The current studies were initially designed to further evaluate the safety of GRFT, which is being advanced as a candidate vaginal product to prevent HIV infection. An unexpected finding was that GRFT inhibited HSV-2 infection postentry by blocking cell-to-cell spread ( and ) and protected mice from genital herpes disease (). There was little dissemination of virus in mice treated with a single dose of gel prior to vaginal inoculation, as evidenced by the imaging studies with a luciferase-reporter virus (). Importantly, GRFT retained its protective activity in the mice when virus was introduced in human seminal plasma. These results contrast with studies of PRO 2000 and cellulose sulfate, polyanionic vaginal products that failed to prevent HIV or HSV-2 in clinical trials (18
). Seminal proteins interfered with the antiviral activity of these entry inhibitors by competing with their ability to bind to the viral envelope (16
). Thus, GRFT appears to inhibit HSV-2 by a unique postentry mechanism distinct from other candidate topical PrEP agents.
HSV disseminates primarily by direct cell-to-cell spread, a process that enables virus to escape neutralizing antibody. The more potent in vitro
activity of GRFT against cell-to-cell spread compared to entry is somewhat surprising, as both fusion events require cellular receptors/coreceptors and glycoproteins B (gB), gD, and hetero-oligomers of gH-gL. However, several lines of evidence suggest that entry and cell-to-cell spread are not identical processes. First, mutants selected for growth on U(S)11cl19.3 cells, a cell line resistant to entry and cell-to-cell spread because of the absence of a gD receptor, exhibited a marked increase in cell-to-cell spread without a concomitant increase in efficiency of entry of free virus, indicating that these processes are distinct (41
). Second, gE contributes to cell-to-cell spread in epithelial and neuronal cell models but is not required for viral entry (42
). Third, in studies of HSV-1 infection in Ric21 cells, a BHK cell line defective in the enzymes of the Golgi system which add terminal sugars to N-linked glycans, there was a reduction in plaque size and the number of viral progeny released into the culture supernatant, but the infectivity of released virions from both cell types was similar (44
). These findings are consistent with the observation here that GRFT, which targets N-linked glycans, has only modest effects on entry but prevents cell-to-cell spread. The more potent activity against cell-to-cell spread is also reminiscent of the finding that GRFT is the most potent antiretroviral of any class for preventing transmission of cell-associated HIV-1 (45
GRFT may target glycans on the viral envelope glycoproteins and/or cellular receptors/coreceptors involved in cell-to-cell spread. The most extensively characterized HSV glycoprotein, with respect to its N- and O-linked glycosylations, is HSV-1 gC (gC-1), which contains nine sites for N-linked glycosylation and numerous clusters of O-linked glycans (46
). However, while gC-1 mediates binding of HSV-1 to heparan sulfate and is involved in immune evasion through its interactions with complement components, it is not required for cell-to-cell spread (47
). Moreover, for HSV-2, gB-2, not gC-2, plays the major role in promoting viral binding, and gC-2 is dispensable for viral infection in cell culture (48
). Thus, it seems unlikely that GRFT targets gC. It is also possible that GRFT targets carbohydrates on heparan sulfate proteoglycans or other cell-surface components that participate in cell-to-cell spread (31
). The specific structural features of heparan sulfate required for cell-to-cell spread may differ from those required for binding and have not yet been elucidated (49
). Future studies with deletion viruses, recombinant glycoproteins (presuming that they are properly glycosylated), and cell surface molecules are needed to identify precisely what viral and/or cellular components and glycans GRFT targets to prevent cell-to-cell spread.
The other key findings from this study add to the growing safety profile of GRFT. First, mice responded to HSV-2 exposure with a potent IFN response that was similar in both the Carbopol- and GRFT-treated mice (). Thus, GRFT does not appear to prevent protective mucosal responses. Second, although GRFT induced a modest decrease in the TER and a concomitant statistically not significant increase in HIV migration across the epithelial barrier to infect T cells cultured in the lower chamber (), this did not translate to increased susceptibility to HSV-2 when mice were treated for 7 days with GRFT and then challenged with sublethal doses of HSV-2. This contrasts with results obtained here and in previous studies with N-9, which triggered a rapid and profound drop in TER and increased the susceptibility of the mice to HSV-2 following intravaginal challenge with either an LD10
). The results also differ from our studies with cellulose sulfate, which triggered a more sustained decrease in TER and promoted HSV-2 infection in the murine model (14
). Third, GRFT was not cytotoxic and had no deleterious effects on DCs. The drug did not induce DC apoptosis or maturation nor did it prevent DCs from responding appropriately to a maturation signal (LPS). Maintenance of DC function is critical, as these cells play a sentinel role in mucosal defense and link innate and adaptive immune responses.
The protective effects of GRFT against HSV disease, a major cofactor in the HIV epidemic, suggest that GRFT may contribute both directly and indirectly to HIV protection. Despite efforts to limit the spread of HSV, the worldwide prevalence and public health impact of genital herpes continues to increase; HSV-2 seroprevalence rates approach 90 to 95% among HIV-infected individuals and female sex workers in developing countries, where HSV-2 remains the dominant cause of genital ulcerative disease (50
). Two meta-analyses support the conclusion that prevalent HSV-2 infection increases the risk of HIV acquisition 2- to 4-fold, and incident HSV-2 further increases the risk and contributes more to the spread of HIV-1 than number of sex partners or other sexually transmitted infections (STI) (51
). Thus, the protective effects of GRFT against HSV-2 should further augment its potential as a topical PrEP product.
While the concentration of GRFT needed to inhibit HSV in vitro
(high nanomolar to low micromolar) is greater than that observed for other viruses where GRFT is effective in the low nanomolar to mid-picomolar range (e.g., HIV, severe acute respiratory syndrome coronavirus [SARS-CoV]) (53
), the concentrations delivered in topical gels clearly exceed the concentration needed to protect against HSV and were effective in the murine model even with only a single dose and in the presence of human seminal plasma. The higher concentrations may reflect differences in carbohydrates expressed by HSV envelope glycoproteins. GRFT binds to select monosaccharides (mannose, glucose, and N
-acetylglucosamine) in a multivalent manner via its three independent carbohydrate-binding domains and is thus ideally designed to engage multiple triantennary arms of specific high-mannose oligosaccharides, including those expressed by HIV gp120 (54
). Repeated gel applications or alternative sustained release formulations of the drug could provide even greater protection against both HSV and HIV and indicate that GRFT is a strong candidate for further advancement, either alone or in combination with other antiretroviral drugs, as topical PrEP.