specific Nef epitopes contributes to HIV suppression. These data suggest that a re-evaluation of the potential use of Nef in HIV T-cell vaccine candidates would be justified.

Figure 1. Variation in C-clade Nef sequences, and relationship between amino acid variability and presence of HLA-class I selection pressure.

[A] Nef consensus sequence (derived from 739 C-clade sequences from Southern African patients) plotted against Shannon entropy score. Residues at which there is an association with HLA-Class I expression are shown in grey. As previously observed, the sequence is most highly conserved in the central portion of the protein (residues 66-148) [8].. [B] Entropy scores of residues associated with HLA-class I expression vs. those with no HLA association, showing significantly higher variability at sites at which HLA selection operates, particularly in the central conserved region. P-value by Mann Whitney U test.

Figure 2. Relationship between number of HLA associations with Nef sequence polymorphisms and median viral load for subjects expressing that allele, for HLA-A, -B and -C alleles.

Data from lineage-corrected analysis of 739 C-clade Nef sequences. P-values by Mann–Whitney U test.

Figure 3. Confirmation of Nef-KY11 (KEKGGLEGLIY) and Nef-QY9 (QEILDLWVY) as novel HLA-B*44: 03 restricted

CD8 T cell epitopes using HLA-Class I tetramers. HLA-B*44: 03 tetramers were loaded with [A] Nef-KY11 and [B] Nef-QY9 optimal peptide and stained against donor PBMC’s from a C-clade infected B*44: 03 positive individual, subject R129 HLA-A*23: 01/30:04, B*08: 01/44:03, C*03:04/04:01. The number shown above each gate is the percentage of total lymphocytes that are tetramer-specific (tetramer positive cells expressed as a percentage of CD8+ cells were 1.48% for KY11 and 0.65% for QY9).

Figure 4. Map of central region of Nef showing sites of key epitopes and residues at which CD8+ selection pressure operates.

Central conserved region of HIV-1 Nef is shown previously defined as HXB2 residues 81-160 [8]. Corresponding B-clade and C-clade consensus sequences are shown along with SIVmac239 consensus. Positions of epitopes restricted by alleles HLA-B*27 [37], HLA-B*57 [28,64,65] and HLA-B*44 are highlighted in green, orange and blue respectively. Regions homologous to Mamu-B*08 [36] and Mamu-B*17 epitopes [66] are also marked (yellow and purple respectively). SIV 115-129 also highlighted as a region recently associated with SIV control in macaques [41]. Responses to overlapping peptides 79 and 85 are associated with viraemic control, q<0.2 (black boxes). Sites of mutations selected by HLA-B*57, HLA-B*27, HLA-B*44, Mamu-B*08 and Mamu-B*17 are marked with arrows [29,36,67]. This highlights the substantial overlap between HIV and SIV epitopes restricted by alleles that are associated with favourable immune control.