Screening Of Neutralizing Antibody Combinations Against SARA-CoV-2

BackgroundThe global COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection has led to severe public health burden and socioeconomic crises.Neutralizing antibodies have been proven to be highly effective in treating mild and moderate COVID-19 patients.However,a series of Omicron variants resulting from rapid mutation of the virus escape most commercially therapeutic antibodies.In the face of mutating SARS-CoV-2 and the threat of resistance to a single SARSCoV-2 neutralizing antibody,screening or designing a cocktail of broad-spectrum neutralizing antibodies targeting highly conserved sites of SARS-CoV-2 and antibody combinations(Cocktail)may be an effective measure to address immune escape of variant strains of the virus.To date,no cocktail of antibodies or broad-spectrum antibodies has been shown to be fully immune to mutation-mediated antibody escape.Taken together,the development of new cocktail therapies targeting different epitopes could provide additional therapeutic options for COVID-19.In a previous experimental study,we used B cells culture combined with antibody cloning to screen a series of specific antibodies targeting different epitopes of SARS-CoV2 from peripheral blood of convalescent patients infected SARS-CoV-2.The antibodies were classified into NTD,RBD and S2 specific antibodies based on binding to the viral S glycoprotein.This study is intended to investigate the neutralizing activity of specific antibodies against Omicron and their ability to bind to its key mutation sites,and to provide a useful basis for subsequent antibody cocktail therapy to prevent viral immunity escape.Part 1 Effect of key mutation sites in Omicron variant on the recognition of human-derived neutralizing antibodiesObjectiveThe S1 plasmid containing Omicron key mutation sites was constructed using a targeted mutation method,and the binding ability of antibodies 825,843,809,826 and 846 to S1 protein at the mutation sites was analyzed using a capture ELISA to clarify the effect of key mutation sites on antibody recognition and to predict which mutation sites produced escape to the five antibodies.Methods1.Construction and expression of key mutation site plasmidsDesign primers containing key mutation sites,amplify the target sequence containing mutation sites by PCR,transform the target sequence into DH5α,pick clones and send them for sequencing,compare the sequencing results with the template plasmid sequence by SnapGene,and if the sequence is the target mutation site,expand the production plasmid and transfect it into HEK293T cells for mutation site proteins expression.2.Capture ELISA methodThe successfully expressed S1 protein antigen containing mutant locus can be captured by using anti-mouse antibody in 96 ELISA plate,adding the screened neutralizing antibody,and then adding goat anti-human IgG(H+L)with HRP labeling,and developing the color under the catalyst of substrate TMB.The antibody was analyzed for binding ability to the Omicron S1 protein containing key mutation sites.Results1.Selection of a series of key mutation sitesBased on extensive literature reports,this experiment summarizes some of the key mutation loci causing the escape of approved therapeutic antibodies against COVID-19,T19I,del 24-26,A27S,G142D,del 143-145,S371L,S371F,S373P,S375F,D405N,K417N,N440K,G446S,E484A,Q493R,G496S,Q498R,N501Y,Q493R+G496S,Q493R+Q498R.2.Analysis of plasmid sequencing results for a series of key mutant sitesUsing SnapGene software,the sequencing results from Biotech were compared with the template plasmid sequence,and a plasmid containing 20 key mutation sites was successfully constructed.3.Identification of S1 protein expression containing key mutation sitesThe above constructed partial key mutant locus plasmids were transfected with HEK293T cells for expression,and all key mutant locus proteins were successfully expressed with correct protein size by Western Blot analysis for expression identification.4.Selection of antigen dilution at key mutation sitesIn preparation for the ELISA experiments to follow,to avoid over-saturation of the antigen used and to improve the sensitivity of the experiments,gradient dilutions of the antigen at the expressed key mutation sites were performed in this study,and the antigen was quantified with specific antibodies and the appropriate antigen dilution was selected.5.Effect of S1 protein containing Omicron key mutation site on antibody binding ability809,826 and 843 have reduced binding ability to S1 proteins bearing single point mutations S371F,S375F and G446S,and S371F and S375F reduce the binding ability of 809,826,825 and 843 to S1 proteins.T19I,S371F and del143-145 reduced the binding ability of monoclonal antibody 846 to S1 protein in the NTD region,and mutated sites in the RBD region had essentially no effect on the 846 antibody.ConclusionsThe 5 antibodies have good binding ability to most of key mutation sites of Omicron variant.S371F,S375F and G446S reduced the binding ability of antibodies 809,826,825,843,and T19I,del143-145 and S371F reduced the binding ability of antibody 846,further predicting that mutations in these sites could potentially make the 5 antibodies less neutralizing or escaping to the Omicron variant with reduced neutralization or escape.Part 2 Establishment of flow cytometry-based SARS-CoV-2 pseudovirus neutralizing assay and assessment of the neutralizing assay of human-derived neutralizing antibodies against Omicron mutant strainObjectiveTo establish a SARS-CoV-2 pseudovirus neutralization system and explore the neutralization effect of obtained neutralizing antibodies 825,843,809,826 against SARSCoV-2 RBD region and 846 against NTD region against Omicron mutant strain,which provides a valid basis for the subsequent antibody cocktail therapy to prevent virus escape mutation.Methods1.Establishment of flow cytometry-based SARS-CoV-2 pseudovirus neutralization assay The production of a SARS-CoV-2 lentiviral pseudovirus expressing the Enhanced Green Fluorescent Protein(eGFP)was performed using three plasmids psPAX2,pLVXeGFP,and p19eH-IgG1-WT S or pcDNA3.1(+)-Omicron S transfected into HEK293T cells.The conditions for pseudovirus packaging were optimized on the coding sequence of S protein,a truncated S protein and harvesting time post transfection.The titers of pseudovirus were measured by infecting 293T cells with stable expression of hACE2(293T-hACE2)and quantifying the rate of 293T cells with eGFP on flow cytometry.2.Detection of antibody neutralizing activity against pseudovirusThe established neutralization assay was used to analyze the neutralization activity of 825、843、809、826 and 846 antibodies against SARS-CoV-2 WT and Omicron pseudovirus.Results1.Construction of SARS-CoV-2 S protein expression plasmidIn this study,a recombinant expression vector for SARS-CoV-2 WT S protein with a C-terminal truncation of 19 amino acids(p19eH-IgG1-WT S)and a recombinant expression vector for Omicron S protein using a C-terminal truncation of 21 amino acids(pcDNA3.1(+)-Omicron S)were successfully constructed.Sequencing analysis showed that the gene encoding the S protein had been successfully inserted into the vector,and the sequence was correct.Meanwhile,the S protein recombinant expression vectors of both virus strains were verified by double digestion,and the results of agarose gel electrophoresis showed that the size of the digested target S gene was consistent with the expected size,which was about 3.8 kb.2.Expression and identification of SARS-CoV-2 S proteinExpression plasmid p19eH-IgG1-WT S or pcDNA3.1(+)-Omicron S were instantly transfected into HEK293T cells,respectively.The expression of S protein was first identified by Western Blot,which showed that both WT and Omicron showed 2 major protein bands,consistent with the expected size of S protein(approximately 180 kD)and S1 protein(approximately 110 kD).The cell surface S protein expression was further verified by immunofluorescence,and the results showed that both WT and Omicron variants expressed S proteins on the surface of HEK293T cells,whereas HEK293T cells transfected with the empty vector did not express any S protein.These data suggest that recombinant plasmids carrying WT or Omicron S protein can be used for pseudovirus packaging.3.Packaging and optimization of pseudovirusIn this study,three plasmids lentivirus packaging system based on the HIV-1 backbone was used to successfully package a pseudovirus with eGFP by co-transfecting psPAX2,pLVX-eGFP and p19eH-IgG1-WT S or pcDNA3.1(+)-Omicron S plasmids in HEK293T cells.In our study,we observed the effect of transfection of viral fluids collected at 24h,48h and 72h on the titer of packaging pseudovirus.The results showed that the pseudovirus titer of both WT and Omicron variants reached peak levels at 72h,73.03%and 69.67%,respectively.4.Establishment and validation of flow cytometry-based SARS-CoV-2 pseudovirus neutralization assayIn this study,the virus neutralizing ability of the established method system was tested using plasma from patients infected Omicron variant,and the results showed that the established flow-based pseudovirus neutralization assay method is specific.We also determined the antibody neutralizing activity of the same plasma samples using the routinely luciferase assay(LUCA)system and correlated the results with those obtained by the FCM method.The results showed that the established FCM method has the same sensitivity as the routinely LUCA method.We also performed a reproducible assay of SARS-CoV-2 WT pseudovirus packed by the established FCM method using antibody REGN10933 with an average IC50 of 4.358 ng/mL,which is consistent with the previously reported results.5.Detection of antibody neutralizing assay against pseudovirusAntibodies 826,809,825 and 843 all neutralized SARS-CoV-2 WT pseudovirus;826,809 and 825 effectively neutralized the Omicron variant;843 antibody show immune escape against Omicron variant;Epitopes of antibody 825 is conserved;846 antibody did not show neutralizing activity against both WT and Omicron pseudovirus.ConclusionsIn this study,we successfully established an experimental method for the neutralization of SARS-CoV-2 pseudovirus based on flow cytometry,and used this system to detect the neutralizing activity of five antibodies against Omicron variant.Combined with the results of the first part of the study showed that the S371F and S375F mutations reduced the neutralizing activity of 825 antibody against Omicron;the S371L and G446S mutations caused the escape of 843 antibody against Omicron variant;S371L,S371F,S375F and G446S mutation sites are largely unaffected by the 826 and 809 antibodies.The combinations between antibodies 826,843 and 825 with different epitopes and between 826,843 and 846 is expected to be candidate antibodies for the clinical treatment of COVID-19.