The long term commercial objective of the proposed research is to make available to the research community a complete set of monoclonal antibodies to all key epitopes of the major HIV-1 coreceptors CCR5 and fusin. The availability of these antibodies is essential for the basic understanding of molecular interactions between these HIV coreceptors/chemokine receptors and their cognate ligands. These antibodies will aid in the discovery of novel therapeutic drugs for AIDS and other inflammatory diseases. They can also be used judiciously for the identification of HIV-1-resistant individuals in the population.
Specific aims during phase I will include: the immunization of mice with transfected mouse cells expressing CCR5 or fusin on their surfaces; the primary screening of hybridoma supernatants by whole cell ELISA immunoassays using transfected human 293 cells expressing the receptors; the construction of epitope tagged CCR5 and fusin expression clones for subsequent recombinant membrane protein expression and purification; and the preparation of epitope tagged peptides corresponding to the extracellular domain and three extracellular loops of the receptors. During phase II, the primary clones identified during pase I will be further screened, characterized and epitope mapped using biosensor, flow cytometry and functional assays (Ca mobilization, env-mediated fusion etc).
