1876. A Hepacivirus-Like Protein Is Targeted by the Antibody Response to Kawasaki Disease (KD)

Abstract

BackgroundClinical and epidemiologic data support a viral cause of KD, but the etiology has eluded 50 years of study. We previously identified virus-like intracytoplasmic inclusion bodies (ICI) in ciliated bronchial epithelium of KD children but not infant controls, but the antigens within the ICI were unknown. At 1–2 weeks following infection, 75% of peripheral blood plasmablasts (PB) specifically target the infectious agent. We cloned the PB response to KD to identify KD-specific antibodies and their target antigens.MethodsWe isolated single PB from children with KD 1–3 weeks after fever onset by flow cytometry, and amplified immunoglobulin VDJ and VJ genes from each PB by RT-PCR. We sequenced the products and made monoclonal antibodies (Mab) from clonally expanded PB in individual patients. Mab were tested for binding to KD tissues and to a viral peptide array containing 29,939 peptides from known B cell epitopes of animal viruses (www.iedb.org).ResultsWe sequenced 1156 PB from 11 KD patients, and identified 44 clonally expanded sets of PB. We prepared 61 Mab from clonally expanded and highly mutated IgA PB, and found that 33/61 bind to KD ICI, 10 strongly and 23 weakly. Of 10 Mab that strongly bind, 2 were VH3-33 (single patient), 2 VH3-23 (single patient), 1 VH3-15, 1 VH3-74, 3 VH1-46 (2 patients), and 1 VH4-59. These Mab CDR3s varied from 11 to 20 aacids, with 4–28 aacid mutations. Mab KD4-2H4 recognized multiple similar peptides from nonstructural protein 4A of hepacivirus C; pt KD4 sera was negative for hepatitis C by fourth-generation ELISA. Amino acid substitution analysis yielded an optimized peptide, and 6 KD Mab recognized this peptide by ELISA. These 6 Mab derived from 3 KD patients, all of whom had coronary aneurysms, and were VH3-74 (n = 1), VH3-33 (n = 2, single patient), VH1-45 (n = 1), and VH3-72 (n = 2, single patient). Strong binding of KD Mab KD4-2H4 and KD6-2B2 to ICI was totally blocked by pre-incubation with optimized peptide. KD but not control sera react with optimized peptide expressed as a glutathione S-transferase fusion protein by western blot.ConclusionChildren with KD make antibodies to a hepacivirus-like protein, and KD ICI contain this protein. These results strongly suggest that a previously unidentified hepacivirus with a respiratory portal of entry is etiologically related to KD.Disclosures All Authors: No reported Disclosures.