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HERV-K(HML-2) Gag and Env specific T cell responses are 1 infrequently detected in HIV-1-infected subjects using standard 2 peptide-matrix based screening 3 4 R. Brad Jones1, Vivek M. John1, Diana V. Hunter1, Eric Martin1, Shariq Mujib1, Vesna 5 Mihajlovic1, Peter C. Burgers2, Theo M. Luider2, Gabor Gyenes1, Neil C. Sheppard3, Devi 6 SenGupta4, Ravi Tandon4, Feng-Yun Yue1, Erika Benko5, Colin Kovacs5, Douglas F. Nixon4, 7 Mario A. Ostrowski1 8 9 1Department of Immunology, University of Toronto, Medical Sciences Building, Rm 6271, 1 10 King's College Circle, Toronto ON, M5S 1A8, Canada and the Li Ka Shing Knowledge Institute 11 of St. Michael’s Hospital, Toronto, ON, M5B 1W8, Canada. 12 13 2Laboratories of Neuro-Oncology/Clinical and Cancer Protemoics, Department of Neurology, 14 Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. 15 16 3Vaccine Research, Worldwide Research and Development, Pfizer Inc., 10777 Science Center 17 Drive, San Diego, CA 92121, USA. 18 19 4Division of Experimental Medicine, University of California, San Francisco, 1001 Portrero 20 Avenue, Building 3, Room 603, San Francisco, CA 94110, USA. 21 22 5Maple Leaf Medical Clinic, Toronto, ON, M5B 1L6, Canada. 23 24 25 26 To whom correspondence should be addressed: 27 Brad Jones, brad.jones@utoronto.ca 28 29 30 31 32 33 34 35 36 37 38 39 40

Copyright © 2011, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05583-11 CVI Accepts, published online ahead of print on 28 December 2011

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Abstract. 41 T-cell responses to HERV-K(HML-2) Gag and Env were mapped in HIV-1-infected 42 subjects using 15mer peptides. Small peptide pools, and high concentrations were 43 used to maximize sensitivity. In the 27 subjects studied, only three bona-fide HERV-44 K(HML-2)-specific responses were detected. At these high peptide concentrations 45 we detected false positive responses, three of which were mapped to an HIV-1-Gag 46 peptide contaminant. Thus, HERV-K(HML-2) Gag and Env specific T-cell 47 responses are infrequently detected by 15mer peptide mapping. (73 words) 48 Human endogenous retroviruses (HERVs) are the DNA remnants of ancient 49 retroviruses that infected the germ-line of our evolutionary ancestors (2). The 50 complement of HERVs in the human genome is diverse, representing all three major 51 branches of the retroviral tree: gamma-epsilon, spuma and delta-lenti-alpha-beta 52 retroviruses (see (14) for review). The potential for HERVs to serve as a source of 53 antigens was highlighted by a recent study which mapped the epitope specificity of a 54 renal cell carcinoma (RCC) reactive CD8+ T-cell to a HERV-E-derived peptide (26). 55 Previously we presented the hypothesis that the manipulation of an infected cell 56 by HIV-1, to favor retroviral expression, may result in the expression of HERV antigens. 57 Supporting this, we have reported the detection of T-cell responses to peptides derived 58 from diverse HERV families, selected for their predicted binding to common MHC-I 59 molecules, in HIV-1-infected subjects, but not in uninfected controls (12, 27). We have 60 since reported that the presence of strong HERV-specific T-cell responses is associated 61 with control of HIV-1 in chronic infection (25). This has led us to speculate that HERV 62 antigen expression in HIV-1-infected cells may serve as a surrogate marker that could be 63

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targeted in novel T-cell based HIV-1 vaccines. In testing this model, we have decided to 64 focus in on the HML-2 lineage of the HERV-K class II superfamily. Recent proliferation 65 of HERV-K(HML-2) is evidenced by the presence of human-specific and polymorphic 66 insertions. These recently integrated HERV-K(HML-2) proviruses are comparatively 67 intact and many contain complete open reading frames for viral proteins (3, 5, 10, 13, 19-68 21, 23, 28-30). We have recently demonstrated that the expression of HERV-K(HML-2) 69 Gag and Env proteins is induced upon HIV-1-infection of primary CD4+ T-cells, and that 70 a HERV-K(HML-2)-Env-specific CD8+ T-cell clone specifically eliminates cells infected 71 with diverse isolates of HIV-1 (Jones et al., submitted). 72 Here, we took a comprehensive approach to evaluating a potential role for HERV-73 K(HML-2) Gag and Env specific T-cell responses in natural control of HIV-1 infection. 74 T-cell responses to 15mer peptides (manufactured at >70% purity), overlapping by 11 75 amino acids and spanning HERV-K(HML-2) Gag and Env, were measured by IFN-γ 76 ELISPOT. In contrast to our previous reports, which used peptides predicted to be 77 optimal epitopes for common MHC-I alleles, this method allowed for screening of 78 subjects irrespective of their HLA-type. Critically, here we are also focusing on the 79 Gag and Env antigens of the HML-2 lineage of HERVs, based on our evidence for 80 HIV-1-induced expression of these proteins (Jones et al. submitted), whereas 81 previous reports considered responses to diverse HERV families. ELISPOT assays 82 were performed using standard procedures using the Mabtech IFN-γ ELISPOT 83 assay. Briefly, cryopreserved cells were thawed and rested overnight in AIM-V 84 medium (Invitrogen) supplemented with 60U/ml of Benzonase (Sigma). Cells were 85 then plated at 2x106 cells/well in AIM-V medium and cultured for 16 hours with 86

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peptides (final concentration 0.25% DMSO), 0.25% DMSO alone, or 2μg/ml SEB 87 (Sigma). Plates were then washed, probed with Abs following the manufacturer’s 88 instructions and developed with the AP Color Development Reagent (Biorad). Plates 89 were dried overnight and spots were counted using a CTL-Immunospot system. 90 Criteria for positive responses are given in the legend of Fig. 1. Initially, we screened 91 a number of HIV-1-infected and uninfected subjects for HERV-K(HML-2) Gag and Env 92 specific CD8+ T-cell responses using master pools containing 172 peptides (Gag) or 164 93 peptides (Env) at 0.1μg/ml/peptide, but consistently observed a lack of responses. We 94 reasoned that HERV-K(HML-2)-specific CD8+ T-cell responses may be of lower avidity 95 than those specific for exogenous viruses such as HIV-1, as higher avidity clones specific 96 for HERV-K(HML-2) may have been deleted by thymic selection. We therefore moved 97 to a higher sensitivity approach that has been successfully applied for other antigens, and 98 arranged peptides into matrix pools of 10-18 peptides each (1, 4, 6, 11, 17, 22, 24). In 99 addition to allowing us to test higher per-peptide concentrations, this approach allows for 100 mapping of responses to an individual 15mer peptide. PBMC from 27 HIV-1-infected 101 subjects, comprising 11 chronic progressors, 9 viral controllers and 3 subjects in 102 acute/early infection (see (15) for definitions) as well as 6 uninfected controls were 103 screened using these matrix pools (Table 1). We observed a lack of responses to HERV-104 K(HML-2)-Gag and Env, while detecting clear responses to HIV-1-Gag and CMV-pp65 105 peptide pools. To further increase our ability to detect low-avidity responses, we re-106 screened these subjects with matrix pools at 10μg/ml/peptide. Under these conditions, we 107 observed 22 responses to HERV-K(HML-2) in HIV-1-infected subjects, and a lack of 108 responses in HIV-1-uninfected controls. When we attempted to confirm these responses, 109

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however, we observed that the majority of these proved irreproducible with newly 110 synthesized batches of 15mer peptide (while responding to the original batch of 111 individual 15mer peptide in parallel). Only three responses, both detected in viral 112 controllers, were reproduced across multiple batches of peptide. One subject, OM77, 113 made weak responses to one epitope each in Gag and Env: HERV-K(HML-2)-Gag-114 QSAIKPLKGKVPAGS – mean 60 SFU/million PBMC, and HERV-K(HML-2)-Env-115 HKMNKMVTSEEQMKL – mean 65 SFU/million PBMC. A second subject, OM9, 116 made a response to HERV-K(HML-2)-CIDSTFNWQHRILLV – 813 SFU/million 117 PBMC. The latter response has been subsequently fine-mapped to CIDSTFNWQHR 118 (Jones et al., submitted). 119 Intriguingly, the 19 responses which proved irreproducible with new batches of 120 peptide were detected exclusively in HIV-1-infected subjects. This led us to consider that 121 perhaps they may be true HERV-K(HML-2)-specific CD8+ T-cell responses, but specific 122 for some modified version of the relevant HERV-K(HML-2) peptide which was present 123 in only the first batch of peptide. To study this possibility, we generated CD8+ T-cell 124 clones specific for HERV-K-Env-SSVQSVNFVNDWQKN (pep124), HERV-K-Gag-125 GIGQNWSTISQQALM (pep100), and HERV-K-Gag-YENANPECQSAIPL (pep121) 126 using previously described method of IFN-γ capture followed by limiting dilution cloning 127 (16). These responses had been detected from the HIV-1-infected chronic progressor 128 ‘OM125’ by matrix mapping (Fig. 1A, B), and were confirmed at the level of the 129 individual peptide (Fig. 1C and data not shown). The clones failed to respond to newly 130 synthesized batches of their corresponding 15mer peptides (Fig. 2A), as well as to 131 truncated peptides designed to fine-map their epitopes, while responding to the original 132

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peptide in parallel (data not shown). Serendipitously, our experimental setup was such 133 that mismatched epitopes were employed as negative controls for clone responses. For 134 example, the HERV-K(HML-2)-Gag-pep100-specific clone was tested against HERV-135 K(HML-2)-Env-pep124 and HERV-K(HML-2)-Gag-pep121. Surprisingly, we observed 136 that each of these clones responded to all three of these original peptides, while failing to 137 respond to newly synthesized batches (Fig. 2B). A CMV-pp65-specific CD8+ T-cell 138 clone did not respond to the original HERV-K(HML-2)-derived peptides, ruling out a 139 non-specific effect. This finding suggested that all three clones were specific for a foreign 140 peptide contaminant that was common to all three of these peptide preparations. We 141 tested the possibility that this peptide may be derived from HIV-1 by screening these 142 clones with HIV-1 peptide matrix pools (NIH AIDS reagent program cat#: 8117-Gag and 143 6208-Pol) and observed clear recognition of HIV-1-Gag-peptide pools 5, 20 and 21 (Fig. 144 2B). The two 15mer peptides mapped by these responses, AAEWDRLHPVHAGPI and 145 DRLHPVHAGPIAPGQ share the HLA-B*35-restricted T-cell epitope HPVHAGPI – 146 corresponding with the B35+ status of the subject. The possibility that these ‘clones’ may 147 be oligo-clonal, with distinct subsets specific for the HERV-K(HML-2)-derived peptides 148 and HPVHAGPI was ruled out by flow cytometry experiments indicating responsiveness 149 of the entire population to the original HERV-K(HML-2) peptide preparations (Fig. 2C). 150 Thus, the CD8+ T-cell responses identified using our original HERV-K(HML-2) peptides 151 in subject OM125 are specific for an HIV-1-derived peptide contaminant. We have since 152 confirmed the presence of the HPVHAGPI peptide in these peptide preparations by 153 nanoLC-Orbitrap mass-spectrometry. While we did not identify a specific contaminant 154 for the other 16 of 19 irreproducible responses (observed against other HERV-K(HML-2) 155

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peptides), we extrapolate upon this result to propose that these may each be due to low-156 level HIV-1 peptide contaminants, explaining their exclusive presence in HIV-1-infected 157 subjects. 158

We can rule out that the contamination occurred in our hands based on two lines 159 of evidence. First, peptides were pooled by the manufacturer. It was mapping, based on 160 these pre-assembled matrix pools, which led us to test corresponding individual peptides, 161 at which level these responses were confirmed. Second, the original peptides were 162 shipped direct from the manufacturer in two separate aliquots to our labs in Toronto and 163 San Francisco. The contaminant-specific T-cell clones responded to the corresponding 164 HERV-K(HML-2) peptides at both sites. We propose that the contamination issues 165 encountered in this study may be one for which the study of HERV-K(HML-2)-specific 166 T-cell responses is likely to be particularly prone. Here, we are looking for low frequency 167 responses, which when detected require fairly high levels of peptide – IC50 of 2μM for a 168 bona-fide HERV-K(HML-2)-Env-specific (Jones et al., submitted) in subjects infected 169 with HIV-1, a pathogen for which synthetic peptides are frequently produced at 170 manufacturing facilities. Thus, in addition to iterating the need, highlighted by others (7-171 9), for caution in considering the possibility of foreign contaminants in peptide 172 preparations, we emphasize a specific need to consider the possibility of HIV-1 peptide 173 contaminants when screening HIV-1-infected subjects for responses to HERV-K(HML-174 2) and other non-HIV-1 antigens. This can be guarded against by testing at least a 175 subset of identified responses against newly synthesized batches of peptide provided 176 by a different supplier. 177

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Our original presentation of HERV-specific T-cell responses in HIV-1-178 infected subjects (12), used different batches of peptides (from a different 179 manufacturer) than those used for our follow-up studies (25). The fact that we 180 observed comparable magnitudes and frequencies of responses between these 181 studies demonstrates the reproducibility of these earlier results. The discrepancy 182 between the infrequent detection of T-cell responses in the present study, and the frequent 183 HERV-specific T-cell responses that we have previously reported, likely results from a 184 number of factors. Critically, in previous studies we considered T-cell responses to 185 diverse families of HERV, with only six peptides representing HERV-K(HML-2). Of 186 these six, four were from Pol – an antigen which was not included in the present study. 187 Additionally, in previous studies we tested individual peptides predicted to be optimal 188 epitopes in an HLA-matched manner. This is a more sensitive method for detecting CD8+ 189 T-cell responses than using pools of 15mers (4, 6, 18). In the current study we attempted 190 to maximize the sensitivity of screening with 15mer peptides by testing up to 191 10μg/ml/peptide. Although this did yield some bona-fide responses, these were in the 192 minority. The bona-fide and false HERV-K(HML-2)-specific T-cell responses detected in 193 this study titered down similarly, losing responsivness between 1-10 μg/ml, confirming 194 that we are unable to further boost sensitivity of 15mer screening without compromising 195 specificity. As the use of predicted optimal epitopes is not amenable to comprehensive 196 screening of subjects with diverse HLA types, alternative approaches, such as expansion 197 of T-cells with HERV-K(HML-2) gag and env mRNA transfected dendritic cells, will be 198 explored in future studies. At present it is unclear whether the low frequency of HERV-199 K(HML-2) Gag and Env-specific T-cell responses observed in the current study reflects 200

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the ineffectiveness of screening with 15mer peptides, or whether it represents a true 201 dearth of T-cell responses against the Env and Gag antigens of this particular lineage of 202 HERV in natural HIV-1 infection. Given our recent demonstration of anti-HIV-1 activity 203 by a HERV-K(HML-2)-specific T-cell clone (Jones et al. submitted), the finding that 204 such responses are not commonly induced in natural infection would highlight the 205 potential for intervention by HERV-K(HML-2)-targeted therapeutic vaccination 206 strategies. 207 Acknowledments. 208 Funding for this work was provided by Pfizer Inc. through as sponsored research 209 agreement. This work was also supported by funds from the National Institutes of Health 210 (AI076059 and AI084113). The following reagents were obtained from the NIH AIDS 211 Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 212 from Dr. Maurice Gately and peptide sets representing HIV-1: Pol and Gag. RBJ 213 gratefully acknowledges salary support from the Ontario HIV Treatment Network 214 (OHTN). 215 Fig. 1. T-cell responses can be mapped using matrix pools of HERV-K(HML-2) Gag 216 and Env derived peptides. (A,B) Peripheral blood mononuclear cells (PBMC) from the 217 HIV-1-infected subject ‘OM125’ were screened by IFN-γ ELISPOT using peptide-matrix 218 pools. Shown are results depicting mean spot forming units (SFU) / million PBMC. Tests 219 were performed in duplicate and error bars represent standard deviation. Background 220 levels were established by measuring responsiveness to 0.5% DMSO, and a pool of 221 CMV-pp65-peptides was included as a positive control. The horizontal line depicts the 222 cut-off of a positive response based on both: i) >3x background and ii) >50 SFU/million 223

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PBMC after background subtraction. (A) The response to HERV-K(HML-2)-Env peptide 224 matrix pools 7 and 26 maps to HERV-K(HML-2)-Env-pep124. (B) The response to 225 HERV-K(HML-2)-Gag peptide matrix pools 9 and 21 maps to pep100, while the 226 response to HERV-K(HML-2)-Gag peptide matrix pools 4 and 23 maps to pep121 (C) 227 Shown are IFN-γ ELISPOT results with 200,000 OM125 PBMC/well confirming the 228 response to the HERV-K(HML-2)-Env pep124 15mer peptide common to matrix pools 7 229 and 23. The response to these matrix pools are also shown in parallel. The label ‘HK’ is 230 an abbreviation of HERV-K(HML-2). 231 Fig. 2. CD8+ T-cell clones identified by responses to HERV-K(HML-2)-Env-pep124, 232 Gag-pep100 and Gag-pep121 are specific for an HIV-1-Gag peptide contaminant. 233 (A) CD8+ T cell clones responsive to original batches of HERV-K(HML-2) peptides 234 were obtained by IFN-γ capture, followed by two rounds of limiting dilution cloning. 235 Shown are IFN-γ ELISPOT results indicating that these T-cell clones, presumably of 3 236 separate specificities, in fact respond to all three original HERV-K(HML-2) peptides, 237 while failing to respond to newly synthesized batches of peptides with the same 238 sequences. A CMV-pp65-specific CD8+ T-cell clone did not respond to the original 239 HERV-K(HML-2)-specific peptides, ruling out a non-specific effect. All peptides were 240 tested at 100μg/ml in 0.05% DMSO. The label ‘HK’ is an abbreviation of HERV-241 K(HML-2). (B) These CD8+ T-cell clones were tested against HIV-1-derived peptide-242 matrix pools to test the hypothesis that they were specific for a contaminating HIV-1 243 peptide. All three clones responded to matrix pools 5, 20 and 21, mapping to the 15mers 244 AAWDRLHPVHAGPI and DRLHPVHAGPIAPGQ. Shown are results for the clone 245 obtained using HERV-K(HML-2)-Gag-pep100. (C) The CD8+ T-cell clone from B was 246

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mixed with autologous B lymphoblastoid cells and either pulsed with peptide or 247 maintained as a no-peptide control. Shown are flow cytometry data, gated on CD8+ cells, 248 depicting CD107a (y-axis) by IFN-γ (x-axis). 249 References. 250 1. Addo, M. M., X. G. Yu, A. Rathod, D. Cohen, R. L. Eldridge, D. Strick, M. N. 251

Johnston, C. Corcoran, A. G. Wurcel, C. A. Fitzpatrick, M. E. Feeney, W. R. 252 Rodriguez, N. Basgoz, R. Draenert, D. R. Stone, C. Brander, P. J. Goulder, E. 253 S. Rosenberg, M. Altfeld, and B. D. Walker. 2003. Comprehensive epitope 254 analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell 255 responses directed against the entire expressed HIV-1 genome demonstrate 256 broadly directed responses, but no correlation to viral load. J Virol 77:2081-92. 257

2. Bannert, N., and R. Kurth. 2006. The evolutionary dynamics of human 258 endogenous retroviral families. Annu Rev Genomics Hum Genet 7:149-73. 259

3. Barbulescu, M., G. Turner, M. I. Seaman, A. S. Deinard, K. K. Kidd, and J. 260 Lenz. 1999. Many human endogenous retrovirus K (HERV-K) proviruses are 261 unique to humans. Curr Biol 9:861-8. 262

4. Beattie, T., R. Kaul, T. Rostron, T. Dong, P. Easterbrook, W. Jaoko, J. 263 Kimani, F. Plummer, A. McMichael, and S. Rowland-Jones. 2004. Screening 264 for HIV-specific T-cell responses using overlapping 15-mer peptide pools or 265 optimized epitopes. Aids 18:1595-8. 266

5. Berkhout, B., M. Jebbink, and J. Zsiros. 1999. Identification of an active 267 reverse transcriptase enzyme encoded by a human endogenous HERV-K 268 retrovirus. J Virol 73:2365-75. 269

6. Betts, M. R., D. R. Ambrozak, D. C. Douek, S. Bonhoeffer, J. M. Brenchley, 270 J. P. Casazza, R. A. Koup, and L. J. Picker. 2001. Analysis of total human 271 immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: 272 relationship to viral load in untreated HIV infection. J Virol 75:11983-91. 273

7. Currier, J. R., L. M. Galley, H. Wenschuh, V. Morafo, S. Ratto-Kim, C. M. 274 Gray, L. Maboko, M. Hoelscher, M. A. Marovich, and J. H. Cox. 2008. 275 Peptide impurities in commercial synthetic peptides and their implications for 276 vaccine trial assessment. Clin Vaccine Immunol 15:267-76. 277

8. de Beukelaar, J. W., J. W. Gratama, P. A. Smitt, G. M. Verjans, J. Kraan, T. 278 M. Luider, and P. C. Burgers. 2007. The impact of impurities in synthetic 279 peptides on the outcome of T-cell stimulation assays. Rapid Commun Mass 280 Spectrom 21:1282-8. 281

9. de Graaf, M. T., J. W. de Beukelaar, P. C. Burgers, T. M. Luider, J. Kraan, 282 P. A. Smitt, and J. W. Gratama. 2008. Contamination of synthetic HuD protein 283 spanning peptide pools with a CMV-encoded peptide. Cytometry A 73:1079-85. 284

10. Dewannieux, M., S. Blaise, and T. Heidmann. 2005. Identification of a 285 functional envelope protein from the HERV-K family of human endogenous 286 retroviruses. J Virol 79:15573-7. 287

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11. Draenert, R., M. Altfeld, C. Brander, N. Basgoz, C. Corcoran, A. G. Wurcel, 288 D. R. Stone, S. A. Kalams, A. Trocha, M. M. Addo, P. J. Goulder, and B. D. 289 Walker. 2003. Comparison of overlapping peptide sets for detection of antiviral 290 CD8 and CD4 T cell responses. J Immunol Methods 275:19-29. 291

12. Garrison, K. E., R. B. Jones, D. A. Meiklejohn, N. Anwar, L. C. Ndhlovu, J. 292 M. Chapman, A. L. Erickson, A. Agrawal, G. Spotts, F. M. Hecht, S. Rakoff-293 Nahoum, J. Lenz, M. A. Ostrowski, and D. F. Nixon. 2007. T cell responses to 294 human endogenous retroviruses in HIV-1 infection. PLoS Pathog 3:e165. 295

13. Harris, J. M., R. H. Haynes, and E. M. McIntosh. 1997. A consensus sequence 296 for a functional human endogenous retrovirus K (HERV-K) dUTPase. Biochem 297 Cell Biol 75:143-51. 298

14. Jern, P., G. O. Sperber, and J. Blomberg. 2005. Use of endogenous retroviral 299 sequences (ERVs) and structural markers for retroviral phylogenetic inference and 300 taxonomy. Retrovirology 2:50. 301

15. Jones, R. B., L. C. Ndhlovu, J. D. Barbour, P. M. Sheth, A. R. Jha, B. R. 302 Long, J. C. Wong, M. Satkunarajah, M. Schweneker, J. M. Chapman, G. 303 Gyenes, B. Vali, M. D. Hyrcza, F. Y. Yue, C. Kovacs, A. Sassi, M. Loutfy, R. 304 Halpenny, D. Persad, G. Spotts, F. M. Hecht, T. W. Chun, J. M. McCune, R. 305 Kaul, J. M. Rini, D. F. Nixon, and M. A. Ostrowski. 2008. Tim-3 expression 306 defines a novel population of dysfunctional T cells with highly elevated 307 frequencies in progressive HIV-1 infection. J Exp Med 205:2763-79. 308

16. Jones, R. B., F. Y. Yue, X. X. Gu, D. V. Hunter, S. Mujib, G. Gyenes, R. D. 309 Mason, R. Mohamed, K. S. MacDonald, C. Kovacs, and M. A. Ostrowski. 310 2009. Human immunodeficiency virus type 1 escapes from interleukin-2-311 producing CD4+ T-cell responses without high-frequency fixation of mutations. J 312 Virol 83:8722-32. 313

17. Kiecker, F., M. Streitz, B. Ay, G. Cherepnev, H. D. Volk, R. Volkmer-Engert, 314 and F. Kern. 2004. Analysis of antigen-specific T-cell responses with synthetic 315 peptides--what kind of peptide for which purpose? Hum Immunol 65:523-36. 316

18. Luckey, C. J., G. M. King, J. A. Marto, S. Venketeswaran, B. F. Maier, V. L. 317 Crotzer, T. A. Colella, J. Shabanowitz, D. F. Hunt, and V. H. Engelhard. 318 1998. Proteasomes can either generate or destroy MHC class I epitopes: evidence 319 for nonproteasomal epitope generation in the cytosol. J Immunol 161:112-21. 320

19. Magin, C., R. Lower, and J. Lower. 1999. cORF and RcRE, the Rev/Rex and 321 RRE/RxRE homologues of the human endogenous retrovirus family 322 HTDV/HERV-K. J Virol 73:9496-507. 323

20. Mayer, J., M. Sauter, A. Racz, D. Scherer, N. Mueller-Lantzsch, and E. 324 Meese. 1999. An almost-intact human endogenous retrovirus K on human 325 chromosome 7. Nat Genet 21:257-8. 326

21. Mueller-Lantzsch, N., M. Sauter, A. Weiskircher, K. Kramer, B. Best, M. 327 Buck, and F. Grasser. 1993. Human endogenous retroviral element K10 328 (HERV-K10) encodes a full-length gag homologous 73-kDa protein and a 329 functional protease. AIDS Res Hum Retroviruses 9:343-50. 330

22. Nixon, D. F., A. R. Townsend, J. G. Elvin, C. R. Rizza, J. Gallwey, and A. J. 331 McMichael. 1988. HIV-1 gag-specific cytotoxic T lymphocytes defined with 332 recombinant vaccinia virus and synthetic peptides. Nature 336:484-7. 333

on May 27, 2021 by guest

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23. Reus, K., J. Mayer, M. Sauter, D. Scherer, N. Muller-Lantzsch, and E. 334 Meese. 2001. Genomic organization of the human endogenous retrovirus HERV-335 K(HML-2.HOM) (ERVK6) on chromosome 7. Genomics 72:314-20. 336

24. Rodda, S. J. 2002. Peptide libraries for T cell epitope screening and 337 characterization. J Immunol Methods 267:71-7. 338

25. SenGupta, D., R. Tandon, R. G. Vieira, L. C. Ndhlovu, R. Lown-Hecht, C. E. 339 Ormsby, L. Loh, R. B. Jones, K. E. Garrison, J. N. Martin, V. A. York, G. 340 Spotts, G. Reyes-Teran, M. A. Ostrowski, F. M. Hecht, S. G. Deeks, and D. F. 341 Nixon. Strong Human Endogenous Retrovirus-Specific T Cell Responses Are 342 Associated with Control of HIV-1 in Chronic Infection. J Virol 85:6977-85. 343

26. Takahashi, Y., N. Harashima, S. Kajigaya, H. Yokoyama, E. Cherkasova, J. 344 P. McCoy, K. Hanada, O. Mena, R. Kurlander, A. Tawab, R. Srinivasan, A. 345 Lundqvist, E. Malinzak, N. Geller, M. I. Lerman, and R. W. Childs. 2008. 346 Regression of human kidney cancer following allogeneic stem cell transplantation 347 is associated with recognition of an HERV-E antigen by T cells. J Clin Invest 348 118:1099-109. 349

27. Tandon, R., D. Sengupta, L. C. Ndhlovu, R. G. Vieira, R. B. Jones, V. A. 350 York, V. A. Vieira, E. R. Sharp, A. A. Wiznia, M. A. Ostrowski, M. G. 351 Rosenberg, and D. F. Nixon. Identification of Human Endogenous Retrovirus-352 Specific T Cell Responses in Vertically HIV-1-Infected Subjects. J Virol 353 85:11526-31. 354

28. Tristem, M. 2000. Identification and characterization of novel human 355 endogenous retrovirus families by phylogenetic screening of the human genome 356 mapping project database. J Virol 74:3715-30. 357

29. Turner, G., M. Barbulescu, M. Su, M. I. Jensen-Seaman, K. K. Kidd, and J. 358 Lenz. 2001. Insertional polymorphisms of full-length endogenous retroviruses in 359 humans. Curr Biol 11:1531-5. 360

30. Yang, J., H. P. Bogerd, S. Peng, H. Wiegand, R. Truant, and B. R. Cullen. 361 1999. An ancient family of human endogenous retroviruses encodes a functional 362 homolog of the HIV-1 Rev protein. Proc Natl Acad Sci U S A 96:13404-8. 363

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75010001250

SFU

/ m

illio

n PB

MC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

0.5% DMSO

CMV-pp65

HERV-K(HML-2)-Gag peptide matrix pools

0.5% DMSOHK-Env pep 124

100mg/mlHK-Env pep 124

10mg/ml

HK-Env pep 1241mg/ml

HK-Envmatrix pool 7

HK-Envmatrix pool 23

A B

C

HERV-K(HML-2)-Env peptide matrix pools HERV-K(HML-2)-Gag peptide matrix pools

Fig. 1

CMV-pp65pool

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HK-Envpep124

HK-Gagpep100

HK-Gagpep121 CMVpp65

Clones added

0.05%DMSO

Orig. HK-Envpep124

New HK-Envpep124

New HK-Gagpep100

New HK-Gagpep121

Orig. HK-Gagpep100

Orig. HK-Gagpep121

PHA

Ant

igen

s ad

ded

A

B

‘HERV-K(HML-2)-Gag-pep100-speci�c’ T cell clone

HIV-1-Pol peptidem

atrix poolsH

IV-1-Gag peptide

matrix pools

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32 PHA

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 Gag pool

DMSO DMSO

HK-Envmatrixpool 7

HK-Envmatrix

pool 23

HK-Envmaster

pool

HK-Gagmatrixpool 9

HK-Gagmatrix

pool 21

HK-Gagmatrixpool 4

HK-Gagmatrix

pool 23

HK-Gagmaster

pool

Fig. 2

100 101 102 103 104100

101

102

103

104

<FL2-H>: CD107A-PE

1.28

100 101 102 103 104100

101

102

103

104

<FL2-H>: CD107A-PE

88.6

No peptide orig. HK-Gag-pep100

IFN-g

CD10

7a

B

C

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Table 1. Clinical data of subjects screened for HERV-K(HML-2)-specific T-cell responsesSubject ID Classification Viral Load (copies/ml) Abs CD4 count (cells/mm3) Estimated Duration of Infection (months)

1 HIV negative N/A N/A N/A71 HIV negative N/A N/A N/A176 HIV negative N/A N/A N/A484 HIV negative N/A N/A N/AVM HIV negative N/A N/A N/AEM HIV negative N/A N/A N/A2 Acute/early 332 360 2

314 Acute/early 10560 780 2348 Acute/early 4.00E+05 1317 17 Chronic Progressor 1.00E+05 240 1110 Chronic Progressor 22415 132 Not avail (> 1 year)12 Chronic Progressor 1.00E+05 510 Not avail (> 1 year)17 Chronic Progressor 4.00E+05 660 Not avail (> 1 year)19 Chronic Progressor 2.00E+05 600 Not avail (> 1 year)125 Chronic Progressor 1.00E+05 580 90378 Chronic Progressor 5.36E+04 460 97380 Chronic Progressor 1.34E+04 490 66411 Chronic Progressor 3.36E+04 430 Not avail (> 1 year)412 Chronic Progressor 1.45E+04 710 86415 Chronic Progressor 2.20E+04 530 Not avail (> 1 year)4 Viral Controller 499 803 1209 Viral Controller <50 760 Not avail (> 1 year)77 Viral Controller 79 670 Not avail (> 1 year)148 Viral Controller <50 1070 Not avail (> 1 year)194 Viral Controller <50 1440 97374 Viral Controller <50 990 239410 Viral Controller 876 740 Not avail (> 1 year)419 Viral Controller 648 390 Not avail (> 1 year)481 Viral Controller <50 670 35

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