Immunodominant Epitopes in Kawasaki Disease

描述（由申请方提供）：川崎病（KD）是一种婴幼儿急性炎症性血管炎，已成为美国和日本儿童获得性心脏病的主要原因。对KD的病因和发病机制的有限了解迫使依赖于临床诊断标准，导致非典型病例的治疗延迟，并阻碍了新疗法和预防策略的开发。KD的临床病程和流行病学模式表明，该疾病是由感染引起的，并且有证据表明在KD的急性期存在特异性抗原驱动的IgA抗体应答。测量KD相关抗体的能力将为鉴定病原体的努力提供强有力的工具，并可形成KD诊断试验的基础。本项目的目标是开发检测KD特异性抗体的方法，具体目的是鉴定这些抗体识别的免疫显性表位的肽模拟物。该策略是使用随机肽的噬菌体展示文库作为肽模拟物的来源，以鉴定候选肽，然后验证候选肽作为KD相关抗体识别的表位的模拟物。发现阶段（子目标1）将通过使用3种通用方法来筛选噬菌体展示文库中的感兴趣的肽来鉴定候选肽。(1)将用从患有急性未治疗KD的患者的血清纯化的多克隆IgA抗体筛选肽文库。(2)将用合成单克隆抗体筛选肽文库，所述合成单克隆抗体衍生自急性KD动脉病变中普遍存在的α重链和κ轻链可变序列，并且识别支气管上皮和病变巨噬细胞中的KD相关细胞内抗原（由Dr. A.罗利）。(3)将用来自KD血清的多克隆IgA抗体和合成的单克隆抗体依次筛选肽文库。在对筛选抗体进行几轮选择后，将克隆免疫选择的噬菌体，并将克隆用于创建基于噬菌体的抗原微阵列。这些微阵列将用来自急性未经治疗的KD患者和年龄匹配的发热对照的库存血清进行探测。将确定与每个噬菌体克隆反应的抗体的KD的灵敏度和特异性，并设计使用多相克隆的分类方案。基于该信息，将选择候选肽。验证阶段（子目标2）将确定候选肽是否为KD相关抗原的模拟物。将使用KD患者和年龄匹配的发热对照（子目标1中使用的除外）验证抗体与候选肽反应性KD的特异性和灵敏度。还将组合分析单个测试的结果，以确定是否可以组合对不同肽模拟物的抗体反应性的测量，以进一步增强KD的灵敏度和特异性。

DESCRIPTION (provided by applicant): 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.