埃博拉假病毒突变体感染性及靶向抗体中和作用评价

Evaluation of infectivity of Ebola pseudovirus mutants and neutralization of targeted antibodies

目的:利用埃博拉假病毒(EBOV)报告系统,体外评价埃博拉病毒突变体对4种细胞系的相对感染力及3株单抗对突变体的中和能力。方法:构建表达埃博拉假病毒及其突变体GP蛋白的重组质粒GP-pcDNA3.1,与含有萤火虫荧光素酶报告基因的HIV骨架载体pSG3.ΔEnv.CMV.Fluc共转染HEK293T细胞获得假病毒上清。利用Western blot法及HIV p24 ELISA抗原定量试剂盒对假病毒进行鉴定及定量,采用等量的假病毒感染HEK293T、Huh7、A549及THP-1细胞,36 h后,裂解细胞测荧光素酶活性(RLU),计算突变体与母本的相对感染力。将mAb114、ADI-15946、rEBOV-520与EBOV及其突变体假病毒预孵育1 h,将假病毒上清和抗体混合液感染HEK293T细胞,36 h后测RLU值,计算抑制率。结果:相对于母本,所有突变体感染细胞的能力均有不同程度增强。单抗mAb114、ADI-15946能有效中和母本及14株突变体,rEBOV-520对N107D-P330S-G480D中和作用减弱。结论:体外实验证明14株突变体入侵靶细胞能力增强。3株中和抗体对突变体中和作用良好,为应对EBOV突变引发的疫情提供了一定参考。

Objective:To evaluate the relative infectivity of Ebola virus(EBOV)mutants against four cell lines and the neutralizing ability of three monoclonal antibodies against mutants in vitro using the Ebola pseudovirus reporter system.Methods:The recombinant plasmid GP-pcDNA3.1 expressing Ebola pseudovirus and its mutant glycoprotein GP protein were constructed respectively and co-transfected with HIV backbone vector pSG3.ΔEnv.CMV.Fluc containing firefly luciferase to obtain pseudovirus supernatant.The pseudoviruses were identified and quantified by Western blot and HIV p24 ELISA kit method.HEK293T,Huh7,A549 and THP-1 cells were infected with the same amount of pseudoviruses.After 36 h,the cells were lysed to measure the luciferase activity(RLU).Relative infectivity of the mutants compared with the parents was calculated.mAb114,ADI-15946,rEBOV-520 were pre-incubated with EBOV and its mutants for 1 h,and the mixtures was added into the culture of HEK293T cells.After 36 h,the RLU value was measured and the inhibition rate was calculated.Results:All mutants had enhanced ability to infect host cells at variable degrees compared to the parents.mAb114 and ADI-15946 exhibited neutralizing activity to the parents and all 14 mutants.rEBOV-520 also showed neutralizing activity to the mutants,except for N107D-P330S-G480D.Conclusion:Fourteen mutants have enhanced ability to invade host cells in vitro.The neutralizing antibodies of the three strains have prominent effects on the mutants,which provides potential therapeutic cargos to deal with the outbreak of EBOV mutants in future.