Research On Mechanism Of A Specific AB 82A6 Neutralized SARS-CoV-2 Via S1 Shedding

Background and Objective:COVID-19 caused by SARS-CoV-2 seriously threatens the safety of human life.The development of the neutralizing Abs begins since the outbreak of SARS-CoV-2 infection and is well underway.The developed neutralizing monoclonal Abs（n Abs）exerted their neutralizing function by targeting SARS-CoV-2 spike（S）protein,the viral membrane expressed protein responsible for the viral entry into host cells.S protein is composed of two subunits,S1 subunit and S2 subunit.The RBD on S1 subunit is the core region for the host cell receptor ACE2 engagement,thus is used as the major target of neutralizing Abs for blocking the virus entry.Except their neutralizing mechanism of the occlusion of ACE2 for RBD,some of the RBM specific Abs were reported to trigger S1 shedding to elevate the total neutralization capacity.The epitopes for the majority of these neutralizing Abs were mainly distributed on RBM,the ACE2 binding sites.As another proportion of RBD specific Abs,the non-RBM specific Abs can also neutralize the virus.But the neutralizing mechanism of the non-RBM n Abs remain poorly understood.S protein in the membranes of SARS-CoV-2-infected cells drives neighboring cells expressing ACE2 to fuse and form syncytia and it is unknown whether the non-RBM n Abs can also influence Spike mediated cell-cell fusion and syncytia formation.We obtained more than 200 SARS-CoV-2 RBD specific Abs from the COVID-19 convalescent patients,and verified the function of these antibodies.Among these RBD specific Abs,82A6 was found to efficiently neutralize SARS-CoV-2 via independent of receptor-binding inhibition.Therefore,82A6 is a non-RBM specific antibody.The main purpose of this study is to investigated the neutralizing mechanism of 82A6 and to provide a research basis for the prevention and diagnosis of COVID-19 and the development of antiviral drugs.Methods:In this study,the antibody sequence was cloned into the expression plasmid and expressed m Ab in Expi293^TM cells.The purity of 82A6 was detected by silver stain.Then the binding ability was detected by ELISA and the affinity of the antibody was determined by SPR.The neutralization ability of 82A6 was evaluated by pseudovirus neutralization assay and plaque reduction neutralization test（PRNT）.The receptor-blocking assay was evaluated by competitive ELISA,SPR and Flow cytometry.We identified the binding ability of 82A6 to the denatured RBD via Western blot analysis.Then,we utilized previously designed and synthesized 20-mer peptides（RBD1 to RBD15）to elucidate the antigenic site recognized by 82A6.These peptides overlapped with 5 amino acids to cover the entire RBD,as amino acids 319-541 of SARS-CoV-2 S(S^319-541).To further determine the essential amino acid residues accounted for 82A6 binding in the region represented by RBD13,to which 82A6 bound with higher capability,we then individually replaced each amino acid residue in RBD13 with alanine（A）.We further explore the neutralization mechanism of 82A6.First,we transfected SARS-CoV-2 S protein into 293T cells（293T/S）to construct the S expressed cells and incubated 82A6 with these cells to assess its ability to trigger S1 dissociation.Then,we utilized the binding ability of 293T/ACE2 cells to the S protein to confirm the presence of the S1 subunit in the cell supernatant from the incubation system of293T/S cells and 82A6.In parallel,the presence of the S1 subunit in the cell supernatant was tested via Western blot.Finally,we perform cell-cell fusion experiment to explore whether antibodies inhibit the formation of syncytia.Results:The plasma of COVID-19 convalescent patients was highly responsive to SARS-CoV-2 S1 protein and RBD protein.We identified the RBD specific antibody 82A6 from the peripheral blood of COVID-19convalescent patients.82A6 efficiently neutralized both authentic SARS-CoV-2 virus(IC₅₀ was 0.100μg/m L)and pseudovirus bearing SARS-CoV-2 S protein(IC₅₀ was 0.06μg/m L)with high binding affinity to RBD(EC₅₀=0.012μg/m L,Kd=2 n M).We found that 82A6 did not interrupt the interaction of ACE2 and RBD.Then we further investigated the precise epitope recognized by 82A6 and found that 82A6 was capable to bind to the denatured RBD,and 82A6 mainly bound to RBD2(S^334-353)in the spatial structure.In order to further investigated the neutralization mechanism of 82A6,we found that the expression level of the S1 subunit on 293T/S cells in 82A6 group was decreased in a time-dependent manner,whereas CR3022 group,as negative control,failed to promote the S1dissociation.The cell supernatant was found to be capable to interact with293T/ACE2 cells,and the detected protein was increased with the elevated incubation quality of 82A6.The addition of 82A6 into 293T/S cells led to the formation of S1 band at approximately 130 k Da via Western blot.Finally,EGFP labeled 293T/S and co-incubated with 293T/ACE2 in the presence of 82A6,we found that there was no syncytia formation in the82A6 incubation group,but the syncytia formation was observed in the cells incubated with CR3022.Conclusion:1.82A6 efficiently neutralized both authentic SARS-CoV-2 virus and pseudovirus bearing SARS-CoV-2 S protein with high binding affinity to RBD.2.82A6 was capable to bind to the denatured RBD,and 82A6 mainly bound to RBD2（S334-353）in the spatial structure,far away from the receptor-binding interface.3.82A6 did not interrupt the interaction of SARS-CoV-2 RBD-ACE2.4.82A6 triggered S1 shedding and blocked the syncytia formation.5.The neutralizing mechanism of 82A6 against SARS-CoV-2 is independent of receptor-binding inhibition.