Identification of Novel HLA-A2-Restricted Human ... - Journal of Virology

JOURNAL OF VIROLOGY, Feb. 2001, p. 1301–1311 0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.3.1301–1311.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 3

Identification of Novel HLA-A2-Restricted Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Epitopes Predicted by the HLA-A2 Supertype Peptide-Binding Motif MARCUS A. ALTFELD,1 BRIAN LIVINGSTON,2 NEHA RESHAMWALA,1 PHUONG T. NGUYEN,1 MARYLYN M. ADDO,1 AMY SHEA,1 MARK NEWMAN,2 JOHN FIKES,2 JOHN SIDNEY,2 PEGGY WENTWORTH,2 ROBERT CHESNUT,2 ROBERT L. ELDRIDGE,1 ERIC S. ROSENBERG,1 GREGORY K. ROBBINS,1 CHRISTIAN BRANDER,1 PAUL E. SAX,3 STEVE BOSWELL,4 THERESA FLYNN,1 SUSAN BUCHBINDER,5 PHILIP J. R. GOULDER,1 BRUCE D. WALKER,1 ALESSANDRO SETTE,2 AND SPYROS A. KALAMS1* Partners AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School,1 Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School,3 and Fenway Community Health Center,4 Boston, Massachusetts, and Epimmune, Inc., San Diego,2 and AIDS Office, Department of Public Health, San Francisco,5 California Received 1 August 2000/Accepted 30 October 2000

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2restricted CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines. The logic behind characterizing the cellular immune response is that HIV-1-specific cytotoxic T lymphocytes (CTL) are considered to play a central role in the immune response against HIV-1 (2, 11, 33). High levels of HIV-1-specific CTL are detectable in subjects with asymptomatic chronic infection (38–40, 63, 81) but generally decline with disease progression (48). Furthermore, in vitro studies have demonstrated potent inhibition of viral replication by HIV-1-specific CTL, mediated by both lytic and nonlytic mechanisms (86), and in vivo there is strong evidence that AIDS viruses evolve to escape CTL recognition by epitope-specific mutations (1, 8, 21, 29, 32, 49, 62). The critical role of virus-specific CTL responses for the control of viremia has been directly demonstrated by CD8⫹ T-cell depletion studies in simian immunodeficiency virus infection in macaques, showing that CD8⫹ T cells effectively suppress viral replication (42, 72). Recent data derived from HIV-1-infected individuals who were treated during acute HIV-1 infection showed enhancement of both CTL and T-helper cell responses against HIV-1 associated with subsequent viral control following supervised treatment interruptions (65). Furthermore, HIV-1-specific CTL responses (66–68), as well as the possession of HLA class I alleles of the HLA-A2 superfamily (51), have been associated with resistance to HIV-1 infection in heavily exposed sex workers. These data suggest that the induction of HIV-1-specific CTL responses in vivo may help to prevent infection of uninfected individuals or attenuate HIV-1 disease in infected individuals. The precise characterization of epitope-specific CTL responses is important for assessing HIV pathogenesis and vac-

Efforts to control disease progression associated with infection by human immunodeficiency virus type 1 (HIV-1) have led to the use of a combination of antiviral drugs, referred to as highly active antiretroviral therapy (HAART). Treatment with HAART has dramatically decreased the morbidity and mortality caused by HIV-1 (15, 56). HAART effectively lowers viral load and allows for a partial reconstitution of immunological function in a majority of HIV-1-infected patients (5, 6, 19, 47, 50, 55, 75, 83). However, the restoration of HIV-1specific immune responses is normally not observed in individuals treated during chronic HIV-1 infection (5, 60). In fact, HIV-1-specific cellular immune responses may actually decline as HAART continues, an effect thought to be associated with the lack of exposure of the immune system to viral antigen (46, 54, 61). Despite the initial success of HAART in controlling HIV-1, this therapeutic approach also has limitations. The initial antiretroviral treatment fails in 20 to 55% of patients, due to adverse side effects of the drugs, the development of resistant virus, and problems with adherence (14, 16, 23, 57, 58, 71). Secondary salvage regimens have even higher failure rates (17, 22, 23, 36, 64). In addition, most people infected by HIV-1 worldwide have no access to antiretroviral treatment due to the high cost of this regimen. Therefore, the need for a vaccine preventing HIV-1 infection or attenuating disease remains paramount.

* Corresponding author. Mailing address: MGH-East, CNY, 5th Floor, 149 13th St., Charlestown, MA 02129. Phone: (617) 726-8166. Fax: (617) 726-5411. E-mail: [email protected]. 1301

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TABLE 1. Subjects evaluated for CTL responses against HLA-A2 binding peptides CD4 cell count (cells/mm3)

Viral load (copies/ml) Subject

HLA type

At the time of screening

At the time of confirmation

At the time of screening

At the time of confirmation

AC01 AC03 AC04 AC07 AC09 AC13 AC14 AC22 AC26 AC29 AC35 AC41 PG 16842 14142 157i 11442 15760 161j RRP-004 16732 11324 12801 17548 17697 13070 14279 13010 021-22IL 19921 115I 011-441G MA010 13499

Aⴱ0201/3, B35/Aⴱ0201/3, B7/44 Aⴱ0201/11, B18/44 Aⴱ0201/30, B62/63 Aⴱ222/31, B61/Aⴱ0201/68, B44/14 A2/3, B8/62 Aⴱ0201/28, B60/63 A2/23, B44/A1/ⴱ0201, B8/15 Aⴱ0203/74, B18/72 A2/11, B35/44 A2/30, B44/51 Aⴱ0201/11, B27/58 Aⴱ0201/-, B37/44 A2/3, B40/51 A1/2, B14/57 Aⴱ0201/-, B44/51 Aⴱ0201/3, B7/60 Aⴱ0201 A2/3, B7/8 Aⴱ0201/3, B7/62 Aⴱ0201/11, B7/35 A2/3, B35/62 Aⴱ0201/25, B7/18 Aⴱ0201/3, B7/61 Aⴱ0201/11, B13/35 Aⴱ0201/33, B ND A2/-, B7/8 A2/3, B35/51 Aⴱ0201/28, B14/52 A2/24, B18/55 A2/68, B7/44, Cw5/7 Aⴱ0201/24, B27/39

⬍50 ⬍50 ⬍50 ⬍50 28,700 ⬍50 ⬍50 ⬍50 ⬍50 3,420 37,300 ⬍50 500 977 ND ⬍500 851 1,200 ⬍500 ND ⬍500 697 2,665 ⬍500 ND 5,390 89,900 ⬍500 201,000 116,600 ND ND ⬍50 32,170

⬍50 ⬍50 ⬍50 ⬍50 ⬍50 ⬍50 ⬍50 ⬍50 ⬍50 ⬍50 ⬍50 110 ⬍50 28,907 26,943 6,090 1,714 963 ⬍500 ND 86 ND 915 ⬍500 10,280 ND 531 ⬍500 ND ⬍500 ND 2,740 ⬍50 19,080

964 1,019 754 641 365 814 961 486 577 870 340 NDa 500 685 ND 414 862 692 ⬎500 ND 670 576 200 153 770 358 277 712 723 308 524 ND 563 810

1,025 781 740 554 567 815 778 554 684 998 527 815 565 781 523 506 873 514 ⬎500 ND 489 ND 249 180 523 ND 475 967 ND 422 528 792 584 789

a b c

Treatmentb

2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, 2 NA, None None None 2 NA, None None None 2 NA, 2 NA, 2 NA, 2 NA, None 2 NA 2 NA, 2 NA, None 2 NA, None 2 NA, 2 NA, None

1 1 1 1 1 1 1 1 1 1 1 1 1

PI PI PI PI PI PI PI PI, HU PI NNRTI PI PI PI

1 PI

1 1 1 1

PI PI PI PI

1 PIc 1 PI 1 PIc 1 PI 1 PI

ND, not done. NA, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor; PI, protease inhibitor; HU, hydroxyurea. These patients had no treatment at the time of screening and the indicated treatment at the time of confirmation.

cine candidates. In this study, potential epitopes were identified by scanning HIV-1 proteins for peptides containing the HLA-A2 supermotif. The use of major histocompatibility complex (MHC) class I binding motifs to identify CTL epitopes facilitates identification of those peptides that will bind to numerous different HLA class I antigens and therefore provide for an increased population coverage (28). Conserved motifbearing peptides were tested for binding to HLA-A2 supertype alleles, and peptides with cross-reactive HLA-A2 binding affinity were tested for CTL recognition by HIV-1-infected individuals. Utilizing this process, seven previously unreported HIV-1-derived epitopes were identified. MATERIALS AND METHODS Subjects. A total of 41 individuals were included in this study (Table 1). The studied subjects were divided into three groups. Group A included 22 individuals with chronic HIV-1 infection. In group B, 12 individuals who were diagnosed and treated very early after HIV-1 infection were included. Eight of these subjects had a positive HIV-1 plasma RNA result with a negative HIV-1 enzyme-linked immunosorbent assay (ELISA) result, two subjects had a weakly positive HIV-1 ELISA result and an evolving HIV-1 Western blot result, with less than three bands, and two subjects had fully HIV-1 seroconverted, but HIV-1 infection

within the previous 180 days had been confirmed by an adapted ELISA (41) and a recent syndrome compatible with acute HIV infection (45). All these individuals were treated with HAART at the time of the study. Group C consisted of seven HIV-1-negative control subjects. All 41 individuals studied expressed the HLA-A2 allele, and of the 21 HIV-1-infected individuals who were subtyped for HLA-A2, 19 (91%) expressed HLA-Aⴱ0201 (Table 1). For each HIV-1-infected subject, samples from two independent time points were analyzed, and one sample was analyzed from the HIV-1-negative individuals of group C. The median time interval between analyzed samples was 13 months (range, 0 to 31 months) for group A and 6 months (range, 2 to 18 months) for group B. For the same patient, each peptide that was recognized significantly by CTL gave a positive result at both time points analyzed, and CTL frequencies are given as an average of both time points. HLA class I alleles for the A and B loci, HIV-1 RNA plasma load, and CD4 cell counts at the time points analyzed, as well as antiretroviral treatment for the subjects studied, are shown in Table 1. Prior to inclusion in this study, all subjects signed informed consent approved by the Institutional Review Board. HLA typing. HLA class I molecular typing was performed at the Massachusetts General Hospital Tissue Typing Laboratory using sequence-specific primer PCR (13). HLA-A2 supertype motif searching and conservancy analyses of putative HIV-1 CTL epitopes. Multiple intact HIV-1 sequences in the Los Alamos data base were analyzed using a text string search software program, MotifSearch, 1.4, to identify amino acid sequences of 8 to 11 amino acids in length containing the HLA-A2 supertype motif (73). Nine HIV-1 antigens, Gag, Pol, Env, Nef, Rev, Tat, Vif, Vpr, and Vpu, were scanned for motif-bearing peptides. Peptides in

VOL. 75, 2001 which the contiguous amino acid sequence was conserved in ⬎50% of clade B isolates were tested for binding to HLA-A2 supertype alleles. Measurement of peptide binding to HLA class I antigens. Putative epitopes of 8 to 11 amino acids in length were synthesized as free acids on an Applied Biosystems 430A peptide synthesizer using standard 9-fluorenylmethoxycarbonyl chemistry and were purified by reversed-phase high-pressure liquid chromatography. HLA class I molecules were purified from detergent lysates of EpsteinBarr virus-transformed homozygous cell lines (78) for use in the competitive binding assays. Peptides known to bind to particular HLA antigens with high affinity were iodinated using the chloramine-T method and used as standards for binding assays. To measure HIV-1 peptide binding to HLA molecules, 5 to 50 nM purified HLA molecules were incubated with the HIV-1 test peptides, at concentrations ranging from 120 ␮g/ml to 1.2 ng/ml, along with 1 to 10 nM radiolabeled standard peptides, for 48 h in phosphate-buffered saline (PBS) containing 0.05% NP-40. All assays were run at pH 7 in a cocktail of protease inhibitors. Following incubation, HLA-peptide complexes were separated from free peptide by gel filtration on 7.8-mm by 15-cm TSK200 columns (TosoHaas 16215) with PBS (pH 6.5) containing 0.5% NP-40 and 0.1% NaN3. The radioactivity in the column eluates was measured using a Beckman 170 radioisotope detector, and the fraction of bound HIV-1 peptide was calculated. Binding of peptides to HLA-Aⴱ0201 was determined first, and peptides that bound with high affinity (defined as a 50% inhibitory concentration [IC50] of ⬍500 nM) were evaluated for their ability to bind HLA-Aⴱ0202, -Aⴱ0203, -Aⴱ0206, and -Aⴱ6802 (70, 77). Supertype or degenerate binding peptides were defined as those that bound three or more HLA-A2 supertype alleles (76, 80, 84). Cell lines and media. Epstein-Barr virus-transformed B lymphoblastoid cell lines were established and maintained in R20 medium (RPMI 1640 medium [Sigma, St. Louis, Mo.] supplemented with 2 mM L-glutamine, 50 U of penicillin per ml, 50 ␮g of streptomycin per ml, 10 mM HEPES, and 20% heat-inactivated fetal calf serum [Sigma]), as previously described (81). For culture of CTL clones, medium containing 10% fetal calf serum (R10) supplemented with 50 U of recombinant interleukin-2 (kindly provided by M. Gately, Hoffmann-La Roche, Nutley, N.J.) per ml was used. Generation of CTL clones. CTL clones were isolated by limiting dilution as previously described (44, 82), using the anti-CD3-specific monoclonal antibody (MAb) 12F6 as a stimulus for T-cell proliferation. Developing clones were screened for HIV-1-specific CTL activity by a chromium 51 release assay (81) against autologous B-cell lines pulsed with the peptides recognized in the enzyme-linked immunospot (Elispot) assays. HIV-1-specific clones were maintained by stimulation every 14 to 21 days with an anti-CD3 MAb and irradiated allogeneic peripheral blood mononuclear cells (PBMC). HLA restriction of CTL epitopes was determined using a panel of target cells matched through only one of the HLA-A, HLA-B, or HLA-C class I alleles expressed by the effector cells (82). Chromium 51 release assay. Three million each of T1 and T2 cells were infected with the molecular HIV-1 clone NL4-3 at a multiplicity of infection of one 50% tissue culture infectious dose in a volume of 0.5 ml for 4 h at 37°C. Five milliliters of medium was then added to the cells and cultured overnight. During the following 4 days, the cells were washed daily and resuspended in fresh medium at 0.5 ⫻ 106 cells/ml. The supernatants of these cells were saved for measurement of p24 antigen by ELISA (NEN, Boston, Mass.). On day 4 after initial infection, the cells were used as targets in a standard chromium 51 release assay as previously described (85). The control target cells used in the assay were uninfected T1 and T2 cells and infected T1 and T2 cells pulsed with the cognate peptide. Elispot assay. HIV-1-specific CTL responses were quantified using the Elispot assay, as described previously (4). Briefly, frozen or fresh PBMC (0.5 ⫻ 105 to 1 ⫻ 105) and individual peptides (10⫺5 M) were added to wells of 96-well polyvinylidene difluoride-backed plates (MAIP S45; Millipore, Bedford, Mass.) that had been previously coated with 0.05 ␮g of anti-gamma interferon (IFN-␥) MAb 1-D1k (Mabtech, Stockholm, Sweden). For each individual peptide, the assay was run in duplicate. For negative and positive controls, PBMC were incubated with medium and phytohemagglutinin, respectively. The plates were incubated at 37°C, 5% CO2, overnight and then processed as described previously (4). IFN-␥-producing cells were counted by direct visualization and are expressed as spot-forming cells (SFC) per 106 PBMC. The number of specific IFN-␥-secreting T cells was calculated by subtracting the negative control value from the established SFC count. Negative control values were always ⬍20 SFC per 106 input cells. Results were considered positive when at least 50 SFC/106 PBMC were detected. Flow cytometric detection of antigen-induced intracellular IFN-␥. Intracellular cytokine staining assays were performed as described elsewhere, with minor modifications (34, 59). Briefly, 0.5 to 1.0 million PBMC were incubated in 24-well

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plates with 2 ␮M peptide and 1 ␮g each of the MAbs anti-CD28 and anti-CD49d (Becton Dickinson) per ml at 37°C, 5% CO2, for 1 h before the addition of 10 ␮g of brefeldin A (Sigma) per ml. Following a further 5-h incubation at 37°C, 5% CO2, the cells were placed at 4°C overnight. PBMC were then washed with PBS–1% bovine serum albumin and stained with surface antibodies anti-CD8 and anti-CD4 (Becton Dickinson) at 4°C for 20 min. Following three further washes, the PBMC were fixed and permeabilized using a Caltag fixation and permeabilization kit (Caltag, Burlingame, Calif.), and anti-IFN-␥ MAb (Becton Dickinson) was added. Cells were then washed and analyzed on a FACSort flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) using peridinin chlorophyll protein, allophycocyanin, and fluorescein isothiocyanate as fluorescent parameters. Control conditions were established by the use of autologous PBMC which had not been stimulated with peptide but otherwise had been treated identically. Cell population boundaries were established by exclusion of 99.97% of the control lymphocytes.

RESULTS Identification of conserved HLA-A2 binding peptides. Of the approximately 12,000 HLA-A2 supermotif-bearing peptide sequences identified, 233 were conserved in ⬎50% of the clade B isolates analyzed. These peptides were synthesized and tested for their capacity to bind purified HLA-Aⴱ0201; 30 peptides were found to be moderate (IC50, 50 to 500 nM)- or high (IC50, ⬍ 50 nM)-affinity HLA-Aⴱ0201 binders. Twenty of the 30 (67%) HLA-A2 binders were found to bind to at least three of the five HLA-A2 supertype alleles tested (Table 2). These 20 HLA-A2 binders included two previously described HLAA2-restricted CTL epitopes, ILKEPVHGV (IV9) in RT (82) and SLLNATDIAV (SV10) in gp41 (20). Three additional HLA-A2-restricted CTL epitopes (SLYNTVATL [SL9] in p17 [44], VIYQYMDDL [VL9] in RT [37], and AFHHVAREL [AL9] in Nef [9]) which have been reported in infected persons but did not fulfill the above criteria in terms of significant binding to at least three HLA-A2 subtypes were also analyzed for the ability to induce peptide-specific HLA class I-restricted T-cell responses in HIV-1-infected individuals. Detection of CD8ⴙ T-cell CTL responses in persons with chronic HIV-1 infection. The detection of antigen-specific IFN-␥ production of CD8⫹ cells by Elispot allows for a rapid and comprehensive assessment of CD8⫹ T-cell responses in an overnight assay and requires no further in vitro expansion or stimulation of PBMC. Furthermore, the results of this assay correlate well with more cumbersome and less sensitive cytolytic assays for CTL detection (34). The Elispot assay was therefore chosen to measure HIV-1-specific CTL frequencies in this cohort of 41 subjects, using the 23 peptides described above. A total of 75 independent samples from 41 individuals were analyzed for CTL responses directed against a panel of predicted HLA-A2 peptides (Table 1). The individuals studied included 22 individuals with chronic HIV-1 infection (group A), 12 individuals who were treated during acute HIV-1 infection (group B), and 7 HIV-1-negative controls (group C). The recognition of the 23 HLA-A2-restricted peptides was first analyzed in individuals with chronic HIV-1 infection (group A) and compared to HIV-1-seronegative controls (group C). None of the seven seronegative control subjects recognized any of the tested peptides (data not shown). However, 18 of the 22 individuals (82%) with chronic HIV-1 infection in group A had detectable CTL responses to at least one of the 23 tested HLA-A2 peptides (range, 1 to 6 peptides recognized; median, 2 peptides recognized) (Fig. 1a). Eleven individuals (50%) had

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TABLE 2. In vitro binding of conserved HIV-1-derived peptides to HLA-A2 supertype alleles Protein

Sequence

Gag-271 Gag-386 Pol-132 Pol-163 Pol-182 Pol-183 Pol-220 Pol-316 Pol-448 Pol-772 Pol-774 Env-134 Env-651 Env-814 Nef-221 Nef-221a Vpr-59 Vpr-62 gp41 SV10 RT IV9

MTNNPPIPV VLAEAMSQV KMIGGIGGFI LVGPTPVNI CTLNFPISPI TLNFPISPI ALVEICTEM YTAFTIPSI KLVGKLNWA RAMASDFNL MASDFNLPPV KLTPLCVTL LLQLTVWGI KAACWWAGI LTFGWCFKL LTFGWCFKLV AIIRILQQL RILQQLLFI SLLNATDIAV ILKEPVHGV

Conservation (%)

A2 supertype binding capacity (IC50 [nM])

Total

Clade B

Aⴱ0201

Aⴱ0202

Aⴱ0203

Aⴱ0206

Aⴱ6802

Alleles bound

31 52 97 84 94 97 23 58 95 64 39 81 53 49 61 55 61 56 22 64

89 74 95 100 100 100 79 68 95 79 68 95 63 79 74 74 74 74 68 79

166.6 66.6 172.4 454.5 147 75.7 217.3 26.3 59.5 217.3 62.5 102 9.8 277.7 35.7 294.1 333.3 19.2 9.8 192.3

7,166.7 82.7 54.4 153.6 23.9 1,482.8 187 6.1 12.6 116.2 22.6 126.5 215 1,075 33.1 48.9 22.6 1,535.7 1,303 2,388.9

33.3 15.2 4.8 19.2 30.3 1.1 140.8 9.1 5.9 25,000 55.6 66.7 43.5 83.3 4,545.5 185.2 41.7 125 238.1 6.7

1,608.7 115.6 770.8 2,846.2 8.4 1,947.4 264.3 7 39.8 52.1 33.6 185 24.7 160.9 205.6 57.8 38.5 37 28.5 37,000

12.1 363.6 3,333.3 67.8 100 57.1 2,857.1 16.7 3,076.9 3,076.9 18.2 20,000 645.2 2,666.7 5.6 6.2 547.9 1,818.2 5,479.4 363.6

3 5 3 4 5 3 4 5 4 3 5 4 4 3 4 5 4 3 3 3

detectable CTL responses against at least one of the 18 newly identified HLA-A2 binders, recognizing a total of 7 newly defined HLA-A2 binders. Of the previously described HLAA2-restricted CTL epitopes, the classic immunodominant epitopes in p17 (SL9) and RT (IV9) were the most frequently targeted CTL epitopes in this study. SL9 was recognized by 12 of 22 individuals and IV9 by 7 of 22 individuals, with mean CTL frequencies of 223 and 164 SFC/106 PBMC, respectively. Of the newly identified HLA-A2 binders, the Vpr-59 peptide was the most frequently recognized peptide (5 of 22) but reached only low CTL magnitudes (range, 50 to 123 SFC/106 PBMC; mean, 85 SFC/106 PBMC). The Gag-386 peptide was recognized by four individuals in this group, and CTL responses directed against this peptide reached higher magnitudes (range, 63 to 500 SFC/106 PBMC; mean, 255 SFC/106 PBMC), representing the immunodominant HLA-A2-restricted response in three of the four individuals recognizing this epitope. The third most frequently recognized peptide was Vpr-62, recognized by 3 of 22 individuals at a low mean CTL magnitude of 72 SFC/106 PBMC (range, 58 to 108 SFC/106 PBMC). Taken together, the most frequently recognized HLA-A2-restricted CTL epitopes in this group of chronically HIV-1-infected individuals were the previously described peptides SL9 and IV9. However, three of the newly identified HLA-A2 binders were frequently recognized, including epitopes in Vpr and Gag, and represented the immunodominant HLA-A2-restricted responses in three individuals. Recognition of predicted HLA-A2-restricted epitopes in acute infection. In order to determine whether some of the HLA-A2 binders were recognized early during HIV-1 infection, the 23 HLA-A2-restricted peptides were subsequently tested in a cohort of 12 HIV-1-infected individuals who were treated during or shortly after acute HIV-1 infection (Fig. 1b). Six of the 12 individuals (50%) had CTL responses directed against at least one HLA-A2 peptide (range, 1 to 2 peptides recognized; median, 1 peptide recognized). CTL from two individuals recognized the Nef-221 peptide at low frequencies

(60 and 200 SFC/106 PBMC), and CTL from two individuals targeted the Vpr-59 peptide, which was recognized at higher frequencies, reaching CTL frequencies of 940 and 600 SFC/106 PBMC. In contrast, the classically targeted HLA-A2-restricted epitopes SL9 and IV9 were rarely recognized (1 of 13 and 1 of 13 individuals, respectively) by the subjects of this cohort with treated acute HIV-1 infection, as described previously (Goulder, et al., submitted). Combining this data with the data for the persons with chronic HIV-1 infection, 9 of the 20 predicted HLA-A2 epitopes (45%) induced peptide-specific T-cell responses in HIV-1-infected subjects, and 7 of these epitopes had never been described before as HLA-A2-restricted epitopes (Fig. 1). The Vpr-59 peptide was recognized by PBMC from 4 of 22 chronically and 2 of 12 acutely HIV-1-infected individuals and induced high CTL responses in acutely infected individuals. The Gag-386 peptide was only recognized in chronically infected individuals (4 of 22) and was the immunodominant HLA-A2-restricted CTL response in three of them. None of the 23 HLA-A2 binding peptides induced IFN-␥ production by PBMC in the 7 HIV-1-negative individuals of control group C (data not shown), indicating that responses to these newly identified peptides are unique to HIV-1-infected individuals. Vpr-59-specific CTL responses contribute importantly to the total CTL response directed against HIV-1. The above studies show the relative strength of responses to predicted HLA-A2-restricted epitopes compared to previously defined HLA-A2-restricted responses. However, those studies do not indicate the relative contribution of these novel responses to the entire CTL response in infected individuals. Assessment of antigen-specific IFN-␥ production by flow cytometry allows for sensitive and specific quantification of relative CTL frequencies (34). This assay was thus used to determine the contribution of the CTL response directed against the most frequently recognized peptide, Vpr-59, to the total HIV-1-specific CTL response. These assays were performed for the two individuals of group B (AC04 and AC13) for whom sufficient samples were

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FIG. 1. Peptide-specific CD8⫹ T-cell responses to HLA-A2 binding peptides as measured by IFN-␥ Elispot assay. CD8⫹ T-cell responses were tested for 23 different peptides at two different time points for each subject studied and are shown as mean frequencies. Only subjects with positive responses (⬎50 SFC/106 PBMC [SFC/Mill PBMC]) are shown. (a) Individuals with chronic HIV-1 infection (n ⫽ 18). (b) Individuals with acute HIV-1 infection (n ⫽ 6). The maximum number of epitopes targeted was six, by subject SF17697.

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FIG. 2. CD8⫹ T-cell frequencies against described optimal CTL epitopes for the corresponding HLA type were measured by intracellular IFN-␥ staining on flow cytometry for two individuals (AC04 [A] and AC13 [B]). Responses to the negative control without peptide (a) and five positive responses to Vpr-59 (b) and four other described optimal CTL epitopes for the corresponding HLA type (c to f) are demonstrated, and percentages of activated CD8⫹ T cells are shown. FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll protein.

available, using a panel of all described optimal CTL epitopes for the corresponding HLA type (9). AC04 and AC13 each recognized a total of five different CTL epitopes, restricted by three and four different alleles, respectively. For AC04, 0.6% of the CD8⫹ T cells were specific for the Vpr-59 peptide (Fig. 2A). The total CTL response for this person was 2.2% of the CD8⫹ T cells, so CD8⫹ cells directed against Vpr-59 thus contributed 30% to the total HIV-1-specific CD8⫹ T-cell responses, representing the second strongest individual response against HIV-1. For AC13, 0.8% of the CD8⫹ T cells were specific for peptide Vpr-59, representing also in this individual the second strongest individual response and contributing 30% to the total HIV-1-specific CD8⫹ response (Fig. 2B). For both individuals, samples from the time of acute HIV-1 infection, prior to HIV-1 seroconversion and treatment with HAART, were available. Vpr-59-specific CTL responses were already detectable in AC13 at that early time point but developed later in AC04 (data not shown). Taken together, these data demonstrate that the epitope Vpr-59 played a substantial role in the total CTL response against HIV-1 of these individuals

treated during acute infection and that responses to this epitope can be detected in the acute stage of infection. Lytic activity of CTL directed against Vpr-59 from HIV-1infected individuals. Of the newly defined HLA-A2-restricted epitopes, the Vpr-59 peptide was the most frequently recognized epitope. The functionality of the cellular immune responses directed against this epitope was thus subsequently analyzed in more detail. CTL clones specific for the Vpr-59 peptide were generated from the two subjects of group B who recognized this peptide (AC04 and AC13). CTL clones from these subjects specifically lysed B cells pulsed with the Vpr-59 peptide in a standard chromium 51 release assay, but not B-cell lines pulsed with control peptides (Fig. 3a and b). This cytotoxic response was restricted to B-cell lines expressing the HLA-A2 allele (Fig. 3c). After demonstrating that the Vpr-59specific CTL clones were able to lyse B cells pulsed with the Vpr-59 peptide, it was investigated whether Vpr-59 was adequately processed and presented on HLA class I molecules in cells infected with the molecular HIV-1 clone NL4-3. Therefore, HLA-A2-positive, TAP-deficient B-cell lines (T2), which

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FIG. 3. Percent specific lysis of target cells pulsed with the Vpr-59 peptide or no peptide (control) by epitope-specific clones in chromium release assay. Results for subject AC13 (a) and subject AC04 (b) are shown at different effector-to-target ratios (E:T). In panel c, determination of HLA restriction using Vpr-59-specific CTL clones in a chromium release assay is shown for individual AC13. Targets were either pulsed with no peptide (white bars) or pulsed with the relevant peptide Vpr-59 (black bars). The HLA class I types of the targets used were as follows: Aⴱ0201/3, B7/60; A28/29, B14/44; and A34/68, B57/71 (matching HLA class I alleles are shown in bold).

are not able to process and bind viral antigen to HLA class I molecules, and HLA-A2-positive, TAP-competent B-cell lines (T1) were either infected with NL4-3 alone or additionally pulsed with the Vpr-59 peptide. HIV-1 NL4-3-infected T1 cells were lysed in a chromium 51 release assay by Vpr-59-specific CTL clones, as were the T1 cells that were additionally pulsed with the Vpr-59 peptide (Fig. 4a). In contrast, TAP-deficient T2 cells, which are not able to process the viral antigen, were not lysed by Vpr-59-specific CTL when infected with NL4-3 alone, but only when the Vpr-59 peptide was added (Fig. 4b). These data show that the Vpr-59 peptide is adequately pro-

cessed and presented by HLA class I molecules by in vitro HIV-1-infected T1 cells and thus demonstrate that the strategy employed to detect novel CTL epitopes is able to identify epitopes processed in HIV-1-infected cells. DISCUSSION This study focuses on the identification and characterization of HIV-1-derived CTL epitopes that are restricted by the HLA-A2 superfamily. Eighteen new HLA-A2 supertypic peptides were identified based on their high degree of conserva-

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FIG. 4. Percent specific lysis of HLA-A2-positive, TAP-competent (T1) (a) and TAP-deficient (T2) (b) B-cell line target cells by Vpr-59-specific CTL clones. T1 and T2 B-cell lines were infected with the molecular HIV-1 clone NL4-3 HIV (⫹NL4-3). The control target cells used in the assay were uninfected T1 and T2 cells and infected T1 and T2 cells pulsed with the Vpr-59 peptide (⫹NL4-3⫹peptide). Results are shown at different effector-to-target ratios (E:T) on day 4 after infection, following chromium 51 labeling of the cells, as described previously (85).

tion within HIV-1 clade B strains and their strong binding to different alleles of the HLA-A2 superfamily. These peptides were tested for specific recognition by cytotoxic T cells in 34 HIV-1-infected individuals and 7 HIV-1-negative controls expressing HLA-A2 alleles. Seven of the 18 novel HLA-A2 binders induced peptide-specific T-cell responses in at least one of the HIV-1-infected subjects. In some cases these responses were stronger than those to previously identified HLA-A2restricted epitopes. These results include the first description of CTL responses directed against HIV-1 Vpr and demonstrate that it can play a major role in the total CTL response against HIV-1 in acutely infected individuals. The identification of CTL epitopes can be a difficult and labor-intensive process. Overlapping peptides spanning the length of a given protein may be used to identify a region within the protein that is recognized, and the optimal sequence can be defined using shorter, truncated peptides (4, 26–28, 44, 52). Basing epitope identification on MHC class I binding motifs simplifies the process by decreasing the number of peptides that require binding or immunogenicity screening. Rather than testing all possible peptides from a given antigen, analysis can be focused on a select subset of motif-bearing peptides. Further screening of these motif-bearing peptides on the basis of MHC binding affinity has been shown to markedly increase the frequency of epitope recognition (74). Similarly, recent data from HLA-B60 (4) and HLA-B53 (M. M. Addo et al., submitted) showed a very high concordance between the HLA class I binding motif and the novel HIV-1-specific CTL epitopes. While the association between the peptide-binding motif and the amino acid sequence of the actual CTL epitopes is not always absolute (18, 30, 43, 69), the identification of seven HLA-A2-restricted HIV-1 CTL epitopes in this study provides further evidence that the prediction of CTL epitopes

from the binding motif of the corresponding HLA class I molecule is an efficient approach to epitope identification. Increasing data demonstrate that virus-specific CTL responses play a crucial role in the immune response against HIV-1 (2, 11, 33). Vaccines inducing strong CTL responses against HIV-1 may therefore represent a possible avenue to prevent infection or attenuate HIV-1 disease. However, the large degree of HLA polymorphism (7) represents a significant challenge for this approach, if specific epitopes for a large number of different HLA class I specificities have to be defined. The identification of peptides capable of binding to multiple HLA class I molecules may simplify the epitope selection (73). This study therefore focused on the identification of CTL epitopes that have high binding affinities for different HLA class I molecules of the HLA-A2 superfamily, which includes HLA-Aⴱ0201, -Aⴱ0202, -Aⴱ0203, -Aⴱ0206, and -Aⴱ6802. CTL responses against the identified HLA-A2 binders were analyzed in a Caucasian population expressing predominately HLA-Aⴱ0201. However, six of seven novel epitopes recognized by individuals expressing HLA-Aⴱ0201 showed higher binding capacities for HLA-A2 subtypes other than HLA-Aⴱ0201, suggesting that they may also be recognized by these subtypes. This should be evaluated in future studies on populations expressing different HLA-A2 subtypes. In order to evaluate the role of the novel epitopes within the HLA-A2-restricted CTL response against HIV-1, the CTL responses induced by these novel epitopes were compared to CTL responses directed against SLYNTVATL (SL9), the immunodominant HLA-A2-restricted CTL response in chronic HIV-1 infection (10, 31, 35, 53). In contrast to SL9, which is recognized by 70% of persons with chronic HIV-1 infection but did not meet the inclusion criteria used here for predicted epitopes, most of the newly identified HLA-A2 epitopes were

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recognized by only one or two individuals and were of low magnitude, indicating a subdominant role for these CTL epitopes. As described previously, individuals with chronic HIV-1 infection recognized more CTL epitopes than individuals treated during acute or early HIV-1 infection (3). Two novel epitopes, Vpr-59 and Gag-386, were recognized by seven and four subjects, respectively, and played a major role in the HLA-A2-restricted CTL response against HIV-1 in the individuals studied. However, the quantitative hierarchy of CTL activity may not necessarily correlate with the ability to protect against infection (24). Furthermore, immunodominance of CTL epitopes is not a static property, as subdominant epitopes can take over a dominant and protective function after escape from an immunodominant epitope (25, 79). It may therefore be important to include both dominant and subdominant CTL epitopes in a prophylactic or therapeutic vaccine in order to provide immune protection after emergence of escape variants from the immunodominant epitopes. The CTL epitopes identified in this study represent potential candidates for a vaccine including epitopes recognized by the HLA-A2 superfamily. These studies are also important because they include the first description of CTL responses directed against minimal epitopes in HIV-1 Vpr and show that strong responses against Vpr can be generated in acute HIV-1 infection. HIV-1 Vpr plays an important role in HIV-1 replication (reviewed in reference 12). The responses directed against Vpr-59, the most frequently recognized novel CTL epitope in this study, were therefore analyzed in more detail. It was demonstrated that this HLA-Aⴱ0201-restricted epitope was efficiently processed in cell lines infected with HIV-1, inducing lysis of the infected cells by CTL clones specific for this peptide. Interestingly, Vpr-59 contributed importantly to the total CTL response directed against HIV-1 in acute infection, when CTL responses directed against the Gag-386 and SL9 epitopes were absent or rarely detectable (unpublished data). In contrast, CTL responses directed against Vpr-59 were weak and subdominant in chronic infection, when SL9 and Gag-386 represented the immunodominant HLA-A2-restricted responses. These findings indicate differences in the recognition of HLA-A2-restricted CTL epitopes between acute and chronic HIV-1 infection and show that CTL responses against the Vpr-59 epitopes may be induced more readily and earlier by HIV-1 infection. This may have implications for prophylactic and therapeutic vaccine design, as highly immunogenic CTL epitopes would be preferably included in these vaccines. In conclusion, 18 HLA-A2 supertypic peptides were identified based on their conservation within HIV-1 clade B strains and their high binding to different alleles of the HLA-A2 superfamily. Seven of these peptides, including the first described optimal CTL epitopes in the accessory protein HIV-1 Vpr, were recognized by CTL in HIV-1-infected individuals, indicating that the prediction of CTL epitopes using the binding motif of the corresponding HLA class I molecule may be a highly efficient approach to define novel CTL epitopes. ACKNOWLEDGMENTS M.A.A. and B.L. contributed equally to this work. The authors are greatly indebted to the San Francisco City Clinic Cohort and the Boston Acute Infection Collaborative for provision of blood samples and clinical data in order to study the CTL responses described above.

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This work was supported by grants to M.A.A. from the Deutscher Akademischer Austauschdienst (DAAD) (grant D/99/08826), to S.A.K. from the National Institutes of Health (AI39966, AI38858, and U19 AI38584), to M.M.A. from the Deutsche Forschungsgemeinschaft (AD-161), to B.L. from the National Institutes of Health (NO1-AI95362), and to B.D.W. through the National Institutes of Health (R37 AI28568, R01 AI44656, R01 AI40873, U01 AI 41531, and U01 AI 48023) and the Doris Duke Charitable Foundation (B.D.W. and E.S.R.). P.J.R.G. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation. B.D.W. is a Doris Duke Distinguished Clinical Science Professor. REFERENCES 1. Allen, T. M., D. H. O’Connor, P. Jing, J. L. Dzuris, B. R. Mothe, T. U. Vogel, E. Dunphy, M. E. Liebl, C. Emerson, N. Wilson, K. J. Kunstman, X. Wang, D. B. Allison, A. L. Hughes, R. C. Desrosiers, J. D. Altman, S. M. Wolinsky, A. Sette, and D. I. Watkins. 2000. 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