Sexual transmission of HIV-1 (HIV) occurs across a mucosal surface, most commonly the cervicovaginal mucosa of the female genital tract. The per-contact probability of male to female sexual transmission is relatively low (<1%), although this varies depending on the type of sex, status of the mucosal epithelium, viral load of the infected partner and endogenous properties of the seminal fluid [1–3]. Thus, in most cases of sexual exposure to HIV, the innate defences of the genital mucosa are sufficient to prevent acquisition. Although repeatedly HIV-exposed individuals may mount adaptive immune responses against HIV in the absence of established infection (reviewed in ), relatively few studies have examined the innate immune milieu of the female genital tract as it relates to HIV susceptibility and infection (reviewed in [5,6]).
Numerous antimicrobial peptides are present in the mucosal secretions that coat the vaginal and cervical epithelia. Although some of these innate molecules demonstrate anti-HIV activity in vitro [7–12], little is known about their in-vivo biological role in preventing HIV acquisition. The two main families of genital mucosal antimicrobial peptides are defensins and cathelicidins, both expressed by leukocytes and epithelial cells at several mucosal sites, including the female genital tract [13,14]. The defensins are further divided into the α-defensin and β-defensin subfamilies . The α-defensins human neutrophil peptides (HNP) 1-3 and the β-defensins 1-3 have been extensively investigated in relation to inhibition of HIV [11,12,16–20]. Both HNP-1-3 and human β defensin (HBD)-2-3 are able to inhibit HIV by a direct effect on the virion, as well as through target cell modification [11,12,20]. The cathelicidin LL-37 has been reported to inhibit HIV replication in vitro  and interestingly, LL-37 may induce the expression and release of α-defensins from neutrophils, demonstrating a link between these two peptides .
Other genital innate immune factors with HIV inhibitory capacity include the β-chemokine regulated upon activation normal T-cell expressed and secreted (RANTES)  and secretory leukocyte protease inhibitor (SLPI) [10,22]. These innate immune factors have been found at elevated levels in the genital tract of highly exposed, persistently seronegative (HEPS) individuals (reviewed in ). However, the high-risk sexual behavior that defines the HEPS phenotype means that these HEPS individuals often have a relatively high prevalence of other sexually transmitted infections (STIs), which may in turn induce inflammation and increased host production of antimicrobial peptides . Therefore, it is not clear whether increased levels of mucosal innate antimicrobial peptides are responsible for reduced HIV susceptibility, or whether they indicate STI-associated inflammation that would be expected to increase HIV susceptibility. Indeed, genital levels of RANTES in high-risk women have been associated with local inflammation and increased numbers of HIV-susceptible activated CD4+ T cells in the cervical epithelium .
In order to address these issues, we have assayed several innate immune molecules in the cervicovaginal fluid of high-risk female sex workers (FSWs), elucidated their association with genital coinfections and the ability of cervicovaginal fluid to neutralize HIV in vitro and finally examined their prospective association with HIV acquisition.
Material and methods
Study population and sample preparation
From 1998–2002, a randomized trial of antibiotic prophylaxis to prevent STIs including HIV infection was performed in a cohort of 466 Kenyan FSWs from Kibera, a slum area of Nairobi . At enrolment, all participants completed a well validated questionnaire regarding medical symptoms and sexual risk taking . After trial completion, 113 participants were included in a prospective, nested case–control study that examined the association between HIV-specific adaptive immune responses and HIV acquisition . This work has now been extended to examine the associations of mucosal innate factors. The study protocol was approved by institutional review boards at the Kenyatta National Hospital (Nairobi, Kenya), the University of Toronto (Toronto, Canada), the University of Manitoba (Winnipeg, Manitoba) and Karolinska Institutet (Stockholm, Sweden).
A cervicovaginal swab was collected at study enrolment with a cotton-tipped swab that was rotated 360° in the cervical os; a second swab was used to collect secretions from the posterior vaginal fornix. Both swabs were transferred into a single vial containing 5 ml of phosphate-buffered saline (PBS), transported to the laboratory on ice within 2 h, spun down at 1500 rpm for 5 min to remove cellular debris and cryopreserved at −80°C. IgA1 depletion of the samples was performed by adding 800 μl of undiluted cervicovaginal secretion (CVS) to 400 μl of jacalin-agarose beads (Immunkemi, Stockholm, Sweden), which was mixed for 2 h at +4°C followed by centrifugation (2000 rpm, 5 min, 4°C). The unbound (IgA1-depleted) fraction was collected and stored at −80°C; this fraction was used for the innate immune studies now described.
LL-37, human neutrophil peptide-1-3, human β defensin-2-3, secretory leukocyte protease inhibitor, regulated upon activation normal T-cell expressed and secreted, and IFNα quantification
Commercial enzyme-linked immunosorbent assay (ELISA) kits were used, according to the manufacturers protocols, to quantify the immune molecules of interest (additional CVS dilution in brackets): the α-defensins HNP-1-3 (1: 500), LL-37 (1: 10) (both Hycult biotechnology, Uden, the Netherlands), SLPI (1: 100–1: 1000) (RD Systems Europe Ltd, Abingdon Oxon, UK), the β-defensins HBD-2 (1: 10–1: 1000), HBD-3 (1: 10) (both Phoenix Pharmaceuticals, INC., Burlingame, California, USA), RANTES (1: 1) (Biosource International, Camaraillo, California, USA) and IFNα (1: 1–1: 10) (PBL Biomedical Laboratories, Piscataway, New Jersey, USA).
Clinical protocol and genital infection screening
At enrolment, all FSWs underwent a complete physical examination and STI testing and treatment . Cervical swabs were obtained for Neisseria gonorrhoeae and Chlamydia trachomatis polymerase chain reaction assays (Amplicor PCR Diagnostics, Roche Diagnostic Systems, Ontario, Canada) and for N. gonorrhoeae culture. Trichomonas vaginalis cultures were performed using In Pouch TV (Biomed Diagnostics, San Jose, California, USA). A vaginal Gram was performed; bacterial vaginosis was defined as a Nugent score of 7–10, and vaginal candidiasis as the finding of yeast on gram stain. Blood samples were obtained for HIV, syphilis and herpes simplex type 2 (HSV-2) serology. The rapid plasma reagin test (RPR test, Becton Dickinson, Groot-Bijgaarden, Belgium) was performed to diagnose syphilis, positive samples confirmed by Treponema pallidum haemagglutination assay (TPHA) (Randox Laboratories, Crumlin, UK). HIV screening ELISA was performed using the Detect-HIV kit (BioChem ImmunoSystems Inc, Montreal, Canada), positive tests confirmed with Recombigen HIV-1/HIV-2 EIA (Cambridge Biotech Corporation, Galway, Ireland). Testing for HSV-2 was performed using an HSV-2 IgG enzyme immunoassay (Kalon Biological Ltd, Aldershot, UK).
HIV neutralization assay
HIV neutralization assays were performed according to a predefined protocol and neutralization cut-off . Two R5 tropic primary isolates (clade A: 92RW024 and clade C: ZA003, both obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were collected from phytohemagglutinin (PHA-P) and IL-2 (both Sigma-Aldrich Sweden AB, Stockholm, Sweden) stimulated PBMC. The TCID50 value was determined and supernatants were aliquoted and stored at −80°C. As the TCID50 may differ between PBMC donors, at least two viral dilutions were used in each assay. The IgA1-depleted CVS were not further diluted or concentrated when tested for neutralizing capacity. Duplicate wells of 75 μl of each virus dilution and 75 μl of each sample fraction were incubated for 1 h at 37°C followed by addition of a mixture of 1 × 105 phytohemagglutinin P (PHA-P)-stimulated peripheral blood mononuclear cell (PBMC) from two to three donors. After 24 h incubation at 37°C, the cells were centrifuged, unbound virus was washed away and 200 μl of fresh RMPI 1640 medium (Invitrogen AB, Lidingö, Sweden) with bovine serum albumin and IL-2 were added to each well. On day 3, 120 μl of medium was discarded and replaced with new medium, and on day 6 supernatants were collected for analysis of virus production with a p24 antigen ELISA (Vironostika HIV-1 Antigen; Electra-Box Diagnostica AB, Stockhom, Sweden). As previously described , neutralization was defined as at least 67% reduction of p24 antigen in the supernatant as compared with p24 antigen content when the virus isolate was incubated in the presence of the low-risk HIV seronegative samples. Positive control samples (HIV IgG positive serum) were included in each assay.
All analyses were performed with SPSS version 16.0 software (Chicago, Illinois, USA). Samples with undetectable levels of innate factors were assigned a value at the assay cut-off. Levels were log10 transformed, and baseline (cross-sectional) associations of innate immune factors were examined: continuous variables were compared between groups using the independent samples t-test (after Levene's test for equality of variances) and categorical variables using Fisher's exact test. FSWs acquiring HIV during subsequent clinical follow-up (cases) were matched by an external biostatistician to controls who had remained HIV uninfected, as previously described . Matching was based on time of study enrolment, study arm (azithromycin vs. placebo) and (c) duration of HIV seronegative follow-up. Univariate associations of HIV acquisition were assessed in a matched case–control format, using Mantel–Haenszel analysis (dichotomous variables) or conditional logistic regression (continuous variables). Stratified multivariable analyses of the associations of HIV acquisition were performed by conditional logistic regression using a Cox proportional hazards model, with the inclusion of variables associated with HIV acquisition in univariate analysis (P ≤ 0.10). SPSS does not offer conditional logistic regression per se, and so these analyses were performed using an established adaptation of Cox regression to obtain equivalent results.
Levels of innate immune factors in the genital tract of female sex workers
The demographics and sexual risk taking profile of the 113 participants are outlined in Tables 1 and 2 . The CVS levels of innate immune molecules are reported as measured in the original 5 ml dilution. Median concentrations were: HNP-1-3: 255 ng/ml (n = 111, range 0.05–4658); HBD-2-3: 5 ng/ml (n = 112, range 0.16–220); LL-37: 13 ng/ml (n = 111, range 2–191); SLPI: 205 ng/ml (n = 109, range 15–602) and RANTES: 5 pg/ml (n = 112, range 5–2027) (Fig. 1). The most abundant β-defensin was HBD-2 (HBD-2: median 4 ng/ml, range 0.1–220 ng/ml; HBD-3 median 0.65 ng/ml, range 0.06–42 ng/ml, P < 0.0001). IFNα quantifications were performed in 50% of the CVS, and in all cases, levels were below the detection limit of the assay (10 pg/ml). The remaining study material was thus not analyzed. Previously reported minimum HIV inhibitory levels of these factors are: HNP1-3: 1000 ng/ml ; HBD2-3: 9000–20000 ng/ml ; LL-37: 50000 ng/ml ; SLPI: 100 ng/ml  and RANTES: 3 ng/ml .
Prevalent genital infections and the innate immune environment
Genital infections were common at enrolment (Table 1) and were correlated with increased levels of several innate immune molecules, including HNP-1-3, LL-37 and HBD-2-3 (Table 2). Infection by C. trachomatis and N. gonorrhoeae were associated with higher levels of LL-37 (P = 0.006 and P = 0.026, respectively), HNP-1-3 (P = 0.1 and P = 0.021, respectively) and HBD-2-3 (P = 0.027 and P = 0.023, respectively). Vaginal candidiasis was associated with higher levels of LL-37 and HBD-2-3 (P = 0.002 and P = 0.08, respectively); T. vaginalis infection was associated with an increased level of HBD-2-3 (P = 0.019). The presence of several simultaneous coinfections was associated with increased levels of both defensins and LL-37 (Fig. 2). Bacterial vaginosis was present in 59% of participants, but was not associated with differences in the level of any innate factors. No women had clinically evident genital ulceration, and neither HSV-2 nor syphilis seropositivity were associated with innate immune alterations (data not shown).
Levels of human neutrophil peptide-1-3 and LL-37 correlated with HIV neutralization capacity
To examine the anti-HIV capacities of the innate immune molecules, IgA1 antibodies were depleted prior to neutralization assays. Two R5-tropic primary isolates were used: clade A, representing the most common clade in Kenya and clade C, representing the most common clade globally. Although all women were HIV IgG seronegative, 23 of the 113 (20%) IgA1-depleted CVS samples could neutralize the clade A isolate, and 13 of the 113 (12%) could neutralize both the clade A and clade C isolates. Single clade neutralizing activity did not correlate with the measured levels of any innate immune molecules. However, those samples that could neutralize both the clade A and the clade C HIV primary isolates (n = 13), representing a more robust neutralizing response, had higher concentrations of HNP-1-3 (P = 0.007) and LL-37 (P = 0.002) (Table 3). HIV neutralizing capacity did not correlate with the presence of specific genital infections (data not shown).
Additive effect of innate immune factors on HIV neutralization
Levels of innate immune factors often fell below previously reported HIV inhibitory levels [7,8,10–12]. However, as large variations in innate immune factor levels were noted within our study cohort, we examined the possibility of additive HIV inhibitory effects in this context. Each level of innate immune factor dataset was divided into quartiles and assigned a score (0–3), and for each participant these scores were aggregated. The ability to neutralize HIV was strongly associated with a higher ‘innate aggregate score’ (Mann–Whitney, P = 0.003; Fig. 3), demonstrating an additive effect of innate antimicrobial peptides below in-vitro assay inhibitory levels.
Innate immune factors and HIV acquisition
Having defined the cross-sectional associations of these innate mucosal factors at the cohort enrolment visit, we then examined their association with subsequent HIV acquisition in a stratified case–control format, comparing levels in women who subsequently acquired HIV with matched controls (matched 1: 4) who remained HIV uninfected. Despite the association of HNP-1-3 and LL-37 with an increased HIV neutralizing ability of cervicovaginal secretions, in univariable analysis the levels of these innate factors tended to be increased in participants who subsequently acquired HIV [odds ratio (OR) 1.98; P = 0.074 and OR 2.63; P = 0.075, respectively)]. No associations were observed with levels of RANTES, SLPI or HBD-2-3 (OR 0.78; P = 0.46, OR 3.9; P = 0.12 and OR 0.76; P = 0.42, respectively). No individual bacterial STI at baseline was associated with HIV acquisition, and neither was a composite endpoint of ‘any STI’ [(9/24 cases vs. 35/89 controls (Mantel–Haenszel, OR 0.98; P = 0.97)].
Alcohol intake, IgA with HIV neutralizing capacity and HIV-specific T-cell proliferation were previously associated with differential HIV acquisition in this cohort . Therefore, we constructed stratified multivariable models that incorporated these factors, as well as levels of the innate immune factors LL-37 and HNP-1-3. The first model incorporated HNP-1-3 levels, HIV-neutralizing genital IgA, HIV-specific proliferation and alcohol intake; in this model, HNP-1-3 concentration was independently associated with increased HIV acquisition (adjusted OR 8.2; P = 0.02) without affecting the previously described immune/behavioral associations (all P < 0.05; data not shown). A second model incorporated LL-37 levels, HIV-neutralizing genital IgA, HIV-specific proliferation and alcohol intake; LL-37 was also independently associated with acquisition (adjusted OR 6.9; P = 0.05), although significance was lost if both these innate factors were incorporated into the model simultaneously.
The increased strength of the association between innate factors and HIV acquisition in the multivariable model implies interaction with other factors in the model; this was confirmed by the finding of reduced HNP1-3 levels in participants with genital HIV-neutralizing IgA (2.17 vs. 2.60; P = 0.03). All women acquiring HIV were HSV-2 infected, precluding the incorporation of HSV-2 into the multivariable model. In a stratified case–control analysis that included only HSV-2 infected participants, the association between HIV acquisition and both LL-37 and HNP-1-3 lost significance (P = 0.16 for both), likely due to reduced statistical power.
Numerous mucosal innate factors demonstrate anti-HIV activity in vitro [7–12], and several current microbicide candidates comprise analogues of such innate immune molecules, including RANTES , lactoferrin  and θ-defensins . Therefore, it will be essential to characterize baseline levels of naturally occurring innate immune molecules (not θ-defensins, which are not naturally occurring) in populations similar to those in which phase 3 efficacy trials may be performed, and to assess their ‘real world’ HIV inhibitory capacity in relevant mucosal samples. Levels of mucosal innate factors have been evaluated in low-risk patients , but few comprehensive studies have been also performed in high-risk populations [32–35].
We have now examined the levels of several innate immune molecules with anti-HIV activity in a large cohort of sex workers at a high risk of contracting both HIV and other STIs. The α-defensins HNP-1-3 and SLPI were plentiful, and the β-defensins HBD-2-3 and the cathelicidin LL-37 levels were lower but easily assayed. RANTES was only found in picogram levels, and IFNα was undetectable. Therefore, any in-vivo antiviral effect of the latter two factors may be more likely to be exerted at the level of the cervicovaginal epithelium or submucosa , rather than in the genital secretions. IgA1-depleted mucosal samples from a significant minority of women were able to neutralize relevant primary HIV isolates, and this neutralization capacity was significantly correlated with levels of HNP-1-3 and LL-37, confirming the HIV inhibitory capacity of these molecules. Additive effect of innate factors was observed, confirming previous findings , with enhanced HIV neutralization seen in samples with subinhibitory levels of multiple different innate factors.
However, we found that several STIs were associated with substantial increases in the mucosal concentration of innate immune factors with anti-HIV activity. This association makes the interpretation of cross-sectional studies very difficult in HEPS cohorts, and emphasizes the need for full coinfection diagnostics to allow for meaningful interpretation of results. The definition of the HEPS phenotype involves high-risk sexual activity, meaning that such individuals have a high STI prevalence. Therefore, increased levels of mucosal innate antimicrobial peptides in these HEPS individuals could be interpreted as a possible cause of immune protection against HIV, but might also be a secondary effect of STI-associated inflammation.
This means that mucosal innate molecules with anti-HIV activity in vitro could theoretically have either beneficial or detrimental effects in vivo. Effects of innate immune molecules include the blockade of virus entry and subsequent infection [17,18,20,29,38,39], and/or the augmentation of adaptive immune responses through the recruitment of immune cells [40,41]. As these recruited cells, including T cells, are also potential target cells for HIV, this could increase the risk of HIV acquisition. Whether the HIV inhibitory effects of these factors, either naturally occurring or exogenously applied, overrides the potentially negative effects of HIV susceptible target cell recruitment requires careful study. To explore these issues further, we performed a case–control study comparing levels of innate factors with anti-HIV activity between participants who did or did not subsequently acquire HIV. Unexpectedly, we found that higher baseline genital levels of both the α -defensins HNP-1-3 and the cathelicidin LL-37 were associated with HIV acquisition, independent of the behavioral and immune correlates of HIV acquisition previously defined in this cohort .
These findings certainly do not prove that these mucosal innate factors directly enhanced HIV acquisition. Levels were only measured once, at cohort enrolment, with no subsequent assays performed closer to the time of HIV acquisition, making it difficult to define causation. More importantly, during prospective follow-up in the clinical trial in which this immune study was nested, a bacterial STI (N. gonorrhoeae or C. trachomatis or both) in the past 3 months was associated with HIV acquisition in time-dependent analysis . Therefore, it is perhaps more plausible that higher levels of these genital innate factors at enrolment identified a subgroup of FSWs with increased rates of genital infections, and it were the infections (rather than the innate immune factors) that were responsible for increased HIV susceptibility. We did not incorporate bacterial STIs into our multivariable model based on enrolment parameters, as the presence or absence of an STI at the enrolment visit was not associated with HIV acquisition. Nonetheless, the possibility that these innate factors may have directly caused increased HIV susceptibility, either through local enhancement of HIV replication  or through recruitment of HIV susceptible target cells , must be considered. Given the potential application of innate immune factor analogues as microbicide candidates, these issues urgently require further study.
Bacterial vaginosis is a prevalent and often recurrent imbalance of the vaginal bacterial flora that has been associated with an increased susceptibility to HIV and other STIs  and with decreased levels of defensins and SLPI . Bacterial vaginosis was common in our study population, but was not associated with altered levels of any innate factors. The reasons for this discrepancy are not immediately clear, although participants in the prior study were generally at a low risk for HIV acquisition, and those with other STIs were excluded . As bacterial vaginosis predisposes to several STIs, including C. trachomatis and T. vaginalis  and as these STIs were associated with increased levels of genital innate factors in our population, this may have reduced our power to detect any bacterial vaginosis associated reductions. This re-emphasizes the importance of performing mucosal immune studies in different target populations.
An array of innate immune molecules in the cervicovaginal mucosa constitutes the first line of immune defense against the sexual acquisition of HIV by women. Our present study confirms that high levels of several innate immune factors are present in the cervicovaginal secretions, and correlate with HIV neutralizing capacity. However, their levels are strongly influenced by local factors, particularly concomitant STIs, and this confounds the ability to assess their in-vivo role in protection against HIV acquisition. Neither the level nor the HIV inhibitory capacity of innate immune factors in the cervicovaginal secretions can be assumed to be a direct reflection of mucosal HIV susceptibility. Careful prospective studies are urgently needed to define the influence of these and other innate immune factors on natural protection against HIV infection.
We acknowledge the substantial contribution of the late Job J. Bwayo (Department of Medical Microbiology, University of Nairobi, Kenya) to this work. We thank Nico Nagelkerke for statistical support; Klara Hasselrot for scientific advice and Mia Ehnlund for technical support. In addition, we acknowledge the support of the Nairobi City Council, and give special thanks to all the Kibera study participants for their enthusiasm and support. T.H. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
The Kibera HIV Study Group: Dr Florence Keli, Dr Isaac Malonza, Dr Francis Mwangi, Dr Karolien Fonck, Dr Marleen Temmerman, Dr Allan R. Ronald.
Authors' contributions: Study concept and design: T.H., R.K., K.B., P.L., S.M., J.K., E.N. and K.S.M.. Analysis and interpretation: T.H., R.K., K.B., P.L.. Drafting of manuscript: T.H., R.K., K.B., P.L.. Critical review of manuscript: T.H., R.K., K.B., P.L., S.M., J.K., E.N., K.S.M.. Study supervision: T.H., R.K., K.B., P.L., S.M., J.K., E.N., K.S.M.. Obtained funding: T.H., R.K., K.B, K.S.M..
The authors do not have a commercial or other association that might pose a conflict of interest.
Financial support: The study was supported by grants from the Rockefeller Foundation (2000 HE 025); the European Commission (DG VIII/8, Contract No. 7-RPR-28); the Canadian Research Chair Program (R.K.); Ontario HIV Treatment Network (K.S.M.; Career Scientist and ROG); the Canadian Institutes of Health Research (R.K.; HET-85518 and S.M.; Investigator Award); SIDA/SAREC (K.B., T.H.) and the Swedish Research Council (K.B.).
Part of this work has previously been presented at the XVII International AIDS conference in Mexico City, 3–8 August 2008.
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