The placental trophoblastic epithelium constitutes a barrier between free HIV or HIV-infected cells in the maternal blood and potentially susceptible blood cells of the fetus. This proposal is based on our recent observation that in vitro physical contact between latent HIV-infected monocytes or lymphocytes and CD4-negative epithelia triggers rapid assembly and release of mature HIV into an enclosed space between donor and acceptor cells. Virions sequestered in these sites are rapidly internalized within phagocytic endosomes. HIV escape into host cytoplasm either by fusion with endosomal membrane or by dissolving lysosomal membrane. Apart from phagocytosis, we have observed that HIV may enter the epithelium by direct fusion with the plasma membrane or via coated pits. We propose to screen human trophoblastic placental cell lines to determine the mode of HIV infection. Virus detection by electron microscopy, in situ hybridization, and immunocytochemistry will be employed initially. In experiments involving antibody neutralization we will examine the role of various molecules such as CD4, Fc receptor, complement receptor, LFA-1, HLA, TA1, TA2, and mannose receptor in viral attachment and entry into placental cells. In situ hybridization, reverse transcriptase assay, ELISA, and virus culture will be employed to determine if HIV productively infects the target epithelium. Virus transepithelial sorting will be monitored by electron microscopy and immunolocalization. Since adhesion between infected leukocytes and placenta appears to be the initial step in infection, we will study adhesion factors such as cell adhesion molecules and carbohydrates. Adherence assays will include counting of isotope- labeled cells and rose bengal dye uptake by bound cells. We will evaluate the effect of maternal antibody on cell-mediated and cell-free HIV spread with specific reference to Fc receptor and complement. Lastly, we will examine the effect of hormones and other bioactive molecules of pregnancy such as hCG, lactogen, and steroids on cell attachment and viral infection of the trophoblast epithelium. We believe that the carefully controlled in vitro experiments which we are proposing will yield insights into how the AIDS-causing virus may cross the placenta and infect the fetus in utero.
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