Materials and methods

Construction, expression, and purification of recombinant protein
The construction, expression, and purification of recombinant RBD proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV were performed as previously described with some modifications.27,28 Briefly, genes encoding residues 331-524 of SARS-CoV-2 S protein, residues 318-510 of SARS-CoV S protein, or residues 377-588 of MERS-CoV S proteins, were amplified by PCR using codon-optimized SARS-CoV-2 S protein (GenBank accession number: QHR63250.1), SARS-CoV S protein (GenBank accession number: AY278488.2), or MERS-CoV S protein (GenBank accession number: AFS88936.1), as respective template, and fused into pFUSE-hIgG1-Fc2 expression vector (hereinafter named hFc; InvivoGen, San Diego, CA). The RBD proteins were expressed in human embryonic kidney (HEK)293T cells, secreted into cell culture supernatants, and purified by protein A affinity chromatography (GE Healthcare, Marlborough, MA).

SDS-PAGE and Western blot
The purified RBD proteins were analyzed by SDS-PAGE and Western blot as previously described.27,29 Briefly, proteins were separated by 10% Tris-glycine SDS-PAGE and stained with Coomassie brilliant blue or transferred to nitrocellulose membranes. The blots were blocked with 5% fat-free milk in PBS containing 0.5% Tween-20 (PBST) for 2 h at 37 °C and further incubated with SARS-CoV RBD-specific polyclonal antibody (mouse sera, 1:3,000),30 or MERS-CoV RBD-specific antibody (mouse sera, 1:3,000),31 overnight at 4 °C. The blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature and then visualized with ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).

Flow cytometry analysis
Flow cytometry analysis was performed to detect the binding of SARS-CoV-2 RBD protein to hACE2 receptor in 293T cells stably expressing hACE2 (hACE2/293T).26,32 SARS-CoV and MERS-CoV RBDs, as well as 293T cells stably expressing hDPP4 receptor (hDPP4/293T), were used as controls. Briefly, cells were incubated with respective RBD of SARS-CoV-2, SARS-CoV, or MERS-CoV containing a C-terminal hFc at 20 μg/ml for 30 min at room temperature, which was followed by incubation with FITC-labeled goat anti-human IgG antibody (1:500; Thermo Fisher Scientific) for 30 min and analyzed by flow cytometry. The blockage of RBD-receptor binding was performed by incubation of soluble human ACE2 (sACE2; 5 μg/ml; R&D Systems, Minneapolis, MN) receptor with respective RBD of SARS-CoV-2, SARS-CoV, or MERS-CoV (20 μg/ml), followed by the same procedure as that described above. hIgG-Fc protein (hFc: 20 μg/ml), or soluble human DPP4 (sDPP4; 5 μg/ml; R&D Systems) receptor, was included as control.
Detection of hACE2 protein expression in hACE2/293T, or hDPP4 protein expression in hDPP4/293T, stable cell lines was performed by flow cytometry analysis, as described above, except that the cells were sequentially incubated with hACE2- or hDPP4-specific goat antibody (0.5 μg/ml; R&D Systems) at room temperature for 20 min and FITC-labeled anti-goat IgG antibody (1:200; Abcam, Cambridge, MA) for 1 h at 4 °C.
Flow cytometry analysis was also performed to detect the binding between SARS-CoV-2 RBD and hACE2, or bat-ACE2 (bACE2), receptor in transiently transfected 293T cells. Briefly, 293T cells were transfected with hACE2 or bACE2 plasmid using the calcium phosphate method, and 48 h later, they were incubated with SARS-CoV-2 RBD protein at various concentrations for 30 min at room temperature. SARS-CoV and MERS-CoV RBDs were included as controls. After staining with FITC-conjugated goat anti-human IgG antibody (1:500; Thermo Fisher Scientific), the mixture was analyzed by flow cytometry as described above.

Immunofluorescence staining
This was performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor in hACE2/293T stable cell lines.33 SARS-CoV and MERS-CoV RBDs were used as controls. Briefly, cells were sequentially incubated with Fc-fused SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (20 μg/ml) and hACE2-specific goat antibody (5 μg/ml) for 30 min at room temperature. After three washes, the cells were incubated with FITC-labeled goat anti-human IgG (Fc) antibody (1:500; Thermo Fisher Scientific), or Alexa-Fluor 647-labeled anti-goat antibody (1:200 dilution; Abcam) for 30 min at room temperature. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min and mounted in VectaMount Permanent Mounting Medium. The samples were imaged on a confocal microscope (Zeiss LSM 880), and the images were prepared using the ZEN software.

ELISA
ELISA was performed to detect the binding of SARS-CoV-2 RBD protein to sACE2 receptor, as previously described.27,32,34 SARS-CoV and MERS-CoV RBDs, as well as sDPP4 protein, were used as controls. Briefly, ELISA plates were precoated with SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (1 μg/ml) overnight at 4 °C and blocked with 2% fat-free milk in PBST for 2 h at 37 °C. Serially diluted sACE2, or sDPP4, protein was added to the plates and incubated for 2 h at 37 °C. After four washes, the bound protein was detected using hACE2- or hDPP4-specific goat antibody (0.5 μg/ml, R&D system) for 2 h at 37 °C, followed by incubation with HRP-conjugated anti-goat IgG antibody (1:5,000, Thermo Fisher Scientific) for 1 h at 37 °C. The reaction was visualized by addition of substrate 3,3’,5,5’-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) and stopped by H2SO4 (1 N). The absorbance at 450 nm (A450) was measured by an ELISA plate reader (Tecan, San Jose, CA).
The cross-reactivity of SARS-CoV-2 RBD protein to SARS-CoV RBD-specific antibody was performed by coating ELISA plates with SARS-CoV-2 RBD (1 μg/ml), as well as SARS-CoV RBD or MERS-CoV RBD (as controls, 1 μg/ml), followed by sequential incubation with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera and HRP-conjugated anti-mouse IgG (1:5,000; Thermo Fisher Scientific) antibodies.

Pseudovirus neutralization and inhibition assays
SARS-CoV-2 pseudovirus was generated, as previously described, with some modifications.25,27,29 Briefly, 293T cells were cotransfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding SARS-CoV-2 S protein using the calcium phosphate method. SARS-CoV and MERS-CoV pseudoviruses were packaged as controls. The transfected medium was changed into fresh Dulbecco’s Modified Eagle’s Medium (DMEM) 8 h later, and pseudovirus-containing supernatants were collected 72 h later for single-cycle infection in target cells. Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2, SARS-CoV, or MERS-CoV pseudovirus with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera for 1 h at 37 °C, followed by addition of the mixture into hACE2/293T (for SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus) or hDPP4/293T (for MERS-CoV pseudovirus) target cells. Fresh medium was added 24 h later, and the cells were lysed 72 h later in cell lysis buffer (Promega, Madison, WI). The lysed cell supernatants were incubated with luciferase substrate (Promega) and detected for relative luciferase activity using the Infinite 200 PRO Luminator (Tecan). The 50% MERS pseudovirus neutralizing antibody titer (NT50) was calculated using the CalcuSyn computer program, as previously described.29,35
Inhibition of pseudovirus entry by SARS-CoV-2 RBD protein was carried out, as previously described, with some modifications.31 Briefly, SARS-CoV-2 RBD protein at serial dilutions was incubated with hACE2/293T target cells for 1 h at 37 °C. After removing medium containing the protein, the cells were infected with SARS-CoV-2 pseudovirus. SARS-CoV RBD and MERS-CoV RBD, as well as SARS-CoV pseudovirus and MERS-CoV pseudovirus, were used as controls. Fresh medium was added 24 h later, and the cells were lysed and analyzed, as described above. The 50% inhibitory concentration (IC50) of the RBD protein was calculated using the CalcuSyn computer program, as described above.

Statistical analysis
Values were expressed as mean and standard error (s.e.m). Statistical significance between different groups was calculated by GraphPad Prism Statistical Software. Two-tailed Student’s t test was used. ∗∗∗ represents P < 0.001.