Our laboratory and others have demonstrated the ability of CD8+ cells from HIV-infected individuals to suppress virus replication in CD4+ cells. We have determined that this non-lytic, non-MHG-restricted activity is mediated in part by a novel cytokine produced solely by CD8+ cells. This CD8+ cell antiviral factor (CAF) is made at highest levels by CD8+ cells from asymptomatic individuals and its production decreases with advancement to disease. Our studies indicate that CAF is heat and pH stable, and has a size of approximately 30 kD or less. It is resistant to trypsin and lyophilization and does not directly inactivate HIV. It is sensitive to staph V8 protease. The recent removal of CAF by cation columns suggests that this small protein is positively charged at pH 6.0. 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 used will include standard protein purification methods (i.e., column chromatography, isoelectric focusing and gel electrophoresis) and the development and use of a factor-specific monoclonal antibody. The monoclonal antibody would also be helpful in the final purification of the factor and also provide a means for derivation of a CAF-specific ELISA to rapidly assay for the factor, help identify cells producing CAF, and evaluate methods for enhancing its production. Other studies to assist in CAF purification include approaches to establish cell lines producing CAF, to enhance CAF production by CD8+ lymphocytes, and to develop more rapid assays for detection of CAF. The purification and characterization of this antiviral factor could have important therapeutic value in HIV infection.
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