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

Abstract

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 HIV_Ba-L was significantly associated with the protonated form of lactic acid in cervicovaginal fluid. The HIV_Ba-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.

Keywords: HIV transmission; Lactobacillus; human immunodeficiency virus; metabolite; microbiota; vagina.

FIG 1

Most native, minimally diluted CVF samples fully inactivate HIV_Ba-L (A) and the transmitted/founder HIV_RHPA strain (B); however, inactivation is lost upon neutralization of CVF. Lines connect HIV_Ba-L and HIV_RHPA infectivity measurements from native and neutralized aliquots of the same CVF sample. HIV_12 and HIV_25 CVF samples are indicated. Data represent CVF samples from 22 distinct samples (from n = 20 women) (A) and 4 distinct samples (from n = 4 women) (B). Note that data points overlay for several samples in panel A.

FIG 2

HIV_Ba-L inactivation in CVF is significantly associated with percent d+l-protonated lactic acid but not with percent d+l-lactate anion or with pH. Generalized linear mixed modeling (GLMM) was used to estimate the association between HIV_Ba-L infectivity per milliliter and CVF sample percent d+l-protonated lactic acid (A), percent d+l-lactate anion (B), and pH (C). Observed data from native, minimally diluted, 3-fold diluted, 9-fold diluted, 27-fold diluted, and neutralized CVF samples are depicted by open black circles and predicted values in filled red circles. Given the discrete (nonnormal) nature of the HIV_Ba-L infectivity outcome data, linear modeling was generalized by a Poisson distribution and log-link function. To account for inherent dependency in the data from repeated measures of HIV_Ba-L infectivity after CVF treatment, GLMM analyses specified a participant-specific random intercept effectively modeling the data corresponding to the heterogeneity between participants in person-specific HIV_Ba-L infectivity levels which induce correlation across serial measurements. Modeling data from unadjusted (univariable) analyses are shown where associations reached statistical significance for infectivity per milliliter versus percent d+l-protonated lactic acid (P < 0.001) but not versus percent d+l-lactate anion (P = 0.280) or pH (P = 0.139). Data represent CVF samples from 22 distinct samples (from n = 20 women).

FIG 3

HIV_Ba-L inactivation is significantly associated with both percent d-protonated lactic acid and percent l-protonated lactic acid. Generalized linear mixed modeling (GLMM) was used to estimate the association between HIV_Ba-L infectivity per milliliter and CVF sample percent l-protonated lactic acid (A) and percent d-protonated lactic acid (B). Observed data from native, minimally diluted, 3-fold diluted, 9-fold diluted, 27-fold diluted, and neutralized CVF samples are depicted by open black circles and predicted values in filled red circles. Given the discrete (nonnormal) nature of the HIV_Ba-L infectivity outcome data, linear modeling was generalized by a Poisson distribution and log-link function. To account for inherent dependency in the data from repeated measures of HIV_Ba-L infectivity after CVF treatment, GLMM analyses specified a participant-specific random intercept effectively modeling the data corresponding to the heterogeneity between participants in person-specific HIV_Ba-L infectivity levels which induce correlation across serial measurements. Modeling data from unadjusted (univariable) analyses are shown where associations reached statistical significance for infectivity per milliliter versus percent l-protonated lactic acid (P < 0.001) and percent d-protonated lactic acid (P < 0.001). Data represent CVF samples from 22 distinct samples (from n = 20 women).

FIG 4

Anti-HIV_Ba-L activity was present in the acidic <3-kDa-MW CVF fraction and was retained after digestion with pepsin. (A) Data represent anti-HIV_Ba-L activity of pooled native CVF, including unmodified (Native) activity and activity at neutral pH (Native pH 7), separated into a low-molecular-weight <3-kDa fraction by centrifugation through a 3-kDa-MWCO membrane at the original acidic pH (Filtrate) or neutralized pH (Filtrate pH 7) or the ≥3-kDa retentate that was extensively washed with PBS− and resuspended in medium to the original native CVF volume (Pellet pH 7). Data represent results from an individual (n = 1) assay of three separate sets of pooled CVF samples. (B) Anti-HIV_Ba-L activity of pooled acidic CVF (pH 4.13, clarified by centrifugation, i.e., supernatant), including unmodified (Neat), neutralized pH (Neat pH 7), pepsin-digested, and <3-kDa filtrate recovered by centrifugation and tested at the original acidic pH 4.15 (Pepsin filtrate) or adjusted to neutral pH (Pepsin filtrate pH 7). HIV_Ba-L infectivity was normalized to infectivity measured in DMEM-50. The limit of detection was 1.5 × 10³ infectious units/ml. Error bars denote standard deviations from n = 3 independent assays.

FIG 5

16S rRNA gene sequencing of CVF samples reveals groups with distinct vaginal microbiota, dominated by Lactobacillus crispatus (CST I), L. iners (CST III), or L. jensenii (CST V). Colored bars indicate the abundance of different bacterial species as a proportion of all species in the sample. CVF sample numbers (from n = 13 participants) are indicated at the bottom of the heat map.

FIG 6

Anti-HIV_Ba-L activity and percent d+l-protonated lactic acid (wt/vol) levels of CVF samples with distinct vaginal microbiota dominated by Lactobacillus spp. (A) HIV_Ba-L infectivity after incubation in CVF samples (HIV_15 to HIV_27) with community state types (CST) dominated by L. crispatus (CST I), L. iners (CST III), or L. jensenii (CST V). Samples represented below the dotted horizontal line were below the detection limit of the infectivity assay. (B) Levels of percent d+l-protonated lactic acid in CVF samples (HIV_15 to HIV_27) categorized as CST (I), CST III, and CST V. Solid horizontal lines represent the means.