Comparison of Human Immunodeficiency Virus (HIV)-Specific T-Cell Responses in HIV-1- and HIV-2-Infected Individuals in Senegal

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1 JOURNAL OF VIROLOGY, Dec. 2004, p Vol. 78, No X/04/$ DOI: /JVI Copyright 2004, American Society for Microbiology. All Rights Reserved. Comparison of Human Immunodeficiency Virus (HIV)-Specific T-Cell Responses in HIV-1- and HIV-2-Infected Individuals in Senegal N. N. Zheng, 1,2 N. B. Kiviat, 1 P.S.Sow, 3 S. E. Hawes, 4 A. Wilson, 2 H. Diallo-Agne, 3 C. W. Critchlow, 4 G. S. Gottlieb, 5 L. Musey, 2 and M. J. McElrath 2,5,6 * Departments of Pathology, 1 Epidemiology, 4 Medicine, 5 and Laboratory Medicine, 6 University of Washington, and Program in Infectious Diseases, Clinical Research Division, Fred Hutchinson Cancer Research Center, 2 Seattle, Washington, and University of Dakar, Dakar, Senegal 3 Received 19 February 2004/Accepted 12 August 2004 Human immunodeficiency virus type 2 (HIV-2) infection is typically less virulent than HIV-1 infection, which may permit the host to mount more effective, sustained T-cell immunity. We investigated antiviral gamma interferon-secreting T-cell responses by an ex vivo Elispot assay in 68 HIV-1- and 55 HIV-2-infected Senegalese patients to determine if differences relate to more efficient HIV-2 control. Homologous HIV-specific T cells were detected in similar frequencies (79% versus 76%, P 0.7) and magnitude (3.12 versus 3.08 log 10 spot-forming cells/10 6 peripheral blood mononuclear cells) in HIV-1 and HIV-2 infection, respectively. Gag-specific responses predominated in both groups (>64%), and significantly higher Nef-specific responses occurred in HIV-1-infected (54%) than HIV-2-infected patients (22%) (P < 0.001). Heterologous responses were more frequent in HIV-1 than in HIV-2 infection (46% versus 27%, P 0.04), but the mean magnitude was similar. Total frequencies of HIV-specific responses in both groups did not correlate with plasma viral load and CD4 T-cell count in multivariate regression analyses. However, the magnitude of HIV-2 Gag-specific responses was significantly associated with lower plasma viremia in HIV-1-infected patients (P 0.04). CD4 T-helper responses, primarily recognizing HIV-2 Gag, were detected in 48% of HIV-2-infected compared to only 8% of HIV-1-infected patients. These findings indicate that improved control of HIV-2 infection may relate to the contribution of T-helper cell responses. By contrast, the superior control of HIV-1 replication associated with HIV-2 Gag responses suggests that these may represent cross-reactive, higher-avidity T cells targeting epitopes within Gag regions of functional importance in HIV replication. The two types of human immunodeficiency virus, HIV-1 and HIV-2, which cocirculate in West Africa typically induce different patterns of HIV disease. Compared to HIV-1, HIV-2 infection is characterized by a much longer asymptomatic stage, lower plasma viral load, slower decline in CD4 T-cell count, and lower mortality rate attributable to AIDS (11, 14, 19, 28, 36). Infected patients produce higher levels of HIV-1 RNA than HIV-2 RNA but notably similar levels of proviral DNA (11). In addition, recent findings demonstrate similar cytopathogenicity in vitro (29, 34). Thus, the key difference between the two HIV types lies in the degree of viral replication, and it is presumed that host immunity largely contributes to the more successful control of HIV-2 infection. Evidence that T-cell immunity plays a central role in control of HIV infection stems primarily from assessment of patients with acute and nonprogressive subtype B HIV-1 infection. During acute infection, HIV-1-specific CD8 cytotoxic T lymphocytes emerge as plasma viremia declines (5, 15), and such responses are predictive of subsequent lower viral loads at set point and higher CD4 T-cell counts (20). Further studies indicate that the frequency of individual immunodominant HIV-1-specific CD8 T cells identified by major histocompatibility complex/peptide tetramer binding may correlate with * Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, D3-100, 1100 Fairview Ave. North, P.O. Box 19024, Seattle, WA Phone: (206) Fax: (206) E- mail: kd@u.washington.edu. N.N.Z. and N.B.K. contributed equally to this work. lower levels of plasma viremia (23). However, in recent comprehensive, quantitative analyses of T-cell responses in larger patient populations, the results fail to link the total frequency of gamma interferon (IFN- )-secreting CD8 T cells with viral load in either acute or chronic infection (1, 4, 7). In addition to CD8 T cells, the induction and maintenance of proliferating HIV-1-specific CD4 T cells correlate with lower viral loads (30). In the absence of treatment, these responses are infrequently detected except in patients with long-term nonprogressive HIV-1 disease (17, 21). By contrast, comprehensive, quantitative analyses of HIVspecific T-cell immunity in HIV-2 infection are lacking. In one of the few previous studies, Ariyoshi et al. examined 16 HIV-2 and four dually HIV-1/HIV-2-infected patients and found an inverse correlation between the total and dominant cytotoxic T lymphocyte activities recognizing HIV-1 Gag, Pol, and Nef and HIV-2 proviral load (2). In addition, Sarr et al. reported lower HIV-2 viral load in association with T cells, presumably CD4 T cells, recognizing the p26 core protein (33). Taken together, these studies suggest that HIV-2 infection induces T cells with antiviral properties similar to those identified in HIV-1 infection. However, it remains unclear if the HIV-2-specific T cells are more efficient in controlling viral replication and, if so, by what mechanism. One hypothesis that has received increasing attention is that the ability to recognize specific epitopes that are cross-reactive between HIV-1 and HIV-2 strains may be an important determinant of immune-mediated control (3, 14). Given the relatively high degree of homology that exists between many con

2 VOL. 78, 2004 T-CELL IMMUNITY IN HIV-1 AND HIV-2 INFECTION served regions of proteins encoded by HIV-1 and HIV-2, it is perhaps not surprising that infection with one HIV type induces T cells recognizing epitopes within the other HIV type. This indeed has been shown to be the case in HIV-2 infection (3), but has not been extensively studied in HIV-1 infection. Moreover, it is not known if the induction of CD8 T memory cells recognizing major histocompatibility complex class I epitopes that are cross-reactive between HIV-1 and HIV-2 provide a more efficient antiviral response. This issue deserves further examination in patients already infected with HIV-1 or HIV-2, particularly since T cells recognizing cross-reactive epitopes may tolerate amino acid changes and thus limit the emergence of escape mutations. Thus, to understand the distinguishing features of T cells induced in HIV-1 and HIV-2 infection that confer enhanced viral control, we examined responses in 123 untreated Senegalese patients with either HIV-1 or HIV-2 infection. In particular, we evaluated the specificity and frequency of IFN- -secreting T-cell responses to Gag, Env, Tat, and Nef epitopes within both HIV types as well as the T-cell subset mediating the response. The analysis was restricted to recognition of the four gene products based on the availability of overlapping peptides for the two HIV types and the number of peripheral blood mononuclear cells (PBMC). We also assessed the association of these parameters with levels of plasma viremia and CD4 T-cell counts. We provide evidence that the frequency of responses to the same or the heterologous subtype is not associated with control of viremia. However, improved viral suppression is linked with cross-reactive HIV-2 Gag-specific T-cell responses in HIV-1 infection, and helper IFN- -secreting T cells were common in HIV-2-infected patients. MATERIALS AND METHODS Study population. Beginning in October 2000, all patients 16 years of age and older who presented to the University of Dakar Infectious Disease Clinic (Fann Hospital, Dakar, Senegal, West Africa), as well as commercial sex workers who presented to a sexually transmitted disease clinic in Dakar (Institut d Hygiene Sociale) were offered screening for HIV infection. HIV testing was performed after obtaining written informed consent, and HIV-seropositive patients were offered HIV/AIDS counseling in compliance with the University of Washington Human Subjects Institutional Review Board and Senegalese AIDS National Committee. Subjects were invited to enroll in a longitudinal immunological study if they tested positive by HIV-1/HIV-2 enzyme immunoassay (Genscreen, version 2, HIV-1/HIV-2; Bio-Rad) and were confirmed as positive by a synthetic peptide-based membrane immunoassay (Immunocomb II, bispot; PBS). Furthermore, while all individuals seropositive for HIV-2 infection were invited to participate, only those infected with HIV-1 who had CD4 T-cell counts over 350 cells/ l were eligible to participate in the study. All consenting participants underwent a physical examination, a standardized interview, and venipuncture for CD4 counts, PCR confirmation of their HIV status, and immunologic assays. We report here the overall T-cell responses from their first visit and the CD4 T-cell helper responses from those returning for a follow-up visit. By November 2002, 157 HIV-infected patients were enrolled into the study and had completed the first study procedures. This included 17 individuals with dual HIV-1 and HIV-2 who were excluded from the present analyses. Eleven subjects (nine with HIV-1 and two with HIV-2) were enrolled but initiated antiviral treatment prior to completing the first study visit procedures and were excluded. In addition, HIV-1 infection could not be confirmed by PCR methods in five HIV-1-seropositive individuals, and they were excluded. Finally, one HIV-2-infected subject had missing CD4 and plasma viral load data and was excluded. In total, data from 68 HIV-1- and 55 HIV-2-infected patients were used in the final analysis. Virological and T-cell subset analyses. Plasma HIV-1 RNA was determined by the Amplicor Monitor HIV-1 test (Roche Diagnostics) (9), with the exception that two additional primers, SK145 and SK151, were added to ensure accurate and reliable quantification of non-subtype B HIV-1 RNA. The lower level of sensitivity was 50 copies/ml, with reliable quantitation at levels above 400 copies/ ml. Plasma HIV-2 RNA was determined by the quantitative HIV-2 assay based on the Amplicor Monitor test as previously described (11). Briefly, primers RAR1000 (5 -GCTGGCAGATTGAGCCCTGGGAGGTTCTCT-3 ) and RAR04 (5 -GAATGACCAGGCGGCGACTAGGAGAGAT-3 ) were used to amplify a 201-bp fragment of the HIV-2 long terminal repeat. The HIV-2 assay has a limit of detection of 40 HIV-2 RNA copies/ml, with reliable quantitation at levels above 200 HIV-2 RNA copies/ml. The HIV-2 assay is comparable to the Amplicor Monitor HIV-1 test in accuracy, linearity, and reproducibility. Peripheral blood CD4 and CD8 T-cell counts were measured by a consensus flow cytometry method (9). Synthetic peptides. To express HIV epitopes in Elispot assays, peptides of 15 amino acids (15-mers) were synthesized (Chiron Corp, Emeryville, Calif.) that overlapped by 10 amino acids spanning the entire HIV-1CRF-02 (HIV-1 A/G recombinant virus IbNG, accession no. AJ251056) Gag (n 97), Env (n 171), Nef (n 40), and Tat (n 19) and HIV-2 ROD (accession no. M15390) Gag (n 103), Env (n 170), Tat (n 24), and Nef (amino acids 1 to 180, n 34). The HIV-2 Nef (amino acids 181 to 256, n 15) peptides were synthesized by the Shared Resources Center at Fred Hutchinson Cancer Research Center (Seattle, Wash.). The peptides were reconstituted at a concentration of 10 mg/ml in 100% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) and used at a final concentration of 2 g/ml. Peptides corresponding to each protein were used initially in pools for Gag (five pools), Env (nine pools), Nef (four pools) and Tat (two pools) for both HIV-1 and HIV-2. Subsequently, positive responses were deconvoluted by stimulating with either individual peptides within a pool or two serially overlapping 15-mers for the CD4 T helper cell studies. IFN- Elispot assay. To evaluate T-cell responses recognizing HIV-1 and -2 epitopes, PBMC were isolated from anticoagulated blood and evaluated fresh in IFN- Elispot assays. In subsequent studies in a subset of patients, responses of CD4 and CD8 enriched T cells were analyzed by purification of the subsets with anti-cd4 magnetic microbeads (Miltenyi Biotec, Auburn, Calif.) and a paramagnetic separation column according to the manufacturer s instructions. This typically resulted in approximately 97% CD4 T cells in the positively selected fraction, and the CD4-depleted fraction contained predominantly CD8 T cells ( 95%). Cells were suspended in complete medium (RPMI with 10% fetal calf serum) at a concentration of 10 5 or cells/100 l and added to individual wells of 96-well hydrophobic polyvinylidene difluoride-backed plates (Millipore, Bedford, Mass.) previously coated with 50 l of10- g/ml anti-ifn- monoclonal antibody (1-D1K, mouse immunoglobulin G1; Mabtech, Nacka, Sweden) overnight at 4 C. HIV-1 and HIV-2 peptides and the positive control, phytohemagglutinin (Sigma, St. Louis, Mo.), were added to the wells at a final concentration of 1 g/ml. The wells with no added peptides served as negative controls. All responses were tested in duplicate. Plates were incubated overnight (16 to 20 h at 37 C in 5% CO 2 ), washed with phosphate-buffered saline containing 0.05% (Tween 20), and incubated at room temperature for 2 h with biotinylated anti- IFN- monoclonal antibody at 1 g/ml (7-B6-1, mouse immunoglobulin G1; Mabtech). The avidin-biotinylated enzyme complex from the Vectastain ABC Elite kit (PK-6100; Vector Laboratories, Burlingame, Calif.) was added at room temperature for 1 h, followed by the Vectastain AEC peroxidase substrate. Spots formed by IFN- -secreting cells were counted with an automated ImmunoSpot plate reader (Cellular Technologies, Cleveland, Ohio), and results are presented as spot-forming cells (SFC) per 10 6 PBMC. A response was considered positive when the mean SFC for the experimental wells was at least twofold greater than the mean SFC for the negative control wells and the mean SFC/10 6 PBMC in the experimental wells was 100 after subtraction of the mean SFC/10 6 PBMC of the control wells. IFN- SFC frequencies over 50 per 10 6 CD4 T cells were scored positive. Duplicate experimental and control wells were used in each experiment. Statistical methods. Values of HIV-1 and HIV-2 plasma RNA viral loads as well as counts of IFN- SFC were log 10 transformed in order to normalize the distribution of these measurements for statistical analyses. Two-sided Mantel- Haenszel 2 or Fisher s exact tests were performed to assess univariate associations between HIV type (HIV-1 compared to HIV-2) and factors of interest. Associations with ordered categorical factors such as CD4 T-cell count level ( 500, 200 to 500, and 200 cells/ l) were tested with Mantel-Haenszel 2 tests for trend, and Student s t tests or analysis of variance was used to compare groups with respect to continuous variables. Multivariable regression analyses were performed to evaluate the independent magnitude of associations between specific IFN- SFC responses and HIV viral loads or CD4 T-cell counts after adjusting for the possible confounding effects of age, gender, and work as a commercial sex worker. A two-sided 0.05 level test determined statistical signif-

3 13936 ZHENG ET AL. J. VIROL. TABLE 1. Baseline demographic, clinical, and virological profiles of study participants Characteristic icance for all analyses. Data analyses were conducted with SAS 8.2 for Windows (SAS Institute, Cary, N.C.). RESULTS HIV-1-infected patients (n 68) HIV-2-infected patients (n 55) Study clinic populations [no. (%)] Fann, male 8 (12) 9 (16) Fann, female 36 (53) 26 (47) IHS, female sex workers 24 (35) 20 (36) Mean age, yr (range) 34.5 (16 55) 40.6 (26 56) CD4 cells Mean cells/ l No. (%) of patients with counts (cells/ l) of: (47) 36 (65) (43) 8 (15) (10) 11 (20) Mean CD8 count (cells/ l) HIV plasma RNA Mean log 10 copies/ml No. (%) of patients with loads (copies/ml) of: (16) 28 (51) 100 1,000 2 (3) 14 (25) 1,000 10, (43) 9 (16) 10, , (29) 3 (5) 100,000 6 (9) 1 (2) Study population. In total, 68 HIV-1- and 55 HIV-2-infected, antiretroviral drug-naïve subjects from the infectious disease and sexually transmitted disease clinics were recruited and enrolled in this study. The demographic, clinical, and virological profiles of the participants at study entry are shown in Table 1. With one known exception, patients with HIV-2 were infected with subtype A (data not shown). HIV-1 infection in the cohort was predominantly with subtype CRF-02 (A/G) and subtype A (10, 39). Eighty-six percent of the study participants were female, and approximately one-third were commercial sex workers (Table 1). Subjects with HIV-1 were younger (P 0.001), with a mean age of 34.5 years (range, 16 to 55 years) compared to those with HIV-2 infection, who had a mean age of 40.6 years (range, 26 to 56 years). Due to the study inclusion criteria, the patient population was generally asymptomatic and demonstrated relatively high CD4 T-cell counts and low viral loads (Table 1). Mean CD4 counts, obtained on the first study visit once enrolled, were marginally lower in those with HIV-1 (554 cells/ l) compared to HIV-2 (665 cells/ l; P 0.09). By contrast, mean CD8 T-cell counts were significantly higher in those with HIV-1 (961 cells/ l; range, 260 to 2,000 cells/ l) compared to HIV-2 infection (739 cells/ l; range, 128 to 1,804 cells/ l, P 0.005). Of those enrolled, 47% of the HIV-1-infected and 65% of the HIV-2-infected cases had CD4 T-cell counts over 500 cells/ l. As expected, the mean plasma HIV RNA was higher in those with HIV-1 (3.59 log 10 copies/ml) compared to HIV-2 (1.57 log 10 copies/ml; P 0.001). Although (as noted above) the presence of HIV infection was confirmed by qualitative PCR, 11 (16%) patients with HIV-1 and 28 (52%) patients with HIV-2 infection had plasma RNA levels below the level of HIV RNA quantification. Defining HIV-specific IFN- -secreting T cells. To determine if HIV-1 and HIV-2 infection induced an antiviral T-cell response in the Senegalese patients, fresh PBMC were stimulated ex vivo with HIV-1 and HIV-2 peptide pools and examined for IFN- secretion in an Elispot assay. A typical response profile is depicted in Fig. 1. The strongest T-cell responses noted in this HIV-2-infected patient are those recognizing the HIV-2 GagC pool containing peptides spanning amino acids 211 to 320, with frequencies of 740 IFN- -secreting cells per million PBMC (Fig. 1A). To define the T-cell subset secreting IFN- and further identify the specificity of each response, we fractionated PBMC into CD4 and CD4 cells and then stimulated with each peptide within a recognized pool, testing two peptides (adjacent in sequence) per well. HIV-2-specific T-cell responses were observed in both the CD4 T-cell-enriched and depleted populations in some study participants, as illustrated in the HIV-2-infected patient in Fig. 1B. In this case, the highest frequencies were directed to HIV-2 GagC4, spanning amino acids 241 to 260 (230 SFC/10 6 CD4-depleted T cells) and to HIV-2 GagC7, spanning amino acids 271 to 290 (220 SFC/10 6 CD4 T cells). Thus, as demonstrated here, this approach provides a means of assessing the overall frequency, magnitude, and specificity of the homologous and cross-reactive HIV-specific T-cell responses induced by HIV-1 and HIV-2 infection. Frequency and magnitude of HIV-specific IFN- -secreting T cells. The majority of patients exhibited IFN- T cells recognizing epitopic peptides within at least one peptide pool of homologous gene products (i.e., HIV-1 peptides in HIV-1- infected patients or HIV-2 peptides in HIV-2-infected patients) (Fig. 2A). The percentage of responders was similar between the HIV-1-infected (79%) and HIV-2-infected (76%) groups (P 0.7). Gag-specific T cells predominated in both groups (Fig. 2A), with 65% of HIV-1- and 64% of HIV-2- infected patients recognizing homologous Gag epitopes (P 0.9). A significantly higher percentage of subjects with HIV-1 (54%) compared to HIV-2 (22%) exhibited Nef-specific responses (P 0.001). By contrast, HIV-2 infection was marginally associated with a higher frequency of Env-specific responses (42%) than HIV-1-infection (27%) (P 0.07). Recognition of HIV Tat was uncommon and similar in frequency (4% in HIV-1 and 11% in HIV-2; P 0.2) between the two study groups. We next compared the magnitude of IFN- -secreting T-cell frequencies recognizing peptide pools within the individual gene products (Fig. 2B). Overall, the mean frequencies of homologous HIV responses were similar in the two HIV-infected groups, with 3.12 log 10 SFC/10 6 PBMC in HIV-1-infected patients, compared to 3.08 log 10 SFC/10 6 PBMC (P 0.7) in HIV-2-infected patients. In addition, the magnitude of homologous responses to the individual Env, Gag, and Tat gene products was similar among those with HIV-1 and HIV-2 infection (P 0.05 in all cases). However, the HIV-1 Nefspecific T-cell frequencies were significantly higher in HIV-1- infected patients than HIV-2 Nef-specific frequencies in HIV- 2-infected patients (2.45 versus 1.58 log 10 SFC/10 6 PBMC, respectively; P 0.001). Thus, T cells from patients with

4 VOL. 78, 2004 T-CELL IMMUNITY IN HIV-1 AND HIV-2 INFECTION FIG. 1. HIV IFN- Elispot response profile of PBMC from an HIV-2-infected patient. The bars depict the average number of IFN- spot-forming cells (SFC) per 10 6 PBMC. (A) Initial mapping of the HIV-specific T-cell responses with peptide pools spanning Gag, Env, Nef, and Tat of HIV-1 and HIV-2. For HIV-1, there are five Gag peptide pools (pool 1, peptides spanning amino acids 1 to 110; pool 2, 100 to 210; pool 3, 200 to 310; pool 4, 300 to 410; pool 5, 400 to 495); nine Env pools (pool 1, 1 to 110; pool 2, 100 to 210; pool 3, 200 to 310; pool 4, 300 to 410; pool 5, 400 to 510; pool 6, 500 to 610; pool 7, 600 to 710; pool 8, 700 to 810; pool 9, 800 to 861); two Tat pools (pool 1, 1 to 65; pool 2, 55 to 101), and four Nef pools (pool 1, 1 to 65; pool 2, 55 to 120; pool 3, 110 to 175; pool 4, 165 to 209). For HIV-2, there are five Gag pools (pool A, 1 to 110; pool B, 100 to 210; pool C, 200 to 310; pool D, 300 to 410; pool E, 400 to 522); nine Env pools (pool A, 1 to 110; pool B, 100 to 210; pool C, 200 to 310; pool D, 300 to 410, pool E, 400 to 510; pool F, 500 to 610; pool G, 600 to 710; pool H, 700 to 810; pool I, 800 to 858); two Tat pools (pool A, 1 to 75; pool B, 65 to 130), and four Nef pools (pool A, 1 to 65; pool B, 55 to 120; pool C, 110 to 175; pool D, 165 to 256). Phytohemagglutinin (PHA)-stimulated cells served as the positive control (not shown), and cells stimulated with no peptides served as the negative control. (B) Determination of the T-cell subset (CD4 or CD4 ) responsible for IFN- secretion and recognition of the individual 20-mer peptides within HIV-2 Gag pool C.

5 13938 ZHENG ET AL. J. VIROL. FIG. 2. Frequency and magnitude of T-cell responses to HIV peptides recognizing epitopes within Gag, Env, Nef, and Tat. (A) Frequency of homologous T-cell responses recognizing HIV Gag, Env, Nef, and Tat (i.e., responses to HIV-1 Gag, Env, Nef, and Tat in HIV-1-infected patients, and responses to HIV-2 Gag, Env, Nef, and Tat in HIV-2-infected patients). (B) Magnitude of homologous T-cell responses. (C) Frequency of cross-reactive T-cell responses to HIV Gag, Env, Nef, and Tat in HIV-1- and HIV-2-infected patients (i.e., responses to HIV-2 Gag, Env, Nef, and Tat in HIV-1-infected patients, and responses to HIV-1 Gag, Env, Nef, and Tat in HIV-2-infected patients). (D) Magnitude of cross-reactive T-cell responses to HIV Gag, Env, Nef, and Tat in HIV-1- and HV-2-infected patients. HIV-2 infection were less likely to recognize Nef epitopic peptides, and when detected, the frequency of responses was lower. Frequency and magnitude of heterologous T-cell responses to HIV-1 and HIV-2 gene products. We then investigated the ability of the T cells from HIV-1- and HIV-2-infected patients to recognize heterologous epitopes within the four gene products of HIV-2 and HIV-1. IFN- -secreting T cells recognizing HIV-2 epitopic peptides were observed in 46% (31 of 68) HIV-1-infected patients (Fig. 2C). By contrast, only 27% of HIV-2-infected patients (15 of 55) exhibited cross-reactive T- cell responses to HIV-1 peptides (P 0.04). Thus, in our study population, HIV-1-infected patients were more likely to have cross-reactive responses than HIV-2-infected patients. The frequency of cross-reactive responses to specific gene products differed by HIV type. The HIV-1-infected group was more likely to exhibit HIV-2 Gag-specific responses (34%) in comparison to the HIV-2-infected group to HIV-1 Gag (13%) (P 0.007). Moreover, in those with HIV-1 infection, the dominant heterologous responses were directed to Gag epitopes, while among the HIV-2-infected patients, the crossreactive responses were distributed more equally across Env, Gag, and Nef (Fig. 2C). Heterologous responses directed to Env, Nef, and Tat were detected in 12% of study subjects and did not differ by HIV type (P 0.05). Of note, those with HIV-1 infection who exhibited HIV-2 Gag-specific responses also had HIV-1 Gag-specific responses. The mean magnitude of total cross-reactive responses across all four gene products spanning HIV-1 and HIV-2 were similar in HIV-1- and HIV-2-infected patients (2.61 log 10 compared to 2.55 log 10 SFC/10 6 PBMC, respectively; P 0.6) (Fig. 2D). Those with HIV-1 and HIV-2 had similar mean magnitudes of cross-reactive responses to HIV Env peptides (2.01 versus 2.08 log 10 SFC/10 6 PBMC, respectively; P 0.6). We observed a trend toward stronger cross-reactive Gag-specific responses in those with HIV-1 compared to HIV-2 (mean difference, 0.25 log 10 SFC/10 6 PBMC; P 0.07). In addition, those with HIV-2 had marginally higher levels of cross-reactive responses to HIV Nef (mean difference, 0.30 log 10 SFC/10 6 PBMC; P 0.06) and HIV Tat (mean difference, 0.25 log 10 SFC/10 6 PBMC; P 0.10) compared to those with HIV-1. Thus, the dominant cross-reactive epitopes that HIV-1-infected patients recognize are in HIV-2 Gag. Moreover, HIV-2-infected patients are less likely to develop cross-reactive responses to HIV-1, but in those who do, the response is broader than in HIV-1-infected patients and of similar magnitude. Associations between antiviral T-cell responses and HIV disease. To assess the immunologic responses that may impact HIV disease progression in our study population, we performed both univariate and multivariate regression analyses, taking into consideration the demographic patient profiles and utilizing CD4 T-cell counts and plasma viral load as markers of clinical disease. As commonly shown in subtype B HIV-1 infection, a lower level of plasma HIV RNA was associated with increased CD4 T cells in multivariate regression analysis among both HIV-1- and HIV-2-infected patients (Table 2). Viral load was not impacted by age, gender, and commercial sex work (Table 2). In the univariate analyses among HIV-1 patients, no associations were observed between total frequencies of HIV-1-

6 VOL. 78, 2004 T-CELL IMMUNITY IN HIV-1 AND HIV-2 INFECTION specific T-cell responses and either HIV-1 plasma viral load (P 0.3) or CD4 T-cell count (P 0.3; data not shown). Similar analyses among those with HIV-2 infection showed that a greater magnitude of total HIV-2-specific T-cell responses was associated with higher CD4 T-cell counts ( 267 cells per log 10 increase in HIV-2-specific IFN- SFC; P 0.006) and lower log 10 HIV-2 plasma RNA copy number ( 0.84 log 10 plasma RNA copies per log 10 increase in HIV-2 specific SFC; P 0.02; data not shown). However, after adjusting for age, gender, and commercial sex work in the multivariate regression analyses, these associations were somewhat attenuated and no longer remained statistically significant ( 117 cells, P 0.17; and 0.62 log 10 plasma RNA copies per log 10 increase in HIV-2-specific IFN- SFC, P 0.12; data not shown). We next examined the relationship between the magnitudes of responses to specific gene products of HIV-1 or HIV-2 to CD4 T-cell counts. In the multivariate analysis adjusting for gender, age, and commercial sex work, the magnitude of responses to peptide pools of individual HIV-1 and HIV-2 proteins was not associated with CD4 T-cell counts among those with HIV-1 infection (P 0.4). However, similar multivariate analyses among those with HIV-2 infection showed that each log 10 increase in IFN- -secreting HIV-2 Nef-specific frequencies was associated with a 170-cell/ l increase in CD4 T-cell counts (P 0.02; data not shown). Furthermore, in the multivariate analysis among those with HIV-1, lower HIV-1 viral load was associated with greater frequencies of HIV-2 cross-reactive Gag-specific T-cell responses ( 0.57 log 10 plasma RNA copies per log 10 increase in HIV-2 Gag-specific SFC; P 0.04; Table 2). Similarly, in those with HIV-2 exhibiting a cross-reactive HIV-1 Gag-specific response, there was a trend toward a lower HIV-2 viral load ( 0.58 log 10 plasma RNA copies per log 10 increase in HIV-1 Gag-specific SFC; P 0.11). No other HIV-1- or HIV-2-associated gene-specific responses were significantly associated with HIV-1 RNA levels. In summary, the ability to mount a cross-reactive HIV-2 but not a (homologous) HIV-1 Gag-specific T-cell response among those with HIV-1 infection correlates with improved viral suppression. By contrast, neither the total frequency of responses nor the responses to individual gene products of HIV-1 or HIV-2 impacted viral load in patients with HIV-2 infection. HIV-specific T helper responses in HIV-1 and HIV-2 infection. To determine if the HIV-specific T cells elicited in either type of infection provided CD4 help, we examined fresh CD4 T cells from 24 HIV-1- and 27 HIV-2-infected patients for their ability to secrete IFN- in response to the HIV peptide pools. As shown in Table 3, only two (8%) of the HIV-1- infected patients mounted an HIV-1-specific IFN- response. By contrast, 13 (48%) of 27 HIV-2-infected patients exhibited an HIV-1-specific T helper response (P 0.002) (Table 3). The majority (84%) of responses in the HIV-2-infected group were directed to epitopes within HIV-2 Gag, and the median IFN- SFC frequency was 325 SFC/10 6 CD4 cells. Although those HIV-2-infected patients demonstrating a CD4 T-cell response tended to have higher CD4 T-cell counts (P 0.07) in the univariate analysis, there was no association of these responses with HIV-2 plasma viral load. TABLE 2. Associations between demographic profiles and HIVspecific IFN- SFC frequencies with HIV viral load a Characteristic HIV-1-infected patients (n 68) Log 10 plasma P HIV-1 RNA DISCUSSION HIV-2-infected patients (n 55) Log 10 plasma HIV-2 RNA Intercept Age (per 10 yr) Male gender Commercial sex worker CD4 count (per 100) IFN- SFC frequency (log 10 ) HIV-1 Env HIV-1 Gag HIV-1 Nef HIV-1 Tat HIV-2 Env HIV-2 Gag HIV-2 Nef HIV-2 Tat a Regression coefficients and P values from two separate multivariable regressions modeling HIV-1 (or HIV-2) plasma RNA levels (dependent variable) against all factors in the table (independent variables). This investigation is one of the first to extensively examine the antiviral properties of T cells in conjunction with clinical disease in a relatively large African population with HIV-1 or HIV-2 infection. Remarkably, although HIV-2-infected patients had approximately 100-fold lower mean plasma viral loads than the HIV-1 patients, the percentage of T cells recognizing HIV epitopes and the frequencies of IFN- -secreting T cells were similar in the two groups. Furthermore, these immune parameters were not associated with the level of viremia in either type of infection. These results indicate that patients with HIV-1 are just as likely as those with HIV-2 infection to mount an antiviral IFN- -secreting T-cell response, and the magnitude of the response induced by either type of infection does not impact the ability to control viral replication. We explored the possibility that the specificity, breadth, and function of memory T cells may be more relevant than their absolute quantity. As observed in persons with HIV-1 infections of other subtypes, T cells from our HIV-1 and HIV-2 cohort predominantly recognized HIV Gag (1, 4, 7, 22). However, apart from this, distinct differences were noted. HIV-1- infected Senegalese patients preferentially recognized HIV-1 Nef epitopic peptides, but surprisingly, Nef-specific HIV-2 responses were found in only 22% of HIV-2-infected patients. Of note, a previous study of cytotoxic T-lymphocyte activity in HIV-2-infected patients also demonstrated a similar lower frequency of HIV-2 Nef-specific cytolytic responses (25%) in comparison to recognition of targets expressing other HIV-2 gene products (2). There was also a trend toward greater Env-specific T-cell responses in HIV-2-infected patients (42%) in comparison to the HIV-1-infected patients (27%). We recognize that differences may occur in recognition of epitopes within the other HIV gene products and those based upon autologous HIV sequences, but these investigations were beyond the scope of this initial study. We and others have P

7 13940 ZHENG ET AL. J. VIROL. TABLE 3. CD4 T helper responses recognizing HIV-1 or HIV-2 gene products a CD4 helper responses HIV-1-infected patients No. (%) Mean CD4 T- cell count (cells/ l [range]) HIV-2-infected patients No. (%) Mean CD4 T cell count (cells/ l [range]) Present 2 (8) 561 ( ) 13 (48) 769 ( ) Absent 24 (92) 432 ( ) 14 (52) 625 ( ) a Responses in HIV-1-infected patients were directed to HIV-1 gene products only. Similarly, HIV-2-infected patients recognized HIV-2 gene products only. shown that multiple HIV-1 subtypes are circulating in Senegal, but the most abundant is the CRF-02 recombinant (about 45%), followed by subtype A (20 to 30%) (10, 39). By phylogenetic analysis, CRF-02 gag is subtype A, gp120 is predominantly subtype A, gp41 is predominantly subtype G, nef is predominantly subtype G, and tat is both subtypes A and G ( This suggests that although there was likely some HIV-1 peptide subtype mismatching in HIV-1-infected subjects, the overall effect was likely small. The lower frequency of HIV-2 Nef-specific T-cell responses is not understood at present. We speculated that the HIV-2 in our patients might have a higher frequency of deleted or truncated nef genes, as noted in a previous study of asymptomatic HIV-2-infected patients (38). However, preliminary findings from our study cohort (data not shown) and a recent report of a Portuguese HIV-2 cohort (24) suggest little Nef disruption. Thus, this is unlikely to explain the low level of Nef-specific responses in our patients. HIV-2 Nef typically has greater genetic diversity than HIV-1 Nef, as assessed by comparing mean pairwise genetic distances between HIV-1 subtype A or subtype CRF-02 Nef sequences and those of HIV-2 subtype A Nef sequences from the HIV Sequence Database ( lanl.gov) (data not shown). More diversity may indicate that there are less structural constraints for Nef function in HIV-2. It may also indicate that HIV-2 isolates have more nonfunctional Nef genes than HIV-1. Nonetheless, escape mutants in HIV-2 Nef may occur with less impact on viral fitness, leading to decay of Nef-specific CD8 T cells in HIV-2-infected patients who have been infected for many years, which may be the case in our study population. This may be important in the pathogenesis of HIV-2 infection, particularly in view of recent studies suggesting that Nef-specific cytotoxic T lymphocyte more readily induce cytotoxic T lymphocyte escape (40) and exhibit greater antiviral effects. Another possibility is that processing and/or presentation of HIV-2 Nef peptides with class I major histocompatibility complex molecules is less efficient than HIV-1 Nef peptides, resulting in lower expression of Nef epitopes. Since our study populations were of similar ethnic background and HLA allele frequencies, host effects are less likely and rather point to differences in the HIV types and even suggest that HIV-1 contains more immunodominant Nef epitopes than HIV-2. These considerations bear further study, which we are currently undertaking. In addition, a survey of responses recognizing Nef epitopes within autologous HIV-2 will provide a means of understanding the extent of the lower responsiveness to HIV-2 Nef in contrast to those represented in the HIV-2 ROD reference strain. Greater Env-specific T-cell responses were detected in the HIV-2-infected group in comparison to the HIV-1-infected group. We speculate that the lower viral loads noted in the majority of patients with HIV-2 infection may be associated with less viral quasispecies diversity in HIV-2 Env, similar to that noted in previous studies of controllers of HIV-1 infection (18). In fact, molecular epidemiologic studies indicate that HIV-1 Env is more genetically diverse than HIV-2 (25, 32) (G. S. Gottlieb, unpublished observation). Thus, the low diversity of HIV-2 Env may permit greater cytotoxic T-lymphocyte epitope recognition than is seen in HIV-1. Interestingly, T cells from HIV-1 patients recognized HIV-2 epitopes in a higher percentage than T cells from HIV-2 patients recognized HIV-1 epitopes. Since HIV-1 diversifies more in a given individual than HIV-2, infection with HIV-1 may elicit broader responses over time (31). Indeed, we have observed that in patients followed longitudinally after diagnosis of primary HIV-1 subtype B infection (7, 8), a greater number of epitopes are recognized over time in the absence of antiretroviral treatment. Of note, broadening specificity does not necessarily correlate with improved viral suppression (8). Obviously, this may be balanced by the diversity of the T-cell repertoire associated with each epitopic response, which may be greater in HIV-2 than HIV-1 infection (16). There is also the remote possibility that patients with HIV-1 infection have prior immunity to HIV-2 from previous exposures, yet they never manifested overt HIV-2 infection. In this investigation, the only IFN- -secreting T-cell response that correlated with control of viremia in the multivariate analysis was the ability to elicit HIV-2 Gag-specific T cells among those with HIV-1 infection. In fact, each log 10 increase in the number of spot forming cells was associated with a greater than 0.5 log decrease in plasma RNA copies/ l. We speculate that the HIV-2-specific T cells represent high avidity immunodominant responses and that they may recognize essential structural components within Gag, which are conserved between HIV-1 and HIV-2 and consequently are less likely to undergo escape mutation. Fine mapping, identification of the major histocompatibility complex-restricting molecules, and avidity studies are in progress in consenting volunteers at additional time points. These investigations will define the extent of similarity in amino acid sequence between the recognized HIV-2 epitope and flanking regions and HIV-1 strains and their contribution to the function of the Gag protein. Obviously, the ability to elicit responses to these epitopes may be an important component of HIV immunotherapeutic strategies and T-cell based preventive vaccines. Recent studies on CD4 T-cell help in generating functional CD8 T-cell memory indicate that the quality of memory CD8 T cells depends on help from CD4 T cells (6, 12, 35, 37). Although memory CD8 T cells are generated in the absence of CD4 T cells, their function may be impaired. Forty-eight percent of our HIV-2 patients exhibited HIV-specific IFN- -secreting CD4 T helper responses, and the majority of these were Gag-specific responses. This finding confirms previous studies in HIV-2 infection in which Gag-specific proliferative responses were noted (26, 27). It is unclear why T-cell help is preferentially maintained in HIV-2 than in

8 VOL. 78, 2004 T-CELL IMMUNITY IN HIV-1 AND HIV-2 INFECTION HIV-1 infection. It is possible that acute HIV-2 infection conveys less damage to the memory CD4 T-cell pool, as our HIV-2 patients had higher mean CD4 count than HIV-1-infected patients. Of note, even with attempts to recruit HIV-1- infected patients with higher CD4 T-cell counts, they were lower, albeit nonstatistically, than those with HIV-2 infection. Moreover, variability in HIV-2 Nef over time may compromise its ability to down-regulate class II molecules, which may sustain presentation of class II HIV-2 epitopes to CD4 T cells. It remains unclear if the sustained T-cell help in HIV-2-infected patients permits the CD8 effectors to function more efficiently. This issue is under further exploration in longitudinal studies, including a more extensive analysis of the cytokine profiles, e.g., interleukin-2 and tumor necrosis factor alpha, of the HIV-2 helper T cells. Recently, a similar study comparing the cellular immune responses to HIV-1 and HIV-2 antigens in Gambian patients was reported (13). The Gambian study (13) was limited to the assessment of immune responses recognizing homologous Gag and Pol proteins in a smaller population, and the methods used to detect both CD4 and CD8 T-cell responses were different than in our study. These dissimilarities may account for the lower frequencies of T helper responses they noted in HIV-2-infected patients. However, in agreement with our findings, the study demonstrated that the level of CD8 T-cell responses and the proportion of responders were similar in the HIV-2-infected patients. Moreover, we recognize that measurement of IFN- secretion alone may not be sufficient to enumerate HIV-specific T cells and that other antiviral effector responses may be more relevant in controlling viral replication. In summary, our findings indicate that despite the superior control of HIV-2 infection in comparison to HIV-1 infection, the T-cell response induced is not significantly different in magnitude and in the percentage of responders. Differences may lie in the ability to recognize key epitopes within HIV-2 Gag for HIV-1-infected patients and the ability to mount a CD4 T helper response among HIV-2-infected patients. Additional studies in these two HIV-infected populations, analyzing the fine specificities of the CD8 T cells and their major histocompatibility complex restricting alleles and the phenotypic profile of the CD4 T cells recognizing HIV epitopes, will provide clues toward strategies to improve control of HIV replication and vaccine development. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants R01-AI48470, R01-AI47086, and U01-AI48017 and a Burroughs Wellcome Clinical Scientist Award in Translational Research (M.J.M.). 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