Antigen-Specific b-chemokine Production and CD8 + T-Cell Noncytotoxic Antiviral Activity in HIV-2-Infected Individuals

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1 Antigen-Specific b-chemokine Production and CD8 + T-Cell Noncytotoxic Antiviral Activity in HIV-2-Infected Individuals R. K. S. Ahmed*, H. Norrgreny, Z.daSilvaz, A.Blaxhult,E.-L.Fredriksson{, G. Biberfeld*, S. Andersson* & R. Thorstensson* *Tumorbiology Center, Karolinska Institute, Solna; ydepartment of Infectious Diseases, University Hospital, Lund, Sweden; znational Public Health Laboratory, Bissau; Guinea-Bissau; Karolinska Hospital, Solna; and {South Hospital, Stockholm, Sweden Received 1 July 24; Accepted in revised form 1 October 24 Correspondence to: Dr R. K. S. Ahmed, Swedish Institute for Infectious Disease Control, Solna SE , Sweden. raija.ahmed@smi.ki.se Abstract Human immunodeficiency virus-2 (HIV-2) is less pathogenic than HIV-1, and the disease progression in HIV-2-infected individuals seems to be similar to that seen in HIV-1-infected long-term nonprogressors. Cell-mediated immune responses and the production of noncytotoxic CD8 + T-cell antiviral factors (CAF) and b-chemokines have been correlated to protection against HIV-1 and associated with asymptomatic infection and slower disease progression. We investigated the antigen-induced b-chemokine production in HIV-2- infected patients living in Sweden and in Guinea-Bissau. We also compared in vitro CD8 + T-cell-mediated noncytotoxic antiviral activity against b-chemokinesensitive R5 virus (HIV-1 Bal ) and b-chemokine-insensitive X4 virus (HIV-1 IIIB ) in HIV-2-infected patients with that in HIV-1-infected patients. HIV-2-specific b-chemokine production was demonstrated in a majority of the HIV-2-infected subjects. CD8 + T cells of both HIV-1 and HIV-2-infected individuals suppressed R5 virus replication in vitro in a similar manner, while the inhibition of X4 virus replication seemed to be more frequent and of a higher magnitude among HIV-2-infected patients compared to HIV-1-infected subjects. Taken together, our results indicate that the production of CD8 + T-cell noncytotoxic antiviral factors may contribute to the low transmission of the virus and slower disease progression in HIV-2-infected patients. Introduction Human immunodeficiency virus-2 (HIV-2), a second retrovirus causing AIDS, is generally less pathogenic compared to HIV-1. The heterosexual [1] and perinatal [2, 3] transmission rates are lower for HIV-2 than for HIV-1 and the disease progression in most HIV-2-infected individuals seems to be similar to that seen in HIV-1 long-term nonprogressors (LTNP) [4 6]. HIV-2, like HIV-1, uses primarily CCR5 and CXCR4 as coreceptors for cell entry [7, 8] but in contrast to HIV-1, HIV-2 isolates are more promiscuous in coreceptor usage [9 11]. Cell-mediated immune responses to HIV-1 are believed to be critical for the control of the infection. Besides the cytotoxic activity of the CD8 + T cells, they also produce soluble antiviral factors that are able to inhibit or control HIV infection in vitro [12, 13]. Cytotoxic [14 16] as well as noncytotoxic [17, 18] CD8 + T-cell antiviral responses have been demonstrated in HIV-1-exposed uninfected individuals and in HIV-1-infected LTNP. Our previous studies [19 21] and several other studies [18, 22, 23] suggest that high production of b-chemokines and CD8 + T-cell noncytotoxic antiviral response may protect against simian immunodeficiency virus (SIV) and HIV infection or delay disease progression. There is a limited knowledge about CD8 + T-cell noncytotoxic antiviral activity and the production of b-chemokines in HIV-2-infected individuals most likely, as the number of HIV-2-infected subjects has remained low worldwide compared to HIV-1 [24]. HIV-2 is confined mainly to West Africa, and Guinea- Bissau has the highest prevalence of HIV-2 infection in the world [24]. There are also a small number of HIV-2- infected individuals living in Sweden representing a valuable study population for understanding why and how HIV-2 induces a milder disease than HIV-1. # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61,

2 64 Antiviral Factors in HIV-2-infected Individuals R. K. S. Ahmed et al. In the present study, we compared in vitro CD8 + T-cellmediated noncytotoxic antiviral activity in HIV-2-infected individuals with that in HIV-1-infected patients. We also investigated the antigen-specific production of the b-chemokines RANTES, macrophage-inflammatory protein (MIP)-1a and MIP-1b in HIV-2-infected individuals living in Sweden and in Guinea-Bissau. Materials and methods Subjects. Blood was obtained from 13 HIV-2-infected patients (five males and eight females) and 11 patients infected with HIV-1 (all males). Eight HIV-2-infected patients were living in Sweden at the time of the study as were all the patients infected with HIV-1. Five HIV-2- infected individuals and five HIV-negative subjects were living in Guinea-Bissau. The mean age of the HIV-2- infected individuals from Sweden and Guinea-Bissau were 39 years (range: years) and 46 years (range: years), respectively. The mean age was 43 years for the HIV-1-infected patients (range: years). The median CD4 + T-cell count was 627 cells/ml (range: cells/ ml) and 661 cells/ml (range: cells/ml) in the HIV-2- infected patients from Sweden and Guinea-Bissau, respectively. The 11 HIV-1-infected patients were matched to have similar numbers of CD4 + T cells as the HIV- 2-infected subjects, thus their median CD4 + T-cell count was 628 cells/ml (range cells/ml). Four of the Swedish HIV-2-infected patients were treated with antiretroviral drugs (patients 4, 13, 16 and 17) and four were drug naïve (patients 2, 1, 11 and 15), so were all subjects from Guinea-Bissau and the patients infected with HIV-1. The median CD8 + T-cell count was 116 cells/ml (range: cells/ml) in HIV-1-infected patients and 673 cells/ml (range: cells/ml) and 194 cells/ml (range: cells/ml) in HIV-2-infected patients from Sweden and Guinea-Bissau, respectively. All samples were collected after informed consent was given. Ethical permission for the study was received from the Ethical Committee at the Karolinska Institute, Stockholm, Sweden and from the Ministry of Health, Guinea-Bissau. Fluorescence-activated cell sorter analysis. T-lymphocyte subsets were determined by flow cytometry according to the instructions of the manufacturer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The following antihuman monoclonal antibodies were used for the Swedish patients: CD3 fluorescein isothiocyanate (FITC)/CD8 phycoerythrin (PE)/CD45 PerCP/CD4 allophycocyanin (APC) and CD3 FITC/CD16 + CD56 PE/CD45 PerCP/CD19 APC. The lymphocyte subsets of the patients in Guinea-Bissau were determined by using two-colour immunofluoresence reagents CD3 FITC/CD4 PE and CD3 FITC/CD8 PE (Simulset; Becton Dickinson). CD8 + cell noncytotoxic anti-hiv response. The CD8 + T-cell noncytotoxic anti-hiv activity of HIV-1 and HIV-2-infected patients was investigated in coculture experiments in which blood donor CD4 + T cells were infected with two different virus isolates (HIV-1 Bal or HIV-1 IIIB ) and then cultured with the patients CD8 + T cells. b-chemokines are able to inhibit HIV-1 Bal (R5 virus) replication but have no effect on HIV-1 IIIB (X4 virus). In contrast, CD8 + T-cell antiviral factor (CAF) is active against both R5 and X4 virus. Briefly, peripheral blood mononuclear cells (PBMC) from healthy blood donors were separated on Ficoll Paque. CD4 + T cells were enriched (negative selection) from mononuclear cells by using Dynabeads (Dynal, Oslo, Norway) and stimulated with phytohaemagglutinin (PHA) (1 mg/ml, Difco, Detroit, MI, USA) for 3 days. The cells were then washed and infected with 1 TCID 5 of HIV-1 Bal or HIV-1 IIIB for 2 h at 37 C and thereafter washed twice to remove free virus and re-suspended in RPMI-164 growth medium containing interleukin (IL)-2 (2 U/ml, Advance Biotechnologies, Silver Springs, MD, USA). The infected CD4 + T cells were cultured alone or with various amount of CD8 + T cells purified from previously PHA-stimulated PBMC of HIV-2-(n ¼ 1) and HIV-1-(n ¼ 11) infected individuals. The CD8 + :CD4 + cell ratios used were 1 : 1,.5 : 1 and.25 : 1, and all the ratios were tested in duplicates. The cell-culture fluid was changed at 3 4 days interval and monitored for reverse transcriptase (RT) activity (HS-kit Lenti RT, Cavidi, Uppsala, Sweden). Per cent suppression of HIV-1 infection was calculated by comparing the average of RT activity in culture fluids from the CD8 + cell/cd4 + cell cocultures with the average of RT activity in several wells containing only infected CD4 + T cells. To investigate the nature of the CD8 + T-cell-mediated HIV-suppressive activity in vitro, R5 and X4 virus-infected CD4 + T cells were also cultured in the presence of CD8 + T-cell-culture supernatants of three HIV-2-infected individuals as described elsewhere [21]. In brief, previously PHA stimulated (1 mg/ml) CD8 + T cells were cultured in RPMI-164 medium supplemented with IL-2 (2 U/ml). At 2 days intervals, the culture fluids were collected and passed through a.45-mm filter. The anti-hiv activity in these fluids was evaluated when PHA-stimulated CD4 + T cells of blood donors were infected with R5 or X4 virus as described above and cultured in the presence of CD8 + T-cell-culture supernatants (collected on day 7). Cellculture fluids of cocultures were collected and new CD8 cell-culture supernatants were added every second day. RT activity in culture fluids was determined and the per cent inhibition was calculated by comparing the average value of RT activity in fluids in wells containing infected CD4 + T cells and CD8 + T-cell-culture supernatants, with the average of RT activity from several control wells containing only infected CD4 + T cells. # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61, 63 71

3 R. K. S. Ahmed et al. Antiviral Factors in HIV-2-infected Individuals 65 Antigen-specific b-chemokine production. In order to study the production of RANTES, MIP-1a and MIP-1b in response to HIV-2-specific antigens, PBMC of HIV-2- infected individuals were infected with attenuated recombinant vaccinia virus (rvv) (NYVAC) [25] expressing HIV-2 gag/pol (vp145) and env genes (vp92) or the parental vaccinia virus alone (vp866). One million cells were incubated for 9 min with five plaque-forming units per cell of different vaccinia constructs in medium containing 2% fetal calf sera (FCS) at 37 C and 6% CO 2. Thereafter, the cells were washed once with phosphate-buffered saline (PBS) containing 5% FCS. The cells were re-suspended in complete medium (1 1 6 million/ml) containing IL-2 (2 U/ml) and IL-7 (25 ng/ml; R&D Systems) and incubated at 37 C. On day 7, the supernatants were collected and monitored in b-chemokine ELISAs [21]. Statistical analysis. Non-parametric Spearman rank test and Mann Whitney U-test were used for statistical calculations. Results CD8 + T-cell noncytotoxic anti-hiv activity The presence of CD8 + T-cell noncytotoxic antiviral response in HIV-1- and HIV-2-infected patients were tested against R5 (HIV-1 Bal ) and X4 (HIV-1 IIIB ) virus. CD8 + T cells from both HIV-1 and HIV-2-infected patient suppressed b-chemokine-sensitive R5 virus replication in vitro in a similar manner with all CD8 : CD4 cell ratios tested (Fig. 1). In contrast, the suppression of X4 virus infection by CD8 + T cells seemed to be more frequent and of a higher magnitude among HIV-2-infected patients (Fig. 2A) compared to that among HIV-1-infected individuals (Fig. 2B), although the difference did not reach statistical significance. In general, more CD8 + T cells of HIV-1-infected patients were required to control X4 virus replication in vitro compared to that of those infected with HIV-2 (Fig. 2). Three out of 1 HIV-2-infected individuals and none of the 11 HIV-1- infected patients suppressed X4 virus infection >9% in vitro at the highest CD8 : CD4 cell ratio used (1 : 1). CD8 + T-cell-noncytotoxic antiviral activity was also detected in cell-free culture supernatants of the three HIV-2-infected individuals tested (patients 2, 4 and 1). Direct cell-to-cell contact resulted in more efficient suppression of both virus isolates in comparison to the inhibition by CD8 + T-cell-culture supernatants in one of these patients (Table 1). HIV-2-specific production of b-chemokines b-chemokine production was analysed after infection of PBMC with rvv (NYVAC) expressing HIV-2 gag/pol and env genes. NYVAC constructs have been shown to elicit specific CD8 + T-cell responses by the MHC class I pathway [26]. The relationship between the CD4 + T-cell count and b-chemokine production in HIV-2-infected individuals is shown in Fig. 3. A significant correlation (Spearman rank correlation) was found between HIV-2- specific RANTES, MIP-1a and MIP-1b production and the absolute number of CD4 + T cells (Fig. 3). Higher production of all three b-chemokines by PBMC of HIV- 2-infected individuals was associated with higher CD4 + T-cell counts. In general, production of MIP-1a and MIP-1b in response to recombinant NYVAC-expressing HIV-2 env and gag/pol was higher compared to RANTES production (Fig. 4). There was no apparent difference in the production of b-chemokines in HIV-2-infected patients treated with antiviral drugs (patients 4, 13, 16 and 17) or in nontreated patients (patients 2, 1, 11 and 15) (Fig. 4). The lowest levels of b-chemokines produced were observed in the three patients (15, 17 and 186) having the lowest CD4 + T-cell counts (144, 72, 271 cells/ml, respectively). Measurable levels of all three b-chemokines were also produced in response to the wildtype vaccinia virus, but the levels were lower compared to the levels produced in response to vaccinia virus expressing HIV-2 proteins. As expected, the cells obtained from five HIV-2-negative individuals produced low amounts of all three b-chemokines in response to HIV- 2-specific stimulation (Fig. 4). There was no correlation between the b-chemokine production and the number of B cells, NK or CD8 + T cells (data not shown). Discussion The key finding in this study was the presence of strong CD8 + T-cell-mediated noncytotoxic antiviral activity in HIV-2-infected individuals. An interesting observation was that this antiviral response seemed to be more frequent and of a higher magnitude among HIV-2-infected individuals compared to HIV-1-infected patients matched with regard to CD4 + T-cell counts. The noncytotoxic anti-hiv immune response involves still unknown CAF and b-chemokines. While b-chemokines inhibit virus binding to CCR5 [27 29], CAF inhibits the virus replication at the level of transcription [3 32]. Moreover, in contrast to b-chemokines, which are active against R5 virus, CAF is active against both R5 and X4 virus isolates. The clinical relevance of b-chemokine production in the pathogenesis of HIV infection is still poorly understood. In some studies, high levels of b-chemokines have been correlated to protection against HIV-1 infection [33] and associated with asymptomatic HIV-1 infection [22] and slower disease progression [34]. In most of the studies reported thus far, b-chemokine levels have been measured in response to nonspecific activation of the cells obtained from HIV-1-infected # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61, 63 71

4 66 Antiviral Factors in HIV-2-infected Individuals R. K. S. Ahmed et al. A HIV-2 infected B HIV-1 infected 12 1:1 12 1: : : : : Figure 1 Per cent inhibition of human immunodeficiency virus (HIV)-1 Bal (R5 virus) by CD8 + T cells of (A) HIV-2- and (B) HIV-1-infected individuals. CD8 : CD4 ratios of 1 : 1,.5 : 1 and.25 : 1 were used. individuals. In only few studies, the production of b-chemokines in response to the specific antigens has been reported [35, 36]. Importantly, not much is known about b-chemokine production in HIV-2-infected individuals. Our results herein show higher antigen-induced RANTES, MIP-1a and MIP-1b production in HIV-2-infected individuals as compared to HIV-2-seronegative subjects (Fig. 4). This antigen-specific b-chemokine production correlated to the absolute number of CD4 + cells. In previous studies, antigen-specific b-chemokine production in HIV-1-infected individuals has been associated with reduced viral replication [35] and AIDS-free status [36]. In agreement with our study, antigen-induced b-chemokine levels in HIV-1-infected individuals were correlated with CD4 + cell counts, but not with CD8 + cell counts [36]. In contrast, mitogen-induced MIP-1a and MIP-1b production by CD8 + T cells correlated with both CD4 + and CD8 + T-cell counts in another study [22]. The difference between the relationship of b-chemokine production and CD4 + T cells or CD8 + T cells in different # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61, 63 71

5 R. K. S. Ahmed et al. Antiviral Factors in HIV-2-infected Individuals 67 A HIV-2 infected B HIV-1 infected 12 1:1 12 1: : : :1.25: Figure 2 Per cent inhibition of HIV-1 IIIB (X4 virus) by CD8 + T cells of human immunodeficiency virus (A) (HIV)-2- and (B) HIV-1-infected individuals. CD8 : CD4 ratios of 1 : 1,.5 : 1 and.25 : 1 were used. Table 1 Suppression of human immunodeficiency virus (HIV)-1 4 and HIV-1 R5 virus replication by CD8 + cells (CD8 : CD4 cell ratios of 1 : 1) and cell-culture supernatants from three HIV-2-infected patients Culture supernatant (% inhibition) Cell-to-cell contact (% inhibition) Patient X4 R5 X4 R studies is most likely due to methodological differences and the use of different cell populations (PBMC or CD8 + cells)or different agents for stimulation (PHA or specific antigen) of the cells. It was reported recently that different CD8 + T-cell subpopulations differ in their ability to produce different b-chemokines [37]. There was no correlation between b-chemokine production and CD8 + T-cell counts in our present study even though the antigens we used have been shown to activate CD8 + T cells [26]. However, that might be a reflection of the state of # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61, 63 71

6 68 Antiviral Factors in HIV-2-infected Individuals R. K. S. Ahmed et al. A env gag/pol ρ =.86 P <.1 ρ =.94 P <.1 B env gag/pol 4, 4, 35, 35, 3, 3, 25, 25, 2, 2, 15, 15, 1, 1, C 7, 6, 5, 4, 3, 2, 1, ρ =.73 P =.7 ρ =.81 P =.1 env , 6, 5, 4, 3, 2, 1, gag/pol ρ =.64 P =.2 ρ =.71 P =.9 Figure 3 Relationship between RANTES (A), macrophage-inflammatory protein (MIP)-1a (B) and MIP-1b (C) production in response to recombinant vaccinia virus (NYVAC) expressing human immunodeficiency virus (HIV)-2 gag/pol or env genes and the absolute number of CD4 + cells in HIV-2-infected individuals. immunodeficiency, as the lowest levels of b-chemokines produced were observed in the patients having the lowest CD4 + T-cell counts (Fig. 4). There was no apparent difference in b- chemokine production in patients treated with antiviral drugs or in nontreated patients in our study, although antiretroviral therapy has been shown to have an effect on b-chemokine levels [38]. HIV-2 infection is characterized by a long clinical latency period comparable to that seen in HIV-1-infected LTNP [5]. The presence of strong and stable CD8 + T-cell noncytotoxic anti-hiv activity and production of CAF have been reported in LTNP [39, 4] and has been correlated with the clinical stage of the HIV-1-infected individuals [4, 41]. In our study, CD8 + T cells of HIV- 2-infected individuals had strong anti-hiv-1 activity against both b-chemokine-sensitive (R5) and -insensitive (X4) virus isolates, indicating that this antiviral activity was not mediated solely by b-chemokines. Similarly, CD8 + T cells of uninfected individuals highly exposed to HIV-1 has been shown to inhibit the growth of HIV-1 regardless of coreceptor usage [42] and the resistance to HIV-1 infection seems to be associated with production of antiviral factors different from b-chemokines [18]. In contrast to the above observations, Kokkotou et al. [43] reported that PBMC of HIV-2-infected sex workers resisted the infection with b-chemokine-sensitive R5 virus, but not X4 virus. In our study, we were not able to show any correlation between antigen-induced b-chemokine production and the presence of noncytotoxic CD8 + T-cell antiviral activity in HIV-2-infected patients. The noncytotoxic CD8 + T-cell antiviral activity seems to be independent of b-chemokine production and apparently b-chemokines and CD8 + T-cell soluble factor (CAF) are produced by different subpopulations of CD8 + T cells [38]. Furthermore, in addition to CD8 + T cells, many other cells including NK cells produce b-chemokines [44, 45]. Interestingly, NK cell-culture supernatants have been shown to suppress both R5 and X4 virus replication in vitro, and the b-chemokines were only partly responsible for this antiviral activity [44]. # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61, 63 71

7 R. K. S. Ahmed et al. Antiviral Factors in HIV-2-infected Individuals 69 A B env gag/pol Vector control Negative control 4, 35, 3, 25, 2, 15, 1, 5 C Negative control 7, 6, Figure 4 Production of RANTES (A), macrophage-inflammatory protein (MIP)- 1a (B) and MIP-1b (C) by peripheral blood mononuclear cells of 12 human immunodeficiency virus (HIV)-2-infected individuals in response to recombinant vaccinia virus (NYVAC) expressing HIV-2 gag/pol and env genes and a vector control. Neg control, mean of five HIV-negative individuals. 5, 4, 3, 2, 1, Negative control Our present study provides further evidence of the complex role of CD8 + T-cell noncytotoxic antiviral activity and the production of b-chemokines in controlling HIV infection. It seems that CD8 + T cells from HIV- 2-infected individuals are more likely to control HIV replication in vitro than CD8 + T cells from HIV- 1-infected individuals. It is possible that infection with the less pathogenic HIV-2 may lead to the production of b-chemokines together with still unidentified CAF. The disease progression in many HIV-1-infected individuals is associated with a switch from CCR5 using R5 virus to X4 virus that use CXCR4 [46]. The production of CAF and b-chemokines may inhibit or delay this switch and may in turn be related to the slower disease progression in HIV- 2-infected individuals compared to the individuals infected with HIV-1. In addition, some individuals may have innate capacity to produce different HIV-suppressive factors, as the production of these antiviral factors is not restricted to HIV infection [47]. Acknowledgments This work was supported by grants from the Swedish International Development Cooperation Agency, Department for Research Cooperation (Sida/SAREC). We thank Helén Linder, Sanna Björkman and clinical staff in Guinea-Bissau and Sweden for assistance in collecting the specimens. We also thank Jan-Olov Persson for the statistical calculations. References 1 Kanki PJ, Travers KUM, Boup S et al. Slower heterosexual spread of HIV-2 than HIV-1. Lancet 1994;343 (893): Poulsen A, Kvinesdal B, Aaby P et al. Lack of evidence of vertical transmission of human immunodeficiency virus type 2 in a sample of the general population in Bissau. J Acquir Immune Defic Syndr 1992;5 (1): Andreasson PA, Dias F, Naucler A, Andersson S, Biberfeld G. A prospective study of vertical transmission of HIV-2 in Bissau, Guinea-Bissau. AIDS 1993;7 (7): # 25 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 61, 63 71

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