一种水溶性佐剂CS-GTA-ML对H7N9亚型禽流感灭活疫苗免疫效果评价  

Preparation and immune evaluation of water-soluble CS-GTA-ML adjuvant based on H7N9 vaccine

作　　者：尹文竹 许泽玉 邓碧华[1,2] 马芳 周明旭 王海燕 卢宇[1,2,3] 张金秋 YIN Wenzhu;XU Zeyu;DENG Bihua;MA Fang;ZHOU Mingxu;WANG Haiyan;LU Yu;ZHANG Jinqiu(National Research Center of Engineering and Technology for Veterinary Biologicals,Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base,Ministry of Science and Technology,Nanjing 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地,江苏南京210014 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2023年第8期1618-1626,共9页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32172895);江苏重点研发计划(现代农业)资助项目(BE2021351)。

摘　　要：旨在合成一种直接溶解在生理缓冲溶剂中、可注射的功能化修饰的壳聚糖衍生物作为兽用佐剂,用于替代铝盐佐剂。壳聚糖中的氨基与环氧丙基三甲基氯化铵(GTA)中的环氧基团发生加成开环反应,偶联成CS-GTA中间体;再与4-(2-氯乙基)吗啉(ML)发生氮烷基化反应生成终产物CS-GTA-ML。产物通过FI-IR、1H NMR、Zeta电位等表征手段检测其结构及重要成分的嫁接率;通过溶血试验、细胞活性计数(CCK-8)和组织病理分析(HE)方法评估了CS-GTA-ML的生物安全性,为注射剂型的使用提供了安全保障;配伍H7N9亚型流感灭活病毒,对雌性ICR小鼠和SPF鸡进行皮下免疫,测定对其血凝抑制(HI)效价,并通过流式分析仪初步探索脾淋巴细胞活化能力。结果显示,作为抗原递送系统,CS-GTA-ML在不同pH值的水溶液中均可溶解,其中GTA和ML的嫁接率分别约为53%,15%,且带有15.8 mV的正电荷。当质量浓度在1 g/L以下时,体外脾细胞存活率在88%以上,且对机体重要组成器官无损伤;当质量浓度高达10 g/L时,体外红细胞溶血率仅0.5%左右,具有良好的生物相容性。与单一的H7N9疫苗和ISA206+H7N9疫苗对比,CS-GTA-ML有显著的血凝抑制作用。通过相关细胞因子及流式细胞分析发现CS-GTA-ML倾向于CD4^(+)T细胞分化趋势,具有体液免疫和细胞免疫双重免疫效力。To obtain water-soluble vaccine adjuvant for replacing of aluminum adjuvant.A functionalized chitosan derivative(CS-GTA-ML),which can be directly dissolved in physiological buffer,was synthesized by opening ring reactions of amino group with epoxy group in epoxy propyl trimethylammonium chloride(GTA)and N-alkylation with 4-(2-chloroethyl)morpholine(ML).The structure and properties of as-prepared CS-GTA-ML were analyzed through FT-IR,NMR and Zeta potential.The biosafety of CS-GTA-ML was evaluated via hemolysis,CCK-8 assay and histopathological analysis(HE).CS-GTA-ML with H7N9 influenza inactivated virus were injected in mice and SPF chicken and the hemagglutination inhibition(HI)potency was detected.Furthermore,the activation ability and differentiation of splenic lymphocytes were preliminarily explored by flow cytometry.As an antigen delivery system,CS-GTA-ML,with a positive charge(15.8mV),could be directly dissolved in water at different pH.The grafting rates of GTA and ML in CSGTA-ML were 53%and 15%,respectively.CCK-8and HE experiments suggested that CS-GTAML were non-toxic when its concentrations was below 1g/L,which showed well biosafe in vitro and in vivo.Moreover,hemolysis rate of erythrocytes in vitro was only about 5%as the concentration of 10g/L,which also indicated the excellent biocompatibility of CS-GTA-ML.Compared with pristine H7N9 vaccine or ISA206+H7N9 vaccine,CS-GTA-ML had significant hemagglutination inhibition.The results of cytokines and flow cytometry demonstrated that CS-GTA-ML tends to favor the CD4^(+)T cell differentiation trend,with dual humoral and cellular immunity pathway.

关 键 词：壳聚糖 吗啉环 水溶性 H7N9禽流感病毒 佐剂 

分 类 号：S852.65[农业科学—基础兽医学]