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

• 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
• 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

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.

项目总结 过敏性疾病发病机制的核心是免疫球蛋白E分子。在敏感的个体中，Re- 暴露于令人讨厌的过敏原会导致免疫球蛋白E参与，导致Fcε受体交联和 肥大细胞和嗜碱性粒细胞的激活。这会触发介质释放到局部组织中，从而导致 与过敏性疾病相关的大量症状，包括过敏性休克。到目前为止，研究 对人类IgE分子及其过敏原的靶向表位的研究一直非常有限。我们的大多数人 对这一过程的了解来自于使用过敏患者血清的研究，这种血清含有一种 许多抗体，具有许多特异性，针对许多不同的表位，并且有许多 不同的亲和力；因此，对IgE与靶变应原分子相互作用的研究很大程度上 有缺陷。研究这一过程的理想方法是使用自然产生的人类免疫球蛋白E单抗 (单抗)，从过敏对象中分离出来。不幸的是，由于许多无法逾越的内在技术障碍，没有 以前也曾制造过这样的抗体。我们现在已经建立了一种方法来生长、识别和 从过敏性疾病患者外周血制备人杂交瘤永生化编码B细胞的IgE 个人。在这项提案中，我们开发了第一个全面的天然花生面板 用变应原特异性人IgE单抗来确定最有效的诱导剂的分子相互作用 过敏反应。我们已经开始了全面的图谱研究，以确定关键的免疫优势 花生主要变应原蛋白Arah1、2、3和6的抗原部位。 花生过敏反应的定位将通过人FcεRI的被动敏化来完成 用IgE单抗转基因小鼠。在目标2中，将选择与独特抗原位点结合的原型单抗 并以重组免疫球蛋白单抗的形式表达，用作高级图谱研究的工具 血清。Ig G单抗与制造它们的Ig E单抗具有相同的结合力，将用于 一组花生过敏研究对象冰冻血清和免疫CAP的阻断研究 诊断。这将定义每个抗原性部位特异性IgE抗体群体在 以及花生过敏者之间的关系。这些信息将被用来得出疾病的临床相关性。 并选择具有未被我们的小组阻止的IgE抗体的研究对象，考虑到 在目标1中产生新的位点特异性IgE单抗。最后，在目标3中，我们将获得原子分辨率 结构，以精确地确定第一个自然产生的人类免疫球蛋白E表位的Arah2和6通过X- 射线结晶学。这项工作的目标是创建一个确定的、完整的和全面的分子 花生主要变应原蛋白的人IgE抗体反应图。结果将作为 一个亟需的路线图，以便设计新的免疫疗法和过敏疫苗。