Research And Development Of Human Monoclonal Antibody Drugs Against SARS-CoV-2

Background and Aims:Since 2020,Corona Virus Disease 2019(COVID-19)has become a global epidemic infectious disease,seriously to threat to human health.Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)is a pathogenic virus that causes COVID-19,which has spawned Delta and Omicron mutants,posing a higher challenge to the fight against COVID-19.Search for safer,more effective and stable antiviral drugs has become an urgent need for clinical treatment of COVID-19 at this critical time of the global epidemic.Spike protein S,an important surface protein of coronavirus,is composed of S1 subunit and S2 subunit,which is closely related to the infectivity of the virus.The RBD region of the S1 protein is currently used as the primary target for the development of antibody drugs and vaccines.By comparing the gene sequences,we found that the sequence of S1 protein is similar to that of coronaviruses related to Severe Acute Respiratory Syndrome(SARS)in 2003 and Middle East Respiratory Syndrome(MERS)in 2012.However,the gene sequence of the S2 protein is highly conserved to all known pathogenic coronaviruses including SARS-Co V-2,suggesting that the S2 protein has a great potential for the development of broad-spectrum monoclonal antibody drugs against SARS-Co V-2.Therefore,this study aims to use the S2 protein of SARS-Co V-2 as an antigen to screen human monoclonal antibodies against all pathogenic coronavirus including SARS-Co V-2from the whole blood from survivors infected with COVID-19 with the "magnetic bead sorting single cell sequencing" and "phage library display" techniques,in order to rapidly develop monoclonal antibodies that can effectively neutralize and kill these viruses.Methods and Results:1.Preparation of S2 protein of SARS-Co V-2: Firstly,the DNA sequence of S2 protein of SARS-Co V-2 was synthesized by searching the literature and comparing with NCBI libraries.Then the c DNA sequence was cloned into the expression vector and the optimal conditions for protein expression were determined through changing the induction temperature and time of the protein.Finally,the S2 antigenic protein was successfully prepared,and it was verified that the S2 protein could be used for subsequent monoclonal antibody screening.2.Screening and preparation of monoclonal antibodies: Whole blood was collected from the survivors infected with SARS-Co V-2 and the S2 protein was used as the antigen protein for screening monoclonal antibody using "magnetic bead sorting single cell sequencing" and "phage library display" techniques respectively.The screened monoclonal antibodies were further validated by enzyme-linked immunosorbent assays(Elisa),and expressed and purified for further experiments.3.A neutralization assay for SARS-Co V-2 pseudovirus: The SARS-Co V-2pseudovirus was prepared by constructing a stable HEK293 T cell line specifically overexpressing ACE2 and using HIV lentiviral vector and S2 protein of SARS-Co V-2as the basis.The screened monoclonal antibodies were subjected to pseudovirus neutralization assay.The final IC50 values were determined to evaluate the neutralization efficiency of the antibodies.Conclusions.1.We successfully constructed a prokaryotic expression plasmid for S2 protein of the SARS-Co V-2 virus,in which the S2 protein was expressed and purified using E.coli system.S2 protein will be used as an antigen for subsequently screening monoclonal antibodies.2.The whole human monoclonal antibodies have been screeded by "highthroughput sequencing by magnetic bead screening" and "phage library display" technologies,and the antibodies were validated by SARS-Co V-2 pseudovirus neutralization assay.Finally,we successfully obtained two specific whole human monoclonal antibodies against SARS-Co V-2 pseudovirus with IC50 of 12.11 μg/m L and 25.40 μg/m L,respectively.