Novel Antigens & T & B Cell Responses in Biliary Atresia

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DESCRIPTION (provided by applicant): Multiple lines of evidence have suggested that viral infection may play a pathogenetic role in biliary atresia (BA). However, searches for putative viral pathogens have been restricted to a small number of selected viruses and have not focused on virus-specific immune responses. The hypothesis underlying this proposal is that viral infection plays an etiopathologic role in biliary atresia. The purpose of the present proposal is three-fold: 1) Establish a Clinical Biliary Atresia Center. Five referral centers including the Johns Hopkins Pediatric Liver Center will recruit 10-20 new BA patients/year to add to the 73 BA patients in the present database. The database will gather demographic and clinical information as well as biological specimens pertinent to the viral infection hypothesis. 2) BA Peptide Project, (3-years) will employ a powerful new technique, bacteriophage peptide display, to search for novel antigenic epitopes specific to BA. BA sera will be utilized to "biopan" random peptide libraries to identify epitopes not recognized by control sera. One patient/control group from each of the Clinical Centers will be studied, for the greatest possible geographic, seasonal, and ethnic diversity. "BA peptides" will be sequenced and synthesized. Peptides will be used to immunoaffinity purify the corresponding monospecific antibodies from BA sera. Protein databases will be searched for homologous viral proteins. To determine if the BA peptides exhibit cross reactivity as HLA-A2+ restricted T cell epitopes, the peptides will be incubated with dendritic cells, antigen-presenting cells (APC) harvested from HLA-A2+ maternal peripheral blood lymphocytes (PBL). Maternal PBL will be incubated with the peptide-pulsed APC to identify peptide-specific CTL using target cell lysis assays. 3) The BA-Dimer Project, (1-year) will employ a sensitive new "dimer" technique to determine if HLA-A2+ biological mothers of BA (MBA) patients exhibit unique peptide-specific CTL. The dimer technique uses immunoglobulin as a molecular scaffold to produce a divalent peptide-HLA-A2-Ig complex. Dimers will be loaded with BA or homologous viral peptides and PBL from MBA and control mothers from each Clinical Center will be tested for dimer reactivity using FACS analysis. Novel BA-specific peptides, monospecific antibodies, and BA-dimers could be used for early diagnosis, identification of cellular targets and intrahepatic T cells.