CD+8 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 antiviral factor (CAF) produced by CD8+ cells. CAF is made at highest levels by CD8+ cells from asymptomatic individuals and long-term survivors. Its production decreases with progression to disease. Previous studies indicate that CAF is heat- and low pH-stable, has a size of 30 kD or less, and is a protein resistant to trypsin and sensitive to staph V8 protease. We I- lave since determined that CAF can be produced by cells grown in serum- free medium and the protein appears glycosylated based on its recovery from ConA columns. In comprehensive studies, we have shown that CAF lacks identity to other known cytokines including the beta-chemokines. We have also demonstrated that lL2 and antibodies to CD28 can increase the CD8+ cell antiviral response. Toward recovery and identification of CAF, we have established Herpes virus saimiri-transformed CD8+ cells producing this factor as well as T cell hybridoma lines releasing CAF. The major objective of the present proposal is to purify CAF so that its amino acid sequence can be determined and subsequent cloning and expression of the factor can be undertaken. The approaches include standard protein purification methods (i.e., column chromatography, isoelectric focusing, and gel electrophoresis) using an algorithm that removes albumin from the serum-free medium and permits concentration of the factor. Monoclonal antibodies to CAF will be produced to help in purification. With these antibodies, an ELISA assay will be developed to assess the presence of 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 and quantitate CAF-producing cells by flow cytometry.A molecular approach to identify CAF is also proposed in which representational difference analysis(RDA) will be utilized using either CAF-producing and non-producing cell clones or CD8+ cells from an individual before and after production of CAF. These molecular studies could lead to the detection of CAF-specific mRNA. Subsequent cloning of the gene and expression in mammalian cells would be undertaken. The ultimate objective is to evaluate CAF as a therapeutic approach to control HIV infection.
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