H9亚型禽流感病毒mRNA疫苗的构建与效力评价  

Construction and Efficacy Evaluation of mRNA Vaccines against H9 Subtype Avian Influenza Virus

作　　者：黄程 杨志远[1] 林健[1] 程慧敏 王米 毛惠琳 王国良 刘贵明 赵际成 刘月焕[1] HUANG Cheng;YANG Zhiyuan;LIN Jian;CHENG Huimin;WANG Mi;MAO Huilin;WANG Guoliang;LIU Guiming;ZHAO Jicheng;LIU Yuehuan(Institute of Animal Husbandry and Veterinary Medicine,Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097,China;Novoprotein Scientific Inc,Suzhou 215200,China;Institute of Biotechnology,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China)

机构地区：[1]北京市农林科学院畜牧兽医研究所,北京100097 [2]苏州近岸蛋白质科技股份有限公司,苏州215200 [3]北京市农林科学院生物技术研究所,北京100097

出　　处：《畜牧兽医学报》2025年第4期1843-1853,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：北京市农林科学院院长基金(YZJJ202102);北京市农林科学院青年科研基金(QNJJ202234);现代农业产业技术体系北京市家禽创新团队(BAIC06-2024)。

摘　　要：H9亚型禽流感(avian influenza, AI)对养鸡业尤其肉鸡产业危害巨大,灭活疫苗不能有效阻止病毒在鸡上呼吸道感染复制,有必要研究H9亚型禽流感病毒(avian influenza virus, AIV)血凝素(hemagglutinin, HA)基因的mRNA疫苗。参考GenBank 2022年公开的HA基因序列,优化HA全长(HA)和HA胞外区(HAe)序列,连接至pUC57载体构建体外转录模板,经体外转录、加帽、纯化,采用脂质纳米颗粒(Lipid nanoparticles, LNP)包封mRNA,并转染人胚肾细胞(293T)、仓鼠肾细胞(BHK-21)和鸡胚成纤维细胞(DF-1),Western blot检测HA表达。20只3周龄SPF鸡,随机分成4组,每组5只,分别经腿部肌肉接种0.3 mL PBS、25μg HA mRNA LNP、25μg HAe mRNA LNP和0.3 mL H9亚型AI灭活疫苗。免疫后4周采集血清,检测血凝抑制(hemagglutination inhibition, HI)抗体,每只鸡滴鼻点眼H9N2亚型AIV 0.2 mL(含10^(6.0)EID_(50)),攻毒后5日采集咽喉拭子和外周血单个核细胞,分别检测病毒分离情况及IFN-γ、IL-4的表达水平,观察气管组织病理学变化。Western blot结果显示两种mRNA HA蛋白均成功表达,经LNP包封后可有效在细胞内完成翻译。HA mRNA LNP和HAe mRNA LNP免疫SPF鸡后HI抗体效价(log2)平均值分别为5.6和3。H9亚型AI灭活疫苗免疫后HI抗体效价(log2)平均值为9.8,极显著高于2个mRNA LNP组(P<0.01)。非免疫对照、HA mRNA LNP、HAe mRNA LNP和H9亚型AI灭活疫苗组病毒分离结果分别为5/5、1/5、4/5和2/5。HA mRNA LNP组IFN-γ表达水平显著高于其他组(P<0.05),HAe mRNA LNP组IL-4表达水平显著低于其他组(P<0.05)。气管组织病理学变化结果表明HA mRNA LNP和H9亚型AI灭活疫苗免疫可有效减轻H9亚型AIV对SPF鸡气管上皮细胞的损伤。构建的H9亚型AIV HA全长mRNA疫苗免疫鸡能提供有效保护,而HA胞外区mRNA疫苗不能提供有效的免疫保护。H9 subtype of avian influenza(AI)poses a significant threat to the poultry industry,particular the broiler industry.As current inactivated vaccine cannot effectively prevent virus in-fection and replication in the upper respiratory tract of chickens,it is necessary to generate an mRNA vaccine targeting the hemagglutinin(HA)gene of H9 subtype avian influenza virus(AIV).Based on the HA gene sequences available on GenBank in 2022,full-length of the HA sequences(HA)and its extracellular domain(HAe)were optimized,and cloned into the pUC57 vector to construct transcription templates in vitro.After transcription in vitro,capping,and purification,the mRNAs were encapsulated in lipid nanoparticles(LNPs)and transfected into human embryonic kidney cells(293T),baby hamster kidney cells(BHK-21),and chicken em-bryo fibroblast cells(DF-1).HA protein expression was confirmed via Western blot.Twenty 3-week-old SPF chickens were randomly divided into four groups(n=5).Each chicken was im-munized with 0.3 mL PBS,25μg HA mRNA LNP,25μg HAe mRNA LNP and 0.3 mL H9 subtype AI inactivated vaccine,respectively.Serum samples was collected 4 weeks post-immuni-zation to assess hemagglutination inhibition(HI)antibody titers.All animals were intranasally and ocularly challenged with 0.2 mL of H9N2 subtype AIV(containing 10^(6.0)EID_(50)).Oral swabs were collected 5 days post-challenge to detect virus isolation,and peripheral blood mononuclear cells(PBMCs)were collected for the measurement of expression levels of IFN-γand IL-4,along-side observing pathological changes of tracheal tissue.Western blot analysis showed that both 2 mRNA HA proteins were successfully expressed and could be effectively translated into cells fol-lowing LNP encapsulation.The mean HI antibody titers(log2)post immunization with HA mR-NA LNP and HAe mRNA LNP in SPF chickens were 5.6 and 3,respectively.The H9 subtype AI inactivated vaccine induced significantly higher antibody titer(9.8),compared to both two mRNA LNP groups(P<0.01).Virus isolation rates of the challenge c

关 键 词：H9亚型 禽流感病毒 mRNA疫苗 LNP 免疫保护 

分 类 号：S852.659.5[农业科学—基础兽医学]