Molecular basis for anaphylaxis due to galactose-alpha-1,3-galactose (alpha-gal)

半乳糖-α-1,3-半乳糖（α-gal）引起的过敏反应的分子基础

• 批准号: 10177870
• 负责人: Scott Alan Smith
• 金额: $ 18.7万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-02 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10177870
• 关键词: Adaptive Immune System; Affinity; Allergens; Allergic; Allergic Disease; Anaphylaxis; Antibodies; Antibody Response; Antigens; B-Lymphocytes; Basophils; Binding; Biological Assay; Blood Circulation; Cells; Clinical; Clone Cells; Column Chromatography; Data Set; Developed Countries; Developing Countries; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Epidemic; Epitopes; Exhibits; Exposure to; Food; Gene Amplification; Generations; Genes; Genetic; Germ Lines; Glycoproteins; Goals; Helminths; Human; Hybridomas; Hypersensitivity; IgE; Immediate hypersensitivity; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunohistochemistry; Immunoprecipitation; Incidence; Individual; Kinetics; Knowledge; Length; Maps; Mass Spectrum Analysis; Measurement; Meat; Mediating; Mediator of activation protein; Methods; Molecular; Molecular Target; Monoclonal Antibodies; Mutate; Oligosaccharides; Pathogenesis; Pathogenicity; Pathologic; Patients; Peripheral Blood Mononuclear Cell; Phylogeny; Polysaccharides; Population; Process; Reaction; Recombinants; Research Subjects; Risk; Serum; Specificity; Surface Plasmon Resonance; Techniques; Technology; Testing; Ticks; Tissues; Transgenic Mice; Variant; Western Blotting; alpha-gal syndrome; associated symptom; base; crosslink; experience; functional group; galactosyl-(1-3)galactose; human monoclonal antibodies; immunoaffinity chromatography; insight; mast cell; neoantigens; novel; peripheral blood; receptor; response; vector tick

PROJECT SUMMARY The IgE molecule lies at the center of the pathogenesis of allergic diseases. In sensitized individuals, re-exposure to the offending allergen results in IgE engagement, causing Fcε receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, resulting in the vast array of symptoms associated with allergic diseases, including anaphylactic shock. Studies of the human IgE molecule, and its targeted allergens, have been very limited. Nearly all of our knowledge of this process has come from studies using allergic patient serum, which contains a mixture of many antibodies, with many specificities, directed toward many different epitopes, and having many different affinities; thus studies of the molecular interactions of IgE with target allergens are greatly flawed. The ideal way to study this process is to use naturally- occurring human IgE monoclonal antibodies (mAbs), isolated from allergic subjects. Unfortunately, due to many impassible intrinsic technical hurdles no such antibodies have previously ever been made. We have established a method to grow, identify and immortalize IgE encoding B cells by making human hybridomas from the peripheral blood of allergic individuals. In this proposal, we develop the first panel of naturally-occurring alpha- gal-specific human IgE mAbs from subjects with red meat allergy. We intend to define the precise molecular basis for IgE-mediated reactions to red meat, from both the allergen and the antibody perspective. We will begin by generating human hybridomas from peripheral blood B cells of a few highly characterized research subjects. Purified IgE mAbs then will be used to precisely define critical molecular details of human anti-alpha-gal IgE binding. We will employ this panel of antibodies in various molecular assays to assist in localizing, identifying, and ultimately characterizing the allergen(s) found in ticks, responsible for sensitization of humans. As these IgE mAbs represent the B cells which are induced to undergo class-switch recombination, we will use their variable gene sequences to interrogate Illumina sequencing datasets from the research subjects from which the mAbs were obtained. This will allow for a keen understanding of the B cell population(s) underlying this allergic disease and may provide insights needed to predict individuals’ risk of sensitization. Together, this proposal will begin studies to define the exact glycoprotein targets of the human IgE B cell response, and the origin of the B cells themselves, which will provide much needed details underlying the sensitization and development of hypersensitivity to this common oligosaccharide.

项目概要 IgE 分子是过敏性疾病发病机制的核心。对于敏感个体，再次暴露 与有害过敏原结合导致 IgE 结合，导致 Fcε 受体交联并激活肥大 细胞和嗜碱性粒细胞。这会触发介质释放到局部组织中，从而产生大量的 与过敏性疾病相关的症状，包括过敏性休克。人类 IgE 分子的研究， 及其针对的过敏原非常有限。我们对这个过程的所有知识几乎都来自于 使用过敏患者血清进行的研究，其中含有多种抗体的混合物，具有多种特异性， 针对许多不同的表位，并具有许多不同的亲和力；因此分子研究 IgE 与目标过敏原的相互作用存在很大缺陷。研究这个过程的理想方法是自然地使用- 从过敏受试者中分离出的人类 IgE 单克隆抗体 (mAb)。不幸的是，由于许多 由于无法逾越的内在技术障碍，以前从未制造过此类抗体。我们已经建立了 一种通过从 B 细胞中制备人类杂交瘤来培养、鉴定和永生化编码 IgE 的 B 细胞的方法 过敏个体的外周血。在这个提案中，我们开发了第一组天然存在的α- 来自红肉过敏受试者的半乳糖特异性人 IgE 单克隆抗体。我们打算定义精确的分子 从过敏原和抗体的角度来看，这是 IgE 介导的红肉反应的基础。我们将开始 通过从一些高度特征化的研究对象的外周血 B 细胞中产生人类杂交瘤。 纯化的 IgE mAb 将用于精确定义人类抗 α-gal IgE 的关键分子细节 绑定。我们将在各种分子测定中使用这组抗体来协助定位、识别、 并最终表征蜱虫中发现的导致人类过敏的过敏原。由于这些 IgE mAb 代表被诱导进行类别转换重组的 B 细胞，我们将使用它们的变量 基因序列，用于查询来自研究对象的 Illumina 测序数据集，单克隆抗体来自这些研究对象 获得。这将使我们能够深入了解这种过敏性疾病背后的 B 细胞群 并可能提供预测个人过敏风险所需的见解。这项提案将一起开始 研究确定人类 IgE B 细胞反应的确切糖蛋白靶标以及 B 细胞的起源 本身，这将为宣传和发展提供非常需要的细节 对这种常见的寡糖过敏。