Identifying Specific Antigenic Targets of Kawasaki Disease

识别川崎病的特定抗原靶点

基本信息

批准号：
10118994
负责人：
ANNE H ROWLEY
金额：
$ 50.36万
依托单位：
LURIE CHILDREN'S HOSPITAL OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-22 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10118994
关键词：
Amino Acid Substitution Aneurysm Antibodies Antibody Response Antigen Targeting Antigens Autoantigens Binding Biological Assay Cells Cessation of life Child Childhood Clinical Coronary artery Data Developed Countries Development Diagnosis Diagnostic tests Disease Enzyme-Linked Immunosorbent Assay Epidemiology Epitopes Etiology Fever Future Geographic Locations Goals Health Care Costs Heart Diseases High-Throughput Nucleotide Sequencing Human Immune Targeting Immune response Immunoglobulin A Immunoglobulin G Immunoglobulin M Immunoglobulins Inclusion Bodies Infant Infection Investigation Lead Libraries Mass Spectrum Analysis Messenger RNA Monoclonal Antibodies Mucocutaneous Lymph Node Syndrome Myocardial Infarction Nucleic Acids Pathogenesis Patients Pediatrics Peptides Phage Display Plasmablast Prevention Protein Array Proteins RNA RNA Sequences Reverse Transcriptase Polymerase Chain Reaction Risk Serologic tests Serological Testing Time Tissues Viral Virus Western Blotting bronchial epithelium cDNA Library diagnostic assay disorder control immunogenic improved laser capture microdissection novel therapeutics peripheral blood response screening

项目摘要

PROJECT SUMMARY/ABSTRACT Kawasaki Disease (KD) is the leading cause of acquired heart disease in children in developed nations. KD can result in coronary artery aneurysms that can lead to lifelong heart disease, myocardial infarction, and death. The clinical and epidemiologic features support an infectious etiology in genetically susceptible children, but the cause has eluded more than 50 years of study. Delayed and missed diagnoses increase the risk of coronary artery aneurysms. The development of urgently needed diagnostic tests and improved therapies are dependent upon identifying the etiology. In preliminary studies, we analyzed the peripheral blood plasmablast response at 1-3 weeks after KD fever onset using single cell RT-PCR and made 60 monoclonal antibodies (Mab) from these plasmablasts. We used these Mab to determine their target antigens. We found that 32/60 Mab, derived from 9/11 KD patients, identify intracytoplasmic virus-like inclusion bodies (ICI) in ciliated bronchial epithelium of KD children but not infant controls. Using peptide arrays to identify binding epitope(s), amino acid substitution arrays, and ELISA, we found that 5 of the 32 (16%) Mab, derived from 3 KD patients with coronary artery aneurysms, recognize KD peptide epitope 1. KD peptide 1 completely blocks binding of these Mab to KD ICI, indicating that a protein with the binding epitope sequence is present in the ICI. Moreover, pre-treatment sera from 5/8 KD patients >day 8 after fever onset but 0/17 infant control sera have IgG antibody to the peptide epitope. We recently identified two additional epitopes using this approach that require further study. These findings support our hypothesis that at least a subset of KD cases results from infection with a presently unidentified, ubiquitous viral causative agent containing the KD peptide epitope 1 sequence. In this proposal, we hypothesize that identification of antigens targeted by the plasmablast response to KD can lead to identification of the etiology(ies) and development of KD serologic tests. To test these hypotheses, we will: 1) identify the specific proteins recognized by our panel of KD Mab that bind to KD tissues, 2) screen for additional antigenic targets recognized by the immune response to KD, and 3) evaluate the antibody responses of KD and control sera to KD antigens. Our findings will inform pathogenesis of KD, with the long-term goals of improving diagnosis and treatment of KD, enabling prevention, and reducing healthcare costs from the long-term consequences of coronary artery aneurysms arising in young childhood.

项目概要/摘要川崎病（KD）是发达国家儿童获得性心脏病的主要原因。肯德基可能导致冠状动脉瘤，从而导致终生心脏病、心肌梗塞和死亡。临床和流行病学特征支持遗传易感儿童的传染性病因，但其原因却一直未能得到超过 50 年的研究。延迟和漏诊会增加风险冠状动脉瘤。开发迫切需要的诊断测试和改进的治疗方法取决于确定病因。在初步研究中，我们分析了外周血浆母细胞使用单细胞 RT-PCR 检测 KD 发热后 1-3 周的反应并制备 60 个单克隆抗体 (Mab) 来自这些浆母细胞。我们使用这些单克隆抗体来确定它们的靶抗原。我们发现 32/60 Mab 源自 9/11 KD 患者，可识别纤毛中的胞浆内病毒样包涵体 (ICI) 川崎病儿童的支气管上皮，但不是婴儿对照。使用肽阵列来识别结合表位，通过氨基酸替换芯片和 ELISA，我们发现 32 种 Mab 中的 5 种 (16%) 来自 3 名 KD 患者冠状动脉瘤，识别 KD 肽表位 1。KD 肽 1 完全阻断结合这些 Mab 为 KD ICI，表明 ICI 中存在具有结合表位序列的蛋白质。此外，发烧后第 8 天，5/8 的 KD 患者的治疗前血清有变化，但 0/17 的婴儿对照血清有针对肽表位的 IgG 抗体。我们最近使用这种方法鉴定了两个额外的表位需要进一步研究。这些发现支持我们的假设，即至少一部分 KD 病例是由感染一种目前未知、普遍存在的含有 KD 肽表位 1 的病毒病原体顺序。在这个提议中，我们假设浆母细胞反应所针对的抗原的鉴定对川崎病的认识可以导致川崎病病因的鉴定和川崎病血清学检测的发展。为了测试这些假设，我们将： 1) 鉴定我们的 KD Mab 组识别的与 KD 结合的特定蛋白质组织，2) 筛选由 KD 免疫反应识别的其他抗原靶标，以及 3) 评估 KD 和对照血清对 KD 抗原的抗体反应。我们的研究结果将为川崎病的发病机制提供信息，长期目标是改善川崎病的诊断和治疗、预防和减少幼儿时期出现的冠状动脉瘤的长期后果导致医疗费用。