Human monoclonal antibodies block the SARS-CoV-2 RBD protein-hACE2 protein interaction a ELISA binding assay of COVID-19 patient sera to ELISA plate coating of SARS-CoV-2 S1 protein. b ELISA binding assay of COVID-19 patient sera to ELISA plate coating of SARS-CoV-2 RBD protein. c COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 S1 protein binding to hACE2 protein by ELISA. d An overall strategy of anti-SARS-CoV-2 RBD mAbs. e Flow cytometry analysis of SARS-CoV-2 RBD-specific IgG+ B cells in PBMCs of healthy donor and patient XFQ. f Specificity of mAbs (311mab–31B5, −32D4 and −31B9 clones) to SARS-CoV-2 RBD protein by ELISA. g ELISA analysis of SARS-CoV-2 RBD-hACE2 interaction inhibited by 311mab–31B5, −32D4, and −31B9 mAbs. h Flow cytometry analysis of SARS-CoV-2 RBD-hACE2 interaction inhibited by 311mab–31B5, −32D4, and −31B9 mAbs. The numbers adjacent to the outlined areas indicate the percentages of anti-mouse IgG+ hACE2-plasmid transiently transfected 293T cells, which are summarized in i (left panel). i (right panel) Mean fluorescence intensity (MFI) of Alexa Fluor 647 anti-mouse IgG in anti-mouse IgG+ hACE2-plasmid transiently transfected 293T cells. j Antibody-mediated blocking of luciferase-encoding SARS-Cov-2 typed pseudovirus into hACE2/293T cells. NC, negative control. HD, healthy donor. The data are representative of two independent experiments with three replicates per group (f, g, i, j; error bars in f, g, i, j indicate the SD)
Next, we set out to clone human mAbs using the blood samples from three COVID-19 recovered patients, of which their sera showed potent hACE2 receptor binding inhibition. To this end, we sorted each SARS-CoV-2 RBD-specific, IgG class-switched memory B cell into a single well of the 96-well microplates. Subsequently, we used reverse transcription polymerase chain reaction to amplify IgG variable heavy chain (VH) and light chain (VL) from each single memory B cell. After cloning both VH and VL, we inserted both sequences into expression plasmids that encoding constant regions of human IgG1 heavy chain and light chain (Fig. 1d).21 We found that SARS-CoV-2 RBD-specific, IgG-positive memory B cells only enriched in COVID-19 recovered patients, but not in healthy controls (Fig. 1e), suggesting the specificity of our sorting strategy. After antibody cloning, we acquired three pairs of IgG VHs and VLs inserted expression plasmids.
Finally, we expressed these paired plasmids encoding IgG VH and VL sequences and named these three mAbs as 311mab–31B5, 311mab–32D4 and 311mab–31B9, respectively. We first examined whether these human mAbs were able to bind to SARS-CoV-2 RBD protein by ELISA. The results showed that all three mAbs strongly and specifically bind to the RBD protein (Fig. 1f). Next, we tested whether these mAbs can block the interaction between SARS-CoV-2 RBD and hACE2. We found that both 311mab-31B5 and 311mab-32D4 could efficiently block SARS-CoV-2 RBD-hACE2 interaction (IC50 = 0.0332, and 0.0450 μg/ml, respectively), while 311mab–31B9 clone failed to inhibit such an interaction (Fig. 1g). The 31B5- and 32D4-mediated inhibition of RBD-hACE2 interaction was also evidenced by flow cytometry analysis (Fig. 1h, i). Furthermore, we determined the neutralization of these three mAbs using a SARS-CoV-2 S pseudotyped lentiviral particle.22 In line with ELISA- and flow cytometry-based blockade results, both 311mab-31B5 and 311mab-32D4 effectively neutralized pseudovirus entry to host cells ectopically expressing hACE2 (IC50 = 0.0338, and 0.0698 μg/ml, respectively). As expected, 311mab-31B9 clone failed to show any neutralization activities (Fig. 1j).
In conclusion, we have successfully cloned two human blocking mAbs using SARS-CoV-2 RBD-specific memory B cells isolated from recovered COVID-19 patients. These two mAbs can specifically bind to SARS-CoV-2 RBD, block the interaction between SARS-CoV-2 RBD and hACE2 receptor, and lead to efficient neutralization of SARS-CoV-2 S protein pseudotyped virus infection. Such human anti-SARS-CoV-2 RBD-hACE2 blocking mAbs are first reported, and hold great promise to be exploited as specific prophylactic and therapeutic agents against ongoing SARS-CoV-2 pandemic.
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