Screening And Qualifying Immunogenic Epitopes In SARS-CoV-2 For Neutralizing Odies Identification Based On Phage Display Technology

The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its associated COVID-19 disease pose an emerging threat to global public health.The human-to-human transmission was evident in the early stages of the COVID-19 outbreak,and the number of confirmed cases is increasing daily.SARS-CoV-2 can spread among the population through various transmission routes such as contact infection,droplet transmission,and aerosol transmission.Infected persons will exhibit flu-like symptoms such as fever,fatigue,sore limbs,dry cough,etc.During the progression of the disease,symptoms such as pneumonia and difficulty breathing can lead to death in severe cases.Currently,it has been confirmed that SARS-CoV-2 mediates virus entry through the interaction of the Spike protein on its surface with the cell surface receptor ACE2.Among these proteins,the RBD domain plays a significant role.A recognition and cleavage site of TMPRSS2 at the S1/S2 site in the Spike protein can effectively hydrolyze the S protein,promote membrane fusion,and subsequently promote virus infection.Therefore,the S protein has essential research significance.Innate immunity is the body’s first line of defense against virus infection.It can effectively identify the common characteristics of various viruses and generate corresponding antiviral responses.In response to SARS-CoV-2 infection,patients develop polyclonal antibodies that recognize numerous epitopes across the viral proteome.This pool of polyclonal antibodies(including IgM,IgG,etc.)provides humoral immunity against SARS-CoV-2.Researchers have also found that virus particles isolated from the bronchoalveolar lavage fluid(BALF)of infected patients could be neutralized by the serum of infected patients,indicating that neutralizing antibodies were effectively induced by the virus.In addition,sera from recovered COVID-19 patients can neutralize SARS-CoV-2 in an in vitro assay and have been successfully used to treat other COVID-19 patients.Therefore,neutralizing antibodies against SARS-CoV-2 is promising.Here,we utilized various techniques and methods,such as screening phage display library,conducting genomic and protein structure analysis,and performing neutralization experiments,to identify immunogenic epitopes across the spike protein and develop neutralizing antibodies for SARS-CoV-2 infection treatment.The genome and protein structure analysis revealed that the epitopes were mainly found in the receptor binding domain(RBD)and the S1/S2 protease cleavage site.Specifically,we identified immunogenic epitopes located in the RBD and S1/S2 protease cleavage site of the SARS-CoV-2 S protein,tested their antibody levels in COVID-19 patients,and evaluated their potential to inhibit SARS-CoV-2 virus infection in vitro.Additionally,a humanized monoclonal antibody phage display library was constructed using PBMCs from convalescent COVID-19 patients.Monoclonal antibodies that specifically bind to these peptides have therapeutic potential to neutralize SARS-CoV-2 infection.The systematic identification of immunogenic peptides and direct identification of human monoclonal antibodies from patients can provide valuable diagnostic and therapeutic tools for COVID-19 and other emerging infectious diseases.Neutralizing antibodies are approved drugs to treat coronavirus disease-2019(COVID-19)patients,yet mutations in severe acute respiratory syndrome coronavirus(SARS-CoV-2)variants may reduce the antibody-neutralizing activity.In the face of the constantly emerging and rapidly spreading SARS-CoV-2 variants,the titers of neutralizing antibodies developed against the original strain of SARS-CoV-2 have been affected to varying degrees.Research has shown that most neutralizing antibodies against SARS-CoV-2 lose their neutralizing ability against mutant strains carrying E484K,K417N/T mutations in the RBD domain.Therefore,after proposing methods for screening and identifying SARS-CoV-2 immunogenic epitopes and neutralizing antibodies,we are committed to exploring new methods for antibody development and transformation,and developing neutralizing antibodies against various mutant strains.We identified multiple monoclonal antibodies from an antibody phage display library from COVID-19 patients.We further characterized the R3P1-E4 clone,which effectively suppressed SARS-CoV-2 infection and rescued the lethal phenotype in mice infected with SARS-CoV-2.Subsequently,the neutralization effect of the R3P1-E4 clone on the mutant strain of SARS-CoV-2 was tested in vitro,and we found that the neutralization effect of the clone on the mutant strain carrying the K417 mutation was significantly reduced.More importantly,Crystal structural analysis explained why R3P1-E4 had selectively reduced binding and neutralizing activity to SARS-CoV-2 variants carrying K417 mutations and allowed us to engineer mutant antibodies with improved neutralizing activity against these variants.Thus,we screened out R3P1-E4 mAb,which inhibits SARS-CoV-2 and related transformations in vitro and in vivo.Antibody engineering improved neutralizing activity of R3P1-E4 against K417 mutations.In summary,using phage display technology,we first identified four immunodominant epitopes of the SARS-CoV-2 S protein.We found that serological detection based on these antigenic peptides can reflect the clinical course of COVID19 patients,which has high sensitivity and accuracy for diagnosing COVID-19.In addition,we have established a rapid and efficient screening method for different targets,which can screen out humanized monoclonal antibodies with neutralizing activity.Finally,we propose an antibody engineering-based antibody remodeling strategy that can respond to infection with various variants.These studies are expected to provide an effective control strategy in the early stages of the outbreak of COVID19 disease and other novel infectious diseases.