CD8+ lymphocytes from healthy HIV-infected individuals show cytotoxic and non-cytotoxic anti-HIV activities. Our laboratory has focused on the CD8+ cell noncytotoxic response (CNAR) that appears to be part of the innate immune system responding to HIV infection. When CD8+ cells from an asymptomatic infected individual are co-cultivated with HIV acutely infected CD4+ cells, suppression of virus replication takes place without killing the CD4+ cells. CNAR is not HLA-restricted, not specific for a particular retrovirus (inhibits all HIV-1, HIV-2 and SIV isolates tested), appears very early in HIV infection, and is associated with secretion of a CD8+ cell antiviral factor (CAF) that has yet to be identified. CAF is a protein stable to heat and low pH and inhibits HIV transcription. We have recently found that protease inhibitors block CAF activity suggesting that a protease may be involved in the mediation of CNAR. The clinical importance of CNAR/CAF activity has been shown in several studies of protection of individuals from HIV infection and disease progression. The specific objective of the present proposal is to identify the polypeptide(s) that mediate CAF activity. Our ultimate goal is to clone/sequence and produce CAF for evaluation in therapeutic and diagnostic trials. Biochemical and molecular approaches are proposed. The biochemical approach involves mass spectrometric analysis of biochemically fractionated proteins from CAF-containing fluids. For these studies we propose to use: a) multi dimensional chromatography-tandem mass spectrometric analysis and an isotope-coded affinity tag (ICAT) methodology to identify peptides unique or over expressed in CAF-active fluids relative to control fluids; b) protease inhibitor-based affinity chromatography; and, when necessary, c) standard protein purification procedures. By the molecular approach, DNA microarray and kinetic RT-PCR procedures will be used to identify the gene(s)encoding CAF. With CD8+ cells that suppress HIV replication and those that do not, we have identified about 42differentially expressed genes in CD8+ cells showing CNAR. These genes will be further evaluated to verify that they are upregulated in CD8+ cells that suppress HIV replication. Candidate genes will be expressed in human cells and the secreted proteins evaluated for anti-HIV activity. Monoclonal antibodies will also be derived to establish a CAF specific ELISA and to use for flow cytometric studies in developing a diagnostic test to measure disease progression.
