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Viricidal effect of Lactobacillus acidophilus on human immunodeficiency virus type 1: possible role in heterosexual transmission.

Peroxidase, H2O2, and a halide form a powerful antimicrobial system in phagocytes and tissue fluids, and certain microorganisms can serve as the source of H2O2 for this system. H2O2-generating Lactobacillus acidophilus (LB+) is present in the vagina of most normal women and peroxidase has been detected in vaginal fluid. LB+ at high concentration is viricidal to HIV-1, and, at levels where LB+ is ineffective alone, the addition of peroxidase (myeloperoxidase, eosinophil peroxidase) and a halide (chloride, iodide, bromide, thiocyanate) restore viricidal activity. LB+ can be replaced by H2O2, but not by non-H2O2-producing LB, and viricidal activity is inhibited by azide and catalase. The survival of HIV in the female genital tract and thus the likelihood of transmission may be influenced by the activity of the LB(+)-peroxidase-halide system in the vagina.

H eterosexual transmission of HIV is occurring with increasing frequency in the United States (1) and remains the most prevalent mode of transmission in Africa (2, 3). In heterosexual transmission, HIV released into the vagina by either partner survives the local defense mechanisms to infect sexual partners. However, the efficiency of sexual transmission is extremely variable, suggesting that there may be factors that can affect the survival of HIV in the vagina .
Peroxidases, when combined with H202 and a halide, form a powerful antimicrobial system which is effective against a variety of microorganisms (4), including viruses (5) . Mammalian peroxidases effective in this way include myeloperoxidase (MPO) present in neutrophils and monocytes, eosinophil peroxidase (EPO) in eosinophils, and lactoperoxidase (LPO) in milk and saliva. A peroxidase with antimicrobial (6) and spermicidal (7) properties is also present in the uterine fluid of estrogen-primed rats. Although peroxidases are generally effective with several halides, the physiologic halide for MPO is believed to be chloride (4), for EPO bromide (8,9) or the pseudohalide thiocyanate (10), and for LPO thiocyanate (11,12) . The source of H202 for the peroxidasemediated antimicrobial system includes the respiratory burst of stimulated phagocytes and the metabolism of certain bacteria .
Bacteria designated as lactic acid bacteria, e.g., lactobacilli, streptococci and pneumococci, release H202 (13) which can be autoinhibitory (13) and, when mixed cultures are employed, can be toxic to other bacteria (6,14), fungi (14), viruses (15), spermatozoa (16), or tumor cells (17), particularly in the presence of peroxidase and a halide. The ability of lactobacilli to provide H202 for a peroxidase-dependent cidal system is pertinent to the female genital tract, since the predominant bacterial species in normal human vaginal secretions is Lactobacillus acidophilus, and the vaginal overgrowth ofa number of organisms in bacterial vaginosis is associated with a decrease in H202-generating lactobacilli (18). Thus, the production of H202 by lactobacilli in the vigina appears to be a nonspecific host defense mechanism which can be potentiated by peroxidases of leukocytic or uterine origin. In this study, we demonstrate the viricidal effect of the peroxidase-H202-halide system on HIV, and the ability of H202-generating lactobacilli to provide the H202 required for this effect.  6.8) . HIV in the cell supernatant was diluted to 106 tissue culture infective dose 50 (TCIDso)/ml in CEM growth medium and frozen in liquid nitrogen gas phase. Stock virus was diluted 100fold with 0.1 M sodium sulfate prior to a further 10-fold dilution in the reaction mixture (final concentration, 1000 TCIDso/ml). In addition to the components listed, the reaction mixture contained the components of CEM growth medium diluted 1,000-fold.

Materials and Methods
Peroxidase. MPO was purified from human leukocytes (19) and EPO from horse eosinophils (20). Peroxidase activity was determined by guaiacol oxidation (21). Lactobacilli. Hospital cultures of L. acidophilus obtained from human vaginal swabs were initially tested for H202 production by exposure to air of colonies which had been grown anaerobically as surface colonies on agar containing horseradish peroxidase and tetramethylbenzidine (18). Colonies of H20a-generating organisms (LB*) formed a blue pigment, whereas organisms which did not form H202 (LB -) remained white. LB+ and LB -cultures were maintained by aerobic growth on HBT agar plates (01-478 Remel, Lenexa, KS) at 37°C in a C02 incubator for no longer than 2 d. Before the experiment, the organisms were transferred to peptone yeast extract medium (22) containing 1% heat-inactivated FCS (Hyclone Laboratories, Logan, UT) and grown aerobically at 37°C for approximately 6 h with tumbling for LB+ and 24 h in stationary tubes for LB -. The lactobacilli were washed twice and suspended in 0.1 M sodium sulfate. H202 generation by LB' was determined before each experiment by the scopoletin method (23).
MeasurementofKricidal Activity. The reaction mixtures described in the legends to the figure and table were incubated in sterile screwcap microtubes with sealing O-rings (4.3 x 10.8 mm, 1.5 ml capacity #75 .692.005; Sarstedt, Inc., Princeton, NJ) for 30 min at 37°C. The tubes were centrifuged at 2,940,g for 2 min to sediment the bacteria and 10 pl aliquots of the supernatant fraction were added to triplicate wells (48-well plate #3548 ; Costar, Cambridge, MA) containing 2 x 105 CEM cells in 1 ml of CEM grown medium per well. Following incubation at 37°C for 6 d in a C02 incubator (5% C02-95% air), a 200 Etl aliquot was removed for measurement of HIV P24 antigen by a solid phase sandwich-type ELISA (Abbott Laboratories, Chicago, IL) . If an optical density of >2.000 was observed, the supernatant fraction was diluted and remeasured . The data are expressed as the P24 antigen level above background. A positive HIVP24 antigen cutoff of 10pg/ml was used. In general, culture supernatant levels ofHIV P24 antigen were either zero (<10 pg/ml), indicating no replication, or very high (>1,000,000 pg/ml), indicating unrestricted replication, with the level reached being related to the number of CEM cells (2 x 101), the volume of the reaction mixture added to the CEM cells (10 ul) and the growth period (6 d).

Results and Discussion
H202-generating L. acidophilus (LB') alone at a concentration of 101 CFU/ml was viricidal to HIV in lactate buffer pH 5.0 as measured by a decrease in viral replication in CEM cells (Fig. 1). When the LB' concentration was reduced to a level where it was ineffective alone (2 x 105-5 x 106 CFU/ml), the addition of MPO and chloride resulted in a return of HIV viricidal activity (Fig. 1).
The HIV viricidal activity of high concentrations of LB + alone was variable, with complete loss ofHIV infectivity observed in five of nine wells, giving a median P24 level of <10 and a mean of 123 pg/ml (Table 1). An anti-HIV effect was not seen when the LB' was heated at 100°C for 15 min or was replaced by L. acidophilus which did not generate H202 (LB -). This toxic effect on HIV was inhibited by catalase, but not by heated catalase, implicating H202. Table 1 also demonstrates the viricidal effect of low levels of LB' combined with MPO and chloride, the requirement for each component of the system, and the inhibition by heat-treatment of LB +, by the substitution of LB -for LB + and by the ad- dition of azide (which inhibits peroxidase) or catalase, but not heated catalase or superoxide dismutase, again implicating H202. Chloride could be replaced by iodide, bromide or thiocyanate ions, LB+ could be replaced by reagent H202 and MPO could be replaced by EPO. However, chloride was less effective with EPO than with MPO. The inhibition of HIV infectivity was not due to direct toxicity to the CEM cells. The evidence is as follows : (a) the reaction mixture was diluted 100-fold on addition to the CEM cells ; (b) the CEM cells continue to metabolize and divide at a normal rate as indicated by acid production and increase in cell number; (c) identical results were observed when catalase was used to stop the reaction before the addition of an aliquot to the CEM cells ; and (d) the toxicity of the peroxidase system is known to be strongly inhibited by protein and low mol wt components present in the CEM growth medium .
L. acidophilus has been detectedin the vagina ofmost normal women with the level of H202-producing lactobacilli averaging 8.4 x 106 organisms per ml in one study (18). Under our in vitro conditions, the amount of H202 generated by 107 organisms was sufficient to inactivate HIV in the absence of peroxidase, and when MPO and chloride were added, concentrations of LB+ as low as 2 x 105 CFU/ml were effective. Thus, lactobacilli appear to be present in the vagina in adequate numbers to exert an antiviral effect. Peroxidase has also been detected in human vaginal fluid (24). Ofthe halides, chloride is present in cervical mucus in high concentration (25) and iodide and thiocyanate are concentrated there following intravenous administration to humans (26). Their presence in vaginal fluid would thus be anticipated . The pH employed here is approximately that ofvaginal fluid. Additional studies are needed to evaluate the influence of potential inhibitors in vaginal fluid (catalase, protein, low mol wt-reducing substances) . The reaction mixture was as described in Fig. 1, except that the additions were : LB+ and LB -at the concentrations indicated; 23 mU/ml MPO ; 23 mU/ml EPO ; 0.1 M sodium chloride; 10 -4 M sodium iodide ; 10  We have concentrated on the destruction of cell-free HIV HIV in semen is both cell-associated and free in seminal plasma (27) and it is not clear which form is responsible for male to female vaginal transmission . The infection of primates by intravaginal infusion of cell-free HIV or SIV, however, has been described (28,29).
Our findings suggest that the presence and level of H202producing lactobacilli in vaginal fluid may affect the heterosexual transmission of HIV by the release of H202, which can act either alone or in conjuction with a halide and peroxidase ofleukocytic or uterine orgin to inactivate HIV Normal individuals, or persons with sexually transmitted vaginal disease, in whom vaginal H202-producing lactobacilli are few or absent, may thus be at greater risk of infection and, as a corollary, may benefit from vaginal colonization with H202-generating lactobacilli. The systemic treatment of AIDS patients with orally-administered live lactobacilli has been proposed (30). The presence of peroxidase in other locations, e.g., LPO in milk and saliva, MPO in neutrophils and monocytes, EPO in eosinophils and extracellular MPO or EPO in inflammatory loci containing degenerating or stimulated phagocytes, raise the possibility that the peroxidase system utilizing H202 formed by microorganisms, phagocytes or soluble enzymes also contributes to the host defense against HIV at other sites .