一株降低了抗体依赖性增强效应的西尼罗病毒中和抗体的表达与体外活性研究  

Expression and in vitro activity of a neutralizing antibody against West Nile virus that reduces antibody-dependent enhancement

作　　者：郝相君 陈楠[2] 朱婉露 王晶 陈国江 乔春霞 李新颖 沈倍奋 冯健男 柴立辉[2] 肖鹤 Hao Xiangjun;Chen Nan;Zhu Wanlu;Wang Jing;Chen Guojiang;Qiao Chunxia;Li Xinying;Shen Beifen;Feng Jiannan;Chai Lihui;Xiao He(Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Joint National Laboratory for Antibody Drug Engineering,Henan University,Kaifeng 475004,China)

机构地区：[1]军事科学院军事医学研究院毒物药物研究所,北京100850 [2]河南大学抗体药物开发技术国家地方联合工程实验室,开封475004

出　　处：《中华微生物学和免疫学杂志》2024年第1期44-49,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家科技重大专项(2018ZX10101003-005-009)。

摘　　要：目的建立降低抗体依赖性增强(antibody-dependent enhancement,ADE)效应的抗体表达系统,以期降低目标抗体的ADE效应。方法对哺乳细胞抗体表达载体Fc区进行L234A和L235A点突变,建立抗体表达载体pFRT-IgG1κ-FcM。选取前期工作中获得的1株具有显著ADE效应的抗体Wt-WNV利用pFRT-IgG1κ-FcM系统进行表达,获得突变后抗体FcM-WNV。ELISA检测FcM-WNV与靶抗原西尼罗病毒包膜蛋白DⅢ(West Nile virus envelope protein-DⅢ,WNV E-DⅢ)的结合,流式细胞术分析FcM-WNV与人高亲和力IgG Fc受体hFcγRⅠ(hCD64)的结合。假病毒感染宿主细胞(BHK21和K562)试验检测FcM-WNV的体外中和活性。结果FcM-WNV与Wt-WNV表达水平相当,能识别结合WNV E-DⅢ,且呈浓度依赖效应;与Wt-WNV相比,FcM-WNV与hCD64的结合力明显减弱,表现为荧光强度明显降低;与前期实验结果一致,Wt-WNV在5μg/ml的浓度下具有明显增强WNV假病毒感染K562细胞的作用,而FcM-WNV在5μg/ml浓度下,能有效阻断假病毒感染K562和BHK21细胞。结论本研究建立的抗体表达系统可有效降低目标抗体的ADE效应。Objective To establish an antibody expression system to reduce the antibody-dependent enhancement(ADE)effect of target antibody.Methods Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM.An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV.The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ(WNV E-DⅢ)was detected by ELISA,and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ(hCD64)was analyzed by flow cytometry.The neutralizing activity of FcM-WNV in vitro was detected by pseudovirus infection of host cells(BHK21 and K562).Results The expression levels of FcM-WNV and Wt-WNV were comparable,and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner.Compared with Wt-WNV,the binding ability of FcM-WNV to hCD64 was significantly weakened,showing a significant decrease in fluorescence intensity.Consistent with the previous experimental results,Wt-WNV at a concentration of 5μg/ml significantly enhanced the infection of K562 by WNV pseudovirus,while FcM-WNV at a concentration of 5μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells.Conclusions The established antibody expression system can effectively reduce the ADE effect of the target antibody.

关 键 词：中和抗体 西尼罗病毒 抗体依赖性增强效应 

分 类 号：R392[医药卫生—免疫学]