A New Human Hepacivirus as an Etiologic Agent of Kawasaki Disease (Falk Trust Catalyst Award)

Kawasaki Disease (KD) is the leading cause of acquired heart disease in children in developed nations and at Lurie Children’s Hospital in Chicago, we diagnose 60-70 new cases/year. KD can result in coronary artery aneurysms that lead to lifelong heart disease, myocardial infarction, and death. The clinical and epidemiologic features support an infectious etiology, but the cause has eluded more than 50 years of study, significantly hampering the development of a diagnostic test and specific therapies. KD morbidity and mortality remains an ongoing clinical problem. To identify antibodies and antigens relevant to KD pathogenesis, we performed single cell sequencing of immunoglobulin VDJ heavy chain and VJ light chain genes cloned from 1156 peripheral blood plasmablasts (PB) isolated from 11 children 1-3 weeks after KD fever onset. We identified 44 sets of clonally expanded sequences and from these we prepared 61 KD monoclonal antibodies (Mab). By immunohistology, 33 of the Mab detect virus-like intracytoplasmic inclusion bodies (ICI) in KD ciliated bronchial epithelium. Moreover, 6 of the 33 (18%) Mab, which were derived from 3 KD patients with coronary aneurysms, recognize hepacivirus NS4A-like peptides by peptide array and/or ELISA. The KD NS4A peptide completely blocks binding of these Mab to KD ICI, indicating that a hepacivirus NS4A-like protein is present in KD ICI. Our primary hypothesis is that at least a subset of KD cases result from infection with a previously unidentified hepacivirus in genetically susceptible children. We propose these specific aims: Aim 1. Identify the KD-associated hepacivirus RNA sequence by bioinformatics analysis of our KD RNAseq dataset. A. Using HMM analyses, identify hepacivirus-like protein-coding genes in the KD RNAseq dataset. B. Find upstream and downstream RNA sequences of HMM hits and of the known NS4A sequence. Aim 2. Identify additional hepacivirus protein epitopes recognized by KD monoclonal antibodies (Mab) that identify KD intracytoplasmic inclusion bodies (ICI). A. Test 8 Mab that strongly bind to ICI but do not recognize NS4A for binding to viral peptide arrays and to a hepacivirus phage display library. These Mab are likely to yield additional viral proteome data. Aim 3. Test KD patients for serologic response to and for the presence of KD-associated hepacivirus. A. Test KD and childhood control sera for IgM, IgA, and IgG antibodies to hepacivirus proteins. B. Identify viral RNA in KD and control tissues by real-time RT-PCR. These studies will identify genomic and proteomic sequences of the new hepacivirus and enable development of diagnostic assays that can establish the relationship of the virus to KD.