Immunodominant Epitopes in Kawasaki Disease

描述（由申请人提供）：川崎病（Kawasaki disease， KD）是婴幼儿的一种急性炎症性血管炎，在美国和日本已成为儿童获得性心脏病的主要原因。对KD病因和发病机制的了解有限，迫使对临床诊断标准的依赖，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发展。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的急性期有特异性的抗原驱动的IgA抗体反应。测量KD相关抗体的能力将为确定病因提供一个强有力的工具，并可能形成KD诊断测试的基础。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。该策略是使用噬菌体展示的随机肽库作为肽模拟物的来源，鉴定候选肽，然后验证候选肽是否与kd相关抗体识别的表位相似。发现阶段（子目标1）将通过使用3种一般方法筛选感兴趣的噬菌体展示文库来确定候选肽。(1)利用从急性未治疗KD患者血清中纯化的多克隆IgA抗体筛选肽库。(2)肽库将用合成单克隆抗体进行筛选，这些单克隆抗体来源于急性KD动脉病变中普遍存在的α重链和kappa轻链可变序列，并识别支气管上皮和病变巨噬细胞中KD相关的细胞内抗原（由a . Rowley博士提供）。(3)用KD血清中IgA多克隆抗体和合成的单克隆抗体依次筛选肽库。经过几轮筛选抗体的选择，免疫选择的噬菌体将被克隆，克隆的噬菌体将用于创建基于噬菌体的抗原微阵列。这些微阵列将与急性未经治疗的KD患者和年龄匹配的发热对照者的血清进行检测。将确定与每个噬菌体克隆反应的抗体KD的敏感性和特异性，并设计使用多期克隆的分类方案。根据这些信息，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。KD抗体对候选肽反应性的特异性和敏感性将通过KD患者和年龄匹配的发热对照进行验证，而不是在第1项中使用的对照组。单个测试的结果也将被联合分析，以确定是否可以将抗体对不同肽模拟物的反应性测量结合起来，以进一步提高KD的敏感性和特异性。

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.