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+ cell antiviral factor (CAF). CAF is made at highest levels by CD8 divided by 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 30 kD 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 also demonstrated that IL-2 and CD28/CD3 costimulation increases this CD8+ cell antiviral response. The major objective of this proposal is to determine the structure of CAF. Two approaches are undertaken: biochemical purification and molecular biological procedures. In the biochemical approach, a variety of standard protein purification methods will be used in combination with SELDI mass spectrometry to purify CAF to homogeneity for peptide sequencing. The molecular approach involves both 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, and a DNA array analysis which can identify differently expressed genes. Cloning of the putative CAF gene followed by expression in mammalian cells will be undertaken to establish the identity of the protein. Primary polyclonal CD8+ cells and suppressing and non-suppressing CD8+ cell clones as well as T cell hybridomas will be used as the source of CAF and the RNA for SSH-PCR and microarray procedures. 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
