Feline immunodeficiency virus, a lentivirus of cats, provides a natural model for human immunodeficiency virus (HIV) infection and disease. This proposal is designed to further develop this model by exploring the feasibility of a gene therapy approach for preventing vaginal transmission of lentivirus infections. If successful in the FIV model, this approach will be applicable to the prevention of vaginally transmitted HIV infections in women. The FIV model is used to test the hypothesis that lentivirus infections can be prevented at the mucosal site of entry by introduction into cells of the vaginal mucosa of lentiviral Rev-responsive element (RRE) linked to a toxin (suicide) gene. Infection of cells harboring this RRE/suicide gene construct will result in expression of the toxin and death of the infected cells. The interaction of the Rev protein with the RRE is a critical step in the replication cycle of both FIV and HIV. This interaction can be exploited by linking a suicide gene (diphtheria toxin A) to the FIV-RRE in an expression plasmid. The gene will not be expressed unless the FIV Rev protein is present. In theory, once a cell containing the construct is infected with FIV, the plasmid's RRE will interact with the viral Rev, allowing expression of the toxin and resulting in death of the cell before virus can be disseminated beyond the mucosal surface. The objectives of the work detailed in this proposal are: 1) to employ and enhance the FIV model for vaginal transmission of HIV and mucosal defenses against HIV, and 2) to determine if an RRE/toxin construct can be used effectively to terminate lentivirus infection, in vitro and in vivo. These objectives will be achieved by concentrating on the following three specific aims: 1) To determine the effects on tissue- culture cells of an FIV-RRE/diphtheria toxin A expression plasmid, in the presence or absence of infectious FIV, by transfection of the FIV- RRE/toxin plasmid into cultured cells, infection of transfected cells with FIV, and determination of cell viability and virus production; 2) To introduce the FIV-RRE/toxin construct into the cells of the vaginal mucosa of cats and to identify the cells that are transfected, by exposure of the vaginal mucosa of female cats to endotoxin free plasmid DNA and cytologic examination of mucosal cells for the presence of plasmid DNA; and 3) To determine the ability of the FIV-RRE/toxin construct to protect against vaginal transmission of FIV, by exposure of the vaginal mucosa of female cats to the FIV-RRE/toxin plasmid DNA, vaginal introduction of infectious FIV, and examination of cats for evidence of FIV infection.
