| Objective: To prepare monoclonal antibodies(m Ab)with neutralizing activity against the Marburg virus(MARV)and to provide therapeutic drugs for possible large-scale infections caused by MARV.Methods: AF-03 and MARV GP plasmids were transfected into CHO-S cells using transient transfection.After purification,the m Ab AF-03 and protein MARV GP were obtained.The concentration was determined by the quantitative BCA method and the purity was identified by SDS-PAGE.The neutralization effect of the antibody on MARV pseudovirus was evaluated by VSV and HIV pseudovirus systems.Then,a mouse model infected with MARV pseudovirus was established and the role of the antibody was evaluated in mice.The sites of antibody-GP interaction were obtained by computer modeling and molecular docking,which were subjected to point mutation to identify antibody epitopes.The m Ab AF-04 was expressed in the CHOK1-AF knockout host cell line using fucose gene and was identified by glycoform analysis.ELISA and SDS-PAGE,with no functional change compared to AF-03;the binding action to FcγRIII was verified by NK cell and Jurkat-CD16a-NFAT-Luc cell activation assay.Results:(1)Highly pure AF-03,AF-04,and MARV GP were successfully obtained.(2)AF-03 showed strong neutralizing ability in vitro evaluation.(3)AF-03 showed a good blocking effect in a mouse model of MARV pseudovirus infection.(4)The epitopes of AF-03 were predicted by molecular docking(Q128-N129,Q204-T205-Q206,Y218,K222,C226)with validation by binding and neutralization assays.(5)AF-03 prevented in vitro GP enzymatic cleavage of EBOV,SUDV,and BDBV pseudoviruses from infecting cells.(6)AF-04 has higher binding to FcγRIIIα and NK activation.Conclusions: AF-03 effectively inhibited the entry of MARV pseudoviruses into host cells both in vivo and in vitro of mice as well as crossneutralizing EBOV and other pseudoviruses.Enhanced activity of the m Ab AF-04 ADCC with fucose knockout. |
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