基于DDA/TDB阳离子脂质体为佐剂的新型流感疫苗构建及经鼻粘膜免疫效应与机制研究

Influenza may cause serious fatal diseases and constitute a grave threat to human healthy. Vaccination is still one of the effective means which can prevent the influenza virus. With the critical roles that adiuvant and vaccine delivery system play in adaptive immunity, new strategies are being developed to improve adjuvant and route of influenza split vaccine immunization. At present, cationic liposomes are particularly advantageous as adjuvants due to their ability to enhance the uptake of the vaccine by antigen presenting cells (APC) which include the more potent dendritic cells (DCs) and to induce APC activation. DDA/TDB has previously been reported as an effective adjuvant for eliciting cell-mediated and humoral responses. It is a novel candidate for adjuvant of influenza split vaccine. In the previous study, liposome were prepared by film evaporation, then mixed with influenza split vaccine.in addition,the vesicle size, Zeta potential and encapsulated efficiency of DDA/TDB liposomal adjuvant for influenza vaccine were investigated. Quantum dots (QDs) fluorescent nanoprobes were prepared, which can detect DCs migration following vaccine immunization. Therefore, we wondered whether DDA/TDB could also be used effectively as influenza vaccine adjuvants. To address this question, DDA/TDB combined with influenza vaccine (CLIV) was prepared by film hydration method. The properties of CLIV will be investgated, including particles size, Zeta potential, stability and so on. We also investigated the ability of CLIV to elicit humoral and cellular immune responses, delivered by the intranasal route. Furthermore, QD was loaded into hydrophobic membrane of CLIV. Experiments were conducted to identify DCs responsible for antigen uptake. We used QD Fluorescent nanoprobes to follow the migration of DCs from local tissue to draining lymph by the intranasal route. The achievements in research will be provided some valuable reference for further study on liposomal influenza vaccine, and the foundational basis for study on new generation influenza vaccine.

流感传染性强，传播迅速，可引起较高的发病率及死亡率，严重威胁人类健康，接种疫苗仍是现阶段防治流感最为有效的措施之一。免疫佐剂与有效的传递系统在疫苗免疫应答中发挥着至关重要的作用，而现有疫苗佐剂及免疫途径限制了流感疫苗免疫效应。已有研究结果显示，DDA/TDB阳离子复合物脂质体作为一种新型佐剂具有较大的研究潜力和应用价值，这为新型流感疫苗研究提供了思路。本课题在前期制备量子点（QD）荧光探针和阳离子脂质体流感疫苗的工作基础上，首先，拟构建DDA/TDB阳离子复合物脂质体新型流感疫苗传递系统，探讨其形成条件、理化特性及经鼻粘膜免疫后的体液与细胞免疫效应；其次，结合QD纳米荧光标记及示踪技术，用QD标记该脂质体疫苗传递系统，探讨其体外树突状细胞(DC)抗原摄取作用,进而动态示踪DC摄取抗原后在体内淋巴结迁移情况，为疫苗免疫效应及作用机理研究提供有价值的数据资料，为新型疫苗研制提供新思路与新方法。

流感传染性强，可引起较高的发病率及死亡率，严重威胁人类健康，接种疫苗仍是现阶段防治流感最有效的措施。目前我国使用的流感病毒灭活疫苗存在免疫原性较弱、接种途径单一，接种剂量大，副反应多等缺点，因此研究新型疫苗佐剂及有效的传递系统具有重要意义。DDA/TDB阳离子复合物脂质体作为一种新型佐剂，其效果不断得到证明。此外，研究报道TPGS修饰的脂质体可增加其在细胞的摄取从而增强疫苗诱导的免疫效力。本研究构建了DDA/TDB阳离子脂质体流感疫苗，且对TPGS修饰的DDA阳离子脂质体复合物进行了相关研究，为新型流感疫苗的开发提供了新的思路。.基于以上科学假设，本研究制备了DDA/TDB阳离子脂质体及TPGS修饰的DDA阳离子复合物脂质体流感疫苗，透射电镜观察脂质体均呈圆球形或椭球形。DDA/TDB阳离子脂质体流感疫苗体外摄取实验结果显示，树突细胞（DCs）对DDA-TDB脂质体摄取能力明显增强，与中性脂质体组和其他阳离子组相比，有极显著性差异。DCs细胞成熟实验结果显示，DDA-TDB脂质体能显著上调DCs表面共刺激分子CD80、CD86及MHC-II的表达，表明其能有效促进DCs的分化成熟。动物实验结果显示，经鼻黏膜免疫后，DDA-TDB脂质体流感疫苗组能显著提高黏膜免疫、体液免疫及细胞免疫应答，是较有潜力的新型疫苗佐剂。而的TPGS修饰的DDA阳离子脂质体其摄取率低于对照组，并未达到预期效果。.研究采用薄膜分散法制备了包封率较高、稳定性较好的DDA-TDB阳离子脂质体流感疫苗，通过体外的BMDCs摄取及刺激成熟实验发现该阳离子脂质体流感疫苗可被BMDCs有效摄取并促进树突细胞分化成熟，有利于DCs将抗原呈递给B细胞和T细胞的活化，从而诱导较强的体液免疫和细胞免疫应答。阳离子脂质体流感疫苗经鼻黏膜免疫后，显著提高了黏液sIgA和血清各抗体滴度，表明DDA-TDB脂质体流感疫苗经鼻黏膜免疫可同时诱导黏膜免疫、体液免疫及细胞免疫应答效应，为新型流感疫苗的开发提供了实验依据。