具有温度/pH双重响应和甘露糖受体靶向功能的微凝胶疫苗

Vaccines are considered to be one of the most effective modalities for preventing major infectious diseases and improving cancer therapy. But insufficient effectiveness of some inactivated vaccines or sub-unit vaccines and the weak antigen presenting cells (APCs) targeting ability make the repeat injections necessary, which usually result in a high cost. Therefore, we aim to prepare inactivated mink enteritis virus (MEV) loaded multifunctional microgels, which would carry high percentage of vaccines, smartly target APCs and release antigen in controlled manner. The microgels are obtained from surfactant free emulsion polymerization with N-vinylcaprolactam (N-VCL) and vinyl mannose as the monomers respectively and acid-sensitive DMAEP as the crosslinker. In order to effectively pass through the barriers of cell membrane and prevent protein adsorption (such as serum albumin), further modification with DSPE-PEG-Tat will be conducted on the microgels. The thermo-responsive ability is expected to make the controlled and sustainable release of vaccines in blood available. On the other hand, mannose groups on the microgels bind with mannose receptor (MR) on APCs, facilitating the accumulation of vaccines on APCs. With the help of Tat, the microgels are able to reach the target site more easily. After pH responsive degradation in endosome, more antigens are supposed to release from the microgels and activate fixed immunity and adaptive immunity. It is possible to optimize experimental procedures to obtain microgels with uniform sizes, desirable distribution and high stability. They are of great potential to be used as delivery system for more vaccines and create more economical benefits.

针对现有灭活或亚单位疫苗免疫效果不理想的问题，申请人拟分别以N-乙烯基己内胺(N-VCL）和乙烯基化的甘露糖作为共聚单体、以酸敏感的缩酮DMAEP作为交联剂，通过温和的无皂化乳液聚合一锅制备温度/pH双响应智能微凝胶疫苗，实现灭活疫苗（如水貂肠炎细小病毒）的高含量包裹、保持疫苗高效活性及靶向抗原呈递细胞（APCs）的功能。利用磷脂-聚乙二醇-穿膜肽（DSPE-PEG-Tat）在微凝胶表面进一步修饰，延长体内循环时间。该微凝胶疫苗一部分发生温度响应性的抗原缓慢释放，延长疫苗作用时间；另一部分通过甘露糖与甘露糖受体（MR）的结合实现APCs的靶向和富集，在Tat的协助下易于到达细胞内有效部位，随后在内含体较低的pH值下发生响应性降解并释放抗原，激活固有免疫和适应性免疫系统，放大免疫效应。通过实验条件的优化有望获得分散性良好、稳定性高的普适性疫苗担载担载体系，创造一定的经济价值。

疫苗的出现极大的推动了人类健康的发展及社会的进步。传统的减毒疫苗和灭活疫苗，易导致毒性增加、过度炎症、剧烈疼痛等问题。新型疫苗依据其抗原不同主要分为核酸类疫苗或蛋白质类疫苗，其中对于蛋白亚单位疫苗的研究多聚焦于抗原或佐剂的设计，而对疫苗在体内生效的过程却少有探究。疫苗在体内发挥免疫保护能力之前需经过固有免疫和适应性免疫的过程，这两个过程中均存在多种免疫细胞的参与，抗原呈递细胞(APCs)作为沟通固有免疫和适应性免疫的桥梁，在促进机体产生免疫记忆的过程中具有很重要的作用。基于此，本项目利用甘露糖能够靶向结合APCs表面的甘露糖受体的性质，设计并制备了一种甘露糖修饰的微凝胶用于包裹蛋白质抗原，实现对亚单位疫苗的高效递送，提高亚单位疫苗的免疫效果。. 本项目利用微凝胶载体本身三维交联的网状结构，实现了所包裹抗原缓慢而持续的释放，保障抗原的持续刺激，增强免疫响应。选取了甘露糖的同分异构体葡萄糖作为对照，以此评估出抗原呈递细胞表面的甘露糖受体对两种亚单位疫苗的摄取效率的影响。通过内吞实验、甘露糖受体封闭实验以及腹股沟淋巴结的淋巴细胞募集实验，证明了该甘露糖修饰微凝胶确实是通过甘露糖受体与配体的识别来实现对亚单位疫苗摄取效率的提高。通过活体荧光成像实验和注射部位的H&E染色进一步验证了微凝胶能够延长抗原在体内的滞留时间并且能够长时间的募集免疫细胞。脾脏淋巴细胞增殖活力和增殖分析证明，制备的微凝胶能够有效的诱导机体产生免疫记忆，实验组中脾脏淋巴细胞的增殖活力和增殖比例最高。特异性抗体、细胞因子和脾脏淋巴细胞亚群分析则证明MMG-OVA能够有效的诱导机体产生适应性免疫，具有免疫保护能力。以上细胞和动物实验充分证实了甘露糖修饰的微凝胶体系能够靶向APCs表面的甘露糖受体、促进细胞内吞、提高亚单位疫苗疗效，显示了在亚单位疫苗递送领域的优势和潜在应用前景。