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JAIDS Journal of Acquired Immune Deficiency Syndromes:
doi: 10.1097/QAI.0b013e31823f2aeb
Clinical Science

Measurement of Mucosal Biomarkers in a Phase 1 Trial of Intravaginal 3% StarPharma LTD 7013 Gel (VivaGel) to Assess Expanded Safety

Moscicki, Anna-Barbara MD*; Kaul, Rupert PhD; Ma, Yifei MS*; Scott, Mark E. MD*; Daud, Ibrahim I. MS; Bukusi, Elizabeth A. MBChB, MMed (ObGyn), MPH, PhD; Shiboski, Stephen PhD§; Rebbapragada, Anuradha PhD; Huibner, Sanja BSc; Cohen, Craig R. MD, MPH

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Author Information

*Department of Pediatrics, University of California, San Francisco, CA

Departments of Medicine and Immunology, University of Toronto, Ontario, Canada

Center for Microbiology Research, Kenya Medical Research Institute, Nairobi, Kenya

§Department of Biostatistics, University of California, San Francisco, CA

Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, CA

Correspondence to: Anna-Barbara Moscicki, MD, University of California, San Francisco, 3333 California Street, Suite 245, San Francisco, CA 94118 (e-mail: moscickia@peds.ucsf.edu).

Supported by the sexually transmitted infection Clinical Trials Group (National Institutes of Allergy and Infectious Diseases Division of Microbiology and Infectious Diseases HHSN266200400074C). In addition, this study was supported by National Institutes of Health/NCRR University of California, San Francisco-CTSI grant number UL1 RR024131, NIH R37 CA51323-21 (A.B.M., salary support), and the Canada Research Chair Program (R.K., salary support).

The 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.

The authors have no conflicts of interest to disclose.

Received May 18, 2011

Accepted October 25, 2011

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Abstract

Objective: The aim of this study was to examine the effect of the 3% StarPharma LTD 7013 gel (VivaGel) on mucosal immune markers hypothesized to be associated with HIV-1 acquisition.

Design: Phase 1, placebo-controlled, randomized, double-blind clinical trial was performed in 54 young women in the United States and Kenya. Participants used carbopol gel with and without (placebo) StarPharma LTD 7013 twice daily over 14 days. Cervical specimens were collected for cytokines, chemokines, T cells, and dendritic cells at days 0, 7, 14, and 21. A negative binomial regression model was used to assess differences between study arms.

Results: Several mucosal immune parameters were increased in the VivaGel arm compared with placebo. For cytokines D7, IL-6 (P = 0.05); D 14, interferon gamma (P = 0.03), IL-2 (P = 0.04), IL-5 (P = 0.003), and IL-10 (P = 0.001) were increased. On D7, CD8+/CD69+ T cells tended to be increased (P < 0.08); limiting analysis to visits without blood or bacterial vaginosis, these findings were stronger as follows: at D7, CD8+/CD69+ T cells were increased in the VivaGel arm (P < 0.005), as were CD4+/CD69+ cells (P = 0.001) and CD4+/CCR5+ T cells (P = 0.01). The changes described for D7 and 14 were no longer seen at D21.

Conclusions: Markers associated with inflammation and epithelial damage were reversibly elevated in the VivaGel arm compared with the placebo arm after 7–14 days of twice daily product use.

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INTRODUCTION

The HIV pandemic has motivated the research community to develop self-applied vaginal microbicides. An ideal vaginal microbicide would prevent HIV and other sexually transmitted infections (STIs), be safe, acceptable, and affordable. However, defining such products as safe has proven difficult. Several microbicides that were determined to be safe in phase 1/2 trials based on clinical criteria were subsequently found to increase the risk of HIV acquisition in phase 2 and 3 clinical trials. One example is the trial examining the spermicide nonoxynol-9 (N-9), which was thought to be a safe product until 1 trial showed N-9 resulted in an increased risk of acquiring HIV.1

Possible means by which microbicides may increase HIV risk include physical breach of epithelial integrity, mucosal immune cell activation (ie, enhancing the number of target cells and/or their expression of HIV-binding receptors), and immune inhibition.2 All these modes of injury are associated with the complex expression of immune markers including cytokines, chemokines, antimicrobial peptides, and local influx of activated B and T cells. Consequently, researchers have gone back to preclinical in vitro and animal studies with previously unsuccessful microbicides to better define biomarkers which may reflect the observed harm associated with their use. Studies of N-9 found mucosal elevation of numerous cytokines and chemokines including macrophage inflammatory protein 2, interleukin (IL)-1α, IL-1β, IL-6, and IL-8, and tumor necrosis factor (TNF).3–5 These cytokines have been associated with cell damage and the local recruitment of immune cells, including those expressing HIV-adhesion molecules such as CCR5+ and dendritic cell (DC)–specific intercellular adhesion molecule-3-grabbing nonintegrin. Fichorova et al4 also showed that N-9 was associated with diminished genital levels of secretory leukocyte protease inhibitor (SLPI), a factor that is thought to be important in host defense against bacterial pathogens. These results suggest that phase 1 microbicide trials should incorporate the measurement of these mucosal biomarkers in the genital tract as a more sensitive measure of harm to screen investigational products.

We recently published results from a phase 1 placebo-controlled, randomized, double-blinded clinical trial of StarPharma LTD 7013 (VivaGel) applied twice daily over 14 days.6 VivaGel is a unique dendrimer compound that showed efficacy in preventing simian immunodeficiency virus infection in nonhuman primates and herpes simplex virus (HSV)-2 in the guinea pig model and was shown to be safe in a phase 1 trial using a single daily dose over 7 days. Our results demonstrated no grade 3 or 4 adverse events (AEs) and good tolerability.6 However, participants in the VivaGel arm in comparison with the placebo arm had a greater number of grade 1 and 2 genitourinary AEs and superficial colposcopic findings. Similar findings were reported in another phase I trial of VivaGel.7 One of the secondary objectives of this project was to examine the effect of VivaGel on immune markers that may be associated with HIV acquisition among participants enrolled in the phase 1 trial, including cytokines, chemokines, activated T cells, and DCs expressing HIV-adhesion molecules and SLPI. This article reports these findings. In addition, we examine the correlation of these biomarkers with visual evidence of epithelial disruption (ED) in the lower genital tract.

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METHODS

This was a Phase 1, placebo-controlled, randomized, double-blind clinical trial in sexually abstinent young women, conducted at the Clinical Research Center at the University of California, San Francisco, CA, and the Research Care and Training Program unit of the Center for Microbiology Research at the Kenya Medical Research Institute in Kisumu, Kenya. The women used 3.5 g carbopol gel with and without (placebo) StarPharma LTD 7013 (VivaGel) twice daily over 14 days. The study protocol and informed consent forms were approved by the Committee on Human Research at University of California, San Francisco and the National Ethical Review Committee at Kenya Medical Research Institute. Safety oversight was provided by independent safety monitors and a safety monitoring committee. This trial is registered at www.ClinicalTrials.gov (NCT00331032).

Enrolment and design were described in detail previously.6 Briefly, women were eligible if they were 18–24 years of age, sexually experienced, not pregnant within 3 months, known to have regular menstrual cycles, and had not started a new long-acting contraception (eg, depomedroxyprogesterone) within the past 3 months. Women were also excluded if at screening they had a positive test for urinary tract infection, HIV or herpes simplex virus 2 antibodies, syphilis, vaginal candidiasis, symptomatic bacterial vaginosis using Amsel criteria, vaginal nugent score ≥7,8 Trichomonas vaginalis, Neisseria gonorrhoeae, or Chlamydia trachomatis, and abnormal cervical cytology. Women who had epithelia disruptions of the anogenital tract were also excluded.

Women were asked to be sexually abstinent one week before enrollment and throughout the 21 days of study participation. The enrollment visit was scheduled to fall within 5–14 days after the first day of the next menses. A pelvic examination including a visual examination, colposcopy, vaginal pH, vaginal sample for semen exposure, and vaginal wet mount were performed. The following were collected for immune parameters as follows: a cervicovaginal lavage (CVL) was performed with 5 mL of phosphate buffered saline and reaspirated for cytokine analysis; a cervical cytobrush was placed into 5 mL of cellular transport medium [RPMI with 10% FBS (Sigma, St Louis, MO)] for cell analysis by flow cytometry. After collection, cytobrush specimens were stored at 4°C and transported to the laboratory on ice within 2 hours. Women were asked to return for follow-up examinations and collection of immune parameters on days 7, 14, and 21. Women were instructed not to insert study gel at least 6 hours before any of the examinations. Details of examinations were previously reported and included colposcopic examinations and testing for vaginal and cervical infections.6

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Laboratory Methods
Analysis of Cell Types and Surface Markers

Personnel at each site were trained to process samples by a single person, and all samples were received blinded to randomization status. Upon arrival at each site's laboratory, cytobrush samples for cell analysis were vortexed vigorously to dissociate cell clumps and mucus before removing the cytobrush, and the cell suspension was filtered through a 100-micron nylon cell strainer (BD Biosciences, Franklin Lakes, NJ). The resulting suspension was centrifuged at 15 rpm for 10 minutes, the supernatant decanted and the cell pellet reconstituted into 1 mL of freezing medium [heat inactivated fetal bovine serum (Sigma) with 10% dimethyl sulfoxide]. The cryovial was placed into a freezing chamber that had been prechilled overnight to −20°C with isopropanol (Mister Frosty, Fisher Scientific, Nepean, Canada) and cooled overnight to −80°C before transfer into a −150°C freezer. Samples were transported to a central laboratory in a liquid nitrogen dry shipper (MVE Biological Systems [Cleveland, OH] Vapour Shipper) for analysis. Cells were thawed, washed, and divided into 2 equal aliquots for staining with a panel of DC or T-cell markers. Cells in the T-cell aliquot were stained with CD69-FITC, CCR5-PE, CD4-PerCP, and CD3-APC (BD Pharmingen, San Jose, CA); cells in the DC aliquot were stained with CD1a-FITC (Imgenex San Diego, CA), CD11c-PE, CD14-PerCP, and DC-SIGN-APC (eBioscience, San Diego, CA) according to manufacturer instructions. Appropriate isotype control antibodies were used in parallel. Samples were processed using FACSCalibur (Becton-Dickinson Immunocytometry Systems). Cell numbers enumerated by flow cytometry (in the T-cell or DC aliquot) were multiplied by 2 to determine “cells per cytobrush.”

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Analysis of Cytokines and SLPI

CVL specimens were cryopreserved at −80°C within 2 hours of collection and transported to a central laboratory in a liquid nitrogen dry shipper (MVE Biological Systems Vapour Shipper) for analysis by laboratory staff blinded to randomization status. Cell-free supernatants of the CVL specimens were tested for cytokines as previously described9 using the LINCOplex high-sensitivity human cytokine immunoassay kit (Millipore, Billerica, MA) and the Luminex-100 platform (Luminex, Austin, TX) in duplicate according to the manufacturer instructions. The following cytokines and chemokines were included: Granulocyte macrophage colony-stimulating factor (GMCSF), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, and IL-13, TNF, and interferon gamma (IFN-γ). SLPI levels were measured by enzyme-linked immunosorbent assay (Quantikine Human SLPI kit, R&D Systems), according to the manufacturer instructions.

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Statistical Analysis

We treated all the outcome variables (ie, cytokines, and T-cell and DC markers) as counts and used negative binomial regression to examine differences in outcomes between VivaGel and placebo groups. Alternate models fitted using Poisson regression provided similar results but did not fit the data as well. Because this analysis was a secondary objective of the primary phase I trial and considered exploratory, the regression analyses described below did not adjust for multiple testing for different cytokines.

For baseline (day 0) comparison between VivaGel and placebo arms, we fitted all the models with arm as the independent variable, and values of cytokines T-cell and DC markers as dependent variables. Because the cells expressing the early activation marker depend on the initial number of DC or T-cell counts, we included the total cell counts (in logarithm form, such as CD4, CD14) as offsets in regression models. Resulting coefficients can be interpreted as the log relative proportion of cytokine counts (with denominators given by the total cell counts) compared between treatment groups.

For longitudinal observations of cytokine levels, we fitted additional negative binomial models including time on study grouped as days 7, 14, and 21 (and using day 0 as reference) and evaluated between group differences via an interaction between time and treatment group. The exponentiated coefficients of the interaction terms can be interpreted as time-specific relative treatment comparisons similar to those described above. Within-individual correlation in outcomes were accounted for using robust standard errors estimated using generalized estimating equation methods.

For purposes of data presentation, we present for each day the percent of samples in each arm that were above the 75% quartile. Quartiles were calculated by combining the 2 arms for each day. The median, 25th and 75th percentiles for the cytokines and cell markers are shown in Table 1. Because 80% of the samples had undetectable levels for IL-5, the median, 25th and 90% percentile are presented for IL-5. We found 2 outliers in analysis of IL-10 at day 14 in placebo group, which substantially affected the result of negative binomial model. Therefore, we removed them from the model. Although outliers were found for other cytokines, removing them did not alter any of the findings and consequently were not removed.

Table 1
Table 1
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The similar modeling approaches were adopted to examine the effect of AEs on outcome variables indicated above, except that we included the variable AEs as the sole independent variable in the models. All analyses were performed using SAS 9.2 (SAS Institute Inc, Cary, NC).

Because the immune markers were obtained from the CVL specimen (soluble markers) and cervix (cell markers), AEs included in this analysis were limited to evidence of ED in the vagina and cervix. This included vaginitis, cervicitis, abrasions, petechiae, lacerations, and erythema. Vulvar and perianal findings and reported genito-urinary symptoms (eg, pain, discharge) were not included for the purposes of this analysis. Women who were noncompliant with study product at any of the visits, defined as using <80% of expected doses, were excluded from all analyses. Because blood from menstrual bleeding, trauma from the speculum, or presence of bacterial vaginosis could alter cytokines, we also performed a sensitivity analysis excluding those visits if blood was noted on examination or bacterial vaginosis was diagnosed. Three participant visits had bacterial vaginosis diagnosed and 10 had blood visualized in the vaginal vault. Data is not shown for these results, only P values.

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RESULTS

In summary, a total of 54 women were enrolled.6 Of the 54 women, 35 women were enrolled in the VivaGel arm and 19 in the placebo arm. The mean age (20.8 years) was similar in both arms. In addition, lifetime number of sexual partners and number of sexual partners in the last 3 months was also similar.6 The San Francisco site was predominantly white, and the Kisumu arm was 100% black African.

Most (83%) participants, including 30 (86%) in the VivaGel arm and 15 (79%) in the placebo arm, reported taking at least 80% of doses (P = 0.52). Thirty-three of 205 (16%) total follow-up visits had a cervical and/or vaginal finding suggestive of ED—of these, 25 (19%) were in the VivaGel arm and 8 (11%) in the placebo arm, P = 0.17.

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Cytokine/Chemokine Results

No differences were seen at day 0 between the VivaGel and placebo arms for GMCSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, and IL-13, and TNF. Using negative binomial regressions models, we examined differences between arms at days 7, 14, and 21 using day 0 as the comparison day. At day 7, higher levels of IL-6 were found in the VivaGel arm. These findings are summarized in figure showing the percent of samples from each day in each arm that was in the top quartile. For IL-5, percent of samples that were 90th percentile or greater is shown. On day 21 (1 week after discontinuation of study product), no differences were found except that IL-4 (P = 0.007) and GMCSF (P = 0.002) were lower in the VivaGel arm. No differences were seen for SLPI on any of the days between study arms.

When we excluded the visits with blood or bacterial vaginosis, in the sensitivity analysis, the findings were similar. At day 7, there was a trend for IL-6 as it was higher in the VivaGel arm (P = 0.1). At day 14, similar changes were also found in the VivaGel arm in comparison to the placebo arm as follows: higher levels of IFN-γ (P = 0.03), IL-2 (P = 0.04), IL-5 (P = 0.003), and IL-10 (P = 0.008). At day 21, IL-4 (P = 0.16) and GMCSF (P = 0.02) were similarly lower in the VivaGel arm in comparison with the placebo arm.

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T Cells and DC Cell Markers

We found no differences between VivaGel and placebo for any of the T-cell and DC markers at day 0 except for total number of CD8+ T cells, which were greater in the VivaGel arm (P = 0.02). At day 7, VivaGel had a trend for greater number of CD8+/CD69+ T cells (P < 0.08) (Fig. 1). On day 14, no differences were seen for any of the T cell or DC cell markers. At day 21, CD11+/DC-SIGN+ cells were higher in the VivaGel arm (P = 0.05).

Figure 1
Figure 1
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In the sensitivity analysis with the visits with blood or bacterial vaginosis excluded, the findings remained similar. At day 7, CD8+/CD69+ T cells were significantly greater in the VivaGel arm (P < 0.005). In addition, CD4+/CD69+ cells (P = 0.001) and CD4+/CCR5+ T cells (P = 0.01) were higher in the VivaGel arm. At day 21, there was a similar trend for greater numbers of CD11+/DC-SIGN+ cells in the VivaGel arm (P = 0.14).

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Correlation of Vaginal and Cervical ED With Immune Markers

The presence of cervical and vaginal ED was associated with the elevations of several cytokines including IL-4 (P = 0.001), IL-10 (P = 0.007), IL-13 (P < 0.001), IL-1β (P = 0.007), and TNF (P = 0.012). A trend was seen for IFN-γ (P = 0.1). The total number of CD4+ and CD8+ T cells were greater on cytobrushes collected from participants with ED (P = 0.02 and P = 0.009, respectively), and decreases were noted for CD4+/CD69 + T cells (P = 0.07), CD4+/CCR5 + T cells (P = 0.012), and CD4+/CD69+/CCR5+ T cells (P = 0.02). To see if the biomarkers added information beyond visible EDs, we performed another sensitivity analysis by excluding all visits with ED. Although the sample size was diminished significantly, the P values for all the biomarkers were similar (data not shown) except for IL-6 at day 7 which demonstrated a similar trend toward higher levels in the VivaGel arm, but the P value was no longer significant (P = 0.3).

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DISCUSSION

The ideal microbicide should retain its in vitro activity against HIV and other STIs in vivo without disrupting the integrity of the epithelial barrier, inducing inflammation or adversely interfering with innate immunity. In this phase 1 study, several findings associated with VivaGel were of concern. These included increases in genital cytokines and T-cell subsets that were associated with clinical findings of ED, and that have been associated with an increased risk for HIV acquisition in women.

Epithelial damage can result in the release of several cytokines and chemokines including IL-1β, IL-6, and TNF, IL-8, and IL-10.5,10 In accordance with this, visible epithelia damage in the vagina and/or cervix observed in our cohort was associated with increases in IL-1β, IL-10, and TNF. We also saw elevations in the Th2 cytokines, IL-4 and IL-13, which have also been implicated in tissue damage.11,12 In comparison, the findings associated with VivaGel compared with placebo also showed a mixed picture of cytokine alterations, which included greater levels of IFN-γ.

In looking at the trend of cytokine expression across the 4 visit dates, the difference between the VivaGel arm and placebo arm at days 7 and 14 was in part due to the fact that the women in the placebo arm had lower levels on these days than seen at baseline. Because of the protocol design, which enrolled women between days 5 and 14 of their menstrual cycle, the day 7 visit normally occurred mid-cycle and the day 14 visit occurred during the luteal phase. Several studies have shown that there are significant differences in cytokine levels seen during the menstrual cycle,13 with consistent changes seen in IL-6 and IL-8 levels. Shrier et al14 found mid-cycle dips, whereas Al-Harthi et al15 found much lower levels of IL-6 and IL-8 in the luteal phase compared with the follicular phase. Not all studies showed such contrasts during the menstrual cycle.15–17 The absence of mid-cycle and luteal cytokine dips in the VivaGel arm may be interpreted as altering the normal immune cycle and hence detrimental. The impact of such variations on HIV susceptibility in vivo is not clear.

Epithelial damage also can lead to lymphocyte extravasation which, in the case of HIV, may enhance infection. Myer et al18 showed that visible evidence of cervical ED increased the risk of HIV acquisition. After epithelial injury, CD69+/CD8+ and CCR5+/CD4+ T cells would be normally found in the epithelial mucosa as part of the host defense. Unfortunately, CD4+ T cells are also target cells for HIV.19 Interestingly, the number of CD4+ and CD8+ T cells in our study were increased in those with evidence of epithelial damage but activated CD4+ T cells (CD69+ and CCR5+) were decreased for reasons not understood. Studies with N-9 demonstrate that the product was associated with an influx of CD4+ T cells. Although we did not observe increases in the sheer number of CD4+ or CD8+ T cells with VivaGel, we did observe greater numbers of activated CD8+ T cells in the VivaGel arm at day 7 compared with placebo; however, this difference was not sustained at day 14.

Except for N-9, few other microbicide studies have incorporated immune markers. Bollen et al20 examined the effects of Carrageen, a relatively inert substance without an apparent effect on HIV acquisition. In their study, Carrageen and the placebo gel caused a decrease in the levels of IL-8 and IL-6, suggesting that the gels may have caused some inhibition in the assays. Decreases in IL-8 levels have been observed with the use of several microbicides, including cellulose sulfate, Pro2000, and BufferGel.21–23 Pro2000 and BufferGel were also associated with lower levels of IL-1β and SLPI. Because the placebo in our trial was the same substance (Carbopol) used as the vehicle for VivaGel, the dampening observed in the placebo arm for many of the cytokines was not likely due to inhibition by the vehicle itself.

One of the strengths of this investigation was that participants were all enrolled and subsequently followed at similar times of their menstrual cycle. A second strength of the study is that women were screened for numerous STIs and were excluded if any of the tests were positive. Third, the close observation of the women allowed us to control for possible factors such as visible blood and bacterial vaginosis which may also influence immune markers. Last, women agreed to abstain from sexual intercourse 1 week before enrollment and throughout follow-up, and very few women had objective evidence of semen exposure.6

Limitations include our small sample size, which was consistent with most phase 1 trials. Also, the clinical interpretation of these findings is limited because there are no human trials that have yet shown that these immune markers define absolute risks for female HIV acquisition, although results from CAPRISA-004 are forthcoming.24 Rather, the evidence for risk of HIV acquisition related to altered innate and adaptive immunity in the female genital tract are based primarily on in vitro and animal studies.3–5 In addition, the study involved 2 different populations. The impact of race and ethnicity could not be studied in this small sample size. However, in a previous publication,9 we found significant immune differences between the 2 groups at baseline before product exposure. Future studies should include the potential impact of race and ethnicity on the association of microbicide products on mucosal immune changes in the genital tract.

In summary, in this phase 1 clinical trial, markers associated with epithelial damage were elevated in the VivaGel arm compared with the placebo arm after 7–14 days of twice-daily product use. Most of the changes were reversed 7 days after discontinuation of the product. These findings need to be taken with a certain degree of caution due to their exploratory nature and small sample size. However, these findings and the observed increase in AEs reported in the 2 phase 1 trials should be taken into consideration in planning any future clinical trials.

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ACKNOWLEDGMENTS

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; National Institutes of Allergy and Infectious Diseases Division of Microbiology and Infectious Diseases; the Director, Kenya Medical Research Institute; and the study participants for volunteering their time and information.

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REFERENCES

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17. Scott ME, Ma Y, Farhat S, et al.. Covariates of cervical cytokine mRNA expression by real-time PCR in adolescents and young women: effects of Chlamydia trachomatis infection, hormonal contraception, and smoking. J Clin Immunol. 2006;26:222–232.

18. Myer L, Wright TC Jr, Denny L, et al.. Nested case-control study of cervical mucosal lesions, ectopy, and incident HIV infection among women in Cape Town, South Africa. Sex Transm Dis. 2006;33:683–687.

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21. Anderson DJ, Williams DL, Ballagh SA, et al.. Safety analysis of the diaphragm in combination with lubricant or acidifying microbicide gels: effects on markers of inflammation and innate immunity in cervicovaginal fluid. Am J Reprod Immunol. 2009;61:121–129.

22. Keller MJ, Zerhouni-Layachi B, Cheshenko N, et al.. PRO 2000 gel inhibits HIV and herpes simplex virus infection following vaginal application: a double-blind placebo-controlled trial. J Infect Dis. 2006;193:27–35.

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Keywords:

mucosal immune markers; vaginal microbicide

© 2012 Lippincott Williams & Wilkins, Inc.

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