Ex vivo comparison of microbicide efficacies for preventing HIV-1 genomic integration in intraepithelial vaginal cells

Abstract

Vaginally applied microbicides hold promise as a strategy to prevent sexual HIV transmission. Several nonspecific microbicides, including the polyanion cellulose sulfate, have been evaluated in large-scale clinical trials but have failed to show significant efficacy. These findings have prompted a renewed search for preclinical testing systems that can predict negative outcomes of microbicide trials. Moreover, the pipeline of potential topical microbicides has been expanded to include antiretroviral agents, such as reverse transcriptase, fusion, and integrase inhibitors. Using a novel ex vivo model of vaginal HIV-1 infection, we compared the prophylactic potentials of two forms of the fusion inhibitor T-20, the CCR5 antagonist TAK-778, the integrase inhibitor 118-D-24, and cellulose sulfate (Ushercell). The T-20 peptide with free N- and C-terminal amino acids was the most efficacious compound, causing significantly greater inhibition of viral genomic integration in intraepithelial vaginal leukocytes, measured by an optimized real-time PCR assay, than the more water-soluble N-acetylated T-20 peptide (Fuzeon) (50% inhibitory concentration [IC50], 0.153 microM versus 51.2 microM [0.687 ng/ml versus 230 ng/ml]; P<0.0001). In contrast, no significant difference in IC50s was noted in peripheral blood cells (IC50, 13.58 microM versus 7.57 microM [61 ng/ml versus 34 ng/ml]; P=0.0614). Cellulose sulfate was the least effective of all the compounds tested (IC50, 1.8 microg/ml). These results highlight the merit of our model for screening the mucosal efficacies of novel microbicides and their formulations and potentially rank ordering candidates for clinical evaluation.

FIG. 1.

HIV-1 infection of leukocytes residing in vaginal epithelial sheets obtained by EDTA treatment. (A and B) Confocal microscopy of HIV-1 binding to intraepithelial lymphocytes and Langerhans cells. Epithelial sheets were separated from the underlying vaginal stroma by overnight incubation in 5 mM EDTA at 4°C, placed in Hanks buffered salt solution containing 5 mM calcium chloride for 1 h on ice, washed in PBS, and spinoculated for 2 h with either GFP-Vpr-tagged HIV-1_JRCSF or HIV-1 ΔEnv at room temperature. Subsequently, the sheets were stained with anti-CD1a antibody and analyzed by confocal microscopy. Virus is shown in green, and the round staining pattern is typical for virus that is bound to intraepithelial T lymphocytes (arrows) (26). The arrowheads point to virus that localized to CD1a⁺ Langerhans cells. The overlap between green-fluorescence-labeled virus and red-fluorescent CD1a staining is in yellow. The blue staining shows the TOPRO-3 nuclear counterstain. The images depict tissues from two different individuals. Exposure to HIV-1 ΔEnv exhibited no specific binding (not shown). (C) Calcium replenishment after EDTA treatment of vaginal epithelial sheets improves HIV-1 infectivity. EDTA-separated vaginal epithelial sheets were incubated with or without the addition of 5 mM calcium chloride for 1 h, spinoculated with HIV-1_JRCSF for 2 h, and cultured for 48 h. Cells that had emigrated from the epithelium into the culture supernatant were harvested, stained for HIV-1 Gag and CD3 using anti-HIV-1 Gag p55/p24 and anti-CD3 antibodies, and analyzed by flow cytometry. The gating in CellQuest was set to exclude 7-AAD⁺ dead cells, and HIV-1 Gag p24/p55 expression is plotted on the x axis versus CD3 on the y axis. The percentages of Gag-positive CD3⁺ T cells are given in the right upper quadrants. One tissue donor out of two donors examined is shown.

FIG. 2.

Ex vivo preexposure prophylaxis inhibits HIV-1 genomic integration in vaginal intraepithelial cells. Vaginal epithelial sheets from six tissue donors were incubated with the indicated compounds for 1 h at 37°C, spinoculated with 100 ng/ml HIV-1 Gag p24 of the CCR5-tropic laboratory clone HIV-1_JRCSF (A and B) or the primary mucosal isolate HIV-1_M1 (C) for 2 h, washed extensively, and cultured for 2 days. Total DNA was isolated from the sheets and emigrated cells, and the amount of integrated HIV-1 DNA in tissues treated with microbicides before infection was determined relative to the amount of integrated HIV-1 DNA in tissue not treated with microbicides before infection (= 100%). (A) In initial experiments, the PCR for the housekeeping gene β-actin, which was used to normalize variability in DNA input, was performed in separate wells (singleplex PCR assay). (B and C) Subsequently, conditions were optimized to run the Alu-LTR and β-actin PCR in the same wells (multiplex PCR assay). Each PCR assay was performed independently, either by four operators for the singleplex assay (A) or by two operators for the multiplex assay (B and C). Individual PCRs were performed in quadruplicate and averaged. The values depicted represent means, and the error bars depict the standard deviations across individual operators. The concentrations of the compounds used to treat the tissues were based on previously published in vitro studies (11, 13, 15, 43) and were as follows: CXCR4 antagonist AMD-3100, 0.794 μg/ml (1 μM); fusion inhibitor T-20 (manufactured by the Division of AIDS, NIAID), 1 μg/ml (0.223 μM); CCR5 antagonist TAK-779, 0.531 μg/ml (1 μM); and integrase inhibitor 118-D-24, 7.416 μg/ml (30 μM). The viral inoculum contained negligible amounts of Alu-LTR DNA.

FIG. 3.

Titration and comparison of the HIV-inhibitory effects of N-acetylated T-20 (Fuzeon), free T-20, and cellulose sulfate in the vaginal epithelium. (A) T-20 titrations. Vaginal epithelial sheets from 6 tissue donors were incubated with either the T-20 peptide manufactured by Roche (Fuzeon) (T-20 Roche) or the T-20 peptide produced by the Division of AIDS (NIAID) (T-20 DAIDS) at the indicated concentrations and then infected with R5-tropic HIV-1_JRCSF (100 ng/ml HIV-1 Gag p24). (B) Cellulose sulfate titration. Vaginal epithelial sheets from 4 tissue donors were incubated with cellulose sulfate (Ushercell) at the indicated concentrations and then infected with R5-tropic HIV-1_JRCSF (100 ng/ml HIV-1 Gag p24). In panels A and B, infections and measurement of integrated HIV-1 DNA were performed as outlined in the legend to Fig. 2. Individual PCRs were performed in quadruplicate and averaged. The colors represent measurements derived from different donor tissues. Solid and dashed lines of similar colors represent two independent experiments performed with the same donor tissue. (C) IC₅₀ determination for T-20 Roche, T-20 DAIDS, and cellulose sulfate. Dose-response curves were fitted to the data in panels A and B by nonlinear regression, and IC₅₀ concentrations were determined using Prism 4.0. Mean values of the individual data points in panels A and B are depicted; the error bars represent the standard deviations. Concentrations were log₁₀ transformed, and relative viral integration values were normalized to percentages of the maximum response. (D) IC₅₀ determinations for T-20 Roche, T-20 DAIDS, and cellulose sulfate in PHA-activated T cells pretreated in vitro with the indicated microbicides and infected with R5-tropic HIV-1_JRCSF, similarly to the vaginal epithelial sheets (n = 2 blood donors; means and standard deviations are shown).