1 In the presence of highly diluted vaginal fluid, even 1 Molar H 2 O - PDF document

Only a minority of women have a ‘normal’ lactobacillus -dominated microbiota When lactobacilli dominate they Nugent scores 0-3 ‘ Normal ’ : Viscoelastic Mucus inactivate HIV and BV bacteria with lactic acid Light gram positive monoculture pH ~3.5 ~1% Lactic acid ‘ Normal ’ ~ 38% No acetic acid D.E. O’Hanlon (JHU), G. Tachedjian (Burnet), Nugent scores 7-10 BV: T.R. Moench (ReProtect), and R.A. Cone (JHU and ReProtect) Intermediate Watery discharge ~ 33% Heavy polyculture pH ≥4.5 BV ~ 29% Succinic acid Acetic acid Butyric acid Microbicides 2012, Sydney, Australia Putrescine Cadaverine % of U.S.women Tyramine on any given day (fishy odor) Supported in part by NIH grants Allsworth and Peipert, 2007 AI45967, AI60598, and AI66726. 3,727 women, demographically balanced Increased risks if BV is present on day of entry into n prospective trials BV increases several factors that may increase susceptibility to infections, e.g., inflammatory cytokines; degraded mucus; weak acidity. BV increases several factors that may increase susceptibility to infections, e.g., inflammatory cytokines. Do lactobacilli protect against infections? Do lactobacilli protect against infections? Some lactobacilli produce hydrogen peroxide (H 2 O 2 ). Does the H 2 O 2 they produce protect against infections? 1

In the presence of highly diluted vaginal fluid, even 1 Molar H 2 O 2 fails to kill BV bacteria 1% vaginal fluid Unfortunately, lactobacilli can only produce H 2 O 2 when oxygen is present. In the hypoxic environment of the vagina, as well as in antioxidant rich vagina fluid, they produce < 1 micro-molar H 2 O 2 (our threshold of detection). 0.1% vaginal fluid ( O’Hanlon , Lanier, Moench, and Cone, BMC Infect Dis 2010) H 2 O 2 kills lactobacilli more potently than BV bacteria: How can lactobacilli use it to prevent BV? It is improbable that H 2 O 2 produced by lactobacilli in the hypoxic vagina, and immersed in antioxidant rich vaginal fluid, can protect against BV, or HIV, or any other STD pathogen. Semen is also antioxidant rich. Lactobacilli  However, H 2 O 2 producing lactobacilli are strongly associated with reduced BV, and many other infections, but it is likely that H 2 O 2 producing lactobacilli are best at producing something else - - - like lactic acid??? When lactobacilli dominate (Nugent Scores 0-3) they acidify the vagina with lactic acid 4.4 4.2 Does lactic acid produced by lactobacilli protect against infections? Fresh undiluted samples of cervical vaginal fluid from (As believed for most of the past century, but not after H 2 O 2 emerged.) 4.0 24 women with Nugent scores 0-3 3.8 pH 3.6 3.4 3.2 3.0 0.6 0.8 1 1.2 1.4 Lactic acid concentration (% w/v) 2

When lactobacilli dominate (Nugent Scores 0-3) they acidify the vagina with lactic acid 4.4 When lactobacilli dominate (Nugent scores 0-3): 4.2 4.0 In air, no CO 2 • Vaginal pH = 3.5 ± 0.3 (pH range 3.0-3.9) 3.8 • Vaginal lactic acid concentration = 1.0 ± 0.2% pH 3.6 • Acetic acid < 0.003% 3.4 If corrected for • H 2 O 2 < 1 x 10 -6 molar 3.2 5% vaginal CO 2 3.0 0.6 0.8 1 1.2 1.4 Lactic acid concentration (% w/v) The presence of vaginal fluid does not diminish the microbicidal effect of lactic acid Lactic acid potently inactivates BV bacteria while sparing lactobacilli 1000000000 L.crispatus acidity alone (HCl, pH 4.5) L.jensenii 100000000 L.gasseri lactobacilli L.iners Colony forming units/mL after 2 hours 10000000  G.vaginalis Lactobacilli P.levii 1000000 P.bivia P.corporis 100000 A.prevotii F.nucleatum 10000 B.ureolyticus M.micros 1000 P.acnes M.elsdenii 100 P.anaerobius E.lenta Lactic acid with BV bacteria A.teradius 10 healthy flora BV bacteria A.vaginae 1.0 + 0.2% U.urealyticum 1 M.curtisii M.mulieris 0 M.hominis 0 0 1 10 100 1000 pH 7.0 pH7.0 ----------------------[lactic acid] mM at pH4.5------------------- no lactic acid Lactic acid preserves food. Carcasses are sprayed with lactic acid as a preservative. Gram negative bacteria Gram negative bacteria 3

Effect of lactic acid on Neisseria gonorrhoeae (incubated anaerobically at pH 4.5 for 2 hours at 37 o C) 10 10 1 Effect of lactic acid on HSV-2 incubated at pH 4.5 for 20 minutes at 37C 10 1 as discovered by Deirdre O’Hanlon growth Proportion of infected indicator cells 10 0 stasis 1  10 0 (cfu at t=2hrs) / (cfu at t=0) 10 -1 10 -2 10 -1 0 10 -3  Average 10 -2 killing vaginal 0 10 -4 concentration Average 1.0% vaginal  10 -5 ≤ 10 -3  concentration  0 1.0% 0.0 0.0 0.1 0.5 1.0 5.0 10.0 pH 7.0 pH 4.5 10 -6 0.0 0.0 0.1 0.5 1.0 5.0 10.0 <10 -7 Lactic acid (%) pH 7 | ------------------ pH 4.5---------------------- | 10.0 0.0 0.1 0.5 1.0 5.0 Lactic acid (%) HIV is trapped in acidic cervicovaginal mucus (CVM), Fraction of HSV-2 that remains infectious after 30 min exposure to pH 3.8 but not in neutralized mucus with HCl, D-lactic acid, and L-lactic acid L-lactic acid is ___ 10-fold more potent than HCl 4-fold more potent than D-LA P<0.0001 ___ H-Cl (also, acetic acid) ___ D-lactic acid ___ L-lactic acid Used 1/10 vaginal LA to slow rate of inactivation Lai, Hida, Shukair, Wang, Figueiredo, Cone, Hope, Hanes: J Virol. 2009 HIV Ba-L inactivation by 1% L-LA is more rapid and potent L-LA inactivates different HIV-1 subtypes, X4 and than low pH alone and 1% acetic acid R5 strains, patient isolates and HIV-2 37 o C n=3 n=4 30min at 37 o C 37 C Gilda Tachedjian Gilda Tachedjian 4

SUMMARY CONCLUSIONS • Lactic acid as produced by lactobacilli in the vagina, but not H 2 O 2 , can inactivate BV bacteria without inactivating lactobacilli. • At pH 3.5, vaginal lactic acid will likely inactivate most • Even at pH 4.5, 1% lactic acid completely inactivates all 17 BV-associated acid-sensitive pathogens shed vaginally by infected females and bacteria tested to date. reduce female-to-male transmission of infections. • When lactobacilli dominate, they produce lactic acid rapidly enough to • Semen transiently alkalinizes the vagina, but lactobacilli may restore maintain the vagina at a mean pH of 3.5 with 1% lactic acid. acidity in the epithelium rapidly enough to help reduce male-to-female transmission of acid-sensitive pathogens. • Lactic acid potently inactivates HIV, HSV, and Neisseria gonorrhoeae. • Lactic acid inactivates HIV in the presence of seminal and vaginal fluid, and BV-associated bacteria in the presence of vaginal fluid. HIV in normal vaginal mucus with lactic acid HIV in neutralized vaginal mucus Human immunodeficiency virus type 1 is trapped by acidic but not by neutralized human vaginal mucus. Lai, Hida, Shukair, Wang, Figueiredo, Cone, Hope, Hanes. J Virol. 2009 Nov;83(21):11196-200. 5