By alignment of the RBD sequences of SARS-CoV and SARS-CoV-2, we identified the region of SARS-CoV-2 RBD at residues 331 to 524 of S protein (Fig. 1a). We then constructed a recombinant RBD protein containing codon-optimized RBD sequences with a C-terminal Fc of human IgG1 (hFc) using pFUSE-hIgG1-Fc2 expression vector, expressed the protein in mammalian cell 293T, and purified it from cell culture supernatant using protein A affinity chromatography. Similar to SARS-CoV and MERS-CoV RBD protein controls, SARS-CoV-2 RBD protein had high expression with strong purity (Fig. 1b). Notably, only SARS-CoV-2 and SARS-CoV RBDs were recognized by SARS-CoV RBD-specific, but not MERS-CoV RBD-specific, polyclonal antibodies (Fig. 1c), whereas only MERS-CoV RBD was recognized by MERS-CoV RBD-immunized polyclonal antibodies (Fig. 1d), suggesting the cross-reactivity of SARS-CoV RBD-specific antibodies with SARS-CoV-2 RBD protein.
Four experiments were performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor. First, we tested if stably transfected hACE2/293T cells expressed hACE2 by flow cytometry analysis. Since 293T cells alone did not express either hACE2 or hDPP4, they could not be recognized by anti-hACE2 or anti-hDPP4 antibodies (Fig. 2a (left panel)). Only hACE2/293T cells, but not hDPP4/293T cells, expressed hACE2, which was recognized by an anti-hACE2 antibody (Fig. 2a (middle panel)), whereas only hDPP4/293T cells, but not hACE2/293T cells, expressed hDPP4 and was, correspondingly, recognized by an anti-hDPP4 antibody (Fig. 2a (right panel)). These data confirmed the expression of hACE2 in hACE2/293T cells and the expression of hDPP4 in hDPP4/293T cells. Second, we used these hACE2/293T cells to detect the binding of SARS-CoV-2 RBD protein to cell-associated hACE2 by flow cytometry analysis and immunofluorescence staining. Similar to SARS-CoV RBD, SARS-CoV-2 RBD bound to hACE2/293T cells expressing hACE2 (Fig. 2b (left and middle panels)), but not to hDPP4/293T cells expressing hDPP4 (Fig. 2c (left and middle panels)). Furthermore, the binding between SARS-CoV-2 RBD and hACE2-expressing 293T cells was much stronger than the binding between SARS-CoV RBD and hACE2-expressing 293T cells (Fig. 2b (left and middle panels)). MERS-CoV RBD did not bind to hACE2-expressing 293T cells (Fig. 2b (right panel)), but rather bound to hDPP4-expressing 293T cells (Fig. 2c (right panel)). The results from immunofluorescence staining revealed positive signals for both hACE2 and hFc on hACE2/293T cells treated with SARS-CoV-2 RBD and SARS-CoV RBD, both of which contained a C-terminal hFc tag, whereas hACE2/293T cells treated with MERS-CoV RBD (containing a C-terminal hFc tag) showed positive signals for hACE2, but not for hFc, indicating that there is no binding of MERS-CoV RBD to the hACE2-expressing cells (Fig. 2d). These data suggest that SARS-CoV-2 RBD and SARS-CoV RBD can bind to cell-associated hACE2, but not to hDPP4. Third, we detected the binding of SARS-CoV-2 RBD to soluble hACE2 protein (sACE2) by ELISA. The results indicated that SARS-CoV-2 RBD bound to sACE2 in a dose-dependent manner and that the binding between SARS-CoV-2 RBD and sACE2 with 50% effective dose (EC50) of 1.07 μg/ml was stronger than that between SARS-CoV RBD and sACE2 (EC50: 1.66 μg/ml). In contrast, MERS-CoV RBD did not bind to sACE2 (Fig. 2e). While neither SARS-CoV-2 RBD nor SARS-CoV RBD bound to sDPP4, MERS-CoV RBD strongly bound to sDPP4 (EC50: 0.92 μg/ml) (Fig. 2f). These data suggest that both SARS-CoV-2 RBD and SARS-CoV RBD could bind to hACE2 in solution, but not to hDPP4 in solution. Fourth, flow cytometry analysis further indicated that the binding between SARS-CoV-2 RBD and cell-associated hACE2 receptor could be significantly blocked by sACE2 protein (Fig. 2g, i), but not by sDPP4 protein (Fig. 2h, i). Taken together, the above results confirm that the identified SARS-CoV-2 RBD could bind to both cell-associated and soluble hACE2 proteins.
Like SARS-CoV and MERS-CoV, SARS-CoV-2 also originates from bats.22,23,24 Next, we detected the binding affinity of the identified SARS-CoV-2 RBD to bat ACE2 (bACE2) and compared this binding with that of SARS-CoV RBD. We transiently transfected a bACE2-expressing plasmid into 293T cells and included a hACE2-expressing plasmid as a control, followed by detection of fluorescence intensity 48 h later. Results indicated that SARS-CoV-2 RBD bound strongly to 293T-expressed bACE2 with intensity similar to that of its binding to 293T-expressed hACE2 (Fig. 3a, c), and that this binding occurred in a dose-dependent manner (Fig. 3e, f). In addition, the binding affinity between SARS-CoV-2 RBD and 293T-expressed bACE2 (EC50: 0.08 μg/ml) or hACE2 (EC50: 0.14 μg/ml) was significantly higher than that between SARS-CoV RBD and 293T-expressed bACE2 (EC50: 0.96 μg/ml) or hACE2 (EC50: 1.32 μg/ml) (Fig. 3b, d-f). Nevertheless, MERS-CoV RBD bound neither bACE2- nor hACE2-expressing 293T cells (Fig. 3). These data suggest that SARS-CoV-2 RBD can bind to both bACE2 and hACE2 with significantly stronger binding than that of SARS-CoV RBD to either bACE2 or hACE2, supporting the bat origin of SARS-CoV-2. These results may partially explain why SARS-CoV-2 is more transmissible than SARS-CoV.
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We then evaluated the potential of the identified SARS-CoV-2 RBD protein as an inhibitor of viral entry. To accomplish this, we first generated a pseudotyped SARS-CoV-2 by cotransfection of a plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and a plasmid expressing S protein of SARS-CoV-2 into 293T cells, followed by collection of pseudovirus-containing supernatants. We then incubated serially diluted SARS-CoV-2 RBD protein with hACE2/293T target cells, followed by the addition of pseudovirus and detection of inhibitory activity of infection. With the capacity for only one-cycle infection, S protein-expressing pseudovirus cannot replicate in the target cells.25,26 Therefore, the inhibition of pseudovirus infection represents inhibition of viral entry, as mediated by viral S protein. As expected, SARS-CoV-2 RBD protein inhibited SARS-CoV-2 pseudovirus entry into hACE2-expressing 293T cells in a dose-dependent manner with 50% inhibition concentration (IC50) as low as 1.35 µg/ml. Interestingly, it also blocked the entry of SARS-CoV pseudovirus into hACE2-expressing 293T cells with IC50 of 5.47 µg/ml (Fig. 4a). Similarly, SARS-CoV RBD protein blocked the entry of both SARS-CoV pseudovirus and SARS-CoV-2 pseudovirus into hACE2-expressing 293T cells with IC50 of 4.1 and 11.63 µg/ml, respectively (Fig. 4b). In addition, neither SARS-CoV-2 RBD nor SARS-CoV RBD blocked the entry of MERS-CoV pseudovirus into hDPP4-expressing 293T cells (Fig. 4c). MERS-CoV RBD did not block the entry of SARS-CoV-2 pseudovirus or SARS-CoV pseudovirus into hACE2-expressing 293T cells, but it did block the entry of MERS-CoV pseudovirus into hDPP4-expressing 293T cells (IC50: 22.25 µg/ml) (Fig. 4a-c). These results suggest that SARS-CoV-2 RBD protein could be developed as an effective therapeutic agent against SARS-CoV-2 and SARS-CoV infection.
Since SARS-CoV-2 is more phylogenetically related to SARS-CoV than MERS-CoV,22 we further detected the cross-reactivity of SARS-CoV RBD-specific antibodies with SARS-CoV-2 RBD and cross-neutralizing activity of SARS-CoV RBD-specific antibodies against pseudotyped SARS-CoV-2. First, we performed an ELISA to detect the cross-reactivity of SARS-CoV RBD-immunized mouse sera with SARS-CoV-2 RBD. The results showed that SARS-CoV-2 RBD reacted strongly with anti-SARS-CoV RBD IgG with antibody titer of 1:2.4 × 104 (Fig. 4d), but it did not react with anti-MERS-CoV RBD IgG (Fig. 4e). As expected, SARS-CoV RBD reacted strongly with anti-SARS-CoV RBD IgG (antibody titer: 1:1.4 × 105) (Fig. 4d), but not with anti-MERS-CoV RBD IgG (Fig. 4e). MERS-CoV RBD did not react with anti-SARS-CoV RBD IgG (Fig. 4d), but instead reacted with anti-MERS-CoV RBD IgG (antibody titer: 1:1.3 × 105) (Fig. 4e). Second, we performed a pseudovirus neutralization assay to detect the cross-neutralizing activity of SARS-CoV RBD-immunized mouse sera against SARS-CoV-2 pseudovirus infection. Results revealed that SARS-CoV RBD-specific antisera could neutralize SARS-CoV-2 pseudovirus infection with a neutralizing antibody titer of 1:323, while these antisera could neutralize SARS-CoV pseudovirus infection with higher neutralizing antibody titer (1:1.2 × 104) (Fig. 4f). MERS-CoV RBD-inducing mouse sera only neutralized MERS-CoV pseudovirus infection in hDPP4-expressing cells with a neutralizing antibody titer of 1:4 × 104 (Fig. 4g), but failed to neutralize infection by either SARS-CoV-2 pseudovirus or SARS-CoV pseudovirus (Fig. 4f). These data suggest that SARS-CoV RBD-specific antibodies can cross-react with SARS-CoV-2 RBD and cross-neutralize SARS-CoV-2 pseudovirus infection.
In summary, we have characterized the SARS-CoV-2 RBD protein which exhibits strong binding to its cell-associated and soluble ACE2 receptors with human and bat origin. This RBD protein also demonstrated significantly higher binding affinity to ACE2 than SARS-CoV RBD. SARS-CoV-2 RBD protein could block S protein-mediated SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus entry into their respective ACE2 receptor-expressing target cells, suggesting the potential of SARS-CoV-2 RBD protein as a viral attachment or entry inhibitor against SARS-CoV-2 and SARS-CoV. SARS-CoV RBD-induced antibodies could cross-react with SARS-CoV-2 RBD and cross-neutralize SARS-CoV-2 pseudovirus infection, indicating that SARS-CoV RBD-specific antibodies may be used for treatment of SARS-CoV-2 infection and that either SARS-CoV RBD protein or SARS-CoV-2 RBD protein may be used as a candidate vaccine to induce cross-reactive or cross-neutralizing antibodies for prevention of SARS-CoV-2 or SARS-CoV infection. Taken together, this study provides an essential foundation for the design and development of SARS-CoV-2 RBD-based vaccines and therapeutics.