The objective of this project is the production and characterization of recombinant HIV-1 and SIV MAC antigens effective in stimulating a protective mucosal and systemic immune response. These antigens will be developed for immunization systems using novel microencapsulated and cholera toxin conjugated oral antigen delivery systems which have the proven capacity to stimulate mucosal as well as systemic immunity. Using a vaccinia virus recombinant which we have developed, that expresses the HIV-1 envelope protein and results in secretion of large amounts of the gp120 glycoprotein into culture fluids, we will purify the glycoprotein for use as an antigen in candidate vaccines. We will additionally use baculovirus recombinants to obtain large quantities of the viral gylcoproteins from insect cells. We will construct similar recombinants which express the SIV MAC glycoproteins, including an uncleaved form of the protein, and a secreted, truncated form which lacks membrane-anchor sequences. These forms of the glycoprotein, as well as synthetic peptides, will be evaluated as candidate microencapsulated and/or cholera toxin- conjugated vaccines in both Simian and murine animal model systems (Projects 2-4). To further identify HIV/SIV antigen preparations that are likely to be important for inclusion in ultimate vaccine preparations, we will characterize the oligomeric structure of the HIV-1 and SIV MAC envelop protein complexes, and develop procedures which enable purification of the glycoproteins in their native form. Using the purified HIV-1 and SIV MAC glycoproteins as antigens, we will prepare panels of monoclonal antibodies to these glycoproteins and determine their effects on viral biological activities including infectivity and cell fusion activity. The antibodies will be used to develop procedures for affinity purification of glycoproteins from infected cells and to analyze the extent of antigenic variation among various HIV-1 and SIV MAC isolates. Finally, to obtain a better understanding of events in viral pathogenesis especially relating to this Program involving mucosal immunity, we will investigate the infection process of HIV-1 and SIV MAC in polarized epithelial cells, a potential model for natural routes of infections at mucosal surfaces.
