CD8+ cells from HIV-infected individuals can control virus replication in CD4+ lymphocytes and macrophages by a noncytotoxic mechanism. We, and others, have determined that this non-MHC restricted antiviral response is mediated, at least in part, by a novel CD8+ antiviral factor (CAF). CAF is made at highest levels by CD8+ cells from asymptomatic individuals and long term survivors. Its production decreases with progression to disease. This antiviral factor suppresses all types of HIV-1, HIV-2, and SIV tested. It blocks the expression of viral RNA but does not affect steps prior to virus integration. CAF is stable to heat and low pH, has a size of 30Kd or less and is inactivated by certain proteases. In comprehensive studies, we have shown that CAF lacks identity to other known cytokines including the beta-chemokines and IL-16. We have demonstrated that IL-2 and CD28/CD3 costimulation increases this CD8+ cell antiviral response. The major objective of this proposal is to identify the structure of CAF. Two approaches are undertaken: biochemical purification and molecular biological procedures. In the biochemical approach, a variety of protein purification methods will be used to purify CAF to homogeneity for peptide sequencing. The molecular approach involves a PCR-based hybridization technique known as subtractive suppression hybridization-PCR (SSH-PCR) which selectively amplifies cDNAs that are differentially expressed by CAF producing cells. Cloning of the putative CAF gene followed by expression in mammalian cells will be undertaken to establish the identify of the protein. Primary polyclonal CD8+ cells (from HIV discordant twins) and suppressing and nonsuppressing CD8+ cell clones transformed by Herpesvirus samiri or derived form T cell hybridomas will be used as the source of CAF and the RNA for SSH-PCR. Once CAF has been purified or cloned, monoclonal antibodies to CAF will be produced. These antibodies will be used to develop a CAF-specific ELISA to measure the factor in blood and body fluids and to evaluate methods for enhancing its production in the host. The antibody can also be useful to detect CAF producing cells by flow cytometry. Ultimately, we would like to evaluate CAF in therapeutic approaches to control HIV infection and pathogenesis.
