基于流感病毒载体的人偏肺病毒疫苗的构建及制备  

Construction and preparation of human metapneumovirus vaccine based on influenza virus vector

作　　者：高梦雪 刘晓曼 郭丽茹[2,3] 孔梅[2,3] 庄志超 于爱萍 李蕊[1] 李晓燕[1,2,3] Gao Mengxue;Liu Xiaoman;Guo Liru;Kong Mei;Zhuang Zhichao;Yu Aiping;Li Rui;Li Xiaoyan(Tianjin Medical University,Tianjin 300070,China;Institute of Microbiology,Tianjin Center for Disease Control and Prevention,Tianjin 300011,China;Tianjin Key Laboratory of Pathogenic Microbiology of Infectious Disease,Tianjin Centers for Disease Control and Prevention,Tianjin 300011,China)

机构地区：[1]天津医科大学,天津300070 [2]天津市疾病预防控制中心微生物检验检测所,天津300011 [3]天津市疾病预防控制中心天津市传染病病原微生物重点实验室,天津300011

出　　处：《中华实验和临床病毒学杂志》2024年第1期77-85,共9页Chinese Journal of Experimental and Clinical Virology

基　　金：天津市科技局重点项目(20JCZDJC00130);天津市卫生健康委重点项目(TJWJ2022ZD010);天津医学重点学科(TJYXZDXK-50A)。

摘　　要：目的构建和制备以流感病毒为载体嵌合人偏肺病毒(human metapneumovirus, HMPV)抗原表位的重组病毒株。方法利用8质粒系统共转染293 T/MDCK细胞, 制备嵌合有HMPV抗原表位的重组流感病毒株。重组流感病毒株以A/PR/8/34的内部基因(PB1、PB2、PA、NP、NS、M、HA、NA)作为骨架, 同时通过基因改造, 将HMPV的B细胞表位插入HA基因中, 将HMPV的CTL+Th细胞表位插入到NA基因中。采用"7+1"模式, 利用反向遗传学制备重组流感病毒株。制备成功后, 通过全基因组重测序, 测定血凝素(HA)的活性、半数组织培养感染剂量(TCID50)及生长曲线对重组病毒株进行评价。结果成功将HMPV的抗原表位插入到流感病毒基因组中, 并制备出两株嵌合有HMPV表位的重组流感病毒株, 分别命名为FLU/HMPV/B和FLU/HMPV/CTL+Th, 其中HA均为27, TCID50分别为105.2/ml和105.0/ml。经SPF鸡胚传代3次, 两株重组病毒株可稳定增殖。经全基因组测序鉴定, FLU/HMPV/B嵌合有HMPV的B细胞表位, FLU/HMPV/CTL+Th嵌合有HMPV的CTL和Th细胞表位。生长曲线结果表明两株重组病毒株均可在鸡胚中有效复制。结论成功制备出两株嵌合有HMPV表位的重组流感病毒株, 重组病毒株从生长特性以及遗传稳定性的结果来看, 其符合继续开展下一步动物实验的要求, 后续将通过小鼠实验对重组疫苗株的免疫原性以及免疫保护效果进行进一步的评价, 最终为实现"一苗两用"或"一苗多用"提出新的思路, 也为HMPV疫苗发展提供一种新的策略。Objective To construct and prepare recombinant virus strains chimeric with human metapneumovirus(HMPV)antigenic epitopes.Methods Recombinant influenza virus vectors which chimeric with different HMPV antigenic epitopes were rescued by reverse genetics using eight-plasmid system.The recombinant influenza virus strain used the internal genes of A/PR/8/34(PB1,PB2,PA,NP,NS,M,HA,and NA)as a backbone,with concomitant genetic modifications to insert the B-cell epitopes of HMPV into the HA gene,and the CTL+Th cell epitopes of HMPV into the NA gene.Preparation of recombinant influenza virus strains using reverse genetics in a"7+1"model.The recombinant virus strains were evaluated by measuring hemagglutinin(HA)titers,half tissue culture infectious dose(TCID50)and growth curves.Sequencing analysis was conducted to verify whether the rescued viruses carried the chimeric HMPV epitopes.Results The epitopes of HMPV were inserted into the influenza virus genome and two recombinant influenza virus strains were rescued successfully,named as FLU/HMPV/B and FLU/HMPV/CTL+Th.HA titers of the recombinant strains were both 27,their TCID50 were 105.2/ml and 105.0/ml,respectively.After cultured for three passages in chick embryo,these two recombinant strains could proliferate steadily.Whole genome sequencing verified that the FLU/HMPV/B carried the B-cell epitopes of HMPV,the FLU/HMPV/CTL+Th carried the CTL and Th cell epitopes of HMPV.Growth curve tests also verified that the recombinant strains could proliferate steadily in eggs.Conclusions Two recombinant influenza virus vector strains carrying the B cell,CTL and Th epitopes of HMPV were rescued successfully.The result of the recombinant virus strains in terms of growth characteristics as well as genetic stability indicate that they meet the requirements for proceeding to the next step of animal experiments.The immunogenicity and immunoprotective effect will be further evaluated by mouse experiments.Ultimately new ideas for the realization of"one vaccine for two uses"or"one vaccine for

关 键 词：流感病毒载体 人偏肺病毒 反向遗传学 重组病毒株 

分 类 号：R392[医药卫生—免疫学]