Lorna K. Rabe

HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis

Summary Background In-vitro research has suggested that bacterial vaginosis may increase the survival of HIV-1 in the genital tract. Therefore, we investigated the association of HIV-1 infection with vaginal flora abnormalities, including bacterial vaginosis and depletion of lactobacilli, after adjustment for sexual activity and the presence of other sexually transmitted diseases (STDs). Methods During the initial survey round of our community-based trial of STD control for HIV-1 prevention in rural Rakai District, southwestern Uganda, we selected 4718 women aged 15–59 years. They provided interview information, blood for HIV-1 and syphilis serology, urine for detection of Chlamydia trachomatis and Neisseria gonorrhoeae , and two self-administered vaginal swabs for culture of Trichomonas vaginalis and gram-stain detection of vaginal flora, classified by standardised, quantitative, morphological scoring. Scores 0–3 were normal vaginal flora (predominant lactobacilli). Higher scores suggested replacement of lactobacilli by gram-negative, anaerobic microorganisms (4–6 intermediate; 7–8 and 9–10 moderate and severe bacterial vaginosis). Findings HIV-1 frequency was 14·2% among women with normal vaginal flora and 26·7% among those with severe bacterial vaginosis (p Interpretation This cross-sectional study cannot show whether disturbed vaginal flora increases susceptibility to HIV-1 infection. Nevertheless, the increased frequency of HIV-1 associated with abnormal flora among younger women, for whom HIV-1 acquisition is likely to be recent, but not among older women, in whom HIV-1 is likely to have been acquired earlier, suggests that loss of lactobacilli or presence of bacterial vaginosis may increase susceptibility to HIV-1 acquisition. If this inference is correct, control of bacterial vaginosis could reduce HIV-1 transmission.

Colonization of the Rectum by Lactobacillus Species and Decreased Risk of Bacterial Vaginosis

Lactobacilli colonizing the rectum may be a reservoir for vaginal lactobacilli. In a cross-sectional study of 531 females, vaginal and rectal colonization by lactobacilli were assessed by culture methods. A subset of isolates was identified to the species level by use of whole-chromosomal DNA probes. Lactobacillus crispatus (16%), L. jensenii (10%), and L. gasseri (10%) were the prevalent lactobacilli colonizing the rectums of 290 females. Only 13 (9%) of 147 females colonized by L. crispatus or L. jensenii vaginally and/or rectally had bacterial vaginosis (BV), compared with 12 (44%) of 27 females colonized by other H(2)O(2)-producing lactobacilli (P < .001). Cocolonization of the vagina and rectum by H(2)O(2)-producing lactobacilli was associated with the lowest prevalence of BV (5%), whereas females colonized only vaginally, only rectally, or at neither site had a successively increased risk of BV (P < .001). Lactobacillus species in the rectum may contribute to the maintenance of vaginal microflora.

First Phase 1 Double-Blind, Placebo-Controlled, Randomized Rectal Microbicide Trial Using UC781 Gel with a Novel Index of Ex Vivo Efficacy

Objectives Successful control of the HIV/AIDS pandemic requires reduction of HIV-1 transmission at sexually-exposed mucosae. No prevention studies of the higher-risk rectal compartment exist. We report the first-in-field Phase 1 trial of a rectally-applied, vaginally-formulated microbicide gel with the RT-inhibitor UC781 measuring clinical and mucosal safety, acceptability and plasma drug levels. A first-in-Phase 1 assessment of preliminary pharmacodynamics was included by measuring changes in ex vivo HIV-1 suppression in rectal biopsy tissue after exposure to product in vivo. Methods HIV-1 seronegative, sexually-abstinent men and women (N = 36) were randomized in a double-blind, placebo-controlled trial comparing UC781 gel at two concentrations (0.1%, 0.25%) with placebo gel (1∶1∶1). Baseline, single-dose exposure and a separate, 7-day at-home dosing were assessed. Safety and acceptability were primary endpoints. Changes in colorectal mucosal markers and UC781 plasma drug levels were secondary endpoints; ex vivo biopsy infectibility was an ancillary endpoint. Results All 36 subjects enrolled completed the 7–14 week trial (100% retention) including 3 flexible sigmoidoscopies, each with 28 biopsies (14 at 10 cm; 14 at 30 cm). There were 81 Grade 1 adverse events (AEs) and 8 Grade 2; no Grade 3, 4 or procedure-related AEs were reported. Acceptability was high, including likelihood of future use. No changes in mucosal immunoinflammatory markers were identified. Plasma levels of UC781 were not detected. Ex vivo infection of biopsies using two titers of HIV-1BaL showed marked suppression of p24 in tissues exposed in vivo to 0.25% UC781; strong trends of suppression were seen with the lower 0.1% UC781 concentration. Conclusions Single and 7-day topical rectal exposure to both concentrations of UC781 were safe with no significant AEs, high acceptability, no detected plasma drug levels and no significant mucosal changes. Ex vivo biopsy infections demonstrated marked suppression of HIV infectibility, identifying a potential early biomarker of efficacy. (Registered at ClinicalTrials.gov; #NCT00408538)

Phase 1 Safety, Pharmacokinetics, and Pharmacodynamics of Dapivirine and Maraviroc Vaginal Rings: a Double-Blind Randomized Trial

Background:Variable adherence limits effectiveness of daily oral and intravaginal tenofovir-containing pre-exposure prophylaxis. Monthly vaginal antiretroviral rings are one approach to improve adherence and drug delivery. Methods:MTN-013/IPM 026, a multisite, double-blind, randomized, placebo-controlled trial in 48 HIV-negative US women, evaluated vaginal rings containing dapivirine (DPV) (25 mg) and maraviroc (MVC) (100 mg), DPV only, MVC only, and placebo used continuously for 28 days. Safety was assessed by adverse events. Drug concentrations were quantified in plasma, cervicovaginal fluid (CVF), and cervical tissue. Cervical biopsy explants were challenged with HIV ex vivo to evaluate pharmacodynamics. Results:There was no difference in related genitourinary adverse events between treatment arms compared with placebo. DPV and MVC concentrations rose higher initially before falling more rapidly with the combination ring compared with relatively stable concentrations with the single-drug rings. DPV concentrations in CVF were 1 and 5 log10 greater than cervical tissue and plasma for both rings. MVC was consistently detected only in CVF. DPV and MVC CVF and DPV tissue concentrations dropped rapidly after ring removal. Cervical tissue showed a significant inverse linear relationship between HIV replication and DPV levels. Conclusions:In this first study of a combination microbicide vaginal ring, all 4 rings were safe and well tolerated. Tissue DPV concentrations were 1000 times greater than plasma concentrations and single drug rings had more stable pharmacokinetics. DPV, but not MVC, demonstrated concentration-dependent inhibition of HIV-1 infection in cervical tissue. Because MVC concentrations were consistently detectable only in CVF and not in plasma, improved drug release of MVC rings is needed.

The vaginal microflora of pig-tailed macaques and the effects of chlorhexidine and benzalkonium on this ecosystem.

Background and Objectives: To characterize normal vaginal microflora of pig‐tailed macaques and to evaluate two commonly used intravaginal compounds, chlorhexidine (CHG), a vaginal antiseptic (Surgilube, E. Fougera, Melville, NY), and benzalkonium chloride (BZK) (spermicidal contraceptive) in this monkey model to assess effects on the vaginal microflora. Study Design: Vaginal swabs were collected for microbiologic analysis to characterize normal flora. Subsequently, the vagina was exposed to either CHG or BZK twice at 24‐hour intervals. Results: The vaginal microflora of 26 pig‐tailed macaques was found to be remarkably similar to the vaginal flora of the human with respect to frequency of vaginal colonization by H2O2‐producing lactobacilli, Prevotella species, and several other microorganisms. After two vaginal applications at 24‐hour intervals, CHG had only small effects on the vaginal microflora of five animals. By contrast, BZK applied by the same protocol had profound adverse effects on the lactobacilli and Peptostreptococcus and more transient effects on vaginal Prevotella and viridans streptococci of six animals. Conclusions: These observations demonstrate that the vaginal microflora of the pig‐tailed macaque is a useful model in which to further evaluate newly developed intravaginal contraceptives that may be microbicidal and/or virucidal before widespread intravaginal use in women.

Comparative Genomic Analyses of 17 Clinical Isolates of Gardnerella vaginalis Provide Evidence of Multiple Genetically Isolated Clades Consistent with Subspeciation into Genovars

Gardnerella vaginalis is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen G. vaginalis isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the G. vaginalis strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strains genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets. G. vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.

The Effects of Three Nonoxynol-9 Preparations on Vaginal Flora and Epithelium

To evaluate the effects of nonoxynol-9 (N-9) on the vaginal flora and epithelium, 48 women (16 in each group) were evaluated by use of quantitative vaginal cultures and colposcopy. at baseline and at 0.5, 4, 24, 48, and 72 h after insertion of one of three N-9 preparations (4% gel [Conceptrol], 3.5% gel [Advantage-24], or a 28% vaginal contraceptive film). The proportion positive for H2O2+ or H2O2- lactobacilli did not change significantly with any of the preparations, but lactobacilli concentrations decreased transiently. Both the proportion of women with Gardnerella vaginalis and the concentration of G. vaginalis decreased transiently. The proportion of women with Escherichia coli increased with the 4% gel, and the concentration increased with all preparations. The number with anaerobic gram-negative rods increased, although the concentrations decreased. Symptoms and colposcopic abnormalities were rare. Changes in levels of vaginal bacteria were transient after single applications of N-9, but adverse effects may be enhanced with frequent, chronic use.

Safety and Efficacy Evaluations for Vaginal and Rectal Use of BufferGel in the Macaque Model

Background: The nonhuman primate model allows for safety and efficacy testing of topical microbicide products. Goal: The goal of this study was to evaluate the safety and efficacy of vaginal and rectal applications of BufferGel (ReProtect, Inc.). Study Design: The safety of repeated product applications was evaluated by microflora, pH, vaginal colposcopy, and rectal lavage. To test efficacy in preventing chlamydia, infection was documented by culture and nucleic acid amplification tests. Results: Repeated vaginal or rectal applications of BufferGel were not associated with significant changes in microflora. BufferGel use had a transient acidifying effect on vaginal and rectal pH. Colposcopic observations remained relatively normal in all test animals. A slightly increased incidence of epithelial desquamation was noted after rectal product use compared with the control group. BufferGel did not prevent cervical or rectal chlamydial infection. Conclusion: BufferGel has an acceptable safety profile after repeated vaginal and rectal use, but does not prevent chlamydial infection in the macaque models.

Safe and Sustained Vaginal Delivery of Pyrimidinedione HIV-1 Inhibitors from Polyurethane Intravaginal Rings

ABSTRACT The potent antiretroviral pyrimidinediones IQP-0528 (PYD1) and IQP-0532 (PYD2) were formulated in polyurethane intravaginal rings (IVRs) as prophylactic drug delivery systems to prevent the sexual transmission of HIV-1. To aid in the selection of a pyrimidinedione candidate and the optimal loading of the drug in the IVR delivery system, four pyrimidinedione IVR formulations (PYD1 at 0.5 wt% [PYD10.5wt%], PYD11wt%, PYD24wt%, and PYD214wt%) were evaluated in pigtail macaques over 28 days for safety and pyrimidinedione vaginal biodistribution. Kinetic analysis of vaginal proinflammatory cytokines, native microflora, and drug levels suggested that all formulations were safe, but only the high-loaded PYD214wt% IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 μg/ml to vaginal fluid and 1 μg/g to vaginal tissue, a level over 1,000 times the in vitro 50% effective concentration. The in vitro release of PYD1 and PYD2 under nonsink conditions correlated well with in vivo release, both in amount and in kinetic profile, and therefore may serve as a more biologically relevant means of evaluating release in vitro than typically employed sink conditions. Lastly, the pyrimidinediones in the IVR formulation were chemically stable after 90 days of storage at elevated temperature, and the potent nanomolar-level antiviral activity of both molecules was retained after in vitro release. Altogether, these results point to the successful IVR formulation and vaginal biodistribution of the pyrimidinediones and demonstrate the usefulness of the pigtail macaque model in evaluating and screening antiretroviral IVR formulations prior to preclinical and clinical evaluation.

Effects of nonoxynol-9 on vaginal microflora and chlamydial infection in a monkey model.

Background and Objectives: Nonoxynol‐9, an intravaginal microbicide, is chlamydiacidal in vitro but also cytotoxic. This study examines the effects of nonoxynol‐9 in vivo, using a pigtail macaque model of chlamydial cervicitis. Goals: To establish a minimum infectious dose of Chlamydia trachomatis in the macaque, and to observe the effects of a single dose of nonoxynol‐9 on efficiency of chlamydial infection, vaginal microflora, and cervicovaginal irritation. Study Design: The effects of 4% nonoxynol‐9, C. trachomatis (5,000 or 10,000 IFU) or both nonoxynol‐9 application and chlamydial infection were studied in 17 macaques. Results: Following a single application of nonoxynol‐9, chlamydial infection was prevented in 4 of 6 monkeys infected with 10,000 IFU; there was a transient decrease in anaerobic gram‐negative rods (P < 0.05) and Peptostreptococci (P > 0.05), but no change in Lactobacillus. Mild cervicovaginal irritation was observed in the monkeys. Conclusions: A single dose of nonoxynol‐9 causes minimal vaginal flora and epithelial irritation, and may be useful for prevention of chlamydial infection.