aClinical Science Division, Department of Medicine, University of Toronto, Toronto, Ontario, Canada
bDepartment of Medical Microbiology, University of Nairobi, Nairobi, Kenya
cDepartment of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
dDepartment of Medicine, University Health Network, Canada
eOntario Public Health Laboratories, Ministry of Health and Long Term Care, Toronto, Ontario, Canada.
Correspondence to Dr Rupert Kaul, Clinical Science Division, #6356 Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S1A8, Canada. Tel: +1 416 978 8607; fax: +1 416 978 8765
Despite the scale of the HIV pandemic, mucosal innate immune defenses prevent HIV infection after most sexual exposures. Several soluble immune factors with in-vitro HIV inhibitory activity are present at the genital mucosa in physiologically relevant concentrations, including secretory leukocyte protease inhibitor (SLPI), defensins and regulated upon activation, normal T cell expressed and secreted (RANTES) . However, the impact of such factors on in-vivo HIV susceptibility has been poorly defined . A better understanding of the mucosal immune correlates of HIV susceptibility is needed, particularly as several potential microbicide candidates with in-vitro HIV inhibitory activity have been unexpectedly associated with an increased HIV incidence in phase 3 trials . Genital RANTES levels are increased in HIV-exposed, persistently seronegative (HEPS) women [4–6], suggesting a possible intermediate endpoint for reduced HIV susceptibility. However, RANTES is produced by activated immune cells including HIV-susceptible CD4+ T cells, and levels are increased in conditions such as bacterial vaginosis that may increase HIV susceptibility . Therefore, we examined the correlation between genital levels of soluble immune factors with HIV inhibitory activity, including RANTES, and cervical immune cell populations in high-risk, HIV-uninfected women.
Participants were enrolled through a female sex worker (FSW) clinic in Nairobi, Kenya . Informed consent was obtained, and the protocol was approved by Research Ethics Boards at the University of Toronto, the University of Manitoba and Kenyatta National Hospital (Nairobi, Kenya). A cervicovaginal lavage (CVL), cervical cytobrush specimen and scraping of the external cervical orifice (Benzi Jinshuo Applicator Co., Liaoning, China) were collected, and diagnostic testing performed for HIV, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, syphilis, herpes simplex type 2 (HSV-2) infection/shedding, cytomegalovirus infection/shedding and bacterial vaginosis . All infections were treated according to Kenyan national guidelines. Cervical samples were filtered, washed and stained with a panel of dendritic cell and T-cell markers, including CD69-FITC, CCR5-PE, CD3-PerCP, CD4-APC and CD1a-FITC, (BD Pharmingen, San Jose, California, USA); TLR9-PE (Imgenex, San Diego, California, USA); DC-SIGN-APC (eBioscience, San Diego, California, USA); and isotype controls. Populations were enumerated by flow cytometry. Levels of RANTES and other cytokines/chemokines were assayed by Cytokine Bead Array (CBA; BD Biosciences, San Diego, California, USA). SLPI levels were measured by enzyme-linked immunosorbent assay (Quantikine Human SLPI kit, R&D Systems, Minneapolis, Minnesota, USA).
Fifty-five HIV-uninfected FSWs participated in the study. RANTES was detectable in the CVL of 51 out of 55 participants (93%). The median RANTES level was 12.9 pg/ml (range 0–743.3 pg/ml). There was a strong positive correlation between RANTES levels and the number of cervical CD4 T cells (r = 0.53; P = 0.00006; Fig. 1a), cervical CD4+ T cells expressing the HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5) (r = 0.31; P = 0.028), cervical CD8+ T cells (r = 0.52; P = 0.00005), immature dendritic cells (iDCs; r = 0.38; P = 0.006; Fig. 1b), and iDCs expressing the TLR9 receptor (r = 0.40; P = 0.004). Genital RANTES levels also correlated with increased pro-inflammatory cytokines [IL1, IL6, IL8 and tumor necrosis factor (TNF)-alpha; all P ≤ 0.01] and SLPI (r = 0.34; P = 0.018).
Classical sexually transmitted infections (STIs) were uncommon (N. gonorrheae, n = 1; C. trachomatis, n = 0; syphilis, n = 2; T. vaginalis, n = 4) and were not associated with differences in RANTES levels (data not shown). Although most participants were HSV-2 infected (42/55, 76%), RANTES levels did not vary with HSV-2 infection status (1.14 log10 pg/ml, infected vs. 1.28, uninfected; P = 0.3). HSV-2 shedding was not detected in any participants. Bacterial vaginosis was present in 15 out of 33 participants with an available Gram stain (45%). Genital RANTES levels tended to be higher in bacterial vaginosis (1.24 vs. 0.95 log10 pg/ml; P = 0.054), and were positively correlated with the Nugent score (r = 0.42; P = 0.016).
Low rates of HIV acquisition after sexual exposure complicate the performance of HIV microbicide trials , and compounds with in-vitro HIV inhibitory activity may actually increase HIV susceptibility . Well validated mucosal immune correlates of HIV susceptibility would permit the establishment of much-needed intermediate endpoints for microbicide safety and efficacy studies , but these correlates remain poorly defined. We demonstrate that increased genital levels of RANTES, a soluble immune factor with demonstrated HIV suppressive activity, could actually reflect an increase in HIV susceptibility, as indicated by the number of HIV-susceptible target cells present in the cervical mucosa. Therefore, considerable caution must be exercised when using genital levels of this or other soluble immune factors to make assumptions regarding HIV susceptibility. Previous studies demonstrating increased RANTES levels in the genital tract of HEPS women were not able to control for the increased prevalence of bacterial vaginosis and/or other genital coinfections that may result from the high-risk sexual practices themselves, rather than representing a mechanism of immune protection from HIV acquisition during unprotected sex. Future studies of genital innate immune factors and HIV susceptibility should control for the effects of a wide array of genital infections, including classical bacterial STIs and HSV2, and for disturbances in the vaginal flora.
Jane Kamene and the Pumwani clinic nurses for their clinical assistance; Ann Maingi, Nyakio Chinga and the laboratory staff at the University of Nairobi Microbiology Annex for specimen processing and performing diagnostic assays; the women of the Pumwani cohort for their continued participation and support of our studies.
Canadian Institutes of Health Research (R.K.; HOP-75350 and HET-85518); the Bill and Melinda Gates Foundation (F.A.P.; BMG-77515) and Canadian Institutes of Health Research Grand Challenges in Global Health (F.A.P.; #37873); National Institutes of Health (F.A.P.; R01 AI056980); Canada Research Chair Programme (R.K. and F.A.P.).
R.K., A.R., T.H., C.W., T.B.B., F.A.P., J.K., and W.J. designed and planned the experiments. R.K. and F.A.P. obtained the funding. Recruitment, sampling and immune assays were done by A.R. and C.W. Data analysis was done by R.K. and A.R. Initial manuscript draft was prepared by R.K. Manuscript revisions were done by R.K., A.R., T.H., C.W., T.B.B., F.A.P., J.K. and W.J.
These data were presented in part at the 15th Conference on Retroviruses and Opportunistic Infections, Boston (February 2008).
1. Spear GT, Sha BE, Saarloos MN, Benson CA, Rydman R, Massad LS, et al
. Chemokines are present in the genital tract of HIV-seropositive and HIV-seronegative women: correlation with other immune mediators. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 18:454–459.
2. Iqbal SM, Kaul R. Mucosal innate immunity as a determinant of HIV susceptibility. Am J Reprod Immunol 2008; 59:44–54.
3. van de Wijgert JH, Shattock RJ. Vaginal microbicides: moving ahead after an unexpected setback. AIDS 2007; 21:2369–2376.
4. Hirbod T, Nilsson J, Andersson S, Uberti-Foppa C, Ferrari D, Manghi M, et al
. Upregulation of interferon-a and RANTES in the cervix of HIV-1-seronegative women with high-risk behavior. J Acquir Immune Defic Syndr 2006; 43:137–143.
5. Iqbal SM, Ball TB, Kimani J, Kiama P, Thottingal P, Embree JE, et al
. Elevated T cell counts and RANTES expression in the genital mucosa of HIV-1-resistant Kenyan commercial sex workers. J Infect Dis 2005; 192:728–738.
6. 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.
7. 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.
8. Feldblum PJ, Adeiga A, Bakare R, Wevill S, Lendvay A, Obadaki F, et al
. SAVVY vaginal gel (C31G) for prevention of HIV infection: a randomized controlled trial in Nigeria. PLoS ONE 2008; 3:e1474.
9. Keller MJ, Herold BC. Impact of microbicides and sexually transmitted infections on mucosal immunity in the female genital tract. Am J Reprod Immunol 2006; 56:356–363.