Effect of anti-SARS S1 antibodies T62 on SARS-CoV-2 S protein
Since SARS-CoV-2 S reacted weakly with polyclonal rabbit anti-SARS S1 antibodies T62 in western blot (Fig. 1d), we investigated whether SARS-CoV-2 S protein in native conformation could be recognized by anti-SARS S1 antibodies T62. HEK293T cells transiently expressing SARS-CoV-2 S proteins were incubated with polyclonal anti-SARS S1 antibodies T62 and analyzed by flow cytometry. SARS-CoV S was used as a positive control. As expected, expression of SARS-CoV S proteins on 293T cell surface were readily detected by polyclonal anti-SARS S1 antibodies T62 (Fig. 5a). In contrast, only low level of binding of SARS-CoV-2 S protein to polyclonal rabbit anti-SARS S1 antibodies T62 was detected (Fig. 5a). To further delineate where the conformational epitopes for this polyclonal antibodies are located, two new constructs were generated, a SARS-CoV S protein backbone with RBD from SARS-CoV-2 (SARS-CoV S/nRBD) and a SARS-CoV-2 S protein backbone with RBD from SARS-CoV (SARS-CoV-2 S/sRBD). Replacement of SARS-CoV RBD with SARS-CoV-2 RBD (SARS-CoV S/nRBD) decreased binding of polyclonal antibodies T62 to S protein, whereas substitution of SARS-CoV-2 RBD with SARS-CoV RBD (SARS-CoV-2 S/sRBD) increased the affinity of S protein to polyclonal antibodies T62 (Fig. 5a). All chimera proteins were expressed at levels similar to SARS-CoV-2 S protein (Fig. 5b, top panel). These results suggest that at least one major conformational epitope for polyclonal antibodies T62 might be located in RBD where the sequences differ between SARS-CoV and SARS-CoV-2 (Fig. 5c). Of note, while SARS-CoV-2 S/sRBD bound to polyclonal antibodies T62 better than SARS-CoV S/nRBD in flow cytometry, the signal for SARS-CoV S/nRBD was stronger than SARS-CoV-2 S/sRBD in western blot by polyclonal antibodies T62, indicating that major linear epitopes of polyclonal antibodies T62 were located on SARS-CoV NTD. Among residues in SARS-CoV RBD critical for receptor binding and virus entry43,44, seven of them are different between SARS-CoV and SARS-CoV-2 RBDs (Fig. 5c). We then mutated each of them in SARS-CoV S protein to the corresponding residues in SARS-CoV-2 S. With exception of SARS-CoV mutant Y442L, which had reduced protein expression (Supplementary Fig. 3), binding of SARS-CoV S mutants to polyclonal rabbit anti-SARS S1 antibodies T62 was not affected (Fig. 5d). This suggests that these residues might not be direct binding sites for polyclonal antibodies T62. Next, we tested whether polyclonal rabbit anti-SARS S1 antibodies T62 could inhibit entry of SARS-CoV-2 S pseudovirions. CoV S protein pseudovirions were incubated with polyclonal antibodies T62 on ice for 1 h, and their transduction was measured according to luciferase activities. As expected, polyclonal antibodies T62 neutralized SARS-CoV S pseudovirion effectively in a dose dependent manner (Fig. 5e). In contrast, even at a concentration of 50 μg/ml, polyclonal antibodies T62 did not have marked effect on transduction by SARS-CoV-2 S proteins (Fig. 5e).
Fig. 5 Characterization of polyclonal rabbit anti-SARS S1 antibodies T62.
a Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to SARS-CoV-2, SARS-CoV S, and chimeric S proteins. HEK293T cells transiently expressing either SARS-CoV-2 S, SARS-CoV S, SARS-CoV S/nRBD, or SARS-CoV-2 S/sRBD proteins were incubated with polyclonal rabbit anti-SARS-CoV S1 antibody T62 for 1 h on ice, followed by a FITC-conjugated secondary antibody, then cells were analyzed by flow cytometry. Experiments were done three times and one representative is shown. b Expression of SARS-CoV-2 S, SARS-CoV S, or chimeric S proteins on 293T cells. Cells from panel A were lyzed and blotted with anti-FLAG M2 antibody and polyclonal anti-SARS S1 antibody T62. c Amino acid sequence alignment of SARS-CoV and SARS-CoV-2 S RBDs. Stars (*) indicate the seven critical residues different between SARS-CoV-2 and SARS-CoV RBDs. d Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to mutant SARS-CoV S proteins. e Neutralization of SARS-CoV-2 S and SARS-CoV S pseudovirions by polyclonal rabbit anti-SARS S1 antibody T62. Pseudovirons were pre-incubated with serially diluted polyclonal rabbit anti-SARS S1 antibodies T62 on ice, then virus-antibody mixture was added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.