No abstract

Topical microbicides are self-administered, prophylactic products for protection against sexually transmitted pathogens. A large number of compounds with known anti-human immunodeficiency virus type 1 (HIV-1) inhibitory activity have been proposed as candidate topical microbicides. To identify potential leads, an in vitro screening algorithm was developed to evaluate candidate microbicides in assays that assess inhibition of cell-associated and cell-free HIV-1 transmission, entry, and fusion. The algorithm advances compounds by evaluation in a series of defined assays that generate measurements of relative antiviral potency to determine advancement or failure. Initial testing consists of a dual determination of inhibitory activity in the CD4-dependent CCR5-tropic cell-associated transmission inhibition assay and in the CD4/CCR5-mediated HIV-1 entry assay. The activity is confirmed by repeat testing, and identified actives are advanced to secondary screens to determine their effect on transmission of CXCR4-tropic viruses in the presence or absence of CD4 and their ability to inhibit CXCR4-and CCR5-tropic envelopemediated cell-to-cell fusion. In addition, confirmed active compounds are also evaluated in the presence of human seminal plasma, in assays incorporating a pH 4 to 7 transition, and for growth inhibition of relevant strains of lactobacilli. Leads may then be advanced for specialized testing, including determinations in human cervical explants and in peripheral blood mononuclear cells against primary HIV subtypes, combination testing with other inhibitors, and additional cytotoxicity assays. PRO 2000 and SPL7013 (the active component of VivaGel), two microbicide products currently being evaluated in human clinical trials, were tested in this in vitro algorithm and were shown to be highly active against CCR5-and CXCR4-tropic HIV-1 infection.

The first product to be clinically evaluated as a microbicide contained the nonionic surfactant nonoxynol-9 (nonylphenoxypolyethoxyethanol; N-9). Many laboratories have used N-9 as a control compound for microbicide assays. However, no published comparisons of the results among laboratories or attempts to establish standardized protocols for preclinical testing of microbicides have been performed. In this study, we compared results from 127 N-9 toxicity and 72 efficacy assays that were generated in five different laboratories over the last six years and were performed with 14 different cell lines or tissues. Intra-assay reproducibility was measured at two-, three-, and fivefold differences using standard deviations. Interassay reproducibility was assessed using general linear models, and interaction between variables was studied using step-wise regression. The intra-assay reproducibility within the same N-9 concentration, cell type, assay duration, and laboratory was consistent at the twofold level of standard deviations. For interassay reproducibility, cell line, duration of assay, and N-9 concentration were all significant sources of variability (P < 0.01). Half-maximal toxicity concentrations for N-9 were similar between laboratories for assays of similar exposure durations, but these similarities decreased with lower test concentrations of N-9. Results for both long (>24 h) and short (<2 h) exposures of cells to N-9 showed variability, while assays with 4 to 8 h of N-9 exposure gave results that were not significantly different. This is the first analysis to compare preclinical N-9 toxicity levels that were obtained by different laboratories using various protocols. This comparative work can be used to develop standardized microbicide testing protocols that will help advance potential microbicides to clinical trials.