Expression And Characterization Of SARS-CoV-2 S-related Proteins In Mammalian Cells And Screening Of Neutralizing Monoclonal Antibodies

The Coronavirus disease 2019(COVID-19)pandemic is the result of the rapid spread of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).SARSCoV-2 is highly contagious and highly mutagenic.Symptoms of varying severity will appear after the virus infects the human body,and even cause death.As of June 2,2021,there have been more than 170 million infections worldwide,causing more than 3.54 million deaths from COVID-19-related diseases.Therefore,the research on the SARS-CoV-2 vaccine and its neutralizing antibodies is essential to overcome the COVID-19 disease.Similar to other coronaviruses,the envelope glycoprotein S trimer protein on the surface of the SARS-CoV-2 virus can bind to the receptor to mediate the membrane fusion between the virus and the host cell.It is the main target of the immune system and also the main target of the new coronavirus vaccine research and the neutralizing antibody research.We designed four spikes S-related proteins based on the full-length S gene sequence of SARS-CoV-2:The pre-fusion trimer form of S-2P is a proline mutation at the 986 and 987 amino acid sites and a replacement of the furin cleavage site"AGAG"(residues 682-685),with 19 amino acids trunked at the C-terminal,NTD fragment,RBD fragment and S2 subunit.Besides,we synthesized SARS-CoV S-2P gene based on the S gene sequence of SARS-CoV,and these genes were expressed into proteins in eukaryotic cell,which used for monoclonal antibody screening and property identification.In addition,based on the SARS-CoV-2 S-2P gene,four amino acid sites of F817P,A892P,A899P and A942P were introduced,and six proline mutant S-6P proteins were designed and constructed.After purification,the protein expression,trimer morphology,protein purity and activity were verified by SDS-PAGE,Western Blot,negative staining electron microscope observation and HPLC.Besides,the identification of the antigenic properties using convalescent patient serum and mouse and rabbit antibodies showed that these proteins have complete epitopes.Secondly,we used the SARS-CoV-2 S-2P trimer protein as the immunogen,the indirect ELISA method and the SARS-CoV-2 pseudovirus neutralization system have verified that different adjuvants and different immunization strategies can induce protein binding.It is different from the virus-neutralizing antibody.Among them,the new composite adjuvants FH-002C and Mn-004 can stimulate the body to produce an immune response to the 293F-S-2P protein,the dose 50 μg/mouse of 293F-S-2P is better than 5μg/mouse.The effect of 293F-S-2P to stimulate antibody production is better than that of Bac-S-2P protein,which verifies the advantages of eukaryotic cells in expressing protein processing and modification.Analyzing the characteristics of the cellular immune response of immunized mice,all immune groups stimulated IgG1,IgG2a and IgG2b responses,of which IgG1 responses were the main one,and the IgG1/IgG2a ratio showed that the immune response was biased towards Th2,which proved the protein immunization induces a humoral immune response mediated by B cells.Then,34 monoclonal antibodies were obtained through multiple rounds of antibody screening.The antibody subclass,antibody purity,antigen binding capacity,pseudovirus neutralization activity and other properties of these antibodies were analyzed and identified.Among them,16 monoclonal antibodies were directed against RBD epitope,5 monoclonal antibodies targeting S2 protein,10 monoclonal antibodies targeting NTD epitopes,and 3 monoclonal antibodies only binding SARS-CoV-2 S-2P protein.The obtained monoclonal antibodies all have good binding activity with SARS-CoV-2 S-2P trimer.The SARS-CoV-2 pseudovirus neutralization test results show that the RBD-targeting antibody has a higher titer of neutralization ability,which verifies that the RBD epitope is the advantage of the S trimer and epitopes can stimulate the body to produce strong neutralizing antibodies.Then,we used ELISA blocking experiment verify the blocking of RBD antibodies with each other and with the hACE2 receptor.Through further analysis of the structure of the monoclonal antibody,the epitope features of 1C4,8H12,9G11,and 13H7 with a resolution of 7.44(?),5.94(?),6.79(?) and 7.54(?) were obtained.Among them,1C4 is combined with 3 RBDs in the close state,and 9G11 and 8H12 are combined with 3 RBDs in the open state respectively.1C4 does not block the interaction between hACE2 receptor and RBD,but 8H12 and 9G11 block the binding of the receptor to varying degrees.13H7 binds to NTD table similarly to the reported 4A8(targeting NTD antibody).These provide certain information for studying the neutralization mechanism of antibodies.In summary,this study verified that the S-2P trimeric protein expressed by mammalian HEK 293F cells with high purity,obvious trimer morphology and good immunogenicity,34 monoclonal antibodies were screened by hybridoma technology,and the ability of monoclonal antibodies to bind proteins and neutralize pseudoviruses was identified,and 4 neutralizing antibodies were analyzed.The epitope information lays the foundation and provide certain guiding significance for the development of neutralizing antibodies and antibody drugs against SARS-CoV-2.