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目的筛选肠道病毒71型(Enterovirus 71,EV71)灭活疫苗免疫小鼠后诱导的细胞免疫抗原表位,探讨EV71疫苗诱导的细胞免疫应答机制。方法以EV71 BJ08株(C4基因型)病毒结构蛋白氨基酸序列为模板,合成覆盖EV71 VP1～VP3的256条合成肽(每条肽12个氨基酸),分别以其为刺激物(80μg/ml),采用ELISPOT法测定EV71灭活疫苗免疫小鼠脾单个核细胞(Mononuclear cell,MNC)诱导IFNγ分泌的斑点形成细胞数(Spot-forming cell,SFC),筛选细胞免疫抗原表位。从脾MNC中去除CD4+或CD8+T细胞及封闭MHCⅡ类分子或MHCⅠ类分子,分析抗原提呈途径。结果通过对256条合成肽的筛选,获得3条可刺激小鼠脾MNC高水平分泌IFNγ的合成肽。以3条合成肽作为刺激物时,小鼠脾MNC分泌IFNγSFC均值(单针免疫组:22.3、12.8、17.4;两针免疫组:29.3、44.1、28.5)较乙肝表面抗原对照肽S28-3(9单针:2.1;两针:5.2)显著升高(P<0.01)。当MNC中去除CD4+T细胞或封闭MHCⅡ类分子后,IFNγSFC均值显著下降(P<0.05);而去除CD8+T细胞或封闭MHCⅠ类分子时,IFNγSFC均值未见显著性差异(P>0.05)。结论已成功筛选出3个EV71细胞免疫抗原表位,分别位于VP1、VP2和VP3区域,3条合成肽均属MHCⅡ类限制性多肽。
Objective To screen the cellular immune epitope induced by Enterovirus 71 (EV71) inactivated vaccine and to explore the cellular immune response mechanism induced by EV71 vaccine. Methods A total of 256 synthetic peptides (12 amino acids per peptide) covering EV71 VP1 ~ VP3 were synthesized based on the amino acid sequence of structural protein of EV71 BJ08 strain (C4 genotype) ELISPOT method was used to determine the spot-forming cell (SFC) of IFNγ secreted by spleen mononuclear cells (MNCs) immunized with EV71 inactivated vaccine, and the cellular immune epitopes were screened. The CD4 + or CD8 + T cells were removed from the splenic MNC and the MHC class II or MHC class I molecules were blocked and the antigen presentation pathway was analyzed. Results By screening 256 synthetic peptides, three synthetic peptides that stimulated the secretion of IFNγ from mice spleen MNC were obtained. When three synthetic peptides were used as stimuli, the average of IFNγSFCs secreted by mouse spleen MNCs (single-needle immunized group: 22.3,12.8,17.4; two-needle vaccinated groups: 29.3,44.1,28.5) was higher than that of hepatitis B surface antigen control peptide S28-3 9 single-needle: 2.1; two-needle: 5.2) was significantly increased (P <0.01). When CD4 + T cells were removed or the MHC class II molecules were blocked in MNC, the average value of IFNγSFC was significantly decreased (P <0.05). However, no significant difference was found in the average of IFNγSFCs when CD8 + T cells were blocked or MHC class I molecules were blocked (P> 0.05) . Conclusion Three EV71 cell epitopes have been successfully screened, which are located in the VP1, VP2 and VP3 regions, respectively. All three synthetic peptides belong to MHC class Ⅱ restricted peptides.