Efforts to define immune correlates of protection against HIV propose a key role to some major histocompatibility complex (MHC) class I alleles and to qualitative parameters of HIV-specific CD8+ T cells [1–6]. The human leukocyte antigen (HLA)-B27, B57, or the closely related B5801 (HLA-B57/5801), alleles dictate robust HIV-specific CD8+ T cells that are associated with HIV control [7–12]. Antigen specificity of CD8+ T cells also appears to be critical with a stronger protection conferred by Gag-specific T cells [13–18], but mechanisms involved appear to differ and are not yet fully elucidated. HLA-B27 drives the superior antiviral efficacy of Gag-specific CD8+ T cells in long-term nonprogressors by allowing higher cell avidity, polyfunctionality and clonal turnover . In contrast to HLA-B27, which presents to CD8+ T cells an immunodominant epitope in Gag but not in Nef, HLA-B57/5801 present several Gag and Nef epitopes [20,21]. In HLA-B57/5801+ individuals Gag-specific T cells appear to mediate a superior antiviral control than Nef ones , although the latter was shown to be preserved in HLA-B57/5801+ nonprogressors . However, little attention has been paid thus far to the differentiation triggered by specific HIV antigens in HLA-B57/5801+ nonprogressors and their association with disease protection. Therefore deciphering the relative properties of HIV-specific CD8+ T cells against the Gag and Nef epitopes in HLA-B57/5801+ nonprogressors should help further understand the protective effect of HIV-specific CD8+ T cells.
In order to identify attributes of the CD8+ T-cell-mediated protection against HIV in HLA-B57/5801+ nonprogressors, we investigated in HLA-B57/5801-positive and negative individuals, whether antigen specificity and HLA restriction trigger distinct patterns of cell differentiation that could explain the association between HLA-B57/5801 and virus control.
Materials and methods
Samples were obtained from 53 French ALT-ANRS-CO15 cohort patients (Supplemental Table 1, http://links.lww.com/QAD/A70). As described [23,24], inclusion criteria in the cohort were: HIV seropositivity for at least 8 years, CD4 cell counts above 600/μl for the last 5 years without symptoms or antiretroviral therapy. The median length of HIV seropositivity was 9 years at entry into the cohort . The study was approved by the institutional review board at the Pitié-Salpêtrière Hospital, and all patients provided written informed consent.
HLA genotyping was performed by amplification refractory mutation system PCR, with sequence-specific primers .
Parameters of HIV production
Plasma HIV-RNA were quantified as described [23,24]. The HIV-DNA in PBMCs was quantified with a modified Amplicor Monitor assay (Roche Laboratories) with a cut-off value of 5 copies of HIV-DNA/106 PBMC .
Synthetic 15-mer peptides overlapping by 11 amino acids (Neosystem, France) and spanning the HIV-HxB2 Gag and Nef sequences were combined into 11 and 3 pools, respectively. Eight CD8+ T-cell epitopes derived from HIV-HxB2 Gag or Nef were synthesized according to the Los Alamos HIV database. The B57-restricted peptides were: ISPRTLNAW (Gag147-155, IW9), KAFSPEVIPMF (Gag162-172, KF11), TSTLQEQIGW (Gag240-249, TW10), and HTQGYFPDW (Nef116-124, HW9). The A3-restricted peptides were: KIRLRPGGK (Gag18-26, KK9), and QVPLRPMTYK (Nef73-82, QK10). The A26-restricted peptide was EVIPMFSAL (Gag167-175, EL9). Lastly, the B35-restricted peptide was TPGPGVRYPL (Nef128-137, TL10).
IFN-γ enzyme-linked immunospot assay
PBMCs were incubated with phytohaemagglutinin (positive control), medium alone (negative control), Gag and Nef peptide pools as described . Spots were considered positive if above 50 SFC/106 PBMCs after subtracting background. Functional avidity was assessed by testing serial 10-fold dilutions ranging from 100 to 0.0001 μg/ml of optimal peptides and determining the peptide concentration inducing half-maximal responses in enzyme-linked immunospot (ELISpot) assays .
Intracellular cytokine staining, phenotyping and tetramer staining
PBMCs were incubated overnight with staphylococcal enterotoxin β (positive control), medium (negative control), Gag and Nef pools found to be positive in the ELISpot assay. Brefeldin-A was added as described . Cells were stained with anti-CD8-PE-Cy7, anti-CD45RA-ECD (Beckman-Coulter), anti-CCR7-PE (R&D System), anti-CD27-FITC and anti-IFN-γ-APC and/or anti-IL-2-PE (Becton Dickinson). Cells were analyzed on a Beckman Coulter FC500 flow cytometer using the CXP Analysis (Beckman Coulter) software. The CD3+CD8+CD45RA-CD27+/-CCR7+/-IFN-γ+ cells accounted for up to 99% of the CD8+CD45RA-CD27+/-CCR7+/-IFN-γ+ cells as analyzed in parallel on a FACSCanto-I flow cytometer (Becton Dickinson), thus assessing the T-cell nature of the IFN-γ-producing CD8+ cells. CD27 expression on CD8+ T cells binding the B57/KF11 tetramer (Beckman-Coulter) was also analyzed in combination with anti-CCR7-PE-Cy7, anti-CD27-APC, anti-CD8-APC-Cy7 (Becton Dickinson), and anti-CD45RA-FITC (Beckman-Coulter) by a FACSCanto-I flow cytometer and the FlowJo (version 8.0, TreeStar) software on three HLA-B57/5801+ individuals.
Statistics were conducted with SPSS 13.0 software (SSPS Inc., Chicago, Illinois, USA). The Wilcoxon rank sum test, Mann–Whitney U test and Spearman's rank correlation were used. The Benjamini–Holberg procedure was used to correct for the multiplicity of tests  and mentioned as ‘corrected P’ value.
Frequencies of Gag-specific CD8+ T cells but not Nef-specific ones correlates negatively with cell-associated HIV-DNA
In this group of 53 nonprogressors, the five-fold higher numbers of IFN-γ-producing Gag-specific T cells than their Nef-specific counterparts (median: 2527 vs. 480 SFC/106 PBMC, corrected P < 0.0001; Supplemental Table 1, http://links.lww.com/QAD/A70 and Fig. 1a), negatively correlated with cell-associated HIV-DNA loads (r = −0.395, corrected P = 0.004; Fig. 1b), whereas the Nef-specific ones did not (r = −0.151, P = 0.294; Fig. 1c). Despite HIV-DNA loads highly correlated with plasma HIV-RNA loads (r = 0.801, corrected P < 0.0001; Fig. 1f), HIV-specific cell frequencies did not correlate with plasma HIV-RNA levels (Fig. 1d and e).
HLA-B57/5801 molecules are frequent in nonprogressors and associated with virus control. They restrict CD8+ T-cell recognition of epitopes in both Gag and Nef. We further investigated whether such differences could be triggered by HLA-B57/5801. We therefore performed a head-to-head comparison of Gag and Nef-specific CD8+ T cells in samples from 11 HLA-B57/5801+ individuals and 11 HLA-B57/5801− individuals with simultaneous responses to Gag and Nef peptide pools containing the HLA-B57/5801-restricted epitopes (Supplemental Table 1, http://links.lww.com/QAD/A70 and Supplemental Fig. 1, http://links.lww.com/QAD/A70).
Recognition of the corresponding Gag and Nef epitopes restricted by HLA-B57/5801 and other HLA types (Supplemental Fig. 2, http://links.lww.com/QAD/A70) confirmed differences in magnitude between these sets of cells. A multifunctional flow cytometry analysis showed few of the Gag and Nef-specific CD8+ T cells producing IL-2 (Supplemental Fig. 3, http://links.lww.com/QAD/A70), regardless of HLA.
HLA-B57/5801 dictates a preferential CD27 expression of Gag-specific central memory CD8+ T cells compared to Nef ones
The superior magnitude of Gag-specific IFN-γ+CD8+ T cells compared to Nef-specific ones did not reflect a distinct repartition of the classical central and effector memory cells [central memory T cells (TCM) and effector memory T cells (TEM), respectively] in either HLA group (Supplemental Fig. 4a, http://links.lww.com/QAD/A70). However, the HLA-B57/5801+ Gag-specific IFN-γ+IL-2−CD8+ T cells differed from their Nef-specific counterparts by a higher proportion of IFN-γ+CD45RA-CD8+ cells displaying CD27 (38 vs. 26%, corrected P = 0.007; Fig. 2a and Supplemental Fig. 4b, http://links.lww.com/QAD/A70). This difference was maintained in HLA-B57/5801+ individuals between CD27 expression on 39% [interquartile range (IQR) 31–56%] of Gag-specific CD8+ TCM cells and 33% (IQR 16–42%) of Nef-specific ones (corrected P = 0.007; Fig. 2c), but not in the HLA-B57/5801− population (Fig. 2b and d). The preferential CD27 expression of HLA-B57/5801+ TCM was observed as well on HLA-B57-tetramer binding Gag-specific CD8+ TCM (Supplemental Fig. 5, http://links.lww.com/QAD/A70). A similar but not significant trend was observed on the Gag-specific TEM whatever the HLA (Fig. 2c and d). In contrast effector Gag and Nef-specific cells displayed similar CD27 expression (data not shown).
The preferential CD27 expression on HLA-B57/5801+ Gag-specific CD8+ TCM correlates with virus control
The CD27+ Gag-specific IFN-γ+CD8+ TCM negatively correlated with both plasma HIV-RNA (r = −0.502, corrected P = 0.024) and HIV-DNA (r = −0.567, corrected P = 0.018; Fig. 2e) in the total group. This negative correlation was maintained in the HLA-B57/5801+ (r = −0.683, corrected P = 0.042; Fig. 2e) but not in the HLA-B57/5801− group. The Nef-specific counterpart was not correlated with either the HIV-DNA (Fig. 2f) or the plasma HIV-RNA loads, regardless of HLA. In addition, no significant correlation was found between CD27+ Gag or Nef-specific IFN-γ+ TEM and HIV burden in either HLA group (Supplemental Table 2, http://links.lww.com/QAD/A70).
These results show that a 37.5% Gag-specific CD27+ TCM are required to be associated with a 1 log reduction in HIV-DNA load in the whole group. In the HLA-B57/5801+ group, however, a 29% Gag-specific CD27+ TCM is sufficient.
Our findings demonstrate HLA-B57/5801 confer HIV-Gag-specific T cells an advantage to control HIV by allowing higher numbers of Gag-specific central memory CD8+ T cells to display CD27 compared to Nef-specific ones. These results provide new insights into the mechanisms by which protective HLA-B57/5801 alleles support superior control exerted by Gag-specific compared to Nef-specific T cells.
In addition, we provide novel evidence that the cell-associated HIV-DNA load is a better surrogate marker for cell-mediated virus control than the plasma HIV-RNA load . Indeed the negative correlation observed in the whole group of nonprogressors between total Gag-specific CD8+ T cells and cell-associated HIV-DNA is re-inforced in HLA-B57/5801+ individuals in whom Gag-specific CD27+ TCM correlated with cell-associated HIV-DNA load. As HIV-specific CD8+ T cells detect infected cells producing HIV antigens but not free virus particles, and assuming the number of HIV-DNA copies per million cells roughly represents the numbers of infected cells, the cell-associated HIV-DNA more accurately reflects the infected cell targets of CD8+ T cells than the plasma HIV load. It remains unclear yet whether the HIV-DNA load is determined by the CD8+ T-cell phenotype or vice versa.
The new characteristics we demonstrate here for HLA-B57/5801+ Gag-specific CD8+ T cells contrasts with previous study suggesting persistent CD27 expression explain the failure of HIV-specific CD8+ T cells to control the virus . The known property of CD27 to promote cell survival and proliferation required for the generation and long-term maintenance of antigen-specific T-cell immunity  fits, however, with a better control of HIV mediated by these Gag-specific CD8+ TCM. Alternatively, stronger CD27 expression on HLA-B57/5801+ Gag-specific CD8+ TCM might simply reflect preferential survival in the context of a low antigen burden, although both HLA groups were comparable for CD4 cell counts and plasma viral loads.
Those distinct stages of differentiation reached by HLA-B57/5801+ Gag and Nef-specific cells are reminiscent of a distinct differentiation pattern reported for CD8+ T cells specific for lytic and latent Epstein-Barr virus (EBV) proteins, suggesting modalities of antigen stimulation and kinetics of antigen expression may account for differences in the differentiation profiles. HIV-Nef indeed differs from Gag since expressed only in virus-producing cells, whereas the abundantly released capsid antigens can be cross-presented by professional antigen-presenting cells. Alternatively, more frequent viral escape from the Nef-specific immune response than against the Gag-specific one might account for such differences [32–34]. More differentiated T cells, as observed for Nef-specific cells, might exert a stronger selective pressure and facilitate emergence of variants. On the contrary, structural constraints on Gag epitopes might allow specific T cells to persist in a more quiescent central-memory status.
Altogether our findings provide new insight into the immune correlates of HIV control and the mechanisms of protection conferred by HLA-B57/5801 and confirm the need for closer attention to the nature of the HIV antigens included in vaccines against HIV.
The study was supported by grants of the French Agency of AIDS Research (ANRS), the European Union-funded GISHEAL (Genomic and Immunologic Studies of HIV-infected European and African long-term nonprogressors) (EU N96X) consortium (LSHP-CT-2007-037616), INSERM and the Japanese National Institute of Health. J.X. and W.L. were supported by the French Embassy in China, the European CHIVAC-III (ICA4-CT-2002-10042) and GISHEAL consortia. M.L. was supported by a grant from the European Union (ATTACK project LHS-CT-2005-018914). We thank Mr and Mrs André Lagadec for their kind and continuous support to this work. We are also indebted to Dr Y. Yokota from the Japan NID for her support and the ALT patients for participating in this study.
J.X. performed the research, analyzed data, and wrote the manuscript. W.L. performed the research and participated in writing the manuscript. A.S. participated in data management and coordination of the French ALT cohort. D.C. provided supervision for statistical analysis. A.S., B.S., C.B. and M.A. participated in performing the research. I.T. oversaw the HLA genotyping. C.R. supervised the viral load quantification. B.A. managed and designed the research, coordinated the French ALT cohort, and provided critical review of the manuscript.
The ALT study group: Agut, H. Laboratoire de Virologie, Hôpital Pitié-Salpêtrière; Debré, P., Almeida, M. and Pellé O. Laboratoire d'Immunité et Infections, INSERM UMR-S 945, Hôpital Pitié-Salpêtrière, UPMC Paris 06, F-75013, Paris, France; Clauvel, J.P. Service d'Immuno-Pathologie, Hôpital St. Louis; Sicard, D. Service de Médecine Interne Hôpital Cochin, Paris, France.
1. Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, et al
. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 2001; 410:106–111.
2. Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, et al
. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002; 8:379–385.
3. Migueles SA, Laborico AC, Shupert WL, Sabbaghian MS, Rabin R, Hallahan CW, et al
. HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat Immunol 2002; 3:1061–1068.
4. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, et al
. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 2006; 107:4781–4789.
5. Klein MR, van Baalen CA, Holwerda AM, Kerkhof Garde SR, Bende RJ, Keet IP, et al
. Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics. J Exp Med 1995; 181:1365–1372.
6. Harrer T, Harrer E, Kalams SA, Barbosa P, Trocha A, Johnson RP, et al
. Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load. J Immunol 1996; 156:2616–2623.
7. Gao X, Bashirova A, Iversen AK, Phair J, Goedert JJ, Buchbinder S, et al
. AIDS restriction HLA allotypes target distinct intervals of HIV-1 pathogenesis. Nat Med 2005; 11:1290–1292.
8. Migueles SA, Sabbaghian MS, Shupert WL, Bettinotti MP, Marincola FM, Martino L, et al
. HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors. Proc Natl Acad Sci U S A 2000; 97:2709–2714.
9. Magierowska M, Theodorou I, Debre P, Sanson F, Autran B, Riviere Y, et al
. Combined genotypes of CCR5, CCR2, SDF1, and HLA genes can predict the long-term nonprogressor status in human immunodeficiency virus-1-infected individuals. Blood 1999; 93:936–941.
10. Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, Chetty S, et al
. Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 2004; 432:769–775.
11. O'Brien SJ, Gao X, Carrington M. HLA and AIDS: a cautionary tale. Trends Mol Med 2001; 7:379–381.
12. Tang Y, Huang S, Dunkley-Thompson J, Steel-Duncan JC, Ryland EG, St John MA, et al
. Correlates of spontaneous viral control among long-term survivors of perinatal HIV-1 infection expressing human leukocyte antigen-B57. AIDS 2010; 24:1425–1435.
13. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, et al
. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 2007; 13:46–53.
14. Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, Strick D, et al
. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol 2003; 77:2081–2092.
15. Allen TM, O'Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, et al
. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 2000; 407:386–390.
16. van Baalen CA, Pontesilli O, Huisman RC, Geretti AM, Klein MR, de Wolf F, et al
. Human immunodeficiency virus type 1 Rev- and Tat-specific cytotoxic T lymphocyte frequencies inversely correlate with rapid progression to AIDS. J Gen Virol 1997; 78(Pt 8):1913–1918.
17. Haas G, Plikat U, Debre P, Lucchiari M, Katlama C, Dudoit Y, et al
. Dynamics of viral variants in HIV-1 Nef and specific cytotoxic T lymphocytes in vivo. J Immunol 1996; 157:4212–4221.
18. Lichterfeld M, Yu XG, Cohen D, Addo MM, Malenfant J, Perkins B, et al
. HIV-1 Nef is preferentially recognized by CD8 T cells in primary HIV-1 infection despite a relatively high degree of genetic diversity. AIDS 2004; 18:1383–1392.
19. Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, Bornstein E, et al
. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med 2007; 204:2473–2485.
20. Altfeld M, Kalife ET, Qi Y, Streeck H, Lichterfeld M, Johnston MN, et al
. HLA alleles associated with delayed progression to AIDS contribute strongly to the initial CD8(+) T cell response against HIV-1. PLoS Med 2006; 3:e403.
21. Bailey JR, Brennan TP, O'Connell KA, Siliciano RF, Blankson JN. Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors. J Virol 2009; 83:88–97.
22. Navis M, Schellens IM, van Swieten P, Borghans JA, Miedema F, Kootstra NA, et al
. A nonprogressive clinical course in HIV-infected individuals expressing human leukocyte antigen B57/5801 is associated with preserved CD8+ T lymphocyte responsiveness to the HW9 epitope in Nef. J Infect Dis 2008; 197:871–879.
23. Candotti D, Costagliola D, Joberty C, Bonduelle O, Rouzioux C, Autran B, Agut H. Status of long-term asymptomatic HIV-1 infection correlates with viral load but not with virus replication properties and cell tropism. French ALT Study Group. J Med Virol 1999; 58:256–263.
24. Martinez V, Costagliola D, Bonduelle O, N'go N, Schnuriger A, Theodorou I, et al
. Combination of HIV-1-specific CD4 Th1 cell responses and IgG2 antibodies is the best predictor for persistence of long-term nonprogression. J Infect Dis 2005; 191:2053–2063.
25. Bunce M, O'Neill CM, Barnardo MC, Krausa P, Browning MJ, Morris PJ, Welsh KI. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 1995; 46:355–367.
26. Schnuriger A, Dominguez S, Guiguet M, Harfouch S, Samri A, Ouazene Z, et al
. Acute hepatitis C in HIV-infected patients: rare spontaneous clearance correlates with weak memory CD4 T-cell responses to hepatitis C virus. AIDS 2009; 23:2079–2089.
27. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc Series B (Methodological) 1995; 57:289–300.
28. Rouzioux C, Hubert JB, Burgard M, Deveau C, Goujard C, Bary M, et al
. Early levels of HIV-1 DNA in peripheral blood mononuclear cells are predictive of disease progression independently of HIV-1 RNA levels and CD4+ T cell counts. J Infect Dis 2005; 192:46–55.
29. van Baarle D, Kostense S, Hovenkamp E, Ogg G, Nanlohy N, Callan MF, et al
. Lack of Epstein-Barr virus- and HIV-specific CD27- CD8+ T cells is associated with progression to viral disease in HIV-infection. AIDS 2002; 16:2001–2011.
30. Hendriks J, Gravestein LA, Tesselaar K, van Lier RA, Schumacher TN, Borst J. CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol 2000; 1:433–440.
31. Mallard E, Vernel-Pauillac F, Velu T, Lehmann F, Abastado JP, Salcedo M, Bercovici N. IL-2 production by virus- and tumor-specific human CD8 T cells is determined by their fine specificity. J Immunol 2004; 172:3963–3970.
32. Brumme ZL, Brumme CJ, Heckerman D, Korber BT, Daniels M, Carlson J, et al
. Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1. PLoS Pathog 2007; 3:e94.
33. Brumme ZL, Tao I, Szeto S, Brumme CJ, Carlson JM, Chan D, et al
. Human leukocyte antigen-specific polymorphisms in HIV-1 Gag and their association with viral load in chronic untreated infection. AIDS 2008; 22:1277–1286.
34. Miura T, Brumme CJ, Brockman MA, Brumme ZL, Pereyra F, Block BL, et al
. HLA-associated viral mutations are common in human immunodeficiency virus type 1 elite controllers. J Virol 2009; 83:3407–3412.