Screening And Identification Of Human Neutralizing Monoclonal Antiboby Against SARS-CoV-2

Coronavirus Disease 2019(COVID-19)is a zoonotic,severe,acute and highly contagious respiratory disease caused by the novel Coronavirus(SARS-COV-2).Undoubtedly,COVID-19 outbreak is the greatest public health threat today and causes disastrous consequences in many countries and regions.Therefore,it is significant to understand the characteristics of immune changes in COVID-19 survivors and explore potential treatment options.However,there are currently no anti-COVID-19 antibody drugs approved globally.For the rapidly evolving SARS-CoV-2 virus,a mixture of several noncompetitive antibodies may provide the greatest therapeutic effect.Therefore,the development of new potential antibodies is still very valuable.In this study,blood samples from 16 COVID-19 patients were collected on the day of discharge for peripheral blood monocyte cells(PBMCs)isolated.PBMCs were divided into three aliquots for separate data generation:single cell RNA sequencing,human leukocyte antigen(HLA)genotyping and TCR and BCR immune repertoire sequencing.To reveal the changes in human immune cells after SARS-COV-2 infection,the blood samples from eight healthy individuals were collected for single-cell sequencing analysis.Using t-SNE processing and unsupervised clustering,we found 28 different clusters representing different cell types.The number of monocytes and plasma cells were significantly increased in COVID-19 patients compared with that of healthy controls.Meanwhile,we found regulatory CD4+T cells and cytotoxic CD8+T cells in COVID-19 patients.Moreover,the β chain CDR3s on TCR was sequenced at the mRNA level to explore the cellular immunity changes in COVID-19 survivors.In total,we selected 493,317 βCDR3s from both COVID-19 patients and healthy control.Using pairwise alignment,we identified a total of 916 groups shared by COVID-19 recoveries.These sequences are candidates for SARS-CoV-2 specific TCRs.At the repertoire level,T cells had significantly lower diversity in COVID-19 patients than healthy control,but the cytotoxic CD8+T cells in COVID-19 patients had a higher enrichment,consistent with the clonal expansion upon antigen exposure.This indicates that the T cell subsets in the COVID-19 patients are mainly cytotoxic short-lived effector T cells shortly after virus clearance.The identification of COVID-19 TCR groups also allowed us to uncover the candidate antigenic epitopes from the virus genome,and 114 epitopes were found on the SARS-CoV-2 genome,of which more than 91%of the epitopes were distributed in proteins ORF1ab,S,N,and ORF3a.In summary,our analysis revealed a number of candidate peptides as promising targets for SARS-CoV-2 vaccine development.We next compared the abundance of different B cell clusters between COVID-19 patients and controls.We observed average of 3.1 folds increase of plasma cells and significantly lower diversity of BCR in COVID-19 patients compared to healthy controls,indicating widespread B cell clonal expansions upon antigen recognition.With deep BCR sequencing,we found immunoglobulin(Ig)heavy chain isotypes IgM presents the largest fraction in the peripheral repertoire for COVID-19 patients and controls,and significantly higher abundance of IgGl and IgAl antibodies were observed in COVID-19 patients.These changes are consistent with the anti-viral responses mounted by the adaptive immune system to clear viral particles.We assembled a total of 74,634 BCRs derived from COVID-19 patients based on the BCR heavy chain CDR3s.Combined with the scRNA-seq data,eventually 347 natural antibodies were selected that were highly expanded in the patient’s blood with evidence of antigen selection.100 antibodies were randomly selected for in vitro verification,and of which 14 antibodies had strong binding to S protein of SARS-CoV-2.Interestingly,the GD1-69 antibody was identified to effectively neutralize SARS-CoV-2 pseudovirus(IC50:1.77 μg/mL)and live virus(IC50:0.44 μg/mL).Taken together,our study provides a valuable SARS-CoV-2 neutralizing monoclonal antibody named GD1-69,which is an important scientific basis for the treatment and vaccine development of COVID-19 worldwide.