The cervicovaginal mucus barrier to HIV-1 is diminished in bacterial vaginosis.

Bacterial vaginosis (BV), a condition in which the vaginal microbiota consists of community of obligate and facultative anaerobes rather than dominated by a single species of Lactobacillus, affects ~30% of women in the US. Women with BV are at 60% increased risk for HIV acquisition and are 3-times more likely to transmit HIV to an uninfected partner. As cervicovaginal mucus (CVM) is the first line of defense against mucosal pathogens and the home of the resident vaginal microbiota, we hypothesized the barrier function of CVM to HIV may be diminished in BV. Here, we characterized CVM properties including pH, lactic acid content, and Nugent score to correlate with the microbiota community composition, which was confirmed by 16S rDNA sequencing on a subset of samples. We then quantified the mobility of fluorescently-labeled HIV virions and nanoparticles to characterize the structural and adhesive barrier properties of CVM. Our analyses included women with Nugent scores categorized as intermediate (4-6) and BV (7-10), women that were either symptomatic or asymptomatic, and a small group of women before and after antibiotic treatment for symptomatic BV. Overall, we found that HIV virions had significantly increased mobility in CVM from women with BV compared to CVM from women with Lactobacillus crispatus-dominant microbiota, regardless of whether symptoms were present. We confirmed using nanoparticles and scanning electron microscopy that the impaired barrier function was due to reduced adhesive barrier properties without an obvious degradation of the physical CVM pore structure. We further confirmed a similar increase in HIV mobility in CVM from women with Lactobacillus iners-dominant microbiota, the species most associated with transitions to BV and that persists after antibiotic treatment for BV. Our findings advance the understanding of the protective role of mucus and highlight the interplay between vaginal microbiota and the innate barrier function mucus.

Vaginal cytokine profile and microbiota before and after lubricant use compared with condomless vaginal sex: a preliminary observational study.

BACKGROUND: Limited data suggest that personal lubricants may damage the vaginal mucosal epithelium, alter the vaginal microbiota, and increase inflammation. We compared vaginal cytokine profiles and microbiota before and after vaginal lubricant use and condomless vaginal sex. METHODS: Reproductive-age women were recruited to a 10-week observational cohort study and were asked to self-collect vaginal samples and behavioral diaries daily. This nested case-control analysis utilized samples collected before and after self-reported condomless sexual activity with lubricants (22 case participants) and without lubricants (22 control participants). Controls were matched to cases on race/ethnicity. Microbiota composition was characterized by sequencing amplicons of the 16S rRNA gene V3-V4 regions. Cytokine concentrations were quantified using a magnetic bead 41-plex panel assay and read using a Bio-Plex 200 array reader. Wilcoxon signed-rank tests were used to assess baseline differences in vaginal cytokines between cases and controls as well as differences pre- and post-exposure. Linear mixed effects models were used to examine differences in relative post-to-pre change in each individual cytokine between matched cases and controls. Similar analyses were conducted for the microbiota data. RESULTS: Mean age was 29.8 years (SD 6.8), and 63.6% were African American. There were few statistically significant changes in cytokines or microbiota before and after exposure in cases or controls. In mixed-effects modeling, the mean relative post-to-pre change of cytokines was higher in cases vs. controls for macrophage derived chemokine (MDC) (p = 0.03). The microbiota data revealed no significant changes when measured by similarity scores, diversity indexes and descriptive community state types (CST) transition analyses. However, post sexual activity, the mean relative abundance of L. crispatus decreased for those who used lubricants (particularly those who were L. iners-dominated prior to exposure). CONCLUSIONS: Although there were overall few differences in the vaginal microbiota and cytokine profiles of lubricant users and controls before and after condomless vaginal sex, there was a trend toward decreases in relative abundance of L. crispatus following use of lubricant. Future larger studies that take into account osmolarity and composition of lubricants may provide additional insights.

Engineering monoclonal antibody-based contraception and multipurpose prevention technologies†.

Sexually transmitted infections are highly prevalent, and over 40% of pregnancies are unplanned. We are producing new antibody-based multipurpose prevention technology products to address these problems and fill an unmet need in female reproductive health. We used a Nicotiana platform to manufacture monoclonal antibodies against two prevalent sexually transmitted pathogens, HIV-1 and HSV-2, and incorporated them into a vaginal film (MB66) for preclinical and Phase 1 clinical testing. These tests are now complete and indicate that MB66 is effective and safe in women. We are now developing an antisperm monoclonal antibody to add contraceptive efficacy to this product. The antisperm antibody, H6-3C4, originally isolated by Shinzo Isojima from the blood of an infertile woman, recognizes a carbohydrate epitope on CD52g, a glycosylphosphatidylinositol-anchored glycoprotein found in abundance on the surface of human sperm. We engineered the antibody for production in Nicotiana; the new antibody which we call "human contraception antibody," effectively agglutinates sperm at concentrations >10 µg/ml and maintains activity under a variety of physiological conditions. We are currently seeking regulatory approval for a Phase 1 clinical trial, which will include safety and "proof of principle" efficacy endpoints. Concurrently, we are working with new antibody production platforms to bring the costs down, innovative antibody designs that may produce more effective second-generation antibodies, and delivery systems to provide extended protection.

Vaginal microbiota and sexually transmitted infections that may influence transmission of cell-associated HIV.

Vaginal microbiota and sexually transmitted infections (STIs) are likely to influence the transmission of cell-associated human immunodeficiency virus (HIV). Lactic acid produced by Lactobacillus-dominated microbiota (Nugent score 0-3) will likely inhibit transmission, especially female-to-male transmission. In contrast, polymicrobial microbiota (Nugent score 4-10), community state types IV-A and IV-B, and STIs will likely increase transmission of cell-associated HIV.

Evaluation of microbicide gel adherence monitoring methods.

BACKGROUND: An objective and accurate method that measures adherence to vaginal microbicide gel regimens during clinical trials could provide more accurate estimates of microbicide efficacy, aid in targeting adherence promotion resources, and enable objective assessment of adherence promotion strategies. METHODS: We evaluated 4 methods to assess whether or not gel applicators had been vaginally inserted. At the study site, 50 women inserted hydroxyethylcellulose universal placebo gel through a polypropylene vaginal applicator and handled, but did not insert a second "sham-inserted" applicator. Applicators were discarded into a container capped with a medical event monitor system (MEMS) that recorded the time and date of opening. Fifteen additional participants did likewise at 2 study site visits, and administered gel on 6 intervening days at home. Applicators were scored as inserted, or not, by direct inspection under ambient light, ultraviolet (UV) light, staining with Alcian blue, and microscopic detection of vaginal cells stained with iodine. RESULTS: Mean sensitivity/specificity of 2 readings each by 3 test readers for UV, Alcian blue, ambient light, and iodine methods were 84/83, 79/83, 76/63, and 65/80%, respectively. Sensitivity of all methods was significantly higher in applicators inserted after one or more prior insertions of gel, with the highest sensitivity (95%) obtained with UV. MEMS caps accurately recorded applicator disposal time. CONCLUSIONS: The modest accuracy of all 4 methods for applicator insertions without prior gel applications may limit their accuracy in monitoring coital regimens. However, for daily dosing regimens, MEMS monitoring and UV inspection should provide a rapid, reliable, and quantitative assessment of adherence.

In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide.

BACKGROUND: Hydrogen peroxide (H2O2) produced by vaginal lactobacilli is generally believed to protect against bacteria associated with bacterial vaginosis (BV), and strains of lactobacilli that can produce H2O2 are being developed as vaginal probiotics. However, evidence that led to this belief was based in part on non-physiological conditions, antioxidant-free aerobic conditions selected to maximize both production and microbicidal activity of H2O2. Here we used conditions more like those in vivo to compare the effects of physiologically plausible concentrations of H2O2 and lactic acid on a broad range of BV-associated bacteria and vaginal lactobacilli. METHODS: Anaerobic cultures of seventeen species of BV-associated bacteria and four species of vaginal lactobacilli were exposed to H2O2, lactic acid, or acetic acid at pH 7.0 and pH 4.5. After two hours, the remaining viable bacteria were enumerated by growth on agar media plates. The effect of vaginal fluid (VF) on the microbicidal activities of H2O2 and lactic acid was also measured. RESULTS: Physiological concentrations of H2O2 (< 100 µM) failed to inactivate any of the BV-associated bacteria tested, even in the presence of human myeloperoxidase (MPO) that increases the microbicidal activity of H2O2. At 10 mM, H2O2 inactivated all four species of vaginal lactobacilli but only one of seventeen species of BV-associated bacteria. Moreover, the addition of just 1% vaginal fluid (VF) blocked the microbicidal activity of 1 M H2O2. In contrast, lactic acid at physiological concentrations (55-111 mM) and pH (4.5) inactivated all the BV-associated bacteria tested, and had no detectable effect on the vaginal lactobacilli. Also, the addition of 10% VF did not block the microbicidal activity of lactic acid. CONCLUSIONS: Under optimal, anaerobic growth conditions, physiological concentrations of lactic acid inactivated BV-associated bacteria without affecting vaginal lactobacilli, whereas physiological concentrations of H2O2 produced no detectable inactivation of either BV-associated bacteria or vaginal lactobacilli. Moreover, at very high concentrations, H2O2 was more toxic to vaginal lactobacilli than to BV-associated bacteria. On the basis of these in vitro observations, we conclude that lactic acid, not H2O2, is likely to suppress BV-associated bacteria in vivo.

Observational cohort study of the effect of a single lubricant exposure during transvaginal ultrasound on cell-shedding from the vaginal epithelium.

The outer layers of the vaginal epithelium (VE) are important because they accumulate glycogen which, under optimal conditions, Lactobacillus spp. consume to grow and acidify the vaginal microenvironment with lactic acid. We hypothesized that exposure to lubricant, for example in the conduct of a transvaginal ultrasound (TVUS), may contribute to the shedding of mature epithelial cells, exposing immature cells. Cervicovaginal fluid (CVF) was sampled at four time points by menstrual cup (Softdisc™) from 50 women referred for TVUS, during which a controlled volume of lubricant was applied to the TVUS wand. Samples were collected (1) immediately before TVUS and (2) 6-12 hours, (3) within one week, and (4) two weeks after TVUS. Clinical vaginal lubricants are similar to commercial lubricants, and often have a high osmolality or pH, and contain bactericides such as methylparaben and propylparaben. The number and maturity of epithelial cells in each CVF sample were measured by quantitative and differential fluorimetry (maturity index, MI). Comparisons of cell-counts and maturity were made by paired Wilcoxon signed-rank tests. Among women with a high pre-TVUS MI (> 3), there was a decrease in median cell-count and mean MI in the sample collected 6-12 hours after TVUS (p<0.001, n = 26 and p < 0.001, n = 26, respectively). For these women, cell-count and MI remained lower in the sample collected within the subsequent week (p<0.001, n = 29 and p<0.01, n = 29, respectively), and MI remained lower in the sample collected within two weeks of TVUS (p<0.01, n = 25), compared to the pre-TVUS sample. Among participants with a low pre-TVUS MI (< 3), cell-count was higher in the sample collected within two weeks of TVUS compared to the pre-TVUS sample (p = 0.03, n = 15), but no significant changes in MI were observed. Results were similar when restricted to reproductive-age women. This preliminary data indicates hypertonic vaginal lubricants may increase vaginal epithelial cell shedding.

Rapid fluctuation of the vaginal microbiota measured by Gram stain analysis.

BACKGROUND: The aetiology of bacterial vaginosis (BV) remains unknown. OBJECTIVE: To describe longitudinal changes in vaginal microbiota. METHODS: Thirty-nine women (mean age 36.8 years; 22 (56.4%) African-American) self-collected vaginal specimens twice weekly for 16 weeks as part of a vaginal douching cessation study. In an analysis where each woman serves as her own control, conditional logistic regression was used to evaluate daily, time-varying factors associated with a woman's incident BV episode(s) as compared with her persistently BV-negative sample(s). BV was defined by a Nugent's Gram stain score >or=7. RESULTS: 46.2% of participants had BV in the first 4 weeks of observation. Rapid fluctuation of vaginal microbiota was observed in 226 transitions to BV or spontaneous remission. Duration of BV was often short: 51% of the episodes lasted for only one sample interval (3 days). Among women who had at least one BV episode, the median number of episodes per woman was 8.7 (SD 7.4, range 1-22). Lubricant use 1 day before specimen collection (adjusted OR (aOR)=11.75, 95% CI 1.96 to 70.27) and rectal sex 2 days before (aOR=4.48, 95% CI 2.79 to 7.17) were associated with BV onset. CONCLUSION: Rapid fluctuation of the vaginal microbiota was seen. Longitudinal studies with long intervals between sampling are likely to miss episodes of BV. Recent report of lubricant use and rectal sex were associated with incident BV.

Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli.

BACKGROUND: H2O2 produced by vaginal lactobacilli is believed to protect against infection, and H2O2-producing lactobacilli inactivate pathogens in vitro in protein-free salt solution. However, cervicovaginal fluid (CVF) and semen have significant H2O2-blocking activity. METHODS: We measured the H2O2 concentration of CVF and the H2O2-blocking activity of CVF and semen using fluorescence and in vitro bacterial-exposure experiments. RESULTS: The mean H2O2 measured in fully aerobic CVF was 23 +/- 5 microM; however, 50 microM H2O2 in salt solution showed no in vitro inactivation of HSV-2, Neisseria gonorrhoeae, Hemophilus ducreyii, or any of six BV-associated bacteria. CVF reduced 1 mM added H2O2 to an undetectable level, while semen reduced 10 mM added H2O2 to undetectable. Moreover, the addition of just 1% CVF supernatant abolished in vitro pathogen-inactivation by H2O2-producing lactobacilli. CONCLUSIONS: Given the H2O2-blocking activity of CVF and semen, it is implausible that H2O2-production by vaginal lactobacilli is a significant mechanism of protection in vivo.

Microbicide excipients can greatly increase susceptibility to genital herpes transmission in the mouse.

BACKGROUND: Several active ingredients proposed as vaginal microbicides have been shown paradoxically to increase susceptibility to infection in mouse genital herpes (HSV-2) vaginal susceptibility models and in clinical trials. In addition, "inactive ingredients" (or excipients) used in topical products to formulate and deliver the active ingredient might also cause epithelial toxicities that increase viral susceptibility. However, excipients have not previously been tested in susceptibility models. METHODS: Excipients commonly used in topical products were formulated in a non-toxic vehicle (the "HEC universal placebo"), or other formulations as specified. Twelve hours after exposure to the excipient or a control treatment, mice were challenged with a vaginal dose of HSV-2, and three days later were assessed for infection by vaginal lavage culture to assess susceptibility. RESULTS: The following excipients markedly increased susceptibility to HSV-2 after a single exposure: 5% glycerol monolaurate (GML) formulated in K-Y® Warming Jelly, 5% GML as a colloidal suspension in phosphate buffered saline, K-Y Warming Jelly alone, and both of its humectant/solvent ingredients (neat propylene glycol and neat PEG-8). For excipients formulated in the HEC vehicle, 30% glycerin significantly increased susceptibility, and a trend toward increased HSV-2 susceptibility was observed after 10% glycerin, and 0.1% disodium EDTA, but not after 0.0186% disodium EDTA. The following excipients did not increase susceptibility: 10% propylene glycol, 0.18%, methylparaben plus 0.02% propylparaben, and 1% benzyl alcohol. CONCLUSIONS: As reported with other surfactants, the surfactant/emulsifier GML markedly increased susceptibility to HSV-2. Glycerin at 30% significantly increased susceptibility, and, undiluted propylene glycol and PEG-8 greatly increased susceptibility.

Formulating a sulfonated antiviral dendrimer in a vaginal microbicidal gel having dual mechanisms of action.

SPL7013 is the sodium salt of a sulfonated dendrimer that has potent antiviral properties. VivaGel, a topical gel containing 3% (wt/wt) SPL7013, is in development as a vaginal microbicide. BufferGel is a Carbopol-based acidic buffering gel that enhances the natural protective action of the vagina to produce a broad-spectrum microbicidal environment. The positive attributes of both gels were combined into a combination vaginal microbicidal gel having dual mechanisms of action. A 3% (wt/wt) SPL7013 combination gel, pH 3.7, was developed and fully characterized and was shown to have more than twofold greater acidic buffering capacity than BufferGel. Ultracentrifugation experiments demonstrated that SPL7013 was not sequestered or entropically trapped in the viscous gel, thereby confirming, along with viral challenge studies, that SPL7013 has sufficient mobility in the viscous gel to exert antiviral properties.

Conceptual Design of a Universal Donor Screening Approach for Vaginal Microbiota Transplant.

The success of fecal microbiota transplant (FMT) in treating recurrent Clostridioides difficile infection has led to growing excitement about the potential of using transplanted human material as a therapy for a wide range of diseases and conditions related to microbial dysbiosis. We anticipate that the next frontier of microbiota transplantation will be vaginal microbiota transplant (VMT). The composition of the vaginal microbiota has broad impact on sexual and reproductive health. The vaginal microbiota in the "optimal" state are one of the simplest communities, dominated by one of only a few species of Lactobacillus. Diversity in the microbiota and the concomitant depletion of lactobacilli, a condition referred to as bacterial vaginosis (BV), is associated with a wide range of deleterious effects, including increased risk of acquiring sexually transmitted infections and increased likelihood of having a preterm birth. However, we have very few treatment options available, and none of them curative or restorative, for "resetting" the vaginal microbiota to a more protective state. In order to test the hypothesis that VMT may be a more effective treatment option, we must first determine how to screen donors to find those with minimal risk of pathogen transmission and "optimal" vaginal microbiota for transplant. Here, we describe a universal donor screening approach that was implemented in a small pilot study of 20 women. We further characterized key physicochemical properties of donor cervicovaginal secretions (CVS) and the corresponding composition of the vaginal microbiota to delineate criteria for inclusion/exclusion. We anticipate that the framework described here will help accelerate clinical studies of VMT.

Development of rectal enema as microbicide (DREAM): Preclinical progressive selection of a tenofovir prodrug enema.

HIV pre-exposure prophylaxis (PrEP) strategies have the potential to prevent millions of incident HIV infections each year. However, the efficacy of PrEP strategies has been plagued by issues of non-adherence, likely because of the difficulty in motivating otherwise healthy people to adhere to treatment regimens that require significant behavioral changes and daily discipline. An alternative approach to PrEP is to focus on strategies that fit in to normal, and even desirable, sexual behaviors, such as the use of cleansing enemas by men who have sex with men (MSM) prior to receptive anal intercourse (RAI). Here, we describe preclinical efforts toward optimizing a tenofovir (TFV)-based enema formulation for rectal PrEP. Using a murine model, we compared the plasma and tissue pharmacokinetics of TFV and various TFV prodrugs, including tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), and hexadecyloxypropyl tenofovir (CMX157), after dosing as enema formulations with varying osmolality and ion content. We observed that the enema vehicle composition played a more important role than the TFV prodrug properties in achieving rapid and therapeutically relevant tenofovir diphosphate (TFV-DP) concentrations in mouse colorectal tissue. Our results support the next steps, which are further preclinical (non-human primate) and clinical development of a hypo-osmolar TFV enema product for rectal PrEP.

Hyperosmolal vaginal lubricants markedly reduce epithelial barrier properties in a three-dimensional vaginal epithelium model.

Most of the widely used vaginal lubricants in the U.S. and Europe are strongly hyperosmolal, formulated with high concentrations of glycerol, propylene glycol, polyquaternary compounds or other ingredients that make these lubricants 4 to 30 times the osmolality of healthy vaginal fluid. Hyperosmolal formulations have been shown to cause marked toxicity to human colorectal epithelia in vivo, and significantly increase vaginal transmission of genital herpes infections in the mouse/HSV model. They also cause toxicity to explants of vaginal epithelia, to cultured vaginal epithelial cells, and increase susceptibility to HIV in target cells in cell cultures. Here, we report that the osmolality of healthy vaginal fluid is 370 ±â€¯40 mOsm/Kg in women with Nugent scores 0-3, and that a well-characterized three-dimensional human vaginal epithelium tissue model demonstrated that vaginal lubricants with osmolality greater than 4 times that of vaginal fluid (>1500 mOsm/Kg) markedly reduce epithelial barrier properties and showed damage in tissue structure. Four out of four such lubricants caused disruption in the parabasal and basal layers of cells as observed by histological analysis and reduced barrier integrity as measured by trans-epithelial electrical resistance (TEER). No epithelial damage to these layers was observed for hypo- and iso-osmolal lubricants with osmolality of <400 mOsm/Kg. The results confirm extensive reports of safety concerns of hyperosmolal lubricants and suggest the usefulness of reconstructed in vitro vaginal tissue models for assessing safety of lubricants in the absence of direct clinical tests in humans.

Anti-HIV-1 Activity of Lactic Acid in Human Cervicovaginal Fluid.

Women of reproductive age with a Lactobacillus-dominated vaginal microbiota have a reduced risk of acquiring and transmitting HIV and a vaginal pH of ~4 due to the presence of ~1% (wt/vol) lactic acid. While lactic acid has potent HIV virucidal activity in vitro, whether lactic acid present in the vaginal lumen inactivates HIV has not been investigated. Here we evaluated the anti-HIV-1 activity of native, minimally diluted cervicovaginal fluid obtained from women of reproductive age (n = 20) with vaginal microbiota dominated by Lactobacillus spp. Inhibition of HIVBa-L was significantly associated with the protonated form of lactic acid in cervicovaginal fluid. The HIVBa-L inhibitory activity observed in the <3-kDa acidic filtrate was similar to that of the corresponding untreated native cervicovaginal fluid as well as that of clarified neat cervicovaginal fluid subjected to protease digestion. These ex vivo studies indicate that protonated lactic acid is a major anti-HIV-1 metabolite present in acidic cervicovaginal fluid, suggesting a potential role in reducing HIV transmission by inactivating virus introduced or shed into the cervicovaginal lumen.IMPORTANCE The Lactobacillus-dominated vaginal microbiota is associated with a reduced risk of acquiring and transmitting HIV and other sexually transmitted infections (STIs). Lactic acid is a major organic acid metabolite produced by lactobacilli that acidifies the vagina and has been reported to have inhibitory activity in vitro against bacterial, protozoan, and viral STIs, including HIV infections. However, the anti-HIV properties of lactic acid in native vaginal lumen fluids of women colonized with Lactobacillus spp. have not yet been established. Our study, using native cervicovaginal fluid from women, found that potent and irreversible anti-HIV-1 activity is significantly associated with the concentration of the protonated (acidic, uncharged) form of lactic acid. This work advances our understanding of the mechanisms by which vaginal microbiota modulate HIV susceptibility and could lead to novel strategies to prevent women from acquiring HIV or transmitting the virus during vaginal intercourse and vaginal birth.

Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent.

Because both HIV-1 virions and HIV-infected cells are present in the semen and cervical mucus of infected individuals, HIV-1 prevention strategies must consider both cell-free and cell-associated virus. Antibodies that target HIV-1 virions have been shown to prevent vaginal transmission of cell-free virus in macaques, but since cell-associated transmission has not been reliably demonstrated in this model system, no strategies to prevent such transmission have been tested. We have employed a mouse model in which SCID mice carry human peripheral blood leukocytes (HuPBLs). In these mice, vaginal transmission of cell-associated, but not cell-free, HIV-1 transmission occurs, mediated by transepithelial migration of HIV-infected cells. Topical application of beta-cyclodextrin (beta-CD), a cholesterol-sequestering agent that interferes with cell migration and budding of virus from lipid rafts, blocks transmission of cell-associated HIV-1. The HuPBL-SCID model of vaginal HIV-1 transmission should prove useful for investigating cell-associated HIV-1 transmucosal HIV-1 transmission, as well as for screening reagents for their potential efficacy in preventing sexual HIV-1 transmission.

The role of lactic acid production by probiotic Lactobacillus species in vaginal health.

Vaginal eubiosis is characterised by beneficial lactobacillus-dominated microbiota. In contrast, vaginal dysbiosis (e.g. bacterial vaginosis, BV), characterised by an overgrowth of multiple anaerobes, is associated with an increased risk of adverse urogenital and reproductive health outcomes. A major distinguishing feature between the vaginal environment in states of eubiosis and dysbiosis is a high concentration of lactic acid, produced by lactobacilli, that acidifies the vagina in eubiosis versus a sharp drop in lactic acid and an increase in pH in dysbiosis. Here we review the antimicrobial, antiviral and immunomodulatory properties of lactic acid and the use of lactic acid and lactobacilli probiotics in preventing or treating BV.

Vaginal microbicides: detecting toxicities in vivo that paradoxically increase pathogen transmission.

BACKGROUND: Microbicides must protect against STD pathogens without causing unacceptable toxic effects. Microbicides based on nonoxynol-9 (N9) and other detergents disrupt sperm, HSV and HIV membranes, and these agents are effective contraceptives. But paradoxically N9 fails to protect women against HIV and other STD pathogens, most likely because it causes toxic effects that increase susceptibility. The mouse HSV-2 vaginal transmission model reported here: (a) Directly tests for toxic effects that increase susceptibility to HSV-2, (b) Determines in vivo whether a microbicide can protect against HSV-2 transmission without causing toxicities that increase susceptibility, and (c) Identifies those toxic effects that best correlate with the increased HSV susceptibility. METHODS: Susceptibility was evaluated in progestin-treated mice by delivering a low-dose viral inoculum (0.1 ID50) at various times after delivering the candidate microbicide to detect whether the candidate increased the fraction of mice infected. Ten agents were tested - five detergents: nonionic (N9), cationic (benzalkonium chloride, BZK), anionic (sodium dodecylsulfate, SDS), the pair of detergents in C31G (C14AO and C16B); one surface active agent (chlorhexidine); two non-detergents (BufferGel, and sulfonated polystyrene, SPS); and HEC placebo gel (hydroxyethylcellulose). Toxic effects were evaluated by histology, uptake of a 'dead cell' dye, colposcopy, enumeration of vaginal macrophages, and measurement of inflammatory cytokines. RESULTS: A single dose of N9 protected against HSV-2 for a few minutes but then rapidly increased susceptibility, which reached maximum at 12 hours. When applied at the minimal concentration needed for brief partial protection, all five detergents caused a subsequent increase in susceptibility at 12 hours of approximately 20-30-fold. Surprisingly, colposcopy failed to detect visible signs of the N9 toxic effect that increased susceptibility at 12 hours. Toxic effects that occurred contemporaneously with increased susceptibility were rapid exfoliation and re-growth of epithelial cell layers, entry of macrophages into the vaginal lumen, and release of one or more inflammatory cytokines (Il-1beta, KC, MIP 1alpha, RANTES). The non-detergent microbicides and HEC placebo caused no significant increase in susceptibility or toxic effects. CONCLUSION: This mouse HSV-2 model provides a sensitive method to detect microbicide-induced toxicities that increase susceptibility to infection. In this model, there was no concentration at which detergents provided protection without significantly increasing susceptibility.

Diffusion of Immunoglobulin G in Shed Vaginal Epithelial Cells and in Cell-Free Regions of Human Cervicovaginal Mucus.

Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments.