Investigations into the failures of N-9, C31G, Ushercell, Carraguard, and PRO2000 continue to provide valuable insights into factors that may adversely impact in vivo microbicide efficacy. Studies of N-9, which began shortly after the phase III trial failures , focused on cervicovaginal tissue damage and inflammation following application of N-9. These studies implicated cervical tissue damage , induced recruitment of immune cell populations , and the production of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and IL-8 [51–53] as explanations for the general failure of an N-9-based microbicide, as well as increases in HIV-1 infection after high-frequency application . In contrast, factors that can be invoked to explain the failures of the remaining microbicides have not yet been clearly identified. It is apparent, however, that multiple mechanisms are likely associated with the failures of these microbicides.
The present in vitro studies, which were conducted to examine microbicide loss following topical application as a potential mechanism of microbicide failure, demonstrated significant increases in HIV-1 infection following the application and removal of the polyanionic compounds DS, CS, and LC. Increases in infection, which were observed in experiments using an HIV-1-susceptible cell line as well as primary human immune cells, were found to be dependent on the target cell and co-receptor usage by the infecting virus.
Although enhancement of HIV-1 infection following polyanionic compound washout was clearly demonstrated in the HIV-1 indicator cell line and in primary human immune cells, specific results were not always comparable between experiments using these two cell populations. For example, LC washout at its IC50 had no effect on HIV-1 IIIB infection of P4-R5 MAGI cells (Figure 3A) but had a significant effect on HIV-1 IIIB infection of PBMCs (Figure 5A). Conversely, LC application and removal at its IC90 (Figure 2A) resulted in enhancement of HIV-1 BaL infection that increased with post-exposure time in P4-R5 MAGI cells, whereas the comparable experiment in PBMCs (Figure 4A) demonstrated no HIV-1 BaL enhancement as a consequence of LC pre-exposure. These apparently disparate results are likely due to differences inherent in the cells used in these experiments, including cell type (recombinant cervical carcinoma cell line versus primary human immune cell) and levels of receptor and co-receptor expression (over-expressed on the P4-R5 MAGI cells). In addition, a considerably lower multiplicity of infection (MOI) was used in the PBMC experiments. Any or all of these factors could have affected the outcome of these experiments. Future mechanism-focused experiments may provide more definitive insights into the underlying causes of these differences in outcome.
The effects of compound washout were also dependent on the interval between compound removal and introduction of virus, suggesting both immediate and delayed mechanisms of enhanced infection. Enhancement following a short (1- to 2-h) interval between compound removal and infection suggests immediate changes on the cell surface that enhance the early stages of the HIV-1 replication cycle. Cell surface changes initiated by microbicide exposure may be induced by the retention of residual amounts of compound at the cell surface or may persist after the compound has been removed. Interactions between compound and cell surface may enhance HIV-1 infection by favoring increased viral binding and entry. Although numerous cell surface molecules have been shown to participate in HIV-1 binding and entry, the impact of co-receptor usage on enhancement implicates the involvement of the HIV-1 co-receptors CCR5 and CXCR4. However, the specificity of this effect remains to be demonstrated.
Alternatively, enhancement after a relatively long (6- to 7-h) interval after washout may suggest more indirect mechanisms potentially involving signal transduction events initiated by compound exposure. As evidence that polyanionic molecules can initiate cell signaling cascades, studies of innate immune activation using both human and murine model systems have demonstrated that carrageenan-induced inflammation requires signaling through Toll-like receptor 4 [55, 56] as well as MyD88-dependent signal transduction . Polyanion-induced signal transduction and inflammation do not appear to be limited to carrageenan, because DS has been used for 20 years as an agent that reliably induces experimental colitis in mice . The demonstrated activities of these polyanionic molecules raise the possibility that delayed increases in HIV-1 infection observed in the present studies were the result of signaling initiated by DS, CS, or LC. Because of the time required to initiate events downstream of compound-initiated signal transduction, the effects of this mechanism of enhancement were not immediately evident but did affect HIV-1 infection after a period of delay. A specific target of polyanion-mediated signaling within the viral replication cycle was not apparent from these results and will need to be determined as part of future investigations.
In the experiments involving primary human PBMCs cells, observations of post-exposure, time-dependent enhancement may also have been complicated by cell population heterogeneity. Differences in the levels and mechanisms of enhancement between T lymphocytes and cells of monocyte lineage, which also differ greatly in frequency within the total cell population, may have biased the observed time-dependent enhancement of HIV-1 infection relative to results obtained using the homogeneous P4-R5 MAGI cell line. For example, washout of LC at its BaL IC50 (Figure 4A) resulted in enhanced infection at 1, 3, 5, 6, and 7 h post-exposure and no enhancement at 2 and 4 h. This unusual pattern of enhancement might be explained as an overlay of time-dependent enhancement in T cells and monocytes/macrophages, with different peaks of infection arising from different HIV-1-infected cell populations. Furthermore, if compound-mediated intracellular signaling results in the downstream release of cytokines and chemokines, the combined pattern of time-dependent enhancement might be altered further because the release of these soluble factors subsequently affects the susceptibility to infection of uninfected neighboring cells. The contributions of these factors will need to be resolved in experiments using enriched target cell populations.
The present results, as well as the results of previously published studies, indicate that enhancement under these conditions is also dependent on the identity of the compound. This same assay design was previously used to evaluate the antiviral activity of the biguanide-based cationic entry inhibitor NB325  as well as the activities of the polyanionic compounds poly (styrene-alt-maleic acid) (alt-PSMA), poly(styrene sulfonate) (PSS), and DS . In direct contrast to the present studies, which were focused on DS, CS, and LC, washout experiments involving NB325 demonstrated persistent protection from infection by HIV-1 IIIB after compound removal. Furthermore, studies involving alt-PSMA and PSS demonstrated that enhancement of infection following compound washout is not an effect that can be generally ascribed to polyanionic compounds. Although alt-PSMA and PSS are similarly based on polystyrene backbones, they derive their polyanionic charges from different moieties: maleic acid in the case of alt-PSMA and sulfonic acid in PSS. In washout assays, alt-PSMA removal had no effect (enhancement or inhibition) on HIV-1 infection, whereas PSS significantly increased levels of infection relative to infection controls. The results of our previous studies with DS are consistent with previously published studies [29, 60, 61], which demonstrated that DS could enhance HIV-1 infection in monocyte-derived macrophages but not in CD4+ T lymphocytes . The current experiments demonstrated DS-mediated enhancement in PBMCs but not in P4-CCR5 MAGI cells, presumably due to the inclusion of monocyte-derived macrophages in the former cell population.
How does one reconcile the present results with the clinical failures of these compounds? Low concentrations of compound will be achieved clinically by dilution during sexual intercourse or time-dependent compound leakage after application. Early phase I and phase II safety and acceptability trials of carrageenan and CS indicated, either through direct measurement or through self-reporting by the women in the trials, that the product leaked after application [15, 31, 34, 62, 63]. Leakage would decrease the concentration of active compounds available in the cervicovaginal environment, negating the antiviral activity of the microbicide and potentially favoring mechanisms that promote HIV-1 infection. Therefore, the efficacy of a polyanionic compound such as carrageenan or CS would be dictated by concentration. At high concentrations (as found after the initial application of the microbicide product), the antiviral activity of the compound will override any enhancing activity. At low concentrations (or after the complete loss of microbicide), the compound would be unable to inhibit infection or offset the mechanisms that enhance infection, and its overall effect would be to increase the risk of infection. The activity of such a compound in a clinical trial would be driven by variables that affect the amount of product in the cervicovaginal tract, including the elapsed time between product application and sexual intercourse, dilution by seminal fluid, and the degree of product leakage following application.
These results also suggest that plans to use carrageenan as a potential "active excipient" in formulations of other active agents may not be advisable. Although the safety of Carraguard was demonstrated in early clinical trials and no enhancement was evident in the results of the failed phase III trial [8–10, 12, 13, 64], the possibility remains that, under different circumstances, the use of carrageenan in a formulated microbicide may result in an increased risk of HIV-1 transmission. In light of these results, the use of carrageenan in future microbicide products should be carefully considered. These results, however, do not broadly rule out the use of polyanionic molecules in antiviral formulations, because enhancement of HIV-1 infection does not appear to be a universal property of polyanionic compounds. Previous studies of the polyanionic alt-PSMA demonstrated that its washout did not result in increased infection .