具有双重免疫增强特性的Pickering乳液构建及其调控机制研究

Traditional emulsion adjuvant systems, which was elicited to induce potent humoral immunity responses, have been employed in various commercial vaccine formulations. However, insufficient enhancement on cellular immunity hinders them from combatting against pandemic intracellular infectious diseases, such as AIDS, tuberculosis, hepatitis, seasonal influenza and etc. Moreover, surfactant, which is the key component of emulsion, was proved to harbor the hazard of hemolysis and agitating the stability of vaccine formulations after long-term co-storage with antigens. To grabble with this formidable obstacle, biodegradable polylactide-based micro/nano-particles stabilized emulsion is proposed in this proposal, which will guarantee the synergy between emulsion adjuvant (humoral immunity potentiator) and micro/nano-particles (which is proved to possess the potential in intensifying the cellular immune responses) to simultaneously induce both humoral and cellular immune responses. In addition, properties of micro/nano-particles (hydrophilicity, charges, sizes and etc.) will be optimized to obtain surfactant-free and stable emulsion (Pickering emulsion), which will not only improve the safety profile and stability of the adjuvant, but also enables the versatility of the emulsion, in order to be harnessed into various applications with different kinds of antigens and administration routes. Furthermore, Shirasu porous membrane emulsification technique will also be introduced to this systems, in order to produce micro/nano-particles and Pickering emulsion with uniform and controllable sizes, which may secure the satisfactory repeatability and stability from patch to patch, and shed light on the exploration of the mechanism and structure-activity relationship of this novel adjuvant.

油乳佐剂由于其优异的抗体水平提升效果，已在多种商品化疫苗制剂中应用。然而，常用油乳佐剂难以有效诱导细胞免疫应答，导致对于胞内感染抗原，油乳佐剂无法发挥有效的免疫增强作用。同时，油乳佐剂中所含表面活性剂具有一定的毒性及溶血作用，并易导致膜糖蛋白发生变性，影响制剂稳定性。为此，本研究拟构建由可生物降解的聚乳酸类纳微颗粒稳定的Pickering油乳佐剂，利用颗粒佐剂的细胞免疫增强优势，结合油乳佐剂在诱导体液免疫方面的特性，获得具有双重免疫增强特性的新型油乳佐剂。并通过调节颗粒性质（亲疏水性、粒径、表面电荷等），使颗粒稳定吸附在油水界面上，得到不含表面活性剂的稳定乳液（Pickering乳液），提高佐剂的生物安全性。此外，本研究还将引入快速膜乳化技术到Pickering乳液的制备，获得粒径均一的乳滴，提高乳液稳定性，并为乳液性质的可控调节、构效关系及作用机制的系统研究提供可能。

针对现有油乳佐剂难以有效诱导细胞免疫应答，并且油乳佐剂中所含表面活性剂具有一定的毒性及溶血作用等问题，本研究构建了由可生物降解的聚乳酸类纳微颗粒稳定的Pickering油乳佐剂。将所制备的Pickering乳液用于OVA、H1N1流感抗原以及MUC1抗肿瘤短肽疫苗制剂，通过细胞实验与动物实验，发现Pickering乳液可以有效激活体液及细胞免疫应答。在预防性疫苗中，Pickering乳液组可以实现实验各组中最高的攻毒保护率；在肿瘤治疗性疫苗中，Pickering乳液组可以显著杀伤肿瘤，抑制肿瘤生长，并延长荷瘤小鼠的生存期。此外，还从抗原储库、细胞摄取、细胞活化等多个方面对Pickering乳液的免疫作用机制进行了表征分析。本项目共发表SCI论文7篇（包括Nature Materials, Advanced Materials, ACS Nano, Small等国际顶级期刊），申请3项发明专利（含1项欧洲发明专利），获4项发明专利授权（含1项日本发明专利），培养博士生3名，硕士生2名。