Antigenic determinants of asthma-associated allergens for design of immunotherapy

项目摘要/摘要：在美国，对蟑螂和尘螨的过敏反应是重要的健康问题，影响着市中心地区高达83%的哮喘儿童，也是哮喘患者进入急诊室的一个危险因素。美国的主要蟑螂种德国小蠊（Blattella germanica）产生Bla g 2，其致敏率比猫和螨的致敏率低10-100倍，在蟑螂致敏率中最高（40-70%）。Der p1和Der f1分别是尘螨和尘螨产生的半胱氨酸蛋白酶。螨虫过敏患者对1组过敏原的IgE致敏率为80%。Der p1的蛋白水解活性可能会增加IgE抗体的产生，并有助于哮喘的肺部炎症，但Bla g2是一种无活性的天冬氨酸蛋白酶同源物。本项目的主要目的是研究与哮喘相关的蛋白水解（螨虫组1）和非蛋白水解（Bla g 2）过敏原的抗原结构。将过敏原与单克隆抗体共结晶，鉴定抗体（IgE和mAb）结合的关键氨基酸。另外，IgE抗体结合表位的鉴定将通过噬菌体展示技术进行。具体目标是：1)通过晶体学绘制blag2中的抗原决定因子；2)利用晶体学方法定位1群螨过敏原抗原决定因子，并分析Der p1与Der f1交叉反应的结构基础；3)通过定点诱变研究定位抗体结合表位的关键氨基酸，并鉴定具有T细胞反应性的低过敏性突变体，可用于免疫治疗。IgE抗体与表位突变体的结合将通过ELISA、多重阵列技术和细胞介质释放试验进行分析。将突变体与选择低致敏形式进行比较，以设计用于螨虫和蟑螂过敏免疫治疗的疫苗。公共卫生相关性：蟑螂和螨虫过敏与哮喘的发展有关，在美国有1700万人受到哮喘的影响。Bla g 2和1组螨虫过敏原的抗原决定因素将被确定，以阐明这些过敏原内在特性对过敏性疾病的重要性。低致敏性突变体将被生产出来，并测试IgE抗体结合和T细胞增殖，获得的信息将有助于合理设计过敏疫苗。

DESCRIPTION (provided by applicant): Antigenic determinants of asthma-associated allergens for design of immunotherapy Project Summary/Abstract Allergic reactions to cockroach and dust mite are important health problems in the U.S. affecting up to 83% of asthmatic children in inner city areas and are a risk factor for emergency room admission with asthma. The main cockroach species in the U.S., Blattella germanica, produces Bla g 2 which induces sensitization at exposure levels 10-100 times lower than cat and mite allergens, and has the highest prevalence of sensitization among cockroach allergens (40-70%). Der p 1 and Der f 1 are cysteine proteases produced by the dust mites Dermatophagoides pteronyssinus and D. farinae, respectively. IgE sensitization to these Group 1 allergens is >80% among mite allergic patients. Proteolytic activity of Der p 1 may enhance IgE antibody production and contribute to lung inflammation in asthma but Bla g 2 is an inactive aspartic protease- homolog. The main goal of this project is to investigate the antigenic structure of proteolytic (mite Group 1) and non-proteolytic (Bla g 2) allergens associated with asthma. Allergens will be co-crystallized with monoclonal antibodies, and the key amino acids involved in antibody (IgE and mAb) binding will be identified. Alternatively, identification of IgE antibody binding epitopes will be performed by phage display technology. The specific aims are: 1) mapping of antigenic determinants in Bla g 2 by crystallography; 2) mapping of antigenic determinants of Group 1 mite allergens by crystallography and analysis of the structural basis for the cross-reactivity between Der p 1 and Der f 1; and 3) localization of the key amino acids involved in the antibody binding epitopes by site-directed mutagenesis studies and identification of hypoallergenic mutants with T cell reactivity that could be used for immunotherapy. IgE antibody binding to the epitope mutants will be analyzed by ELISA, multiplex array technology and cell mediator release assays. Mutants will be compared to select hypoallergenic forms for the design of vaccines for immunotherapy of mite and cockroach allergy. PUBLIC HEALTH RELEVANCE: Cockroach and mite allergy is associated with the development of asthma, which affects 17 million people in the U.S. The antigenic determinants of Bla g 2 and Group 1 mite allergens will be identified to elucidate the importance of the intrinsic properties of these allergens on allergic disease. Hypoallergenic mutants will be produced and tested for IgE antibody binding and T cell proliferation, and the information obtained will facilitate a rational design of allergy vaccines.