Antigenic landscape of the human helminth IgE antibody response

人抗蠕虫IgE抗体应答的抗原性图谱 摘要 尽管人们认为它在蠕虫免疫中起着核心作用，但人们对自然的 发生人IgE抗体应答时，优势蠕虫靶蛋白甚至大小和 功能抗体库的复杂性。我们对免疫球蛋白E抗体靶向的大部分知识 蠕虫感染和/或过敏原来自使用多克隆血清的研究或从 鼠源性单抗(MAbs)。我们假设人类抗蠕虫IgE抗体 反应是复杂的，但由针对蠕虫蛋白的B细胞克隆组成 已知变应原蛋白的同源物，并能够介导关键效应细胞 在体外发挥作用。 在这里，我们使用了我的实验室新创建的人类B细胞杂交瘤方法，使生长不朽 有史以来第一次产生记忆B细胞，自然产生全长的人类IgE单抗。 患者临床信息被用来选择包含针对重要细胞的样本 蠕虫病原体。在这项提案中描述的初步研究中，我们选择了三种蠕虫 感染者，一名患有钩端螺旋体病，两名患有丝虫病。免疫球蛋白E编码的频率 B细胞被发现大约是每百万个单核细胞中有3个，在 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 以极大地扩展这项工作，制备数百株IgE单抗。由于资金限制，我们最初 对6个班氏柳杉和3个班克罗夫提IgE的小小组进行了特征研究 粗面类圆线虫IgE单抗。纯化的IgE单抗也被发现具有不同的反应性 线虫裂解物在ELISA法和Western印迹法中均有表达。通过鉴定特定的蠕虫蛋白靶标 免疫亲和层析/质谱仪，属于已知的过敏原蛋白家族。 使用Phadia诊断技术进行粗变应原蛋白交叉反应研究 在某些情况下，发现是阳性的，这表明过敏原可能类似蠕虫蛋白。 免疫球蛋白E序列分析显示，体细胞发生了显著程度的超突变。初始泛函 研究表明，每一种IgE蠕虫特异性单抗都能产生剂量依赖性的肥大细胞介质。 在裂解物存在的情况下释放-功能效力研究正在进行中。最终的蠕虫- 将组装特定的IgE单抗面板，以反映免疫优势蠕虫蛋白的层次结构 靶点和效应器细胞功能。除了提高我们对这一问题的基本认识之外 研究人类免疫和过敏致敏的分支，通过研究获得的信息 这项提案中概述的将允许设计和开发新的蠕虫疫苗。

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.