Vaginal lactic acid elicits an anti-inflammatory response from human cervicovaginal epithelial cells and inhibits production of pro-inflammatory mediators associated with HIV acquisition.

Inflammation in the female reproductive tract (FRT) is associated with increased HIV transmission. Lactobacillus spp. dominate the vaginal microbiota of many women and their presence is associated with reduced HIV acquisition. Here we demonstrate that lactic acid (LA), a major organic acid metabolite produced by lactobacilli, mediates anti-inflammatory effects on human cervicovaginal epithelial cells. Treatment of human vaginal and cervical epithelial cell lines with LA (pH 3.9) elicited significant increases in the production of the anti-inflammatory cytokine IL-1RA. When added simultaneously or prior to stimulation, LA inhibited the Toll-like receptor agonist-elicited production of inflammatory mediators IL-6, IL-8, TNFα, RANTES, and MIP3α from epithelial cell lines and prevented IL-6 and IL-8 production by seminal plasma. The anti-inflammatory effect of LA was mediated by the protonated form present at pH≤3.86 and was observed with both L- and D-isomers. A similar anti-inflammatory effect of LA was observed in primary cervicovaginal cells and in an organotypic epithelial tissue model. These findings identify a novel property of LA that acts directly on epithelial cells to inhibit FRT inflammation and highlights the potential use of LA-containing agents in the lower FRT as adjuncts to female-initiated strategies to reduce HIV acquisition.

Diffusion of macromolecules and virus-like particles in human cervical mucus.

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.

Origins of vaginal acidity: high D/L lactate ratio is consistent with bacteria being the primary source.

BACKGROUND: The origin of the lactic acid that acidifies the vagina is not well established. It is widely accepted that during times of high oestrogen (during the neonatal period and again during a woman's reproductive years) large amounts of glycogen are deposited in the vaginal epithelium and that the glycogen is anaerobically metabolized to lactic acid. What is not established is whether lactic acid is primarily produced by vaginal bacteria or by vaginal epithelial cells. Human cells can make only L-lactate, while bacteria can produce both D- and L-, thus the D- to L-lactate ratio can indicate the relative contribution of bacterially derived lactic acid. METHODS: In this study, we used chiral HPLC to examine the percentages of D- and L-lactate in vaginal secretions, in primary cultures of bacteria from these vaginal secretions, and in cultures of lactobacillus isolates of vaginal origin. RESULTS: We found that in most vaginal secretion samples, >50% of the lactic acid was the D-isoform (mean 55%, range 6-75%, n = 14). CONCLUSIONS: Our results thus support the hypothesis that vaginal bacteria, not epithelial cells, are the primary source of lactic acid in the vagina.

Tests of Buffergel for contraception and prevention of sexually transmitted diseases in animal models.

BACKGROUND: BufferGel is a novel spermicidal and microbicidal gel formulated to maintain the natural protective acidity of the vagina by acidifying semen, which otherwise alkalinizes the vagina. GOAL: To test the efficacy of BufferGel for preventing sexually transmitted infections and pregnancy in animal models. STUDY DESIGN: Animals were challenged with pathogens or sperm after pretreatment with both test and control agents, or after no pretreatment, then evaluated for infection or pregnancy using standard methods. RESULTS: BufferGel provided significant contraceptive efficacy in the rabbit, and significant protection against vaginal and rectal transmission of herpes simplex virus type 2 (HSV-2) in the mouse, vaginal transmission of Chlamydia trachomatis in the mouse, and skin transmission of cottontail rabbit papillomavirus in the rabbit. It did not protect against vaginal transmission of Neisseria gonorrhoeae in the mouse. CONCLUSIONS: The protective efficacy of BufferGel in five of the six animal models suggests that this microbicide warrants clinical evaluation for both contraception and disease prevention.

Preventing infectious disease with passive immunization.

Antibodies can prevent infectious diseases by providing passive immune protection. Here we review successful clinical trials of passive immunization and consider some of the unique qualities monoclonal antibodies are now beginning to offer for developing methods for passive immunization against a wide range of infectious diseases.

The rate at which human sperm are immobilized and killed by mild acidity.

OBJECTIVE: To determine the rate at which mild acidity immobilizes and kills human sperm and to evaluate an acidic microbicide, BufferGel, for sperm immobilization. DESIGN: Controlled in vitro study. SETTING: An academic research university and hospital andrology lab. PATIENT(S): Eight volunteer male sperm donors. INTERVENTION(S): Semen samples were treated with hydrochloric acid (HCl) or BufferGel. MAIN OUTCOME MEASURE(S): Sperm motility was measured by using a computerized automated semen analyzer and video microscopy. Sperm membrane permeability and intracellular pH were measured by using fluorescent techniques. RESULT(S): In semen acidified with HCl to pH 4.0, sperm were rapidly immobilized (within 1 min) and were irreversibly immobilized (killed) within 10 minutes. The speed of immobilization and of killing were both linearly proportional to hydrogen ion activity over a pH range of 7.5-4.0. Across the same range, the intracellular pH of human sperm equilibrated to within 0.5 pH units of extracellular pH within 1-2 minutes. BufferGel immobilized sperm significantly faster than HCl from pH 4.0-6.0. CONCLUSION(S): Exposure to mild acidity rapidly acidifies the intracellular pH of human sperm and is rapidly spermicidal. BufferGel accelerates acid immobilization of sperm.

Acid production by vaginal flora in vitro is consistent with the rate and extent of vaginal acidification.

Perinatally, and between menarche and menopause, increased levels of estrogen cause large amounts of glycogen to be deposited in the vaginal epithelium. During these times, the anaerobic metabolism of the glycogen, by the epithelial cells themselves and/or by vaginal flora, causes the vagina to become acidic (pH approximately 4). This study was designed to test whether the characteristics of acid production by vaginal flora in vitro can account for vaginal acidity. Eight vaginal Lactobacillus isolates from four species-L. gasseri, L. vaginalis, L. crispatus, and L. jensenii-acidified their growth medium to an asymptotic pH (3.2 to 4.8) that matches the range seen in the Lactobacillus-dominated human vagina (pH 3.6 to 4.5 in most women) (B. Andersch, L. Forssman, K. Lincoln, and P. Torstensson, Gynecol. Obstet. Investig. 21:19-25, 1986; L. Cohen, Br. J. Vener. Dis. 45:241-246, 1969; J. Paavonen, Scand. J. Infect. Dis. Suppl. 40:31-35, 1983; C. Tevi-Bénissan, L. Bélec, M. Lévy, V. Schneider-Fauveau, A. Si Mohamed, M.-C. Hallouin, M. Matta, and G. Grésenguet, Clin. Diagn. Lab. Immunol. 4:367-374, 1997). During exponential growth, all of these Lactobacillus species acidified their growth medium at rates on the order of 10(6) protons/bacterium/s. Such rates, combined with an estimate of the total number of lactobacilli in the vagina, suggest that vaginal lactobacilli could reacidify the vagina at the rate observed postcoitally following neutralization by the male ejaculate (W. H. Masters and V. E. Johnson, Human sexual response, p. 93, 1966). During bacterial vaginosis (BV), there is a loss of vaginal acidity, and the vaginal pH rises to >4.5. This correlates with a loss of lactobacilli and an overgrowth of diverse bacteria. Three BV-associated bacteria, Gardnerella vaginalis, Prevotella bivia, and Peptostreptococcus anaerobius, acidified their growth medium to an asymptotic pH (4.7 to 6.0) consistent with the characteristic elevated vaginal pH associated with BV. Together, these observations are consistent with vaginal flora, rather than epithelial cells, playing a primary role in creating the acidity of the vagina.

Using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases.

Passive immunization with antibodies has been shown to prevent a wide variety of diseases. Recent advances in monoclonal antibody technology are enabling the development of new methods for passive immunization of mucosal surfaces. Human monoclonal antibodies, produced rapidly, inexpensively, and in large quantities, may help prevent respiratory, diarrheal, and sexually transmitted diseases on a public health scale.

Comparison of an anti-HSV-2 monoclonal IgG and its IgA switch variant for topical immunoprotection of the mouse vagina.

An IgG2a monoclonal antibody (Mab) directed against glycoprotein D of herpes simplex virus 2 (HSV-2) was compared with an IgA heavy chain Mab switch variant to investigate the effect of isotype for topical immunoprotection of the murine vagina. The IgA Mab, a mixture of monomeric and polymeric IgA, was indistinguishable from its IgG parent in an in vitro HSV-2 neutralization assay. When these class switched Mabs were delivered to the mouse vagina, we also found no significant difference between the IgG and IgA for preventing vaginal transmission of HSV-2 infection. The implications of these results for active and passive immunization strategies against vaginal transmission of genital herpes infections are discussed.

A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes.

The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosal immunoprotection against sexually transmitted diseases. To investigate the suitability of plant-expressed Mabs for vaginal preventive applications, we compared a humanized anti-herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soybean. We found these Mabs to be similar in their stability in human semen and cervical mucus over 24 h, their ability to diffuse in human cervical mucus, and their efficacy for prevention of vaginal HSV-2 infection in the mouse.

Contraceptive testing of vaginal agents in rabbits.

Development of new vaginal products, such as microbiocides and contraceptives, requires in vivo testing of their effect on fertility. Rabbits, unlike smaller laboratory animals such as rats and mice, which inseminate in the uterus, inseminate vaginally and thus are valuable as animal models for testing vaginal agents for contraceptive effects. Rabbits are inexpensive and easy to handle compared to nonhuman primates, and have frequently been used for testing the effects of vaginal agents on fertility. We review the pertinent literature and report findings that provide guidance for effectively using and improving the rabbit contraceptive model in testing new vaginal products.

Antigen-releasing polymer rings and microspheres stimulate mucosal immunity in the vagina.

Several recent studies suggest that direct application of antigen to the vaginal surface may enhance local IgA secretion, but the most effective methods for stimulating immunity at the vaginal surface have not been identified. We used antigen-loaded, biocompatible, vaginal rings to provide controlled and sustained antigen delivery directly to the vaginal mucosal surface. Mice were primed with ferritin, either subcutaneously or orally by ferritin-loaded polymer microspheres, and vaginally boosted by insertion of a ferritin-loaded polymer ring. We found that the vaginal rings were a convenient method for providing controlled antigen delivery to the vagina. Subcutaneously primed mice receiving ferritin-loaded vaginal rings had ferritin-specific IgA in their mucus secretions, while mice receiving blank rings did not. Oral priming with ferritin-loaded poly(lactic acid) microspheres also produced significant levels of ferritin-specific IgA in the vaginal secretions, but required the presence of cholera toxin. Controlled ferritin delivery to mucosal surfaces, either by oral, biodegradable microspheres or vaginal rings, provides a convenient and reliable method for enhancing vaginal IgA production in mice.

Contraceptive effect of sperm-agglutinating monoclonal antibodies in rabbits.

Immune infertility in humans correlates clinically with the presence of anti-sperm antibodies that trap (agglutinate) sperm in semen and cervical mucus. To test whether sperm-agglutinating antibodies can be effective contraceptive agents, several mouse anti-rabbit sperm (MARS) sperm-agglutinating monoclonal antibodies (mAbs) were developed that rapidly and completely agglutinate sperm: MARS-M3 (IgM), MARS-G16 (IgG3), and MARS-G17 (IgG3). Contraceptive efficacy of these mAbs was tested by mixing the mAb with 0.1 ml semen (approximately 1/5 of a whole ejaculate) immediately before artificially inseminating rabbits paracervically. This paracervical dose of semen provided a rigorous test since it delivered several thousand times more fertilizing doses than does a human ejaculate. All of the mAbs were contraceptively effective, and MARS-G16 reduced the number of fetuses per animal by 88% and 95% with doses of 150 microg and 2 mg, respectively. The contraceptive efficacy of the MARS mAbs in the rabbit suggests that human sperm-agglutinating mAbs may be effective agents for vaginal contraception in humans.

Tests of vaginal microbicides in the mouse genital herpes model.

Microbicide candidates were selected that have demonstrated activity against sperm or sexually transmitted disease pathogens in vitro, and the efficacy of these agents for preventing vaginal transmission of genital herpes infection was evaluated in the progestin-treated mouse. Each agent was delivered to the vaginas of mice approximately 20 sec prior to delivering a highly infectious herpes simplex virus-2 inoculum. The following agents provided significant protection: anti-HSV monoclonal antibodies III-174 and HSV8, modified bovine beta-lactoglobulin (beta-69), carrageenan, concanavalin A, chlorhexidine, dextran sulfate (average molecular weight 8,000 and 500,000), fucoidan, neem, nonoxynol-9, polystyrene sulfonate, and povidone-iodine. Two agents, gramicidin and heparan sulfate, though highly effective in vitro, were not protective in vivo at the doses tested.

Topically applied human recombinant monoclonal IgG1 antibody and its Fab and F(ab')2 fragments protect mice from vaginal transmission of HSV-2.

A recombinant human anti-herpes simplex virus monoclonal IgG1, antibody and the corresponding Fab and F(ab')2 fragments were tested for efficacy in preventing vaginal transmission of HSV-2 infection in a well-established mouse model for genital herpes. IgG1, Fab, and F(ab')2 were approximately equally protective; vaginal delivery of 1-5 ng provided approximately 50% protection, and vaginal delivery of 400 ng completely protected mice from genital herpes infection (P < 0.001). These results suggest that topical applications of human monoclonal antibodies may be useful in developing new methods for preventing sexually transmitted disease.

Controlled release of antibodies for long-term topical passive immunoprotection of female mice against genital herpes.

Current methods for sexually transmitted diseases (STD) prophylaxis, which can be disruptive and inconvenient, must be used before each act of sexual intercourse, so a method that provides protection over the course of many acts is desirable. We used a mouse model of vaginally-transmitted herpes simplex virus 2 (HSV-2) infection to test polymeric controlled-release devices for sustained passive immunoprotection. Vaginal disks were prepared by dispersing a monoclonal antibody to HSV-2 (III-174) within a matrix of poly(ethylene-co-vinyl acetate); these disks released 2 to 40 micrograms/day of antibody into buffered water. When disks were placed in the vagina, large amounts of III-174 (5 to 3,000 ng) were recovered from the vaginal fluid over the next 8 days. Mice were vaginally challenged with 10 ID50 of HSV-2 either 3 or 7 days after disk placement; no mice receiving III-174 disks became infected, while 65% of control mice receiving identical disks with nonspecific IgG did. Controlled-release disks with III-174 provided significant protection against HSV-2 infection (p < 0.005). This new technology for long-term STD prophylaxis should increase user compliance, a factor limiting the efficacy of current methods.

Residence half-life of IgG administered topically to the mouse vagina.

Antibodies delivered directly to the vagina can provide passive immunoprotection against pregnancy and sexually transmitted disease. The duration of protection is limited by the residence time of the antibodies in the vagina; to our knowledge such residence times have not been reported. We investigated the time-course of disappearance of IgG delivered to the mouse vagina using three different methods to monitor the amount of administered IgG remaining in the vagina: gamma counting of 125I-IgG, viral neutralization of unlabeled monoclonal anti-herpes virus IgG2a, and ELISA of biotinylated IgG. The test IgG was delivered to the vagina in saline and recovered by lavage. All three methods yielded similar results, suggesting that the residence half-life is not significantly affected by the volume administered, phase of the estrous cycle, or labeling of IgG. In awake mice, a significant fraction of IgG disappeared with a relatively short half-life, (t1/2)alpha, of 0.7 +/- 0.1 h; but this rapid (alpha phase) decrease did not occur in anesthetized mice, suggesting that the movements of awake mice expel some of the test IgG-saline solution from the vagina. Over the next 25 h, the test IgG disappeared with a residence half-life, (t1/2)beta, of 5 +/- 2 h. We believe this slow elimination of IgG may depend on the rate that mucus secretions are shed from the vagina.

Passive immunization of the vagina protects mice against vaginal transmission of genital herpes infections.

Vaginal application of human herpes simplex virus (HSV) antiserum, complement-inactivated antiserum, or IgG purified from antiserum protected mice (P < .001, P < .001, and P < .01, respectively) from visible signs of genital HSV-2 infection after subsequent vaginal inoculation with HSV-2 (10 ID50). Vaginal application of an anti-HSV-2 monoclonal antibody (MAb III-174) also protected mice against infection. This MAb, a neutralizing mouse IgG2A against glycoprotein D, prevented infection as determined by viral shedding from the vagina (P < .05), blocked 50% of visible signs of genital herpes infection at a vaginal dose of approximately 10 ng, and blocked 100% of visible signs of infection at a vaginal dose of 1 microgram (P < .001). These results suggest that vaginal applications of anti-HSV antibodies may help prevent sexual transmission of genital herpes infection.

Antibody diffusion in human cervical mucus.

The mucosal immune system actively transports large quantities of antibodies into all mucus secretions, and these secreted antibodies help prevent infectious entry of many pathogens. Mucus is generally thought to protect epithelial cells by forming a diffusional barrier through which only small molecules can pass. However, electron microscopy indicates that the pore size in mucus is approximately 100 nm, which suggests that antibodies as well as other large molecules might also diffuse through mucus. We measured the diffusion coefficients for antibodies and other proteins within human midcycle cervical mucus using two techniques: fluorescence imaging of concentration profiles and fluorescence photobleaching recovery. The two techniques are complementary, since the rates of diffusion are observed over millimeter distances with fluorescence imaging of concentration profiles and micron distances with fluorescence photobleaching recovery. Both methods yielded essentially the same diffusion coefficients. In contrast to previous reports indicating mucus significantly impedes diffusion of small molecules, antibody diffusion in mucus was relatively unimpeded. In our observations IgG, IgG fragments, IgA, and IgM diffused almost as rapidly in cervical mucus as in water (1.0 > Dmucus/Dwater > 0.7). Simple models for diffusion through water-filled pores suggest that the hydrodynamic pore size for cervical mucus is approximately 100 nm, smaller than the approximately 1000 nm pore size of a collagen gel (at 1 mg/ml) and larger than the approximately 10 nm pore size of gelatin (at 100 mg/ml). This estimated pore size is consistent both with electron micrographs and geometric models of interfiber spacing. Based on these results, we predict that particles as large as viruses can diffuse rapidly through human midcycle cervical mucus, provided the particle forms no adhesive interactions with mucus glycoproteins.