Kawasaki disease (KD), an acute inflammatory vasculitis of infants and young children, has become the leading cause of acquired heart disease among children in the United States and Japan. Limited insight into the cause and pathogenesis of KD forces reliance on clinical criteria for diagnosis, results in delayed treatment of atypical cases, and hampers the development of novel therapies and preventative strategies. The clinical course and epidemiologic pattern of KD suggest that the disease results from an infection, and there is evidence for a specific, antigen-driven IgA antibody response during the acute phase of KD. The ability to measure KD-associated antibodies would provide a powerful tool for efforts to identify the etiologic agent and could form the basis for a diagnostic test for KD. The goal of this project is to develop the means to detect KD-specific antibodies, and the specific aim is to identify peptide mimics of the immunodominant epitopes recognized by these antibodies. The strategy is to use a phage-displayed library of random peptides as a source of the peptide mimics, to identify candidate peptides, and then to validate the candidates peptides as mimics of the epitopes recognized by KD-associated antibodies. The discovery phase (subaim 1) will identify candidate peptides by using 3 general approaches to screen the phagedisplayed library for peptides of interest. (1) The peptide library will be screened with polyclonal IgA antibodies purified from sera of patients with acute, untreated KD. (2) The peptide library will be screened with synthetic monoclonal antibodies that were derived from alpha heavy-chain and kappa light-chain variable sequences prevalent in an acute KD arterial lesion and that recognize a KD-associated intracellular antigen in bronchial epithelia and lesional macrophages (kindly provided by Dr. A. Rowley). (3) The peptide library will be screened sequentially with polyclonal IgA antibodies from KD sera and with the synthetic monoclonal antibodies. After several rounds of selection on the screening antibodies, the immunoselected phage will be cloned, and the clones will be used to create phage-based antigen microarrays. These microarrays will be probed with banked sera from acute untreated KD patients and age-matched febrile controls. The sensitivities and specificities for KD of the antibodies reactive with each phage clone will be determined, and classification schemes using multiple phase clones will be devised. Based on this information, candidate peptides will be selected. The validation phase (subaim 2) will determine whether the candidate peptides are mimics of KD-associated antigens. The specificities and sensitivities for KD of antibody reactivity to the candidate peptides will be validated using KD patients and age-matched febrile controls other than those used in subaim 1. The results of individual tests also will be analyzed in combination to determine whether measurements of antibody reactivity to different peptide mimics can be combined to further enhance sensitivity and specificity for KD. ? ? ?
