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Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies - PubMed

. 2020 Jun;17(6):621-630.

doi: 10.1038/s41423-020-0458-z. Epub 2020 May 15.

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Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Chunyan Yi et al. Cell Mol Immunol. 2020 Jun.

Abstract

Coronavirus disease 2019 (COVID-19), caused by the novel human coronavirus SARS-CoV-2, is currently a major threat to public health worldwide. The viral spike protein binds the host receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD), and thus is believed to be a major target to block viral entry. Both SARS-CoV-2 and SARS-CoV share this mechanism. Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies. The in vivo experiments showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice; however, the cross-neutralizing activity was much weaker, indicating that there are distinct antigenic features in the RBDs of the two viruses. This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2. It is worth noting that a newly developed SARS-CoV-2 human antibody, HA001, was able to neutralize SARS-CoV-2, but failed to recognize SARS-CoV. Moreover, the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD, representing new binding sites for neutralizing antibodies. Overall, our study has revealed the presence of different key epitopes between SARS-CoV and SARS-CoV-2, which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.

Keywords: SARS-CoV-2; cross-neutralizing antibody; receptor binding motif; spike protein; substitution mutation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

Both the SARS-CoV-2 RBD and SARS-CoV RBD bind to hACE2. a Receptor-dependent infection of SARS-CoV-2 and SARS-CoV pseudo-typed virus entry into hACE2+ 293 T cells. 293T cells stably expressing hACE2 were infected with SARS-CoV-2 or SARS-CoV pseudo-typed viruses, and the cells were harvested to detect the luciferase activity. Fold changes were calculated by comparison to the levels in the uninfected cells. VSV pseudo-typed viruses were included as controls. b Syncytia formation between S protein- and hACE2-expressing cells. 293T cells transfected with hACE2 plasmid were mixed at a 1:1 ratio with 293T cells transfected with plasmid encoding S protein from SARS-CoV-2 (bottom left) or SARS-CoV (bottom right). As controls, 293T cells transfected with an empty plasmid were either mixed at a 1:1 ratio with 293T cells transfected with the hACE2 plasmid (top row), S protein from SARS-CoV-2 (middle left) or SARS-CoV (middle right). Images were photographed at ×20 magnification. Representative images are shown. c Dose-dependent binding of the SARS-CoV-2 RBD to soluble hACE2 as determined by ELISA. The binding of both the SARS-CoV-2 RBD and SARS-CoV RBD with an Fc tag on hACE2 was tested. Human Fc was included as a control. Data are presented as the mean OD450 ± s.e.m. (n = 2). d Binding profiles of the SARS-CoV-2 RBD and SARS-CoV RBD to the soluble hACE2 receptor measured by biolayer interferometry in an Octet RED96 instrument. The biotin-conjugated hACE2 protein was captured by streptavidin that was immobilized on a chip and tested for binding with gradient concentrations of the soluble RBD of S proteins from SARS CoV and SARS CoV-2. Binding kinetics were evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 9.0 software

Fig. 2
Fig. 2

The antibody response induced by recombinant RBD of SARS-CoV and SARS-CoV-2 in mice. a Schematic of the vaccine regimen. Five C57BL/6 mice per group were immunized two times (2–3 weeks apart) intramuscularly with 25 µg of the SARS CoV-2 RBD-hFc or SARS CoV RBD-hFc protein in combination with quick adjuvant. Mice immunized without the RBD protein but with hIgG were included as controls. Mice were sacrificed on day 35 after immunization, and antisera were collected for subsequent tests. b Cross-reactivity of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera against the SARS-CoV RBD or SARS-CoV-2 RBD as determined by ELISA. Mouse antisera were serially diluted three-fold and tested for binding to the SARS-CoV RBD or SARS-CoV-2 RBD. The IgG antibody (Ab) titres of SARS-CoV-2 antisera (red), SARS-CoV antisera (blue) and control antisera (black) were calculated at the endpoint dilution that remained positively detectable for the SARS-CoV-2 RBD or SARS-CoV RBD. The data are presented as the mean A450 ± s.e.m. (n = 5). c Cross-competition of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera and hACE2 with the SARS-CoV RBD or SARS-CoV-2 RBD as determined by ELISA. The data are presented as the mean blocking (%) ± s.e.m. (n = 5). Fifty percent blocking antibody titres (BT50) against the SARS-CoV pseudo-typed virus or SARS-CoV pseudo-typed virus were calculated. d Cross-neutralization of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera against SARS-CoV-2 or SARS-CoV pseudo-typed virus entry, measured by pseudo-typed virus neutralization assay. The data are presented as the mean neutralization (%) ± s.e.m. (n = 5). Fifty percent neutralizing antibody titres (NT50) against the SARS-CoV-2 or SARS-CoV pseudo-typed virus were calculated

Fig. 3
Fig. 3

Single amino acid substitution mutagenesis of the SARS-CoV-2-RBD and SARS-CoV-RBD. a Sequence differences in the SARS-CoV and SARS-CoV-2 RBDs. RBM is in red. Previously, identified critical ACE2-binding residues are shaded in green. The conserved residues are marked with asterisks (*), the residues with similar properties between groups are marked with the colon symbol (:) and the residues with marginally similar properties are marked with the period symbol (.). b ACE2 binding with reciprocal amino acid substitutions in the SARS-CoV-2 RBD. Each value is calculated as the binding relative to that of the WT (%). The mean±S.E.M. of duplicate wells is shown for two independent experiments. The two red dotted lines represent 75% and 125% relative to the WT data, respectively. c, d Structural alignment of SARS-CoV-2-RBD and SARS-CoV-RBD binding with ACE2. The SARS-CoV-RBD complex (PDB ID: 2AJF) is superimposed on the SARS-CoV-2 RBD (PDB ID: 6lzj. grey: ACE2, wheat: SARS-CoV-2. Mutants that weaken the SARS-CoV-2 RBD binding with ACE2 are highlighted in cyan (c). The corresponding residues from SARS-CoV are indicated in green and are illustrated in detail (c left). Mutants that enhance ACE2 binding are highlighted in magenta (d). e ACE2 binding with reciprocal amino acid substitutions in the SARS-CoV RBD. Each value is calculated as the binding relative to that of the WT (%). The mean ± S.E.M. of duplicate wells is shown in two independent experiments. The two red dotted lines represent 75 and 125% relative to the WT data, respectively. f Molecular docking of the SARS-CoV 2 RBD carrying the Q498Y mutant in complex with hACE2. Q498Y formed π-π stacking with Y41 in hACE2: left, Y498; right, Q498

Fig. 4
Fig. 4

Cross-reactivity of the RBD-targeting neutralizing mAbs against SARS-CoV and SARS-CoV-2. a Characteristics of the neutralizing mAbs against the SARS CoV-2 RBD and SARS CoV RBD. b, c Dose-dependent binding of SARS-CoV and SARS-CoV-2 mAbs to the SARS-CoV RBD (b) or SARS-CoV-2 RBD (c) as determined by ELISA. Isotype antibody was included as a control. Data are presented as the mean OD450 ± s.e.m. (n = 2). d, e Dose-dependent competition of the SARS-CoV-2 or SARS-CoV mAbs and hACE2 with the SARS-CoV RBD (d) or SARS-CoV-2 RBD (e) as measured by ELISA. Data are presented as the mean OD450 ± s.e.m. (n = 2). f IC50 values were determined for a panel of mAbs neutralizing the SARS-CoV-2 or SARS-CoV pseudo-typed viruses. Representative data are shown

Fig. 5
Fig. 5

Recognition pattern of mAbs to single amino acid substitute mutants of SARS-CoV or SARS-CoV-2 RBD. a Sequence conservation in the SARS-CoV and SARS-CoV-2 RBDs in a surface representation. Red, different; grey, identical. b Site mutagenesis scanning. The SARS-CoV and SARS-CoV-2 RBD mutant panel includes the reported antibody epitope positions and sequence changes within the RBMs. Relative binding to the wild-type: 0–25% presented in black; 25–50%, presented in dark grey; 50–75% presented in light grey; >75%, presented in white. The results shown represent the mean percentage of binding signal for the mAbs bound to the mutants relative to that of the wild-type RBD in at least two independent experiments. c Interaction of Y484 and D480 in the SARS-CoV RBD with 80 R (PDB ID: 2ghw). Polar interactions are indicated by yellow dashed lines. d Interaction of Y484 and T487 in the SARS-CoV RBD with m396 (PDB ID: 2dd8). Yellow: heavy chain, cyan: light chain. The binding surface of m396 is shown by electrostatic surface representations. e The residues that are important for HA001 binding are on the interface of the ACE2 and RBD (PDB ID: 6VW1)

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