Supplementary Material for: Alteration of Allergen Fold of Bos d 5 into a Hypoallergenic Vaccine for Immunotherapy of Cow’s Milk Allergy

Background: Cow’s milk allergy (CMA) is the most common IgE-mediated food allergy and Bos d 5 is the major allergen in cow’s milk proteins. More than 60% of the patients with CMA are sensitized to this protein. Methods and Results: A recombinant protein, encoded by a synthetic gene and consisting of reassembled Bos d 5 fragments, was expressed in E. coli strain BL21 (DE3) cells and purified to homogeneity. The B5M lacked relevant IgE-reactivity and allergenic activity compared with Bos d 5 in dot-blot and basophil activation assays. T-cell proliferation experiments demonstrated that B5M preserved the main T cell epitopes of Bos d 5. Immunization of rabbits with B5M induced protective IgG antibodies that blocked the binding of patients’ IgE antibodies to the wild-type allergen and inhibited the degranulation of basophils induced by Bos d 5. Conclusion: Thus, we developed a new strategy, which was based on rational molecular reassembly for allergen-specific immunotherapy (AIT) of CMA and food allergy.背景：牛奶过敏（Cow’s milk allergy, CMA）是最为常见的IgE介导型食物过敏，主要变应原（Bos d 5）是牛乳蛋白中的核心变应原。超过60%的CMA患者对该蛋白致敏。方法与结果：本研究制备了由合成基因编码、重组装配的Bos d 5片段构成的重组蛋白，该蛋白在大肠杆菌（E. coli）BL21(DE3)菌株细胞中实现表达，并经纯化达到均一性。斑点印迹（dot-blot）与嗜碱性粒细胞活化实验结果显示，相较于Bos d 5，B5M不具备相应的IgE结合活性与变应原性。T细胞增殖实验证实，B5M保留了Bos d 5的主要T细胞表位。用B5M免疫家兔后，可诱导产生保护性IgG抗体，该抗体能够阻断患者IgE抗体与野生型变应原的结合，并抑制Bos d 5诱导的嗜碱性粒细胞脱颗粒。结论：综上，本研究开发了一种基于理性分子重组的新策略，可用于CMA与食物过敏的变应原特异性免疫治疗（allergen-specific immunotherapy, AIT）。