Background: Without an effective vaccine against HIV infection, topical microbicide development has become an urgent priority. Ideally, a vaginally-applied microbicide would be effective against a broad range of pathogens but would not interfere with critical host defence mechanisms. Sulfonated agents including PRO2000 is the largest class of compounds moving forward into clinical studies. Pro2000 blocks HIV infection of cervical tissue in vitro and is active against HSV both in vitro and in vivo. One of the first cell types to contact HIV in genital tract and spread the virus to lymphoid tissue is dendritic cells (DCs). Thus, interfering with virus-DC interactions is a unique challenge to the success of topical microbicides.

Methods: Human monocyte-derived DCs (MDDCs) were generated from CD14⁺ monocytes using CD14 magnetic beads. Immature MDDCs were generated from monocytes in the presence of IL-4 and GM-CSF for 6-8 days. LPS was added to the cells to generate mature MDDC. CD14^- cells were kept in the culture for 5-6 days and then used to isolate CD4⁺ T cells. Cells phenotype was monitored by flow cytometry. CD4⁺ T cells were stimulated with PHA for 24-48h and used in autologous co-cultures with MDDCs. Pseudotyped HIV_JRFL and HIV_BaL were used to assess the effect of Pro2000 on virus infection and transmission in MDDC-CD4⁺CCR5⁺ HeLa and MDDC-CD4⁺ T cell co-cultures. For transmission experiments, viruses were pre-incubated with the compound and then incubated with MDDCs for 2hrs at 37°C, then cells were washed, re-counted and co-cultured with HeLa or CD4⁺ T cells. HIV_JRFL internalization by MDDCs was monitored by p24 ELISA.

Results: No significant changes in MDDC phenotype were detected after cells were exposed to the compound (10 and 100µg/ml) for 1hr and then washed. Pro2000 effectively blocked single cycle HIV_JRFL infection in MDDC-HeLa cell co-cultures (95-99% inhibition at 100µg/ml). Similarly, HIV_BaL infection in MDDC-CD4⁺ T cell co-cultures was inhibited by Pro2000 in a dose dependent manner.  Exposure of viruses to Pro2000 resulted in dose-dependent decrease of infection of both HeLa and CD4⁺ T cells (50-80% and 60-90% at 100µg/ml, respectively) in transmission experiments and did not decrease MDDC associated p24 level.

Conclusion: Efficient block of HIV infection in DC-T cell co-cultures which represent virus permissive environment and inhibition of DC-mediated virus transmission reinforces potential value of Pro2000 as anti-HIV compound.