通用流感mRNA候选疫苗的构建及免疫效果评价  

Construction and evaluation of a universal influenza mRNA vaccine

作　　者：田昱莹 邓卓雅 李聪 孙芳 曹蕊 杨鹏辉 TIAN Yuying;DENG Zhuoya;LI Cong;SUN Fang;CAO Rui;YANG Penghui(College of Basic Medical Science,Inner Mongolia Medical University,Hohhot,Inner Mongolia Autonomous Region,010110;Institute of Hepato-Pancreato-Biliary Surgery,The First Medical Center,Chinese PLA General Hospital,Beijing,100853,China)

机构地区：[1]内蒙古医科大学基础医学院,呼和浩特010110 [2]中国人民解放军总医院第一医学中心肝胆胰外科医学部研究所,北京100853

出　　处：《陆军军医大学学报》2024年第7期725-731,共7页Journal of Army Medical University

摘　　要：目的构建通用流感mRNA疫苗并全面评价其免疫保护效果。方法优化流感病毒株A/California/04/2009的血凝素(hemagglutinin,HA)、核蛋白(nucleoprotein,NP)和基质蛋白2胞外区(matrix protein 2 ectodomain,M2e)抗原序列,并将HA、NP和3个串联的M2e(3M2e)分别克隆至pcDNA3.1载体,通过线性化、体外转录、酶学法加帽、酶促加尾合成mRNA,命名为mRNA-HA、mRNA-NP和mRNA-3M2e。3种mRNA分别转染293T细胞后,通过免疫荧光实验鉴定蛋白的表达。利用脂质纳米颗粒分别包裹mRNA-HA、mRNA-NP和mRNA-3M2e并测量其粒径和电位。再将3种mRNA等体积混合制备成Comb-mRNA疫苗。将28只6周雌性Balb/c小鼠(体质量为18~22 g)按简单随机分组法分为2组:LNP组(n=14)和Comb-mRNA组(n=14)。通过血凝抑制(hemagglutination inhibition,HI)、微量中和(microneutralization,MN)实验评价流感mRNA疫苗诱导小鼠产生的血清抗体滴度;通过流式细胞术评价Comb-mRNA疫苗诱导的细胞免疫反应。采用5LD_(50)野生型H1N1流感病毒株感染小鼠评价Comb-mRNA疫苗的免疫保护效果。结果成功构建mRNA-HA、mRNA-NP和mRNA-3M2e,且3种mRNA均能在293T细胞表达。脂质纳米颗粒包裹mRNA平均粒径为(119.53±6.5)nm,平均电位为(-8.23±1.3)mV。与LNP组比较,Comb-mRNA组疫苗的HI几何平均滴度(geometric mean titer,GMT)达179.6,MN的GMT达201.6,同时诱导IFNγ+CD4+/CD8+T细胞比例升高。Comb-mRNA组在加强免疫后2周能够提供对5LD_(50)野生流感H1N1亚型病毒的保护。结论通用流感疫苗候选疫苗Comb-mRNA能够诱导小鼠免疫反应并保护小鼠免受病毒感染。Objective To construct a universal influenza mRNA vaccine and evaluate its immunogenicity.Methods The antigen sequence of hemagglutinin(HA),nucleoprotein(NP)and matrix protein 2 ectodomain(M2e)in influenza A/California/04/2009 was optimized.HA,NP and 3 tandem M2e(3M2e)were cloned into pcDNA3.1 vector,respectively.Then the mRNAs were synthesized by linearization,in vitro transcription,enzymatic capping and enzymatic tailing,and named as mRNA-HA,mRNA-NP and mRNA-3M2e,respectively.The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay.Comb-mRNA vaccine was prepared by enveloped mRNA-HA,mRNA-NP and mRNA-3M2e with lipid nanoparticles,respectively,and the particle size and potential were identified.Twenty-eight 6-week-old female BALB/c mice(18~22 g)were randomly divided into LNP group(n=14)and Comb-mRNA group(n=14).Hemagglutination inhibition(HI)method and microneutralization(MN)test were used to evaluate the serum antibody titer induced by Comb-mRNA vaccines.The mice were infected by 5LD_(50) wild-type H1N1 influenza virus to evaluate the protective efficacy.Results The mRNA-HA,mRNA-NP and mRNA-3M2e were successfully constructed,and the 3 mRNAs could be expressed in 293T cells.The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm,and the average potential was-8.23±1.3 mV.The geometric mean titer(GMT)of HI and MN in the Comb-mRNA group were 179.6 and 201.6,compared with the LNP group.The ratio of IFN-γ^(+)CD4^(+)/CD8^(+)T cells was increased.The Comb-mRNA group could provide protection against 5LD_(50) wild type influenza H1N1 virus after 2 weeks of booster immunization.Conclusion Comb-mRNA,an influenza vaccine candidate,can induce immune responses and protect mice from influenza virus challenge.

关 键 词：mRNA疫苗 流感疫苗 血凝素 核蛋白 基质蛋白2胞外区 

分 类 号：R392.2[医药卫生—免疫学] R392.7[医药卫生—基础医学] R511.7