口蹄疫病毒O型东南亚拓扑型抗原变异研究

doi:10.3864/j.issn.0578-1752.2024.15.014

摘要/Abstract

摘要：

【目的】O型口蹄疫病毒（foot-and-mouth disease virus，FMDV）不断变异，易导致现用疫苗与流行毒株的抗原匹配性下降引起免疫效果不佳，疫情零星散发。近年来，O型FMDV中SEA（东南亚）拓扑型流行活跃且不断变异，持续对口蹄疫防疫产生压力。系统解析O型FMDV毒株特异性抗原位点以及不同拓扑型毒株的序列差异，明确抗原变异的关键氨基酸，可为FMD抗原分子设计提供依据，为FMD防控提供指导。【方法】利用10株（SEA拓扑型）毒株特异性牛源单克隆中和抗体，对毒株O/GSLX/2010（O/Mya/98谱系）进行抗体压力筛选逃逸突变株，鉴定这10株抗体所识别表位的关键氨基酸。利用牛源多抗血清样本（32份）与逃逸突变毒株进行病毒交叉中和试验，分析SEA拓扑型毒株的免疫优势表位；通过序列比对分析不同拓扑型毒株在该抗原表位上的差异。利用反向遗传技术将差异性抗原表位引入O型FMDV经典疫苗株O/HN/CHA/93（Cathay拓扑型），对拯救的点突变毒株测序分析后进行间接免疫荧光试验鉴定，并通过病毒蚀斑测定与一步生长曲线检测病毒的复制动力学。利用SEA拓扑型毒株特异性单克隆中和抗体，通过中和试验分析拯救突变株的抗原性，确定影响病毒抗原性的关键氨基酸，评估毒株特异性位点氨基酸的改变对抗原谱的影响。【结果】SEA拓扑型特异性抗原表位主要集中于结构蛋白VP1的B-C/C-D环，属于抗原位点3。10株抗体中多数抗体（8/10）识别的抗原表位在VP1上，其中有6株单抗（A19、B55、B74、C5、F53和F166）识别的关键氨基酸位于VP1 B-C环（T43、K45）及C-D环（P58），另外2株单抗（B66和F41）识别的关键氨基酸位于VP1 C末端。病毒交叉中和试验结果表明，VP1上43位和VP3上131位氨基酸的改变显著降低了牛源多抗血清的抗体效价。对O型FMDV不同谱系毒株（26株）序列进行分析，发现三种拓扑型毒株VP1 B-C/C-D环的28、47、56和58位氨基酸不同。利用反向遗传技术成功将毒株特异性抗原表位引入Cathay拓扑型毒株（O/HN/CHA/93），拯救出6 株FMDV的点突变株：POZ-GSLX-M58、POZ-GSLX-M56/58、POZ-GSLX-M28/58、POZ-GSLX-M47/58、POZ-GSLX-M28/58/47和POZ-GSLX-M47/56/58。微量中和试验结果表明，VP1上56/58位对毒株抗原性起到关键作用；然而，进一步增加28位和47位突变会降低抗体B83的中和作用，影响毒株自身的抗原性。因此，VP1上56和58位氨基酸的改变可以扩展SEA拓扑型特异性中和mAbs对O/HN/CHA/93毒株的中和效力与广度。【结论】O型FMDV结构蛋白VP1上56和58位氨基酸是引起SEA拓扑型毒株抗原变异的关键位点，引入这些抗原决定簇可以拓展FMDV O/HN/CHA/93的抗原谱。研究为FMD防控及疫苗设计提供参考。

关键词: 口蹄疫病毒, 毒株特异性中和抗体, 抗原变异

Abstract:

【Objective】The continuous variation of foot-and-mouth disease virus (FMDV) serotype O easily leads to the decrease of antigen matching between the currently used vaccine and the circulating strain, resulting in poor immune effect and sporadic outbreaks. In recent years, Southeast Asia (SEA) topotype epidemic of FMDV serotype O has been active and constantly changing, which has continued to exert pressure on foot-and-mouth disease prevention. Systematic analysis of specific antigen sites of serotype O FMDV strains and sequence differences of different topological strains and identification of key amino acids of antigen variation could provide basis for FMD antigen molecular design, and provide guidance for FMD prevention and control. 【Method】In this study, 10 bovine-derived monoclonal neutralizing antibodies specific for SEA topotype strains were used to screen escape mutants by antibody pressure in strain O/GSLX/2010 (O/Mya/98 lineage) to identify the key amino acids. The viral cross-neutralization tests were performed using bovine polyclonal antiserum samples (32 samples) and escape mutants to analyze the immunodominant epitopes of SEA topotype strains. The differences in this epitope between different topotype strains were analyzed by sequence alignment. Reverse genetic technique was used to introduce differential epitopes into FMDV serotype O classic vaccine strain O/HN/CHA/93 (Cathay topotype), and the rescued point mutant strains were sequenced and identified by indirect immunofluorescence assay. Virus plaque detection and one-step growth curve were used to detect the replication dynamics of the virus. Using SEA topotype strain specific monoclonal neutralization antibodies, the antigenicity of the rescue mutant was analyzed by neutralization test, the key amino acids affecting the antigenicity of the virus were identified, and the influence of the amino acids changes at the specific site of the strain on the antigen profile was evaluated. 【Result】SEA topotype-specific epitopes were mainly concentrated in the B-C and C-D loop of VP1, which belonged to antigenic site 3. Most of the 10 mAbs (8/10) recognized epitopes on VP1, among which 6 mAbs (A19, B55, B74, C5, F53, and F166) recognized key amino acids located in the B-C loop (T43, K45) and C-D loop (P58) of VP1, and the other 2 mAbs (B66 and F41) identified key amino acids at the C-terminus of VP1. The results of virus cross-neutralization test showed that the changes of amino acids 43 of VP1 and 131 of VP3 significantly reduced the antibody titer of bovine polyclonal antisera. The sequence analysis of different lineages of FMDV serotype O (26 strains) revealed that amino acids 28, 47, 56, and 58 in the B-C/C-D loop of VP1 were different in the three topotype. Using reverse genetic technology, the virus-specific epitope was successfully introduced into Cathay topotype (O/HN/CHA/93) and saved 6 FMDV point mutants: POZ-GSLX-M58, POZ-GSLX-M56/58, POZ-GSLX-M28/58, POZ-GSLX-M47/58, POZ-GSLX-M28/58/47, and POZ-GSLX-M47/56/58. The results of microneutralization test showed that 56/58 VP1 played a key role in the antigenicity of the virus. However, further additions of mutations at positions 28 and 47 reduced the neutralization of antibody B83, affecting the antigenicity of the strain itself. Therefore, the changes in amino acids at positions 56 and 58 on VP1 could extend the effectiveness and breadth of SEA topotype specific neutralization of mAbs against O/HN/CHA/93 strains.【Conclusion】Amino acids 56 and 58 on the structural protein VP1 of FMDV serotype O were key sites causing antigenic variation in SEA topotype, and introducing these antigenic determinants could expand the neutralization potency and breadth of SEA topotype-specific neutralizing mAbs, and effectively expand the antigenic profile of FMDV O/HN/CHA/93. This study provided an important reference for FMD prevention and vaccine design.

Key words: foot-and-mouth disease virus, strain specific neutralizing antibody, antigenic variation

王莉, 李坤, 黄书伦, 李凤娟, 王省, 卢曾军, 刘在新. 口蹄疫病毒O型东南亚拓扑型抗原变异研究[J]. 中国农业科学, 2024, 57(15): 3093-3104.

WANG Li, LI Kun, HUANG ShuLun, LI FengJuan, WANG Sheng, LU ZengJun, LIU ZaiXin. Antigenic Variation of Foot-and-Mouth Disease Virus Serotype O in Southeast Asia Topotype[J]. Scientia Agricultura Sinica, 2024, 57(15): 3093-3104.

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单克隆抗体 mAbs	中和抗体效价 Neutralization antibody titer
单克隆抗体 mAbs	O/GSLX/2010	O/CHA/Mya33	O/GZSB	O/Mya/98	O/HNNY/2022
A19	>50.00	>50.00	48.69	4.56	35.41
B55	49.92	>50.00	24.04	2.54	>50.00
B66	37.50	>50.00	>50.00	1.76	>50.00
B74	3.91	2.93	2.81	1.17	>50.00
B83	16.64	27.56	46.42	6.89	>50.00
C5	>50.00	>50.00	>50.00	17.40	>50.00
F41	3.96	6.33	2.64	1.32	>50.00
F53	17.63	13.12	35.25	3.30	47.00
F136	31.57	31.57	15.42	2.59	14.73
F166	4.29	2.87	2.87	0.72	11.47

单克隆抗体 mAbs	亲本毒 Parent virus	突变位置 Residue change	突变频率 Frequency of mutants	中和浓度 Neutralization concentration (μg·mL^-1)
B74	O/GSLX/2010	VP1 T43P	1/8	>600
		VP1 P58S	3/8	>600
		VP1 T43A, P58S	4/8	>600
F166	O/GSLX/2010	VP1 P58S	3/5	>600
F166	O/GSLX/2010	VP1 K45N, P58S	2/5	>600
B55	O/GSLX/2010	VP1 P58S	5/6	>600
B55	O/GSLX/2010	VP1 P58H	1/6	>600
A19	O/GSLX/2010	VP1 P58S	4/4	>600
C5	O/GSLX/2010	VP1 P58S	3/4	>600
C5	O/GSLX/2010	VP1 V209A	1/4	>600
F53	O/GSLX/2010	VP1 K45Q	2/5	>600
F53	O/GSLX/2010	VP1 D99G	3/5	>600
F41	O/GSLX/2010	VP1 A199D	5/8	>600
		VP1 A199D; VP3 E131D	1/8	>600
		VP2 P74S; VP3 E131D	1/8	>600
		VP1 A199D; VP3 E131D	1/8	>600
B66	O/GSLX/2010	VP1 V209A	2/5	>600
		VP1 A207S	1/5	>600
		VP1 Q211R	1/5	>600
		VP1 Q211R; VP2 V199E, N202K; VP3 G196V	1/5	>600
B83	O/GSLX/2010	VP2 P195T	2/6	>600
B83	O/GSLX/2010	VP2 P195S	4/6	>600
F136	O/GSLX/2010	VP3 E131D	6/7	>600
F136	O/GSLX/2010	VP1 A199D; VP3 E131D	1/7	>600