Lactic acid produced by optimal vaginal Lactobacillus species potently inactivates HIV-1 by several mechanisms including promoting inhibition of virion-associated reverse transcriptase activity and viral RNA degradation

doi:10.21203/rs.3.rs-4447264/v1

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Lactic acid produced by optimal vaginal Lactobacillus species potently inactivates HIV-1 by several mechanisms including promoting inhibition of virion-associated reverse transcriptase activity and viral RNA degradation

https://doi.org/10.21203/rs.3.rs-4447264/v1

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Background. An optimal vaginal microbiota dominated by Lactobacillus spp. protects women against acquiring and transmitting HIV in contrast to a nonoptimal vaginal microbiota exemplified by bacterial vaginosis (BV); however, the virucidal activity of carboxylic acid metabolites present in vaginal fluid is not well defined. Here we determined the HIV-1 virucidal activity of lactic acid (LA), short chain fatty acids (SCFAs) and succinic acid under conditions observed in women with a Lactobacillus-dominated vaginal microbiota compared to women with BV and examined the mechanism by which LA inactivates HIV-1. The ability of LA to inactivate HSV-2 and HPV16 was also examined.

Results: LA was >10-fold more potent at inactivating an HIV-1 transmitted/founder strain than SCFAs (acetic, butyric, and propionic acid) and succinic acid when tested at an equivalent 20 mM of protonated acid at pH 4.2 (p£0.05). While LA decreased HIV-1 infectivity by >10³-fold, virions were intact, expressing a similar gp120:p24 ratio, and showed a 2-fold decrease in CD4 binding compared to the untreated control (p£0.05). Treatment of recombinant gp120 with LA revealed no major conformational changes by small angle X-ray scattering. LA treatment of HIV-1 at pH 3.8 resulted in an 80% decrease in virion-associated reverse transcriptase activity compared to untreated virus, which was more potent than acetic acid or HCl-adjusted media at pH 3.8. LA decreased HIV-1 virion-associated RNA levels by ~50% compared to untreated virus (p<0.001), acetic acid or HCl acidified media, with this effect potentiated in the presence of cervicovaginal fluid. In contrast, HSV-2 virucidal activity of LA was similar to acetic acid and HCl-acidified media while HPV16 was acid-resistant.

Conclusions: These findings reveal LA’s potent and specific HIV-1 virucidal activity, mediated by its membrane permeant properties, compared to SCFAs and succinic acid, with implications for the vaginal transmission of HIV-1 to partners and neonates during birth.

HIV

retroviruses

Lactobacilli

optimal vaginal microbiota

bacterial vaginosis

lactic acid

short chain fatty acids

succinic acid

STIs

HSV-2

Heterosexual transmission accounts for the majority of HIV infections worldwide, yet HIV transmission risk in women following receptive vaginal intercourse is low, estimated at 0.08% [1]. This is due in part to the combined physical and immunobiological defences of the lower female reproductive tract (FRT) against invading pathogens, although pathogenic and commensal microorganisms also influence HIV transmission. Sexually transmitted infections (STIs) including herpes simplex virus (HSV), chlamydia, gonorrhoea and fungal infections are associated with increased HIV acquisition in women [2–6]. Commensal microbes also influence HIV transmission risk as women with an optimal vaginal microbiota dominated by Lactobacillus spp. have an approximately 4.4-7.2-fold lower risk of HIV infection as well as a decreased risk of other bacterial and viral STIs as compared to women with a non-optimal microbiota [7–10]. An optimal vaginal microbiota is associated with decreased levels of vaginal HIV-1 RNA and a reduced risk of a women transmitting HIV to male sexual partners and neonates [7, 11–13]. These findings highlight the importance of the vaginal microbiota in modulating HIV acquisition in women; however, the specific microbial components, including their metabolites, involved in modulating this risk and the mechanisms through which these effects are conferred remain poorly understood.

Unlike the gut, where microbial diversity is advantageous, an optimal lower FRT microbiota in women of reproductive age is dominated by Lactobacillus spp., with L. crispatus associated with greatest protection against HIV infection [7, 8, 10]. In contrast, a non-optimal vaginal microbiota, exemplified by the vaginal dysbiotic condition, bacterial vaginosis (BV), comprises a high relative abundance of diverse obligate and facultative anaerobic bacteria such as Prevotella spp., Fannyhessea vaginae (previously Atopobium vaginae) and Gardnerella spp. and is depleted of Lactobacillus spp.[14]. Globally, BV affects approximately one in three women of reproductive age [15–17] and is associated with increased risk of a range of adverse sexual and reproductive health outcomes including spontaneous pre-term birth [18–21], and acquisition of STIs [7, 22] including HIV [2, 23, 24] compared to women with an optimal Lactobacillus-dominated microbiota. One mechanism by which lower FRT microbes influence HIV transmission is through modifying the physical and immunological properties of the cervicovaginal environment. BV is associated with increased genital inflammation, and in some but not all studies, an increase in activated HIV target cells in the cervix [25–31], which likely contributes to a heightened risk of HIV acquisition. In contrast, optimal Lactobacillus spp. are associated with lower genital inflammation and promote epithelial barrier integrity and wound healing [32–34].

A major mechanism through which optimal and non-optimal FRT microbiota exert these effects on the host and/or other microorganisms is through the production of bacterial carboxylic acid metabolites including short chain fatty acids (SCFA), succinic acid and lactic acid (LA; an alpha hydroxy acid). Lactobacillus spp. produce LA, which reaches concentrations of ∼100 mM, and acidify the vagina to a pH as low as 3.5 [35, 36]. Vaginal acidification through LA production is regarded as a key defence in the lower FRT, generating an environment inhospitable to most exogenous microorganisms [37]. LA has a pKa of 3.86, thus in the context of an optimal vaginal microbiota where the pH is ≤4.5, LA is predominantly found in the protonated (uncharged) form in contrast to at pH > 4.5, where the lactate anion (charged) dominates [38].

We have shown that protonated LA elicits a range of protective responses from cervicovaginal epithelial cells including maintaining an anti-inflammatory state and enhancing barrier integrity [34, 39, 40]. Protonated LA also has potent bactericidal and virucidal properties. LA inactivates 17 different BV-associated bacteria but not vaginal Lactobacillus spp. [41], Chlamydia trachomatis [42], Neisseria gonorrhoeae [43] and also is virucidal against herpes simplex virus (HSV) [44, 45] and HIV (HIV-1 and HIV-2) as demonstrated in vitro [38] and ex vivo in cervicovaginal fluid (CVF) [46]. Levels of endogenous LA within CVF from women with an optimal vaginal microbiota correlate positively with HIV-1 virucidal activity [46], suggesting this metabolite contributes to the virucidal properties of CVF from women with an optimal microbiota.

LA exists as L- and D-isomers, which are differentially produced by various Lactobacillus spp. [47, 48], with L-LA exhibiting more potent HIV-1 virucidal activity at threshold concentrations [38]. In contrast, D-LA has greater potency in blocking chlamydia infection of cervicovaginal epithelial cells in vitro [49] and is associated with enhanced trapping of HIV-1 particles in cervicovaginal mucous from women colonised with a L. crispatus-dominated vaginal microbiota [50, 51]. In contrast to the LA-enriched environment of an optimal vaginal microbiota, women with BV exhibit increased vaginal pH > 4.5, reduced vaginal concentrations of LA, including protonated LA, and a concomitant increase in the short chain fatty acids (SCFAs; acetic, propionic, butyric acid) and succinic acid [52–54]. Certain SCFA associated with BV elicit heightened production of inflammatory cytokines including TNFα from FRT epithelial cells in vitro [40], but their effect on the viability of viral STIs such as HIV are not known.

The role of LA and other vaginal microbiota carboxylic acid metabolites and their contribution to BV-associated STI risks, remains poorly understood. The few studies that have sought to determine the role of SCFAs in BV have primarily focused on their potential immune modulatory [40, 54] and barrier abrogating effects [55] on FRT epithelial cells. The study of their potential bactericidal or virucidal properties has been neglected even though many STI causing viruses, including HIV, are known to be acid labile [56–59]. In addition, the mechanism of viral inactivation and the discrete virucidal effects of specific carboxylic acids found in vaginal fluid as compared to low pH alone (HCl adjusted) have not been investigated. Here, we compared the virucidal activity of LA and BV-associated SCFA and succinic acid under physiological conditions and investigated the mechanisms through which LA inactivates HIV-1, to inform the design of therapeutic modulators of the lower FRT environment to protect women from acquiring and transmitting HIV.

Cell culture and virus production

The HIV-1 permissive TZM-bl reporter cell line [60] and 293T cells were obtained through the NIH HIV Reagent Program and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; ThermoFisher, Waltham, MA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS; Sigma-Aldrich, St Louis, MO), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (all from ThermoFisher; DMEM-10). Phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) from HIV seronegative donors were prepared as previously described [38] prior to infection with HIV. Infectious HIV-1 subtype B transmitted/founder strain, HIV_RHPA, was generated from the molecular clone pRHPA.c/2635 (HIV Reagent Program) by calcium phosphate transfection of 239T cells followed by propagation in human PBMCs as described previously [38]. The HIV-1 subtype B CCR5-coreceptor using HIV_Ba−L was propagated by infection of PHA-stimulated PBMCs and purified by ultracentrifugation through a sucrose cushion as previously described [38, 46].

Treatment of HIV-1 with vaginal acids

DL-LA was prepared from an 85% (w/w) solution (Sigma-Aldrich), D-LA was prepared from D-(-)-LA crystalline powder (Chem-Impex International Wood Dale, IL, USA), L-LA was prepared from a 30% (w/w) L-(+)-LA solution (Sigma-Aldrich), acetic acid was prepared from 99.5% (w/w) glacial acetic acid (Merck, Kenilworth, NJ), and sodium lactate was prepared from ~ 98% crystalline powder (Sigma-Aldrich). Solutions were pH-adjusted using hydrochloric acid (HCl) or sodium hydroxide (Sigma-Aldrich). All acids were of American Chemical Society (ACS) or reagent grade. All stock solutions were volumetrically prepared to 1 M stock solutions.

To simulate vaginal carboxylic acid composition associated with an optimal microbiota, HIV-1 virions were treated in DMEM-10 containing DL-lactic acid alone (100 mM, adjusted to pH 3.8) or a mixture (DL-LA + optimal SCFA) of 100 mM lactic acid, 4 mM acetic acid, 1 mM propionic acid, 1 mM butyric acid, and 1 mM succinic acid (adjusted to pH 3.8) to reflect levels of LA, SCFA and succinic acid associated with a Lactobacillus-dominated microbiota (Supplementary Table 1)[54]. To simulate vaginal acid composition associated with BV, HIV-1 virions were treated with acetic acid (100 mM, pH 5.0) or a mixture (Non-optimal SCFA) of acids comprising of 20 mM lactic acid, 100 mM acetic acid, 2 mM propionic acid, 2 mM butyric acid, and 20 mM succinic acid (adjusted to pH 5.0) (Supplementary Table 1). DMEM-10 adjusted to the corresponding pH with HCl alone was included in all experiments as a control. Virus was incubated for 5, 10 and 30 min at 37°C with continuous gentle stirring. At each time-point 200 µL aliquots of treated virus were removed and immediately neutralized by 10-fold dilution in DMEM-10 containing HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Viral infectivity was determined in the TZM-bl reporter cells using the β-galactosidase infectivity assay to quantify blue-stained HIV-infected cells as previously described [38].

To directly compare virucidal activity between acids with different acid dissociation constants (i.e. LA pK_a 3.86; acetic acid pK_a 4.76, propionic acid pK_a 4.88; butyric acid pK_a 4.83; succinic acid pK_a 4.16 and 5.61), HIV_RHPA was treated at 37°C with an equimolar 20 mM concentration of protonated vaginal carboxylic acids, calculated for a specific pH of 4.2 using the Henderson-Hasselbalch equation: % Dissociation = 100/(1 + 10^{(−1(pH−pKa)}) [35]. Since the anion concentration contributes to the osmolality of the treatment solution and varies for each carboxylic acid, sodium lactate was employed as an osmolality control (~ 473 mOsm/kg) where osmolality was greater than the highest osmolality for any of the acid treatments (∼357 mOsm/kg). HIV-1 virucidal activity of the acids and viral infectivity were determined as above.

Iodixanol velocity gradient purification of lactic acid treated HIV-1.

Iodixanol (OptiPrep) solutions (Axis-Shield, Oslo, Norway) were prepared by diluting Optiprep (60% w/v iodixanol in water) with Mg²⁺ and Ca²⁺ free phosphate buffered saline (PBS-) to give four density solutions of 6%, 10%, 14% and 18% (w/v) which were overlayed from most to least dense and allowed to diffuse overnight at 4°C to generate a linear gradient as according to the manufacturer’s recommendations. Virus samples were ultracentrifuged at 250,000 x g for 1.5 h at 4°C, five 2 ml fractions were collected and the top two and bottom three fractions, which contain HIV-1 in the absence of contaminating microvesicles [61], were treated with 0.1% Triton-X100 for Western blot analysis. Viral proteins were precipitated as previously described [62], diluted in SDS loading dye (6.25 mM Tris pH 6.8, 1% w/v SDS, 0.01% v/v bromophenol blue, 10% glycerol, 0.3 M β-mercaptoethanol) and heated for 10 min at 95^oC prior to separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to a Hybond nitrocellulose membrane (GE Healthcare, Chicago, IL), blocked with 2.5% skim-milk powder in PBS- and HIV-1 proteins detected with HIV-1 positive serum provided by Dale McPhee (Burnet Institute, Melbourne, VIC, Australia) and secondary goat anti-human IgG antibody conjugated to IRDye 800 (Invitrogen, Waltham, MA). Membranes were scanned with the Licor Odyssey system (LICOR, Lincoln, NE) and quantified using the Image Studio Lite software.

CD4 binding ELISA

HIV_Ba−L viral supernatant was treated with 1% (w/w) L-LA at pH 3.8, acidified media at pH 3.8 (HCl adjusted) or sodium L-lactate at neutral pH for 60 min at 37°C then binding to CD4 assessed using an in-house CD4 binding ELISA. Plates coated with mouse anti-CD4 antibody (4B4 clone) were incubated with 80 ng of recombinant soluble CD4 (rCD4) engineered with a deletion in the transmembrane region but retaining the cytoplasmic tail (a gift from Nadine Barnes and David Anderson, Burnet Institute) reconstituted in lysis buffer (10% Triton X100, and 1 µg/ml each of leupeptin, aprotinin and pepstatin A in PBS-) for 1 h at room temperature. Excess rCD4 was removed by washing six times with wash buffer (0.05% Tween-20 in PBS-; 300 µl/well per wash). Treated virus solutions (100 µl/well) were added to plates, incubated for 1 h at room temperature and wells subsequently washed three times with wash buffer and three times with PBS-. Bound virus was quantified using the RETRO-TEK HIV-1 antigen ELISA kit (ZeptoMetrix Corporation, Buffalo, NY) according to the manufacturer’s instructions. Viral infectivity was determined in parallel in the TZM-bl cell line as above.

Small angle X-ray Scattering (SAXS) analysis

Soluble monomeric gp120 glycoprotein derived from the subtype B CCR5-utilizing strain NL(AD8) [63] was obtained from media conditioned by a stably transfected HeLa clone expressing cleavable gp140 [64]. Purification was performed using Lentil Lectin affinity chromatography followed by size exclusion chromatography [65]. Purified gp120 protein (0.1–0.2 mg/ml) in PBS- was treated with a concentration series of L-LA or D-LA at a pH found in women with an optimal vaginal microbiota (close to the pKa of LA) from 0.01–1% (w/w) at pH 4 for 10–15 min at room temperature. LA treatments were compared with untreated gp120 protein at pH 7 and gp120 treated with pH 4 (PBS- acidified with HCl). A no protein control was also included as reference. Analyses were performed at the SAXS/WAXS beamline, Australian Synchrotron. Parameters for SAXS data collection [66] are described in Supplementary Table 2. Briefly, samples were flowed at 1 µl/s during data collection and 10 x 1 second exposures were measured to control for radiation damage. Raw scattering images were averaged, and the appropriate reference data was subtracted from each corresponding gp120 sample using the same conditions. ScatterBrain IDL software (Australian Synchrotron) was used for averaging and reference subtraction, and data was analysed using PRIMUS from the ATSAS package [67].

Analysis of virion-associated RT activity and recombinant RT activity

The reverse transcriptase (RT) assay was used to detect RNA dependent DNA polymerase

(RDDP) activity of virion-associated RT from PBMC-derived HIV_RHPA supernatants and recombinant RT treated with acids, followed by neutralisation. Both assays use an exogenous RNA/DNA template/primer to measure RDDP activity. For quantitation of virion-associated RT activity, viral supernatants were lysed with an equal volume of 0.3% IGEPAL CA-630 (Sigma-Aldrich) and then added to the RT reaction mix containing a final concentration of 5 µg/ml poly(rA)/oligo(dT) template/primer, 10 µCi [α-³³P]-deoxythymidine triphosphate (dTTP) (PerkinElmer, Waltham, MA, USA), 50 mM Tris pH 8, 7.5 mM KCl, 2 mM dithiothreitol and 5 mM MgCl₂ for 1 h at 37^oC as published [68]. The reaction was stopped by applying samples to Whatman DE81 anion exchange paper (Sigma-Aldrich), followed by washing in 2x SSC buffer (300 mM sodium chloride, 30 mM sodium citrate), then 95% ethanol and air dried. The incorporated radiolabel was quantified using the Typhoon Trio Phosphorimager (GE Healthcare, Little Chalfont, BUX, UK) and Image Quant software (GE Healthcare). The average signal for the background control, containing media only, was subtracted from quantified samples. For assays using recombinant HIV-1 RT (subtype B), the p66/p51 HIV-1 RT heterodimer, engineered with an N-terminal histidine tag, was expressed and purified from the pRT6H-PR vector (kindly provided by Nicolas Sluis-Cremer) as published [69]. RDDP assays were performed as above except in the presence of recombinant HIV-1 RT (25–100 ng) where RT treated with acids was neutralised before adding to the reaction mix.

Collection and Processing of CVF

Cervicovaginal fluid (CVF) was collected from women of reproductive age (18–45 years old) with optimal Lactobacillus-dominated microbiota (Nugent score 0–3) recruited at the Johns Hopkins University Campus using a menstrual SoftCup (Instead Inc., La Jolla, CA) as previously described [70]. CVF samples from three women were pooled, and the pH of the pooled CVF was 3.83. Ethical approval was obtained from Homewood Institutional Review Board, Johns Hopkins University HIRB00000526, and the Alfred Ethics Committee, Project 80/13.

Analysis of virion-associated RNA following treatment with carboxylic acids

Following acid treatment of HIV-1, viral RNA was extracted from the neutralised supernatant using the QIAamp Viral RNA Extraction Kit, according to manufacturer’s instructions (QIAGEN, Hilden, Germany). Contaminating DNA was removed by treatment with RNase free DNase I and heat inactivated according to the manufacturer’s instructions (Roche, Basel, Switzerland). Complementary DNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and subjected to quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) using the Brilliant II SYBR qPCR Master Mix (Agilent Technologies, Santa Clara, CA) containing 0.3 µM of each primer targeting the HIV-1 long terminal repeat (LTR); hRU5-F2 5’-GCCTCAATAAAGCTTGCCTTGA-3’ and hRU5-R 5’-TGACTAAAAGGGTCTGAGGGATCT-3’ [71]. Cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min with fluorescence detected using the Mx3005P instrument (Agilent). Absolute DNA copies were calculated using a standard curve derived from serial dilutions of plasmid DNA as previously [72].

Production and infectivity of HSV-2

Herpes simplex type 2 (HSV-2) stocks were generated by infection of Vero cells (ATCC CCL-81) cultured in DMEM with 3% FCS (DMEM-3) with the HSV-2 G strain (ATCC item number VR-734) as previously described [73]. Infected cells and supernatants were collected and frozen at -80°C for > 1 h, with subsequent thawing and re-freezing for a total of three times to ensure HSV-2 release from cells. Viral supernatant was clarified by low-speed centrifugation and stored at -80°C. HSV-2 infectivity was determined by plaque assay in Vero cells, where cells were infected for 3–4 h, viral inoculum removed and cells overlayed with 1.6% low viscosity carboxymethyl cellulose (Sigma-Aldrich) prepared in DMEM-3. After incubation for 48 h, cells were fixed with methanol, washed with PBS- then stained with 0.1% Crystal violet, and plaque forming units/ml counted manually using light microscopy.

Production and infectivity of human papilloma pseudovirus

Human papillomavirus type 16 (HPV 16) pseudoviruses (PsV) were generated by transfection of 239TT cells with p16sheLL and pCLucf plasmids (a gift from J. T Schiller) expressing HPV capsid proteins and the luciferase reporter pseudogenome, respectively as previously described [74]. Transfected cells were pelleted and resuspended in PBS- containing 9.5 mM MgCl₂, 4% Triton X-100, RNase A (7 U/mL) and 25 mM ammonium sulfate [75]. Cell lysate was incubated at 37°C for 20–24 h to allow pseudovirus maturation, clarified by centrifugation, and the supernatant containing mature HPV PsV was transferred to siliconised tubes for storage at -80°C.

Statistical analyses

Statistical significance for virucidal, rCD4 binding, RT activity, qRT-PCR to detect viral genomic RNA and western blot analyses were performed using either an unpaired or paired t test as indicated in the text using GraphPad PRISM version 10.1.0 software.

LA potently inactivates HIV-1 at concentrations and pH associated with an optimal vaginal microbiota in contrast to SCFA and succinic acid

LA is virucidal against HIV in vitro [38], but it is not clear whether this activity is unique to LA or can be mediated and/or potentiated by other carboxylic acid metabolites present in cervicovaginal fluid. LA exists as both L- and D- stereoisomers, which are produced preferentially by different Lactobacillus spp., with L. crispatus predominantly producing D-LA while L. iners produces only L-LA [47]. We have previously found both isomers possess potent HIV-1 and HIV-2 virucidal activity [38], enhance the cervicovaginal epithelial barrier [34], and mediate immunomodulatory effects [39, 40]. Accordingly, here we evaluated a racemic mix of DL-LA.

Treatment of a subtype B transmitted/founder strain of HIV-1 (HIV_RHPA) with either DL-LA alone or in combination with a carboxylic acid mixture (DL-LA + optimal SCFA), representative of that found in women with an optimal vaginal microbiota (Supplementary Table 1)[54], at a physiologically relevant pH (3.8) elicited rapid and potent inactivation of HIV-1 infectivity (Fig. 1A). The DL-LA + optimal SCFA treatment, containing 100 mM DL-LA, decreased virus infectivity by almost 1000-fold relative to untreated virus after only 5 min of incubation and by > 10,000-fold after 30 min (p < 0.001 for both; Fig. 1A). This profile was similar to HIV-1 inactivation by 100 mM of DL-LA alone (Fig. 1A) indicating that DL-LA, present in the mixture of carboxylic acids simulating concentrations and pH observed in an optimal vaginal microbiota, was largely responsible for the virucidal effect. Notably, both conditions containing DL-LA were dramatically more virucidal than acidity alone at all time points, where media was adjusted to pH 3.8 with HCl [pH 3.8 (HCl) Fig. 1A, p < 0.05 for all].

We next evaluated the HIV-1 virucidal activity of acetic acid, which is a carboxylic acid present at the highest concentrations in women with BV [54], either alone, or in a combination with a carboxylic acid mixture (Non-optimal SCFA) representative of that found in women with BV (Supplementary Table 1)[54]. No virucidal effect was observed over time following incubation of HIV_RHPA with the same molar concentration of acetic acid (100 mM) alone, or with a vaginal carboxylic acid mixture representative of that found in the non-optimal state of BV where LA levels are dramatically lower (Fig. 1B)(Supplementary Table 1). Overall, these data demonstrate that DL-LA, at a physiologically relevant concentration and pH 3.8, has a marked ability to elicit rapid and potent inactivation of a transmitted/founder strain of HIV-1 irrespective of the presence of other carboxylic acids typically found in cervicovaginal fluid of women with an optimal vaginal microbiota. In contrast, under physiological relevant concentrations and pH found in women with BV, acetic acid and/or SCFAs lacked HIV-1 virucidal activity.

At an equimolar concentration of protonated acid, LA has more potent HIV-1 virucidal activity compared to other carboxylic acids

At equivalent concentration and pH, LA had potent and specific HIV-1 virucidal activity; however, the protonated concentration of each acid differs based on its unique dissociation constant (pK_a). To determine if protonated LA has greater HIV-1 virucidal activity than the protonated versions of SCFAs and succinic acid, equimolar concentrations of protonated carboxylic acids at a given pH were calculated using the Henderson-Hasselbalch equation. For these experiments, a threshold, sub-inhibitory acid concentration of 20 mM was chosen to allow differences in virucidal activity of vaginal acids to be assessed. For these and subsequent experiments comparing different vaginal carboxylic acids, a pH of 4.2 was utilised as it is largely compatible with the pKa of the various vaginal acids being assessed while within the pH range present in an optimal microbiota.

Under these conditions, L-, D- and DL-LA substantially reduced viral infectivity by 40 to 100-fold as compared to untreated HIV_RHPA (p < 0.001 for all, Fig. 2). Furthermore, the HIV-1 virucidal activity of all forms of LA (D-LA, L-LA and DL-LA) were significantly greater than the low pH 4.2 control (HCl), which itself caused only a 3-fold reduction in viral infectivity. In contrast, the BV-associated SCFAs acetic, propionic, butyric as well as succinic acid elicited a modest but significant reduction in HIV_RHPA infectivity of 4-10-fold compared to untreated virus (p < 0.01 for all; Fig. 2) which, except for butyric acid, was not significantly different compared to the low pH 4.2 control, indicating their activity is likely attributable mainly to acidity alone. Sodium lactate (Na + Lactate), employed as a high solute/osmolality and lactate anion control [38], showed no virucidal effects. When adjusted to neutral pH, 100 mM each of L-LA, acetic, propionic, butyric and succinic acid lacked HIV_RHPA virucidal activity (Supplementary Fig. 1), confirming that, similar to LA, it is the protonated form of these acids and not the anion that mediates the virucidal effect. These data demonstrate that potency of the virucidal activity of LA is specific, and greater than other vaginal carboxylic acids, low pH alone (HCl), or osmolality (∼473 mOsm/kg).

LA does not substantially disrupt HIV-1 virion integrity or decrease surface levels of gp120 envelope protein

A previous study reported that HIV_Ba−L treated with either 0.3 or 1% DL-LA and pelleted through a 20% sucrose cushion, followed by Western blot analysis did not show evidence of substantial virion lysis or loss of gp120 from the virion [51]. We have shown previously that HIV_Ba−L is more sensitive to the virucidal activity of L-LA compared to D-LA at concentrations down to 0.3% (33 mM) at low pH (pH < 4.5) [38, 46]. Accordingly, we determined the HIV_Ba−L virucidal mechanism of LA using the L-isomer of LA. However, in contrast to the previous study [51], we examined the effect of L-LA on HIV-1 virion structure and surface envelope proteins by separating treated virus using iodixanol velocity gradient ultracentrifugation. This technique sediments intact viral particles while avoiding contamination with cell-derived microvesicles and viral proteins/debris, which can co-sediment with viral particles during sucrose density-equilibrium gradient or sucrose cushion centrifugation [61]. The effect of L-LA on p24 capsid core protein was assessed to determine whether the virion remained intact after treatment, while the presence of gp120 envelope glycoprotein, which is required for viral entry, relative to p24 was measured to assess gp120 surface protein loss from the viral particle.

Our analyses indicated no significant difference in either the amount of p24 alone or the gp120:p24 ratio (p = 0.54 and 0.85, respectively) in virions after treatment with L-LA relative to untreated virus (Fig. 3), consistent with a previous study [51]. Parallel analysis of viral infectivity following virus treatment with L-LA under the same conditions confirmed a large reduction (> 1000-fold) in viral infectivity relative to untreated virus (Supplementary Fig. 2A). These data show that L-LA has minimal effects on viral structure or surface levels of HIV-1 gp120, indicating that the virucidal activity of L-LA is mediated by other mechanisms.

LA does not grossly alter HIV-1 envelope conformation and minimally impairs CD4 binding

Given the HIV-1 gp120 envelope protein remains virion-associated after L-LA treatment and is readily accessible to acid on the surface of the virion, we examined if L-LA’s virucidal activity could be mediated by promoting substantial conformational changes in the envelope protein in solution. SAXS analysis was therefore employed [76] to assess whether protonated D-LA or L-LA relative to low pH alone (HCl) caused substantial alterations in the conformation of soluble monomeric gp120 protein with regards to molecular size, shape and dimensions. Our analyses indicate that low pH (HCl) caused denaturation of gp120 protein relative to untreated gp120 at pH 7, as observed by a steep slope in the low q range on the scattering plot [q vs I(q); Fig. 4A]. A large signal in this q range indicates that the sample contains a substantial amount of large particles consistent with protein aggregates [66]. In contrast, no aggregation of gp120 was observed following treatment with 0.1% w/w (11 mM) D-LA or L-LA at pH 4, with the scattering curve being similar to that of untreated gp120 (Fig. 4A). To assess the impact of physiologically relevant levels of LA, gp120 was treated with L-LA at a concentration range from 0.01–1% w/w (1.1 to 110 mM) and compared to untreated gp120 at pH 7. These data indicate no detectable changes in the overall structure of monomeric gp120 in solution at any concentration of L-LA tested (Fig. 4B).

We next investigated whether L-LA elicits conformational changes of HIV-1 gp120 envelope structure, in the context of the virion, that may reduce infectivity by impairing binding of the trimeric gp120 to the cell entry receptor CD4. The ability of virion-associated gp120 to bind recombinant soluble CD4 (rCD4) was assessed using a modified CD4 binding ELISA. Treatment of HIV_Ba−L with 1% w/w (110 mM) L-LA (pH 3.8) elicited an almost 2-fold reduction in the ability of HIV-1 to bind to rCD4 relative to untreated HIV-1 (p = 0.004, Fig. 4C). However, a similar reduction in rCD4 binding was observed following treatment with low pH alone (HCl, pH 3.8), which was not significantly different to that observed for L-LA (p = 0.75), indicating that the effect was associated with low pH and not specific to L-LA. HIV-1 binding to rCD4 was also slightly but significantly impacted by treatment with sodium lactate, suggesting a potential effect of osmolality and/or the lactate anion. Analysis of viral infectivity of HIV_Ba−L treated in parallel showed a > 10,000-fold reduction in viral infectivity following treatment with L-LA relative to untreated virus (Supplementary Fig. 2B). Overall, these data indicate that L-LA and HCl at pH 3.8 cause a similar, approximately 2-fold reduction in the rCD4 binding ability of HIV-1 gp120. In contrast, L-LA treatment was associated with near complete inactivation of viral infectivity, suggesting other mechanisms contribute to the potent virucidal activity of LA.

LA exhibits more potent inhibition of virion-associated HIV-1 RT activity than other acids

Protonated LA present at low pH is membrane permeant [77] and thus may potentially penetrate the HIV-1 lipid envelope to alter proteins within the viral core, such as the reverse transcriptase (RT) enzyme [69], that are critical for viral infectivity. To assess this possibility, HIV_RHPA was treated with DL-LA and acetic acid at both the same absolute concentration (37 mM; equivalent to 0.3% DL-LA) or the same concentration of protonated acid (22 mM of acetic acid). Virion-associated RNA-dependent DNA polymerase activity was assessed after samples were brought to neutral pH and intact virions subsequently lysed with a non-ionic detergent to release the RT from the virion.

Our data show that virion-associated RT activity was markedly decreased upon treatment with DL-LA at pH 3.8, resulting in an ~ 75% reduction in HIV-1 RT activity within 5 min of treatment (p < 0.001, Fig. 5A). Acetic acid treatment also reduced virion-associated RT activity; however, the reduction in RT activity by DL-LA was significantly more potent than both acetic acid at the same total concentration (37 mM) or at the same protonated acid concentration (22 mM acetic acid) as DL-LA (p = 0.01 and 0.002, respectively after 10 min of treatment). Treatment with low pH 3.8 alone (HCl) also impaired virion-associated RT activity by approximately 50% (Fig. 5A), although the inhibitory activity of DL-LA at the same pH was significantly greater than HCl (p < 0.05 after both 5 and 10 min of treatment). Treatment of HIV-1 with DL-LA at a neutral pH (7.0) had no detectable effect on virion-associated RT activity (Fig. 5A), confirming the abovementioned effect of LA is specific to the protonated form present at low pH. HIV-1 infectivity was assessed in parallel and displayed a similar pattern of reduction in viral infectivity (Fig. 5B) as observed for virion-associated RT activity (Fig. 5A), with a more potent effect of DL-LA as compared to acetic acid and HCl.

The inhibitory activity of L-LA on virion-associated RT could be due to either a direct effect of the membrane permeant acid [77, 78] penetrating the virion to interact directly with the RT to inhibit its function and/or due to an indirect effect where L-LA subtly permeabilises [79, 80] the HIV-1 lipid envelope enabling penetration of other factors that inhibit the enzyme. To determine if L-LA treatment can directly inhibit RT enzyme function, we performed experiments with purified recombinant HIV-1 RT. Incubation of recombinant HIV-1 RT with L-LA at low pH demonstrated a dose-dependent impairment of RT activity (Supplementary Fig. 3A). However, a similar level of inhibition was also observed with an equivalent concentration of acetic acid and the low pH (HCl) control (Supplementary Fig. 3B), confirming recombinant HIV-1 RT protein is highly sensitive to the non-specific inhibitory effect of acidification. Taken together, these data indicate that while low pH alone has a detrimental effect on recombinant HIV-1 RT activity, LA elicits a significantly more potent inactivation of virion-associated RT activity, and this effect is specific to the protonated form of LA that may be better able to penetrate the virion to target RT.

LA promotes degradation of virion-associated HIV-1 RNA

The above findings with virion-associated RT activity suggest that protonated LA treatment inhibits HIV-1 RT. The membrane permeant properties of protonated LA [77–80] may also lead to targeting of HIV-1 genomic RNA within the viral core and further contribute to inhibition of viral infectivity. To address this question HIV_RHPA was treated with an equivalent 20 mM concentration of protonated L-, D- and DL-LA or acetic acid at pH 4.2 together with a low pH control (HCl, pH 4.2) for 5 min, neutralised and viral RNA extracted and quantified by qRT-PCR. We observed a significant decrease in HIV-1 RNA indicating degradation of HIV-1 genomic RNA following treatment with L-, D- and DL-LA, which all mediated approximately a 50% reduction in levels of amplifiable HIV-1 RNA as compared to untreated virus (p < 0.001 for all forms of LA; Fig. 6A). No significant reduction in HIV-1 RNA levels were detected following treatment with an equimolar concentration of protonated acetic acid at the same pH (4.2) or low pH alone (HCl).

To determine if LA treatment promotes degradation of virion-associated HIV-1 RNA within the context of the cervicovaginal environment in which it is produced, the above analyses were repeated in the presence of pooled CVF derived from women with an optimal vaginal microbiota. Under these conditions, DL-LA treatment not only retained significant viral RNA degrading ability, but the extent of RNA degradation was enhanced in the presence of CVF (p = 0.03 vs no CVF condition; Fig. 6B). This enhancement may be due to the presence of endogenous LA and/or other bacterial components in CVF from women with a Lactobacillus-dominated microbiota that increase viral permeability following LA treatment, which facilitates penetration of virucidal factors within CVF such as proteases and nucleases. Treatment of HIV-1 with acetic acid at pH 4.2, low pH 4.2 (HCl) alone or sodium lactate at pH 7 did not significantly affect HIV-1 RNA levels in the presence or absence of CVF (Fig. 6B). These data indicate that LA treatment has a specific ability to promote rapid degradation of HIV-1 viral RNA and may potentially increase virion lipid envelope permeability to facilitate decreasing HIV-1 infectivity in vivo.

LA inactivates both HIV-1 and HSV-2, but through different mechanisms

In addition to HIV-1 and HIV-2 [38], LA has potent virucidal activity against HSV-1 and HSV-2 at low pH [44, 45], although it is not clear whether the effects against these viral STIs, which also have a lipid envelope, are mediated by the same mechanism. To address this question, HSV-2 was treated with 0.3% w/w (33 mM) L-LA, D-LA and DL-LA or acetic acid at pH 4.2, representing vaginal conditions in the context of an optimal vaginal microbiota. In parallel, HSV-2 was treat with a low pH (4.2) control (HCl). All samples were treated with acid for 5 min at 37°C, neutralised, and HSV-2 infectivity determined using a plaque assay. Assays were performed at pH 4.2 to maximise observing any differences in the virucidal activity of the acids against HSV-2. All acids, including the low pH (HCl) control, mediated a significant and equivalent reduction in HSV-2 infectivity (Fig. 7A, p < 0.001 for all), indicating inactivation is likely due to a low pH environment and not due to any acid-specific activity.

To determine whether LA was virucidal against non-enveloped STI viruses such as HPV the virucidal activity against pseudoviruses, representing the non-enveloped HPV-16, was evaluated. To maximise observing a virucidal effect we treated HPV-16 with a higher physiological concentration (54 mM) of DL-LA and acetic acid, as well as HCl (all at pH 3.8) for 5, 10 and 30 min at 37^oC, and viral infectivity of neutralised samples assessed using a luciferase-based infectivity assay. We observed no virucidal activity of DL-LA, acetic acid or low pH 3.8 alone against the HPV-16 pseudovirus (Fig. 7B). Overall, these data indicate that HSV-2 inactivation is likely mediated primarily by low pH whereas HPV-16 pseudovirus is not inactivated by low pH or vaginal acids, suggesting that the LA virucidal activity against HIV-1 is virus-specific.

This study is the first to show the distinct and potent HIV-1 virucidal activity of LA compared to SCFAs and succinic acid present at physiological concentrations and pH that may be encountered by HIV-1 either shed or deposited in the vagina from women with an optimal vaginal microbiota compared to BV. Our studies demonstrate that LA’s irreversible and potent HIV-1 virucidal activity is not simply due to virion lysis or loss of surface gp120, but rather through multiple effects including those mediated by the membrane permeant properties of the biologically active protonated form of LA [77–80] to promote inhibition of virion-associated HIV-1 RT activity and degradation of viral genomic RNA. These inhibitory effects of LA treatment on HIV-1 RT and viral RNA were significantly more potent than HCl or acetic acid, the smallest carboxylic acid elevated in vaginal fluid in women with BV, indicating an LA-specific effect. We also show that while LA has virucidal activity against HSV-2, another viral STI with a lipid envelope, this was a low pH effect, as similar inhibition was observed with other acids. Finally, we show that LA does not affect the infectivity of HPV-16, that lacks a lipid envelope. These findings extend our initial studies reporting the superior and potent virucidal activity of LA against a range of clinically relevant HIV-1 strains and HIV-2 compared to acetic acid, as well as low pH (i.e. HCl) [38]. We also defined mechanisms by which LA inactivates HIV-1 at the molecular level. Our findings highlight the potentially important role of LA, compared to other carboxylic acid metabolites found in vaginal fluid, in modulating the risk of a women acquiring and transmitting viral STIs.

In the current study we show that LA was responsible for mediating potent HIV-1 virucidal activity against a subtype B transmitted/founder strain, isolated from a female subject [38], in the context of a mixture of carboxylic acids at pH 3.8 observed in women with an optimal vaginal microbiota. In contrast, we showed that a carboxylic acid mixture and conditions simulating BV (at pH 5.0), lacked HIV-1 virucidal activity. This effect was observed despite there being an overall greater concentration of carboxylic acid metabolites (144 mM) in the mixture representing BV conditions relative to the acid mixture for women with an optimal vaginal microbiota (107 mM) [54]. Furthermore, the superior HIV-1 virucidal activity of LA compared to the other vaginal carboxylic acids was demonstrated under stringent conditions where LA, SCFAs (acetic, propionic and butyric acid) and succinic acid were each tested at equivalent concentrations of the protonated active form of the acid. Taken together, these data suggest that an optimal Lactobacillus-dominated vaginal microbiota potentially has the ability to inactivate HIV-1 present in the vaginal lumen. This is supported by an ex vivo study demonstrating the anti-HIV-1 activity of intrinsic LA in cervicovaginal secretions [46].

We performed studies to determine the HIV-1 virucidal mechanism of action of LA. We initially focused on effects of LA compared to HCl on the HIV-1 lipid membrane and the gp120 envelope protein [81], since they are both exposed on the outside of the virion. In contrast to a previous study that used a sucrose cushion to pellet virus [51] we used an OptiPrep gradient to separate viral particles from co-sedimenting microvesicles that may also contain viral proteins. We found that LA-treated HIV-1 particles remain intact, indicating that LA does grossly not disrupt the viral lipid membrane, and that the gp120 envelope remains virion-associated. Our data are consistent with a previous study reporting that LA treatment does not cause HIV-1 viral particle lysis [51].

While we observed that gp120 is present on the intact HIV-1 virion, the possibility remained that LA may have altered its conformation and function. Using SAXS analysis we found that treatment of recombinant monomeric HIV-1 gp120 with HCl (pH 4) results in rapid protein denaturation. In contrast, no major conformational changes were observed when gp120 was treated with either D-LA or L-LA at the same low pH found in women with an optimal vaginal microbiota, under conditions where the biologically active protonated form of LA is present [37]. Our findings are partly consistent with a previous study where the protein, creatine kinase, was treated with LA and HCl, with conformational changes in protein secondary structure measured by circular dichroism [82]. This study found that treatment with HCl resulted in aggregation of creatine kinase, similar to our findings with gp120. In contrast, treatment of creatine kinase with LA (at pH 3–4) elicited protein unfolding in the absence of aggregation while exposing hydrophobic protein regions. Taken together with the known effect of LA on creatine kinase, this suggests that while we did not observe gross changes in gp120 structure by SAXS, that LA may still mediate changes in protein conformation that could impact on its functions. However, analysis of the effect of LA treatment of HIV-1 particles and ability to bind to rCD4 in vitro failed to demonstrate an LA-specific effect, where LA or HCl at pH 3.8 mediated the same modest 2-fold decrease in binding despite an approximately > 10,000-fold decrease in viral infectivity mediated by LA.

LA abolishes the negative surface charge of the HIV-1 lipid membrane with this effect postulated to be due to protonation [50] or hydrogen bonding interactions [83] between the carboxylic acid group on LA and chemical groups on glycoproteins and glycolipid incorporated into the HIV-1 lipid membrane that are derived from the host during viral egress from infected cells [84]. However, these effects are unlikely to explain the modest decrease in LA treated virions binding to rCD4 observed in our study given a similar fold inhibition was observed with HCl, which does not have a carboxylic acid and lacks potent HIV-1 virucidal activity (i.e. Figure 1A and [38]). Regardless, we cannot exclude the possibility that LA may alter the conformation and function of virion gp120 to inhibit binding to HIV coreceptors CCR4 or CCR5 and subsequent gp41 mediated viral fusion and entry (Chen B 2019 Trends in Microbiology 27:878). In addition, the effect of LA on virion incorporated host proteins [e.g. leukocyte function-associated molecule 1 (LFA-1) or intercellular adhesion molecule (ICAM-1)] that promote HIV-1 binding to the cell surface [85, 86] cannot be excluded. Despite these limitations our data indicate that any minor effects of LA on virion gp120 conformation, including its trimeric or protein secondary structure that mediates decreased rCD4-binding, is not LA specific and is unlikely to explain the potent HIV-1 virucidal activity mediated by LA.

Mechanistic insights on the virucidal activity of LA are limited [87] with we and others showing that treatment of HIV-1 with LA is irreversible and does not disrupt the viral particle [38, 51]. Here we now show that treatment with protonated LA, but not the lactate anion, likely penetrates the viral lipid envelope and core to inhibit virion-associated RT activity. This LA treatment effect was more potent compared to equivalent levels of protonated acetic acid (a smaller carboxylic acid than LA) as well as media acidified to the same pH with HCl providing further evidence of the distinct abilities of these acids to penetrate the HIV-1 virion. Previous studies in bacteria have also reported that protonated LA is membrane permeant [77, 78] and permeabilises the outer membrane of gram-negative bacteria resulting in changes in morphology [79, 80]. Accordingly, while our data suggests that LA directly targets HIV-1 RT within the viral particle, we cannot exclude the contribution of indirect effects, where other factors can enter the virion to target the RT, due to subtle permeabilisation (but not lysis) of the viral lipid envelope. Acetic acid is a smaller carboxylic acid compared to LA and has a higher acid dissociation constant (pKa 4.86) compared to 3.86 for LA, which would suggest that at pH 3.8 there would be more protonated acetic acid compared to LA. However, acetic acid is dramatically less efficient at killing BV-associated bacteria [41] as well as inactivating HIV-1 [38]. These findings indicate that LA may act through distinct mechanisms compared to acetic acid, including being more efficient at penetrating and/or subtly permeabilising the lipid envelope and penetrating the viral core.

Levels of HIV-1 genomic RNA are typically quantified by qRT-PCR including determining viral load in the vagina in women with HIV [7, 11–13]. Using qRT-PCR we show that LA treatment of HIV-1 particles results in degradation of the viral genomic RNA, which is potentiated in the presence of CVF. In contrast, we saw little effect on viral RNA degradation when HIV-1 was treated with acetic acid and HCl. The mechanism of this degradation may be due to a direct effect of lactic acid on the viral RNA, or an indirect effect. Regarding the latter, the HIV-1 stock used was in clarified conditioned media, from propagation in cell culture, which may contain factors from the host cell and/or media that contribute to viral RNA degradation in LA-treated virions. These conditions may approximate in vivo conditions where LA acts to permeabilise (but not lyse) the virion and enable molecules in the conditioned medium and CVF, including proteases and ribonucleases, to inactive HIV-1. The ability of LA treatment to degrade viral genomic RNA, a critical template for reverse transcription, as well as inhibit virion-associated RT activity, would be expected to synergise leading to a profound defect on intracellular HIV-1 reverse transcription. This proposed synergy is consistent with the dramatic effect on HIV-1 infectivity of LA treatment as observed in the TZM-bl indicator cell line. However, we cannot exclude the possibility that LA may also alter the conformation and function of other critical viral structural proteins and enzymes. Regardless, LA’s combined effects on RT function and the viral RNA genome template it uses to generate the provirus likely explains the greater decrease in HIV-1 infectivity compared to treatment with the same molar concentration of protonated acetic acid (Fig. 5B).

We extended our analysis to HSV-2, another enveloped viral STI, to determine if there were similarities between inactivation of HIV-1 and HSV-2 by LA compared to acetic acid and low pH alone (HCl). HSV-2 causes genital herpes and possesses a double-stranded DNA genome. In contrast to HIV-1, our data show that both the L- and D-LA isomers have HSV-2 virucidal activity that is similar in potency to acetic acid and acidity alone pH 4.2 (HCl adjusted). Our findings are consistent with a previous study reporting that the HSV-2 virucidal activity of LA and low pH alone are similar [45]. Thus, not all enveloped viral STIs are inactivated by LA and other acids in the same manner with the inactivation of HIV by LA being distinct and specific [38]. This may be related to differences in membrane composition, internal proteins and genomes of HIV-1 and HSV-2.

In contrast to HIV-1 and HSV-2 we found that the HPV-16 pseudovirus, representing a non-enveloped viral STI [88], was not inactivated by DL-LA, acetic acid, or pH 3.8 (HCl). While there are little data on the HPV virucidal activity of acids, nonenveloped viruses (i.e. rhinoviruses) are reported to be acid labile at a pH below 5.3 [89, 90]. The lack of virucidal effect of LA on HPV-16 may be explained by the effect of pH on HPV-16 PsV L1 and L2 capsid proteins, which undergo a maturation process during virus production [75]. This maturation stabilizes the HPV capsid, by reinforcing the intermolecular disulfide bonds between adjacent L1 molecules, forming pentamers, which is disrupted under alkaline conditions [75, 91]. Thus, acidic conditions, such as those in our studies, would be expected to promote the stabilisation of the capsid [75, 92].

The more potent HIV-1 virucidal activity of LA relative to low pH and acetic acid [38] is similar to other studies reporting LA’s microbicidal activity against BV-associated bacteria and Neisseria gonorrhoeae [41, 42, 93]. This suggests that LA, present at high concentrations in women with an optimal vaginal microbiota [35][53], acts to protect the lower FRT from reproductive tract pathogens to a greater extent than acetic acid, which predominates during BV [94, 95]. It also confirms that LA has inherent HIV-1 virucidal activity, that is not simply a low pH effect [41, 42, 93]. These findings indicate that the metabolite shift in the lower FRT that occurs during BV may increase HIV risk in several ways. These include loss of virucidal and bactericidal levels of LA and its replacement with SCFAs and succinic acid that do not target HIV-1, or other viral and bacterial STIs as well as BV-associated bacteria known to promote increased HIV acquisition and transmission [13].

Studies investigating the role of antimicrobials in CVF credited LA with the majority of the observed antimicrobial activity, and epithelial-derived antimicrobial peptides and bacteriocins to a lesser extent [96]. These observations are supported by a more recent study showing that most of the HIV-1 virucidal activity found in native cervicovaginal secretions from women with a Lactobacilllus-dominated vaginal microbiota (Nugent 0–3) can be attributable predominately to the protonated form of LA present in the < 3kDa acidic filtrate of cervicovaginal fluid [46]. Taken together with the findings of the present study, these data support a key role of LA in mediating the HIV virucidal activity of CVF in women colonised with an optimal vaginal microbiota.

The loss of virucidal activity of vaginal microbiota carboxylic acid metabolites during BV has important implications for HIV transmission, particularly in the context of mother-to-child transmission during vaginal birth and potentially during female-to-male transmission and male-to-female transmission [38]. Vaginal HIV load is greater in the presence of BV, which is associated with an increased risk of transmission to a male partner or neonatal child of a HIV-infected female [7, 13, 97, 98]. The potent virucidal activity of LA would be anticipated to directly inactivate HIV shed into the vagina of women with HIV. This notion is supported by our previous study showing that native LA present in CVF from women with a Lactobacillus-dominated microbiota potently inactivates HIV-1 ex vivo [46]. Additionally, it is possible that in the presence of anti-HIV levels of LA in the vagina, that detectable vaginal viral load measured by qRT-PCR may overestimate the levels of infectious HIV-1 that is present.

The virucidal activity of LA, may in part, explain observations that the presence of vaginal lactobacilli negatively correlates with viral load in vaginal fluid [7, 11, 13, 99]. In contrast, our findings show that BV-associated SCFAs have no observable virucidal activity under conditions that prevail during BV. This could explain higher viral load observed in women with HIV who also have BV and increased transmissibility to their male partners [13]. Additionally, LA may indirectly impact HIV, by suppressing growth of BV-associated bacteria [41] and preventing BV and cervicovaginal inflammation, which is associated with an increased risk of HIV acquisition in both males and females [13, 23]. Furthermore, the virucidal activity of LA may synergise with the immunomodulatory effects of LA [39] and vaginal lactobacilli [100–102] to suppress inflammation-related HIV target cell activation (e.g. from resident memory CD4 + T cell reservoirs) [103], and recruitment [104, 105], to further protect from HIV transmission.

The findings on the potent antiviral activity of LA against HIV and other viral and bacterial STIs, along with its direct anti-inflammatory [39, 40] and epithelial barrier integrity strengthening effects on cervicovaginal epithelial cells [34] in vitro and ex vivo need to be confirmed in well-designed clinical studies in women [106]. However, LA could potentially be advanced as an adjunct to antibiotics and/or antiretrovirals to optimise the vaginal microbiota to prevent women acquiring as well as transmitting HIV to their babies and partners. LA could be delivered directly by gel or by sustained intravaginal delivery to decrease infectious HIV shed into the vaginal lumen in pregnant women living with HIV. Alternatively, women with BV could be treated with LA to optimise their vaginal microbiota to foster colonisation with beneficial Lactobacillus spp. that produce LA. Standard of care for BV is treatment is with antibiotics, which while resulting in short-term cure, has a high recurrence rate (58% within 12 months) [107] and does not promote the presence of a stable and non-inflammatory L. crispatus-dominated vaginal microbiota. [108]. Novel strategies are being employed to optimise the vaginal microbiota including the advancement of vaginally-derived Lactobacillus spp. as therapeutics. In this regard, Lactin V, a vaginally applied L. crispatus-based live biotherapeutic, that produces LA, has been shown to prevent BV recurrence in 30% of cases compared to placebo following metronidazole therapy in a phase 2 randomized placebo-controlled trial [109] as well as decreasing genital inflammation [110]. Other investigators are pursuing vaginal microbiome transplants or the use of combinations of more than one L. crispatus strain to maximise vaginal colonisation [6, 111]. Given the link between BV and many adverse health outcomes beyond HIV acquisition including other STIs, preterm birth, pelvic inflammatory disease, endometritis and infertility, if successful, these strategies are anticipated to have a major impact on a women’s sexual and reproductive health.

By utilising a combination of virological, biophysical and molecular biology assays, this study has identified that lactic acid, a major metabolite produced by vaginal lactobacilli, has potent and specific HIV-1 virucidal activity compared to SCFAs and succinic acid found in vaginal fluid. Lactic acid’s potent and irreversible virucidal activity is largely mediated through the ability of the protonated form to penetrate and subtly permeabilise the viral lipid membrane of an intact virus and promote defects in the function and structure of an internal viral enzyme and the viral genomic RNA essential for viral infectivity. These findings provide important mechanistic insights into vaginal carboxylic acid metabolites and their potential role in modulating the risk of women acquiring or transmitting HIV to their partners or neonates in the context of an optimal vaginal microbiota and BV and indicate potential strategies for microbiome-based interventions to decrease vaginal viral load to prevent HIV.

ACKNOWLEDGEMENTS

The following reagents were obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl cells contributed by Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc; human immunodeficiency virus 1 infectious molecular clone pRHPA.c/2635 (ARP-11744) contributed by J Kappes and C Ochsenbauer, anti-human immunodeficiency virus 1 (HIV-1) p24 monoclonal (183-H12-5C, ARP-3537) contributed by Dr. Bruce Chesebro and Kathy Wehrly. P16sheLL, pCLucf and 293TT cells were a kind gift from John. T. Schiller National Cancer Institute, Bethesda, MD, USA. 293T cells were obtained from Richard Axel Columbia University, New York, NY, USA. The pRT6H-PR vector was kindly provided by Nicolas Sluis-Cremer, University of Pittsburgh, PA, USA. We thank Nadine Barnes and David Anderson, Burnet Institute, for the CD4 binding ELISA assay and Dale McPhee, Burnet Institute, for the HIV-1 positive serum. We thank Thomas Moench, Mucommune, LLC, Durham, NC, USA and Richard Cone, John Hopkins University, Baltimore, MD, USA and Mucommune, for providing CVF used in this study.

AUTHORS CONTRIBUTIONS

MA, DT, AJ, CFL, NC, generated the in vitro experimental data, MA, DT, AJ, CFL, NC, PAR, JAH, ACH and GT contributed to data analysis and interpretation. RJC contributed a key reagent to the study. GT, CFL, DT, PAR, and AJ were involved in study design, conception and supervision. ACH, MA, and GT drafted the manuscript. All authors have read and approved the final manuscript.

AUTHOR’S INFORMATION

Not applicable

FUNDING

This work was supported by the National Health and Medical Research Council (NHMRC) (Project Grant 102894). GT was supported by NHMRC Senior Research Fellowship (Grant 543105) and MA was supported by the Australian Postgraduate Award (Monash University) and a Postgraduate Publications Award from Monash University. The authors gratefully acknowledge the contribution to this work of the Victorian Operational Infrastructure Support Program received by the Burnet Institute. The funders did not have a role in the design and collection, analysis, and interpretation of data and in writing the manuscript.

AVAILABILITY OF DATA AND MATERIAL

All data generated during the current study are included in this article and its supplementary information files.

ETHICS APPROVAL AND CONSENT TO PARITCIPATE

Ethical approval for collection of cervical vaginal fluid was obtained from Homewood Institutional Review Board, Johns Hopkins University HIRB00000526, and the Alfred Ethics Committee, Project 80/13.

CONSENT FOR PUBLICATION

Not applicable

COMPETING INTERESTS

GT and ACH are coinventors on a grant US patent on the immunomodulatory effects of lactic acid on cervicovaginal epithelial cells. The other authors declare that they have no competing interests.

CORRESPONDING AUTHOR

Gilda Tachedjian, Life Sciences Discipline and Disease Elimination Program, Burnet Institute, 85 Commercial Rd, Melbourne, VIC 3004, Australia.

Email: [email protected]

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Competing interest reported. GT and ACH are coinventors on a grant US patent on the immunomodulatory effects of lactic acid on cervicovaginal epithelial cells. The other authors declare that they have no competing interests.

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Lactic acid produced by optimal vaginal Lactobacillus species potently inactivates HIV-1 by several mechanisms including promoting inhibition of virion-associated reverse transcriptase activity and viral RNA degradation

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Abstract

Figures

BACKGROUND

MATERIALS AND METHODS

Cell culture and virus production

Treatment of HIV-1 with vaginal acids

CD4 binding ELISA

Small angle X-ray Scattering (SAXS) analysis

Analysis of virion-associated RT activity and recombinant RT activity

Collection and Processing of CVF

Analysis of virion-associated RNA following treatment with carboxylic acids

Production and infectivity of HSV-2

Production and infectivity of human papilloma pseudovirus

Statistical analyses

RESULTS

LA does not grossly alter HIV-1 envelope conformation and minimally impairs CD4 binding

LA exhibits more potent inhibition of virion-associated HIV-1 RT activity than other acids

LA promotes degradation of virion-associated HIV-1 RNA

LA inactivates both HIV-1 and HSV-2, but through different mechanisms

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

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