刘睿伦;吕诗韵;钱莎莎;裴捷;杨祥;汪梦俊;吴杰;申硕;

• 1:武汉生物制品研究所有限责任公司

摘要(Abstract)：

近年来CV-A10已成为手足口病的最主要的病原体之一。本研究对3株不同CV-A10毒株进行生物学特性及体液免疫原性比较分析，筛选最适的CV-A10灭活疫苗候选株。将2014年荆州（JZ）分离株CV-A10-L12、2019江苏沛县（PX）分离株CV-A10-195和CV-A10-300，作为候选疫苗株经10层Vero细胞工厂培养，蔗糖垫底离心、蔗糖梯度离心、氯化铯等密度梯度离心后收获病毒颗粒。对不同候选毒株的滴度、病毒实心颗粒（FP）和空心颗粒（EP）的产量和比例、氯化铯密度、形态及结构蛋白等生物学特性进行比较分析。将纯化后病毒颗粒经甲醛灭活后，免疫Balb/C小鼠评价免疫原性。三株CV-A10候选疫苗株细胞工厂培养、纯化后，收获了高纯度、含病毒结构蛋白、形态完整的EP、FP颗粒。按照细胞培养自然EP和FP比例混合后，免疫6周龄Balb/C小鼠。以同源和非同源的四株CV-A10毒株作为中和毒种，测血清中和抗体滴度，评价3个疫苗候选株的体液免疫原性。CV-A10-195/PX/2019株病毒颗粒产量及免疫原性高于CV-A10-300/PX/2019株，高于CV-A10-L12/JZ/2014株。CV-A10-195/PX/2019株作为三株CV-A10毒株中最适疫苗候选株，可用于单价CV-A10或含CV-A10的多价疫苗的研发。

关键词(KeyWords)： 柯萨奇病毒A组10型;疫苗候选株;体液免疫原性

Abstract:

Keywords:

基金项目(Foundation): 湖北省技术创新专项（重大专项）（项目号：2017ACA078）;题目：手足口病双价灭活疫苗的研制~~

作者(Authors): 刘睿伦;吕诗韵;钱莎莎;裴捷;杨祥;汪梦俊;吴杰;申硕;

DOI: 10.13242/j.cnki.bingduxuebao.004267

参考文献(References)：

• [1] Huang J, Liao Q H, Ooi M H, Cowling B J, Chang Z R, Wu P, Liu F F, Li Y, Luo L, Yu S, Yu H, Wei S.Epidemiology of recurrent hand, foot and mouth disease, China, 2008-2015[J/OL]. Emerg Infect Dis,2018, 24(3):432-442. DOI:10.3201/eid2403.171303.
• [2] Liu L, Mo Z, Liang Z, Zhang Y, Li R, Ong K C,Wong K T, Yang E, Che Y, Wang J, Dong C, Feng M, Pu J, Wang L, Liao Y, Jiang L, Tan S H, David P, Huang T, Zhou Z, Wang X, Xia J, Guo L, Wang L, Xie Z, Cui W, Mao Q, Liang Y, Zhao H, Na R,Cui P, Shi H, Wang J, Li Q. Immunity and clinical efficacy of an inactivated Enterovirus 71 vaccine in healthy Chinese children:a report of further observations[J/OL]. BMC Med, 2015, 13(9):226. DOI:10.1186/s12916-015-0448-7.
• [3] Guan X, Che Y, Wei S, Li S, Zhao Z, Tong Y, Wang L, Gong W, Zhang Y, Zhao Y, Wu Y, Wang S, Jiang R, Huang J, Liu Y, Luo W, Liao Y, Hu X, Zhang W,Dai Y, Jiang G, Min G, Liu F, You X, Xu X, Li J, Li C, Fan S, Hang L, Huang Q, Li Q. Effectiveness and safety of an inactivated Enterovirus 71 vaccine in children aged 6-71 months in a phase IV study[J/OL].Clin Infect Dis, 2020, 71(9):2421-2427. DOI:10.1093/cid/ciz1114.
• [4] Meng X D, Tong Y, Wei Z N, Wang L, Mai J Y, Wu Y, Luo Z Y, Li S, Li M, Wang S, Wei S, Gong W,Zhang W, Hu X, Huang J, Shi J, Yang G, Meng S,Wang Z, Guan X, Shen S. Epidemical and etiological study on hand, foot and mouth disease following EVA71 vaccination in Xiangyang, China[J]. Sci Rep,2020, 10(1):20909.
• [5]徐长征，文丹宁，周金戈，罗剑锋，王泽鋆，申硕，李华东，陈晓琦. 2018年武汉地区手足口病肠道病毒分子流行病学分析[J/OL].中国生物制品学杂志，2020, 33(11):1262-1269. DOI:10.13200/j. cnki.cjb.003196.
• [6]杨祥，徐长征，陈金华，孟胜利，王泽鋆，申硕，陈晓琦. 2019年EV-A71疫苗接种地区手足口病肠道病毒分子流行病学分析[J/OL].中国生物制品学杂志，2021, 34(07):843-848. DOI:10.13200/j. cnki.cjb.003379.
• [7] Mirand A, Henquell C, Archimbaud C, Ughetto S,Antona D, Bailly J L, Peigue-Lafeuille H. Outbreak of hand, foot and mouth disease/herpangina associated with Coxsackievirus A6 and A10 infections in 2010,France:a large citywide, prospective observational study[J/OL]. Clin Microbiol Infect, 2012, 18(5):E110-118. DOI:10.1111/j.1469-0691.2012.03789.x.
• [8] Wu Y, Yeo A, Phoon M C, Tan E L, Poh C L, Quak S H, Chow V T. The largest outbreak of hand; foot and mouth disease in Singapore in 2008:the role of Enterovirus 71 and Coxsackievirus A strains[J/OL].Int J Infect Dis, 2010, 14(12):e1076-1081. DOI:10.1016/j.ijid.2010.07.006.
• [9] Chen M Y, He S Z, Yan Q, Xu X R, Wu W H, Ge S X, Zhang S Y, Chen M, Xia N S. Severe hand, foot and mouth disease associated with Coxsackievirus A10infections in Xiamen, China in 2015[J]. J clin Virol,2017, 93(8)20-24.
• [10]Fuschino M E, Lamson D M, Rush K, Carbone L S,Taff M L, Hua Z, Landi K, George K S. Detection of Coxsackievirus A10 in multiple tissues of a fatal infant sepsis case[J/OL]. J Clin Virol, 2012, 53(3):259-261. DOI:10.1016/j.jcv.2017.05.011.
• [11]柴青，宫金伶，赵丹，史晓燕，于萍，汪照国. 2010-2012年青岛地区手足口病病原谱及柯萨奇A10型的分子流行病学研究[J/OL].中华微生物学和免疫学杂志，2014, 34(10):731-736. DOI:10.3760/cma. j.issn.0254-5101.2014.10.001.
• [12]Lu Q B, Zhang X A, Wo Y, Xu H M, Li X J, Wang X J, Ding S J, Chen X D, He C, Liu L J, Li H, Yang H, Li T Y, Liu W, Cao W C. Circulation of Coxsackievirus A10 and A6 in hand-foot-mouth disease in China, 2009-2011[J/OL]. PloS one, 2012, 7(12):e52073. DOI:10.1371/journal.pone.0052073.
• [13]Zhou Y, Van Tan L, Luo K, Liao Q, Wang L, Qiu Q,Zou G, Liu P, Anh N T, Hong N T T, He M, Wei X,Yu S, Lam T T, Cui J, Van Doorn H R, Yu H.Genetic variation of multiple serotypes of Enteroviruses associated with hand, foot and mouth disease in Southern China[J/OL]. Virol Sin, 2021, 36(1):61-74. DOI:10.1007/s12250-020-00266-7.
• [14]Fu X, Wan Z, Li Y, Hu Y, Jin X, Zhang C. National epidemiology and evolutionary history of four hand, foot and mouth disease-related Enteroviruses in China from2008 to 2016[J/OL]. Virol Sin, 2020, 35(1):21-33.DOI:10.1007/s12250-019-00169-2.
• [15]Zhang Y, Tan X, Cui A, Mao N, Xu S, Zhu Z, Zhou J, Shi J, Zhao Y, Wang X, Huang X, Zhu S, Zhang Y, Tang W, Ling H, Xu W. Complete genome analysis of the C4 subgenotype strains of enterovirus 71:predominant recombination C4 viruses persistently circulating in China for 14 years[J/OL]. PloS one,2013, 8(2):e56341. DOI:10.1371/journal.pone.0056341.
• [16]Plevka P, Perera R, Cardosa J, Kuhn R J, Rossmann M G. Crystal structure of human enterovirus 71[J/OL].Science, 2012, 336(6086):1274. DOI:10.1126/science.1218713.
• [17]Basavappa R, Syed R, Flore O, Icenogle J P, Filman D J, Hogle J M. Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly:structure of the empty capsid assembly intermediate at 2.9 A resolution[J/OL]. Protein Sci, 1994, 3(10):1651-1669. DOI:10.1002/pro.5560031005.
• [18]Baggen J, Thibaut H J, Strating J, Van Kuppeveld F J M. The life cycle of non-polio enteroviruses and how to target it[J/OL]. Nat Rev Microbiol, 2018, 16(6):368-381. DOI:10.1038/s41579-018-0005-4.
• [19]Wen X, Sun D, Guo J, Elgner F, Wang M, Hildt E,Cheng A. Multifunctionality of structural proteins in the enterovirus life cycle[J/OL]. Future Microbiol, 2019,14(9):1147-1157. DOI:10.2217/fmb-2019-0127.
• [20]Prim N, Rodríguez G, Margall N, Del Cuerpo M,Trallero G, Rabella N. Combining cell lines to optimize isolation of human enterovirus from clinical specimens:report of 25 years of experience[J/OL]. J Med Virol,2013, 85(1):116-120. DOI:10.1002/jmv.23426.
• [21]Reed L, Muench H. A simple, method of estimating 50percent end point[J/OL]. American J Hygiene,1938,27:493-497. DOI:10.1093/oxfordjournals.aje.a118408.
• [22]Qian S S, Wei Z N, Jin W P, Wu J, Zhou Y P, Meng S L, Guo J, Wang Z J, Shen S. Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model[J/OL]. Emerg Microbes Infect, 2021, 10(1):763-773. DOI:10.1080/22221751.2021.1906755.
• [23]Jin W P, Lu J, Zhang X Y, Wu J, Wei Z N, Mai J Y,Qian S S, Yu Y T, Meng S L, Wang Z J, Shen S.Efficacy of Coxsackievirus A5 vaccine candidates in an actively immunized mouse model[J/OL]. J Virol,2021, 95(6):763-773. DOI:10.1128/jvi.01743-20.
• [24]杨志辉，胡岗，吕诗韵，吴杰，申硕.柯萨奇病毒A组2型生物化学特性及免疫原性分析[J/OL].病毒学报，2022, 38(05), DOI:10.13242/j. cnki.bingduxuebao.004115.
• [25]毛群颖，胡亚林，孙一晟，高帆，邹强，卞莲莲，范昌发，梁争论. CV-A16疫苗的免疫原性评价方法研究[J/OL].中国病毒病杂志，2018, 8(06):495-500.DOI:10.16505/j.2095-0136.2018.0110.
• [26]高微捷.柯萨奇病毒A组10型生物学特性研究及疫苗候选株的筛选[D].北京协和医学院，2020. DOI:10.27648/d.cnki.gzxhu.2020.000489.