Screening and characterization of anti-SARS-CoV-2 receptor binding domain antibodies with broad-spectrum neutralizing activity
Objective To isolate monoclonal B cells specific for the SARS-CoV-2 receptor binding domain(RBD)from two COVID-19 convalescents,and to screen for broad-spectrum neutralizing antibodies against the SARS-CoV-2 RBD.Methods Using biotinylated RBD as a molecular probe,flow cytometry was employed to perform single-cell sorting of B cells from peripheral blood mononuclear cells(PBMCs)of convalescents.The obtained B cells were lysed and subjected to reverse transcription,followed by nested PCR amplification of the heavy and light chains of antibodies was conducted using random primers.The amplified products were cloned into corresponding expression vectors,and the respective matched heavy-light chain plasmids were co-transfected into 293F cells for expression.Monoclonal antibodies were then purified using Protein A column chromatography.Neutralization experiments were conducted with the wild-type(WT)pseudovirus,and antibodies with IC50＜0.1 μg/mL were selected for further testing of neutralizing breadth and potency against the wild-type(WT),Beta variant(B.1.351),Delta variant(B.1.617.2),and currently prevalent pseudovirus strains(XBB,BA.5,BF.7).Results A total of 21 RBD-specific monoclonal B cells were obtained from two recovered patients,resulting in the isolation of 13 pairs of antibody light/heavy chains.Nine antibodies were successfully expressed,with P1-A1,P1-B6,and P1-B9 exhibiting IC50 values below 0.1 μg/mL against the pseudovirus of the wild-type strain(WT).Specifically,P1-B6 effectively neutralized the wild-type strain(WT),Beta variant(B.1.351),and Delta variant(B.1.617.2),with IC50 values reaching 0.01 μg/mL.P1-B9 demonstrated effective neutralization against the wild-type strain(WT),Beta variant(B.1.351),Delta variant(B.1.617.2),and Gamma variant(P.1)pseudoviruses,with IC50 values of 0.42 μg/mL,0.63 μg/mL,0.28 μg/mL,and 2.50 μg/mL,respectively.Additionally,P1-B6 exhibited good neutralization against BA.5 and BF.7 pseudoviruses,with IC50 values of 0.06 μg/mL and 0.09 μg/mL,respectively.Conclusions Infection with the SARS-CoV-2 WT strain can induce the generation of neutralizing antibodies with broad-spectrum activity.Generating these broadly neutralizing antibodies does not require an excessively high somatic hypermutation.The obtained antibodies can be used as candidates for SARS-CoV-2 diagnosis and prevention.
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