Digallate Dimers of (−)-Epigallocatechin Gallate Inactivate Herpes Simplex Virus

Abstract

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 Х2 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.Herpes simplex virus (HSV) is the leading cause of genital ulcers in the developed world (16). In the United States alone 40 to 60 million people are infected with HSV-2 (23). In some cities in North America and Europe, HSV-1, which is usually found orally, causes more than half of the new cases of genital herpes, particularly in women (16).In the absence of a fully effective HSV vaccine (1), topical microbicides represent an important potential strategy for preventing the sexual and mother-to-child spread of HSV. It has been shown for both HSV (33) and human immunodeficiency virus (HIV) (20) that the viral load in genital secretions is the chief predictor of viral transmission, suggesting that if a microbicide reduces the concentration of excreted HSV, the incidence of viral transmission will be reduced.(Ϫ)-Epigallocatechin gallate (EGCG), the primary catechin in green tea (5), has been previously shown in our laboratory to inactivate multiple clinical isolates of HSV-1 and HSV-2 (12). These studies were carried out at neutral pH. While efficacy at neutral pH would be fine for an anti-HSV compound utilized in the eye (14), a topical microbicide utilized vaginally (27) or on the skin (30) to reduce HSV transmission must function across a pH range that is more acidic than neutral. Vaginal pH will vary over a range of 3.9 to 5.7 (21) and then become more neutral in the presence of semen, rising to pH 6 to 7 (27). The p...

A Small Molecule Inhibits Virion Attachment to Heparan Sulfate- or Sialic Acid-Containing Glycans

“…Isaacs et al tested EGCG (mostly as a control for several oxidative dimerization products of EGCG) against a panel of enveloped and nonenveloped viruses (35). EGCG was active against the heparan sulfate-binding respiratory syncytial virus and Semliki Forest virus, but not against PV (which does not bind heparan sulfate or sialic acid), consistent with our results and model.…”

“…EGCG was active against the heparan sulfate-binding respiratory syncytial virus and Semliki Forest virus, but not against PV (which does not bind heparan sulfate or sialic acid), consistent with our results and model. In those experiments, EGCG failed to inhibit infectivity of measles virus, coxsackie A9 virus, coxsackie B4 virus, and echovirus 6 (35).…”

“…To test incorporation of 35 S, 1 l of the labeled virions or mock preparation was fixed in 100 l of 100% ethanol and added to scintillation vials containing 4 ml of aqueous scintillant. 35 S radioactivity was measured using a Beckman Coulter LS 6500 scintillation counter. Virions were labeled to approximately 0.02 cpm/PFU (HSV-1 and VSV), 1 cpm/ PFU (PV), 14 cpm/PFU (RV), or 7 cpm/PFU (AdV).…”

“…Titers of 35 S-labeled viruses were determined by standard plaquing assays. To test incorporation of 35 S, 1 l of the labeled virions or mock preparation was fixed in 100 l of 100% ethanol and added to scintillation vials containing 4 ml of aqueous scintillant.…”

“…EGCG is the most active compound, with activity against human immunodeficiency virus (HIV), influenza A virus (IAV), enterovirus 71, adenovirus (AdV), hepatitis B virus (HBV), clinical isolates of herpes simplex viruses 1 and 2 (HSV-1 and -2), and hepatitis C virus (HCV), among others (27)(28)(29)(30)(31)(32)(33)(34)(35). Derivatives of EGCG, such as digallate dimers, also have antiviral activities (35). For the most part, EGCG has been shown to inhibit the infectivity of a broad range of unrelated enveloped and nonenveloped viruses.…”