一种具有肥大细胞凋亡作用的新型尘螨变应原疫苗及其作用机制的研究

Allergic diseases, which are induced by exposure to allergen, have become epidemic diseases in the new century. In China, dust mite is considered to be the most common allergen. Until now a long-term cure for allergic diseases can only be achieved through the use of allergen-specific immunotherapy (AIT), which has a disease-modifying effect. However, the efficacy of AIT is limited because of long duration of treatment required and low patient adherence. At present, the allergen vaccines used in clinical are mainly made up of crude extract with complicated ingredient, which may induce side effects of local and system allergic reactions mediated by IgE. It is well known that mast cell is one of the major effector cells in allergy. Thus vaccines, which have an additional effect of inducing allergen specific mast cell apoptosis, may shorten treatment period, and improve the safety and efficacy of AIT. Based on the previously successful construction of MC specific apoptotic agent “Chinese medicine loaded monoclonal antibody conjugated nanoparticles”, this project is to generate a new vaccine with mast cell apoptosis effect by constructing a nanoparticle encapsulated with celastrol and coupled with Group II allergens from Dermatophagoides farinae (Der f II) on its surface. The vaccination scheme, including intervals, doses and treatment course, is explored in a mouse model of allergic asthma sensitized and challenged with Der f2. It is expected to improve the safety and efficacy by several mechanism: ① specifically bind to mast cells sensitized with dust mite specific IgE by Der f2 on the surface of nanoparticles, be internalized and induce apoptosis of the mast cells by celastrol; ② induce apoptosis of memory B cell which recognize Der f2, and reduce IgE production; ③ induced the generation of regulatory T or B cells and protective antibodies. This research may also provide innovative ideas and technical platform for AIT.

过敏性疾病已成为新世纪流行病，尘螨是我国最常见的过敏原。过敏原特异性免疫治疗（AIT）是目前唯一针对病因、长期有效的手段。但是AIT疗程长，病人难以坚持，疗效不佳。临床使用的尘螨疫苗多为粗浸液，成分复杂，存在诱发严重过敏反应的风险。肥大细胞（MC）是过敏反应主要效应细胞，具有过敏原特异性MC凋亡诱导作用的疫苗可缩短AIT疗程、提高安全性和有效性。在前期构建成功纳米包载中药偶联靶向抗体的基础上，本课题拟制备“包载雷公藤红素偶联粉尘螨过敏原（Der f2）纳米粒”作为疫苗，探索尘螨过敏性哮喘小鼠模型免疫接种的最佳方案（时间、计量、疗程），一方面，通过携带雷公藤的纳米粒由Der f2导向尘螨特异性IgE致敏的MC，诱导其凋亡，消除致敏的MC；另一方面，诱导记忆性B细胞凋亡，减少IgE产生；以及诱导调节性T细胞或促进B细胞产生保护性抗体等机制实现安全有效治疗过敏。也为AIT提供创新思路和技术平台。

过敏原特异性免疫治疗（AIT）是目前唯一针对病因、长期有效的过敏性疾病治疗手段。.本研究以羧基修饰的Pluronic P123嵌段共聚物所形成的聚合物胶束作为载体，包载雷公藤红素，偶联模型过敏原卵清蛋白（OVA），构建了包载雷公藤红素偶联OVA的纳米疫苗（OVA-NMs-celastrol），并评价了其对小鼠过敏性哮喘的治疗效果。此外，本研究还以中空介孔氧化硅纳米材料（HMSNs）作为载体，包载粉尘螨过敏原II类组分（Der f2），构建了包载Der f2的纳米疫苗（Der f2-HMSNs），评价了以HMSNs为佐剂/载体的疫苗在过敏性疾病特异性免疫治疗中的作用效果。.研究结果显示，所构建的纳米疫苗OVA-NMs-celastrol为球形、粒径较小（50nm左右）。在体内，该疫苗能有效减少过敏小鼠血清OVA特异性IgE和肺泡灌洗液（BALF）中组胺的水平、降低BALF中Th2细胞因子IL-4和IL-5浓度、减少肺组织炎症细胞浸润，改善过敏性炎症反应。此外，构建的Der f2-HMSNs疫苗也具有一定的改善过敏性炎症的效果。.本项目的研究结果表明：① OVA-NMs-celastrol是一种具有潜力的治疗过敏性疾病的疫苗，后续研究还需要改进其制备技术（去除未偶联的过敏原）、丰富表征结果（明确蛋白交联率）、摸索雷公藤红素的剂量等，以提高疫苗的安全性和疗效，明确其作用机理。② HMSNs作为疫苗的佐剂/载体在特异性免疫治疗中具有一定的应用前景，后续需改善其表征（包括孔径大小）、偶联靶分子实现对树突状细胞的靶向性，以提高其作为疫苗佐剂的效果。