Antigenic determinants of asthma-associated allergens for design of immunotherapy.

 描述(由申请人提供)：对屋尘螨的过敏反应是世界范围内一个重要的健康问题，影响高达85%的哮喘儿童，也是哮喘急诊室入院的危险因素。在尘螨过敏患者中，第1组和第2组尘螨变应原占全部屋尘螨特异性IgE反应的50%以上。粉尘变应原Der p 1和Der f 1分别是由尘螨屋尘螨和粉尘螨产生的半胱氨酸蛋白酶。Der-p1的蛋白分解活性可能促进了IgE抗体的产生，促进了哮喘患者的肺部炎症。相反，第二类变应原是脂多糖结合蛋白。据报道，DER P 2模拟了人类的结构同系物，通过Toll样受体激活天然免疫系统。尽管蛋白水解组1和非蛋白水解组2之间存在这些重要的分子差异，但在美国的尘螨过敏患者中，对这两组过敏原的IgE患病率高达83%。本项目的主要目的是研究两组尘螨变应原的抗原结构，为免疫治疗的设计提供依据。过敏原将与通过噬菌体展示技术选择的IgE抗体构建物共结晶。参与IgE抗体结合的关键氨基酸将被识别和修饰。其具体目的是：1)利用噬菌体展示技术从尘螨过敏患者的血液组合文库中筛选出IgE抗体结构物(ScFv)；2)通过X射线结晶学定位1和2组尘螨变应原上的抗原决定簇并分析IgE抗体结合表位；3)定点突变IgE抗体表位，以表达具有T细胞反应性的低变应突变体作为免疫治疗候选。将利用在前两个目标中获得的信息来分析来自人类的尘螨变应原特异性IgE抗体与这些IgE抗体所识别的表位之间的关联。目的#2将生成B细胞表位的三维结构的实验数据集，这些数据集在当前用于开发B细胞表位预测工具的数据库中缺失。最重要的是，该项目将确定IgE抗体对尘螨变应原的反应，并将为合理设计低变应原提供结构基础。在AIM#3中，将通过ELISA、多重阵列技术和细胞介体释放试验来分析与表位突变相结合的IgE抗体，并将评估T细胞的反应性。将对变异体进行比较，并选择低过敏性形式来设计用于螨类过敏免疫治疗的疫苗。

 DESCRIPTION (provided by applicant): Allergic reactions to house dust mite are an important health problem worldwide, affecting up to 85% of asthmatic children, and are a risk factor for emergency room admission with asthma. Group 1 and 2 mite allergens account for more than 50% of total house dust mite specific IgE reactivity in mite allergic patients. Mite allergens Der p 1 and Der f 1 are cysteine proteases produced by the dust mites Dermatophagoides pteronyssinus and D. farinae, respectively. Proteolytic activity of Der p 1 may enhance IgE antibody production and contribute to lung inflammation in asthma. In contrast, group 2 mite allergens are lipopolysaccharide binding proteins. Der p 2 has been reported to mimic a human structural-homolog that activates the innate immune system through toll like receptors. Despite these important molecular differences between proteolytic group 1 and non-proteolytic group 2, a high IgE prevalence of 83% to allergens from both groups has been observed in mite allergic patients in the US. The main goal of this project is to investigate the antigenic structure of both groups of mite allergens, for the design of immunotherapy. Allergens will be co-crystallized with IgE antibody constructs selected by phage display technology. The key amino acids involved in IgE antibody binding will be identified and modified. The specific aims are: 1) selection of IgE antibody constructs (scFv) from combinatorial libraries made from blood of mite allergic patients using phage display technology; 2) mapping of antigenic determinants on groups 1 and 2 dust mite allergens by X-ray crystallography and analysis of IgE antibody binding epitopes; and 3) site-directed mutagenesis of IgE antibody epitopes for expression of hypoallergenic mutants with T cell reactivity as candidates for immunotherapy. An analysis of the association between mite allergen-specific IgE antibodies from the human repertoire and the epitopes recognized by these IgE antibodies will be performed with the information obtained in the first two aims. Aim #2 will generate experimental data sets of three-dimensional structures of B-cell epitopes that are missing in current databases used for developing tools for B cell epitope prediction. Most importantly, this project will define IgE antibody responses to mite allergens and will provide the structural basis for rational design of hypoallergens. In Aim #3, IgE antibody binding to the epitope mutants will be analyzed by ELISA, multiplex array technology and cell mediator release assays, and T cell reactivity will be evaluated. Mutants will be compared and hypoallergenic forms will be selected for the design of vaccines for immunotherapy of mite allergy.