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。食物过敏反应影响大约8%的儿童和3%的成人。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。我们之前的工作已经明确地确定了卵清蛋白（OA）和卵黏液样蛋白（OVM）是主要的蛋清过敏原。此外，我们已经完成了详细的b细胞和t细胞抗原表位定位，并确定了影响IgE结合活性的关键氨基酸。这一信息将使我们能够设计用于免疫治疗的特异性低过敏原。假设是，专门设计的重组OA和OVM过敏原或肽缺乏ige表位（低致敏性），但具有良好的t细胞抗原性（保留t细胞表位），可以诱导对鸡蛋过敏原的耐受性，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细胞对抗原产生耐受性或能量。过敏原的修饰将通过两种不同的方法进行，1)含有OA或OVM MHC ii类限制性t细胞表位的重组杂交肽与大肠杆菌表达系统，2)位点定向突变改变显性b细胞表位结构或蛋白质的三级结构。采用实验诱导的过敏小鼠（BALB/c），通过腹腔注射抗原来评价鸡蛋过敏的低致敏性和治疗性耐受。Th1/Th2和t调节细胞相关细胞因子（ifn - γ、IL-4、IL-10、IL-12和tgf - 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）进行分析。本课题的意义在于：1)指导我们开发低过敏原或多肽免疫疗法治疗食物过敏性疾病；2)更好地了解重组低过敏原诱导的免疫耐受机制；3)促进培养食品生物技术/免疫学/食品安全领域的高素质人才。

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.