Identifying Kawasaki Disease-Specific Antibodies and Antigens

川崎病(KD)是发达国家儿童获得性心脏病的主要原因。 在芝加哥的安和罗伯特·H·鲁里儿童医院，我们每年诊断60-70个新病例，而美国大部分 儿童医院每年诊断数十名KD患者。KD是一种具有临床特征的急性发热性疾病。 常见于感染性疾病，可导致冠状动脉动脉瘤，尤其是 婴幼儿病情严重，6个月大，幼儿5岁发病率最高，极为罕见。 在成人中，发生在疾病在地理上呈波浪式传播的流行病中，而且很少复发。这些特定的 KD的流行病学特征最好的解释是感染了一种普遍存在的传染病病原体，通常 无症状，但可导致遗传易感儿童的KD。没有已知的感染性病原体与 和KD一起。我的实验室报告了急性KD的抗原驱动的动脉IgA免疫反应。在先前的研究中， 我们利用寡克隆免疫球蛋白α重链和随机轻链制备了合成抗体 KD动脉组织cDNA.不能从组织DNA中确定同源轻链和重链对 对我们来说是可用的。我们的原创KD合成抗体检测到小鼠胞浆内包涵体中的抗原。 免疫组织化学检测急性KD而非婴幼儿对照的支气管上皮，但亲和力不强 在免疫沉淀法中结合抗原。在其他研究中，我们发现I型干扰素诱导的基因 在KD患者的肺和冠状动脉中表达上调，与抗病毒免疫反应一致。 根据这些数据，我们假设KD是一种发生在遗传上的免疫介导的疾病。 容易感染一种目前不明病毒的儿童。在这项提案中，我们将使用新方法来 从单细胞分选外周血中快速克隆和制备抗原特异性合成抗体 浆母细胞，允许识别单个浆母细胞中的同源轻链和重链 产生高亲和力抗体。我们计划准备一组KD特异性的单抗和 使用它们来识别KD特异性抗原。在初步研究中，我们鉴定了一种由抗原驱动的寡克隆 1例发展为冠状动脉动脉瘤的急性KD患者的外周血浆母细胞反应。 我们从最流行的寡克隆中利用同源轻链和重链制备了合成抗体。 这位病人的浆母细胞。该抗体与其他KD患者组织中的抗原结合。这项建议 包括一个由KD、病毒学、免疫学和生物统计学专家组成的多学科团队，以执行 具体目标如下：1)鉴定和克隆KD患者的外周血浆母细胞反应；2)鉴定 克隆性KD外周血浆母细胞的抗原靶点。KD特异性抗体的鉴定和鉴定 抗原是识别KD病因、改进诊断和治疗以及使 预防，目标是从长期降低儿科保健费用、发病率和死亡率 儿童时期获得性冠状动脉瘤的后果。

Kawasaki Disease (KD) is the leading cause of acquired heart disease in children in developed nations. At Ann & Robert H. Lurie Children's Hospital of Chicago, we diagnose 60-70 new cases/year, and most US children's hospitals diagnose scores of KD patients annually. KD is an acute febrile illness with clinical features commonly observed in infectious diseases, can result in coronary artery aneurysms that can be particularly severe in infants <6 months of age, has highest incidence in young children <5 years of age, is extremely rare in adults, occurs in epidemics with geographic wavelike spread of illness, and only rarely recurs. These specific epidemiologic features of KD are best explained by infection with a ubiquitous infectious agent that is usually asymptomatic, but can result in KD in genetically susceptible children. No known infectious agent is associated with KD. My laboratory reported an antigen-driven arterial IgA immune response in acute KD. In prior studies, we made synthetic antibodies using oligoclonal immunoglobulin α heavy chains and random light chains from KD arterial tissue cDNA. Cognate light and heavy chain pairs could not be determined from the tissue cDNA available to us. Our original KD synthetic antibodies detected antigen in intracytoplasmic inclusion bodies in acute KD but not infant control bronchial epithelium by immunohistochemistry, but were not of sufficient affinity to bind antigen in immunoprecipitation assays. In other studies, we found that type I interferon-induced genes were upregulated in lungs and coronary arteries of KD patients, consistent with an antiviral immune response. Based on these data, we hypothesize that KD is an immune-mediated illness occurring in genetically predisposed children infected with a presently unidentified virus. In this proposal we will use new methods to rapidly clone and produce antigen-specific synthetic antibodies from single cell sorted peripheral blood plasmablasts, allowing identification of cognate light and heavy chains in individual plasmablasts and production of high-affinity antibodies. We plan to prepare a panel of KD-specific monoclonal antibodies and use them to identify KD-specific antigens. In preliminary studies, we identified an oligoclonal, antigen-driven plasmablast response in peripheral blood of an acute KD patient who developed a coronary artery aneurysm. We prepared synthetic antibody using cognate light and heavy chains from the most prevalent oligoclonal plasmablasts from this patient. This antibody binds to antigen in tissues from other KD patients. This proposal includes a multidisciplinary team of experts in KD, virology, immunology, and biostatistics to perform the following specific aims: 1) Identify and clone the peripheral blood plasmablast response in KD, and 2) Identify antigenic targets of oligoclonal KD peripheral blood plasmablasts. Identification of KD-specific antibodies and antigens is an important step toward identifying KD etiology, improving diagnosis and treatment, and enabling prevention, with the goals of reducing pediatric health care costs, morbidity, and mortality from the long-term consequences of coronary artery aneurysms that are acquired during young childhood.