Comprehensive antigenic mapping of the human anti-peanut IgE antibody response

人类抗花生 IgE 抗体反应的综合抗原图谱

基本信息

批准号：
10520041
负责人：
Scott Alan Smith
金额：
$ 80.63万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
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：
2020-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10520041
关键词：
Adaptive Immune System Affinity Albumins Allergens Allergic Allergic Disease Allergy to peanuts Anaphylaxis Antibodies Antibody Response Antigenic Specificity B-Lymphocytes Basophils Binding Biological Assay Cells Cellular Assay Circulation Clinical Clinical Data Clone Cells Complex Developed Countries Developing Countries Development Diagnostic Disease Dose Enzyme-Linked Immunosorbent Assay Epidemic Epitopes Exposure to Food Freezing Generations Goals Helminths Human Hybridomas Hypersensitivity IgE Immunoglobulin G Immunotherapy In Vitro Incidence Individual Knowledge Location Maps Measures Mediating Mediator Methods Molecular Monoclonal Antibodies Pathogenesis Pathologic Patients Persons Play Population Process Proteins Recombinants Research Subject Selections Research Subjects Resolution Role Serum Severities Severity of illness Site Specificity Structure Therapeutic Time Tissues Transgenic Mice Vaccines Variant Work X-Ray Crystallography associated symptom clinical predictors clinically relevant cross reactivity crosslink design in vivo insight mast cell novel passive sensitization peripheral blood preservation prototype receptor response tool

项目摘要

PROJECT SUMMARY At the center of the pathogenesis of allergic diseases is the IgE molecule. 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. To date, studies of the human IgE molecule, and its targeted epitopes on allergens, have been very limited. Most 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 the 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 now 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 comprehensive panels of naturally-occurring peanut allergen-specific human IgE mAbs to define the molecular interactions of the most potent inducers of anaphylaxis. We have already begun comprehensive mapping studies to identify key immunodominant antigenic sites on the major peanut allergen proteins Ara h 1, 2, 3, and 6. In Aim 1, functional antigenic site mapping via peanut induced anaphylaxis will be accomplished by passive sensitization of human FcεRI transgenic mice using IgE mAbs. In Aim 2, prototype mAbs that bind unique antigenic sites will be selected and expressed as recombinant IgG mAbs to use as tools for advanced mapping studies using human serum. IgG mAbs, which bind identically as the IgE mAbs from which they were made, will be employed in blocking studies using a panel of peanut allergic research subjects’ frozen serum and ImmunoCAP diagnostics. This will define the role that each antigenic site-specific population of IgE antibodies play within and between peanut allergic individuals. This information will be used to draw clinical correlates of disease and to select research subjects which possess IgE antibodies not blocked by our panels, allowing for the generation of new site-specific IgE mAbs in Aim 1. Finally, in Aim 3, we will obtain atomic resolution structures to precisely define the first ever naturally-occurring human IgE epitopes on Ara h 2 and 6 by X- ray crystallography. The goal of this work is to create a definitive, complete and comprehensive molecular map of the human IgE antibody response to the major allergen proteins of peanut. The results will serve as a much-needed road map to allow for the design of new immunotherapies and allergy vaccines.

项目摘要在过敏性疾病的发病机理的中心是IgE分子。在敏感的个体中，暴露于违规过敏原会导致IgE参与，从而导致FCε受体交联和肥大细胞和嗜碱性的激活。这会触发介体向当地组织的释放，从而与过敏性疾病相关的各种症状，包括过敏性休克。迄今为止，研究人类IgE分子及其在过敏原上的靶向表位非常有限。我们的大多数对此过程的了解是来自使用过敏患者精华素的研究，其中包含许多具有许多特异性的抗体针对许多不同的表位，并且有许多抗体不同的亲和力；因此，对IgE与靶过敏原的分子相互作用的研究极为有缺陷。研究这一过程的理想方法是使用自然存在的人类IgE单克隆抗体（mAb），与过敏受试者分离。不幸的是，由于许多无网络的技术障碍此类抗体以前曾经制作过。我们现在已经建立了一种成长，识别和通过从过敏性的外周血中制造人类杂交瘤来使IgE编码B细胞永生在此提案中，我们开发了第一个自然疾病的花生的综合面板过敏原特异性人IgE mabs，以定义最有效诱导剂的分子相互作用过敏反应。我们已经开始进行全面的映射研究，以识别关键的免疫力优势主要花生过敏原蛋白上的抗原位点ARA H 1、2、3和6。在AIM 1中，功能性抗原位点通过花生诱导的过敏反应的映射将通过人FcεRI的被动灵敏度来实现转基因小鼠使用IgE mabs。在AIM 2中，将选择结合独特抗原位点的原型mab 并表示为重组IgG mabs，用于使用人类的高级映射研究工具血清。 IgG mabs将与其制造的IgE mab相同的绑定，将成为员工使用一组花生过敏研究对象的冷冻血清和免疫方法阻止研究诊断。这将定义每种抗原位点特异性IgE抗体在内的作用以及花生过敏的个体之间。该信息将用于吸引疾病的临床相关性并选择具有未被我们面板阻塞的IgE抗体的研究主题，从而允许在AIM 1中生成新的特定地点IgE mab。最后，在AIM 3中，我们将获得原子分辨率精确定义第一个自然构成的人类IgE表位的结构，x- 射线晶体学。这项工作的目的是创建一个确定，完整和全面的分子人IgE抗体对花生主要过敏原蛋白的反应。结果将作为急需的路线图，以设计新的免疫疗法和过敏疫苗。