Ex vivo organotypic cultures offer a valuable link between in vitro culture systems and the clinic by providing a controlled format in which microbicides can be comparatively evaluated for (i) anti-HIV-1 activity in target cells present within the submucosa of human tissues and (ii) toxicity against the mucosal epithelium. Two other ex vivo ectocervical explant systems have been described (13
). In one method, tissues are completely embedded/submerged and HIV-1 with or without microbicide is applied apically (13
). In the other method, the tissue is exposed to HIV-1 with or without microbicide in a nonpolarized manner, and the explants are cultured while submerged in medium (19
). We describe a cervical explant culture for which tissue is maintained in a polarized state with the epithelial surface positioned at the air/tissue interface and the submucosa (stroma) submerged in medium. Unlike the other explant culture systems (13
), this positioning of the cervical tissue allows application of virus and candidate topical microbicides directly to the epithelium and allows access to the cells in the submucosa. As indicated in the present study and in descriptions of other published explant systems (13
), cervical explant cultures have several limitations. These include lack of hormone modulation, lack of recruitment of immune cells, loss of epithelium, and inability to regenerate/repair. However, the last two attributes make this system sensitive to any potential toxic effect by the topical microbicide. Further, this explant culture demonstrates the capacity to be infected with HIV-1 with the subsequent evaluation of efficacy of several topical microbicide compounds. It is important to note that there is currently no consensus among the laboratories using tissue explants regarding appropriate efficacy and toxicity endpoints. A multisite effort is currently under way to determine the optimum endpoints and to address tissue replicates (14a
As reported elsewhere (19
), there was a loss of the outer stratified layers of epithelium during the initial 24 to 72 h of culture of untreated tissues. Although the basal layer remained intact with gradual regeneration over the culture period, the epithelium of these explants did not recapitulate to that of the normal cervix. Since leakiness of the explant culture is a concern with embedded tissues, other investigators have analyzed the integrity of the system by measuring the transmission of blue dextran or fluorescent beads (7.2-μm diameter) across the mucosa (13
). Given the size of HIV-1 virions (~100-nm diameter), smaller fluorescent beads (26-nm diameter) were used for the permeability studies and allowed to remain for 7 days. Because of the smaller bead size and the modifications to the embedded culture system, a higher rate of transmission of the fluorescent beads compared to the 7.2-μm-diameter beads was observed (20
). Despite this, <10% transmission of the beads was detected by day 3 of culture. Although a true polarized tissue orientation may be difficult to achieve ex vivo, this does not negate use of this system. Tears and disruptions to the mucosal epithelium are likely to occur during sexual intercourse (39
). Moreover, sexually transmitted pathogens can result in ulceration of the mucosa and increased acquisition of HIV-1 (24
). An ideal microbicide should have the capacity to prevent HIV-1 infection when the virus has its best opportunity to achieve infection: during the presence of a compromised epithelium and activated immune targets (i.e., inflammation). The cervical explant culture described here represents a unique system for the preclinical evaluation of candidate microbicides that takes into account the optimal environment for HIV-1 infection.
In this cervical explant culture, T cells, macrophages, and dendritic cells were detected by IHC within cervical tissues. However, HIV-1 p24 antigen only colocalized with macrophages. Macrophages were the primary target for infection in one culture system (19
) while CD4+
T cells were the primary infected cell type in the other culture (20
). Since the sensitivity of the IHC technique and the timing after infection can affect the ability to detect infected cells, the inability to detect T-cell infection based on the IHC data does not exclude infection of these or other nonmacrophage cell types in this cervical explant culture. Regardless of which cell type was infected, HIV-1 infection and replication were consistently observed after infection with HIV-1BaL
, thus allowing for the comparative analysis of six topical microbicides for their anti-HIV-1 activity. Microbicides with significant anti-HIV-1 activity typically demonstrated >90% (1 to 2 log10
) reduction in p24 levels at day 14. In instances where >80% reduction of p24 levels was observed, no p24 antigen could be detected in tissue sections by IHC. While the carrageenan (PC-515)- and peptide (D2A21)-based products showed some anti-HIV-1 activity, the CAP, naphthalene sulfonate (PRO 2000), lysine dendrimer (SPL7013), and NNRTI (UC781) products consistently blocked HIV-1 infection.
In previously described explant cultures, the toxicities of candidate topical microbicides were assessed using either biochemical (MTT) (19
) or histopathological (43
) methods. In the present study, both methods were employed. While MTT represents a convenient method for measuring tissue toxicity, it is a limited approach since it measures general toxicity. Conversely, histopathology allows determination of toxicity specific to the mucosal epithelium or the underlying submucosa. With the exception of CAP, both methods detected tissue toxicity after treatment with N9 and 4% PRO 2000. After treatment with CAP, reduced cell viability was observed by MTT assay, yet histology revealed no physical damage to the epithelium or submucosa. The reduced viability observed in the MTT assay may result from interference by CAP, suggesting a potential artifact of this biochemical assay. Alternatively, CAP could be affecting the mitochondrial metabolism without affecting the overall tissue structure/integrity. While the MTT assay may prove beneficial for initial toxicity testing in tissues, these data suggest that it should be used in conjunction with histology to confirm toxicity.
In the explant culture presented here, virus growth was evaluated by monitoring p24 levels over time (at multiple time points) and determining the presence of p24 antigen by IHC. It should be noted that efficacy data obtained for several other explant cultures are limited to one time point (19
), are sometimes presented as percentages of a virus control (thus not depicting actual p24 levels) (17
), and/or rely on that one endpoint (without confirmation by a secondary method, such as IHC) (42
). In the present study, compounds that were found to be active in both donors showed a >80% decrease in culture supernatant p24 levels at the study endpoint and no p24 staining by IHC.
Six topical microbicides were evaluated for toxicity and efficacy in a modified cervical explant culture system. With the exception of 4% PRO 2000, the remaining microbicides were relatively nontoxic, consistent with toxicity results from other explant cultures (19
), animal studies (5
), and safety and acceptability trials (3
). With the exception of PC-515 and D2A21, the microbicides were efficacious against HIV-1 infection, consistent with efficacy data from other cervical explant cultures (1
) and preclinical and animal studies (16
). The low toxicity and high anti-HIV-1 activity of CAP, 0.5% PRO 2000, SPL7013, and UC781 suggest the need for further testing of these products.
Despite the limitations described above, our study shows that the cervical explant culture is valuable for evaluating the efficacy and toxicity of potential microbicides. In addition, when used as a secondary confirmatory assay for compounds demonstrating activity in cell-based assays (14b
), this cervical explant culture can be a valuable tool for selecting priority candidates to advance to clinical trials.