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.
Fig. 4 Ability of SARS-CoV-2 RBD to inhibit viral entry, as well as its cross-reactivity and cross-neutralizing activity with SARS-CoV. a Dose-dependent inhibition of SARS-CoV-2 RBD protein against pseudotyped SARS-CoV-2 entry into hACE2/293T cells. SARS-CoV and MERS-CoV RBDs, as well as hDPP4/293T cells, were included as controls. SARS-CoV-2 RBD protein inhibited entry of SARS-CoV-2 and SARS-CoV pseudoviruses into their respective target (hACE2/293T) cells (a), but not the entry of MERS-CoV pseudovirus into its target (hDPP4/293T) cells (a). SARS-CoV RBD protein inhibited both SARS-CoV-2 and SARS-CoV pseudovirus entry, but not MERS-CoV pseudovirus entry (b). MERS-CoV RBD inhibited neither SARS-CoV-2 nor SARS-CoV pseudovirus entry, but it did inhibit MERS-CoV pseudovirus entry (c). The data are presented as mean inhibition (%) ± s.e.m. (n = 4), and 50% inhibition concentration (IC50) was calculated for SARS-CoV-2 RBD (a, b, black), or SARS-CoV RBD (a, b, red), protein against SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus and for MERS-CoV RBD protein (green) against MERS-CoV pseudovirus (c). d Cross-reactivity of SARS-CoV-2 RBD protein with SARS-CoV RBD-specific mouse sera by ELISA. Sera of mice immunized with mammalian cell-expressed SARS-CoV RBD protein30 were tested. Sera of mice immunized with mammalian cell-expressed MERS-CoV RBD protein31 were used as control. The data are presented as mean A450 ± s.e.m. (n = 4). The IgG antibody (Ab) titers were calculated as the endpoint dilution that remains positively detectable for SARS-CoV-2 RBD (black), or SARS-CoV RBD (red), binding to anti-SARS-CoV RBD sera (d) and for MERS-CoV RBD (green) binding to anti-MERS-CoV RBD sera (e). f Cross-neutralization of SARS-CoV RBD-immunized mouse sera against SARS-CoV-2 infection by pseudovirus neutralization assay. MERS-CoV RBD-immunized mouse sera were used as control. The data are presented as mean neutralization (%) ± s.e.m. (n = 4). 50% neutralizing antibody titers (NT50) were calculated against SARS-CoV-2 pseudovirus (black), or SARS-CoV pseudovirus (red), (f) infection in hACE2/293T target cells, as well as against MERS-CoV pseudovirus (green) (g) infection in hDPP4/293T cells. Experiments were repeated twice and yielded similar results