Vaginal concentrations of lactic acid potently inactivate HIV

Abstract

Objectives: When Lactobacillus spp. dominate the vaginal microbiota of women of reproductive age they acidify the vagina to pH <4.0 by producing ∼1% lactic acid in a nearly racemic mixture of d- and l-isomers. We determined the HIV virucidal activity of racemic lactic acid, and its d- and l-isomers, compared with acetic acid and acidity alone (by the addition of HCl).

Methods: HIV-1 and HIV-2 were transiently treated with acids in the absence or presence of human genital secretions at 37°C for different time intervals, then immediately neutralized and residual infectivity determined in the TZM-bl reporter cell line.

Results: l-lactic acid at 0.3% (w/w) was 17-fold more potent than d-lactic acid in inactivating HIVBa-L. Complete inactivation of different HIV-1 subtypes and HIV-2 was achieved with ≥0.4% (w/w) l-lactic acid. At a typical vaginal pH of 3.8, l-lactic acid at 1% (w/w) more potently and rapidly inactivated HIVBa-L and HIV-1 transmitter/founder strains compared with 1% (w/w) acetic acid and with acidity alone, all adjusted to pH 3.8. A final concentration of 1% (w/w) l-lactic acid maximally inactivated HIVBa-L in the presence of cervicovaginal secretions and seminal plasma. The anti-HIV activity of l-lactic acid was pH dependent, being abrogated at neutral pH, indicating that its virucidal activity is mediated by protonated lactic acid and not the lactate anion.

Conclusions: l-lactic acid at physiological concentrations demonstrates potent HIV virucidal activity distinct from acidity alone and greater than acetic acid, suggesting a protective role in the sexual transmission of HIV.

Keywords: carboxylic acids; female reproductive tract; vaginal lactobacilli; virucidal.

Figure 1.

Inactivation of HIV_Ba-L by l-, d- and dl-lactic acid. (a) HIV_Ba-L was incubated with different forms of lactic acid at 37°C for 30 min without pH adjustment; data are the mean of four independent assays. (b) HIV_Ba-L was incubated in the presence of different forms of lactic acid adjusted to pH 4.0 prior to virus addition or with media adjusted to pH 4.0 with HCl and incubated at 37°C for 30 min followed by immediate neutralization. The lower limit of detection of virus in these assays was 30 infectious units/mL and the data are the mean of at least six independent assays. Viral infectivity was determined in the TZM-bl reporter cell line and expressed relative to virus incubated in the absence of acid. Error bars denote standard error of the mean. Statistically significant differences were determined using the Wilcoxon rank-sum test. LA, lactic acid.

Figure 2.

HIV-1 and HIV-2 virucidal activity of l-lactic acid. HIV was incubated in the presence of l-lactic acid at 37°C for 30 min without pH adjustment, neutralized and viral infectivity determined in TZM-bl cells. (a) Inactivation of X4 HIV-1 strains CB1-br (subtype B) and CMU02 (subtype E/A), and the X4 HIV-2 strain CDC310319; (b) dual tropic HIV-1 strains MACS1-spln (subtype B) and 93BR020 (subtype F); (c) R5 HIV-1 strains MACS3-LN (subtype B), 92RW016 (subtype A) and 92BR025 (subtype C); and (d) subtype B transmitter/founder strains RHPA (F, isolated from a female subject) and CH058 (M, isolated from a male subject). The pH of the media following the addition of l-lactic acid and incubation is expressed as the mean ± standard error (a). Data were obtained from three independent assays and error bars denote the standard error of the mean.

Figure 3.

Acid inactivation time-course study of HIV_Ba-L at pH 3.8. Media acidified with l-lactic acid, acetic acid or HCl were adjusted to pH 3.8 prior to addition to virus; the pH was monitored and adjusted if required during incubation at 37°C. At indicated times an aliquot was removed, neutralized and viral infectivity determined in TZM-bl cells and expressed relative to virus incubated in the absence of acid. Data were obtained from four independent assays and error bars denote the standard error of the mean.

Figure 4.

l-lactic acid inactivation of HIV-1 in the presence of genital secretions. HIV_Ba-L was incubated in the presence of l-lactic acid at 37°C for 30 min, without pH adjustment, in the absence and presence of 50% CVS (a) or 75% SP (b), neutralized and viral infectivity determined in TZM-bl cells relative to the appropriate control incubated in the absence of acid. The final pH of the media following mixing of SP, virus and lactic acid is shown above the bars (b). The data were obtained from three independent assays and error bars denote the standard error of the mean.

Figure 5.

HIV-1 virucidal activity of protonated l-lactic acid compared with l-lactate anion. (a) Protonated lactic acid exists in equilibrium with the lactate anion at pH 3.9. (b) HIV_Ba-L was incubated at 37°C for 30 min with l-lactic acid adjusted to pH 3.8, l-lactic acid adjusted to pH 7.0 or l-sodium lactate (l-NaLactate) at pH 7.0, with the pH adjusted prior to virus addition. After neutralization, infectivity was determined in TZM-bl cells. The data were obtained from three independent assays and error bars denote the standard error of the mean.