Twice-daily application of HIV microbicides alter the vaginal microbiota

Abstract

Vaginal HIV microbicides offer great promise in preventing HIV transmission, but failures of phase 3 clinical trials, in which microbicide-treated subjects had an increased risk of HIV transmission, raised concerns about endpoints used to evaluate microbicide safety. A possible explanation for the increased transmission risk is that the agents shifted the vaginal bacterial community, resulting in loss of natural protection and enhanced HIV transmission susceptibility. We characterized vaginal microbiota, using pyrosequencing of bar-coded 16S rRNA gene fragments, in samples from 35 healthy, sexually abstinent female volunteer subjects (ages 18 to 50 years) with regular menses in a repeat phase 1 study of twice-daily application over 13.5 days of 1 of 3 gel products: a hydroxyethylcellulose (HEC)-based "universal" placebo (10 subjects), 6% cellulose sulfate (CS; 13 subjects), and 4% nonoxynol-9 (N-9; 12 subjects). We used mixed effects models inferred using Bayesian Markov chain Monte Carlo methods, which showed that treatment with active agents shifted the microbiota toward a community type lacking significant numbers of Lactobacillus spp. and dominated by strict anaerobes. This state of the vaginal microbiota was associated with a low or intermediate Nugent score and was not identical to bacterial vaginosis, an HIV transmission risk factor. The placebo arm contained a higher proportion of communities dominated by Lactobacillus spp., particularly L. crispatus, throughout treatment. The data suggest that molecular evaluation of microbicide effects on vaginal microbiota may be a critical endpoint that should be incorporated in early clinical assessment of microbicide candidates.

Importance: Despite large prevention efforts, HIV transmission and acquisition rates remain unacceptably high. In developing countries, transmission mainly occurs through heterosexual intercourse, where women are significantly more vulnerable to infection than men. Vaginal microbicides are considered to be one of the most promising female-controlled products, in that women themselves insert the microbicides into the vagina to prevent HIV transmission during sexual intercourse. The failure of several microbicides in clinical trials has raised questions concerning the low in vivo efficacy of such anti-HIV molecules. This study was designed to gain insights into the failures of two microbicides by testing the hypothesis that the microbicides negatively affect a critical line of defense against HIV, the vaginal microbiota. The results suggest that in the early assessment of candidate microbicides, culture-independent evaluation of their effect on the vaginal microbiota should be considered and may constitute a critical endpoint.

FIG 1

(A) Schematic of the clinical study design. Twice-daily application of the gel products is indicated in green. Idealized menses are indicated in red. Vaginal swabs were collected at visits 2, 3, 4, 5, and 6. (B) Heatmap of phylotype relative abundances from 146 samples. Ward-linkage hierarchical clustering was performed on the Jensen-Shannon divergences between all pairs of community states to obtain five community state types. Nugent score categories (high, 7 to 10; intermediate [Int], 4 to 6; low, 0 to 3), treatment arms, and visits are indicated. The top 25 most abundant phylotypes are shown. The frequency of each community state type is shown in Table 2.

FIG 2

(A) Interaction plot of the mean values and error bars of the log counts of the community state types versus Nugent score category contingency table (see Table S4 in the supplemental material) stratified by community state type and Nugent category, with error bars indicating 95% credible intervals derived from model 2 (described in Materials and Methods). (B) Interaction plot of the mean log(odds) values and the corresponding 95% credible interval of logistic regression model 1, evaluating the association between CSTs (community state types) at visit V4 compared to visits V2 and V3 for each product use arm. In the N-9 (V4) and CS (cellulose sulfate) (V4 and V5) arms, no communities were assigned to CST II. In addition, in the N-9 arm (V4), no communities were assigned to CST III (Fig. 4).

FIG 3

(A) Nugent score category proportions for each treatment and visit. (B) Nugent score categories (high, 7 to 10; intermediate, 4 to 6; low, 0 to 3). There is no significant difference between the values from the baseline and visits V4, V5, and V6.

FIG 4

An interaction plot of the mean values and error bars of the distance from the center of CST IV-A for different treatment arms and visits, based on the mixed effects model (model 1). Error bars indicate 95% credible intervals. The higher frequency of CST IV-A at visits V4 and V5 for treatments with CS and N-9 is notable.