Antigenic landscape of the human helminth IgE antibody response

人类蠕虫 IgE 抗体反应的抗原图谱

• 批准号: 9897458
• 负责人: Scott Alan Smith
• 金额: $ 35.78万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-06 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897458
• 关键词: Adaptive Immune System; Affect; Affinity; Allergens; Allergic; Allergic Disease; Antibodies; Antibody Repertoire; Antibody Response; Antigen Targeting; B-Lymphocytes; Basophils; Binding; Biological Assay; Blood; Blood Circulation; Cell Culture Techniques; Cell physiology; Cells; Clinical; Clone Cells; Column Chromatography; Complex; Developed Countries; Developing Countries; Development; Diagnostic; Disease; Dose; Effector Cell; Enzyme-Linked Immunosorbent Assay; Epitopes; Filaria bancrofti; Filariasis; Frequencies; Funding; Generations; Goals; Helminth Proteins; Helminths; Homologous Gene; Homologous Protein; Human; Hybridomas; IgE; IgG4; Immune; Immune response; Immune system; Immunity; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; In Vitro; Knowledge; Length; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Memory B-Lymphocyte; Methods; Molecular; Monoclonal Antibodies; Mononuclear; Parasites; Patients; Play; Population; Preparation; Process; Protein Family; Proteins; Recombinants; Role; Sampling; Sequence Analysis; Serum; Signal Transduction; Site; Specificity; Strongyloides stercoralis; Strongyloidiasis; Techniques; Technology; Testing; Time; Vaccines; Variant; Western Blotting; Work; base; cross reactivity; design; falls; group competition; helminth infection; human monoclonal antibodies; immunoaffinity chromatography; improved; insight; mast cell; murine monoclonal antibody; novel; novel vaccines; pathogen; prevent; restraint; screening; vaccine candidate

Antigenic landscape of the human anti-helminth IgE antibody response Scott A. Smith ABSTRACT Despite its perceived central role in helminth immunity, very little is known about the naturally occurring human IgE antibody response, the dominant helminth target proteins or even the size and complexity of the functional antibody repertoire. Most of our knowledge of the IgE antibodies targeting helminth infection and/or allergens has come from studies using polyclonal sera or inferred from murine monoclonal antibodies (mAbs). We hypothesize that the human anti-helminth IgE antibody response is complex but is comprised of B cell clones which target helminth proteins that are homologues of known allergen proteins and are capable of mediating key effector cell functions in vitro. Here we employ a human B cell hybridoma method newly created in my lab, immortalizing growing memory B cells to generate for the first time ever, naturally occurring full-length human IgE mAbs. Patient clinical information is used to select samples that contain cells directed toward important helminth pathogens. In the initial studies described in this proposal, we selected three helminth infected subjects, one having strongyloidiasis and two with filariasis. The frequencies of IgE encoding B cells was found to be approximately three per million mononuclear cells with great variability seen in their reactivity to helminth lysate. These B cell frequencies and our technical efficiencies are sufficient to greatly expand this work and make hundreds of IgE mAbs. Due to funding restraints our initial characterization studies were performed on a small panel of six Wuchereria bancrofti IgE and three Strongyloides stercoralis IgE mAbs. Purified IgE mAbs were also found to have variable reactivity to helminth lysate in both ELISA and Western blot. Specific helminth protein targets were identified by immunoaffinity chromatography/mass spectrometry and fell into known allergen protein families. Crude allergen protein cross-reactivity studies were performed using Phadia diagnostic technologies and found in some cases to be positive, suggesting that allergens can resemble helminth proteins. IgE sequence analysis demonstrates significant degrees of somatic hypermutation. Initial functional studies show that each IgE helminth-specific mAb is capable of dose dependent mast cell mediator release in the presence of lysate - functional potency studies are underway. Ultimately helminth- specific IgE mAb panels will be assembled to reflect a hierarchy of immune dominant helminth protein targets and effector cell functionality. In addition to improving our basic understanding of this poorly studied branch of human immunity and allergic sensitization, the information obtained through studies outlined in this proposal will allow for the design and development of new helminth vaccines.

人类抗蠕虫IgE抗体反应的抗原景观。史密斯 摘要 尽管它在蠕虫免疫中被认为是核心作用，但人们对它的自然免疫机制知之甚少。 发生人IgE抗体应答，主要蠕虫靶蛋白或甚至大小， 功能性抗体库的复杂性。我们对IgE抗体靶向的大部分知识 蠕虫感染和/或过敏原来自于使用多克隆血清的研究或从 鼠单克隆抗体（mAb）。我们假设人类抗蠕虫IgE抗体 应答是复杂的，但由靶向蠕虫蛋白的B细胞克隆组成， 已知过敏原蛋白同源物，并能够介导关键效应细胞 在体外发挥作用。 在这里，我们采用了我实验室新创建的人B细胞杂交瘤方法， 记忆B细胞，以产生有史以来第一次，天然存在的全长人IgE单克隆抗体。 患者临床信息用于选择含有针对重要的细胞的样本。 寄生虫病原体在本建议中描述的初步研究中，我们选择了三种蠕虫 受感染的受试者，一个患有类圆线虫病，两个患有丝虫病。IgE编码频率 发现B细胞约为每百万个单核细胞中有3个， 它们对蠕虫裂解物的反应性。这些B细胞频率和我们的技术效率是足够的 来大大扩展这项工作并制造出数百种IgE单克隆抗体。由于资金限制，我们最初 对一小组六种班氏吴策线虫IgE和三种班氏吴策线虫IgE进行了表征研究。 粪类圆线虫IgE mAbs.还发现纯化的IgE mAb对以下物质具有不同的反应性 ELISA和Western blot检测蠕虫裂解物。特异性蠕虫蛋白质靶点通过 免疫亲和色谱/质谱分析，并落入已知的过敏原蛋白质家族。 使用Phadia诊断技术进行粗变应原蛋白交叉反应性研究 并在某些情况下被发现是阳性的，这表明过敏原可能类似于蠕虫蛋白质。 IgE序列分析表明体细胞高度突变的显着程度。初始功能 研究表明，每种IgE蠕虫特异性mAb都能够通过剂量依赖性肥大细胞介导 溶胞产物存在下的释放-功能效力研究正在进行中。最终寄生虫- 将组装特异性IgE mAb组，以反映免疫优势蠕虫蛋白的层次结构 靶点和效应细胞功能。除了提高我们对这一糟糕的基本认识之外， 研究了人体免疫分支和过敏性致敏作用，通过研究获得的信息 该提案中概述的技术将允许设计和开发新的蠕虫疫苗。