Mapping the Critical Epitopes of Ara h 2 and Ara h 6

描述（由申请人提供）：对食物过敏是工业化世界的一个世界性问题，其患病率似乎正在上升，影响了大约1%的美国人口。ige介导的食物反应，特别是花生，是发生在医院外的过敏反应的最常见原因。避免花生，建议的方法，是不够的，因为经常意外摄入。目前有许多新出现的实验性治疗方法，但它们都有局限性。虽然我们对IgE如何与花生过敏原结合了解很多，但对这些过敏原如何交联IgE/IgE受体复合物（IgE/FceRI）激活肥大细胞和嗜碱性细胞知之甚少。该提案将利用我们小组的三个重要发现：1)相关的和互补的2S白蛋白Ara h 2和Ara h 6是最有效的花生过敏原；2)IgE与两个特定线性表位（每个过敏原上一个）的结合与更严重的临床病史相关；3)重组Ara h 6在毕赤酵母中表达，而不是在大肠杆菌中表达，具有充分的效应活性，使其成为了解效应功能分子基础的强大模型。在拟议的研究中，我们将在三个重要的临床环境中测量患者来源的IgE与Ara h 2和Ara h 6特异性线性表位的结合，临床反应性的自然史，患者对临床指示的花生攻击的反应，以及患者对口服免疫治疗（OIT）的反应。我们将使用这些数据来确定在每种临床环境中与Ara h2和Ara h6肽的明确结合模式的相对风险。然后我们将确定Ara h6和Ara h2的特定氨基酸它们对IgE/Fc的有效交联至关重要？RI。最后，我们将开发兔多克隆抗体、鼠单克隆抗体和人scFv片段作为Ara h 2和Ara h 6介导的IgE/Fc？RI。这些也可作为新型诊断试剂。通过完成这些特定目标，我们将克服理解过敏原如何与IgE相互作用以交联IgE/FceRI复合物的重大障碍，并将推动该领域朝着开发新的诊断和治疗工具的方向发展，以评估和治疗花生过敏。未来的研究方向是充分开发这些工具并将其应用于临床实践。

DESCRIPTION (provided by applicant): Hypersensitivity to foods is a world-wide problem in the industrialized world and its prevalence appears to be increasing, affecting approximately 1% of the US population. IgE-mediated reactions to foods, particularly peanuts, are the most common cause of anaphylaxis occurring outside of the hospital. Avoidance of peanuts, the recommended approach, is inadequate due to frequent accidental ingestion. There are a number of emerging experimental therapies but these all have limitations. Although we understand a great deal about how IgE binds to peanut allergens, little is known about how these allergens cross-link IgE/IgE receptor complexes (IgE/FceRI) to activate mast cells and basophils. This proposal will capitalize on three significant findings from our group: 1) the related and complementary 2S albumins, Ara h 2 and Ara h 6, are the most potent peanut allergens, 2) binding of IgE to two specific linear epitopes (one on each allergen) is associated with more severe clinical histories and 3) recombinant Ara h 6 expressed in Pichia pastoris, but not in Escherichia coli, has full effector activity making this a robust model for understanding th molecular basis of effector function. In the proposed studies we will measure binding of patient-derived IgE to specific linear epitopes of Ara h 2 and Ara h 6 in three important clinical settings natural history of clinical reactivity, response of patients to clinically indicated peanut challenes, and response of patients to oral immunotherapy (OIT). We will use these data to determine the relative risk of having defined binding patterns to peptides of Ara h 2 and Ara h 6 in each of these clinical settings. We will then determine the specific amino acids of Ara h 6 and then Ara h 2 that are critical for effective cross-linking of IgE/Fc?RI. Finally we will develop rabbit polyclnal antibodies, murine monoclonal antibodies, and human scFv fragments as potential inhibitors of Ara h 2 and Ara h 6 - mediated cross-linking of IgE/Fc?RI. These will also be useful as novel diagnostic reagents. By completing these specific aims we will overcome the significant obstacle of understanding how allergens interact with IgE to cross-link IgE/FceRI complexes and will move the field forward towards development of new diagnostic and therapeutic tools for the evaluation and treatment of peanut allergy. The future direction of this research is to fully develop these tools and bring them to clinical practice.