Potent mouse monoclonal antibodies that block SARS-CoV-2 infection

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies.

Keywords: SARS-CoV-2; mouse monoclonal antibody; neutralizing antibody; spike.

Figure 1

Production of six monoclonal antibodies against spike protein.A, schematic of recombinant proteins used to establish anti-spike antibodies. For mammalian expression constructs (SΔTM-SBP and RBD-SBP), the HRV3C cleavage site was placed upstream of the SBP tag so that the SBP tag could be removed by HRV3C protease treatment after protein purification (Fig. S1A). B, Coomassie brilliant blue (CBB) staining of recombinant protein purified from E. coli expression system. GST-RBD and MBP-RBD appeared as bands of 46 kDa and 62 kDa, respectively. C, CBB staining of recombinant proteins purified from the mammalian expression system. The glycosylation of recombinant proteins caused smear bands and a lower migration rate of proteins on SDS-PAGE compared with proteins treated with PNGase. D, ELISA-binding affinity of purified monoclonal antibodies to trimeric SΔTM and RBD glycoproteins purified from the mammalian expression system. Error bars indicate standard deviation (n = 3). E, summary of isotype and EC₅₀ of established monoclonal antibodies. F, Western blotting (WB) against SΔTM and RBD glycoproteins (10 or 50 ng per lane) using purified monoclonal antibodies (1 μg/ml in PBS-T). Clone S1D7 and S3D7 could not detect either SΔTM or RBD in WB. G, detection of nonglycosylated SΔTM using established monoclonal antibodies. Four clones could detect SΔTM (30 ng per lane), regardless of glycosylation. H, detection of spike proteins expressed in 293T cells. Lysates of 293T cells expressing artificial spikes carrying T4 foldon or wild-type spike glycoproteins were separated by SDS-PAGE, followed by WB using antibody R52. Foldon, T4 fabritin trimerization motif; GST, glutathione S-transferase; MBP, maltose-binding protein; n.i., nonimmune mouse IgG; NTD, N-terminal domain; RBD, receptor-binding domain; SΔTM, spike lacking TM domain; SBP, streptavidin-binding peptide; SS, signal peptide; TM, transmembrane domain.

Figure 2

Application for immunoprecipitation and immunofluorescence.A, immunoprecipitation (IP) of trimeric glycosylated spike protein (SΔTM) using established monoclonal antibodies. All clones were capable of pulling down RBD and spike glycoprotein. Higher IP efficiency of spike glycoprotein was observed in clones R22, R31, S1D7, and S3D8. For RBD glycoprotein, clone R22, S1D7, and S3D8 showed higher IP efficiency. B, IP of trimeric spike protein deglycosylated by PNGase F using established monoclonal antibodies. "SΔTM" indicates SΔTM glycoprotein untreated with PNGase F. All clones are capable of pulling down deglycosylated spike protein. Higher IP efficiency was observed in clones R22, R31, S1D7, and S3D8. C, immunofluorescence (IF) staining of spike glycoprotein expressed in HeLa cells with monoclonal antibodies S1D7 and S3D8. Spike protein localized on the apical surface of transfected HeLa cells. Scale bar, 30 μm. HC, IgG heavy chain; In, input; LC, IgG light chain; ni, nonimmune mouse IgG; SΔTM, trimeric spike protein without transmembrane domain; S1, S1D7; S3, S3D8.

Figure 3

Inhibition of ACE2–spike interaction by S1D7 and S3D8.A, a schematic of the spike pull-down assay designed to evaluate inhibition of ACE2–spike binding by monoclonal antibody. Spike glycoprotein lacking TM domain (SΔTM) was mixed with a monoclonal antibody. ACE2-SBP was applied to capture SΔTM onto streptavidin beads competitively. Captured SΔTM was detected by WB as a measurement of the antibody's inhibitory ability. B, WB of spike pull-down assay using antibody R52. In the presence of clones S1D7 and S3D8, ACE2 was not able to pull down SΔTM. C, schematic of bead-based neutralization assay designed to quantify inhibition of ACE2–RBD binding by monoclonal antibody. RBD-SBP glycoprotein immobilized on streptavidin beads was mixed with a monoclonal antibody. ACE2-FLAG was applied to bind competitively with RBD. ACE2–RBD binding was quantified by measuring the signal given by an anti-FLAG antibody conjugated with APC fluorophore using FACS. D, one set of representative FACS results of a bead-based neutralization assay in the presence of 4 μg/ml monoclonal antibodies. Clones S1D7 and S3D8 significantly inhibited ACE2-RBD interaction, shown as lowered fluorescence intensity of APC. E, binding profiles of potent neutralizing antibodies. Error bars indicate standard deviation (n = 3). Clones R22 and R31 showed no inhibition of ACE2-RBD binding, while S1D7 and S3D8 inhibited ACE2-RBD interaction at lower ng/ml levels. ni, nonimmune mouse IgG; S1, S1D7; S3, S3D8.

Figure 4

S1D7 and S3D8 neutralized SARS-CoV-2 infection.A, Spike glycoprotein was expressed in VeroE6/TM2 cells during SARS-CoV-2 infection. Spike glycoproteins were detected by western blots using anti-spike antibodies R22 and R52. B, immunofluorescence staining of spike glycoprotein expressed in VeroE6/TM2 cells infected with SARS-CoV-2 at 7 h postinfection. Scale bar, 20 μm. C, S1D7 and S3D8 are capable of neutralizing live virus infections. Although clone R22 failed to protect VeroE6/TM2 cells from SARS-CoV-2 infection, S1D7 and S3D8 blocked SARS-CoV-2 infection significantly with IC₅₀ values of 405.2 ng/ml and 139 ng/ml, respectively. S1D7 and S3D8 cocktail showed intermediate neutralizing activity (200.1 ng/ml). Error bars indicate standard deviation (n = 3).

Figure 5

Recombinant human/mouse chimeric antibodies R52h, S1D7h, and S3D8h.A, R52h is applicable for WB. Lysates of 293T cells expressing artificial spikes carrying T4 foldon or wild-type spike glycoproteins were separated by SDS-PAGE, followed by WB using human/mouse chimeric antibody R52h, which was secreted by Expi293F cells. B, S1D7h and S3D8h are applicable for IF. Spike glycoprotein expressed in HeLa cells was stained with human/mouse chimeric antibodies S1D7h or S3D8h, which were secreted by Expi293F cells. Scale bar, 20 μm. C, Binding profiles of potent neutralizing antibodies. ni, nonimmune mouse IgG. Error bars indicate standard deviation (n = 3).