Radiana Trifonova

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⁺ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.

Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3alpha, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1beta release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.

PROBLEM: Knowledge of the mucosal immune cell composition of the human female genital tract is important for understanding susceptibility to HIV-1. METHOD OF STUDY: We developed an optimized procedure for multicolor flow cytometry analysis of immune cells from human cervix to characterize all major immune cell subsets in the endocervix and ectocervix. RESULTS: Half of tissue hematopoietic cells were CD14(+) , many of which were macrophages and about a third were CD11c(+) , most of which were CD103(-) CD11b(+) CX3CR1(+) DC-SIGN(+) dendritic cells (DCs). The other dominant population were T cells, with more CD8 than CD4 cells. T cells (both CD8 and CD4) and B cells were more abundant in the ectocervix than endocervix of pre-menopausal women; however, CD8(+) T cell and B cell numbers declined in the ectocervix after menopause, while CD4 T cell counts remained higher. B, NK and conventional myeloid and plasmocytoid DCs each were a few percent of tissue hematopoietic cells. Although the ectocervix had more HIV-susceptible CD4(+) T cells, polarized endocervical explants supported HIV replication significantly better. CONCLUSION: Due to their abundance in the genital tract, CX3CR1(+) DC-SIGN(+) DCs might be important in HIV transmission. Our data also suggest that the columnar epithelium of the upper genital tract might be a preferential site for HIV transmission.