Screening And Functional Verification Of Broad-spectrum Neutralizing Antibodies Against SARS-CoV-2 And SARS-CoV

Coronaviruses are a class of enveloped RNA viruses,and there are currently seven coronaviruses that are capable of infecting humans,three of which are highly pathogenic and four of which are less pathogenic.Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)and Severe Acute Respiratory Syndrome Coronavirus(SARS-CoV)are two highly pathogenic coronaviruses of the genus β-coronavirus with approximately 80%genomic sequence similarity,and both viruses enter cells by binding to angiotensinconverting enzyme II(ACE2)on the cell surface.SARS-CoV-2 is less lethal(3%)than SARS-CoV(10%),but is highly infectious.As of February 10,2023,the cumulative number of confirmed SARS-CoV-2 cases worldwide has exceeded 600 million and the number of deaths has exceeded 6.83 million.For this reason,there is a lot of interest in the development of vaccines and neutralizing antibody drugs against this virus,and there are several vaccines and neutralizing antibody drugs approved for emergency use or on the market.Therefore,screening for broad-spectrum neutralizing antibodies that neutralize SARS-CoV-2 and SARS-CoV is important for the prevention and control of β-coronavirus infections similar to SARS-CoV-2 and SARS-CoV.Coronavirus infections like SARS-CoV-2 and SARS-CoV have important applications.In this study,we focused on the screening of broad-spectrum neutralizing antibodies against SARS-CoV-2 and SARS-CoV to provide some ideas for the subsequent development of broad-spectrum class of neutralizing antibody drugs.To this end,we prepared an m RNA vaccine with SARS-CoV-2 RBD as immunogen to immunize mice and induce the production of a large number of antigen-specific antibodies,and then constructed a SARS-CoV-2-specific antibody library by high-throughput single-cell sequencing technology,from which we synthesized and expressed the top 100 monoclonal antibodies with the highest frequency of occurrence,which were analyzed by motif analysis,The screening of anti-SARS-CoV-2and SARS-CoV broad-spectrum neutralizing antibodies was completed through motif analysis,binding effect analysis,affinity determination,blocking effect evaluation and neutralizing activity evaluation.The results showed that immunization with SARS-CoV-2 RBD m RNA vaccine could rapidly induce antigen-specific antibody library production,and the antibody library was focused in the use of genetic motifs.We selected 100 antibodies from the antibody library for gene synthesis and expression,from which we screened 65 antibodies that specifically bind SARS-CoV-2 RBD and 8 antibodies that bind SARS-CoV-2 RBD and SARS-CoV RBD.From these 8 antibodies,we screened 2 broad-spectrum neutralizing antibodies that could neutralize SARS-CoV-2 and SARS-CoV,R30 and R100 antibodies,respectively.Among them,R30 antibody can completely block the binding of RBD protein of both viruses to the receptor,and R100 antibody can completely block the binding of SARS-CoV-2 RBD to the receptor and partially block the binding of SARS-CoV RBD protein to the receptor.After preliminary analysis of antibody epitope competition experiments,it was found that the binding epitope of R100 neutralizing antibody might have a large overlap with the binding site of class I antibody,while the binding epitope of R30 neutralizing antibody needs to be further identified by resolving the crystal structure of the antigen-antibody complex.In addition,we screened five broad-spectrum neutralizing antibodies,including R30 and R100 antibodies,against SARS-CoV-2 and its Delta and Beta variants from these eight strains,and all of them were able to completely block the binding of SARS-CoV-2 RBD to the receptor.In conclusion,the library construction and sequencing of SARS-CoV-2 specific antibody library were successfully completed by high-throughput single-cell sequencing technology in this study,and two broad-spectrum neutralizing antibodies against SARS-CoV-2 and SARS-CoV could be screened by this large-scale screening method.Although the neutralizing activity of these two antibodies was not strong,the subsequent research on their binding epitopes and neutralization mechanism will provide some new directions for the development of broad-spectrum vaccines and neutralizing antibodies in the future.