This application for funds is submitted in response to RFA AI-03-017 and proposes characterization and cloning of a human gene product as a broad spectrum immunotherapeutic for the treatment of infection with multiple Category A and B priority pathogens. Infection by or exposure to some viruses or Gram-negative bacteria induces inflammatory responses that contribute significantly to disease pathogenesis. Common to many such responses is the activation of nuclear factor kB (NFkB) - directed transcription. In our ongoing investigation of HRF, an anti-HIV-1 factor secreted by a transformed human T cell line, we found that it acts through interference with NFkB binding to kB on chromosomal DNA. In recent studies we confirmed that HRF blocks NFkB induction by lipopolysaccharides of several pathogenic bacteria and NFkB activation of inflammatory genes in human cells. Recent HRF peptide sequence findings enabled us to identify a homologous coding sequence, facilitating molecular cloning. This application is directed toward characterization of HRF as an antagonist of NFkB-directed inflammatory responses to several high priority pathogens and to completion of our ongoing program of cloning HRF. We propose to: 1) determine the activity of HRF in human cells in culture in NFkB-directed responses to Shigella, Salmonella, Campylobacter, and Dengue; 2) clone and express recombinant HRF and evaluate its activity in culture for treatment of common inflammatory response to different pathogens; 3) determine the molecular mechanism of action through which HRF antagonizes NFkB-directed transcription. These studies will employ several prokaryotic-eukaryotic coculture systems, molecular cloning, PCR site-directed mutagenesis, and electrophoretic mobility shift assays of protein-protein and protein-DNA complexes. This program will result in the development of a new broad spectrum immunotherapeutic based upon a human protein for the control of NFkB-directed acute inflammatory responses to multiple pathogens. ? ?
