The effect of menopause on the innate antiviral activity of cervicovaginal lavage.

OBJECTIVE: Reproductive hormones are known to impact innate mucosal immune function of the lower genital tract. Our objectives were to determine the effect of hormonal status on intrinsic antiviral (herpes simplex virus [HSV]-1, HSV-2, and human immunodeficiency virus [HIV]-1) activity of cervicovaginal lavage (CVL). STUDY DESIGN: CVL was collected from 165 asymptomatic women including postmenopausal women (n = 29); women not on contraception in days 1-14 (n = 26) or days 15-28 (n = 27) of the menstrual cycle; and women using the levonorgestrel intrauterine device (n = 28), depot medroxyprogesterone acetate (n = 28), or combined oral contraceptives (n = 27). The anti-HSV-1/-2 and the anti-HIV-1 activity of the CVL were measured using plaque assays and the Jurkat-Tat-CCR5 assay, respectively. RESULTS: CVL from all of the groups had modest antiviral activity. Anti-HIV-1 activity was decreased in CVL from postmenopausal women when compared to premenopausal women (11% vs 34%, P = .002). However, there was no difference in anti-HIV-1 activity among premenopausal women regardless of phase of menstrual cycle or contraceptive use. Anti-HIV-1 activity was associated with the protein content of the CVL (r = 0.44, P < .001). There was no difference in anti-HSV-1 or -2 activity by hormonal group. CONCLUSION: Menopause is associated with decreased innate HIV-1 activity in the lower genital tract, suggesting that factors in the vaginal fluid could play a role in increased susceptibility of HIV-1 infection in postmenopausal women. Hormonal contraceptive use, menopause, and phase of menstrual cycle did not have a measurable impact on the intrinsic anti-HSV-1 or -2 activity.

Theaflavin-3,3'-digallate and lactic acid combinations reduce herpes simplex virus infectivity.

The present study examined the efficacy of using multiple mechanisms as part of a topical microbicide to inactivate herpes simplex virus (HSV) by combining theaflavin-3,3'-digallate (TF-3) and lactic acid (LA) over the pH range of 4.0 to 5.7 to mimic conditions in the female reproductive tract. Six clinical isolates of HSV-2 and two clinical isolates of HSV-1 were almost completely inactivated when TF-3 (100 µM) was present with LA over the pH range of 4.5 to 5.7, whereas four additional HSV-1 clinical isolates required TF-3 concentrations of 250 to 500 µM for comparable virus titer reduction. LA (1%) alone at pH 4.0 reduced the titers of laboratory and clinical isolates of HSV-1 and HSV-2 by ≥ 5 log10, but most LA-dependent antiviral activity was lost at a pH of ≥ 4.5. When HSV-1 and HSV-2 were incubated at pH 4.0 without LA virus titers were not reduced. At pH 4.0, HSV-1 and HSV-2 titers were reduced 5 log(10) in 20 min by LA alone. TF-3 reduced HSV-2 titers by 5 log10 in 20 to 30 min at pH 4.5, whereas HSV-1 required 60 min for comparable inactivation. Mixtures of TF-3 and LA stored at 37 °C for 1 month at pH 4.0 to 5.7 maintained antiviral activity. Semen, but not cervical vaginal fluid, decreased LA-dependent antiviral activity at pH 4.0, but adding TF-3 to the mixture reduced HSV titers by 4 to 5 log10. These results indicate that a combination microbicide containing TF-3 and LA could reduce HSV transmission.

Digallate dimers of (-)-epigallocatechin gallate inactivate herpes simplex virus.

Topical microbicides are potentially an alternative method to vaccines for reducing the spread of herpes simplex virus (HSV). We have previously shown (S. Liu et al., Biochim. Biophys. Acta 1723:270-281, 2005) that the catechin (-)-epigallocatechin gallate (EGCG) inactivates HSV at neutral pH; however, to function in the female genital tract EGCG must also be effective at acidic pH. EGCG inactivated HSV-1 and HSV-2 at pH 8.0 by 3 log(10) to 4 log(10) but was ineffective at pH 5.7. The EGCG digallate dimers theasinensin A, P2, and theaflavin-3,3'-digallate (TF-3) inactivated both viruses by 3 log(10) to 4 log(10) at pH 5.7 and as much as 5 log(10) at pH 8.0. TF-3 inactivated HSV-1 and HSV-2 by 4 to 5 log(10) in the pH range of 4.0 to 5.7. Dimers with one gallate moiety had antiviral activity intermediate between the activities of EGCG and digallate dimers. Confocal and electron microscopy showed that theasinensin A did not damage Vero cells. All EGCG dimers inactivated enveloped viruses with class I, class II, and class III (HSV-1, HSV-2) fusion proteins more effectively than did monomeric EGCG. EGCG had no activity against the nonenveloped viruses tested, but TF-3 reduced the titer of 4 of 5 nonenveloped viruses by ≅2 to 3.5 log(10). Results also showed that HSV-1 glycoprotein B (gB) was aggregated more rapidly by theasinensin A than EGCG, which, when taken together with the nonenveloped virus data, suggests that dimers may inhibit the function of viral proteins required for infectivity. Digallate dimers of EGCG appear to have excellent potential as microbicidal agents against HSV at acidic and neutral pHs.

Characterization of cyclodextrin inclusion complexes of the anti-HIV non-nucleoside reverse transcriptase inhibitor UC781.

The highly potent anti-HIV agent UC781 is being evaluated for use in topical microbicides to prevent HIV transmission. However, UC781 is extremely hydrophobic with poor water solubility, a property that may complicate appropriate formulation of the drug. In this study, we examined the ability of several cyclodextrins, beta-cyclodextrin (beta CD), methyl-beta-cyclodextrin (M beta CD), and 2-hydroxylpropyl-beta-cyclodextrin (HP beta CD), to enhance the aqueous solubility of UC781. Each of the cyclodextrins provided dramatic increases in UC781 aqueous solubility, the order being M beta CD>HP beta CD>beta CD. The complexation constants (K (1:1)) of the inclusion complexes were determined via a phase solubility technique using high-performance liquid chromatography and showed that UC781 solubility increased linearly as a function of cyclodextrin concentration. Ultraviolet spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and 2D (1)H ROESY NMR spectroscopy were used to further characterize these UC781/cyclodextrin complexes. The inhibitory potency of UC781 and its HP beta CD inclusion complex were evaluated using an in vitro HIV-1 reverse transcriptase inhibition assay The inhibitory potency of the UC781/HP beta CD complex was 30-fold greater than that of UC781 alone, showing that the complexed drug is able to provide substantial inhibition of its target. The enhancement of UC781 aqueous solubility is essential for the development of a useful vaginal microbicide dosage form, and our data suggest that UC781/cyclodextrin inclusion complexes may be useful in this context.

Killing of Neisseria gonorrhoeae, Streptococcus agalactiae (group B streptococcus), Haemophilus ducreyi, and vaginal Lactobacillus by 3-O-octyl-sn-glycerol.

The microbicide candidate octylglycerol inactivates sexually transmitted bacterial pathogens at concentrations which spare normal vaginal flora (lactobacillus). Standard minimum microbicidal concentration assays and time-kill assays revealed the drug concentrations and times required for inactivation. Octylglycerol concentrations must exceed the binding capacity of any human serum albumin to be effective.

Epigallocatechin gallate inactivates clinical isolates of herpes simplex virus.

In the absence of a fully effective herpes simplex virus (HSV) vaccine, topical microbicides represent an important strategy for preventing HSV transmission. (-)-Epigallocatechin gallate (EGCG) (molecular weight, 458.4) is the primary catechin in green tea. The present study shows that EGCG has greater anti-HSV activity than other green tea catechins and inactivates multiple clinical isolates of HSV type 1 (HSV-1) and HSV-2. EGCG reduced HSV-2 titers by >or=1,000-fold in 10 to 20 min and reduced HSV-1 titers by the same amount in 30 to 40 min. The anti-HSV activity of EGCG is due to a direct effect on the virion, and incubating Vero and CV1 cells with EGCG for 48 h prior to infection with HSV-1 and HSV-2, respectively, does not reduce HSV production. Electron microscopic (EM) studies showed that purified virions exposed to EGCG were damaged, and EM immunogold labeling of the envelope glycoproteins gB and gD was significantly reduced following EGCG treatment while capsid protein labeling was unchanged. When purified HSV-1 envelope glycoproteins gB and gD were incubated with EGCG and then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lower-molecular-weight gB and gD bands decreased and new higher-molecular-weight bands appeared, indicating the EGCG-dependent production of macromolecular complexes. gB and gD are essential for HSV infectivity, and these results suggest that EGCG could inactivate HSV virions by binding to gB, gD, or another envelope glycoprotein. EGCG is stable in the pH range found in the vagina and appears to be a promising candidate for use in a microbicide to reduce HSV transmission.

Effects of physiological fluids on physical-chemical characteristics and activity of topical vaginal microbicide products.

The increased incidence of HIV infection in women has identified a need to develop a female controlled method to prevent sexually transmitted infections (STIs), including HIV. Formulations have been developed in our laboratory for two potential microbicide drug substances, 3-O-octyl-sn-glycerol (3-OG) and UC-781. A major concern for microbicide product development is dilution by vaginal fluids following application thereby reducing antimicrobial activity. We investigated the effect of product dilution on microbicidal activity and the product's chemical and physical properties by using vaginal fluid (VFS) and cervical mucus simulants (CMS). 3-OG and UC-781 were individually formulated into three semi-solid drug containing products: Hydroxyethylcellulose, Methylcellulose/Carbopol, and Liposome. Viscosity, osmolality, pH and in vitro activity against HIV-1 were evaluated. Results showed that pH was not affected when products were diluted with VFS; however, increases in pH were observed following CMS dilution. Viscosity was significantly decreased for all the dilutions tested excepted for some of the liposome products. Hydrogel products maintained greater activity against HIV-1 than Liposome products. The effect of dilution on anti-HIV activity varied based upon excipient choice and chemical characteristics of the active agent. These in vitro assessments can identify the potential for changes in product's physical-chemical characteristics in vivo which may result in diminished product performance.

Use of frozen-thawed cervical tissues in the organ culture system to measure anti-HIV activities of candidate microbicides.

Cervical tissue-based organ culture system has been used to test the cytotoxicity and antiviral activity of microbicides. One of the problems of using current organ culture methods for routine microbicide testing is the need to continually obtain fresh tissue, which can be limited in access and supply. Use of frozen tissue, stored when available and thawed when needed, would alleviate the need for constant access to new tissue. This study was designed to explore the possibility of using frozen-thawed cervical tissue to test microbicides for their anti-HIV activity. We provided biochemical, histological, and quantitative immunohistochemical data to demonstrate the integrity of the frozen-thawed organ culture system. Significant levels of HIV-1 mucosal transmission were noted with both fresh and frozen-thawed tissue, regardless of the coreceptor usage of the virus isolate. Furthermore, candidate microbicides UC781, beta-cyclodextrin, and octylglycerol inhibited HIV-1 transmission across the mucosa of frozen-thawed tissues with a level of efficiency similar to that of fresh tissues. Therefore, frozen-thawed cervical tissue in the organ culture system provides a practical and convenient model to screen topical microbicides for their ability to block sexual transmission of HIV-1, and reduces the problems associated with procurement of the numerous tissues required for evaluation and comparison of microbicide candidates among different laboratories.

Inactivation of herpes simplex virus clinical isolates by using a combination microbicide.

A combination microbicide using the lipid-ether 1-0-octyl-sn-glycerol (OG) (3 mM) and peptide LSA5 (9 microM) synergistically inactivated six clinical isolates of herpes simplex virus type 2 (HSV-2) by 30- to 100-fold and five of six HSV-1 isolates by 10-fold more than the sum of OG and LSA5 used individually within 30 min. OG plus LSA5 inactivated all HSV clinical isolates by > or = 1,000-fold in 10 to 40 min.

Human milk inactivates pathogens individually, additively, and synergistically.

Breast-feeding can reduce the incidence and the severity of gastrointestinal and respiratory infections in the suckling neonate by providing additional protective factors to the infant's mucosal surfaces. Human milk provides protection against a broad array of infectious agents through redundancy. Protective factors in milk can target multiple early steps in pathogen replication and target each step with more than one antimicrobial compound. The antimicrobial activity in human milk results from protective factors working not only individually but also additively and synergistically. Lipid-dependent antimicrobial activity in milk results from the additive activity of all antimicrobial lipids and not necessarily the concentration of one particular lipid. Antimicrobial milk lipids and peptides can work synergistically to decrease both the concentrations of individual compounds required for protection and, as importantly, greatly reduce the time needed for pathogen inactivation. The more rapidly pathogens are inactivated the less likely they are to establish an infection. The total antimicrobial protection provided by human milk appears to be far more than can be elucidated by examining protective factors individually.

A lipid-peptide microbicide inactivates herpes simplex virus.

A microbicide combining the lipid-ether 1-0-octyl-sn-glycerol (OG; 3 mM) and peptide D2A21 (9 microM) reduced herpes simplex virus type 1 (HSV-1) and HSV-2 titers by at least 1,000-fold, more than the sum of the inactivations produced by OG and D2A21 alone. OG plus D2A21 reduced HSV-1 and HSV-2 titers by > or =1,000-fold in < or =10 and < or =20 min, respectively, whereas OG and D2A21 used alone produced almost no virus inactivation during these times.

Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk.

Lipids previously shown to have antiviral and antibacterial activity in buffers were added to human milk, bovine milk, and infant formulas to determine whether increased protection from infection could be provided to infants as part of their diet. Fatty acids and monoglycerides with chain lengths varying from 8 to 12 carbons were found to be more strongly antiviral and antibacterial when added to milk and formula than long chain monoglycerides. Lipids added to milk and formula inactivated a number of pathogens including respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), Haemophilus influenzae, and Group B streptococcus. The results presented in this study suggest that increased protection from infection may be provided to infants at mucosal surfaces, prior to the digestion of milk and formula triglycerides, by the addition of antimicrobial medium chain monoglycerides to an infant's diet.