Women in sub-Saharan Africa have a higher HIV prevalence than men, and a 80–300 times higher HIV-1 incidence than women in the United States [1–3]. These geographic and sex differences may be multifactorial [4,5], but it is known that mucosal immune activation resulting from genital tract infection or the application of nonoxynol-9 increases mucosal expression of the HIV coreceptor C–C chemokine receptor type 5 (CCR5) , the number of activated CD4+ T cells [7,8], and the levels of proinflammatory cytokines [9,10].
We hypothesized that regional differences in HIV acquisition by young women could be partly due to differences in the genital tract immune milieu, particularly because increased systemic immune activation has been seen in individuals from sub-Saharan Africa . We examined this question in the context of a phase 1 microbicide safety trial with sites in Kenya and the United States, in which all participants had undergone screening for a wide array of genital coinfections. Specifically, we hypothesized that young Kenyan women without genital infections would have increased mucosal-activated CD4+ T cells and proinflammatory cytokines relative to a similar US population, leading to greater HIV susceptibility.
We conducted a cross-sectional study of women at enrollment into a phase 1, placebo-controlled, randomized, double-blind clinical trial in sexually abstinent young women in San Francisco, USA, and Kisumu, Kenya. This trial is registered at www.ClinicalTrials.gov (NCT00331032).
Selection of participants
Participants provided informed consent, and all women enrolled in the trial were included in this substudy. Eligible participants were 18–24 years old, in good health, sexually experienced, sexually abstinent 1 week prior to enrollment, not breastfeeding or pregnant, not within 3 months of last pregnancy, and had regular menstrual cycles of at least 25 days. At screening, women were tested for genital infections and pregnancy. Women who had any of the following were excluded: positive test for human chorionic gonadotropin, urinary tract infection, HIV antibodies, herpes simplex virus (HSV)-2 antibodies, syphilis, vaginal candidiasis, symptomatic bacterial vaginosis, a Nugent score of at least 7 , trichomonas, gonorrhea, chlamydia, abnormal cervical cytology, more than two vaginal infections in the last year, an uncontrolled medical condition or recent acute illness, a recent new systemic or topical medication, or any vaginal product 30 days prior to enrollment. All participants were enrolled between the 5th and 14th day of the menstrual cycle. A pelvic examination, including colposcopy, vaginal pH, Gram stain, and vaginal wet mount, was performed. Vaginal specimens were collected for prostate-specific antigen (PSA) testing . A cervical cytobrush was collected and placed into 5 ml of cellular transport medium (Roswell Park Memorial Institute with 10% fetal bovine serum), stored at 4°C, and transported to the laboratory on ice. A cervicovaginal lavage (CVL) was performed with 5 ml of phosphate-buffered saline, reaspirated, and transported to the laboratory on ice. All samples used were collected prior to administration of the vaginal microbicide.
Cervical samples were tested for 37 human papillomavirus (HPV) genotypes and beta-globin (Roche Molecular Diagnostics, Inc., Alameda, California, USA) . Cervical cells from cryopreserved specimens stored at −150°C were stained in following two aliquots : one with CD69-FITC, CCR5-PE, CD4-PerCP, and CD3-APC (BD Pharmingen, San Jose, California, USA); and the other with CD1a (Imgenex, San Diego, California, USA), CD11c, CD14, and DC-SIGN (eBioscience, San Diego, California, USA). Samples were acquired by FACSCalibur (Becton-Dickinson Immunocytometry Systems). Cell numbers were multiplied by two to determine ‘cells per cytobrush’ and log10 transformed for analysis.
Cytokines were assayed in thawed CVL using the LINCOplex High-Sensitivity Human Cytokine Immunoassay Kit (Millipore, Billerica, Massachusetts, USA) and the Luminex-100 platform (Luminex, Austin, Texas, USA). Secretory leukocyte protease inhibitor (SLPI) was measured by ELISA (Quantikine Human SLPI kit; R&D Systems, Inc., Minneapolis, Minnesota, USA).
Site-specific distributions of cell counts, SLPI, and cytokine values were summarized via median and interquartile range values. Wilcoxon rank-sum tests were used to test factors by site. The proportions of immune cells were defined as the percentage of a specific cell phenotype (e.g. CD4 T cells) that expressed a cell surface ligand(s) (e.g. CD69). Proportions were compared between sites using negative binomial regression. Predictor variables for regression models were selected based on biological plausibility for associations with immune outcomes and observed associations with study site. Variables significantly associated with study site at the 10% significance level were considered as candidates for adjustment. Linear regression models were used to investigate the possible confounding influence of these variables.
Enrollment and participant disposition
A total of 54 participants were enrolled, 18 from San Francisco and 36 from Kisumu (Table 1). Women in Kisumu were younger, less likely to use oral contraceptives, had fewer lifetime number of sex partners, and were less likely to report anal sex and a reproductive tract infection. No differences were seen for recent number of sex partners. No participants had bacterial vaginosis at screening, but by the enrollment visit (up to 30 days later) five (14%) and four (11%) participants from Kisumu had asymptomatic bacterial vaginosis or intermediate flora, respectively, compared with no participant from San Francisco (Table 1). HPV was detected in 11 (33%) and six (35%) women in Kisumu and San Francisco, respectively. No participant had PSA detected (≥1 ng/ml), indicating a lack of recent (within 2 days) semen exposure .
Flow cytometry results
The total number of cervical CD4+ T lymphocytes was slightly higher in women from San Francisco than from Kisumu (304 vs. 187 per cytobrush, P = 0.05). However, the number and proportion of CD4+ cells expressing CD69+, with or without CCR5+, was substantially higher among women from Kisumu (both P < 0.0001, Table 2). Similarly, although the number of CD8+ T lymphocytes tended to be greater in women from San Francisco, the proportion of cervical CD8+ T cells expressing CD69+ was higher in Kisumu (Table 2). The number of CD1a+, CD11c+, and CD14+ cells, but not the proportions expressing DC-SIGN+, were greater in women from San Francisco (Table 2).
In multivariate analysis, controlling for age, age at first sex, and lifetime and past 3-month number of sex partners did not significantly affect the association between site and flow cytometry results (data not shown). As HPV infection could putatively influence genital immunology , we controlled for HPV status in multivariate analysis. The number and proportion of CD4+/CD69+ (P < 0.007, P = 0.003, respectively) and CD4+/CD69+/CCR5+ (P = 0.02, P < 0.002, respectively), and proportion of CD8+/CD69+ (P < 0.003) remained statistically greater in women from Kisumu compared with San Francisco participants.
Current use of combined oral contraceptives was not associated with differences in cervical cell populations in San Francisco participants, the only site where participants reported use of combined oral contraceptives (data not shown). In Kisumu, altered vaginal flora (Nugent score 4–10) was associated with decreased cervical CD1a+ (P = 0.01) and CD1a+/DC-SIGN+ (P = 0.02) cell numbers, but not with other differences. Neither the current use of combined oral contraceptives (San Francisco site only) nor abnormal vaginal flora (Kisumu site only) significantly affected the overall study findings of greater T-cell activation in the cervix of young women from the Kisumu site.
Cervicovaginal levels of secretory leukocyte protease inhibitor and cytokines
The median CVL concentration of SLPI was significantly lower in participants from Kisumu than from San Francisco (190 vs. 474 pg/ml, P = 0.009, Table 2); SLPI concentration was not associated with abnormal vagina flora (170 vs. 196 pg/ml, P = 0.83) at the Kisumu site. No consistent differences were seen in cytokine/chemokine levels between the two sites, although levels of interleukin (IL)-2 (P < 0.02) were higher in Kisumu participants, and IL-8 (P < 0.04) and granulocyte colony-stimulating factor (GM-CSF) (P < 0.004) were higher in San Francisco participants (Table 2). At the San Francisco and Kisumu sites, GM-CSF was below the limit of detection (BLD) in five (28%) and 23 (64%) patients and IL-2 was BLD in 12 (67%) and 14 (39%) patients, respectively. When these were dichotomized as ‘detectable’ or ‘not detectable’, both IL-2 (P < 0.05) and GM-CSF (P < 0.01) remained associated with site.
In this study, young healthy women from Kenya had a higher number and proportion of activated endocervical CD4+ T cells than women from the United States. As a critical mass of activated CD4+ T cells at the site of HIV exposure may be essential for local HIV propagation and subsequent systemic dissemination after sexual exposure , this might explain, in part, the increased risk of HIV acquisition in women from sub-Saharan Africa [1–3]. Although it has been suggested that reasons for the discrepancy in HIV seroprevalence include higher prevalence of sexually transmitted infections (STIs), specifically HSV-2 [18,19], as well as structural and sociocultural factors [4,5], for the first time our observations suggest that differences in the genital tract immune milieu may be an important additional contributor. The cellular findings were somewhat surprising as we also observed an approximately two-fold increased concentration of IL-8, a proinflammatory chemokine , in the CVL of participants in San Francisco, suggesting that these observations were independent. In addition, we found that the young women from Kisumu had lower cervicovaginal levels of SLPI. SLPI, an innate protein, has been shown to protect activated CD4+ T cells from HIV infection in vitro , and is reduced in bacterial vaginosis  and following application of nonoxynol-9 .
The mechanism underpinning this increase in activated genital CD4+ T cells will be an important topic for future investigations. A prior study  has shown increased systemic immune activation in several African populations, but to our knowledge, this is the first to compare the genital immune milieu between African and US participants. Although we controlled for confounders such as genital coinfections, sexual behavior, menstrual phase, and hormonal contraception, it may be that genital immune activation stemmed from differences in host genetics, or from chronic infections that are systemic (e.g. malaria) or affect other mucosal sites (e.g. helminths, gastroenteritis, or schistosomiasis) and are more prevalent in sub-Saharan Africa . In theory, infections at nongenital mucosae such as the gut may not only recruit activated, antigen-specific CD4+ T cells to that site but also to other mucosal surfaces such as the genital tract, perhaps through expression of the mucosal homing receptor integrin α4β7 that is expressed at both sites [23,24]. The cause of reduced SLPI concentrations is less clear, although disturbances in the genital tract flora may affect its concentration .
Although all flow cytometry was conducted at a single laboratory, it is important to consider whether sampling differences between sites might have contributed to the observed differences, particularly given that the overall numbers of most cervical cell types were actually higher at the San Francisco site. However, standard operating procedures were used to collect, cryopreserve, and ship the specimens to the central laboratory, and training at the clinical sites was performed by the same investigator.
Although other factors such as physical, behavioral, and structural conditions certainly put young women in sub-Saharan African at greater risk of HIV acquisition, ours is the first study to demonstrate that mucosal immune differences in the genital tract may also be important. In addition to stimulating further research, we believe that these findings should also start to dispel some of the preconceptions and stigma surrounding HIV acquisition among young women in sub-Saharan Africa.
This study was supported by the STI Clinical Trials Group [National Institutes of Allergy and Infectious Diseases Division of Microbiology and Infectious Diseases (NIAID-DMID) HHSN266200400074C]. In addition, this study was supported by National Institutes of Health (NIH)/NCRR UCSF-CTSI grant number UL1 RR024131 and the Canada Research Chair Program (R.K., salary support). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIH. Information on NCRR is available at http://www.ncrr.nih.gov/. Information on Re-engineering the Clinical Research Enterprise can be obtained from http://nihroadmap.nih.gov/clinicalresearch/overview-translational.asp.
We would like to thank Jonathan Glock, Carolyn Deal, Shacondra Brown, the VivaGel study teams in San Francisco and in Kisumu, and Sanja Huibner for technical assistance; the collaborating laboratories, the STI Clinical Trials Group, NIAID-DMID, and the study participants for volunteering their time and information; and the Director of Kenya Medical Research Institute.
1. Centers for Disease Control and Prevention. Subpopulation estimates from the HIV incidence surveillance system: United States, 2006
. MMWR Morb Mortal Wkly Rep
2. Barnighausen T, Wallrauch C, Welte A, McWalter TA, Mbizana N, Viljoen J, et al
. HIV incidence in rural South Africa: comparison of estimates from longitudinal surveillance and cross-sectional cBED assay testing. PLoS One 2008; 3:e3640.
3. Morrison CS, Richardson BA, Mmiro F, Chipato T, Celentano DD, Luoto J, et al
. Hormonal contraception and the risk of HIV acquisition. AIDS 2007; 21:85–95.
4. Bearinger LH, Sieving RE, Ferguson J, Sharma V. Global perspectives on the sexual and reproductive health of adolescents: patterns, prevention, and potential. Lancet 2007; 369:1220–1231.
5. Pettifor AE, van der Straten A, Dunbar MS, Shiboski SC, Padian NS. Early age of first sex: a risk factor for HIV infection among women in Zimbabwe. AIDS 2004; 18:1435–1442.
6. Poles MA, Elliott J, Taing P, Anton PA, Chen IS. A preponderance of CCR5(+) CXCR4(+) mononuclear cells enhances gastrointestinal mucosal susceptibility to human immunodeficiency virus type 1 infection. J Virol 2001; 75:8390–8399.
7. Patterson BK, Landay A, Siegel JN, Flener Z, Pessis D, Chaviano A, Bailey RC. Susceptibility to human immunodeficiency virus-1 infection of human foreskin and cervical tissue grown in explant culture. Am J Pathol 2002; 161:867–873.
8. Zhu J, Hladik F, Woodward A, Klock A, Peng T, Johnston C, et al
. Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nat Med 2009; 15:886–892.
9. Cohen CR, Plummer FA, Mugo N, Maclean I, Shen C, Bukusi EA, et al
. Increased interleukin-10 in the endocervical secretions of women with nonulcerative sexually transmitted diseases: a mechanism for enhanced HIV-1 transmission? AIDS 1999; 13:327–332.
10. Fichorova RN, Tucker LD, Anderson DJ. The molecular basis of nonoxynol-9-induced vaginal inflammation and its possible relevance to human immunodeficiency virus type 1 transmission. J Infect Dis 2001; 184:418–428.
11. Clerici M, Butto S, Lukwiya M, Saresella M, Declich S, Trabattoni D, et al
. Immune activation in Africa is environmentally-driven and is associated with upregulation of CCR5. Italian–Ugandan AIDS Project. AIDS 2000; 14:2083–2092.
12. Novak RM, Donoval BA, Graham PJ, Boksa LA, Spear G, Hershow RC, et al
. Cervicovaginal levels of lactoferrin, secretory leukocyte protease inhibitor, and RANTES and the effects of coexisting vaginoses in human immunodeficiency virus (HIV)-seronegative women with a high risk of heterosexual acquisition of HIV infection. Clin Vaccine Immunol 2007; 14:1102–1107.
13. Chen MP, Macaluso M, Blackwell R, Galvao L, Kulczycki A, Diaz J, et al
. Self-reported mechanical problems during condom use and semen exposure. Comparison of two randomized trials in the United States of America and Brazil. Sex Transm Dis 2007; 34:557–562.
14. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A, et al
. Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000; 38:357–361.
15. Rebbapragada A, Wachihi C, Pettengell C, Sunderji S, Huibner S, Jaoko W, et al
. Negative mucosal synergy between Herpes simplex type 2 and HIV in the female genital tract. AIDS 2007; 21:589–598.
16. Moscicki AB, Hills N, Shiboski S, Powell K, Jay N, Hanson E, et al
. Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females. JAMA 2001; 285:2995–3002.
17. Li Q, Estes JD, Schlievert PM, Duan L, Brosnahan AJ, Southern PJ, et al
. Glycerol monolaurate prevents mucosal SIV transmission. Nature 2009; 458:1034–1038.
18. Abu-Raddad LJ, Magaret AS, Celum C, Wald A, Longini IM Jr, Self SG, Corey L. Genital herpes has played a more important role than any other sexually transmitted infection in driving HIV prevalence in Africa. PLoS One 2008; 3:e2230.
19. van de Wijgert JH, Morrison CS, Brown J, Kwok C, Van Der Pol B, Chipato T, et al
. Disentangling contributions of reproductive tract infections to HIV acquisition in African Women. Sex Transm Dis 2009; 36:357–364.
20. Trifonova RT, Bajpai M, Pasicznyk JM, Chandra N, Doncel GF, Fichorova RN. Biomarkers of leukocyte traffic and activation in the vaginal mucosa. Biomarkers 2007; 12:608–622.
21. McNeely TB, Dealy M, Dripps DJ, Orenstein JM, Eisenberg SP, Wahl SM. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting antihuman immunodeficiency virus 1 activity in vitro. J Clin Invest 1995; 96:456–464.
22. Stuckler D, King L, Robinson H, McKee M. WHO's budgetary allocations and burden of disease: a comparative analysis. Lancet 2008; 372:1563–1569.
23. Arthos J, Cicala C, Martinelli E, Macleod K, Van Ryk D, Wei D, et al
. HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol 2008; 9:301–309.
24. Cicala C, Martinelli E, McNally JP, Goode DJ, Gopaul R, Hiatt J, et al
. The integrin α 4 β 7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to HIV-1 infection. Proc Natl Acad Sci U S A