We consistently detected integrated HIV-1 provirus in intact, stroma-free epithelial sheets from the human vagina within 2 days of HIV-1 exposure, demonstrating that cells residing within the outer vaginal epithelium are highly susceptible to infection by HIV-1. A microbicide that fails to block this initial step of infection is unlikely to be successful in preventing sexual HIV transmission. Thus, prescreening novel microbicides for HIV-1-inhibitory activities using ex vivo vaginal intraepithelial cells can permit rational choices of which candidates may hold promise in larger-scale in vivo preclinical and clinical studies.
To build a feasible platform for systematic microbicide evaluation, we adapted our ex vivo
vaginal HIV transmission model (26
) to quantify alterations in infectivity with preexposure prophylaxis. First, we improved the efficiency of epithelial-stromal separation, allowing us to harvest 100% of the epithelium from each vaginal-tissue sample, thereby decreasing the total number of samples needed for testing. Second, we adopted a readout of productive infection based on real-time PCR amplification of HIV-1 proviral DNA sequences that had integrated into the genome of infected cells. This method of detecting HIV-1 infection of vaginal intraepithelial cells offers three major advantages: high sensitivity, an indication for an advanced step in the productive viral life cycle, and its ability to reliably quantify the relative antiviral efficacies of a given panel of microbicides. The high sensitivity of the PCR assay can detect a newly occurring HIV-1 infection of cells within the vaginal epithelium as early as 2 days following viral challenge. During this short interval, contamination of the ex vivo
cultures with bacteria or fungi rarely occurs, even if tissue processing is performed under clean, but not sterile, conditions. Previously reported ex vivo
human explant studies of microbicide efficacy have employed whole mucosal organ cultures (7
), which usually require longer culture periods for detection of HIV infection, potentially increasing the risk of tissue degradation and pathogen contamination. The greater sensitivity of the real-time PCR assay also ensures that a number of different microbicides with wide titration ranges can be tested in pairwise comparisons within the same donor tissues, as each experimental condition requires only a relatively small amount of epithelium.
The real-time format of the PCR assay allows quantification of the integrated viral copies per cell. To create a standard curve for the calculation of integrated HIV-1 copies, we titrated latently infected ACH-2 cells in parallel with each experimental PCR assay. Because the precise number of proviral copies in a given number of ACH-2 cells was not known, we used the ACH-2 cell standard curve to calculate the relative amounts of integrated provirus under different experimental conditions rather than the absolute number of integrated viral copies. For the purpose of our study, which was to determine whether a given microbicide inhibits viral integration in vaginal cells relative to viral integration in the absence of the microbicide, relative quantification was sufficient. Our comparative dose-response studies clearly demonstrate the power of relative quantification by our PCR assay to discriminate the efficacies of different microbicides for inhibiting viral integration in vaginal target cells.
Of note, the measurement of viral integration is not specific for a particular cell type. Thus, unless mucosal cells are sorted into subpopulations before DNA isolation, the PCR assay does not identify which cells are infected. The specific cell type(s) infected with HIV-1 within the vaginal epithelium could be determined by flow cytometric analysis of isolated cells or by in situ
microscopy techniques, as we have done previously (26
). Compared to real-time PCR, flow cytometry relies on the analysis of a relatively high number of isolated cells in single-cell suspension, and therefore, for enumerating infected cells, it requires a much larger amount of vaginal tissue for each experimental condition. Microscopy techniques, on the other hand, are labor-intensive and harder to accurately quantify than real-time PCR results.
While the PCR assay does not specify the cell type infected with HIV-1, our model ensures that cells within the outer vaginal epithelium, which are the first encountered by HIV during viral penetration in vivo
, are the sole source of the integrated HIV provirus. The vaginal epithelial sheets were completely stroma free and did not contain any microvasculature, which focused the analysis on T lymphocytes and LCs, the sole two leukocyte subtypes consistently residing within the outer vaginal epithelium (17
). Our prior studies had demonstrated that CD4+
T lymphocytes are the main cell type within the vaginal epithelium that is productively infected by HIV-1 (26
). Thus, we presume that integrated provirus detected in our present study is derived mostly or entirely from infected intraepithelial CD4+
Utilizing our vaginal intraepithelial-infection model to compare the HIV-1-inhibitory efficacies of several potential microbicides yielded some relevant findings about the potential activities of microbicides in future studies. The different potencies of these microbicides for preventing HIV-1 integration in intraepithelial target cells, which were consistent in experiments with several donor tissues, demonstrate the potential utility of the model for preclinical microbicide screening. Importantly, we observed a pronounced difference in efficacy between the two different pharmacological versions of the fusion inhibitor T-20 in the tissue model, but not in single target cell suspensions. This underscores two crucial points: (i) Microbicides that show promise after initial testing using PBMC or indicator cell lines require testing in tissue infection models; in vitro
testing alone is not sufficient. (ii) Drugs that are efficacious systemically may be less so when applied as a topical microbicide. The T-20 peptide with free terminal ends likely exhibits higher lipid solubility than the Roche-manufactured N-acetylated T-20 peptide (52
) and thus may penetrate the vaginal epithelium more readily. Compared to our IC50
determination for the Roche-manufactured T-20 (230 ng/ml in tissue; 61 ng/ml in PHA-activated T cells) or the IC50
ranges that have been previously reported for this agent (0.4 to 480 ng/ml for CCR5-tropic viruses in various in vitro
]), the T-20 peptide lacking N-acetylation was highly protective against HIV-1 chromosomal integration in the vaginal epithelium (0.687 ng/ml).
Notably, both T-20 versions inhibited infection of vaginal intraepithelial cells in our model more effectively than cellulose sulfate (Ushercell). Likewise, the CCR5 antagonist TAK-779 and the integrase inhibitor 118-D-24 were markedly more efficacious than cellulose sulfate. Going forward, clear results criteria for comparative efficacy screening in a relevant ex vivo
model like the one presented here need to be formulated to determine whether a product may proceed to further evaluation in vivo
. These criteria will have to incorporate toxicity in the form of a therapeutic index that puts efficacy in relationship to the compound's potential toxicity for the vaginal epithelium. Moreover, screening criteria cannot focus solely on comparing equal dosages of microbicidal agents but will have to consider what concentrations are actually achievable in vivo
and at what cost. Our findings indicate that the T-20 DAIDS peptide with free N- and C-terminal amino acids may be topically effective in the vagina at a much lower dosage than Fuzeon. At 10 ng/ml, >80% inhibition of viral integration in the mucosa was achieved. Extrapolating the amount of tissue we treated in each titration step to the complete surface of the vaginal cavity (39
), we estimate that a total dose of 10 mg T-20 DAIDS could be effective as a vaginal microbicide, costing between $2 and $3. While even less expensive topical microbicides are desirable, this nevertheless indicates that, due to the potent protective efficacy of some fusion inhibitors, as exemplified both in our study for T-20 DIAIDS and in previous work with other compounds (16
), fusion inhibitors could potentially be efficacious in humans as topical microbicides at concentrations that are not prohibitively expensive.
Enhancement of HIV infection of peripheral blood mononuclear cells by cellulose sulfate at low concentrations of around 0.3 μg/ml was suggested by a recently published study (44
). The authors concluded that this may explain why cellulose sulfate appeared to increase the risk of HIV infection in one of two large clinical trials (45
). However, the final statistical analysis comparing the HIV transmission risk between the cellulose sulfate and the placebo groups of the two trials was not significant (23
). Indeed, one of the two studies explicitly concluded that a 6% cellulose sulfate vaginal gel was safe (23
). These clinical observations are consistent with the monophasic dose-response curve observed for cellulose sulfate in our vaginal-infection model: while cellulose sulfate was less efficacious than the other four compounds tested, it also did not enhance infection at any concentration.
In conclusion, we developed and validated an ex vivo
tissue model that uniquely quantifies the sum of the initial events whereby HIV-1 establishes infection of cells embedded in the outer epithelial layer of the human vagina. Mucosal tissues for this model can be easily obtained on a weekly basis from one university medical center as a discarded by-product of vaginal-repair surgeries (4
). We demonstrate that our vaginal-infection model can be utilized to screen topical-microbicide candidates for their efficacy in blocking chromosomal integration of HIV-1, measured by a sensitive real-time PCR assay, in intraepithelial vaginal cells. The relative inefficiency of cellulose sulfate in preventing infection of intraepithelial leukocytes, as well as the superior efficacy of a lipid-soluble over a water-soluble version of T-20 in our model, underscores our model's potential as a screening tool for microbicides in the development pipeline. We propose that this preclinical testing system can be used to narrow down the number of microbicide candidates that progress to human trials.