Association of HIV-Specific and Total CD8 T Memory Phenotypes in Subtype C HIV-1 Infection with Viral Set Point1 Wendy A. Burgers,2* Catherine Riou,2��� Mandla Mlotshwa,��� Pholo Maenetje,��� Debra de Assis Rosa,��� Jason Brenchley,��� Koleka Mlisana,�� Daniel C. Douek,�� Richard Koup, Mario Roederer,# Guy de Bruyn,** Salim Abdool Karim,�� Carolyn Williamson,* Clive M. Gray,3��� and the CAPRISA 002 Acute Infection Study Team Understanding early immunological events during HIV-1 infection that may set the course of disease progression is important for identifying correlates of viral control. This study explores the association of differentiation profiles of HIV-specific and total memory CD8 T cells with viral set point. A cohort of 47 HIV-1-infected individuals, with differing viral set points at 12 mo, were recruited during acute infection. We identified that the magnitude of IFN- T cell responses at 6 mo postinfection did not associate with viral set point at 12 mo. A subset of 16 individuals was further studied to characterize CD8 T cells for expression patterns of markers for memory differentiation, survival (CD127), senescence (CD57), and negative regulation (programmed death-1). We show that viral control and the predicted tempo of HIV disease progression in the first year of infection was associated with a synchronous differentiation of HIV-specific and total CD8 memory subpopulations. At 6���9 mo postinfection, those with low viral set points had a significantly higher proportion of early differentiated HIV-specific and total memory CD8 cells of a central memory (CD45RO CD27 CCR7 ) and intermediate memory (CD45RO CD27 CCR7 ) phenotype. Those with high viral set points possessed significantly larger frequencies of effector memory (CD45RO CD27 CCR7 ) cells. The proportions of memory subsets significantly correlated with CD38 CD8 T cells. Thus, it is likely that a high Ag burden resulting in generalized immune activation may drive differentiation of HIV-specific and total memory CD8 T cells. The Journal of Immunology, 2009, 182: 4751���4761. H uman immunodeficiency virus 1 infection generally leads to a decline of immune function, which in the ab- sence of successful antiretroviral therapy results in pro- gression to AIDS. However, a small group of individuals is able to naturally control viral replication and maintain high levels of CD4 cells, known as long-term nonprogressors or elite control- lers (1). It is now well established that virus fitness and/or host genetic background can contribute to the delay of HIV disease progression (2���5). More controversial is the role of HIV-specific CD4 and CD8 responses in natural viral control. Divergent data show the impact of HIV-specific CD4 T cell responses on disease progression (6, 7) and, in simian models, there is evidence that CD8 T cells may play an important role in the control of viremia (8, 9). These data strengthen the hypothesis that the development of HIV-specific CD8 T cell responses contributes to the delay of disease progression in humans and coincides with a reduction in initial viremia during primary infection (2). Although an inverse relationship has been described between the proportion of HIV- specific CD8 T cells and viral load (10), more recent studies have raised questions about whether the frequency of HIV-specific CD8 T cells is associated with viral control (11���13). Further- more, neither the breadth nor the magnitude of HIV-specific IFN- CD8 T cell responses in chronically infected patients has been shown to be a marker of viral control (12���15). These latter observations collectively infer that the quality, more than the quan- tity, of CD8 T cell responses might play a role in viral control. The hypothesis that the quality of CD8 T cells is important in controlling disease, and would be important to elicit in vac- cine-induced immunity, is supported by data showing that pro- liferation and polyfunctional cytokine responses associate with control of HIV (16���18). Phenotype and function of T cells are integrally linked, and studies have shown that stages of HIV- specific CD8 T cell differentiation may be an important qual- itative assessment. Functionally suboptimal HIV-specific cells accumulate in a pre-terminally differentiated stage (19), and viral control in early and chronic infection is associated with terminally differentiated HIV-specific effector memory CD8 cells (14, 20). The differentiation status of total CD8 memory cells may also be important during HIV infection, where recent data have shown that a late differentiated and aged total CD8 *Institute of Infectious Diseases and Molecular Medicine, Division of Medical Vi- rology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa ���AIDS Research Unit, National Institute for Communicable Diseases, Johan- nesburg, South Africa ���Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 ��Centre for the AIDS Programme of Research in South Africa (CAPRISA), Univer- sity of KwaZulu-Natal, Nelson R. Mandela School of Medicine, Durban, South Af- rica ��Human Immunology Section, Immunology Laboratory, and #ImmunoTech- nology Section, Vaccine Research Center, National Institute for Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892 and **Perinatal HIV Research Unit, University of the Witwatersrand, Johannesburg, South Africa Received for publication November 12, 2008. Accepted for publication February 7, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Department of Health and Human Services Grant U19 A151794 the Canada-Africa Prevention Trials Network and the Bill and Melinda Gates Comprehensive T Cell Vaccine Immune Monitoring Consortium award. W.B. was supported by a Columbia University-Southern Africa Fogarty AIDS International Training Fellowship. 2 W.A.B. and C.R. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Clive M. Gray, National Insti- tute for Communicable Diseases, Private Bag X4, Sandringham 2131, South Africa. E-mail address: cgray@nicd.ac.za The Journal of Immunology www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803801

memory compartment is associated with faster disease progres- sion (21). Few studies have investigated the differentiation profiles of CD8 T cell responses during acute and early infection, with most data having been collected during chronic infection in cross-sec- tional studies. It is well established that plasma viral load at ap- proximately 1 year after infection, known as viral set point, is a strong predictor of subsequent CD4 decline rates and progression to AIDS (22), implying that early events in HIV infection may set the course of viremia and hence for subsequent disease progres- sion. In SIV infection, massive destruction of the CD4 memory T cell compartment occurs in acute infection at mucosal surfaces, particularly in the gut (23). Additionally, phenotypic defects and increased apoptosis manifest in the first few weeks after SIV in- fection (24). As the global vaccine community reflects on the un- successful Ad5 HIV vaccine trial (25), identity of T cell quality and the factors that influence it during the early stages of infection may be important for vaccine development. In this study, we examined the association of T cell responses and memory differentiation with viral set point at 1 year in a sub- type C HIV-1 acute infection cohort. We show that total HIV-1- specific IFN- responses are unrelated to viral control, but a more refined analysis of CD8 T cell memory differentiation revealed associations with viral set point. Materials and Methods Study subjects Acute HIV-infected cohort. A longitudinal cohort of acutely HIV-1-in- fected women were enrolled as part of the Centre for AIDS Programme of Research in South Africa (CAPRISA) acute infection study in Durban, South Africa. This study cohort has been described previously (26, 27). The time postinfection was estimated either by a prospective RNA-posi- tive/Ab-negative measurement or taken as the midpoint between the last Ab-negative test and first Ab-positive ELISA test. Study participants were followed for 12 mo, and follow-up is ongoing. Data from events within the first 3���12 mo are reported herein. None of the study individuals was on antiretroviral therapy during the first year of infection. The University of KwaZulu-Natal, University of Witwatersrand, and University of Cape Town Research Ethics Committees approved this study, and all the subjects provided written informed consent for participation in this study. A total of 47 study participants were analyzed by ELISPOT, and a subset of 16 in- dividuals was analyzed in more depth for HIV-specific memory pheno- types (Table I). HIV-uninfected cohort. We recruited 15 HIV-uninfected individuals from the Perinatal HIV Research Unit, Soweto, South Africa, who were the HIV-uninfected partner in an HIV serodiscordant relationship. Participa- tion in this study was approved by the Human Research Ethics Committee of the University of the Witwatersrand. After providing informed consent, participants were tested for HIV with dual HIV rapid tests performed in parallel. All participants were HIV uninfected by all assays at the enroll- ment and follow-up visits. Calculation of viral set point Viral set point was calculated as the geometric mean of viral load mea- surements at three time points at week 52 6 wk postinfection (between 46 and 58 wk) to minimize possible spurious viral load measurement. In the case where there was an atypical viral load occurring within the 52 6 wk window, defined as a viral load 1 log different from the other measurements within that period, the datum point was removed and re- placed with the next measurement. CAP84 was the only example where a viral load blip at 52 wk was replaced with a measurement beyond the 6 wk period. Plasma viral load determination, CD4 T cell counts, and HLA typing Plasma HIV-1 RNA levels were quantified using the COBAS AMPLICOR HIV-1 monitor test version 1.5 (Roche Diagnostics). Absolute blood CD4 and CD8 T cell counts were measured using a FACSCalibur flow cy- tometer and expressed as cells/mm3. HLA typing was performed as de- scribed in Chopera et al. (28). Synthetic subtype C peptides A set of 432 synthetic overlapping peptides spanning the entire expressed HIV-1 clade C proteome corresponding to gene products from the HIV-1 consensus C (Gag, Vif, Vpr, and Vpu), isolate Du151 (Pol, Nef, Tat, and Rev), and isolate Du179 (gp160 Env) were synthesized using 9-fluorenyl- methoxycarbonyl chemistry and standard based solid phase techniques (Natural and Medical Sciences Institute, University of Tubingen, Tu- bingen, Germany). The nonconsensus synthesized peptides were based on sequences from isolates used for manufacture of a clade C vaccine (29). The estimated purity of peptides was 80% as measured by HPLC and mass spectrometry. Individual peptides were diluted in DMSO (Sigma- Aldrich) and prepared as previously described (13). TABLE I. Characteristics of study participants Patient ID Time Postinfection at Analysis (wk) Viral Set Point (copies/ml)a CD4 Count at Time of Analysis (cells/mm3) HLA-A HLA-B HLA-C % CD8 IFN- Cells (ICS)b Nef Gag Pol Env CAP045 41 472 904 2301/2902 1510/4501 602/1602 0.19 CAP228 37 974 1,411 2301/2601 4403/5101 3c/7 0.97 0.39 0.16 0.07 CAP084 19 4,565 623 2902/7401 1503/4407 2c/7 2.87 0.27 CAP257 30 9,859 489 2301/2902 4202/4403 1701/��� 0.55 CAP271 22 12,732 628 205/2301 1401/4403 303/804 0.04 0.06 CAP217 31 24,836 529 202/2901 1503/5801 210/602 1.15 CAP040 26 26,325 417 30c/��� 1510/4201 304/1701 0.3 CAP229 38 37,686 690 101/��� 5801/��� 602/��� 0.67 CAP088 26 46,304 864 29c/66 4501/5802 602/��� 1.26 0.5 3.5 3.65 CAP030 42 63,338 537 2c/34 4403/4501 4c/16 0.88 CAP129 32 84,959 676 26c/8001 18c/8101 202/4c 0.09 0.08 0.54 CAP174 24 117,279 404 301/7401 4901/5802 6c/7 0.24 CAP239 32 160,922 1,092 101/2902 4201/5801 602/7101 0.41 1.55 CAP256 23 199,234 456 2902/6601 1503/5802 4c/602 1.29 0.7 0.4 CAP206 36 257,196 329 3204/74c 702/4403 210/702 0.13 CAP210 31 289,662 398 6802/��� 1510/��� 304/��� 9.44 Median (n 16) (47)d 31 (24) 41,995 (36,400) 580 (464) 0.69 0.55 0.54 0.335 Range (n 16) 19���42 471���289,662 329���1,411 a Measured by taking the mean of three time points around 52 6 wk. b Peptide pools found to stimulate a positive IFN- response by intracellular cytokine staining (ICS). c Low-resolution HLA typing only. d Median time postinfection at recruitment, viral set point, and CD4 count did not differ significantly between the subgroup studied in detail (n 16) and the larger group (n 47). 4752 CD8 T CELL MEMORY SUBSETS ASSOCIATE WITH VIRAL SET POINT