乙脑病毒为骨架的寨卡病毒减毒活疫苗的构建  

Construction of the chimeric live attenuated Zika vaccine candidate based on Japanese encephalitis virus

作　　者：唐丽萍[1] 任阳 陈岚 李玥珂 冯亚岚[1,2] 黄荣 袁磊 陈蓉 杨健[1,2] Tang Liping;Ren Yang;Chen Lan;Li Yueke;Feng Yalan;Huang Rong;Yuan Lei;Chen Rong;Yang Jian(School of Basic Medicine and Forensic medicine,North Sichuan Medical College,Nanchong 637000,China;Key Laboratory for Drugs for Metabolism and Biologicals of Nanchong,Nanchong 637000,China)

机构地区：[1]川北医学院基础医学与法医学院,南充637000 [2]代谢病药物与生物制品南充市重点实验室,南充637000

出　　处：《中华实验和临床病毒学杂志》2023年第4期435-442,共8页Chinese Journal of Experimental and Clinical Virology

基　　金：南充市川北医学院校地合作项目(20SXQT0338,20SXQT0239)。

摘　　要：目的构建乙脑/寨卡嵌合病毒(ChimZIKV)并评价其是否可成为寨卡候选疫苗株。方法用基因克隆技术将寨卡病毒prM/E基因替换乙脑病毒疫苗株相应区域,构建乙脑寨卡嵌合病毒感染性克隆,体外转录制备RNA,电转染导入BHK-21细胞拯救获得ChimZIKV,通过噬斑试验、免疫荧光、基因测序鉴定病毒;检测其生长特性;通过动物实验分别检测其小鼠脑内神经毒力、病毒血症、中和抗体产生能力及免疫保护效果。结果酶切和测序结果表明ChimZIKV感染性克隆成功构建,病毒测序及免疫荧光证实嵌合病毒拯救成功;噬斑试验显示该嵌合病毒呈小噬斑;生长曲线显示其增殖速度慢于乙脑疫苗株;细胞增殖活性试验显示该嵌合病毒感染组细胞的增殖活性明显高于乙脑病毒疫苗株感染组;动物实验结果显示嵌合病毒对小鼠和乳鼠都表现出极低的神经毒力;病毒血症结果显示该嵌合病毒在小鼠体内没有检测到明显的病毒血症;噬斑减少中和试验显示免疫小鼠血清中和抗体的阳转率达100%,第3周中和抗体效价最高(GMT=85);免疫保护试验显示对ChimZIKV(MR766)的脑内攻击保护率为30%。结论嵌合病毒ChimZIKV具有一定的免疫原性和较好的安全性,有进一步发展为寨卡病毒候选疫苗株的可能。Objective To construct the chimeric virus ChimZIKV of Japanese encephalitis virus and Zika virus and evaluate whether the chimeric virus can be developed into a candidate vaccine strain against Zika virus.Methods The infectious clone of chimeric virus ChimZIKV was constructed by replacement the prM/E of Japanese encephalitis virus vaccine strain with that of Zika virus.RNA of chimeric virus ChimZIKV had been transcribed in vitro and was electro-transfected into BHK-21 cells to rescue chimeric virus.Afterward,the chimeric virus was identified by plaque assay,immunofluorescence and gene sequencing.The growth was shown by the growth curve.The neurobirulence,viremia,neutralizing antibody production and immune protective effect of chimeric virus were tested by animal experiments.Results The result of restriction enzyme digestion showed that the infectious clone of chimeric virus was successfully constructed,and the immunofluorescence assay and sequencing showed that chimeric virus ChimZIKV was successfully rescued.The cell proliferation activity test showed that the cell proliferation activity of the chimeric virus infected group was higher than that of the infected group.The result of animal experiments showed that chimeric virus showed very low neurovirulence to mice and suckling mice,and the result of viremia showed that chimeric virus had no obvious viremia in mice.Plaque reduction neutralization test(PRNT)showed 100%positive conversion of neutralizing antibody in sera of immunized mice,and the highest neutralizing antibody titer(GMT=85)was seen in the third week.The immune protection experiment showed that the protection rate against JE/ZIKV(MR766)was 30%.Conclusions This study showed that the chimeric virus ChimZIKV is immunogenic and with low neurovirulence,and it may be further developed as a candidate vaccine strain against Zika virus.

关 键 词：乙型脑炎病毒 寨卡病毒 嵌合病毒 疫苗 

分 类 号：R392[医药卫生—免疫学]