汉滩病毒Gc的细胞免疫反应性评估和验证

Evaluation and validation on cellular immunoreactivity of Hantaan virus Gc

目的筛选、鉴定并验证汉滩病毒包膜糖蛋白C末端(Hantaan virus glycoprotein C-terminal,HTNV Gc)上免疫反应性表位。方法在预测覆盖率超过全球98%人群主要组织相容性复合物(major histocompatibility complex,MHC)基因基础上,通过免疫表位数据库(Immune Epitope Database,IEDB)、丹麦技术大学(Technical University of Denmark,DTU)等免疫信息学前沿算法系统分析HTNV Gc上9肽和15肽表位的亲和力,使用Vaxijen工具分析免疫原性,使用Blastp工具分析种间种内保守性;双向层次聚类比较MHC-Ⅰ类和Ⅱ类限制性表位的交叉免疫反应性;使用分子对接模拟出优势表位与MHC对接的可能结果;通过酶联免疫斑点(ELISpot)实验验证计算机分析结果。结果将多种生物信息学算法结果整合后,获得高亲和力、种间种内保守和强免疫原性的9肽和15肽表位,分别为8和80个,其中人鼠共同优势表位8个;聚类分析证明H-2基因型与HLA基因型存在部分相似;分子对接结果模拟出下优势表位与多个MHC基因型对接良好,且与亲和力分析结果一致;在非优势表位对比下,优势表位组小鼠脾细胞分泌IFN-γ的量更高。结论使用多种生物信息学算法成功筛选出HTNV Gc上优势表位,并通过ELISpot实验鉴定了上述表位的细胞免疫反应性。

Objective To screen,identify and verify the immunoreactive epitopes on Hantaan virus glycoprotein C-terminal(HTNV Gc).Methods On the basis of predicting the major histocompatibility complex(MHC)gene in more than 98%of the global population,the affinity of 9-peptide and 15-peptide epitopes on HTNV Gc was systematically analyzed by bioinformatics cutting-edge algorithms such as Immune Epitope Database(IEDB)and Technical University of Denmark(DTU).Their immunogenicity was analyzed by using the Vaxijen tool,and the interspecies intraspecific conservation was analyzed with the Blastp tool.Bidirectional hierarchical clustering was employed to compare the cross-immunoreactivity of MHC-classⅠand classⅡrestricted epitopes.Possible results of dominant epitopes docking with MHC were simulated by using molecular docking.Enzyme-linked immunospot(ELISpot)assay was performed to validate the results of computer analyses.Results After integrating the results of various bioinformatics algorithms,we obtained 89-peptide epitopes and 8015-peptide epitopes with high affinity,intraspecific conservation and strong immunogenicity,including 8 human-mouse common dominant epitopes.Cluster analysis showed that H-2 genotype had certain similarity as HLA genotype.The results of molecular docking revealed that dominant epitopes had good docking with various MHC genotypes,which were consistent with the results of affinity analysis.The amount of IFN-γsecreted from the spleen cells was higher in the mice with dominant epitopes than those with non-dominant epitopes.Conclusion With the aids of various bioinformatics algorithms,we successfully screen out the dominant epitopes on HTNV Gc,and validate the cellular immunoreactivity of these epitopes through ELISpot assay.