新型发热伴血小板减少综合征病毒mRNA疫苗的免疫原性研究  

Immunogenicity study of an mRNA vaccine for the novel fever with thrombocytopenia syndrome virus

作　　者：吴鑫宇 钱华 王亚南 张军杰 田莉 刘乐乐 李梦华 郑文文 郑学星[1] 赵忠欣 WU Xinyu;QIAN Hua;WANG Yanan;ZHANG Junjie;TIAN Li;LIU Lele;LI Menghua;ZHENG Wenwen;ZHENG Xuexing;ZHAO Zhongxin(Department of Hygiene Inspection and Quarantine,School of Public Health,Shandong University,Jinan 250012,China;Oral Medicine Center,Shandong University Second Hospital;Department of Pharmacy,Shandong University Second Hospital;Clinical Laboratory Center,Linyi People's Hospital)

机构地区：[1]山东大学公共卫生学院卫生检验与检疫学系,山东济南250012 [2]山东大学第二医院口腔医学中心 [3]山东大学第二医院药学部 [4]临沂市人民医院检验医学中心

出　　处：《中国病原生物学杂志》2025年第5期551-556,562,共7页Journal of Pathogen Biology

基　　金：国家重点研发计划项目(No.2022YFC2305005);国家自然科学基金项目(No.82272335,82102391);山东省自然科学基金项目(No.ZR2021MC010,ZR2021QH140);济南市临床医学科技创新计划项目(No.202328045);山东省口腔组织再生重点实验室开放课题(No.SDDX202103)。

摘　　要：目的了解发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)其囊膜糖蛋白Gn与Gc构建LNP-mRNA疫苗并鉴定其安全性与免疫原性。方法将SFTSV Gn/Gc基因进行密码子优化并合成,体外转录及核苷酸修饰获得mRNA-Gn/Gc;转染HEK-293T细胞,免疫荧光和Western blot验证目的蛋白表达;使用脂质纳米颗粒(LNP)包封mRNA合成LNP-mRNA-Gn/Gc,免疫BALB/c小鼠。检测小鼠血清中SFTSV中和抗体滴度,鼠腹股沟淋巴结B细胞与脾脏T细胞的募集与活化;利用SFTSV攻击免疫小鼠,评价疫苗的保护率。结果免疫荧光与Western blot实验均证明mRNA-Gn/Gc在细胞内可成功表达Gn与Gc蛋白;小鼠免疫LNP-mRNA-Gn/Gc后,可分别诱导产生高水平的特异性中和抗体,LNP-mRNA-Gc峰值可达1∶490,LNP-mRNA-Gn峰值可达1∶938,且两个糖蛋白之间存在显著差异;B细胞和T细胞被显著活化,B细胞活化比例显著增加至20%以上,且IL-4与IFN-γ分泌水平分别达到303 SFU和1435 SFU,显著高于对照组;SFTSV小鼠攻击保护实验显示,LNP-mRNA-Gn/Gc疫苗均能100%保护小鼠免于SFTSV的致死性攻击。结论本研究成功构建了表达SFTSV Gn与Gc蛋白的mRNA疫苗,且Gn蛋白更具有潜力,为SFTSV临床疫苗开发提供理论依据。Objective To construct a lipid nanoparticle(LNP)-encapsulated mRNA vaccine encoding the Gn and Gc glycoproteins of Severe Fever with Thrombocytopenia Syndrome Virus(SFTSV)and evaluate its safety and immunogenicity.Methods The SFTSV Gn and Gc genes were codon-optimized,synthesized,and subjected to in vitro transcription with nucleoside modification to generate mRNA-Gn/Gc.HEK-293T cells transfected with mRNA-Gn/Gc successfully expressed the protein,as confirmed by immunofluorescence and Western blot.Lipid nanoparticles(LNPs)were used to encapsulate the mRNA to produce LNP-mRNA-Gn/Gc,which was then used to immunize BALB/c mice.Serum neutralizing antibody titers,activation of B cells in inguinal lymph nodes,and splenic T cell responses were analyzed.Immunized mice were challenged with SFTSV to evaluate the vaccine's protective efficacy.Results Both immunofluorescence and Western blot experiments confirmed that mRNA-Gn/Gc successfully expressed Gn and Gc proteins within the cells.After immunizing mice with LNP-mRNA-Gn/Gc,high levels of specific neutralizing antibodies were induced.The peak titers of LNP-mRNA-Gc and LNP-mRNA-Gn reached 1:490 and 1:938,with a significant difference between the two glycoproteins.Both B cells and T cells were significantly activated,with the activation rate of B cells increasing to over 20%.Additionally,the secretion levels of IL-4 and IFN-γ reached 303 SFU and 1435 SFU,which were significantly higher than those in the control group.The SFTSV challenge experiment demonstrated that the LNP-mRNA-Gn/Gc vaccine provided 100% protection to the mice,whereas all mice in the control group died.Conclusion This study successfully developed an mRNA vaccine expressing the SFTSV Gn/Gc protein,with the Gn protein showing greater potential.These findings provide a theoretical basis for the clinical development of SFTSV vaccines.

关 键 词：新型发热伴血小板减少综合征病毒 Gn/Gc蛋白 中和抗体 mRNA疫苗 

分 类 号：R392[医药卫生—免疫学]