During HIV-1 Infection Vpr Is Preferentially Targeted By

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Vpr Is Preferentially Targeted by CTL During HIV-1 Infection Marcus Altfeld, Marylyn M. Addo, Robert L. Eldridge, Xu G. Yu, Seddon Thomas, Ashok Khatri, Daryld Strick, Mary This information is current as N. Phillips, George B. Cohen, Sabina A. Islam, Spyros A. of September 26, 2021. Kalams, Christian Brander, Philip J. R. Goulder, Eric S. Rosenberg and Bruce D. Walker J Immunol 2001; 167:2743-2752; ; doi: 10.4049/jimmunol.167.5.2743 http://www.jimmunol.org/content/167/5/2743 Downloaded from

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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 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Vpr Is Preferentially Targeted by CTL During HIV-1 Infection1

Marcus Altfeld,* Marylyn M. Addo,* Robert L. Eldridge,* Xu G. Yu,*‡ Seddon Thomas,* Ashok Khatri,† Daryld Strick,* Mary N. Phillips,* George B. Cohen,* Sabina A. Islam,* Spyros A. Kalams,* Christian Brander,* Philip J. R. Goulder,*§ Eric S. Rosenberg,* Bruce D. Walker,2* and the HIV Study Collaboration3

The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-␥ production was measured by ELISPOT assay and flow-based intracellular cytokine quantifica- Downloaded from tion. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8؉ T cell lines. CD8؉ T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8؉ T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently /targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8؉ T cell responses. These http://www.jimmunol.org findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines. The Journal of Immunology, 2001, 167: 2743–2752.

he human immunodeficiency virus-1 (HIV-1) has infected induce strong virus-specific immune responses should be identi- over 57 million and killed over 22 million individuals fied. To date, the analysis of HIV-1-specific immunity has largely T worldwide since the beginning of the epidemic (1). Over focused on assessment of immune responses directed against the 95% of HIV-1-infected individuals live in developing countries structural HIV-1 proteins Gag, Pol, and Env, as well as the acces-

and have no access to antiretroviral treatment (1). The need for a sory protein Nef (7). Recent data suggest that CTL responses di- by guest on September 26, 2021 vaccine that protects against HIV-1 infection or attenuates disease rected against the early expressed regulatory proteins Tat and Rev has never been more urgent. Understanding the correlates of pro- also play a central role in the HIV-1-specific immune response (5, tective immunity is a logical first step in vaccine development. In 8, 9). However, very little is known about the role of cellular the case of HIV-1 immunity, accumulating data have shown a immune responses directed against the other accessory proteins. central role of HIV-1-specific CTL and T helper cells in control- The relatively small accessory HIV-1 proteins Vpu, Vif, and Vpr ling viral replication (2Ð6). have important functions in viral disassembly, nuclear transport of the To better understand immune control of viremia and for the preintegration complex (PIC),4 viral assembly, and down-regulation design of potential HIV-1 vaccines, those regions of HIV-1 that of CD4 on the cell surface (10Ð14). Thus these accessory proteins appear to be essential for viral replication. However, the extent to which these proteins are targeted by CTL in natural infection remains *Partners AIDS Research Center and Infectious Disease Division, and †Endocrine to be defined, but their cytoplasmic production within infected cells Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129; ‡AIDS Research Center, First Affiliated Hospital China Medical University, suggests that they should be HLA class I presented and candidate Shenyang, China; ¤Nuffield Department of Medicine, John Radcliffe Hospital, Ox- targets for CTL-mediated lysis of infected cells. ford, United Kingdom; ¶Department of Medicine, Brigham and Women’s Hospital ʈ In this study, we performed a detailed characterization of CTL re- and Harvard Medical School, Boston, MA 02115; and Fenway Community Health Center, Boston, MA 02116 sponses to Vpu, Vif, and Vpr, using recently developed techniques ␥ Received for publication March 23, 2001. Accepted for publication June 18, 2001. including IFN- ELISPOT and intracellular flow cytometric analyses, The costs of publication of this article were defrayed in part by the payment of page as well as T cell cloning. Our data provide evidence that the Vpr and charges. This article must therefore be hereby marked advertisement in accordance Vif proteins represent important targets of cellular host defenses, with 18 U.S.C. Section 1734 solely to indicate this fact. whereas Vpu is infrequently recognized. In addition, the Vpr protein 1 The Doris Duke Charitable Foundation, the National Institutes of Health (R37 is one of the most frequently targeted HIV-1 proteins by CTL relative AI128568, R01 AI30914, R01 AI44656, R01 AI40873, U01 AI41535, and U01 AI41531), the Deutscher Akademischer Austauschdienst, the Deutsche Forschungs- to the length of the protein. This report also provides a characteriza- gemeinschaft, the Lloyd Foundation, the Partners/Fenway/Shattuck Center for AIDS tion of discrete CTL epitopes within these proteins, identified using Research, and several private donors supported this research. B.D.W. is the recipient overlapping peptides spanning the entire HIV-1 Vpr, Vpu, and Vif of a Doris Duke Distinguished Clinical Scientist Award and P.J.R.G. is an Elisabeth Glaser Scientist of the Pediatric AIDS Foundation. sequence. 2 Address correspondence and reprint requests to Dr. Bruce D. Walker, Massachusetts General Hospital-East, CNY 5212, 149 13th Street, Charlestown, MA 02129. E-mail address: [email protected] 3 For the HIV Study Collaboration: Nesli Basgoz,* Gregory K. Robbins,* Ben 4 Abbreviations used in this paper: PIC, preintegration complex; RT, reverse tran- Davis,* Paul E. Sax,¶ Steve Boswell,ʈ and Dan S. Cohenʈ. scriptase; rVV, recombinant vaccinia virus; SFC, spot-forming cell; BCL, B cell line.

Materials and Methods final concentration of 1 ϫ 10Ϫ5 molar. Cells were added to the wells at 50,000Ð100,000 cells/well. The plates were incubated at 37¡C, 5% CO Subjects 2 overnight (14Ð16 h) and then processed as described (23, 24). IFN-␥- Sixty HIV-1-infected and 10 HIV-1-negative individuals were studied at producing cells were counted by direct visualization and are expressed as the Massachusetts General Hospital. HIV-1-infected individuals included spot-forming cells (SFCs) per 106 cells. The number of specific IFN-␥- 45 subjects who were treated with highly active antiretroviral therapy secreting T cells was calculated by subtracting the negative control value within 180 days of HIV-1 infection, nine individuals with chronic treated from the established SFC count. The negative controls were always Ͻ20 HIV-1 infection, and six individuals with long-term nonprogressive HIV-1 SFC per 106 input cells. Responses Ն40 SFC per 106 input cells above infection, defined as viremia below 1000 HIV-1 RNA copies per milliliter background were considered positive. The positive control consisted of ϩ for Ͼ6 years in the absence of any antiretroviral treatment. At the time of incubation of 100,000 PBMC with PHA. CD8 T cell dependence of all the CTL analysis, subjects on highly active antiretroviral therapy had been responses to synthetic peptides was confirmed by loss of IFN-␥ production ϩ effectively treated for at least 6 mo and all had viral loads below 50 copies after CD8 T cell depletion using magnetic beads (MACS; Miltenyi Bio- RNA/ml. The study was approved by the Massachusetts General Hospital tech), according to the manufacturer’s protocol. Fine mapping of epitopes Institutional Review Board, and all individuals gave informed consent for by ELISPOT assay was performed as described (23), using peptide trun- participation in the study. cations. Briefly, 100,000 PBMC/well were incubated with concentrations from 10Ϫ4 to 10Ϫ11 M peptide overnight on the ELISPOT plate. All assays HLA typing were run in duplicate. The optimal peptide was defined as the peptide that induced 50% maximal specific IFN-␥ production of T cells at the lowest HLA class I molecular typing was performed at the Massachusetts General peptide concentration (23). Hospital Tissue Typing Laboratory using sequence-specific primer-PCR (15). Synthetic HIV-1 peptides Flow cytometric detection of Ag-induced intracellular IFN-␥ Peptides were synthesized on an automated peptide synthesizer (MBS 396; Intracellular cytokine staining assays were performed as described else- Downloaded from Advanced Chemtech, Louisville, KY) using F-moc chemistry. Seventeen where with minor modifications (25, 26). Briefly, 0.5Ð1.0 million PBMC overlapping peptides spanning the HIV-1 SF2 B clade Vpr sequence, 14 were incubated on 24-well plates with 2 ␮M peptide and 1 ␮g/ml each of overlapping peptides spanning the HIV-1 SF2 B clade Vpu sequence, and the mAbs anti-CD28 and anti-CD49d (BD Biosciences) at 37¡C, 5% CO2, 36 overlapping peptides spanning the HIV-1 SF2 B clade Vif sequence for 1 h, before the addition of 10 ␮g/ml brefeldin A (Sigma). Following

(12Ð18 mers with 10 aa overlap) were generated. In addition, peptides another 5-h incubation at 37¡C, 5% CO2, the cells were placed at 4¡C corresponding to described optimal HIV-1 CTL epitopes (7) and a panel of overnight. PBMC were then washed with PBS/1% BSA and stained with

321 overlapping peptides (15Ð20 mers) spanning the entire p15 Gag, p17 surface Abs, anti-CD8 and anti-CD4 (BD Biosciences) at 4¡C for 20 min. http://www.jimmunol.org/ Gag, p24 Gag, gp41 Env, gp120 Env, reverse transcriptase (RT), Rev, Tat, Following three more washes, the PBMC were fixed and permeabilized and Nef sequence (16), were used. using Caltag Fixation/Permeabilization Kit (Caltag Laboratories, Burlin- game, CA) and anti-IFN-␥ mAb (BD Biosciences) was added. Cells were Cell lines and media then washed and analyzed on a FACSort Flowcytometer (BD Immunocy- EBV-transformed B lymphoblastoid cell lines were established and main- tometry Systems, San Jose, CA) using PerCP, allophycocyanin, and FITC tained in R20 medium (RPMI 1640 medium (Sigma, St. Louis, MO) sup- as fluorescent parameters. Control conditions were established by the use plemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 ␮g/ml strepto- of autologous PBMC, which had not been stimulated with peptide, but mycin, 10 mM HEPES, and 20% heat-inactivated FCS (Sigma)) as otherwise had been treated identically. Cell population boundaries were previously described (17). For culture of CTL clones, medium containing established by exclusion of 99.97% of control lymphocytes. For the determination of HLA class I restriction of responses by flow cytometry, assays

10% FCS (R10) supplemented with 50 U/ml rIL-2 (provided by Dr. M by guest on September 26, 2021 Gately, Hoffmann-LaRoche, Nutley, NJ) was used. using HLA-matched or mismatched BCLs were run as described (27). Briefly, BCLs that were pulsed with 10 ␮M peptide for 1 h were washed Generation of peptide-specific CD8ϩ T cell lines five times before incubation with effectors (105 BCL and 5 ϫ 105 effectors) ϩ in 1 ml of R10. The mAbs anti-CD28 and anti-CD49d were then added, CD8 T cells were nonspecifically expanded from PBMC over 10 days, and the assay was run exactly as described above. using a bispecific CD3/CD4 Ab. Peptide-specific CD8ϩ T cell lines were subsequently isolated using an IFN-␥ catching assay (18Ð20), according to the manufacture’s instructions. Briefly, after expansion 10Ð20 ϫ 106 ϩ Results ␮ ϩ CD8 T cells were incubated on 24-well plates with 20 M peptide and 1 Recognition of Vpr, Vpu, and Vif proteins by CD8 T cells from ␮g/ml each of the mAbs anti-CD28 and anti-CD49d (BD Biosciences, HIV-1-infected persons Mountain View, CA) at 37¡C, 5% CO2, for 6Ð8 h. Cells were subsequently labeled with a bispecific CD45/IFN-␥ catching Ab and incubated for 45 To determine whether the Vif, Vpr, and/or Vpu proteins are tar- min at 37¡C, 5% CO . After several washes, the IFN-␥-producing cells 2 geted by the CTL response in HIV-1 infection, we performed a were stained with a second IFN-␥-PE detection Ab and separated by anti- PE mAb labeled with magnetic beads (MACS; Miltenyi Biotech, Ham- pilot study in individual AC-06 in whom we had previously dem- burg, Germany). The isolated cells were then expanded for 10 days using onstrated responses to a total of nine CTL epitopes in p17, p24, autologous irradiated feeders, as described previously (21). RT, gp41, and Nef, and defined the optimal epitopes recognized Generation of CTL clones within those gene products (16, 28). PBMC from subject AC-06 were analyzed by ELISPOT assay with a set of 388 overlapping CTL clones were isolated by limiting dilution as previously described (21, peptides spanning the expressed HIV-1 clade B sequence. This 22), using the anti-CD3-specific mAb 12F6 as stimulus for T cell prolif- allowed us to assess the relative contribution of Vpr-, Vpu-, and eration. Developing clones were screened for HIV-1-specific CTL activity ϩ by 51chromium-release assay (17) against autologous B cell lines (BCLs) Vif-specific CD8 T cell responses to the overall CD8-mediated pulsed with the peptides recognized in the ELISPOT assays or infected immune response. Fig. 1 shows the comprehensive characteriza- with recombinant vaccinia virus (rVV) expressing either HIV-1 Vpr or Vif tion of HIV-1-specific CTL responses in this person using the en- (provided by G. P. Mazzara, Therion Biologics, Cambridge, MA). HIV- tire panel of overlapping peptides including 17 peptides spanning 1-specific clones were maintained by stimulation every 14Ð21 days with an anti-CD3 mAb and irradiated allogeneic PBMC. HLA-restriction of CTL Vpr, 14 peptides spanning Vpu, and 36 peptides spanning Vif, as epitopes was determined using a panel of target cells matched through only well as peptides spanning p15, p17, p24, RT, gp41, gp120, Nef, one of the HLA-A, HLA-B, or HLA-C class I alleles expressed by the Rev, and Tat. CD8ϩ T cell dependence of all responses was con- effector cells (21). firmed by CD8/CD4 depletion studies (data not shown). In addi- ELISPOT assay tion to the nine previously demonstrated CTL responses, we detected at least two responses to Vif and one to Vpr (Fig. 1). Of all PBMC were plated on 96-well polyvinylidene difluoride-backed plates (MAIP S45; Millipore, Bedford, MA) that had been previously coated with of the responses detected, the response to an epitope contained in 100 ␮l of an anti-IFN-␥ mAb 1-D1k (0.5 ␮g/ml; Mabtech, Stockholm, the Vif peptide THPRVSSEVHIPLG was the third highest in mag- Sweden) overnight at 4¡C. Peptides were added directly to the wells at a nitude (1180 SFC/106 PBMC). These data indicate that multiple The Journal of Immunology 2745

FIGURE 1. HIV-1-speciﬁc CD8ϩ T cell responses determined by screening PBMC in an ELISPOT assay using overlapping peptides (15Ð20 mer) spanning HIV-1 p15 (Gag), p17 (Gag), p24 (Gag), RT, gp41 (Env), gp120 (Env), Nef, Rev, Tat, Vpr, Vpu, and Vif. The amino acid sequences of recognized peptides are shown, and CD8ϩ T cell magnitudes are expressed as SFC/106 PBMC. A total of four different Vif peptides were recognized in this individual; however, these responses were induced by two epitopes contained within the overlap of each of two overlapping peptide pairs (see below). Downloaded from http://www.jimmunol.org/

accessory proteins can be targeted simultaneously by the CTL re- subsequently reconfirmed using a standard 51Cr release assay sponse in a single individual, and failure to assess these responses following the isolation of peptide-specific CTL clones by limiting would lead to an underestimation of total CTL responses. dilution (data not shown). In all, three novel CTL epitopes in two accessory HIV-1 proteins were identified by the use of overlapping Definition of optimal CTL epitopes within the accessory HIV-1 peptides in this single patient (Table I). Of the total of 12 epitopes proteins targeted by individual AC-06, 3 (25%) were located in the acces- The above data indicate that Vif and Vpr were targeted by CD8 sory proteins Vif and Vpr. by guest on September 26, 2021 cells in this individual, but do not indicate the number of epitopes We next determined the contribution of responses directed contained within each of the proteins. For example, the p17 Gag against the individual CTL epitopes contained within the overlap- peptide WEKIRLRPGGKKKYK actually contains two discrete ping peptides to the total HIV-1-specific CTL responses in subject A3-restricted epitopes, KIRLRPGGK and RLRPGGKKK (7), and AC-06, using intracellular IFN-␥ quantification by flow cytometry responses to two peptides in Vif that share a 10-aa overlap could (Fig. 4). Interassay variation, defined as the SD divided by the represent targeting of an epitope shared by both peptides, or two mean of the responses between assays run multiple times in par- discrete epitopes. To determine the contribution of epitopes within allel, was Ͻ12%, as determined in previous experiments to eval- ϩ the accessory proteins to the overall breadth of the CTL response uate this technique (data not shown). Of the CD8 T cells, 15.6% in this person, we next determined the optimal epitopes within were specific for the tested HIV-1 CTL epitopes. The immuno- these longer peptides, using newly adapted techniques (23, 27). dominant response in this subject was directed against the HLA- This is exemplified for the two CTL epitopes within HIV-1 Vif B7-restricted CTL epitope GPGHKARVL (GL9) in p24 Gag. A ϩ (Figs. 2 and 3). The optimal sequences of these novel CTL total of 5.6% of CD8 T cells were directed against this epitope, epitopes were determined by ELISPOT assay, using PBMC and contributing 32% to the total HIV-1-specific CD8 responses. ϩ serial dilutions of truncated peptides (Fig. 2, A and B). The peptide CD8 T cell responses directed against the accessory proteins Vif that induced 50% of maximal specific IFN-␥ production at the and Vpr contributed importantly to the total responses (17% of lowest peptide concentration was defined as the optimal CTL total HIV-1-specific CD8 responses) in this subject. Therefore, epitope (Fig. 2A: RIRTWKSLVK (RK10) for Vif-3; Fig. 2B: these data indicate that the breadth and magnitude of the CTL HPRVSSEVHI (HI10) for Vif-10). The HLA restriction of these responses in this person would have been underestimated by 25 CTL epitopes was subsequently determined by flow cytometric and 17%, respectively, had responses directed against the acces- quantification of peptide-specific IFN-␥ production using PBMC sory HIV-1 proteins Vif, Vpr, and Vpu not been assessed. and partially HLA class I-matched APCs (Fig. 3). The Vif-RK10 peptide induced IFN-␥ production of CD8ϩ T cells only when Frequency of recognition of the HIV-1 proteins Vif, Vpr, and presented by autologous APC or APC expressing HLA-A3, dem- Vpu in HIV-1 infection onstrating that this epitope was restricted by HLA-A3. This pep- The data described above indicate that CD8ϩ T cell responses can tide also conforms precisely to the predicted motif for HLA-A3- target epitopes within the accessory HIV-1 proteins Vif, Vpr, and presented peptides, with a nonpolar residue in position 2 and a Vpu. To characterize CD8ϩ T cell responses directed against these lysine at the C-terminal of the peptide (29, 30). In contrast, the accessory proteins in more detail, 60 HIV-1-infected individuals at Vif-HI10 peptide was restricted by HLA-B7 (Fig. 3), and con- different stages of infection (45 individuals with treated acute formed to the predicted motif for this allele (30). The sequence of HIV-1 infection, 9 individuals with treated chronic infection, and the optimal CTL epitope as well as the HLA class I restriction was 6 individuals with long-term nonprogressive infection) were 2746 CTL RESPONSES DIRECTED AGAINST ACCESSORY HIV-1 PROTEINS Downloaded from

FIGURE 2. Definition of two novel optimal CTL epitopes within HIV-1 Vif. Titration curves using PBMC in an ELISPOT assay incubated with serial http://www.jimmunol.org/ dilutions of truncated peptides within the Vif-3 peptide (A) and Vif-10 peptide (B). The truncated peptides used are shown, and the optimal CTL epitope was defined as the peptide inducing 50% of maximal specific IFN-␥ production of T cells at the lowest peptide concentration (RIRTWKSLVK for Vif-3 and HPRVSSEVHI for Vif-10). screened for CD8ϩ T cell responses against these proteins by using the most frequently targeted. Responses against the Vif peptides overlapping peptides in an IFN-␥ ELISPOT assay. PBMC from range from 40 to 1100 SFC/106 PBMC (median 215), with Vif-6 27/60 (45%) HIV-1-positive study subjects recognized at least one being the most frequently targeted. CD4 depletion assays and overlapping Vpr peptide (Table II), 20/60 (33%) individuals had flow-based analysis of peptide-specific intracellular IFN-␥ pro- responses against one or more Vif peptides (Table III), and only duction determined that all responses against the accessory pro- by guest on September 26, 2021 one individual had responses against the Vpu peptides (individual teins were CD8ϩ T cell mediated (data not shown). Individuals CMW with long-term nonprogressive HIV-1 infection had con- with long-term nonprogressive and treated chronic HIV-1 in- firmed responses against the Vpu-6 peptides at 170 SFC/106 fection targeted the Vpr more frequently than individuals with PBMC). Magnitude of responses against the Vpr peptides ranged treated acute infection ( p ϭ 0.03, two-tailed Fischer’s exact from 40 to 1400 SFC/106 PBMC (median 245), with Vpr-12 being test), but not Vif ( p ϭ 0.8). This observation for HIV-1 Vpr is

FIGURE 3. Determination of the HLA class I restriction of the novel CTL epitopes within HIV-1-Vif (peptide RIRTWKSLVK, RK10; peptide HPRVSSEVHI, HI10), using peptide-pulsed APCs. The HLA class I type of the subject studied (AC-06) was A3/-, B7/-, Cw7/-. PBMC were incubated with either autologous APCs (AC-06-BCL) or APC lines only sharing one HLA class I allele with the subject studied (A3-, B7-, and Cw7- line). APC lines were incubated with the HI10 peptide, the RK10 peptide, or with no peptide as negative controls (Nil). The percentage of IFN-␥-producing CD8ϩ T cells are given in the individual plots. The Journal of Immunology 2747

Table I. Novel optimal CTL epitopes in individual AC-06 within HIV-1, with length-adapted scores of 0.44 and 0.61. Comparing the frequency of recognition of different HIV-1 pro- Amino Acid teins among individuals with acute treated, chronic treated, and HIV-1 Protein Position Sequence HLA Restriction long-term nonprogressive HIV-1 infection, individuals with long- Vif 17Ð26 RIRTWKSLVK A*0301 term nonprogressive infection and individuals with chronic treated ϩ Vif 48Ð57 HPRVSSEVHI B*0702 infection had CD8 T cell responses directed against more epitopes Vpr 34Ð42 FPRIWLHGL B*0702 and more HIV-1 proteins than individuals with acute treated infection, as described previously for the structural HIV-1 proteins (16), but these differences did not reach statistical significance (Fisher’s exact in line with our previous observation that individuals treated test, p Ͼ 0.1). Taken together, these data extend the studies in indi- during acute HIV-1 infection have a narrowly directed CTL vidual AC-06 to show that the accessory proteins are frequently tar- response, compared with individuals treated later in the course geted by CD8ϩ T cells in HIV-1 infection. of infection (16). Responses against the tested Vif, Vpr, and Vpu peptides were Ͻ20 SFC/106 PBMC (range 0Ð20 SFC/106 PBMC, median 0 SFC/106 PBMC) in the 10 HIV-1-negative Identification of additional CTL epitopes within the accessory individuals used as controls. HIV-1 proteins Despite over 140 optimal CTL epitopes defined to date, no Vif and Frequency of recognition of accessory proteins compared with Vpu epitopes have been mapped (34) and only two HLA-A2-re- other HIV-1 proteins stricted epitopes within Vpr, predicted by the HLA-A2 peptide- Downloaded from We next studied how frequently the accessory proteins Vif, Vpr, binding motif, have been described previously (31). In addition to and Vpu were recognized compared with other HIV-1 proteins. the three novel HLA-A3- and B7-restricted CTL epitopes de- Therefore, a subset of 29 HIV-1-infected individuals (4 with long- scribed above in subject AC-06, we characterized the optimal CTL term nonprogressive infection, 17 with acute treated infection and epitopes restricted by different HLA class I molecules for peptides 8 with chronic treated infection) were screened with a panel of 388 recognized frequently in the studied individuals and determined overlapping peptides spanning the entire Gag, gp41, RT, Rev, Tat, the percentage of individuals with the corresponding HLA type http://www.jimmunol.org/ Vpr, Vpu, Vif, and Nef sequence, as described above for subject recognizing the epitope (Table V). For all novel CTL epitopes, the AC-06 (Fig. 1), and frequencies of recognition were compared optimal peptide sequence was defined using serial dilutions of (Table IV). Furthermore, the frequency of recognition was ad- truncated peptides as described above and the HLA class I restric- justed for the length of the proteins by dividing frequency by the tion was determined by using peptides presented by autologous number of amino acids per protein. Adjusted for its length, the and partially HLA-matched cell lines. Optimal CTL epitopes were HIV-1 Vpr and p17 Gag proteins were the most frequently targeted defined by ELISPOT and flow-based assays using PBMC in most HIV-1 proteins in natural HIV-1 infection, whereas HIV-1 Vif was cases and reconfirmed after isolation of peptide-specific cytotoxic targeted as frequently as HIV-1 RT, gp41, or Tat. To exclude a po- CD8ϩ T cell lines in a standard 51Cr release assay (data not tential bias toward an overrepresentation of responses directed against shown). For the HLA-B*1801- and HLA-B*5701-restricted CTL by guest on September 26, 2021 HIV-1 Vpr and p17 Gag due to the high frequency of individuals with responses in Vif and the HLA-B*5701-restricted CTL response in HLA-A2 in the study cohort (40%) and the presence of epitopes re- Vpr, the frequency of peptide-specific CD8ϩ T cells in the periph- stricted by this allele in both proteins (SLYNTVATL in p17 and eral blood was not sufficiently high to determine the optimal AIIRLLQQL in Vpr; Refs. 31Ð33), we repeated the analysis after epitope sequence and the HLA restriction of the response directly the exclusion of individuals expressing HLA-A2. HIV-1 Vpr using PBMC. For the characterization of these epitopes, CD8ϩ T and p17 Gag remained the most frequently targeted proteins cells were nonspecifically expanded using a CD4/CD3-bispecific

FIGURE 4. Quantification of total HIV-1-specific CD8ϩ T cell activity at the single epitope level using intracellular IFN-␥ staining. HLA class I restriction, first and last amino residue, number of amino acids, protein, and percentage of epitope-specific CD8ϩ T cells after subtraction of background activity are shown for each of the 12 CTL epitopes targeted by individual AC-06. Back- ground activity in the negative control (NIL) was 0.09%. 2748 CTL RESPONSES DIRECTED AGAINST ACCESSORY HIV-1 PROTEINS

Table II. Magnitude of peptide-speciﬁc CD8ϩ T cell responses directed against Vpr overlapping peptides as measured by IFN-␥ ELISPOT (SFC/106 PBMC) in persons with treated acute (AC), treated chronic (C), and untreated long-term nonprogressive infection (L)

Vpr-2 Vpr-3 Vpr-4 Vpr-6 Vpr-7 Vpr-9 Vpr-10 Vpr-11 Vpr-12 Vpr-13

AC-04 000000004500 AC-06 0000220000 00 AC-09 280 2600000003100 AC-10 000006000 0 0 0 AC-13 0 0 0 130 00002200 AC-27 0 0 0 310 0000 00 AC-31 00000000 00 AC-38 0000210000 00 AC-43 0 0 0 80 0000 00 AC-46 000000008500 AC-49 0000110000 00 AC-55 800000000 00 AC-59 0000380000 00 AC-60 000001300 1200 0 360 0 AC-63 0000150000 00 AC-TZA 0 0 0 260 0000 00 C-JRH 000000001400 0 Downloaded from C-6002 00000000500 C-6005 00000000700 C-6003 0 0 0 60 0000 00 C-6009 00000400 0 0 0 C-6010 00000 0 01300 0 L-CMW 00000400 0 0 0 L-MJR 000017037001801800

L-TMH#4 0 0 340 250 240 220 0 0 0 0 http://www.jimmunol.org/ L-TMH#6 000000002500 L-CWE 00000000540380

Ab, and peptide-specific CD8ϩ T cell lines were subsequently iso- activity at different E:T ratios in a standard 51Cr release assay (Fig. lated using a newly adapted IFN-␥ catching assay that allows for 5D) and were used to determine the HLA class I restriction and the isolation of peptide-activated CD8ϩ T cells using magnetic minimal sequence of these novel epitopes by standard cytotoxicity beads. This is shown for the CD8ϩ T cells specific for the Vif-20 assays (data not shown). These peptide-specific lines were also peptide in individual AC-04 (Fig. 5). Using fresh PBMC, 0.3% of used in a cytotoxicity assay with autologous B lymphoblastoid by guest on September 26, 2021 CD8ϩ T cells targeted this peptide (Fig. 5A). After a 10-day ex- cells infected with rVV expressing Vpr and Vif to demonstrate that pansion of CD8ϩ T cells using a bispecific CD3/CD4 Ab and IL-2, the novel epitopes defined were effectively processed intracellu- the peptide-specific CD8 population was expanded to 1.3% (Fig. larly, as shown for the HLA-B*1801-restricted CTL response in 5B). The frequency of these Vif-20-specific CD8ϩ T cells was Vif (Fig. 5D). further increased to 67% after isolation of peptide-specific cells Taken together, a total of nine novel CTL epitopes within the using the IFN-␥ catching assay (Fig. 5C). CD8ϩ T cell lines iso- accessory HIV-1 proteins were identified and characterized. Sev- lated this way showed strong (Ͼ80%) peptide-specific cytotoxic eral of these novel epitopes were restricted by HLA class I alleles

Table III. Magnitude of peptide-speciﬁc CD8ϩ T cell responses directed against Vif overlapping peptides as measured by IFN-␥ ELISPOT (SFC/106 PBMC) in persons with treated acute (AC), treated chronic (C), and untreated long-term nonprogressive infection (L)

Vif-2 Vif-3 Vif-4 Vif-6 Vif-7 Vif-8 Vif-9 Vif-10 Vif-12 Vif-13 Vif-14 Vif-15 Vif-16 Vif-18 Vif-20 Vif-27 Vif-28 Vif-29 Vif-30 Vif-31

AC-01 0 0 0 0 0 0 0 0 60 600000000000 AC-04 0 0 0 0 0 0 0 000000088000000 AC-06 0 450 390 0 0 0 550 950 000000000000 AC-07 0 0 0 0 90 0 0 0000000000000 AC-13 0 0 0 0 0 0 0 000000000260000 AC-27 0 0 0 350 0 0 0 0000000000000 AC-31 0 0 0 0 0 0 0 0 160 240 0000000000 AC-36 0 0 0 0 0 0 0 0 50 100 0000000000 AC-42 0 0 0 0 0 0 0 00000013022000000 AC-43 0 0 0 220 0 0 0 0000000000000 AC-46 0 0 0 230 0 0 0 0000000000000 AC-59 0 0 0 1100 170 0 280 840 000000000000 AC-63 0 0 0 0 0 0 0 0 270 310 000005009000 AC-TZA 0 0 0 0 0 0 0 0 0 0 240 110 0 0 0 0 0 0 0 0 C-JRH 490 430 0 0 0 0 0 0000000000000 C-6003 0 0 0 40 0 0 0 0000000000000 C-6009 50 290 170 0 0 50 40 40000000000000 C-6007 0 0 0 0 260 210 0 0000000000000 L-TMH#4 0 0 0 0 0 0 0 00000800000000 L-CMW 0 0 0 0 0 0 0 000001500000000 The Journal of Immunology 2749

Table IV. Subjects with CTL responses against HIV-1

Vpu Vpr Vif Tat Rev p15 p17 p24 gp41 RT Nef

Responders (n ϭ 29) 0 15 (52%) 10 (35%) 5 (17%) 8 (28%) 14 (48%) 22 (76%) 21 (72%) 8 (28%) 16 (55%) 20 (79%) No. amino acids 78 96 191 88 116 137 130 240 350 560 205 Amino acid adjusted scorea 0 0.54 0.18 0.19 0.24 0.34 0.59 0.30 0.08 0.1 0.39

a Frequency of recognition in percentage divided by number of amino acids per protein. common in the studied cohort, including HLA-A*0201, -A*0301, multiple epitopes contained within Vif and Vpr, and include fine and -B*0702 (Table V). The most frequently recognized CTL mapping of nine optimal CTL epitopes within these proteins. epitopes were the HLA-A*0201-restricted epitope AIIRILQQL In these studies we used a comprehensive set spanning ex- (AL9) and the HLA-B*0702-restricted epitope FPRIWLHGL pressed HIV-1 proteins to characterize the relative role of each (FL9) in Vpr. AL9 was targeted by one-third (8/24) of the indi- protein as a target for CD8ϩ T cell responses. Interestingly, HIV-1 viduals expressing the HLA-A2 allele. FL9 was recognized by 4/8 Vpr was the most frequently targeted HIV-1 protein, together with individuals expressing the HLA-B7, as well as by the two indi- p17 Gag, when adapted to the amino acid length of the protein. It viduals in this cohort that expressed HLA-B*8101, an HLA class can be speculated that several factors may contribute to the high I allele common in the African population, indicating a high degree recognition of Vpr and p17 by virus-specific CTL. First, the high of cross-presentation and -recognition between these closely re- frequency of HLA-A2 in our Caucasian study population may Downloaded from lated HLA class I alleles. Also, two novel epitopes restricted by have contributed to an overrepresentation of Vpr- and p17 (Gag)- HLA-B*5701, an allele associated with long-term nonprogressive specific responses due to immunodominant HLA-A2-restricted HIV-1 infection, were identified and frequently recognized in in- CTL epitopes within p17 and Vpr. However, a re-evaluation of our dividuals expressing the corresponding allele (Table V). These data after the exclusion of individuals expressing HLA-A2 from studies not only characterize multiple epitopes within these acces- ϩ the analysis showed that the frequent recognition of HIV-1 Vpr sory proteins, but show that the CD8 T cell responses detected http://www.jimmunol.org/ was independent from the overrepresentation of this allele in the are associated not only with IFN-␥ production but also with study cohort. Another factor influencing the recognition of viral cytolysis. proteins by CTL may be the amount of expression of these proteins during viral infection, with proteins expressed at high frequencies Discussion being more frequently targeted by CTL. Indeed, HIV-1 Gag and HIV-1-specific CTL responses have been studied in great detail HIV-1 Vpr are expressed at higher levels in infected cells com- since their first description (17, 35). Increasing evidence suggests ϩ pared with HIV-1 Pol and also at higher levels compared with that a comprehensive assessment of CD8 T cell responses against HIV-1 Vif (37, 38). However, a potential relationship between HIV-1 is necessary to interpret the entire magnitude and breadth of frequency of recognition by CTL and expression levels of the cor- by guest on September 26, 2021 these responses (8, 16). We have recently demonstrated that the responding protein remain to be determined. Finally, the degree of use of overlapping peptide spanning the HIV-1 structural as well as regulatory proteins allows for a detailed characterization of sequence conservation within a particular HIV-1 protein may in- HIV-1-specific CTL responses and the identification of novel CTL fluence its recognition by CTL, as these proteins are less likely to epitopes within these proteins (9, 16, 23, 24, 27, 36). However, the escape from CTL-mediated immune pressure, leading to the po- relative contribution of CTL responses directed against the acces- tential accumulation of responses directed against these more con- sory HIV-1 proteins Vpr, Vpu, and Vif have not been studied to served proteins. HIV-1 p17 and Vpr are reported to be relatively date. In these studies CD8ϩ T cell responses directed against these highly conserved, but are not more conserved than RT, which was accessory proteins were characterized in 60 HIV-1-infected indi- much less frequently targeted (39). Additional studies, including viduals comprehensively by using overlapping peptides spanning analysis of the autologous viral sequence, are needed to better the entire Vpr, Vpu, and Vif sequence of HIV-1. We demonstrate understand the relation between frequencies of CTL recognition that the accessory HIV-1 proteins Vpr and Vif are frequently tar- and virus variability. Taken together, these data suggest that re- geted by HIV-1-specific CD8ϩ T cells, with 45 and 33% of the sponses directed against the accessory HIV-1 proteins are fre- tested individuals having detectable responses to these proteins, quently detected in natural HIV-1 infection and may contribute ϩ respectively, contributing importantly to the total HIV-1-specific importantly to the total virus-specific CD8 T cell responses. CTL responses. In contrast, HIV-1 Vpu is rarely targeted by CTL Several novel techniques adapted during the last year have al- in infected individuals. These studies also indicate that there are lowed a more rapid characterization of novel CTL epitopes, using

Table V. Novel optimal CTL epitopes within HIV-1 Vpr and Vif and frequency of recognition

HIV-1 Protein Amino Acid Position Sequence HLA Restriction Recognitiona

Vpr 12Ð20 REPHNEWTL B*4002 1/1 Vpr 29Ð38 AVRHFPRIW B*5701 4/6 Vpr 34Ð42 FPRIWLHGL B*8101 2/2 Vpr 34Ð42 FPRIWLHGL B*0702 4/8 Vpr 58Ð66 AIIRILQQL A*0201 8/24 Vif 17Ð26 RIRTWKSLVK A*0301 3/15 Vif 31Ð39 ISKKAKGWF B*5701 2/6 Vif 48Ð57 HPRVSSEVHI B*0702 3/8 Vif 102Ð111 LADQLIHLHY B*1801 2/5

a Individuals with the corresponding HLA class I alleles that responded to the epitope. 2750 CTL RESPONSES DIRECTED AGAINST ACCESSORY HIV-1 PROTEINS

FIGURE 5. Isolation of peptide-specific CD8ϩ T cell lines specific for peptide Vif-20. The percentage of CD8ϩ T cells specific for the Vif-20 peptide in individual AC-04 was quantified using PBMC (A), CD8ϩ T cell lines after 10 days nonspecific expansion with a CD3/CD4 bispecific mAb (B), and following the isolation and expansion of Ag-specific Downloaded from CD8ϩ T cells using the IFN-␥ catching assay (C). The isolated Vif-20-specific CD8ϩ T cells showed high cytotoxic activity in a standard 51Cr release assay against autologous BCL pulsed with the Vif-20 peptide or infected with rVV expressing HIV-1 Vif (D). Background lysis of BCL pulsed with an non- HLA class I-matched peptide (control) or infected http://www.jimmunol.org/ with rVV-lac was below 5%, respectively (D). by guest on September 26, 2021

PBMC with no prior in vitro expansion. These newly adapted tech- the viral life cycle (10Ð14). HIV-1 Vpr is implicated in the nuclear niques include the fine-mapping of novel CTL epitopes using se- translocation of the PIC after infection (40Ð43) and the arrest of rial dilutions of truncated peptides in an ELISPOT assay (23) and the cell cycle in the G2 phase (44, 45). HIV-1 Vif may play an the determination of HLA restriction of these novel epitopes using important role in viral disassembly after infection, the transport APCs only sharing one HLA class I allele with the corresponding of incoming viral PIC to the nucleus, and HIV-1 provirus for- donor in a flow-based restriction assay (27). However, these tech- mation (46Ð50). Different structural regions within these pro- niques require a frequency of CD8ϩ T cells specific for the studied teins have been linked with their function. The Vpr protein epitope of at least 0.3%, in particular for the flow-based HLA contains four structural regions: the N-terminal region, the argi- restriction assay, to provide significant and reliable results. In this nine-rich C-terminal region, and one central domain containing study, we describe an additional technique that allows for the rapid two putative ␣ helices (H␣1 and H␣2) (41, 51Ð53). It has been generation of peptide-specific CD8ϩ T cell lines, including lines shown that the C-terminal basic region appears to be critical for specific for epitopes recognized by CD8ϩ T cells at low frequen- the cell cycle arrest (52Ð54) and that the two ␣ helices are cies. The nonspecific stimulation of CD8ϩ T cells with a bispecific involved in the nuclear localization of the protein as well as the CD3/CD4 Ab allows for the expansion of CD8ϩ T cells to levels incorporation of Vpr into virus particle (41, 51Ð55). The asso- that enable the sorting of peptide-specific CD8ϩ T cells in an ciation between function and structure is less well established IFN-␥ catching assay using magnetic beads. These peptide-specific for HIV-1 Vif (56Ð 60). CD8ϩ T cells can be further expanded after restimulation with Several of the newly defined CTL epitopes map to important autologous feeder cells and exhibit strong peptide-specific cyto- functional domains within the Vpr protein. The most frequently toxic activity in a standard 51chromium release assay. This newly recognized novel CTL epitope AIIRILQQL, restricted by HLA- adapted assay will further facilitate the identification of novel CTL A*0201, is located within the ␣ helix (H␣2) of Vpr. This region epitopes and allow for the rapid generation of epitope-specificT within Vpr is highly conserved (61), and single point mutations, cell lines that can subsequently be used for functional assays. involving either the isoleucine residue in position 60 that repre- The accessory HIV-1 proteins Vpr, Vpu, and Vif are essential sents the P2 anchor residue for the HLA-*0201-restricted epitope for viral replication and have important roles in the early phases of or the C-terminal anchor residue (position 67) of the novel epitope, The Journal of Immunology 2751 have been recently shown to result in a loss of the perinuclear 20. Brosterhus, H., S. Brings, H. Leyendeckers, R. A. Manz, S. Miltenyi, A. Radbruch, M. Assenmacher, and J. Schmitz. 1999. Enrichment and detection localization of the wild-type protein (51), indicating the important ϩ ϩ of live antigen-specific CD4 and CD8 T cells based on cytokine secretion. function of this region. It has been suggested that CTL responses Eur. J. Immunol. 29:4053. directed against functionally important regions within the virus 21. Walker, B. D., C. Flexner, K. Birch-Limberger, L. Fisher, T. J. Paradis, A. Aldovini, R. Young, B. Moss, and R. T. Schooley. 1989. Long-term culture could be more effective, as CTL-induced viral escape mutations and fine specificity of human cytotoxic T-lymphocyte clones reactive with human within these domains are less likely to occur or may lead to re- immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86:9514. duced viral competence (62). In particular, mutations within Vpr 22. Johnson, R. P., A. Trocha, L. Yang, G. P. Mazzara, D. L. Panicali, T. M. Buchanan, and B. D. Walker. 1991. HIV-1 gag-specific cytotoxic T lym- and Vif have been observed in individuals with long-term nonpro- phocytes recognize multiple highly conserved epitopes: fine specificity of the gressive HIV-1 infection and were associated with replication-de- gag-specific response defined by using unstimulated peripheral blood mononu- ficient viruses in human and macaque infection (63Ð65). Longi- clear cells and cloned effector cells. J. Immunol. 147:1512. 23. Altfeld, M. A., A. Trocha, R. L. Eldridge, E. S. Rosenberg, M. N. Phillips, tudinal analysis will be required to evaluate whether the virus is M. M. Addo, R. P. Sekaly, S. A. Kalams, S. A. Burchett, K. McIntosh, et al. 2000. less likely to escape from CTL targeting epitopes located within Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic these accessory proteins and whether these responses have any T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by en- zyme-linked immunospot assay. J. Virol. 74:8541. impact on disease progression. 24. Goulder, P. J., C. Brander, K. Annamalai, N. Mngqundaniso, U. Govender, In conclusion, this study demonstrates that the accessory HIV-1 Y. Tang, S. He, K. E. Hartman, C. A. O’Callaghan, G. S. Ogg, et al. 2000. proteins Vpr and Vif are frequently targeted by virus-specific Differential narrow focusing of immunodominant human immunodeficiency virus ϩ gag-specific cytotoxic T-lymphocyte responses in infected African and Caucasoid CD8 T cells, whereas Vpu is not. Adapted to the length of the adults and children. J. Virol. 74:5679. ϩ proteins, Vpr was preferentially targeted by HIV-1-specific CD8 25. Pitcher, C. J., C. Quittner, D. M. Peterson, M. Connors, R. A. Koup, V. C. Maino, ϩ T cells compared with other viral proteins. Despite the small size and L. J. Picker. 1999. HIV-1-specific CD4 T cells are detectable in most Downloaded from individuals with active HIV-1 infection, but decline with prolonged viral sup- of these proteins, multiple novel CTL epitopes within Vpr and Vif pression. Nat. Med. 5:518. were defined, several of them located within functionally impor- 26. Goulder, P. J., Y. Tang, C. Brander, M. R. Betts, M. Altfeld, K. Annamalai, tant sites of these proteins. These novel CTL epitopes will allow A. Trocha, S. He, E. S. Rosenberg, G. Ogg, et al. 2000. Functionally inert HIV- specific cytotoxic T lymphocytes do not play a major role in chronically infected for a more detailed study of CTL responses directed against HIV-1 adults and children. J. Exp. Med. 192:1819. and may represent potential candidates for future HIV-1 vaccines. 27. Goulder, P. J., M. M. Addo, M. A. Altfeld, E. S. Rosenberg, Y. Tang, U. Govender, N. Mngqundaniso, K. Annamalai, T. U. Vogel, M. Hammond, et al. http://www.jimmunol.org/ 2001. 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