N-2-羟丙基二甲基乙基氯化铵壳聚糖/N,O-羧甲基壳聚糖纳米粒新城疫疫苗的制备及免疫作用机制研究

Using degradable biomaterials to encapsulate vaccine antigens can protect the antigens from degrading by the in vivo acid/base environment and enzymes, improve the efficiency of cross membrane transport and endocytosis of macrophages, fulfil the antigen presentation process, and make the antigen controlled release and targeted delivery possible. Supproted by the fund (31072119) from National Natural Science Foundation of China, we had successfully constructed Newcastle disease virus (NDV) F gene DNA vaccine encapsulated in the N-2-hydroxypropyltimehyl ammonium chloride chitosan/N,O-carboxymethyl chitosan complex nanoparticles, chitosan nanoparticles and Poly (lactic-co-glycolic acid) (PLGA) nanoparticles, attenuated live Newcastle disease vaccine encapsulated in chitosan nanoparticles. The reuslts of mucosal immunization route shown that the constructed nanoparticle vaccines are safe, can induce better celluar, humoral and mucosal immune response, and provide effective immune protection with wild-type viruses challagne. Base on these finished studies, we propose to make the NDV F gene plasmid DNA or NDV La Sota nanopartilces with ourself-synthesised N-2-hydroxypropyldimethyl ammonium chloride chitosan (N-2-HFCC) and N,O-carboxymethyl chitosan (CMC) as delievery carriers by the polyelectrolyte complexation method. Their characteristics including infrared spectra, surface morphology, diameter, particle size distribution, Zeta potential, cytotoxicity, bioactivity and immunological effectiveness in vivo will be investigated. Meantime, the level of immune responses induced by the new nanoparticles vaccine and interactions between the nanoparticles vaccine and intestinal epithelium cells will be investigated. The reults from this proposal will provide a practical solution for the key techniques of enhancing mucosal immune response, controlled release and targeted delivery of antigen, and promote development of new veterinary vaccine products replacement and advance the vaccine industry technology.

利用生物降解材料包裹疫苗抗原，可避免体内酸碱环境和酶类对其破坏，提高跨膜转运及M细胞的胞吞作用，实现黏膜免疫和缓控释及靶向递送。在国家基金资助下，构建了新城疫DNA疫苗N-2-HACC/CMC、壳聚糖和PLGA及新城疫弱毒苗壳聚糖纳米粒黏膜免疫递送系统，该系统安全性好，可刺激机体产生较强的细胞免疫、体液免疫和黏膜免疫。本研究在以前工作基础上，以自行合成的N-2-HFCC和CMC为递送载体、新城疫病毒F基因表达质粒pVAXⅠ-F（o）-C3d6或新城疫病毒株La Sota株病毒液为模型药物，制备新城疫N-2-HFCC/CMC纳米粒疫苗，通过对纳米粒粒径、Zeta电位和包封率及细胞毒性、生物活性和鸡体内外实验的考察，建立疫苗纳米化的安全级黏膜免疫递送系统，实现提高免疫效果、诱导黏膜免疫和缓释目的，并探讨其免疫应答机制。本研究对解决和推动疫苗黏膜免疫和缓控释等关键技术，对促进兽用疫苗产品更新换代具有重要的现实意义。

用可降解生物纳米材料包裹疫苗抗原，保护疫苗抗原完成并高效递送，避免体内酸碱环境和酶类对其破坏，提高疫苗免疫效果，实现抗原的缓控释和靶向递送及诱导黏膜免疫，使生物降解纳米材料成为了疫苗递送系统的优良载体。为此，本项目合成了壳聚糖季铵盐衍生物N-2-羟丙基二甲基乙基氯化铵壳聚糖（N-2-HFCC），并对其FTIR、1H NMR、13C NMR、取代度、溶解性和抑菌活性等进行了分析。与壳聚糖相比，N-2-HFCC水溶性及药物搭载量大大提高。. 用聚电解质复合法制备了载新城疫La Sota株病毒液N-2-HFCC/CMC纳米疫苗（NDV/La Sota-N-2-HFCC/CMC-NPs），纳米疫苗中NDV含量符合《中华人民共和国兽用生物制品规程》中鸡新城疫低毒力活疫苗效力检验标准。NDV/La Sota-N-2-HFCC/CMC-NPs具有持续缓慢释药性能，室温条件下可储存一年以上，大剂量口服接种安全，表明N-2-HFCC/CMC纳米粒能够作为疫苗抗原递送载体制备纳米疫苗。通过滴鼻接种和攻毒保护试验检验了纳米疫苗诱生全身和黏膜免疫应答的能力，结果表明，纳米疫苗滴鼻免疫刺激免疫鸡产生了较强的细胞免疫和体液免疫，实现了疫苗持续缓慢释放和诱导局部黏膜免疫反应，强毒攻击时具有更强的免疫保护力。. 本项目已发表学术论文20篇，其中SCI论文17篇、EI论文2篇；获授权发明专利1项、实用新型专利1项；项目主持人获批了“万人计划”科技创新领军人才和科技部创新人才推进计划“中青年科技创新领军人才”。本项目构建了N-2-HFCC/CMC纳米粒疫苗递送系统，实现了NDV在体内持续缓慢释放，提高了免疫鸡的细胞免疫、体液免疫和局部黏膜免疫水平；同时，本项目也为发展一种安全、高效并具有优良性能的疫苗纳米佐剂及递送载体提供了理论依据，具有一定的科学意义和潜在的推广应用前景。