Disequilibrium with HLA-B*5703 Viremia Is Independent of Its

HLA-A*7401−Mediated Control of HIV Viremia Is Independent of Its Linkage Disequilibrium with HLA-B*5703

This information is current as Philippa C. Matthews, Emily Adland, Jennifer Listgarten, of September 24, 2021. Alasdair Leslie, Nompumelelo Mkhwanazi, Jonathan M. Carlson, Mikkel Harndahl, Anette Stryhn, Rebecca P. Payne, Anthony Ogwu, Kuan-Hsiang Gary Huang, John Frater, Paolo Paioni, Henrik Kloverpris, Pieter Jooste, Dominique Goedhals, Cloete van Vuuren, Dewald Steyn, Lynn Riddell, Fabian Chen, Graz Luzzi, Thambiah Balachandran, Thumbi Downloaded from Ndung'u, Søren Buus, Mary Carrington, Roger Shapiro, David Heckerman and Philip J. R. Goulder J Immunol 2011; 186:5675-5686; Prepublished online 15 April 2011; doi: 10.4049/jimmunol.1003711 http://www.jimmunol.org/ http://www.jimmunol.org/content/186/10/5675

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

HLA-A*7401–Mediated Control of HIV Viremia Is Independent of Its Linkage Disequilibrium with HLA-B*5703

Philippa C. Matthews,* Emily Adland,* Jennifer Listgarten,† Alasdair Leslie,‡ Nompumelelo Mkhwanazi,x Jonathan M. Carlson,† Mikkel Harndahl,{ Anette Stryhn,{ Rebecca P. Payne,* Anthony Ogwu,‖ Kuan-Hsiang Gary Huang,# John Frater,# Paolo Paioni,* Henrik Kloverpris,* Pieter Jooste,** Dominique Goedhals,†† Cloete van Vuuren,‡‡ Dewald Steyn,‡‡ Lynn Riddell,xx Fabian Chen,{{ Graz Luzzi,‖‖ Thambiah Balachandran,## Thumbi Ndung’u,x Søren Buus,{ Mary Carrington,***,††† Roger Shapiro,‖,‡‡‡ David Heckerman,† and Philip J. R. Goulder*,x Downloaded from The potential contribution of HLA-A alleles to viremic control in chronic HIV type 1 (HIV-1) infection has been relatively understudied compared with HLA-B. In these studies, we show that HLA-A*7401 is associated with favorable viremic control in extended southern African cohorts of >2100 C-clade–infected subjects. We present evidence that HLA-A*7401 operates an effect that is independent of HLA-B*5703, with which it is in linkage disequilibrium in some populations, to mediate lowered viremia. We describe a novel statistical approach to detecting additive effects between class I alleles in control of HIV-1 disease, highlighting improved viremic control in subjects with HLA-A*7401 combined with HLA-B*57. In common with HLA-B alleles that are http://www.jimmunol.org/ associated with effective control of viremia, HLA-A*7401 presents highly targeted epitopes in several proteins, including Gag, Pol, Rev, and Nef, of which the Gag epitopes appear immunodominant. We identify eight novel putative HLA-A*7401–restricted epitopes, of which three have been defined to the optimal epitope. In common with HLA-B alleles linked with slow progression, viremic control through an HLA-A*7401–restricted response appears to be associated with the selection of escape mutants within Gag epitopes that reduce viral replicative capacity. These studies highlight the potentially important contribution of an HLA-A allele to immune control of HIV infection, which may have been concealed by a stronger effect mediated by an HLA-B allele with which it is in linkage disequilibrium. In addition, these studies identify a factor contributing to different HIV disease outcomes in individuals expressing HLA-B*5703. The Journal of Immunology, 2011, 186: 5675–5686. by guest on September 24, 2021 uman leukocyte Ag class I genotype is a major de- the allele associated with most effective suppression of viremia in terminant of HIV-1 viremic control and progression to C-clade–infected subjects (6, 14–17). However, an effect on vi- H AIDS (1–5). Much of this effect is mediated by the remic control has also previously been identified in association presentation of HIV-specific epitopes by different HLA class I with some HLA-A alleles (18–21), and with the HLA-Cw class I molecules for recognition of virus-infected cells by CD8+ T cells locus (3, 22, 23). (6–12). HLA-B is the class I locus with the strongest influence on The starting point of these studies was the observation that HLA- median viral load in chronic infection (13), and HLA-B*5703 is A*7401 is associated with the lowest median HIV-1 viral load of

*Department of Paediatrics, University of Oxford, Oxford OX1 3SY, United King- This work was supported by the United Kingdom Medical Research Council (to dom; †Microsoft Research, eScience Group, Los Angeles, CA 90024; ‡Weatherall P.C.M.), the Oxford Radcliffe Hospitals Medical Research Fund (to P.C.M), National Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Head- Institutes of Health Grant 2RO1AI46995, National Institute of Allergy and Infectious ington, Oxford OX3 9DS, United Kingdom; xHIV Pathogenesis Programme, Doris Diseases Contract HHSN272200900045C, the South African AIDS Vaccine Initia- Duke Medical Research Institute, University of KwaZulu-Natal, Durban, 4013 South tive, the Wellcome Trust (to A.L. and P.J.R.G.), the National Cancer Institute, Na- Africa; {Laboratory of Experimental Immunology, Faculty of Health Sciences, Uni- tional Institutes of Health Contract HHSN261200800001E, and the Intramural versity of Copenhagen, DK-2200 Copenhagen, Denmark; ‖Botswana Harvard AIDS Research Program of the National Institutes of Health, National Cancer Institute, Institute Partnership, Gaborone, Botswana; #Nuffield Department of Medicine, Univer- Center for Cancer Research. P.J.R.G. is an Elizabeth Glaser Pediatric AIDS Foun- sity of Oxford, Oxford OX1 3SY, United Kingdom; **Paediatric Department, Univer- dation Scientist. sity of Free State, Kimberley Hospital, Kimberley 8300, Northern Cape, South The content of this publication does not necessarily reflect the views or policies of the Africa; ††Department of Medical Microbiology and Virology, University of the Free Department of Health and Human Services, nor does mention of trade names, com- State/National Health Laboratory Services, Bloemfontein 9300, South Africa; mercial products, or organizations imply endorsement by the U.S. Government. ‡‡Department of Internal Medicine, University of the Free State, Bloemfontein 9300, South Africa; xxDepartment of Genitourinary Medicine, Northamptonshire Healthcare Address correspondence and reprint requests to Prof. Philip J.R. Goulder, University National Health Service Trust, Northampton General Hospital, Cliftonville, Northamp- of Oxford, Peter Medawar Building for Pathogen Research, South Parks Road, Ox- ton NN1 5BD, United Kingdom; {{Department of Sexual Health, Royal Berkshire ford OX1 3SY, United Kingdom. E-mail address: [email protected]. Hospital, Reading RG1 5AN, United Kingdom; ‖‖Sexual Health at Wycombe Clinic, ac.uk Wycombe Hospital, High Wycombe, Bucks HP11 2TT, United Kingdom; ##Depart- The online version of this article contains supplemental material. ment of Sexual Health, Luton and Dunstable Hospital, Luton LU4 0DZ, United King- dom; ***Cancer and Inflammation Program, Laboratory of Experimental Immuno- Abbreviations used in this article: HIV-1, HIV type 1; LD, linkage disequilibrium; logy, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702; LOCI, luminescent oxygen channeling immunoassay; OLP, overlapping peptide; P2, †††Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Tech- position two of epitope; PC, C-terminal position of epitope. nology and Harvard, Boston, MA 02129; and ‡‡‡Department of Immunology and In- fectious Diseases, Harvard School of Public Health, Boston, MA 02115 Received for publication November 8, 2010. Accepted for publication March 4, 2011.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003711 5676 HLA-A*7401–MEDIATED IMMUNE CONTROL OF HIV-1 all the 23 HLA-A alleles expressed at $0.5% phenotypic fre- four-digit type was not resolved in a minority of study subjects. In the quency in a large cohort of HIV-1–infected subjects from Durban, Durban cohort, 100/138 (72%) subjects with HLA-A*74 were typed to 4 South Africa (3). Furthermore, in this C-clade–infected cohort, we digits, and 100/100 (100%) of these subjects typed to 4 digits had HLA- A*7401. Of 81 HLA-A*74+ subjects from the Thames Valley, Botswana, noted that HLA-A*7401 is associated with the selection of escape Kimberley, and Bloemfontein cohorts typed to 4-digits, again 100% had mutations in Gag, Pol, and Nef (3, 5). These two strands of evi- HLA-A*7401 confirmed by high-resolution typing. To test the generality dence both suggest that HLA-A*7401–restricted CD8+ T cell re- of these findings, we also examined a random sample of 90 individuals of sponses may contribute to disease control in chronic HIV-1 African descent with HLA-A*74 recently typed by the National Institutes of Health, and found that .97% had HLA-A*7401 (M. Carrington, un- infection and are in keeping with recently published data doc- published observations). In this study, we have therefore classified all umenting an association between HLA-A*7401 and disease con- HLA-A*74+ subjects together as HLA-A*7401. trol in Tanzania and Kenya (19, 24). Amplification and sequencing of proviral DNA The role of HLA-A*7401 in viremic control in Durban is potentially confounded by its occurrence in linkage disequilibrium Gag sequences were generated from genomic DNA extracted from PBMCs, with HLA-B*5703 (23, 25). This raises the possibility that the amplified by nested PCR to obtain population sequences, as previously described (16, 31). Sequencing for the Durban and Bloemfontein cohorts apparent benefits to viremic control seen among subjects with was undertaken using the Big Dye Ready Reaction Terminator Mix (V3) HLA-A*7401 are, in fact, all mediated by a linked HLA-B*5703– (Applied Biosystems, U.K.) and manually aligned using Se_Al soft- restricted response. Alternatively, it is possible that the impact of ware, as previously described (31, 32). Sequencing of subjects from the HLA-A*7401 is independent of HLA-B*5703 but obscured by the Thames Valley, Botswana, and Kimberley cohorts was performed by stronger effects of the HLA-B allele. Indeed, although HLA- Macrogen (South Korea). Sequences were analyzed using Sequencher v4.8 (Gene Codes Corporation). Online sequences are available at GenBank, B*5703 is the allele most strongly associated with successful FJ198407-FJ199088 (Durban) and FJ497801-FJ497950 (Botswana). Downloaded from control of viremia in sub-Saharan Africa populations, consider- HLA-A*7401 motif prediction and confirmation; HLA–peptide able variation in viral set point is seen among subjects possessing binding studies this allele (23). We therefore hypothesized that some of the stra- tification in disease outcome among subjects with HLA-B*5703 Amino acid sequences for HLA-A*7401 and related HLA-A alleles were might be accounted for by the presence or absence of HLA- downloaded from the International Immunogenetics Project (http://www. ebi.ac.uk/imgt/hla/). HLA-A*7401 optimal epitopes were initially pre- A*7401 in the haplotype. http://www.jimmunol.org/ dicted using motif inference based on the comparison of HLA-A*7401 HLA-A*7401 alleles belong to the HLA-A19 serotype, which with HLA-A alleles that share sequence homology for the B and F pockets comprises HLA-A*29, -A*30, -A*31, -A*32, and -A*74, and has of the peptide binding groove (33). These two pockets determine the been included within the HLA-A*03 supertype (26, 27). Although preferred residues bound at the “anchor” positions of the epitope (corre- HLA-A*7401 is prevalent in sub-Saharan African populations, sponding to position two [P2] and the carboxyl-terminal position [PC]) ∼ (34). The validity of the predicted HLA-A*7401 peptide binding motif was occurring at a phenotypic frequency of 10%, [http://www. tested by two methods. Optimal epitopes were first determined using IFN- allelefrequencies.net (28)], the peptide binding motif for HLA- g ELISPOT assays to test recognition of peptides that corresponded to the A*7401 has yet to be defined. predicted optimal epitope, and of peptides differing by one amino acid in The aims of this study were, therefore, as follows: first, to in- length from each predicted optimal. We used Los Alamos HIV databases vestigate the contribution of HLA-A*7401 to control of HIV-1 (epitopes “A-list”; http://www.hiv.lanl.gov) to search for previously pub- by guest on September 24, 2021 lished HLA-A*7401–restricted epitopes. infection, both alone and in combination with HLA-B*5703 in Binding of these predicted optimal peptides to HLA-A*7401 was tested southern African populations; and second, to investigate the means in HLA–peptide–binding studies, undertaken using a luminescent oxygen by which HLA-A*7401 might contribute to viremic control via the channeling immunoassay (LOCI), as previously described (35). + presentation of HLA-A*7401–restricted CD8 T cell epitopes. IFN-g ELISPOT assays We tested ex vivo PBMCs from 1010 study subjects from the Durban cohort Materials and Methods against a panel of 410 overlapping peptides (OLPs) spanning the entire C- Study cohorts clade HIV-1 proteome to screen for IFN-g ELISOIT responses, as pre- We studied a total of 2126 treatment-naive, adult subjects with chronic HIV- viously described (29). A total of 119 of these subjects had HLA-A*7401 1 C-clade infection from southern Africa. We recruited subjects via five (91 of these 119 [76%] were confirmed to be HLA-A*7401 at 4-digit cohorts as follows: 1) Durban, South Africa (n = 1218), as previously resolution; the remainder were typed to only 2 digits). Fisher’s exact test described (3, 13, 16, 29); 2) Bloemfontein, South Africa [n = 261; in this was performed to identify responses to OLPs that were significantly as- cohort, HLA types were ascertained only for subjects with CD4+ T cell sociated with expression of HLA-A*7401. Analysis was also repeated in counts ,100 or .500 cells/mm3, as previously described (30)]; 3) Kim- the absence of the 28 subjects for whom HLA-A*74 was typed to only 2- berley, South Africa (n = 31 postnatal mothers); 4) Gaborone, Botswana digit resolution to remove any potential bias from subjects not confirmed to have HLA-A*7401 as the high-resolution type. We tested putative optimal (n = 514 antenatal women) (10); and 5) southern African subjects at- + tending outpatient HIV clinics in the Thames Valley area of the U.K. (n = epitopes by ELISPOT using ex vivo PBMCs from HLA-A*7401 subjects 102) originating from Botswana, Malawi, South Africa, and Zimbabwe recruited through the Thames Valley and Durban cohorts. (n = 1, 11, 17, and 73, respectively). Ethics approval was given by Uni- Cell staining and flow cytometry versity of KwaZulu-Natal Review Board and the Massachusetts General Hospital Review Board (Durban cohort); the University of the Free State Cell staining was undertaken from cryopreserved PBMCs using anti–CD3- Ethics Committee (Kimberley and Bloemfontein cohorts); the Office of Pacific orange (Invitrogen), anti–CD8-Alexa Fluor 700 (BD Biosciences), Human Research Administration, Harvard School of Public Health and the and HLA-A*7401-RR9 tetramer conjugated to PE. Dead cells were gated Health Research Development Committee, Botswana Ministry of Health out using “live/dead” viability kit (Invitrogen). (Gaborone cohort); and the Oxford Research Ethics Committee (Durban, Kimberley, and Thames Valley cohorts). Study subjects from all cohorts Statistical analysis gave written informed consent for their participation. Viral load in chronic We used Fisher’s exact test to screen for LD between HLA class I alleles, using infection was measured using the Roche Amplicor version 1.5 assay; CD4+ the online tool available at http://www.hiv.lanl.gov/content/immunology/hla/ T cell counts were measured by flow cytometry (data for each cohort are hla_linkage.html, corrected for multiple comparisons using a Bonferroni shown in Table I). approach. To identify MHC class I haplotypes, we used PyPop software HLA typing [Python for Population Genomics, http://www.pypop.org (36)] (Supple- mental Table I). Other statistical analysis was undertaken using Prism High-resolution HLA typing was performed from genomic DNA by single- GraphPad software v. 5.0a. stranded conformation polymorphism PCR. As in previous studies (3, 5, To assess whether any possible pairs of HLA class I alleles might perform 23), for certain alleles in the Durban cohort, including HLA-A*74, the better than a single allele in mediating disease control in chronic infection, The Journal of Immunology 5677 we devised a novel statistical test for additive effects using pooled data from A*7401+ subjects also carried HLA-B*5703. The HLA-A*74- a total of 2126 C-clade–infected adult subjects from southern Africa. Each B*57 haplotype has previously been identified in African Amer- HLA combination was tested to see whether an additive model for two icans [http://www.allelefrequencies.net (28); both alleles resolved HLA alleles together, irrespective of linkage disequilibrium, performed better in predicting disease control than a model that considered only the to two digits only in this source (3)], but to our knowledge has not most predictive of each single HLA allele. For the purposes of this test, been reported in other African populations to date. each allele was collapsed to a two-digit resolution only, apart from ex- To confirm the haplotypes in which HLA-A*7401 is transmitted, ceptions in which it is known that the four-digit types cross supertype we analyzed HLA data for Durban, Bloemfontein, and Gaborone boundaries or have a strong bearing on outcome (HLA-A*68XX, HLA- B*15XX, and HLA-B*58XX) (13, 27). using PyPop software [http://www.pypop.org (36)] (Supplemental For disease control based on the log of the absolute viral load and the log Table I). In keeping with our analysis of LD (Fig. 1), this dem- of the CD4+ T cell count, we used the log likelihood of linear-regression– onstrates that HLA-A*7401 is transmitted on a haplotype with based models to look for additive effects. In particular, we defined the null HLA-B*1503, HLA-B*3501, and HLA-B*5703 in South Africa. model for a given HLA pair as the one of the two models, pY or pX (defined 0 0 The association of HLA-A*7401 with HLA-B*1503 and HLA- next), that had the maximum likelihood under a maximum likelihood parameter setting. Intuitively, it is a model where at most only one HLA at B*3501 is also seen in Botswana, but in this population, there is a time can have an effect within an individual patient; the HLA chosen is no HLA-A*7401-B*5703 haplotype. In addition, these data show the one with the larger overall effect in such a model. Thus, the null model a weaker haplotype of HLA-A*7401-B*4201 in all three cohorts, pY ð jX; Y Þ¼ ðw þ w9 X9 þ w Y; Þ was either 0 phen normal 0 X y s8 , where X’ = pxð jX; Y Þ¼ but the LD between HLA-A*7401 and this allele was not statis- 0 for people with Y = 1, and otherwise X’ = X,or 0 phen 9 9 tically significant. normalðw0 þ wXX þ wyY ; s8Þ, where Y’ = 0 for people with X = 1, and p ð j otherwise Y’ = Y. The alternative model used is given by 1 phen

HLA-A*7401 is statistically associated with lowered viral load Downloaded from X; Y Þ¼normalðw0 þ wXX þ wyY;s8Þ. The variable “phen” is, for example, the viral load; X and Y are binary in chronic C-clade HIV-1 infection variables representing two HLA alleles; wX and wY are parameters in the To assess the relation between HLA-A*7401 and viral load in model for the effect of each allele; s is the variance parameter of the 8 chronic infection, we compared subjects with and without HLA- normal distribution; and w0 is an offset parameter. Parameters were fit independently for each model. The test statistic for an HLA pair was the A*7401 in four different cohorts: Durban, Gaborone, Bloemfon- difference in log likelihood between these null and alternative models after tein, and the Thames Valley cohorts (excluding Kimberley fitting each by maximum likelihood. The p values were obtained by 50,000 because of small numbers). In each case, HLA-A*7401 was as- http://www.jimmunol.org/ permutations of one HLA allele in the test. In addition, any pair of alleles for which one of the alleles did not have a univariate correlation in the sociated with a reduction in viral load in chronic infection [p = direction of control (as defined by the phenotype of interest) was assigned 0.0007 (Fig. 2A), p = 0.02, p = 0.2, and p = 0.05, respectively, a test statistic of zero. Note that two alleles in perfect linkage disequi- Mann–Whitney U test]. The trend toward lowered viremia in as- librium (LD) (or anti-LD) could not come up as having an additive effect sociation with HLA-A*7401 in Bloemfontein may not have with this test statistic. For binary outcomes (disease control based on cri- + reached statistical significance at least in part because of the small teria of viral load # 2000 RNA copies/ml plasma and CD4 T cell count . 250 cells/mm3), we used an analogous test based on logistic regression. number of HLA-A*7401 subjects in Bloemfontein (n = 8). In all To account for possible differences between cohorts that might cause southern African subjects, HLA-A*7401 was strongly associated artifact either by enhancing or obscuring additive effects between HLA with lowered viremia (median viral set point, 14600 versus 32450 alleles (i.e., confounding), we also corrected for geographical origin of the RNA copies/ml in HLA-A*7401+ versus HLA-A*74012; n = by guest on September 24, 2021 subject by adding a set of binary cohort covariates to each model (one for , recruitment in each of Durban, Kimberley, Bloemfontein, or Gaborone; we 1965 subjects with viral load data available; p 0.0001, Mann– removed Thames Valley subjects who originate from a wide variety of Whitney U test) (Fig. 2B). locations). An LRT test showed these cohort covariates to be highly sig- In subjects from Gaborone (where HLA-A*7401 is not in 259 + nificant (e.g., p =23 10 ) in predicting the log CD4 T cell count, so linkage disequilibrium with HLA-B*5703), CD4+ T cell counts these covariates were used in all models. Note that all HLA pairs coming were statistically higher in subjects with HLA-A*7401 (p = out as significant in these tests had parameter weights in the direction of control in the alternative model, indicating that better predictive power was 0.0005, Mann–Whitney U test) (Fig. 2C). This illustrates that obtained because both alleles were contributing to control rather than one HLA-A*7401 may mediate a protective effect that is independent negating the effect of the other. of linkage disequilibrium with HLA-B*5703. Associations between absolute CD4+ T cell count and expression of HLA-A*7401 did not reach statistical significance in the Durban, Bloemfontein, Results or Thames Valley cohorts (data not shown). However, in pooled HLA-A*7401 is common in African populations and is found in data from all subjects, there was a significant association between linkage disequilibrium with HLA-B*5703 in South Africa the presence of HLA-A*7401 and higher CD4+ T cell count HLA-A*74 was found in 216 subjects from a total of 2126 (median CD4+ cell count, 409 versus 350 in HLA-A*7401+ versus southern African subjects (phenotypic frequency, 10.2%) (Table HLA-A*74012; n = 1962 subjects with CD4+ count available; p = I). These were confirmed to be HLA-A*7401 in 181/216 cases 0.001, Mann–Whitney U test) (Fig. 2D). (84%); the remaining cases were typed only to 2-digit resolution as HLA-A*74. Thus, no four-digit HLA-A*74 allele other than HLA-A*7401 and HLA-B*5703 have an additive effect in HLA-A*7401 was identified in any of our cohorts. lowering viral load in chronic C-clade HIV-1 infection We confirmed LD between HLA-A*7401 and HLA-B*5703 The relation between the presence of HLA-A*7401 and improved among South African subjects from Durban, Kimberley, and disease control of HIV-1 may be confounded by haplotype, Bloemfontein (n = 1510, p = 1.1 3 1029, Fisher’s exact test) (Fig. whereby the observed effect of HLA-A*7401 on viral load arises 1A). In the epidemiologically unlinked study population of adults only as a consequence of linkage with favorable alleles, in par- in Botswana (n = 514), we did not identify statistically significant ticular, with HLA-B*5703 in South African subjects (Fig. 1A). To LD between these two alleles (Fig. 1A,1B). The apparent lack of mitigate this haplotype effect, we next investigated the impact of LD between HLA-A*7401 and HLA-B*5703 in Botswana does HLA-A*7401 on viral load after removing subjects with HLA- not appear to be attributable to a smaller sample size than B*5703 from the analysis. Among subjects with HLA-A*7401, our South African cohorts, because in the Botswana cohort, only there remained an association with lowered viremia in the absence 1/46 (2.2%) HLA-A*7401+ subjects also carried HLA-B*5703, of HLA-B*5703 (p = 0.002 in the pooled cohort, p = 0.09 in the whereas in the South African cohorts, 25/157 (15.9%) HLA- Durban cohort, Mann–Whitney U test) (Fig. 3A,3B). Strikingly, 5678 HLA-A*7401–MEDIATED IMMUNE CONTROL OF HIV-1

Table I. Summary of 2126 C-clade HIV-1–infected study subjects from ﬁve southern African study cohorts

Viral Load (RNA Copies/ml Plasma) CD4 T Cell Count (Cells/mm3) No. of Number (%) with Cohort Location Subjects HLA-A*7401a Number Median IQR Number Median IQR Durban, South Africa 1218 135 (11.1) 1218 38,200 7315–154,250 1159 376 239–519 Bloemfontein, South Africab 261 21 (8.0) 163 84,000 13,000–250,000 261 76 32–548 Kimberley, South Africa 31 1 (3.2) 14 47,000 8800–295,000 29 326 274–477 Gaborone, Botswana 514 46 (8.9) 471 19,100 3920–78,200 415 342 220–476 Thames Valley, southern Africa 102 13 (12.7) 99 5090 942–21,412 98 445 326–562 All Southern Africa 2126 216 (10.2) 1965 29,500 5715–133,500 1962 360 208–520 aThe 4-digit HLA type was determined in 181/216 subjects (84%); in all 181 of these (100%), the 4-digit type was HLA-A*7401. In the remaining 35 cases typed to 2 digits, the 4-digit type was undetermined. bBloemfontein data are for 261 HLA-typed subjects with CD4+ T cell count , 100 or . 500 cells/mm3. In an extended Bloemfontein cohort (including subjects without HLA types), median CD4+ cell count was 229 cells/mm3, with interquartile range (IQR) of 122–366 cells/mm3 (n = 885), and median viral load was 76,000, with IQR of 20,000– 21,0000 cells/mm3 (n = 500).

however, the combination of HLA-A*7401 and HLA-B*5703 African cohort (n = 97) and from Durban (n = 55) according to resulted in significantly lower viral loads than in the presence of viral load. We found significant enrichment of HLA-A*7401 in either allele alone, suggesting an additive effect of these alleles in subjects with viral loads less than the median in each case Downloaded from mediating control of disease (Fig. 3A,3B), as seen previously (23). (p = 0.015 in the whole cohort, p = 0.003 in Durban, Fisher’s exact Additional evidence for the independent role of HLA-A*7401 in test). Division of these HLA-B*5703+ subjects into four pools lowering viremia, irrespective of the presence of HLA-B*5703, by viral load quartiles confirmed enrichment of HLA-A*7401 in comes from the Botswana cohort in which there is no LD between subjects with the lowest viral loads (Fig. 3C,3D). This phenom- these alleles (Fig. 1), but HLA-A*7401 is still associated with enon is not associated with the presence of other favorable HLA-B favorable outcomes (see earlier and Fig. 2C). alleles, such as HLA-B*5702, -B*5801, or -B*8101, in subjects http://www.jimmunol.org/ As HLA-A*7401 is also transmitted in strong LD with HLA- with the lowest viral loads (data not shown), suggesting that HLA- B*1503 and HLA-B*3501 in both Botswana and South Africa A*7401 may indeed be responsible for mediating the effect. (Fig. 1A), we also sought to exclude either of these alleles as the To investigate whether the beneficial effect of HLA-A*7401 driving force behind the apparent benefit of HLA-A*7401. There occurs significantly in combination with HLA alleles other than was no relation between HLA-B*1503 or HLA-B*3501 and HLA-B*5703, we undertook a computational analysis of all 2126 lowered viremia in this cohort (data not shown); indeed, in Dur- southern African subjects. Using this newly developed test, we ban, HLA-B*1503 was actually statistically associated with higher sought evidence for additive effects of any combination of two viral loads (p = 0.03, Mann–Whitney U test; data not shown). The HLA alleles (collapsed to two-digit resolution), irrespective of by guest on September 24, 2021 overall relation between HLA-A*7401 and lowered viral load is, LD in improving disease control compared with the effect of any therefore, not likely to be attributable to linkage with either of these one allele alone. After correction for cohort location, we identified alleles. statistically significant additive effects between HLA-A*74 and To further investigate the possible additive effect of HLA- two favorable HLA-B alleles, HLA-B*57 and HLA-B*81, in me- A*7401 on disease control in subjects with HLA-B*5703, we diating control of viral load (Table II). No significant effect of ranked HLA-B*5703+ subjects from the whole pooled southern HLA-A*74 was determined in conjunction with any other allele in

FIGURE 1. LD between HLA-A*7401 and other class I alleles in southern African populations. A, All HLA-B and HLA-Cw alleles that are significantly linked to HLA-A*7401 in a pooled cohort from South Africa (Durban, Bloemfontein, and Kimberley cohorts, n = 1510) and in Botswana (n = 514) are shown. The p values were calculated by Fisher’s exact test and corrected for multiple comparisons using the Bonferroni approach, using the online tool at: http://www.hiv.lanl.gov/content/immunology/hla/hla_linkage.html. No significant linkage associations were identified for HLA-A*7401 among Zimbab- weans recruited via the Thames Valley cohort (n = 73, data not shown). All subjects with HLA-B*35 are pooled together as HLA-B*35(01). B, Percentage of HLA-A*7401+ subjects with HLA-B*5703 in each of five southern African cohorts. Statistically significant LD between these alleles is present only in the Durban cohort. The Journal of Immunology 5679

predicting preferential binding of the basic residues Arg or Lys (27, 33, 39). Identification of putative HLA-A*7401-restricted epitopes in HIV-1 Having predicted a peptide binding motif for HLA-A*7401, we identified regions of the HIV-1 proteome-containing potential HLA-A*7401–restricted epitopes using two approaches. First, we screened IFN-g ELISPOT responses from 1010 Durban subjects. We identified two statistically significant IFN-g responses made by 119 subjects with HLA-A*7401: these were to OLP-19 in p24 Gag (p = 1.7 3 1024) and OLP-100 in Rev (p = 1.8 3 1025; Fisher’s exact test) (Fig. 4, Table IV). Even after exclusion of the minority of subjects with HLA-A*74 typed to only two digits (n = 28), both of these associations remained statistically significant (p = 2.5 3 1024 and p = 1.8 3 1025, respectively). To define the optimal epitope within each of these OLPs, we identified a putative HLA-A*7401–restricted epitope based on the earlier pre-

dicted binding motif for HLA-A*7401 (L/I/M/Q at P2 and R/K at Downloaded from PC) (Table III). Within OLP-19 (IVQNLQGQMVHQAISPR), the peptides GR11 (GQMVHQAISPR, p24-Gag 8–18) and QR10 (QMVHQAISPR, p24-Gag 9–18) ﬁtted this motif. Within the Rev 17mer OLP-100 (RWRARQRQIHSISERIL), two putative overlapping HLA-A*7401–binding peptides were identiﬁed: Rev-RR9

(RQIHSISER, Rev 50–58) and Rev-RR11 (RQRQIHSISER, Rev http://www.jimmunol.org/ + FIGURE 2. Viral load (VL) and CD4 T cell count in the presence and 48–58). absence of HLA-A*7401 in adults with chronic HIV-1 C-clade infection The second approach we used to identify HIV-specific HLA- in southern African cohorts. A, VL in Durban, South Africa. B,VLin pooled southern Africa cohort (Durban, South Africa; Gaborone, Botswana; A*7401–restricted epitopes involved reference to previously de- Thames Valley subjects from southern Africa [Botswana, Malawi, South termined HLA-A*7401–associated HIV amino acid polymor- Africa, and Zimbabwe]). C, CD4+ T cell count in Gaborone, Botswana. D, phisms within Gag, Pol, and Nef population sequences from the CD4+ T cell count in pooled southern Africa cohort (as above). Median VL Durban cohort (3). Six sites of HIV polymorphism associated (RNA copies/ml plasma) or CD4+ T cell count (cells/mm3) for each group with HLA-A*7401 were identified (3, 40) (Table V). Based on is stated. Boxes show median, 25th and 75th percentile; whiskers 10th– the predicted HLA-A*7401 binding motif, we used these sites 90th percentile. The p values by Mann–Whitney U test. of point mutation to identify further putative HLA-A*7401–re- by guest on September 24, 2021 stricted epitopes, p17-KR9 (p17 12–20, KLDKWEKIR), Nef-SR9 maintenance of CD4+ T cell count. As discussed earlier, HLA- (SQARRHLAR, Nef 188–196), Protease-NR10 (NLAFPQGEAR, A*7401 is the only four-digit HLA-A*74 allele detected in these Pol 6–14), RT-QR9 (QIYPGIKVR, RT 269–277) and the over- populations, so these results are applicable specifically to the ef- lapping RT-SR10 (SQIYPGIKVR, RT 268–277), and RT-SK9 fect of HLA-A*7401. (SLTETTNQK, RT 467–475) (Table V). None of the corre- sponding OLPs (OLPs 2, 91, 145, 202, 227/228) had significant Prediction of the HLA-A*7401 binding motif IFN-g ELISPOT responses associated with the expression of Having established the association between expression of HLA- HLA-A*7401 (data not shown), possibly because of loss of the A*7401 and improved disease control in HIV-1, we next in- CD8+ T cell response after early selection of escape. Of the vestigated the likely mechanism for this effect. To seek evidence ELISPOT responses significantly associated with HLA-A*7401, of CD8+ T cell epitopes restricted by this allele, we predicted the the Gag-GR11 response appears immunodominant, occurring in peptide binding motif for HLA-A*7401 from the residues that .10% of subjects with HLA-A*7401 (Fig. 4). form the B and F pockets into which peptide residues at epitope positions two and PC would bind. This approach has previously Confirmation of optimal HLA-A*7401 epitopes in HIV-1 Gag, been validated for other HLA alleles prevalent in sub-Saharan RT, and Rev African populations, such as HLA-B*8101 and HLA-Cw*1801, To confirm the optimal epitope, we used IFN-g ELISPOT assays to for which the peptide binding motif has not been formally defined quantify CD8+ T cell responses to the optimal peptide and vari- by sequencing of eluted peptides (33). ant truncations of the peptide in individual subjects selected on The amino acid sequence determining the B pocket of the the basis that they made a response to the relevant OLP. Using peptide binding groove of HLA-A*7401 allele is identical to that this method, we acquired data to support the optimal epitope in of HLA-A*3201 and closely related to that of HLA-A*0101 (37) three of our predicted HLA-A*7401–restricted epitopes: p24-Gag- (Table III, top half). HLA-A*0101 preferentially binds epitopes GR11 (p24 Gag 8–18, GQMVHQAISPR), RT-QR9 (RT 269– bearing a small aliphatic residue, such as Thr/Ser/Ile/Met, at po- 277, QIYPGIKVR), and Rev-RR9 (Rev 50–58, RQIHSISER) sition 2 (33, 37, 38), but differs from the HLA-A*7401 allele by (Fig. 5A–C, respectively). In the case of Rev-RR9, we also syn- expressing a Met residue at position 67 of the molecule. In HLA- thesized the peptide–MHC class I tetramer to confirm the HLA A*7401, the smaller m.w. Val residue at position 67 may provide restriction of the response (Fig. 5D). a more capacious B pocket able to accommodate somewhat larger We also used LOCI (35) to confirm binding of HLA-A*7401 to aliphatic residues. At the F pocket (where the C-terminal position optimal peptides Gag-GR11, RT-QR9, and Rev-RR9 (Fig. 6). [PC] of the epitope is accommodated), HLA-A*7401 is identical These binding studies also add to the evidence for two other pu- to HLA-A*3301, -A*6601, and -A*6801 (Table III, bottom half), tative HLA-A*7401–restricted epitopes, p17-KR9 and Nef-SR9. 5680 HLA-A*7401–MEDIATED IMMUNE CONTROL OF HIV-1 Downloaded from http://www.jimmunol.org/

FIGURE 3. Effect of combinations of HLA-A*7401 and HLA-B*5703 on viral load. A and B, Viral load (RNA copies/ml plasma) of C-clade–infected adult study subjects from all study subjects (A) and Durban (B), in the presence and absence of HLA-B*5703 and HLA-A*7401. Boxes show median, 25th and 75th percentile; whiskers 10th–90th percentile. The p values were by Mann–Whitney U test. C and D, Subjects with HLA-B*5703 ranked by viral load by guest on September 24, 2021 and divided by quartiles into four interquartile (IQ) pools for all study subjects (n = 97) (C) and Durban (n = 55) (D). The proportion of each of the four subgroups with HLA-A*7401 is shown, demonstrating that HLA-A*7401 is enriched in the IQ pool with the lowest viral loads. The p values were by Fisher’s exact test.

There was no substantial difference in the strength of binding of binders to HLA-A*7401). We also tested binding of the only overlapping variants of our three conﬁrmed epitopes (p24-Gag HLA-A*7401 HIV-1 epitope that has previously been reported by epitopes p24-GR11/QR10, RT-SR10/QR9, and Rev-RR11/RR9) Los Alamos HIV databases (http://www.hiv.lanl.gov), Prot-IV9 (Fig. 6), suggesting that the optimal peptide for presentation is (Pol 58–67, ITLWQRPLV) (41); this does not match our motif not necessarily determined by differences in HLA class I binding. prediction at the PC, and was also a low binder. Together, these ELISPOT, tetramer, and binding data strongly support three new HLA-A*7401–restricted epitopes (Gag-GR11, In the presence of HLA-A*7401, the HLA-B*5703 Gag RT-QR9, and Rev-RR9) and point to two further likely epitopes footprint is diminished and HLA-B*5703 -restricted IFN-g (Gag-KR9 and Nef-SR9). These data also substantiate our pro- ELISPOT responses are preserved posed motif for HLA-A*7401 as L/I/M/Q at P2. At the PC of the To investigate the way in which HLA-A*7401 may ameliorate epitope, R appears to be the preference (our two putative epitopes disease progression in subjects with HLA-B*5703, we investigated bearing K at this position, p17-SK10 and RT-SK9, were both low the impact of HLA-A*7401 on the selection of polymorphisms and

Table II. Additive effects between HLA-A*74 and other HLA class I alleles in mediating good HIV-1 disease control

Criterion for Disease Control HLA-1 HLA-2 p Value q Value Absolute viral load A*74 B*57 2.00E-05 0.023 A*74 B*81 2.00E-05 0.023 Viral load , 2000 copies/ml A*74 B*57 2.00E-05 0.023 A*74 B*81 2.00E-05 0.023 Absolute CD4+ T cell count A*74 NS CD4 T cell count . 250 cells/mm3 A*74 NS Analysis of HLA type, viral load (n = 1972), and CD4 count (n = 1871) in adults with chronic HIV-1 infection from pooled subjects from southern Africa (South Africa, Botswana, Zimbabwe, and Malawi). All associations between HLA-A*74 and any other allele (q , 0.2) are shown. NS, not signiﬁcant. The Journal of Immunology 5681

Table III. Sequences of HLA-A*7401 and related HLA-A alleles at the B and F pockets of the peptide binding groove and predicted residues bound at these positions

Residues Determining B Pocket

7 9 24 25 34 45 63 66 67 70 99 (Predicted) Residue at P2 HLA-A consensus Y F A V V M E NVHY HLA-A*0101 — — ——————M—— T/S/I/M HLA-A*3201 — — ————————— (I/M) HLA-A*7401 — — ————————— (L/I/M/Q)

Residues Determining F Pocket

77 80 81 84 95 116 123 143 146 147 (Predicted) Residue at PC HLA-A consensus D T L Y I D Y T K W HLA-A*3303 ————— — — — — — R HLA-A*6601 ————— — — — — — R/K HLA-A*6801 ————— — — — — — R/K HLA-A*7401 ————— — — — — — (R/K) Residues bound at the B and F pockets (position 2 and C terminus of the epitope, respectively) are based on those previously published (33, 37).

—, Indicates conservation of the residue shown in the HLA-A consensus sequence. Downloaded from on IFN-g ELISPOT responses associated with HLA-B*5703. from the Durban and Thames Valley cohorts. Although responses HLA-B*5703 is strongly associated with the selection of five to individual OLPs containing HLA-B*5703 epitopes were not mutations in p24 Gag, at positions A146, I147, A163, S165, and statistically associated with the presence or absence of HLA- T242, within and flanking the epitopes ISW9 (p24 Gag 147–155, A*7401, we found overall preservation of an IFN-g response to ISPRTLNAW), KF11 (p24 Gag 162–172, KAFSPEVIPMF), and these OLPs in the presence of HLA-A*7401 (p = 0.006 for re- http://www.jimmunol.org/ TW10 (p24 Gag 240–249, TSTLQEQIAW) (3, 6, 16, 17). sponse to any OLP containing an HLA-B*5703 epitope, Fisher’s In 64 subjects with HLA-B*5703, there was a trend toward exact test) (Fig. 7B). The trend toward maintenance of these IFN-g preservation of the wild-type amino acid in the presence of HLA- ELISPOT responses is in keeping with the sequence data showing A*7401 at 4 of the 5 sites of polymorphism (I147, A163, S165, preservation of wild-type epitopes in the presence of HLA- and T242), although this only reached statistical significance at A*7401. S165 (p = 0.004, Fisher’s exact test; Fig. 7A). The exception was A146, flanking the ISW9 epitope. However, selection at this po- Discussion sition may occur as a consequence of many different alleles be- These studies add to recently published literature (19, 23) sub- by guest on September 24, 2021 cause of the extensive overlap of CD8+ T cell epitopes at this stantiating the beneficial influence of HLA-A*7401 on disease position (including HLA-B*3910, HLA-B*1510, HLA-B*5801, control in adult subjects with HIV-1 infection, both alone and in and B*4801) (10). Subjects with HLA-A*7401 were also less tandem with HLA-B*5703. HLA-A*7401 is common in black likely than HLA-A*74012 subjects to have mutations at all five African populations (occurring at a 10.2% phenotypic frequency . sites (p = 0.03, Fisher’s exact test) (Fig. 7A). in our extended cohort of 2100 southern African subjects), but To determine whether the presence of HLA-A*7401 has an has been relatively understudied compared with many HLA-B impact on HLA-B*5703 responses to Gag epitopes, we studied alleles. ELISPOT data from 46 HLA-B*5703+ southern African subjects The transmission of HLA-A*7401 in LD with HLA-B*5703 in the Durban cohort highlights the difficulties of appreciating the influence of a single allele. Although our calculations to verify LD do not, of course, demonstrate, in an individual subject, on which haplotype the HLA-A*7401 and HLA-B*5703 alleles are located, they show that statistically these alleles are likely to arise on the same haplotype when coexpressed in a particular individual, and this is verified by haplotype analysis using PyPop (36) (Sup- plemental Table I). Given the well-established benefits of HLA-B*5703, it would be easy to dismiss the apparent association between HLA-A*7401 and low viremia as simply caused by linkage with HLA-B*5703. However, in addition to our data demonstrating a significant HLA-A*7401–restricted response through CD8+ T cell epitopes, we also present two further strands of evidence for an independent role for HLA-A*7401. First, the FIGURE 4. Proportion of subjects with and without HLA-A*7401 favorable effect of this allele on viremic control observed in a making IFN-g ELISPOT responses to HIV-1 C-clade OLPs. IFN-g ELI- Botswana cohort (in which these two alleles are not transmitted SPOT responses to OLP-19 and OLP-100 are significantly enriched among in LD) suggests a true independent benefit of HLA-A*7401. subjects with HLA-A*7401 (Durban cohort; n = 1010; p values by Fisher’s Second, we have identified a consistent additive effect of HLA- exact test). Subjects with HLA-B*1510 are removed from the analysis of responses to OLP-100 to avoid potential confounding because of the A*74 and HLA-B*57 through a novel computational approach to presence of an overlapping HLA-B*1510 epitope IL-9 (IHSISERIL, Rev the analysis of all possible pairs of two alleles at the A, B, and C 52–60) (29, 56), accounting for higher than expected rate of responses in loci in .2100 subjects. the HLA-A*74012 population (method as previously described [13]). n = Our results demonstrate that HLA-A*7401 is associated with number making response/total number of subjects. lower viral loads and higher CD4+ T cell counts in several 5682 HLA-A*7401–MEDIATED IMMUNE CONTROL OF HIV-1 Downloaded from http://www.jimmunol.org/

FIGURE 5. In vitro data to support three new HLA-A*7401–restricted epitopes: Gag-GR11, RT-QR9, and Rev-RR9. A, Magnitude of IFN-g ELISPOT responses to Gag-GR11 (GQMVHQAISPR), the alternative epitope QR10 (QMVHQAISPR), and other peptide truncations in a chronically infected adult by guest on September 24, 2021 subject from South Africa (subject ID SK362: HLA-A*0301/A*7401/B*1503/B*1510/Cw*0210/Cw*0401). B, Magnitude of IFN-g ELISPOT responses to RT-QR9 (QIYPGIKVR) and peptide truncations in a chronically infected adult subject from Zimbabwe (subject ID H005: HLA-A*7401/A*3004/B*3501/ B*5802/Cw*0401/Cw*0602). C, Magnitude of IFN-g ELISPOT responses to Rev-RR9 (RQIHSISER), the alternative epitope RR11 (RQRQIHSISER), and the OLP containing these epitopes (OLP-100) in a chronically infected adult subject from Uganda (subject ID R070: HLA-A*0201/A*7401/B*4016/ B*4901/Cw*0701/Cw*0802). D, FACS plot gated on live CD3+ population of PBMCs from subject R070, stained with MHC class I tetramer HLA- A*7401-Rev-RR9 conjugated to PE fluorochrome, showing a tetramer-positive population of CD3+ CD8+ T lymphocytes. southern African cohorts. Although these associations do not presence of epitopes in Gag may contribute to viremic control in reach statistical significance in every cohort, the trends are broadly two specific ways. First, the presence of Gag epitopes suggests the consistent in different settings. There are several possible ex- potential for an HLA-A*7401–restricted response to emerge early planations for the lack of strong correlation with CD4+ T cell in the course of acute infection because of the abundance of Gag counts in cohorts other than Gaborone. First, viral load and CD4+ protein at the initiation of infection (43). Second, of the six se- T cell count are correlated, but not always strongly (typical cor- quence polymorphisms we identified in association with HLA- relation coefficients reported are of r2 = 0.28) (42), so an associ- A*7401, three have previously been predicted to revert to wild- ation between HLA-A*7401 and lowered viremia does not ne- type after transmission to an HLA-mismatched host (Table V), cessarily translate into a statistically significant association with suggesting a fitness cost imposed by the mutation (3). In these higher CD4+ T cell count, especially where cohort size is small. instances, we predict that selection of the mutation itself may Indeed, this same observation has been made in a previous study of contribute to suppression of viremia in subjects with HLA- HLA associations with viral set point or absolute CD4+ T cell A*7401 (3) through an effect on viral infectivity and/or replicative count (23), in which some alleles were significantly associated with capacity. This effect has previously been shown in mutations in low viral set point, for example, but not with high absolute CD4+ HLA-B*57 epitopes that contribute to a reduced fitness virus (6, T cell count. An alternative possible explanation of this observation 17) and are therefore associated with clinical control of disease might be that certain HLA alleles have a stronger impact on CD4 (44–46). In particular, the presence of two reverting sites in Gag count than on viral load, or vice versa, for reasons unknown. highlights specifically that HLA-A*7401 may contribute to vire- Using motif prediction undertaken according to previously mic control through the selection of costly mutations in the highly validated methodology (33), in combination with ELISPOT and structurally constrained Gag protein (17, 47–49). sequence data, we identified eight potential sites of HLA-A*7401– To date, only one putative HIV-1 epitope restricted by HLA- restricted CD8+ T cell epitopes in HIV-1 Gag, Pol, Rev, and Nef A*7401 is listed by Los Alamos HIV databases “A-list” (http:// (Tables IV, V). Putative epitopes at five of these sites are in- www.hiv.lanl.gov), Prot-IV9 (Pol 58–67 ITLWQRPLV) (41). termediate or good binders to HLA-A*7401. In particular, the However, this epitope sequence was determined through pre- The Journal of Immunology 5683

nology Database “A-list.” These criteria include titration curves showing recognition of the optimal epitope at serial peptide dilutions compared with four additional peptides, each differing from the optimal by one amino acid, either longer or shorter by one amino acid at the N or C termini, respectively (see Fig. 5A, 5B), or unequivocal staining with a peptide–MHC tetramer (see Fig. 5D). Peptides have been described that do not precisely fit the established peptide binding motif for the respective HLA molecule (50), but these are the exception rather than the rule. In contrast, more typically, a peptide that does not fit the peptide binding motif (such as Prot-IV9; Fig. 6) proves not to bind once the peptide–MHC binding assays are undertaken. Notably, the approach we adopted using HLA-associated polymorphisms to identify location of an epitope successfully en- abled us to identify optimal HLA-A*7401–restricted epitopes, but this approach is challenging because, by definition, few HLA- A*7401+ individuals make responses to epitopes where escape mutations are so readily selected. Despite screening cryopreserved

PBMCs from 15 HLA-A*7401+ subjects from the Thames Valley Downloaded from cohort against OLPs and optimal peptides for putative epitopes restricted by this allele, we identified only two responses: one each in subjects H005 and R070 (Fig. 5). On a follow-up visit (after an FIGURE 6. Binding of HLA-A*7401 to putative optimal epitopes, in- interval of 23 mo), H005 had lost the IFN-g response to RT-QR9, cluding overlapping variants of epitopes in p17, RT, and Rev and sequence and we were therefore unable to proceed with further restriction + variants of the epitope in Nef. Binding affinity KD (nM) of HLA-A*7401 of this epitope. These findings suggest that HLA-A*7401 CD8 http://www.jimmunol.org/ to optimal peptides assessed by LOCI (35). Strength of binding is classified T cell responses may be short-lived (arising early in the course of on a log scale in accordance with previous methods (35). infection and being lost as a consequence of immune escape), or may be subdominant compared with greater magnitude responses diction, and the HLA restriction has not been demonstrated. The restricted by HLA-B alleles, and therefore difficult to detect us- binding studies shown in this article suggest this peptide is a ing this IFN-g ELISPOT screening method. However, despite the very low binder of HLA-A*7401 (Fig. 6), and together with two challenges of confirming HLA-A*7401–restricted responses, in this of the putative epitopes presented in this article (Prot-NR10 and article, we present in vitro data (binding studies and tetramer data) RT-SK9), it is therefore less likely to be a commonly targeted that support three new epitopes: Gag-GR11 (GQMVHQAISPR), HLA-A*7401–restricted epitope. A further two putative over- RT-QR9 (QIYPGIKVR), and Rev-RR9 (RQIHSISER). by guest on September 24, 2021 lapping HLA-A*7401–restricted epitopes have previously been In these in vitro experiments, HLA-A*7401 constructs were proposed on the basis of an HLA-A*7401–associated poly- used, confirming the binding of this allele (resolved to high res- morphism at position Gag-441 (24). One of these did fit the HLA- olution) to predicted peptides. Although ELISPOT responses to A*7401 binding motif proposed in this article, with Lys at the PC; a particular peptide cannot necessarily be attributed to one specific however, the other lacked a positively charged residue at PC. allele (in this case, HLA-A*7401), the combined approach of No HLA-A*7401 binding data were provided for these proposed analysis of ELISPOT and sequence data, motif inference, in vitro epitopes, and these peptide sequences did not meet the more binding studies, and tetramer binding is a robust method of rigorous criteria for epitopes set out in the Los Alamos Immu- highlighting putative novel epitopes.

FIGURE 7. Relation between HLA-A*7401 status and frequency of HLA-B*5703–restricted CD8+ T cell mutations/IFN-g ELISPOT responses. A, Frequency of HLA-B*5703–selected Gag polymorphisms [deﬁned by previous studies (3, 6, 16, 17)], in 64 subjects with HLA-B*5703 according to the presence/absence of HLA-A*7401. p24-Gag sequences from Bloemfontein (n = 8), Durban (n = 38), Gaborone (n = 11), and Thames Valley cohorts (n = 7), with sequence data available for TW10 (n = 63), KF11 (n = 64), ISW9 (n = 63), and all three epitopes (n = 62). B, Frequency of IFN-g ELISPOT response to three HLA-B*5703 epitopes in p24-Gag in 46 subjects with HLA-B*5703 according to the presence or absence of HLA-A*7401. The p values were by Fisher’s exact test. Data from Durban (n = 31) and Thames Valley cohorts (n = 15). 5684 HLA-A*7401–MEDIATED IMMUNE CONTROL OF HIV-1

Table IV. Putative HLA-A*7401–restricted epitopes predicted using motif inference (32) combined with analysis of IFN-g ELISPOT assays

Predicted HLA-A*7401 Epitope

P2 C Terminus Protein OLP Number C-clade OLP Sequence p Valuea ↓↓Epitope Designation p24-Gag 19 IVQNLQGQMVHQAISPR 1.7 3 1024 Q M VHQAISP R (Gag-QR10) G Q MVHQAISPR (Gag-GR11) Rev 100b RWRARQRQIHSISERIL 1.8 3 1025 R Q IHSISE R (Rev-RR9) R Q RQIHSISER (Rev-RR11) Two C-clade 17-mer OLPs signiﬁcantly associated with IFN-g ELISPOT responses in subjects expressing HLA-A*7401 are shown (Durban and Thames Valley cohorts, as shown in Fig. 4; p values by Fisher’s exact test). Arrows indicate the anchor positions at which the optimal binds the class I allele (position 2 and C terminus); these residues are shown in bold and underlined. aThe p values both remain statistically signiﬁcant if analysis repeated after removal of minority of subjects with HLA-A*74 typed to only two-digit resolution: OLP-19, p = 2.5 3 1024; OLP-100, p =1.83 1025. bResponses to OLP-100 are corrected by removal of subjects with HLA-B*1510 from analysis, because of presence of HLA-B*1510 epitope IL9 (IHSISERIL, Rev 52–60; http://www.lanl.gov).

The mechanism by which the additive effect of HLA-A*7401 effects resulting from LD between HLA-B*57 and other alleles and HLA-B*5703 arises is uncertain. The preservation of wild- may contribute to the B*57 effect. The data presented in this ar- type HLA-B*5703 epitopes and the related maintenance of an ticle demonstrate that, for HLA-A*7401, this is indeed the case. Downloaded from HLA-B*5703–restricted CD8+ T cell response in the presence of As cohort sizes increase, it will become possible to dissect out the HLA-A*7401 may be the cause or effect of lowered viremia. haplotypic effects arising from the presence of several distinct Several potential mechanisms may explain the favorable effect of HLA and non-HLA alleles on the same chromosome. this haplotype. First, there may simply be an additive effect of In addition to HLA-A*7401, there may be other examples of multiple CD8+ T cell responses consequent on the dual pre- HLA-A alleles that are associated with good viremic control but in sentation of epitopes restricted by both alleles on the surface of which the benefit is obscured by the influence of HLA-B alleles http://www.jimmunol.org/ infected cells, particularly given the Gag-directed responses re- with which they are linked. One such example is HLA-A*6601. stricted by both alleles. The preservation of wild-type sequence in This occurs in LD with HLA-B*5802 in Durban (p value for HLA-B*5703–restricted Gag epitopes in the presence of HLA- linkage = 3.5 3 1027, Fisher’s exact test), an allele associated A*7401 suggests the possibility that subdominant responses with poor viremic control and rapid progression to AIDS (13, 55). from the HLA-A allele delay HLA-B–selected escape mutations, When ranked according to median viremia in chronic infection, allowing preservation of responses to epitopes restricted by HLA- HLA-A*6601 appears to be among the worst performing of HLA- B*5703. Further protection might be afforded by HLA-A*7401– A alleles; however, when subjects with HLA-B*5802 are removed peptide complexes as killer Ig-like receptor ligands (51), acti- from the analysis, a significant association between HLA-A*6601 vating a favorable NK response. Finally, we cannot exclude the and good control of viremia emerges (23). by guest on September 24, 2021 presence of another confounding variable, either within or outside In summary, this work represents investigation of HLA-A*7401 the MHC locus, that is transmitted in linkage with HLA-A*7401, and the HLA-A*7401-B*5703 haplotype. Although this allele has which is responsible for enhanced viremic control. recently been recognized to be associated with favorable viremic It is well-known that HLA-B*5703 and other HLA-B*57 alleles control (19, 23, 24), the mechanism for this phenomenon has not are strongly associated with control of HIV. The mechanism for previously been studied. Despite the strong favorable effect me- this control may hinge on the presentation of multiple Gag epit- diated by HLA-B*5703 on control of viremia (3, 6, 13, 16, 23), we opes (3, 8, 52), the selection of escape mutations that impose show in this article that the HLA-A*7401-B*5703 haplotype a cost to viral fitness (3, 15, 17, 48), and the interaction with is more favorable than HLA-B*5703 alone. Our data do not KIR3DS1 to activate a favorable NK cell response (53, 54). demonstrate a single explanation for this effect, but we present However, it remains unclear why some people with HLA-B*57 compelling evidence for an independent CD8+ T cell response have better immune control than others. One possibility is that restricted by HLA-A*7401. These findings highlight the poten-

Table V. Putative HLA-A*7401–restricted epitopes predicted using motif inference (32) combined with analysis of HLA-A*7401 associations with sequence polymorphisms

Durban Consensus Sequencea Predicted HLA-A*7401 Epitope

HXB2 Position of A*7401 Polymorphism P2 C Terminus Epitope Protein Polymorphism ▼ Reversion p Value q Value ↓↓Designation p17 Gag 12 GARASILRGEK LDKWEKIRLR R 2.71E-14 0.00 S I LRGEKLDK (Gag-SK10) p17 Gag 20 GEKLDKWEKIR LRPGGKKHYM R 6.47E-10 0.00 K L DKWEKI R (Gag-KR9) Protease 9 FFRENLAFP QGEAREFPSE 4.83E-05 0.02 N L AFPQGEAR (Prot-NR10) RT 277 ASQIYPGIKVR QLCKLLRGAK 3.13E-04 0.13 Q I YPGIKV R (RT-QR9) S Q IYPGIKVR (RT-SR10) RT 476 IVSLTETTNQK TELQAIQLAL 6.03E-04 0.15 S L TETTNQ K (RT-SK9) Nef 192 KWKFDSSLARR HLARELHPEY R 4.95E-05 0.00 S L ARRHLA R (Nef-SR9) Six sites of HLA-A*7401–associated polymorphism identified from lineage-corrected analysis of sequences from a total of 710 Durban subjects (q , 0.2), as previously published (3). The position of the polymorphism is marked ▼, with consensus sequence 10 aa upstream and downstream of the site of polymorphism. Arrows indicate anchor positions (shown in bold and underlined) as in Table IV. R indicates polymorphisms that are predicted to revert to wild-type after transmission to an HLA-mismatched recipient [methods as previously described (17, 57)]. aSequences based on Durban consensus sequence from Gag (n = 446) and Nef (n = 436) subjects. The Journal of Immunology 5685 tial for HLA class I alleles to work in tandem to influence 18. Kawashima, Y., M. Satoh, S. Oka, T. Shirasaka, and M. Takiguchi. 2008. Dif- ferent immunodominance of HIV-1-specific CTL epitopes among three subtypes HIV-1 disease control, and demonstrate the importance of of HLA-A*26 associated with slow progression to AIDS. Biochem. Biophys. studying HLA class I alleles that arise in different populations; Res. Commun. 366: 612–616. HLA-A*7401 is not found in white subjects, but is common, and 19. Koehler, R. N., A. M. Walsh, E. Saathoff, S. Tovanabutra, M. A. Arroyo, J. R. Currier, L. Maboko, M. Hoelscher, M. L. Robb, N. L. Michael, et al. 2010. appears to exert considerable influence, in black African cohorts. Class I HLA-A*7401 is associated with protection from HIV-1 acquisition and + Furthermore, our data highlight the importance of a broad CD8 disease progression in Mbeya, Tanzania. [Published erratum appears in 2011 J. T cell response, especially targeting Gag peptides, and suggest Infect. Dis. 203: 749.] J. Infect. Dis. 202: 1562–1566. + 20. Schaubert, K. L., D. A. Price, N. Frahm, J. Li, H. L. Ng, A. Joseph, E. Paul, the utility of combining multiple CD8 T cell responses in a po- B. Majumder, V. Ayyavoo, E. Gostick, et al. 2007. Availability of a diversely tential vaccine. avid CD8+ T cell repertoire specific for the subdominant HLA-A2-restricted HIV-1 Gag p2419-27 epitope. J. Immunol. 178: 7756–7766. 21. Tang, J., W. Shao, Y. J. Yoo, I. Brill, J. Mulenga, S. Allen, E. Hunter, and Disclosures R. A. Kaslow. 2008. Human leukocyte antigen class I genotypes in relation to The authors have no financial conflicts of interest. heterosexual HIV type 1 transmission within discordant couples. J. Immunol. 181: 2626–2635. 22. Fellay, J., K. V. Shianna, D. Ge, S. Colombo, B. Ledergerber, M. Weale, K. Zhang, C. Gumbs, A. Castagna, A. Cossarizza, et al. 2007. A whole-genome References association study of major determinants for host control of HIV-1. Science 317: 1. Brumme, Z. L., I. Tao, S. Szeto, C. J. Brumme, J. M. Carlson, D. Chan, C. Kadie, 944–947. N. Frahm, C. Brander, B. Walker, et al. 2008. Human leukocyte antigen-specific 23. Leslie, A., P. C. Matthews, J. Listgarten, J. M. Carlson, C. Kadie, T. Ndung’u, polymorphisms in HIV-1 Gag and their association with viral load in chronic C. Brander, H. Coovadia, B. D. Walker, D. Heckerman, and P. J. Goulder. 2010. untreated infection. AIDS 22: 1277–1286. Additive contribution of HLA class I alleles in the immune control of HIV-1 2. Goulder, P. J., and D. I. Watkins. 2008. Impact of MHC class I diversity on infection. J. Virol. 84: 9879–9888. immune control of immunodeficiency virus replication. Nat. Rev. Immunol. 8: 24. Semeniuk, C. A., L. McKinnon, H. O. Peters, M. Gubbins, X. Mao, T. B. Ball, Downloaded from 619–630. M. Luo, and F. A. Plummer. 2009. Multiple T-cell epitopes overlap positively- 3. Matthews, P. C., A. Prendergast, A. Leslie, H. Crawford, R. Payne, C. Rousseau, selected residues in the p1 spacer protein of HIV-1 gag. AIDS 23: 771–777. M. Rolland, I. Honeyborne, J. Carlson, C. Kadie, et al. 2008. Central role of 25. Catano, G., H. Kulkarni, W. He, V. C. Marconi, B. K. Agan, M. Landrum, reverting mutations in HLA associations with human immunodeficiency virus set S. Anderson, J. Delmar, V. Telles, L. Song, et al. 2008. HIV-1 disease- point. J. Virol. 82: 8548–8559. influencing effects associated with ZNRD1, HCP5 and HLA-C alleles are at- 4. O’Brien, S. J., X. Gao, and M. Carrington. 2001. HLA and AIDS: a cautionary tributable mainly to either HLA-A10 or HLA-B*57 alleles. PLoS ONE 3: e3636. tale. Trends Mol. Med. 7: 379–381. 26. Naugler, C. 2010. Origins and relatedness of human leukocyte antigen class I

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