Cellular Human Immunodeficiency Virus (HIV)Protective Factors: A Comparison of HIV-Exposed Seronegative Female Sex Workers and Female Blood Donors in Abidjan,

¹Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium; ²Projet RETROCôte d'Ivoire, Abidjan, Côte d'Ivoire; ³Division of HIV/AIDS Prevention, National Center for HIV, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia

Received 7 June 2002; revised 16 September 2002; electronically published 30 December 2002.

    Presented in part: XIII International AIDS Conference, Durban, South Africa, 914 July 2000 (abstract WePeA3995).
     Informed consent was given by all study subjects before enrollment. The study was approved by the institutional review board of the Centers for Disease Control and Prevention (CDC) and by the ethical committees of the Côte d'Ivoire Ministry of Health and the Institute of Tropical Medicine.
     Financial support: Belgian Fonds voor Wetenschappelijk Onderzoek Vlaanderen (grant G.0396.99); Division of HIV/AIDS Prevention, CDC.
     Reprints or correspondence: Wim Jennes, Institute of Tropical Medicine, Dept. of Microbiology, Laboratory of Immunology, Nationalestraat 155, 2000 Antwerp, Belgium ().

     In several populations, small proportions of individuals remain human immunodeficiency virus (HIV)seronegative, despite frequent sexual exposure to HIV, which suggests that host factors may contribute to protection against infection [15]. Proposed correlates of protection against HIV infection in HIV-exposed seronegative (ESN) subjects that are well documented include inherited and acquired host factors, such as a homozygous 32-bp deletion in the gene encoding the HIV-1 coreceptor CCR5 [611], particular genetic HLA polymorphisms [12, 13], mucosal and systemic antiHIV IgA [1418], and HIV-specific helper and cytotoxic T cells [1925].

     In addition to CD4, the -chemokine receptor CCR5 is used as a coreceptor by primary macrophage (M)tropic HIV-1 strains that are sexually transmitted [2629], whereas the -chemokine receptor CXCR4 is used by T cell (T)tropic variants of HIV-1 that emerge at later disease stages [3032]. Decreased expression levels of CCR5 on CD4⁺ T cells, in the absence of the 32-bp deletion, have been correlated to decreased susceptibility to M-tropic HIV-1 in vitro [33, 34], to decreased HIV-1 load levels in infected subjects [35], and to increased protection against HIV-1 infection in ESN subjects [36]. However, studies exist in which the latter could not be confirmed [37, 38]. Although susceptibility to T-tropic HIV-1 strains in vitro has been correlated directly to CXCR4 expression levels [3941], no associations have been found between CXCR4 levels and the transmission of HIV-1 in vivo.

     -chemokines are the natural ligands for the CCR5 coreceptor, and, although their HIV-suppressive capacity has been shown in vitro [42], their role in protection against HIV infection in vivo remains controversial. Some studies have found increased -chemokine levels in ESN subjects, compared with those in unexposed control subjects [25, 36, 4345]. However, other studies have not found any difference [37, 38, 46]. Similarly, Th1 cytokines could play a role in protection against infection with HIV in association with cell-mediated immunity [47]. Indeed, among ESN populations, increased levels of Th1 cytokines have been shown in the presence of mucosal and systemic cellular immune activation [48] or together with a decreased Th2 cytokine profile [49, 50]. However, in other studies, decreased Th1 cytokine production was found among ESN subjects [46, 51].

     In the present study, we determined whether African ESN female sex workers (FSWs) differed from African HIV-seronegative female blood donors in one or several immunological aspects with potential HIV-protective capacity. Freshly obtained whole-blood samples were used to measure ex vivo cell surface expression levels of immune activation markers and HIV-1 coreceptors and to detect intracellular -chemokine and cytokine production, all at the level of a single cell, by flow cytometry. In addition, we verified whether these potential correlates of HIV protection were associated with indicative markers of HIV exposure among FSWs, such as the duration of commercial sex work, the number of clients per day, the consistency in using condoms, and the occurrence of sexually transmitted infections (STIs).

SUBJECTS AND METHODS

     Study population.     HIV-seronegative FSWs were enrolled consecutively as part of an ongoing surveillance study at a confidential clinic in Abidjan [52], and blood samples and standard questionnaires with information on sociodemographics and sexual behavior were collected. Voluntary HIV-seronegative female blood donors were enrolled at the national blood transfusion center in Abidjan; only blood samples were collected. The study lasted 2 months and took place in 2 distinct 1-month time periods. In November 1999, 14 FSWs and 15 blood donors were enrolled; in May 2000, 13 FSWs and 12 blood donors were enrolled.

     Laboratory methods for HIV and STI diagnosis.     Whole blood was drawn from FSWs and control subjects into EDTA tubes (Becton Dickinson). Within 4 h of blood collection, plasma was separated from cells by centrifugation at 500 g, aliquoted, and stored at -70°C.

     The HIV-seronegative status of all subjects was determined in plasma by use of the current HIV testing algorithm of Projet RETROCôte d'Ivoire, defined by a combination of ELISAs and Western blots [53, 54]. The HIV-negative status of ESN FSWs was confirmed by reverse HIV-1 reverse-transcriptase (RT) polymerase chain reaction (PCR) in plasma samples [55].

     Bacterial and protozoan STIs were tested in the 27 FSWs: antitreponemal antibodies were detected using the Treponema pallidum hemagglutination assay (TPHA; Fujirebio) and the rapid plasma reagin (RPR) test (Macro-Vue, Becton Dickinson). A diagnosis of syphilis was made if results for both the TPHA and RPR tests were positive [56]. Neisseria gonorrhoeae infection was diagnosed by culture on modified Thayer-Martin medium or by PCR [56]. Chlamydia trachomatis infection was diagnosed by Microtrak EIA (Syva) or by PCR. Trichomonas vaginalis infection was diagnosed by use of wet-mount techniques.

     T cell activation markers.     Phenotypic analyses of peripheral blood lymphocytes were performed on fresh whole blood within 4 h of collection in EDTA. To quantify the levels of CD4⁺ and CD8⁺ T cell activation, whole blood was incubated in the presence of the following monoclonal antibody (MAb) combinations: anti-CD8, anti-CD38, and anti-HLA-DR; anti-CD3, anti-CD8, and anti-HLA-DR; and anti-CD4, anti-CD45RO, and anti-CD62L MAbs. All MAbs were fluorochrome-labeled and purchased from Becton Dickinson. The whole-blood staining procedure was done as described elsewhere [57]. Samples were analyzed using a FACScan flow cytometer and CellQuest software (both from Becton Dickinson). Calibration beads (Dako) were run on a weekly basis, to ensure the stability of the flow cytometer. Percentages of HLA-DR⁺ cells were analyzed within the CD4⁺ and CD8⁺ T cell subsets. Percentages of CD38⁺ and CD38⁺ HLA-DR⁺ double-positive cells were analyzed within the bright CD8⁺ lymphocyte subset. Percentages of CD62L⁺ cells were analyzed within CD45RO⁺ (memory) and CD45RO^- (naive) CD4⁺ lymphocyte subsets. For all analyses, quadrant markers were set using negative isotype controls.

     HIV-1 coreceptor expression.     Levels of HIV-1 coreceptor expression were analyzed on fresh whole blood within 4 h of collection in EDTA. Cells were stained with a combination of peridinin-chlorophyll-proteinlabeled anti-CD4 and fluorescein isothiocyanatelabeled anti-CD45RO (both from Becton Dickinson), and phycoerythrin (PE)labeled anti-CCR5 and anti-CXCR4 MAbs (both from Pharmingen), to assess the membrane expression of CCR5 and CXCR4 on CD45RO⁺ (memory) and CD45RO^- (naive) CD4⁺ lymphocytes. Whole-blood staining was done as described above for T cell activation markers. HIV-1 coreceptor expression levels were calculated as percentages of positive cells or as median logarithmic fluorescence intensity (MFI) levels.

     Intracellular staining of -chemokines and cytokines.     Peripheral blood mononuclear cells (PBMC) were separated from fresh whole blood within 4 h of collection and stained intracellularly for chemokines and cytokines, as described elsewhere [57]. In brief, PBMC were incubated overnight in the presence of monensin (1.5 g/mL; Sigma) for the constitutive intracellular accumulation of the -chemokine RANTES and for 5 h with PMA (0.02 g/mL) and ionomycin (1 g/mL) in the presence of brefeldin A (10 g/mL) (all from Sigma) for the stimulation-induced intracellular accumulation of the -chemokines macrophage inflammatory protein (MIP)1 and MIP-1 and the cytokines interleukin (IL)2, IL-4, interferon (IFN), and tumor necrosis factor (TNF). PBMC were subsequently surface stained for CD3 and CD8, fixed, permeabilized, and stained intracellularly with anti-RANTES or antiMIP-1 (both from Pharmingen); antiIL-2, antiIL-4, antiIFN-, or antiTNF- (all from Becton Dickinson) PE-labeled MAbs; or antiMIP-1 carboxyfluorescein-labeled MAb (R&D Systems) for 30 min at room temperature. Samples were analyzed using a FACScan flow cytometer and CellQuest software (both from Becton Dickinson). Percentages of -chemokinepositive and cytokine-positive cells were analyzed within the CD4⁺ and CD8⁺ T cell subsets. For all analyses, quadrant markers were set using negative isotype controls.

     Statistical analysis.     Because there were 2 different study periods and because we wanted to minimize bias as a result of changing laboratory conditions, ESN FSWs and blood donors with approximately the same date of laboratory analysis were used for comparisons (n = 20 for both groups). Median values were calculated for normal and nonnormal distributions. Differences between groups were tested for statistical significance by use of the nonparametric Mann-Whitney U test. Correlation analyses were performed for all 27 subjects in both study groups by use of the nonparametric Spearman's rank correlation test. The level of significance for all statistical tests was set at P = .05.

RESULTS

     Characteristics of the study population.     The median age of the 27 ESN FSWs was 23 years (range, 1737 years), compared with 23 years (range, 1852 years) for the 27 female blood donors (P = .326). The median duration of commercial sex work reported by 27 ESN FSWs was 6 months (range, 1120 months). Three FSWs (11%) reported doing commercial sex work for 3 years, 11 (41%) reported doing commercial sex work for 6 months to 3 years, and 13 (48%) reported doing commercial sex work for <6 months. The reported number of clients on the last working day ranged from 1 to 14 (median, 3 clients). Ten FSWs (37%) reported "always" using condoms, 14 (52%) reported "often" using condoms, and 3 (11%) reported "rarely" or "never" using condoms. The reported consistency in condom use decreased with prolonged duration of commercial sex work (r = -0.517; P = .006) and increased with higher reported numbers of clients per day (r = 0.554; P = .003). The number of clients per day tended to decrease with prolonged duration of commercial sex work (r = -0.364; P = .062). Of the 27 ESN FSWs, 5 (19%) had 1 STI: 2 tested positive for T. vaginalis, 2 tested positive for C. trachomatis, 1 tested positive for N. gonorrhoea, and none tested positive for T. pallidum.

     ESN FSWs had increased T cell activation, compared with blood donors.     ESN FSWs had significantly higher percentages of CD38⁺ cells in CD8⁺ lymphocytes than did blood donors . Moreover, ESN FSWs tended to have lower percentages of true naive (CD62L⁺ CD45RO^-) CD4⁺ T lymphocytes, compared with blood donors. No differences were found in percentages of HLA-DR⁺ cells in CD4⁺ and CD8⁺ T cell subsets. Since multiple statistical comparisons were performed , there is an increased probability that the statistically significant result for CD38 expression on CD8⁺ T cells has occurred by chance. Among the ESN FSWs, no associations were found between CD38, CD62L, or HLA-DR expression and the duration of commercial sex work, number of clients per day, condom use, or the occurrence of an STI.

fig.ommitted

Table 1.          Expression of T cell activation markers on CD4⁺ and CD8⁺ T cell subsets from human immunodeficiency virus (HIV)seronegative female blood donors and HIV-exposed seronegative (ESN) female sex workers (FSWs).

     ESN FSWs had similar expression levels of the HIV-1 coreceptor CCR5 but significantly decreased levels of CXCR4, compared with blood donors.      shows representative histograms for the analysis of percentages of positive cells and MFI levels of CCR5 and CXCR4 in one blood donor and one ESN FSW for CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocytes. For CCR5 (), the markers M1 and M2 clearly discriminate CCR5^- and CCR5⁺ cell distributions in the CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocyte subsets. Therefore, relative cell numbers of CCR5⁺ cells (marker M2) were recalculated in percentages of CCR5⁺ cells and in MFI levels of the CCR5⁺ distributions within the CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocyte subsets. MFI levels of the CCR5⁺ clusters were calculated as a measurement of CCR5 expression density on CCR5⁺ cells. CXCR4 expression on lymphocytes was not characterized by discrete distributions of positive and negative cells (). Therefore, MFI levels of the whole CXCR4 distributions in the CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocyte subsets were calculated. In both ESN FSWs and blood donors, CCR5 was almost exclusively expressed on CD45RO⁺ (memory) CD4⁺ T cells, whereas the expression of CXCR4 was higher on CD45RO^- (naive) CD4⁺ cells than on CD45RO⁺ (memory) CD4⁺ T cells (), which confirms previous observations [58, 59].

fig.ommitted

Figure 1.        Representative flow cytometry analysis of ex vivo CCR5 and CXCR4 expression on CD4⁺ lymphocytes from one female blood donor and one human immunodeficiency virusexposed seronegative (ESN) female sex worker (FSW). A, For CCR5, the markers M1 and M2 discriminate CCR5^- and CCR5⁺ cell distributions, respectively, in the CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocyte subsets. Relative cell nos. of CCR5⁺ cells (marker M2) were recalculated in percentages of CCR5⁺ cells and in median logarithmic fluorescence intensity (MFI) levels of the CCR5⁺ distributions in both the CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocyte subsets. B, For CXCR4, MFI levels of the whole CXCR4 distributions in the CD45RO⁺ CD4⁺ and CD45RO^- CD4⁺ lymphocyte subsets were calculated.

fig.ommitted

Table 2.          Expression of human immunodeficiency virus (HIV) type 1 coreceptors CCR5 and CXCR4 in CD4⁺ lymphocyte subsets from HIV-seronegative female blood donors and HIV-exposed seronegative (ESN) female sex workers (FSWs).

     Percentages and MFI levels of CCR5⁺ memory, naive and total CD4⁺ T cells were similar between ESN FSWs and blood donors (). Percentages of CCR5⁺ memory CD4⁺ cells among ESN FSWs correlated significantly with percentages of HLA-DR⁺ CD4⁺ T cells (r = 0.621; P = .003), HLA-DR⁺ CD8⁺ T cells (r = 0.502; P = .024), and HLA-DR⁺ CD38⁺ CD8⁺ T cells (r = 0.582; P = .007). None of these correlations was found among blood donors. Among ESN FSWs, no associations were found between CCR5 expression levels and the duration of commercial sex work, number of clients per day, condom use, or the occurrence of an STI.

     MFI of CXCR4 expression on memory, naive, and total CD4⁺ T cells was significantly lower in ESN FSWs than in blood donors (). No associations were found between CXCR4 expression and T cell activation among either ESN FSWs or blood donors. CXCR4 expression levels tended to correlate inversely with the duration of commercial sex work for ESN FSWs, within both naive (r = -0.279; P = .176) and memory (r = -0.276; P = .182) CD4⁺ T cells. To explore further the relationship between CXCR4 expression and the duration of commercial sex work, 10 ESN FSWs who had done sex work for <6 months were compared with 10 ESN FSWs who had done sex work for 6 months, with approximately the same date of laboratory analysis, to eliminate bias as a result of changing laboratory conditions over time (). ESN FSWs who had done sex work for 6 months had lower CXCR4 MFI levels than did ESN FSWs who had done sex work for <6 months, reaching significant levels in the naive (CD45RO^-) CD4⁺ T cell subset but not in the memory (CD45RO⁺) CD4⁺ T cell subset. Although CXCR4 MFI levels on naive CD4⁺ cells tended to correlate inversely with age among ESN FSWs (r = -0.331; P = .106), this was not found to be the case among blood donors. In addition, duration of sex work did not correlate with age (r = 0.183; P = .360) among ESN FSWs, and median age in the groups of ESN FSWs who had done sex work for 6 months or <6 months was not different (P = .239). Therefore, age could be excluded as a significant confounding factor in the analysis. Among ESN FSWs, no associations were found between CXCR4 expression and the number of clients per day, consistency of condom use, or the occurrence of an STI.

fig.ommitted

Figure 2.        CXCR4 median logarithmic fluorescence intensity (MFI) levels of CD45RO^- (naive) and CD45RO⁺ (memory) CD4⁺ cell subsets for 20 female blood donors (white boxes), 10 human immunodeficiency virusexposed seronegative (ESN) female sex workers (FSWs) who had done sex work for <6 months (gray boxes), and 10 ESN FSWs who had done sex work for 6 months (black boxes). Box plots represent the lowest value, the 25th percentile, the 50th percentile (median), the 75th percentile, and the highest value, respectively, indicated by horizontal lines in ascending order. Differences between groups were tested for statistical significance by use of the nonparametric Mann-Whitney U test. P₁, female blood donors vs. all ESN FSWs; P₂, ESN FSWs who had done sex work for <6 months vs. ESN FSWs who had done sex work for 6 months.

     ESN FSWs and blood donors had similar percentages of -chemokinepositive T cells.     RANTES was the most abundant -chemokine detected in both CD4⁺ and CD8⁺ T cells, being constitutively expressed at higher percentages than mitogen-stimulated MIP-1 or MIP-1 (). Percentages of RANTES-, MIP-1 and MIP-1positive cells in CD4⁺ and CD8⁺ T cell subsets were comparable between ESN FSWs and blood donors . Among ESN FSWs, percentages of HLA-DRpositive cells correlated with percentages of RANTES-positive cells in the CD8⁺ T cell subset (r = 0.459; P = .032) and with percentages of MIP-1positive cells in both CD4⁺ (r = 0.478; P = .033) and CD8⁺ (r = 0.549; P = .012) T cell subsets. Among blood donors, percentages of HLA-DRpositive cells correlated with MIP-1positive cells in the CD8⁺ T cell subset (r = 0.522; P = .018). Among ESN FSWs, no correlations were found between proportions of -chemokinepositive T cells and duration of commercial sex work, number of clients per day, the occurrence of an STI, or condom use.

fig.ommitted

Table 3.          Percentages of -chemokinepositive cells and Th1 and Th2 cytokinepositive cells in CD4⁺ and CD8⁺ T cell subsets from human immunodeficiency virus (HIV)seronegative female blood donors and HIV-exposed seronegative (ESN) female sex workers (FSWs).

     ESN FSWs and blood donors had similar percentages of cytokine-positive T cells.     Percentages of CD4⁺ and CD8⁺ T cells that were positive for the Th1 cytokines IL-2, IFN-, and TNF- and the Th2 cytokine IL-4 were comparable among ESN FSWs and blood donors . Percentages of CD38⁺ HLA-DR⁺ CD8⁺ T cells correlated with percentages of IL-2positive (r = 0.538; P = .01), IFN-positive (r = 0.621; P = .002), and TNF-positive (r = 0.525; P = .012) CD8⁺ T cells among ESN FSWs and with IFN-positive CD8⁺ T cells (r = 0.407; P = .06) among blood donors. Among ESN FSWs, no correlations were found between proportions of cytokine-positive T cells and duration of commercial sex work or use of condoms, but the number of clients per day correlated positively with proportions of IL-2positive CD4⁺ T cells (r = 0.512; P = .015) and IL-4positive CD4⁺ T cells (r = 0.479; P = .024). No statistically significant associations were found between proportions of cytokine-positive T cells and the occurrence of an STI among ESN FSWs.

DISCUSSION

     In Abidjan, Côte d'Ivoire, HIV prevalence among FSWs has decreased extensively, from 89% in 1992 to 32% in 1998 [52] and 28% in 2000 (authors' unpublished data), thanks to intensive prevention efforts and increasing condom use. However, although reported condom use is high with clients (79% consistent condom use with clients during the last working day in 1998 [52, 60]), only 20% of the FSWs use condoms consistently with their stable partners (authors' unpublished data). The HIV incidence among FSWs participating in a trial of microbicides during 19982000 was 4% [61], which indicates that seronegative FSWs in Abidjan are still frequently exposed to HIV and are at risk of acquiring infection. In 2000, HIV seroprevalence among first-time blood donors (men and women) in Abidjan was estimated at 6% [62]. Ten percent of pregnant women attending an antenatal clinic in Abidjan during 19972000 were HIV seropositive [63]. These data indicate that, despite targeting a population that is at lower risk for HIV infection than HIV-seronegative FSWs, blood donors, especially young female blood donors, cannot be considered to be HIV unexposed.

     In this study, a group of ESN FSWs was compared with a group of HIV-seronegative female blood donors for the presence of immunological factors that could contribute to decreased susceptibility to HIV infection. Our findings indicate that ESN FSWs and HIV-seronegative female blood donors have comparable expression levels of the HIV-1 coreceptor CCR5, -chemokines, and Th1 or Th2 cytokines, but that ESN FSWs have significantly decreased levels of CXCR4 expression and significantly increased levels of CD8⁺ T cell activation.

     Increased CD8⁺ T cell activation could be a sign of anti-HIV activity by HIV-specific CD8⁺ cytotoxic T cells [48]. Indeed, several studies have described HIV-specific CTL responses in HIV-seronegative subjects in association with frequent sexual exposure to HIV [20, 21, 24, 25]. However, in the present study, CD8⁺ T cell activation among ESN FSWs did not correlate with any marker of sexual HIV exposure. It is possible that CD8⁺ T cell activation is associated with other infections and STIs in ESN FSWs, rather than being related to HIV. Similar to results of a previous study among HIV-infected FSWs in Abidjan [64], we did not observe any statistically significant association between T cell activation markers and the occurrence of protozoan or bacterial STIs. It is more likely that viral non-HIV concurrent infections and STIs (which we have not analyzed in this study), rather than protozoan or bacterial STIs, play a role in T cell activation among ESN FSW.

     Previous studies in Kenya and Ethiopia reported similar expression levels of CCR5 in ESN FSWs, compared with those in unexposed control subjects, as in our study, but found increased rather than decreased levels of CXCR4 [37, 38]. In these studies, however, HIV-1 coreceptor expression was measured on fresh or cryopreserved PBMC, whereas, in the present study, fresh whole blood was used. Variation in technical procedures can highly influence the analysis of HIV-1 coreceptor expression levels. This has been shown for prolonged sample storage before analysis, which causes increased CXCR4 and decreased CCR5 levels [65] and for density gradient isolated PBMC with significantly lower levels of CCR5 [66] and CXCR4 (authors' unpublished data), compared with whole blood. The use of different anticoagulants can also influence laboratory results [65, 66].

     Decreased levels of CXCR4 among ESN FSWs in our study were found to be associated with prolonged duration of sex work. This association was found to be independent from T cell activation, since T cell activation did not correlate with either CXCR4 expression or the duration of sex work. In vitro, HIV-1 envelope gp120 has been shown to down-regulate CXCR4 [67, 68]. Similarly, in vivo, chronic HIV infection results in down-regulation of CXCR4 expression [59, 6971]. Therefore, in the same way as in HIV-infected subjects, chronic exposure to HIV among ESN FSWs could have caused the down-regulation of CXCR4. Alternatively, it is also possible that factors other than HIV are responsible for the down-regulation of CXCR4 in ESN FSWs, as has been reported for infection with human herpesvirus (HHV)6 and HHV-7 [72, 73] or through allogeneic activation [74], both of which could have been acquired gradually by FSWs who had done sex work for a prolonged duration.

     The relevance of CXCR4 down-regulation in ESN FSWs as a correlate of protection against HIV infection remains unclear and can only be addressed in a longitudinal study. Theoretically, it is unlikely that CXCR4 plays a role, since primary HIV-1 strains that are sexually-transmitted, M-tropic strains predominantly use CCR5 as a coreceptor [27, 29, 75], an observation that has been confirmed in Abidjan (authors' unpublished data). Moreover, the few reported cases of HIV-1 transmission via CXCR4 have all occurred in subjects homozygous for the 32-bp deletion in the CCR5 gene [76, 77]. As shown for other African populations [37, 78], this 32-bp deletion has not been detected among ESN FSWs in Abidjan [79]. Still, it would be interesting to investigate whether the down-regulated CXCR4 expression among ESN FSWs could provide a protective mechanism against T-tropic HIV-1 viruses that selectively use CXCR4 as a coreceptor. If this were proven to be the case, as a mechanism, CXCR4 down-regulation could have contributed to the selection of CCR5-using M-tropic HIV-1 strains in sexual transmission.

     In most studies, -chemokine levels in ESN subjects have been measured in plasma samples or in the supernatant of mitogen-stimulated lymphocyte cultures, without providing information on the identity of the cells that produced the -chemokines [36 38, 4346]. Therefore, we measured proportions of -chemokineproducing CD4⁺ and CD8⁺ T cells in ESN FSWs by means of flow cytometry after spontaneous (for RANTES) and mitogen-stimulated (for MIP-1 and MIP-1) intracellular accumulation in vitro. Our results show that proportions of -chemokineproducing CD4⁺ and CD8⁺ T cells were comparable between ESN FSWs and blood donors and did not correlate with any indicative marker of HIV exposure of the FSWs. These data are in agreement with findings from other studies of ESN FSW populations [37, 38]. In a previous study, we found a positive correlation between proportions of -chemokineproducing T cells and HIV-1 plasma virus load in HIV-1infected FSWs, which indicates that intracellular -chemokine expression is a correlate of disease progression rather than of protection [57].

     Proportions of T cells producing the Th1 cytokines IL-2, TNF- and IFN- were similar in ESN FSWs and blood donors. Nevertheless, production of Th1 cytokines correlated more clearly with in vivo T cell activation among the ESN FSWs than among blood donors. Therefore, our data are more in line with augmented T cell activation and Th1 cytokine production in ESN subjects, as reported elsewhere [48] and do not confirm a down-regulated Th1 cytokine profile in ESN subjects in association with a postulated HIV protective low-grade activation state of CD4⁺ T cells [51]. Our data also show comparable percentages of IL-4producing T cells among ESN FSWs and female blood donors and, therefore, do not support the recent observation of a down-regulated Th2 cytokine profile in ESN FSWs [50].

     In summary, we found that ESN FSWs in Abidjan have similar expression levels of the HIV-1 coreceptor CCR5, -chemokines, and Th1 and Th2 cytokines, compared with HIV-seronegative female blood donors, but have significantly decreased levels of CXCR4 expression, together with significantly increased levels of T cell activation. These data indicate that ESN FSWs differ from female blood donors based on immunological factors that have no clear protective potential against HIV transmission.

Acknowledgments

     We thank the community of female sex workers in Abidjan, for their cooperation; Marie-Yolande Borget, Mathieu Maran, Daniel Yavo, and N'Depo Yenon, for technical assistance; Emmanuel Abonga, for providing blood samples from female blood donors; Jef Braem and Serge Blockmans, for logistical support in the field; and Monica Nolan, for critical remarks and discussion.

References

1. 

Clerici M, Giorgi JV, Chou CC, et al. Cell-mediated immune response to human immunodeficiency virus (HIV) type 1 in seronegative homosexual men with recent sexual exposure to HIV-1. J Infect Dis 1992; 165:10129.

2. 

Rowland-Jones S, Sutton J, Ariyoshi K, et al. HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women. Nat Med 1995; 1:5964.

3. 

Fowke KR, Nagelkerke NJ, Kimani J, et al. Resistance to HIV-1 infection among persistently seronegative prostitutes in Nairobi, Kenya. Lancet 1996; 348:134751.

4. 

Mazzoli S, Trabattoni D, Lo CS, et al. HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals. Nat Med 1997; 3:12507.

5. 

Beyrer C, Artenstein AW, Rugpao S, et al. Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand. J Infect Dis 1999; 179:5967.

6. 

Paxton WA, Martin SR, Tse D, et al. Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure. Nat Med 1996; 2:4127.

7. 

Liu R, Paxton WA, Choe S, et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 1996; 86:36777.

8. 

Samson M, Libert F, Doranz BJ, et al. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996; 382:7225.

9. 

Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 1996; 273:185662.

10. 

Paxton WA, Kang S, Koup RA. The HIV type 1 coreceptor CCR5 and its role in viral transmission and disease progression. AIDS Res Hum Retroviruses 1998; 14(Suppl 1):S8992.

11. 

Marmor M, Sheppard HW, Donnell D, et al. Homozygous and heterozygous CCR5-32 genotypes are associated with resistance to HIV infection. J Acquir Immune Defic Syndr 2001; 27:47281.

12. 

Just JJ. Genetic predisposition to HIV-1 infection and acquired immune deficiency virus syndrome: a review of the literature examining associations with HLA. Hum Immunol 1995; 44:15669.

13. 

MacDonald KS, Fowke KR, Kimani J, et al. Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection. J Infect Dis 2000; 181:15819.

14. 

Mazzoli S, Lopalco L, Salvi A, et al. Human immunodeficiency virus (HIV)specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons. J Infect Dis 1999; 180:8715.

15. 

Ghys PD, Belec L, Diallo MO, et al. Cervicovaginal anti-HIV antibodies in HIV-seronegative female sex workers in Abidjan, Cote d'Ivoire. AIDS 2000; 14:26038.

16. 

Belec L, Ghys PD, Hocini H, et al. Cervicovaginal secretory antibodies to human immunodeficiency virus type 1 (HIV-1) that block viral transcytosis through tight epithelial barriers in highly exposed HIV-1seronegative African women. J Infect Dis 2001; 184:141222.

17. 

Kaul R, Trabattoni D, Bwayo JJ, et al. HIV-1specific mucosal IgA in a cohort of HIV-1resistant Kenyan sex workers. AIDS 1999; 13:239.

18. 

Devito C, Hinkula J, Kaul R, et al. Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate. AIDS 2000; 14:191720.

19. 

Clerici M, Levin JM, Kessler HA, et al. HIV-specific T-helper activity in seronegative health care workers exposed to contaminated blood. JAMA 1994; 271:426.

20. 

Rowland-Jones SL, Dong T, Fowke KR, et al. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J Clin Invest 1998; 102:175865.

21. 

Bernard NF, Yannakis CM, Lee JS, Tsoukas CM. Human immunodeficiency virus (HIV)specific cytotoxic T lymphocyte activity in HIV-exposed seronegative persons. J Infect Dis 1999; 179:53847.

22. 

Goh WC, Markee J, Akridge RE, et al. Protection against human immunodeficiency virus type 1 infection in persons with repeated exposure: evidence for T cell immunity in the absence of inherited CCR5 coreceptor defects. J Infect Dis 1999; 179:54857.

23. 

Fowke KR, Kaul R, Rosenthal KL, et al. HIV-1specific cellular immune responses among HIV-1resistant sex workers. Immunol Cell Biol 2000; 78:58695.

24. 

Kaul R, Plummer FA, Kimani J, et al. HIV-1specific mucosal CD8⁺ lymphocyte responses in the cervix of HIV-1resistant prostitutes in Nairobi. J Immunol 2000; 164:160211.

25. 

Skurnick JH, Palumbo P, DeVico A, et al. Correlates of nontransmission in US women at high risk of human immunodeficiency virus type 1 infection through sexual exposure. J Infect Dis 2002; 185:42838.

26. 

Alkhatib G, Combadiere C, Broder CC, et al. CC CKR5: a RANTES, MIP-1, MIP-1 receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996; 272:19558.

27. 

Deng H, Liu R, Ellmeier W, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996; 381:6616.

28. 

Doranz BJ, Rucker J, Yi Y, et al. A dual-tropic primary HIV-1 isolate that uses fusin and the -chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 1996; 85:114958.

29. 

Dragic T, Litwin V, Allaway GP, et al. HIV-1 entry into CD4⁺ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996; 381:66773.

30. 

Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G proteincoupled receptor. Science 1996; 272:8727.

31. 

Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR. Change in coreceptor use correlates with disease progression in HIV-1infected individuals. J Exp Med 1997; 185:6218.

32. 

Simmons G, Wilkinson D, Reeves JD, et al. Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry. J Virol 1996; 70:835560.

33. 

Wu L, Paxton WA, Kassam N, et al. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J Exp Med 1997; 185:168191.

34. 

Blaak H, Ran LJ, Rientsma R, Schuitemaker H. Susceptibility of in vitro stimulated PBMC to infection with NSI HIV-1 is associated with levels of CCR5 expression and -chemokine production. Virology 2000; 267:23746.

35. 

Reynes J, Portales P, Segondy M, et al. CD4⁺ T cell surface CCR5 density as a determining factor of virus load in persons infected with human immunodeficiency virus type 1. J Infect Dis 2000; 181:92732.

36. 

Paxton WA, Liu R, Kang S, et al. Reduced HIV-1 infectability of CD4⁺ lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of -chemokines. Virology 1998; 244:6673.

37. 

Fowke KR, Dong T, Rowland-Jones SL, et al. HIV type 1 resistance in Kenyan sex workers is not associated with altered cellular susceptibility to HIV type 1 infection or enhanced -chemokine production. AIDS Res Hum Retroviruses 1998; 14:152130.

38. 

Messele T, Rinke dWT, Brouwer M, et al. No difference in in vitro susceptibility to HIV type 1 between high-risk HIV-negative Ethiopian commercial sex workers and low-risk control subjects. AIDS Res Hum Retroviruses 2001; 17:43341.

39. 

Tokunaga K, Greenberg ML, Morse MA, Cumming RI, Lyerly HK, Cullen BR. Molecular basis for cell tropism of CXCR4-dependent human immunodeficiency virus type 1 isolates. J Virol 2001; 75:677685.

40. 

BouHamdan M, Strayer DS, Wei D, et al. Inhibition of HIV-1 infection by down-regulation of the CXCR4 co-receptor using an intracellular single chain variable fragment against CXCR4. Gene Ther 2001; 8:40818.

41. 

Jourdan P, Vendrell JP, Huguet MF, et al. Cytokines and cell surface molecules independently induce CXCR4 expression on CD4⁺ CCR7⁺ human memory T cells. J Immunol 2000; 165:71624.

42. 

Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1, and MIP-1 as the major HIV-suppressive factors produced by CD8⁺ T cells. Science 1995; 270:18115.

43. 

Vitale F, Bonura F, Perna AM, Ajello F, Romano N. Serum RANTES levels in HIV-positive individuals and in HIV-negative exposed health-care workers. Infection 1997; 25:3812.

44. 

Zagury D, Lachgar A, Chams V, et al. C-C chemokines, pivotal in protection against HIV type 1 infection. Proc Natl Acad Sci USA 1998; 95:385761.

45. 

Shieh B, Yan YP, Ko NY, et al. Detection of elevated serum -chemokine levels in seronegative Chinese individuals exposed to human immunodeficiency virus type 1. Clin Infect Dis 2001; 33:2739.

46. 

Barretina J, Blanco J, Gutierrez A, et al. Evaluation of the putative role of C-C chemokines as protective factors of HIV-1 infection in seronegative hemophiliacs exposed to contaminated hemoderivatives. Transfusion 2000; 40:4617.

47. 

Clerici M, Shearer GM. The Th1-Th2 hypothesis of HIV infection: new insights. Immunol Today 1994; 15:57581.

48. 

Biasin M, Caputo SL, Speciale L, et al. Mucosal and systemic immune activation is present in human immunodeficiency virusexposed seronegative women. J Infect Dis 2000; 182:136574.

49. 

Barcellini W, Rizzardi GP, Velati C, et al. In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from high-risk HIV-negative intravenous drug users. AIDS 1995; 9:6914.

50. 

Trivedi HN, Plummer FA, Anzala AO, et al. Resistance to HIV-1 infection among African sex workers is associated with global hyporesponsiveness in interleukin 4 production. FASEB J 2001; 15:17957.

51. 

Nicastri E, Sarmati L, Ercoli L, et al. Reduction of IFN- and IL-2 production by peripheral lymphocytes of HIV-exposed seronegative subjects. AIDS 1999; 13:13336.

52. 

Ghys PD, Diallo MO, Ettiegne-Traore V, et al. Increase in condom use and decline in HIV and sexually transmitted diseases among female sex workers in Abidjan, Côte d'Ivoire, 1991-1998. AIDS 2002; 16:2518.

53. 

Nkengasong JN, Maurice C, Koblavi S, et al. Field evaluation of a combination of monospecific enzyme-linked immunosorbent assays for type-specific diagnosis of human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections in HIV-seropositive persons in Abidjan, Ivory Coast. J Clin Microbiol 1998; 36:1237.

54. 

Nkengasong JN, Maurice C, Koblavi S, et al. Evaluation of HIV serial and parallel serologic testing algorithms in Abidjan, Côte d'Ivoire. AIDS 1999; 13:10917.

55. 

Yang C, Pieniazek D, Owen SM, et al. Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma by using highly sensitive and specific generic primers. J Clin Microbiol 1999; 37:25816.

56. 

Ghys PD, Fransen K, Diallo MO, et al. The associations between cervicovaginal HIV shedding, sexually transmitted diseases and immunosuppression in female sex workers in Abidjan, Côte d'Ivoire. AIDS 1997; 11:F8593.

57. 

Jennes W, Sawadogo S, Koblavi-Dème S, et al. Positive association between chemokineproducing T cells and HIV type 1 viral load in HIV-infected subjects in Abidjan, Côte d'Ivoire. AIDS Res Hum Retroviruses 2002; 18:1717.

58. 

Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci USA 1997; 94:192530.

59. 

Ostrowski MA, Justement SJ, Catanzaro A, et al. Expression of chemokine receptors CXCR4 and CCR5 in HIV-1infected and uninfected individuals. J Immunol 1998; 161:3195201.

60. 

Vuylsteke B, Ghys P, Traore M, et al. HIV prevalence and risk factors for infection among clients of female sex workers (FSWs) in Abidjan, Côte d'Ivoire [abstract ThOrC675]. In: Program and abstracts of the XIIIth International AIDS Conference (Durban, South Africa). Stockholm: International AIDS Society, 2000:380.

61. 

Van Damme L, Ramjee G, Alary M, et al. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 2002; 360:9717.

62. 

Yohou L, Ezoua J, Toutou T, et al. Prevalence of human immunodeficiency virus (HIV), infectious hepatitis B (HBV) and hepatitis C virus (HCV) among first time blood donors in Abidjan [abstract 11DT4-337]. In: Program and abstracts of the XIIth International Conference on AIDS and STDs in Africa (Ouagadougou, Burkina Faso). Society on AIDS in Africa, 2001:178.

63. 

Ezoua J, Sassan-Morokro M, Ekra A, et al. Trends in HIV prevalence among pregnant women attending urban antenatal clinics in Côte d'Ivoire, 19972000 [abstract 12PT5416]. In: Program and abstracts of the XIIth International Conference on AIDS and STDs in Africa (Ouagadougou, Burkina Faso). Society on AIDS in Africa, 2001:290.

64. 

Nkengasong JN, Kestens L, Ghys PD, et al. Human immunodeficiency virus type 1 (HIV-1) plasma virus load and markers of immune activation among HIV-infected female sex workers with sexually transmitted diseases in Abidjan, Côte d'Ivoire. J Infect Dis 2001; 183:14058. 

65. 

Shalekoff S, Tiemessen CT. Duration of sample storage dramatically alters expression of the human immunodeficiency virus coreceptors CXCR4 and CCR5. Clin Diagn Lab Immunol 2001; 8:4326. 

66. 

Hultin LE, Lua M, Hultin P, Hausner MA, Giorgi JV. Sample preparation induced variation in chemokines receptor measurements. Cytometry 1999; 38:333.

67. 

Su SB, Gong W, Grimm M, et al. Inhibition of tyrosine kinase activation blocks the down-regulation of CXC chemokine receptor 4 by HIV-1 gp120 in CD4⁺ T cells. J Immunol 1999; 162:712832. 

68. 

Fernandis AZ, Cherla RP, Chernock RD, Ganju RK. CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway. J Biol Chem 2002; 277:181117. 

69. 

Giovannetti A, Ensoli F, Mazzetta F, et al. CCR5 and CXCR4 chemokine receptor expression and -chemokine production during early T cell repopulation induced by highly active anti-retroviral therapy. Clin Exp Immunol 1999; 118:8794. 

70. 

Nicholson JK, Browning SW, Hengel RL, et al. CCR5 and CXCR4 expression on memory and naive T cells in HIV-1 infection and response to highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2001; 27:10515.

71. 

Jennes W, Koblavi-Dème S, Vuylsteke B, et al. HIV-1 coreceptor and intracellular -chemokine expression in HIV-exposed uninfected (EU) and HIV⁺ female sex workers (FSWs) in Abidjan, Côte d'Ivoire [abstract WePeA3995]. In: Program and abstracts of the XIIIth International AIDS Conference (Durban, South Africa). Stockholm: International AIDS Society, 2000:79.

72. 

Secchiero P, Zella D, Barabitskaja O, et al. Progressive and persistent downregulation of surface CXCR4 in CD4⁺ T cells infected with human herpesvirus 7. Blood 1998; 92:45218. 

73. 

Yasukawa M, Hasegawa A, Sakai I, et al. Down-regulation of CXCR4 by human herpesvirus 6 (HHV-6) and HHV-7. J Immunol 1999; 162:541722.

74. 

Ebert LM, McColl SR. Coregulation of CXC chemokine receptor and CD4 expression on T lymphocytes during allogeneic activation. J Immunol 2001; 166:48708.

75. 

Zhu T, Mo H, Wang N, et al. Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 1993; 261:117981.

76. 

Sheppard HW, Celum C, Michael NL, et al. HIV-1 infection in individuals with the CCR5-32/32 genotype: acquisition of syncytium-inducing virus at seroconversion. J Acquir Immune Defic Syndr 2002; 29:30713.

77. 

Naif HM, Cunningham AL, Alali M, et al. A human immunodeficiency virus type 1 isolate from an infected person homozygous for CCR532 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4. J Virol 2002; 76:311424.

78. 

Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion. Nat Genet 1997; 16:1003.

79. 

Koblavi S, Nkengasong JN, Maurice C. Immune activation markers and chemokine receptor 5 (CCR5) genotype in highly exposed but persistently seronegative female sex workers in Abidjan, Côte d'Ivoire [abstract 31147]. In: Program and abstracts of the XIIth International AIDS Conference (Geneva). Stockholm: International AIDS Society, 1998:524.