With a growing epidemic of genital herpes and with more than 40 million people worldwide living with HIV infection, there is an urgent need to identify safe and effective topical microbicides (18
). These studies extend our understanding of one major class of microbicides, SPs, by focusing on their activities against HSV. Using immortalized human endocervical cells, we found that the SPs reduce the levels of HSV infection ~100,000-fold, are active even when the cells are exposed to virus at a very high MOI, and retain their activities against clinical isolates. Additionally, the infectious center studies indicate that SPs also prevent infection by cell-associated virus and cell-to-cell spread. This may be due to the ability of the drugs to bind to the envelope glycoproteins required for cell-to-cell spread. The inhibitory effect on cell-to-cell spread may be important, because the source of sexually transmitted virus may be intracellular.
One of the targets of SP is gB-2. gB-2 plays the predominant role in mediating HSV-2 binding to cells, but it is also required (along with gD and the heterodimeric complex of gH and gL) for penetration and cell-to-cell spread (5
). We used optical biosensor technology to quantify the affinity (KD
) and the stability (Kd
) of the interactions between gB-2 and microbicides. The values obtained show that the complexes of gB-2 and SPs are highly stable (Kd
range, 1.5 × 10−3
to 9 × 10−4
). In an earlier report, biosensor technology was used to investigate the interaction of a truncated form of gB-2 with heparin (39
). The gB-2 ectodomain bound to biosensor surfaces coated with heparin with a KD
of 7.7 × 10−7
M and a Kd
of 4 × 10−4
. The experimental conditions in our studies differed, in that the biosensor surfaces were coated with gB-2. The results obtained, however, suggest that the complexes of microbicides and gB-2 are at least as stable as gB-2 and heparin complexes and have affinities comparable to those observed for gB-2 and heparin. Moreover, the affinities and stabilities are at least as great as those observed for gC-1 and gC-2 with heparan sulfate and greater than those observed for gD and one of its coreceptors, HveA (30
). The stability of the SP-glycoprotein complexes may be critical for their activities as microbicides, since a highly stable complex may function to irreversibly block the binding of HSV to cells. This notion is supported by the observation that anti-HSV activity is retained if virus is first pretreated with the drug, followed by dilution to subinhibitory concentrations (Table ). It should be noted that SPs must also interact with gC-2, gC-1, and gB-1. This follows from the observations that SPs inhibit the binding of HSV-1 to cells, mediated primarily by gC-1 (16
), as well as the binding of variants of both serotypes with gC or gB deletions (data not shown). The affinities of the SPs for these other glycoproteins and the abilities of these compounds to bind to other envelope glycoproteins were not evaluated.
Factors in the genital tract and changes in pH may influence the activities of microbicides. Vaginal secretions from healthy women of reproductive age are characteristically acidic (pH 4 to 5) but are neutralized by alkaline semen (pH 7 to 8). In addition to pH, antimicrobial polypeptides and other components of vaginal fluid could interfere with microbicide activity. These studies demonstrate that SPs retain their activities against HSV over a broad pH range (4.0 to 8.0) and in the presence of cervical fluid. However, these studies should be replicated once an optimal formulation has been identified, as the excipients in the formulation may modify activity or stability.
A fundamental principle in microbicide development is ensuring that candidate compounds are nontoxic to the genital mucosa. The importance of more fully investigating the toxicity profile of any potential agent has been highlighted by a recently completed phase III clinical trial which showed that N-9 may actually increase the rate of HIV transmission (37
). The relevant assays for monitoring toxicity in vitro or in vivo have not been established. Inflammatory responses to topical agents may increase the rates of transmission or acquisition of agents of STIs by several mechanisms. An inflammatory response may recruit target cells into the area; may activate macrophages or quiescent T cells, rendering them more susceptible to HIV or HSV; and may induce HIV replication in the reservoir of latently infected T cells (9
). A recent study showed that N-9 caused IL-1α and IL-1β release and a decrease in SLPI levels by cervical epithelial cells in vitro (8
). These in vitro findings may help explain the observed increase in the rate of HIV acquisition among sex workers who frequently applied N-9 (37
We examined the in vitro effects of the SPs and SAMMA on IL-1α, IL-1β, IL-8, and SLPI production following repeated exposure of immortalized human endocervical cells to these compounds. The most striking finding was that SLPI levels were substantially reduced following exposure to the SPs but not following exposure to SAMMA. SLPI is present in saliva and cervical and bronchial secretions and has been shown to inhibit HIV infection in vitro (24
). Its effects on HSV are unknown. The biological significance of these findings is unclear, particularly in view of the proteinase-inhibitory activity of at least PSS (2
) and PMHS (R. A. Anderson, unpublished data). It remains to be determined whether the reductions in SLPI levels observed in these studies would increase susceptibility to HIV. This possibility needs to be verified in vitro, in animal models, and in pilot clinical studies.
These studies did not uncover substantial differences in the mechanisms of anti-HSV activity between the four SPs and SAMMA, despite the structural dissimilarities between SAMMA and the SPs. Higher concentrations of CS were needed to achieve the same degree of antiviral activity. However, the peak molecular mass of CS is greater than those of the other SPs, which may contribute to the differences noted when the drugs are compared on a weight basis. Notably, differences were observed with respect to cytotoxicity and effects on SLPI levels.
These studies support the concept that SPs should provide protection against HSV by preventing viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the SP-virus complex. Importantly, these compounds retain their antiviral activities in the presence of cervical secretions and over a broad pH range. Prior to the initiation of large-scale clinical trials, more rigorous evaluation of the safety of this class of compounds and all topical formulations should include a thorough investigation of the changes in inflammatory cells and cytokines and the effects on host defenses following repeated applications.