Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

Preclinical evaluation of lime juice as a topical microbicide candidate.

BACKGROUND: The continued growth of the global HIV epidemic highlights the urgent need to develop novel prevention strategies to reduce HIV transmission. The development of topical microbicides is likely to take a number of years before such a product would be widely available. This has resulted in a call for the rapid introduction of simpler vaginal intervention strategies in the interim period. One suggested practice would be vaginal douching with natural products including lime or lemon juice. Here we present a comprehensive preclinical evaluation of lime juice (LiJ) as a potential intervention strategy against HIV. RESULTS: Pre-treatment of HIV with LiJ demonstrated direct virucidal activity, with 10% juice inactivating the virus within 5 minutes. However, this activity was significantly reduced in the presence of seminal plasma, where inactivation required maintaining a 1:1 mixture of neat LiJ and seminal plasma for more than 5 minutes. Additionally, LiJ demonstrated both time and dose-dependent toxicity towards cervicovaginal epithelium, where exposure to 50% juice caused 75-90% toxicity within 5 minutes increasing to 95% by 30 minutes. Cervicovaginal epithelial cell monolayers were more susceptible to the effects of LiJ with 8.8% juice causing 50% toxicity after 5 minutes. Reconstructed stratified cervicovaginal epithelium appeared more resilient to LiJ toxicity with 30 minutes exposure to 50% LiJ having little effect on viability. However viability was reduced by 75% and 90% following 60 and 120 minutes exposure. Furthermore, repeat application (several times daily) of 25% LiJ caused 80-90% reduction in viability. CONCLUSION: These data demonstrate that the virucidal activity of LiJ is severely compromised in the presence of seminal plasma. Potentially, to be effective against HIV in vivo, women would need to apply a volume of neat LiJ equal to that of an ejaculate, and maintain this ratio vaginally for 5-30 minutes after ejaculation. Data presented here suggest that this would have significant adverse effects on the genital mucosa. These data raise serious questions about the plausibility and safety of such a prevention approach.

PRO-2000, an antimicrobial gel for the potential prevention of HIV infection.

Indevus Pharmaceuticals Inc, under license from Paligent Inc, is developing PRO-2000, an antimicrobial gel for the prevention of HIV infection. The company is also investigating its potential to prevent the transmission of other sexually transmitted diseases. In February 2005, Indevus launched a pivotal phase III trial for the prevention of HIV infection in women. At that time, further phase III trials in 12,000 African women were scheduled to begin in 2005. A second phase III trial began for the prevention of sexually transmitted infections, including HIV, herpes, Chlamydia and gonorrhea, in Africa in October 2005.

Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides.

OBJECTIVES: Establishment of an in vitro model to evaluate rectal safety and the efficacy of microbicide candidates. DESIGN: An investigation and characterization of human colorectal explant culture for screening candidate microbicides to prevent rectal transmission of HIV-1 infection. METHODS: Human colorectal explants were cultured at the liquid-air interface on gelfoam rafts. Phenotypic characterization of HIV-1 target cells was performed by fluorescence-activated cell sorter analysis. HIV-1 infection was determined by the measurement of p24 antigen release, viral RNA, and proviral DNA accumulation. RESULTS: Colorectal explant CD4 T cells expressed higher CCR5 and CXCR4 levels compared with blood. Minor differences between the rectal and sigmoid colon were observed with a trend for slightly higher CCR5 and HLA-DR expression in cells from the sigmoid colon. Favourable culture conditions were established for colorectal tissue. Although tissue structure degenerated with time, CD4: CD8 cell ratios remained constant, and tissue supported productive HIV-1 infection. The ability of candidate microbicides to inhibit R5 HIV-1 infection was evaluated. Polyanion candidates, PRO2000 and dextrin sulphate, provided 99% protection at 1 microg/ml and 1 mg/ml, respectively, equivalent to 1/5000 and 1/40 of the vaginal formulations. The nucleotide reverse transcriptase inhibitor (NRTI) 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) provided protection at concentrations 1000-fold lower (10 microg/ml) than the proposed vaginal formulation (1%). Furthermore, non-NRTI UC-781 and TMC-120 provided greater than 99% inhibition at 3.3 or 0.33 microg/ml, respectively. No products demonstrated toxicity to rectal mucosa at inhibitory concentrations. CONCLUSION: Colorectal explant culture was shown to be a useful tool for the preclinical evaluation of potential microbicides. The data suggest that rectally applied microbicides might provide protection from HIV-1 transmission.

Candidate polyanion microbicides inhibit HIV-1 infection and dissemination pathways in human cervical explants.

BACKGROUND: Heterosexual intercourse remains the major route of HIV-1 transmission worldwide, with almost 5 million new infections occurring each year. Women increasingly bear a disproportionate burden of the pandemic, thus there is an urgent need to develop new strategies to reduce HIV-1 transmission that could be controlled by women themselves. The potential of topical microbicides to reduce HIV transmission across mucosal surfaces has been clearly identified, and some agents are currently under evaluation in clinical trials. Many of these "first generation" microbicides consist of polyanionic compounds designed to interfere with viral attachment. Here we have evaluated two candidate polyanion compounds in clinical trials, PRO 2000 and dextrin sulphate (DxS) to determine their safety and efficacy against in vitro HIV-1 and HSV-2 infection using cellular and tissue explant models. RESULTS: PRO 2000 and DxS potently inhibited infection by HIV-1 X4 and R5 isolates when present during viral exposure. However PRO 2000 required 10-fold and DxS 2000-fold more compound to block infection with R5 virus than X4. While both compounds were virucidal for X4 HIV-1, neither was virucidal for R5 virus. PRO 2000 efficiently inhibited infection of cervical explants and dissemination of virus by migratory DC. DxS was less active, able to completely inhibit cervical explant infection, but providing only partial reduction of virus dissemination by DC. PRO 2000, but not DxS, also inhibited HIV-1 binding to DC-SIGN+ cells and trans infection of co-cultured target cells. The inflammatory potential of both compounds was screened by measurement of cytokine production from cervical explants, and statistically significant increases were only observed for IL-1beta and RANTES following treatment with PRO 2000. Both compounds also demonstrated potent activity against HSV-2 infection of cervical epithelial cells. CONCLUSION: Our results demonstrate that PRO 2000 is a potent inhibitor of R5 HIV-1 infection and dissemination pathways in human cervical explants. DxS, while demonstrating significant inhibition of R5 infection, was less active against DC mediated dissemination pathways. PRO 2000 has now entered human phase III efficacy trials.

In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories.

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.