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

描述(申请人提供)：在工业化世界中，对食物过敏是一个世界性的问题，其流行率似乎正在增加，影响到大约1%的美国人口。免疫球蛋白介导的食物反应，特别是花生，是发生在医院外的过敏反应的最常见原因。避免食用花生，这是推荐的方法，由于经常意外摄入，这是不够的。有许多新兴的实验疗法，但这些都有局限性。虽然我们对IgE如何与花生变应原结合有很多了解，但对于这些变应原是如何将IgE/IgE受体复合体(IgE/FceRI)交联以激活肥大细胞和嗜碱性粒细胞却知之甚少。这一建议将利用我们小组的三个重要发现：1)相关和互补的2S白蛋白Ara h 2和Ara h 6是最有效的花生过敏原，2)IgE与两个特定的线性表位(每个过敏原上一个)的结合与更严重的临床病史有关，3)在毕赤酵母中表达的重组Ara h 6具有完整的效应活性，这使该模型成为了解效应功能的分子基础的可靠模型。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与特定的线性表位arah2和arah6的结合，临床反应性的自然历史，患者对临床指示的花生烯的反应，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个临床设置中定义与Ara h2和Ara h 6的多肽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，这些氨基酸是IgE/Fc？RI有效交联的关键。最后，我们将开发兔多克隆抗体、小鼠单抗和人源ScFv片段作为潜在的Arah2和Arah6介导的IgE/Fc？RI交联物的抑制剂。这些也将是有用的新型诊断试剂。通过完成这些特定的目标，我们将克服理解过敏原如何与IgE相互作用以使IgE/FceRI复合体交联的重大障碍，并将推动该领域向前发展，为花生过敏的评估和治疗开发新的诊断和治疗工具。本研究的未来方向是充分开发这些工具并将其应用于临床。

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.