Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

Abstract

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

Keywords: COVID-19; SARS-CoV-2; coronaviruses; effector functions; immunity; neutralizing antibodies.

Graphical abstract

Figure S1

Description of the Cohorts of SARS-CoV-2-Infected Individuals, Related to Figures 1 and 2 (A) Summary of patient demographics. (B) Age distribution of hospitalized, symptomatic and asymptomatic individuals. (C) Time interval between the date of sample collection and the date of symptom onset.

Figure 1

Analysis of the Specificity of IgG, IgA, and IgM Serum/Plasma Abs from a Panel of 647 Hospitalized, Symptomatic, and Asymptomatic SARS-CoV-2-Infected Individuals (A–C) Binding titers (ED50) of antigen-specific IgG (A), IgA (B), or IgM (C) were measured in plasma or sera from convalescent SARS-CoV-2 patients (47 hospitalized, 556 symptomatic, and 44 asymptomatic) and from pre-pandemic healthy donors (n = 32). A cut-off of 30 was determined based on signal of pre-pandemic samples and binding to uncoated ELISA plates. (D) Binding titers (ED50) of S- and N-specific IgGs measured in sera from symptomatic and asymptomatic SARS-CoV-2-infected individuals from the Ticino healthcare workers cohort (n = 459) categorized according to symptoms severity, as described in the methods. (E) IgG binding titers to SARS-CoV-2 RBD (left) and SARS-CoV-2 S pseudovirus neutralizing titers (ID80, center) before and after depletion of RBD-specific Abs from 21 SARS-CoV-2 immune plasma samples. The percentage of depletion of binding and neutralizing Abs (right) for each sample tested is shown on the right. (F) Ab-mediated inhibition of SARS-CoV-2 RBD binding to solid phase ACE2, as determined by ELISA. Shown is the reciprocal plasma or serum dilution that blocks 80% binding (BD80) of RBD to human ACE2. (G) Ab-mediated inhibition of SARS-CoV-2 RBD binding to solid phase ACE2 in the Ticino healthcare workers cohort determined as in (F). A cut-off of 10 was used to separate neutralizing from non-neutralizing titers. (H) Correlation analysis between levels of plasma/serum RBD-specific IgG (ED50) and the titers of Abs blocking RBD attachment to ACE2 (BD80). (I) Correlation analysis between plasma/serum neutralizing Ab titers (ID80) and the titers of Abs blocking RBD attachment to ACE2 (BD80).

Figure S2

Analysis of Serum/Plasma IgG Binding Titers to SARS-CoV-2 and SARS-CoV Antigens, Related to Figures 1 and 2 (A–C) IgG (A), IgA (B) and IgM (C) binding titers to SARS-CoV-2 S, RBD and N from 67 and 154 samples collected from hospitalized and symptomatic individuals, respectively, whose date of symptom onset was known. (D) Correlation between SARS-CoV-2 S- and N-specific IgG binding titers (ED₅₀). (E and F) IgG binding titers to SARS-CoV-2 and SARS-CoV S (E) and RBD (F) from 19 hospitalized, 130 symptomatic and 8 asymptomatic individuals. (G) Ratios of SARS-CoV-2/SARS-CoV S and RBD IgG binding titers.

Figure 2

Kinetics of IgG Responses Specific for the SARS-CoV-2 RBD and Blocking RBD Attachment to ACE2 (A) Binding titers (ED50) of serum or plasma IgG to the SARS-CoV-2 RBD measured at two time points separated by an average time of 44 days in 368 subjects. T1, time of first blood draw; T2, time of second blood draw. (B) Variation of RBD-specific IgG binding titers from T1 to T2. (C) Kinetics of RBD- and N-specific IgG responses in serum or plasma from 24 convalescent individuals (red, hospitalized; blue, symptomatic non-hospitalized). The starting time point corresponds to the date of collection of the first sample. (D) Model predicted longitudinal decline of RBD- and N-specific IgG binding titers from 18 convalescent individuals with respect to the onset of symptoms from infection. Symbols, observations; shaded region, 90% prediction interval; line, median prediction. (E) Serum or plasma titers of Abs blocking RBD attachment to ACE2 (BD80) measured at T1 and T2. (F) Variation of RBD-specific IgG binding titers and titers of Abs blocking RBD attachment to ACE2 (BD80) from T1 to T2. (G) Avidity index of serum IgG binding to RBD (%) measured at T1 and T2. (H) Variation of avidity index of IgG binding to RBD (%) from T1 to T2.

Figure S3

Characteristics of the Six Probe mAbs Used for Structural and Epitope-Mapping Studies, Related to Figures 3, 4, 5, 6, and 7 (A) V(D)J usage, percentage identity to germline, number of somatic mutations, source and time interval between sample collection and mAb isolation, RBD site recognized and neutralization potency of the 6 mAbs. B mem, memory B cell; PC, plasma cells. (B) Binding of the 6 mAbs to the SARS-CoV-2 (up) or SARS-CoV (down) RBD analyzed by ELISA. (C) Competition matrix for binding of each of the six mAbs in presence of another mAb evaluated by biolayer interferometry. (D) mAb-mediated inhibition of RBD binding to ACE2 analyzed by ELISA. (E) mAb-mediated S₁ subunit shedding from cell-surface expressed SARS-CoV-2 S as determined by flow-cytometry. (F) Conservation of RBM and epitope residues in ∼74,000 SARS-CoV-2 sequences (GISAID, August 11^th, 2020). RBM and epitope residues are shown as gray bars. Black bars indicate variant prevalence for epitope residues with at least 2 variants. RBM residues were determined from PDB 6M0J using a 5.0 Å distance cutoff between RBD and ACE2 residues using MOE. (G) Western-blot analysis (top) of the prefusion-stabilized SARS-CoV-2 S ectodomain trimer in presence of S2A4, S304 or S2X35 Fab after incubation for the indicated amount of times. Red ponceau staining (bottom) of the SDS-PAGE gel used for carrying out the western blot confirming the presence of added Fabs when indicated. (H) Analysis of activation of FcγRIIIa (V158 allele) expressed on Jurkat cells by SARS-CoV-2 S stably transfected CHO cells incubated with mAbs. GRLR indicates an antibody Fc variant carrying mutations that abolish binding to FcγRs. (I) Analysis of activation of FcγRIIa (H131 allele), expressed on Jurkat cells by SARS-CoV-2 S stably transfected CHO cells incubated with mAbs. (J) Killing of SARS-CoV-2 S stably transfected CHO cells by mAbs in the presence of complement (CDC assay).

Figure 3

The S2H13 mAb Inhibits SARS-CoV-2 by Blocking Attachment to ACE2 via Recognition of an Epitope Accessible in the Open and Closed S Conformations (A) SARS-CoV-2 S pseudovirus neutralization assay indicating an IC50 of 500 ng/mL. (B and C) Molecular surface representation of the SARS-CoV-2 S/S2H13 Fab complex structure with three RBDs closed shown in two orthogonal orientations. (D) Molecular surface representation of the SARS-CoV-2 S/S2H13 Fab complex structure with one RBD open. Each SARS-CoV-2 protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S2H13 light and heavy chain variable domains are colored magenta and purple, respectively. (E) S2H13 recognizes a crevice formed by the SARS-CoV-2 RBM. Selected side chains at the interface are shown. (F) S2H13 and ACE2 (dark green) bind overlapping RBM epitope. The red star indicates steric clashes. (G) BLI binding competition between S2H13 and ACE2 for binding to the SARS-CoV-2 RBD. (H) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S2H13 epitope colored by residue conservation across SARS-CoV-2 isolates and SARS-CoV.

Figure S4

Cryo-EM Data Processing and Validation of the S/S2H13 and S/S2H14 Complex Datasets, Related to Figures 3 and 4 (A and B) Representative electron micrograph (A) and class averages (B) of SARS-CoV-2 S in complex with the S2H13 Fab embedded in vitreous ice. Scale bar: 400Å. (C) Gold-standard Fourier shell correlation curves for the closed S2H13-bound trimer (black solid line), partially open S2H13-bound trimer (gray solid line) and locally refined RBM/S2H13 variable domains (black dashed line). The 0.143 cutoff is indicated by horizontal dashed lines. (D and F) Local resolution maps calculated using cryoSPARC for the closed (D) and partially open (E) reconstructions as well as for the locally refined RBM/S2H13 variable domains (F). (G and H) Representative electron micrograph (G) and class averages (H) of SARS-CoV-2 S in complex with the S2H14 Fab embedded in vitreous ice. Scale bar: 400Å. (I) Gold-standard Fourier shell correlation curves for the S2H14-bound trimer with one RBD closed (black solid line) or three RBDs open (gray solid line). The 0.143 cutoff is indicated by horizontal dashed lines. (J and K) Local resolution maps calculated using cryoSPARC for the reconstructions with one RBD closed (J) and three RBDs open (K).

Figure S5

Analysis of Fab and IgG Binding to the Prefusion SARS-CoV-2 S Ectodomain Trimer and Recombinant RBD at Neutral and Acidic pH Analyzed by Surface Plasmon Resonance, Related to Figures 3, 4, 5, 6, and 7 (A and B) SARS-CoV-2 S or RBD was captured on the sensor chip surface and binding at multiple mAb concentrations was measured. Neutral pH measurements were performed in multi-cycle format (A) and acidic pH measurements in single-cycle format (B). All data have been fit to a 1:1 binding model, which is an approximation for the S-binding data, since the kinetics incorporate conformational dynamics between open and closed RBD states, and because IgG binding involves avidity. The solid gray horizontal line gives the predicted maximum signal (saturation) based on each fit; the dashed line shows the S309 maximum binding for comparison. Asterisk indicates where a high concentration of S304 IgG was binding to the reference surface (fit was to the first two concentrations only). All mAbs bind similarly to the RBD at both pHs, but the mAbs that bind to only open RBD show a maximum below S309 in the context of the S trimer. This difference is dramatic at acidic pH where RBDs are primarily in the closed state (Zhou et al., 2020b). S2X35 was an exception, likely because its very slow off rate allows it to bias the S equilibrium toward open RBD.

Figure 4

The S2H14 mAb Inhibits SARS-CoV-2 by Blocking Attachment to the ACE2 Receptor (A) SARS-CoV-2 S pseudovirus neutralization assay indicating an IC50 of 900 ng/mL. (B and C) Molecular surface representation of the SARS-CoV-2 S/S2H14 Fab complex structure with two RBDs open and one RBD closed viewed along two orthogonal orientations. (D and E) Molecular surface representation of the SARS-CoV-2 S/S2H14 Fab complex structure with three RBDs open shown in two orthogonal orientations. Each SARS-CoV-2 protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S2H14 light and heavy chain variable domains are colored magenta and purple, respectively. (F) S2H14 binds to an epitope within the SARS-CoV-2 RBM. (G) S2H14 and ACE2 (dark green) bind overlapping RBM epitope. The red star indicates steric clashes. (H) BLI binding competition between S2H14 and ACE2 for binding to the SARS-CoV-2 RBD. (I) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S2H14 epitope colored by residue conservation across SARS-CoV-2 isolates and SARS-CoV.

Figure S6

Conservation Analysis across Clades of Sarbecoviruses, Related to Figures 3, 4, 5, and 6 (A) S glycoprotein residues making contact with S304, S2H13, S2H14 or S2A4 across sarbecovirus clades. Residue numbers for both SARS-CoV-2 S and SARS-CoV S are shown. Multiple sequence alignment was performed using MAFFT. A dash represents the same residue, a strikethrough represents a gap. Asterisk (^∗) indicates manually aligned residues. Civet SARS-CoV is SARS-CoV HC/SZ/61/03 and raccoon dog SARS-CoV is SARS-CoV A031G. (B) Identity and similarity of SARS-CoV-2 S, RBD, RBM and mAb epitopes across select sequences of the 3 sarbecovirus clades. Values were calculated using EMBOSS Needle. The insertion in the S2A4 epitope for the Clade 1 sarbecoviruses was not included in the calculation.

Figure 5

The S2A4 mAb Promotes SARS-CoV-2 S Opening through Binding to a Cryptic Epitope (A) SARS-CoV-2 S pseudovirus neutralization assay indicating an IC50 of 3.5 μg/mL. (B and C) Molecular surface representation of the SARS-CoV-2 S/S2A4 Fab complex cryo-EM structure with three RBDs open viewed along two orthogonal orientations. Each SARS-CoV-2 protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S2A4 light and heavy chains are colored magenta and purple, respectively. (D and E) Zoomed-in views of the contacts formed between S2A4 and the RBD with selected side chains shown. (F) S2A4 and ACE2 (dark green) bind distinct RBD epitopes but would clash via steric hindrance. The red star indicates steric clashes. (G) BLI binding competition between S2A4 and ACE2 for binding to the SARS-CoV-2 RBD. (H) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S2A4 epitope colored by amino acid residue conservation with SARS-CoV. The position of the SARS-CoV N357 glycan is indicated with red dotted lines.

Figure S7

Cryo-EM Data Processing and Validation of the S/S2A4 and S/S304 Complex Datasets, Related to Figures 5 and 6 (A and B) Representative electron micrograph (A) and class averages (B) of SARS-CoV-2 S in complex with the S2A4 Fab embedded in vitreous ice. Scale bar: 400Å. A 2D class average corresponding to an S₁ subunit trimer (with disordered S₂) bound to three S2A4 Fabs is highlighted in red. (C) Gold-standard Fourier shell correlation curves for the S2A4-bound trimer (black solid line) and locally refined RBD/S2A4 variable domains (black dashed line). The 0.143 cutoff is indicated by a horizontal dashed line. (D and E) Local resolution maps calculated using cryoSPARC for the whole reconstruction (D) as well as for the locally refined RBD/S2A4 variable domains (E). (F) Superimposition of the three distinct open conformations of the S trimer, with three bound S2A4 Fabs and RBDs swung out to various extent. The arrows indicate the distinct positions of the Fabs in the maps. (G and H) CryoEM reconstruction of the S₁ subunit trimer (with disordered S₂) bound to three S2A4 Fabs viewed along two orthogonal orientations and the corresponding atomic model fit in density. Each SARS-CoV-2 S₁ protomer is colored distinctly (cyan, pink and gold). The S2A4 light and heavy chains are colored magenta and purple, respectively. (I and J) Representative electron micrograph (I) and class averages (J) of SARS-CoV-2 S in complex with the S304 Fab embedded in vitreous ice. Scale bar: 400Å. (K) Gold-standard Fourier shell correlation curve for the S304-bound S trimer reconstruction. The 0.143 cutoff is indicated by a horizontal dashed line. (L) Local resolution map calculated using cryoSPARC. (M) Superimposition of the three distinct open conformations of the S trimer, with three bound S304 Fabs and RBDs swung out to various extent. The arrows indicate the distinct positions of the Fabs in the maps. (N and O) CryoEM reconstruction of the S₁ subunit trimer (with disordered S₂) bound to three S304 Fabs viewed along two orthogonal orientations and the corresponding atomic model fit in density. Each SARS-CoV-2 S₁ protomer is colored distinctly (cyan, pink and gold). The S304 light and heavy chains are colored magenta and purple, respectively.

Figure 6

The S304 mAb Promotes SARS-CoV-2 S Opening through Binding to a Cryptic Epitope Conserved within the Sarbecovirus Subgenus (A and B) Molecular surface representation of the SARS-CoV-2 S/S304 Fab complex cryo-EM structure with three RBDs opened viewed along two orthogonal orientations. Each SARS-CoV-2 S protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S304 light and heavy chains are colored magenta and purple, respectively. (C) Cryo-EM reconstruction of the S₁ subunit trimer (with disordered S₂) bound to three S304 Fabs viewed along two orthogonal orientations and the corresponding atomic model fit in density. Each SARS-CoV-2 S₁ protomer is colored distinctly (cyan, pink, and gold). The S304 light and heavy chains are colored magenta and purple, respectively. (D) Ribbon diagram of the crystal structure of S304 (pink and purple), S2H14, and S309 in complex with the SARS-CoV-2 RBD (light blue). Only the S304 variable domains are shown, whereas S2H14 and S309 were omitted for clarity. (E) Positioning of ACE2 (dark green) relative to the S304 Fab bound to the SARS-CoV-2 RBD. ACE2 N-linked glycans at position N322 and N546 are indicated, as they could putatively clash with S304. (F) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S304 epitope colored by residue conservation with SARS-CoV. (G and H) Positioning of ACE2 (dark green) relative to the S2A4 (G) and S2X35 (H) Fabs bound to the SARS-CoV-2 RBD. The red stars indicate steric clashes.

Figure 7

Structure-Guided High-Resolution Serology (A) Composite model of the SARS-CoV-2 S trimer with three open RBDs viewed along two orientations with all six mAbs used for competition ELISA shown bound to one RBD. (B–G) Epitopes recognized by each mAb are shown on the surface of the RBD for S2H14 (teal, B), S2H13 (orange, C), S2X35 (red, D), S2A4 (yellow, E), S304 (magenta, F), and S309 (purple, G). The glycan at position N343 is rendered as blue spheres and the RBM is shown as a black outline. (H–J) Competition ELISA (blockade-of-binding) between individual mAbs and sera or plasma from hospitalized (H), symptomatic (I), and asymptomatic (J) COVID-19 convalescent subjects. Each plot shows the magnitude of inhibition of binding to immobilized RBD in the presence of each mAb, expressed as reciprocal sera or plasma dilution blocking 80% of the maximum binding response. (K) Correlation analysis of titers of serum Abs blocking RBD binding to ACE2 and Abs blocking each of the six probe mAbs. (L) Comparison of RBD-specific IgG titers between sera containing Ab blocking at least one probe mAb and sera that do not contain Ab blocking any of the six probe mAbs.