肠道病毒71型衣壳蛋白VP1特异性识别和激活手足口病患儿CD4<sup>+</sup> T细胞免疫活化

doi:10.3877/cma.j.issn.1674-1358.2018.05.003

中华实验和临床感染病杂志(电子版) ›› 2018, Vol. 12 ›› Issue (05) : 427 -433. doi: 10.3877/cma.j.issn.1674-1358.2018.05.003

所属专题：文献；

论著

肠道病毒71型衣壳蛋白VP1特异性识别和激活手足口病患儿CD4⁺ T细胞免疫活化

高宁¹, 李梅¹, 李亚萍¹, 王文俊¹, 石娟娟¹, 贾晓黎¹, 党双锁¹^,^†(

)

^1. 710004　西安市，西安交通大学第二附属医院感染科

收稿日期:2018-05-29 出版日期:2018-10-15
通信作者: 党双锁
基金资助:
国家自然科学青年科学基金项目(No. 81701632); 西安交通大学第二附属医院人才培养专项科研青年基金(No. YJ（QN）201520); 陕西省重点研发计划项目(No. 2017ZDXM-SF-071)

Enterovirus 71 capsid protein VP1 specifically recognizes and activates CD4⁺ T cell immune activation in children with hand, foot and mouth disease

Ning Gao¹, Mei Li¹, Yaping Li¹, Wenjun Wang¹, Juanjuan Shi¹, Xiaoli Jia¹, Shuangsuo Dang¹^,^†()

^1. Department of Infectious Diseases, the Second Affiliated Hospital of Medical School of Xi’an Jiaotong University, Shannxi 710004, China

Received:2018-05-29 Published:2018-10-15
Corresponding author: Shuangsuo Dang
About author:
Corresponding author: Dang Shuangsuo, Email: dang212@126.com

全文 ( ) 下载PDF ( ) 导出引用

引用本文：

高宁, 李梅, 李亚萍, 王文俊, 石娟娟, 贾晓黎, 党双锁. 肠道病毒71型衣壳蛋白VP1特异性识别和激活手足口病患儿CD4⁺ T细胞免疫活化[J]. 中华实验和临床感染病杂志(电子版), 2018, 12(05): 427-433.

Ning Gao, Mei Li, Yaping Li, Wenjun Wang, Juanjuan Shi, Xiaoli Jia, Shuangsuo Dang. Enterovirus 71 capsid protein VP1 specifically recognizes and activates CD4⁺ T cell immune activation in children with hand, foot and mouth disease[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2018, 12(05): 427-433.

目的

探讨肠道病毒病毒71型衣壳蛋白VP1诱导EV71感染患儿外周血T淋巴细胞产生干扰素-γ(IFN-γ)应答特征。

方法

收集2010年9月至2011年12月西安交通大学医学院第二附属医院和西安市儿童医院住院的EV71感染手足口病患儿共31例，根据临床特征分为3组：急性感染期组(16例)、临床恢复期组(9例)和感染恢复后1个月组(6例)；另外，选取10例健康儿童为对照组。通过重叠肽技术合成覆盖EV71衣壳蛋白VP1 36条重叠肽段作为抗原，采用IFN-γ酶联免疫斑点分析法(ELISPOT)和磁珠分选法(MACs)体外检测各组患儿EV71 VP1特异性CD4⁺ T细胞和CD8⁺ T细胞免疫应答特征。

结果

急性感染期组有4例(25%)患儿对VP1蛋白存在T细胞免疫反应，临床恢复期组5例(56%)患儿对VP1蛋白存在T细胞免疫反应，其中EV71感染临床恢复期组患者反应强度最强，显著高于急性感染期，差异有统计学意义(Z=－2.042、P = 0.041)，而感染恢复后1个月组和对照组均未检测到T细胞免疫反应，临床恢复期组患者T细胞反应强度显著高于感染恢复后1个月组(Z=－2.105、P = 0.035)和对照组(Z=－2.633、P= 0.008)，差异均有统计学意义；提示EV71感染患儿VP1蛋白被识别的频率(即例数)随着临床症状及体征消失而消失。VP1蛋白诱导的细胞免疫应答以特异性CD4⁺T细胞为主导；筛选确定了8个EV71 VP1蛋白区CD4⁺ T细胞表位。

结论

EV71感染患儿在发病期对VP1蛋白存在特异性T细胞反应，且以特异性CD4⁺ T细胞为主导；筛选确定EV71感染患儿VP1蛋白的CD4⁺ T细胞表位有助于阐明EV71感染的细胞免疫学发病机制。

关键词: 肠道病毒71, T细胞免疫, 衣壳蛋白VP1, 酶联免疫斑点分析, T细胞表位

Objective

To investigate the characteristics of interferon-γ(IFN-γ) response induced by enterovirus 71 (EV71) capsid protein VP1 in peripheral blood T lymphocytes of children with EV71 infection.

Methods

From September 2010 to December 2011, a total of 31 patients with hand, foot and mouth disease (HFMD) infected with EV71 were collected from the Second Affiliated Hospital of Medical College of Xi’an Jiaotong University and Xi’an Children’s Hospital. According to the clinical features, 31 cases were divided into three groups: acute infection group (16 cases), clinical convalescence group (9 cases) and one month after recovery of infection group (6 cases), in addition, 10 healthy children were selected as control group. Total of 36 overlapping peptides of EV71 capsid protein VP1 were synthesized by overlapping peptide technique as antigens. The immunological responses of EV71 VP1 specific CD4⁺T and CD8⁺T cells were detected by IFN-γenzyme-linked immunoblot assay (ELISPOT) and magnetic cell sorting (MACs) in vitro.

Results

In the acute infection group, 4 cases (25%) had T cell immune response to VP1 protein, 5 cases (56%) in the clinical convalescence group had T cell immune response to VP1 protein. The strength of T-cell responses in clinical convalescence group had the strongest, significantly higher than acute infection group (Z=-2.042,P= 0.041), and one month after recovery of infection group (Z=-2.105,P = 0.035) and control group (Z=-2.633,P = 0.008), with significant differences. The T cell immune response was not detected in the control group and one month after recovery of infection group. The results suggested that the frequency of VP1 protein recognition (the number of cases) disappeared with the disappearance of clinical symptoms and signs in children with EV71 infection. The cellular immune response induced by VP1 protein was dominated by specific CD4⁺T cells. Eight CD4⁺T cell epitopes of EV71 VP1 protein region were identified.

Conclusions

There was a specific T cell response to VP1 protein in children with EV71 infection, and the specific CD4⁺T cells were dominant. The CD4⁺T cell epitopes of VP1 protein were identified in EV71 infected children. It is helpful to elucidate the cellular immunopathogenesis of EV71 infection.

Key words: Enterovirus 71, T cell-mediated immunity, Capsid protein VP1, Enzyme-linked immunospot assays, T cell Epitope

表1 EV71感染HFMD患儿入院时临床特征

临床特征	急性感染期组(16例)	临床恢复期组(9例)	感染恢复后1个月组(6例)
发病年龄(月)^a	23.63(8～49)	18.22(9～41)	28.17(13～43)
性别(女/男，例)	8/8	2/7	4/2
采血时间(发病天数)^a	3(2～4)	11(8～16)	30
病程(d)^a	11.56(8～20)	11.56(8～17)	10.17(8～11)
合并中枢神经系统症状[例(%)]	7(43.75)	1(11.11)	0(0.00)
轻型/重型(例)	6/10	7/2	6/0
hsCRP(mg/L)^a	2.80(0.8～4.7)	8.10(4.7～11.9)	—
最高体温(℃)^a	38.4(38.0～40.0)	38.5(38.0～39.2)	38.2(37.8～38.5)
白细胞计数(10⁹/L)^a	10.98(8.30～14.70)	11.06(6.70～19.90)	7.21(3.81～11.20)

表1 EV71感染HFMD患儿入院时临床特征

临床特征	急性感染期组(16例)	临床恢复期组(9例)	感染恢复后1个月组(6例)
发病年龄(月)^a	23.63(8～49)	18.22(9～41)	28.17(13～43)
性别(女/男，例)	8/8	2/7	4/2
采血时间(发病天数)^a	3(2～4)	11(8～16)	30
病程(d)^a	11.56(8～20)	11.56(8～17)	10.17(8～11)
合并中枢神经系统症状[例(%)]	7(43.75)	1(11.11)	0(0.00)
轻型/重型(例)	6/10	7/2	6/0
hsCRP(mg/L)^a	2.80(0.8～4.7)	8.10(4.7～11.9)	—
最高体温(℃)^a	38.4(38.0～40.0)	38.5(38.0～39.2)	38.2(37.8～38.5)
白细胞计数(10⁹/L)^a	10.98(8.30～14.70)	11.06(6.70～19.90)	7.21(3.81～11.20)

图1 EV71病毒衣壳蛋白VP1特异性T细胞应答强度个体分布

表2 EV71 VP1蛋白在EV71感染患儿的不同阶段T细胞反应强度比较

组别	Z值	P值
急性感染期组vs.临床恢复期组	－2.042	0.041^a
急性感染期组 vs.感染恢复后1个月组	－1.315	0.189
急性感染期组 vs.对照组	－1.678	0.093
临床恢复期组 vs.感染恢复后1个月组	－2.105	0.035^a
临床恢复期组 vs.对照组	－2.633	0.008^a
感染恢复后1个月组 vs.对照组	0.000	1.000

表2 EV71 VP1蛋白在EV71感染患儿的不同阶段T细胞反应强度比较

组别	Z值	P值
急性感染期组vs.临床恢复期组	－2.042	0.041^a
急性感染期组 vs.感染恢复后1个月组	－1.315	0.189
急性感染期组 vs.对照组	－1.678	0.093
临床恢复期组 vs.感染恢复后1个月组	－2.105	0.035^a
临床恢复期组 vs.对照组	－2.633	0.008^a
感染恢复后1个月组 vs.对照组	0.000	1.000

图2 EV71 VP1蛋白在EV71感染患儿的不同阶段特异性T细胞反应强度和频率

表3 EV71 VP1蛋白重叠肽库中含有的CD4⁺ T细胞表位

患者编号	重叠肽段^a	氨基酸位置	应答强度^b(SFUs/10⁶cells)	肽段序列^c	CD4/CD8为主导
1	VP1-22	734～751	711	LAWQTATNPSVFVKLSDP	4
	VP1-23	742～759	736	PSVFVKLSDPPAQVSVPF	?
	VP1-25	758～775	658	PFMSPASAYQWFYDGYPT	?
2	VP1-19	710～727	40	EVVPQLLQYMFVPPGAPK	4
	VP1-28	782～799	46	EKDLEYGACPNNMMGTFS	?
	VP1-30	798～815	53	FSVRTVGSSKSKYPLVIR	?
	VP1-33	822～839	53	HVRAWIPRPMRNQNYLFK	?
	VP1-34	830～847	33	PMRNQNYLFKSNPNYAGD	?

表3 EV71 VP1蛋白重叠肽库中含有的CD4⁺ T细胞表位

患者编号	重叠肽段^a	氨基酸位置	应答强度^b(SFUs/10⁶cells)	肽段序列^c	CD4/CD8为主导
1	VP1-22	734～751	711	LAWQTATNPSVFVKLSDP	4
	VP1-23	742～759	736	PSVFVKLSDPPAQVSVPF	?
	VP1-25	758～775	658	PFMSPASAYQWFYDGYPT	?
2	VP1-19	710～727	40	EVVPQLLQYMFVPPGAPK	4
	VP1-28	782～799	46	EKDLEYGACPNNMMGTFS	?
	VP1-30	798～815	53	FSVRTVGSSKSKYPLVIR	?
	VP1-33	822～839	53	HVRAWIPRPMRNQNYLFK	?
	VP1-34	830～847	33	PMRNQNYLFKSNPNYAGD	?

[1]	Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system[J].J Infect Dis,1974,129(3):304-309.
[2]	高宁,党双锁,李梅.肠道病毒71型手足口病致神经系统发病机制的最新进展[J/CD].中华实验和临床感染病杂志(电子版),2012,6(2):162-164.
[3]	Weng KF, Chen LL, Huang PN, et al. Neural pathogenesis of enterovirus 71 infection[J]. Microbes Infect,2010,12(7):505-510.
[4]	Zhang W, Dai W, Zhang C, et al. A virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity[J]. Emerg Microbes Infect,2018,7(1):94.
[5]	Ooi MH, Wong SC, Lewthwaite P, et al. Clinical features, diagnosis, and management of enterovirus 71[J]. Lancet Neurol, 2010,9(11):1097-1105.
[6]	Solomon T, Lewthwaite P, Perera D, et al. Virology, epidemiology, pathogenesis, and control of enterovirus 71[J]. Lancet Infect Dis,2010,10(11):778-790.
[7]	Ho M, Chen ER, Hsu KH, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group[J]. N Engl J Med,1999,341(13):929-935.
[8]	McMinn PC. An overview of the evolution of enterovirus 71 and its clinical and public health significance[J]. FEMS Microbiol Rev,2002,26(1):91-107.
[9]	Tseng FC,Huang HC,Chi CY, et al. Epidemiological survey of enterovirus infections occurring in Taiwan between 2000 and 2005: Analysis of sentinel physician surveillance data[J].J Med Virol,2007,79(12):1850-1860.
[10]	中华人民共和国国家卫生和计划生育委员会.手足口病的诊疗指南(2010年版)[J].国际呼吸杂志,2010,30(24):1473-1475.
[11]	Dang SS, Li M, Gao N, et al. Clinical feature of 36 critical cases of hand foot and mouth disease[J]. Int J Infect Dis,2011,15(11):S41-S42.
[12]	Wang Y, Feng ZJ, Yang Y, et al. Hand, foot, and mouth disease in China patterns of spread and transmissibility[J]. Epidemiology, 2011,22(6):781-792.
[13]	Chang LY,Hsiung CA,Lu CY, et al. Status of cellular rather than humoral immunity is correlated with clinical outcome of enterovirus 71[J].Pediatr Res,2006,60(4):466-471.
[14]	Dang SS, Gao N, Li M. The pathogenesis during enterovirus 71-induced HFMD[J]. Int J Infect Dis,2011,15(11):S108-S108.
[15]	Fujii K, Sudaka Y,Takashino A, et al. VP1 amino acid residue 145 of enterovirus 71 is a key residue for its receptor attachment and resistance to neutralizing antibody during cynomolgus monkey infection[J].J Virol,2018. [Epub ahead of print].
[16]	Kataoka C,Suzuki T,Kotani O, et al. The role of VP1 amino acid residue 145 of enterovirus 71 in viral fitness and pathogenesis in a cynomolgus monkey model[J].PLoS Pathog,2015,11(7):e1005033.
[17]	Victorio CB,Xu Y,Ng Q, et al. Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)[J].Emerg Microbes Infect,2016,5:e60.
[18]	Foo DGW, Alonso S, Phoon MC, et al. Identification of neutralizing linear epitopes from the VP1 capsid protein of enterovirus 71 using synthetic peptides[J]. Virus Res,2007,125(1): 61-68.
[19]	Foo DGW, Ang RX, Alonso S, et al. Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in western blots[J].Clin Microbiol Infect,2008,14(3):286-288.
[20]	Yang C, Deng C, Wan J, et al. Neutralizing antibody response in the patients with hand, foot and mouth disease to enterovirus 71 and its clinical implications[J]. Virol J,2011,8(16):1261-1263.
[21]	Bae JY, Moon SH, Choi JA, et al. Recombinant DNA and protein vaccines for foot-and-mouth disease induce humoral and cellular immune responses in mice[J]. Immune Netw,2009,9(6):265-273.
[22]	Lindner J, Barabas S, Saar K, et al. CD4(+) T-cell responses against the VP1-unique region in individuals with recent and persistent parvovirus B19 infection[J]. J Vet Med B Infect Dis Vet Public Health,2005,52(7-8):356-361.
[23]	Lundstig A, Eliasson L, Lehtinen M, et al. Prevalence and stability of human serum antibodies to simian virus 40 VP1 virus-like particles[J]. J Gen Virol,2005,86(Pt 6):1703-1708.
[24]	Yuan J,Shen L,Wu J, et al. Enterovirus A71 proteins: structure and function[J].Front Microbiol,2018,9(21):286.
[25]	Foo DGW, Macary PA, Alonso S, et al. Identification of human CD4(+) T-cell epitopes on the VP1 capsid protein of enterovirus 71[J].Viral Immunol,2008,21(2):215-224.

[1]	刘红梅, 郑梅玲, 贺江梅, 张桂林, 化爱玲, 王治鸿. T细胞免疫球蛋白黏蛋白分子-3在女性生殖系统疾病中的研究现状[J]. 中华妇幼临床医学杂志(电子版), 2017, 13(03): 256-260.
[2]	张丽帆, 杨峥蓉, 刘晓清. 程序性死亡受体1/程序性死亡配体在结核分枝杆菌感染免疫中的作用和宿主导向治疗前景[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(03): 145-148.
[3]	袁娟, 邓慧玲, 李亚萍, 吴媛, 李梅, 王军, 张玉凤, 党双锁. 雾化吸入重组人干扰素α1b治疗肠道病毒71型手足口病患儿的疗效[J]. 中华实验和临床感染病杂志(电子版), 2019, 13(06): 478-484.
[4]	阎永红, 刘顺爱, 成军, 梁璞. 人肠道病毒71型感染及其防治研究进展[J]. 中华实验和临床感染病杂志(电子版), 2019, 13(05): 366-369.
[5]	邹莉, 梅文静. EV71感染手足口病患儿T细胞亚群表达[J]. 中华实验和临床感染病杂志(电子版), 2018, 12(04): 397-401.
[6]	王海燕, 王银铃, 朱莉, 陈慧, 钱峰, 李明, 朱伟, 张雪华, 胥萍, 朱传武. 干扰素疗效与慢性乙型肝炎患者外周血CD8⁺ T细胞程序性死亡受体-1和T细胞免疫球蛋白黏蛋白分子3表达的关系[J]. 中华实验和临床感染病杂志(电子版), 2018, 12(01): 40-45.
[7]	王军, 邓慧玲, 袁娟, 张玉凤, 李亚萍, 李梅, 王文俊, 高宁, 党双锁. 淋巴细胞亚群失衡与EV71型手足口病重症化的相关性[J]. 中华实验和临床感染病杂志(电子版), 2017, 11(02): 156-161.
[8]	朱名超, 朱娅, 韩利蓉, 孙莉. 天门地区2014年手足口病病原学检测[J]. 中华实验和临床感染病杂志(电子版), 2016, 10(03): 342-345.
[9]	陈莹, 陈维进. 2010至2013年黄石市手足口病的流行特征及病原学分析[J]. 中华实验和临床感染病杂志(电子版), 2016, 10(01): 73-77.
[10]	邹容容, 张国良, 李建明, 刘映霞. miRNA在肠道病毒71型手足口病诊断及治疗中的应用[J]. 中华实验和临床感染病杂志(电子版), 2016, 10(01): 10-12.
[11]	中华医学会器官移植学分会, 国家肾脏移植质控中心. 肾移植受者人类微小病毒B19感染临床诊疗技术规范(2022版)[J]. 中华移植杂志(电子版), 2022, 16(04): 193-200.
[12]	周梦婕, 梁馨月, 吉前成. TIM-1、TIM-3联合检测对活动性肺结核的诊断意义[J]. 中华肺部疾病杂志(电子版), 2021, 14(06): 788-790.
[13]	梁卓政, 郭程, 孟平, 李洪涛, 张天托. TIM-3-574G>T基因多态性与中国汉族人群支气管哮喘的相关性分析[J]. 中华肺部疾病杂志(电子版), 2019, 12(01): 63-67.
[14]	谢曼芬, 高燕, 蔡晨露, 王肖, 郭会敏. 静注人免疫球蛋白对EV71型感染重症手足口病患儿炎症因子、免疫功能的影响[J]. 中华临床医师杂志(电子版), 2022, 16(02): 136-140.
[15]	张剑, 郭彦伟, 张自森, 王利娟, 张红巧. 胃癌患者外周血T细胞免疫球蛋白黏蛋白-3的表达及临床意义[J]. 中华临床医师杂志(电子版), 2021, 15(08): 621-625.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Enterovirus 71 capsid protein VP1 specifically recognizes and activates CD4⁺ T cell immune activation in children with hand, foot and mouth disease

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Enterovirus 71 capsid protein VP1 specifically recognizes and activates CD4+ T cell immune activation in children with hand, foot and mouth disease

Enterovirus 71 capsid protein VP1 specifically recognizes and activates CD4⁺ T cell immune activation in children with hand, foot and mouth disease