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During HIV-1 Infection Vpr Is Preferentially Targeted By 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- 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 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- 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 (2–6). 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 (10–14). 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. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2744 CTL RESPONSES DIRECTED AGAINST ACCESSORY HIV-1 PROTEINS Materials and Methods final concentration of 1 ϫ 10Ϫ5 molar. Cells were added to the wells at 50,000–100,000 cells/well. The plates were incubated at 37°C, 5% CO Subjects 2 overnight (14–16 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- Advanced Chemtech, Louisville, KY) using F-moc chemistry. Seventeen where with minor modifications (25, 26). Briefly, 0.5–1.0 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 (12–18 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.
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