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. 2012 Dec;86(23):12643-54.
doi: 10.1128/JVI.01381-12. Epub 2012 Sep 12.

Differential clade-specific HLA-B*3501 association with HIV-1 disease outcome is linked to immunogenicity of a single Gag epitope

Philippa C Matthews  1 , Madoka Koyanagi, Henrik N Kløverpris, Mikkel Harndahl, Anette Stryhn, Tomohiro Akahoshi, Hiroyuki Gatanaga, Shinichi Oka, Claudia Juarez Molina, Humberto Valenzuela Ponce, Santiago Avila Rios, David Cole, Jonathan Carlson, Rebecca P Payne, Anthony Ogwu, Alfred Bere, Thumbi Ndung'u, Kamini Gounder, Fabian Chen, Lynn Riddell, Graz Luzzi, Roger Shapiro, Christian Brander, Bruce Walker, Andrew K Sewell, Gustavo Reyes Teran, David Heckerman, Eric Hunter, Søren Buus, Masafumi Takiguchi, Philip J R Goulder
Affiliations

Differential clade-specific HLA-B*3501 association with HIV-1 disease outcome is linked to immunogenicity of a single Gag epitope

Philippa C Matthews et al. J Virol. 2012 Dec.

Abstract

The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8(+) T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10(-5)). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8(+) T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8(+) T-cell response in all individuals, irrespective of HLA type.

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Figures

Fig 1
Fig 1
Ranking of HLA-B alleles with respect to median viral load (VL) in chronic HIV-1 infection in B- and C-clade-infected study cohorts. Boxes show median and 25th and 75th centiles; whiskers show 10 to 90% confidence intervals. HLA-B*3501 is highlighted in gray. Dashed lines indicate median VL for the whole cohort. P values by Mann-Whitney test, comparing VL for subjects with each allele to the whole population: ***, P < 0.0001; **, P < 0.001. Alleles represented are those occurring at ≥0.5% phenotypic frequency and for which a minimum of 5 subjects had VL data available. (A) Kumamoto, Japan (median VL, 19,500 RNA copies/ml). (B) Gaborone, Botswana (median VL, 19,150 RNA copies/ml). For equivalent data for Durban, South Africa, see reference .
Fig 2
Fig 2
Median viral load in subjects with and without HLA-B*3501 in B- and C-clade-infected study cohorts. Boxes show median and 25th and 75th centiles; whiskers show 10 to 90% confidence intervals. (A) Kumamoto, Japan (B clade); (B) Mexico City, Mexico (B clade); (C) Gaborone, Botswana (C clade); (D) Durban, South Africa (C clade). P values are by the Mann-Whitney test.
Fig 3
Fig 3
Optimization of the NY10 Gag epitope (NPPIPVGDIY). (A) IFN-γ ELISpot responses to titrated amounts of the 9-mer PPIPVGDIY versus the 10-mer NPPIPVGDIY peptides made by an HLA-B*3501-positive adult subject with chronic B-clade HIV-1 infection (Thames Valley subject H033, HLA-A*3601, -A*7401, -B*3501, -B*5301, -Cw*0401, -Cw*0401). (B) Unequivocal definition of the correct HLA-B*3501-restricted optimal epitope NY10 using an HLA-B*3501–NY10 tetramer to stain the NY10 responder PBMCs from the same subject (H033) as used for panel A. Results from one representative of two independent experiments are shown.
Fig 4
Fig 4
Percentage of HLA-B*3501-positive subjects making IFN-γ ELISpot responses to 13 HLA-B*3501-restricted epitopes in B- and C-clade infection and the impact on viral load to HA9 Gag, NY10 Gag and VY/VF8 Nef responses controlled by HLA-B*3501 matched nonresponding individuals. (A) Responses expressed as protein specific were obtained by pooling the percentage of adult HLA-B*3501-positive subjects with B-clade infection (Kumamoto, Japan) making IFN-γ ELISpot responses to individual HLA-B*3501-restricted optimal peptides (n = 30 subjects) pooled with another B-clade cohort (23, 44) screened against 18-mer overlapping peptides containing the optimal epitopes (n = 44 subjects) (blue) (total of 74 B-clade-infected subjects) and compared to adult subjects with C-clade infection (southern African subjects) tested against C-clade consensus overlapping peptides containing the corresponding optimal peptides (n = 42 subjects) (red). (B) Responses as in panel A but shown for individual epitopes within Gag and Pol proteins. (C) Responses as in panel A but shown for individual epitopes within Rev, Env, and Nef proteins. (D) Comparison of viral load between responders and nonresponders for B-clade-infected Japanese subjects (n = 30), based on responses to optimal peptides, HA9 Gag (left), NY10 Gag (middle), and VY/VF8-Nef (right) (top panels) and C-clade southern African subjects based on responses to OLPs containing the corresponding optimal peptides (bottom panels). In each case, a positive ELISpot response is defined as >100 SFC/106 PBMCs; P values are by Fisher's exact test (A, B, and C) (and for B and C are shown only when significant after correction for multiple comparisons) or by Mann-Whitney U test (D).
Fig 5
Fig 5
Selection of Gag-D260E substitution in C-clade infection and effect of this polymorphism on CD8+ T-cell recognition and lack of NY10-260E specific CD8+ T cells. (A) Selection of Gag-D260E polymorphism in subjects with HLA-B*3501 from an extended B-clade data set as previously published (24) (n = 1,077; total subjects with HLA-B*3501, n = 135 [12.5%]) (left) and selection of Gag-D260E polymorphism in subjects with HLA-B*3501 from an extended southern African data set (Durban, n = 695; Botswana, n = 298; Thames Valley Africans, n = 59; total subjects with HLA-B*3501, n = 45 [4.3%]) (right). (B) IFN-γ ex vivo ELISpot responses made by an HLA-B*3501-positive Japanese subject with chronic B-clade infection (subject KI705, HLA-A*2402, -A*2601, -B*3501, -B*5201, -Cw*0303, -Cw*1202) to optimal epitope NY10 (NPPIPVGDIY) and an escape variant containing the D260E substitution (NPPIPVGEIY) and IFN-γ intracellular cytokine staining of CD8+ T cells in vitro expanded and tested against titrated amounts of NY10-260E and NY10-260D peptides. One experiments was performed. (C) IFN-γ ex vivo ELISpot responses made by an HLA-B*3501-positive subject with chronic B-clade infection (subject OX035, HLA-A*0201, -A*1101, -B*1801, -B*3501, -Cw*0401, -Cw*0501) to optimal epitope NY10-260D and an escape variant containing the D260E substitution NY10-260E and dual NY10-260E and NY10-260D HLA-B*3501 tetramer staining of ex vivo PBMCs controlled by HLA-B*4201 mismatch tetramer. Results from one representative of two independent experiments are shown. (D) In vitro-expanded PBMCs from subject OX035 using NY10-260D (top) and NY10-260E (bottom) peptides and stained with titrated amounts of dual HLA-B*3501 tetramers (260D/260E) gated on CD8+ T cells (dot plots) and expressed as CD3+/Tet+ positive cells for all tetramer titrations (right) controlled by HLA-B*4201 mismatch tetramers. P values are by Fisher's exact test. One experiments was performed. (E and F) HLA-negative and HLA-B*3501-expressing target cells were infected with either Gag-260E or Gag-260D virus and tested for epitope recognition by specific CD8+ T cells determined by IFN-γ production after coculture and shown for Gag-NY10 epitope processing (left) or the control Pol-EY10 epitope (right) by fluorescence-activated cell sorter (FACS) plots (E) and shown as horizontal bar graphs (F). Peptide-pulsed target cells (PEP) were included as a positive control for optimal epitope presentation.
Fig 6
Fig 6
Binding of NY10 Gag and PY9-Gag to the HLA-B*3501 molecule. Strength of binding affinity (Kd, nM) of HLA-B*3501 was determined using the luminescent oxygen channeling immunoassay, as previously described (29) (top panels), and stability half-life (t1/2) of binding (h) was determined using scintillation proximity assay, as previously described (30) (bottom panels), for NY10 (A) and PY9 (B). Results from one representative of four independent experiments are shown.
Fig 7
Fig 7
Modeled B3501-NY10 structure using the B3501-EPLPQGQLTAY complex (71). (A) HLA-B*3501 (shown in gray cartoon)-NPPIPVGDIY (shown in blue sticks), looking down at the MHC-binding groove. Position P8D in the peptide is circled. (B) HLA-B*3501 (shown in gray cartoon)-NPPIPVGEIY (shown in red sticks), looking down at the MHC-binding groove. P8E is circled. (C) Modeled interaction with NY10 residue P8D (blue stick) and MHC residues A150 and V152 (green sticks). (D) Modeled interaction with NY10 residue P8E (red stick) and MHC residues A150 and V152 (green sticks). The longer side chain of E in the escape mutant NY10 compared to D in the wild-type NY10 could generate steric hindrance with MHC residue A150. This could destabilize, and change the conformation of, the NY10 escape mutant peptide.
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