The dual-chamber culture model and the experiments with monocytic cells (U1 cells and PBMCs) uncovered unanticipated adverse effects associated with CS that, in hindsight, might have predicted the clinical trial outcomes. After the failure of the N-9 clinical trial, subsequent studies suggested that the increased risk of HIV acquisition could be attributed to a proinflammatory response. This resulted in the addition of cytokine measurements to phase 1 clinical studies [24
]. CS gel underwent extensive phase 1 evaluations that included such measurements, and no significant increases in levels of proinflammatory cytokines were observed [7
]. The results of the present in vitro experiments are consistent, as we observed only a modest increase in inflammatory cytokines in epithelial cell culture supernatants, and these had no significant effect on HIV replication in U1 cells. However, direct exposure of U1 cells to CS at 100 μg/mL, a concentration likely to be present in the genital tract after gel application [30
], resulted in a significant increase in HIV replication, which was associated with NF-κB activation. Significant NF-κB activation was also observed after exposure of PBMCs to CS. Whether these in vitro findings contributed to the clinical trial outcomes is not known. CS could have increased HIV replication in incoming infected PBMCs in semen, thereby increasing the risk of transmission. Notably, a recent study also demonstrated that low doses of CS (<1 μg/mL) significantly increased HIV replication in vitro, although the mechanisms were not elucidated [31
The dual-chamber culture model results suggest a completely different mechanism by which microbicides might inadvertently increase HIV acquisition. Consistent with the results of studies demonstrating that HIV crosses an intact mucosal epithelium poorly [23
], we found that polarized HEC-1-A cells provide a relatively impervious barrier to HIV migration. Disruption of this barrier after N-9 exposure was predicted by its known surfactant properties. However, the results obtained with CS were surprising. Notably, no significant or persistent drop in TER was observed after exposure to PRO2000, indicating that this is not a property shared by all sulfated or sulfonated polymers. Differences in chemical composition and molecular weight between CS and PRO2000 may have contributed to the differential response. CS has a peak molecular mass of 2300 kDa, whereas PRO2000 has a peak molecular mass of only 5 kDa [21
The sustained drop in TER (), which persisted even after removal of the drug, is consistent with the observed down-regulation of junctional protein gene expression (). Regulation of junctional proteins is incompletely understood, and there is a paucity of data focusing on genital tract epithelium. Junctional complexes contain a large number of cytoplasmic proteins, which serve as adaptors that link the integral membrane proteins to the actin cytoskeleton and play roles in transcription, polarity, and other signaling functions. It is possible that CS-induced epithelial disruption and activation of NF-κB are linked through signaling pathways. Consistent with this notion is the finding that junctional adhesion molecules are redistributed away from junctions in response to inflammation [33
The results of ongoing studies in mice are consistent with the results obtained in the dual-chamber culture model here. We and others have previously shown that exposure to N-9, but not PRO2000, increases the susceptibility of mice to herpes simplex virus (HSV) infection [18
]. More recently, we found that the increase in susceptibility to HSV infection is associated with the disruption of epithelial tight junctions (S.S.W., N.C., E.F., P.M.M.M., M.J.K., and B.C.H., unpublished data). Notably, CS gel also increased the susceptibility of mice to genital herpes in a pilot experiment—8 of 10 mice developed genital herpes when challenged with a low dose of HSV type 2 twelve hours after the seventh daily dose of CS, compared with 4 of 10 mice treated with hydroxyethylcellulose (P
< .05). Further studies of CS gel are planned in collaboration with CONRAD.
These experiments were conducted with active drugs and should be expanded to evaluate formulations, which could also affect the epithelial barrier because of osmolarity or the effects of other excipients [35
]. These findings suggest that phase 1 trials should be expanded to include an assessment of the effect that microbicides have on epithelial integrity by obtaining biopsy specimens. This is important for gels, as well as for ring formulations. Comparable experiments should also be conducted to address the effect of rectally applied microbicides, for which the columnar epithelium may be more vulnerable.
The findings of the current models are consistent with the safety observed in the recently completed HIV Prevention Trials Network 035 trial, in which a 30% reduction in HIV incidence was observed in women who applied 0.5% PRO2000 gel [36
]. However, it is important to note that a modest inflammatory response, a reduction in SLPI level, and a transient drop in TER were observed with higher doses of PRO2000. It is possible that this contributed to the finding that the 2% PRO2000 gel had little or no chance of showing efficacy and the discontinuation of the 2% gel arm of the Microbicide Development Programme trial (MDP 301). Importantly, the current results predict that PMPA will not increase HIV acquisition through disruption of the epithelium or activation of NF-κB pathways. The results of ongoing clinical trials will need to be translated back to the bench to optimize these and other biomarkers of safety.