N-2-羟丙基三甲基氯化铵壳聚糖-N,O-羧甲基壳聚糖为佐剂制备H9N2禽流感病毒基因纳米疫苗经黏膜递送的效果及机制

Veterinary vaccines have the poor immunogenicity, low bioavailability of antigen, poor safety or not obvious effect of vaccine adjuvants, and can't induce both humoral, cellular and mucosal immune response or poor immune response etc. Because nano-materials have the functions of delivery antigens, enhancing the immune response and protecting antigens from degradation, so we have synthesized the chitosan derivatives N-2-hydroxypropyl trimethyl ammonium chloride chitosan-N,O-carboxymethyl chitosan (N-2-HACC-CMC) with a good water soluble in our group. In order to study the biodegradable polymer N-2-HACC-CMC as a adjuvant and delivery carrier for veterinary vaccines, we prepared the N-2-HACC-CMC nanoparticles containing the attenuated live Newcastle disease vaccine (strain La Sota) against Newcastle disease, oil emulsion inactivated Newcastle disease vaccine against Newcastle disease and Newcastle disease virus F gene-containing DNA vaccine against Newcastle disease, respectively. The results confirmed that the N-2-HACC-CMC could be used to prepare microcapsule sustained-release vaccine as an efficient and safe adjuvant and delivery carrier for NDV vaccine to induce mucosal immunity. Base on these finished studies, here, we will use N-2-HACC-CMC as a adjuvant, H9N2 avian influenza virus eukaryotic plasmid expressing HA protein (pCAGGS-opti441-HA) as a model antigen for the formulation of an intranasal N-2-HACC-CMC-based vaccine against H9N2 avian influenza virus (N-2-HACC-CMC/pCAGGS-opti441-HA), and in vitro and in vivo assays will be conducted to evaluate its efficacy and explore the possible underlying mechanism of action, including safety, nasal uptake, epithelial cell tight junctions, dendritic cells (DCs) antigen uptake and maturation, in vivo immunization etc. We will establish a platform of veterinary vaccine mucosal immune delivery system in our study, which can improve the bioavailability of vaccine antigen, protect the antigen or plasmid DNA from being degradated under unfavorable conditions, enhance the immune effect, slow release and induce mucosal immune. The study will lay the foundation for the rational development of new and improved vaccines and application of chitosan derivatives in animal or human microcapsule sustained-release vaccines and drugs.

兽用疫苗具有免疫原性差，抗原生物利用度低，佐剂安全性差或效果不明显，不能同时诱导体液、细胞、黏膜免疫或免疫反应较差等问题。纳米材料具有运载、增强免疫应答、避免抗原降解等功能，课题组合成了N-2-HACC-CMC，用其包裹新城疫弱毒苗、油乳剂灭活苗、新城疫DNA疫苗，证实了其可做为疫苗佐剂和递送载体制备微胶囊缓释疫苗。本研究以N-2-HACC-CMC为佐剂和递送载体、H9N2亚型禽流感病毒HA基因真核表达质粒为模型抗原，制备经鼻递送的禽流感N-2-HACC-CMC纳米疫苗，通过安全性、黏膜吸附性、树突细胞摄取抗原和成熟、效力检验等体内外实验评价N-2-HACC-CMC的佐剂和载体效应及作用机制，建立兽用疫苗黏膜免疫递送系统的技术平台，以提高抗原生物利用度、避免抗原降解、增强免疫效果、缓释及诱导黏膜免疫。本研究将为合理开发新型或改进疫苗及壳聚糖衍生物在其它生物制品中应用的研究奠定基础。

纳米生物载体材料具有运载、避免抗原降解、提高疫苗免疫效力等功能。本项目改进了负责人前期确定的N-2-HACC合成工艺，确定了合成N-2-HACC/CMC纳米粒的最佳工艺，试制了控制纳米粒大小和分散度的装置，N-2-HACC/CMC纳米粒血液相容性、生物降解性、生物安全性和生物黏附性均较好。. 构建了H9N2 AIV HA基因真核表达质粒，制备了载pβH5N1、pβH5N1SH9和6个重组质粒DNA疫苗，合成了N-2-HACC/CMC纳米油佐剂和纳米水佐剂，制备了H9N2禽流感N-2-HACC/CMC灭活疫苗。该灭活苗的性状、无菌、安全等检验结果符合《中国兽药典》规定的质量标准，免疫持续期比商品灭活苗延长了四分之三，攻毒保护率为100%。. 比较了N-2-HACC/CMC水佐剂、N-2-HACC/CMC油佐剂、N-2-HACC-Al、GEL01、ISA 78VG、VSP70、CpG、poly(I:C)、铝胶增强H9N2禽流感灭活疫苗的免疫效果。N-2-HACC/CMC水佐剂和油佐剂灭活疫苗组、N-2-HACC-Al灭活疫苗组免疫鸡血清抗体效价差异不显著，但均显著高于其它佐剂灭活苗组。另外，为了建立兽用疫苗N-2-HACC/CMC纳米粒黏膜免疫递送系统的技术平台，本项目制备了猪流行性腹泻纳米黏膜疫苗，该疫苗肌注和口服接种均刺激豚鼠产生了较强的细胞、体液及肠道黏膜免疫反应，免疫持续期比商品灭活疫苗延长了四分之一以上。. 阐明了N-2-HACC/CMC纳米粒滴鼻免疫诱导免疫应答的作用机制。该纳米粒主要通过MHC II途径递呈抗原，引起以Th2为主的免疫反应，促进淋巴细胞增殖和分化，刺激机体产生了高效的特异性系统免疫和黏膜免疫应答。N-2-HACC/CMC纳米粒通过TLR4受体介导激活NF-κB通路，提高促炎刺激的NF-κB响应，进而激活并提高其下游相应促炎刺激分子、促炎细胞因子及巨噬细胞中关键的调节促炎因子表达。. 本项目发表论文22篇（SCI论文16篇、中文6篇），申请发明专利2件、实用新型专利3件（授权发明专利1件、实用新型专利3件），培养了1名助理研究员晋升为副研究员和10名硕士研究生，获得地市级一等奖1项，申请了2项中国兽药学会团体标准，参加学术会议17次（做报告7次）。本研究为开发疫苗新剂型及壳聚糖衍生物在其它生物制品中应用奠定了理论基础。