Engineered hypo-allergens for specific immunotherapy for food allergy

食物过敏是儿童和成人的主要健康问题。大约8%的儿童和3%的成年人会受到食物过敏反应的影响。蛋清是最常见的过敏反应来源之一，主要发生在儿童身上。大约三分之二被诊断为食物过敏的儿童对蛋清有反应。我们以前的工作已经明确地确定卵清蛋白(OA)和类卵粘蛋白(OVM)是主要的蛋清过敏原。此外，我们已经完成了两种过敏原的详细B细胞和T细胞表位图，并确定了影响IgE结合活性的关键氨基酸。这些信息将使我们能够为免疫治疗设计特定的低过敏原。我们的假设是，设计的缺乏IgE表位(变态反应性低)，但具有良好的T细胞抗原性(保留T细胞表位)的重组OA和OVM变应原或多肽可以诱导对鸡蛋变应原的耐受，这是预防和治疗鸡蛋过敏的一种有吸引力的解决方案。这可能导致特异性T细胞对抗原的耐受性或无效性。变应原的修饰将通过两种不同的方法进行，1)含有OVMMHC II类限制性T细胞表位与大肠杆菌表达系统的重组杂交肽，2)改变蛋白质的优势B细胞表位结构或三级结构的定点突变。鸡蛋过敏的减敏和治疗耐受性将利用实验诱导的过敏小鼠(BALB/c)通过腹腔注射抗原的方法进行评估。Th1/Th2和T调节细胞相关细胞因子(干扰素-γ、IL-4、IL-10、IL-12和转化生长因子-β)、特异性抗体(IgE、IgG1、IgG2a和IgG4)和组胺水平的分析将通过酶联免疫吸附试验(ELISA)(脾细胞和血清)或实时RT-PCR技术(粘膜免疫)进行。该项目的意义是：1)指导我们开发针对食品过敏性疾病的低过敏原或基于多肽的免疫疗法；2)更好地了解重组低过敏原诱导免疫抵抗的机制；3)促进食品生物技术/免疫学/食品安全领域高素质人才的培训。

Food allergy is a major health concern in both children and adults. Food-hypersensitivity reactions affect about 8% of children and 3 % of adults. Hen egg white is one of the most common sources of allergic reactions, mainly in children. Approximately, two-thirds of children diagnosed with food allergies are reactive to egg white. Our previous work has been clearly identified ovalbumin (OA) and ovomucoid (OVM) as major egg white allergens. Furthermore, we have completed detailed B-cell and T-cell epitope mapping of both allergens and identified critical amino acids that affect IgE binding activity. This information will enable us to design specific hypo-allergens for immunotherapy. The hypothesis is that specifically designed recombinant OA and OVM allergens or peptides that lack of IgE-epitopes (low allergenicity), but show good T-cell antigenecity (retain T-cell epitopes) could induce tolerance to egg allergens which represent an attractive solution to prevent and treat egg allergy. This could result in specific T-cell tolerance or anergy against the antigens. Modification of allergens will be performed by two different approaches, 1) recombinant hybrid peptides comprising OA or OVM MHC class II-restricted T-cell epitopes with E. coli expression system, and 2) site-directed mutagenesis altering dominant B-cell epitope structure or tertiary structure of the proteins. Hypo-sensitization and therapeutic tolerance of egg allergy will be evaluated using experimentally-induced allergy mice (BALB/c) by means of intraperitoneal of antigens. Analysis of Th1/Th2 and T-regulatory cells-associated cytokines (IFN-gamma, IL-4, IL-10, IL-12 and TGF-beta), specific antibodies (IgE, IgG1, IgG2a and IgG4) and histamine levels will be analyzed by enzyme-linked immunosorbent assay (ELISA) (splenocytes and sera) or real-time RT-PCR technique (mucosal immunity). The significance of the proposed project are 1) to direct us to develop hypo-food allergens or peptide-based immunotherapy for food allergic diseases, 2) better understanding mechanism of recombinant hypoallergen-induced immunotorelance, 3) to facilitate training of highly qualified personnel in food biotechnology/immunology/food safety area.