Figures and data
Anti-RBD antibodies recognizing Class I and Class II epitopes
(A) Cryo-EM reconstruction for spike-Fab V3-9 complex. (B) Fab V3-9 targets an epitope which overlaps with the ACE2 binding site on RBD and belongs to Class I. (C) Expanded view highlights interactions of V3-9 with RBD and illustrates hydrogen bonding and hydrophobic contacts of the interface (D) rotated 180° along the y-axis relative to C. (E) Cryo-EM reconstruction for spike-Fab V6-4 complex. (F) Fab V6-4 belongs to Class II and its epitope overlaps with the ACE2 binding site on the RBD. (G) Expanded view highlights interactions of V6-4 with RBD and illustrates hydrogen bonding and hydrophobic contacts of the interface (H) rotated 180° along the y-axis relative to G. Spike is colored grey, RBD red, V3-9 light chain green, V3-9 heavy chain blue, V6-4 light chain orange and V6-4 heavy chain teal.
NTD top binding antibodies recognize supersite I
(A) Cryo-EM reconstruction for spike-Fab V5-6 complex. (B) Fab V5-6 binds the supersite I epitope on NTD. (C) Expanded view highlights interactions of V5-6 with NTD and illustrates hydrogen bonding and hydrophobic contacts at the interface (D) rotated 180° along the y-axis relative to C. (E) Cryo-EM reconstruction for spike-Fab V6-7 complex. (F) Fab V6-7 binds the supersite I epitope on NTD. (G) Expanded view highlights interactions of V6-7 with NTD and illustrates hydrogen bonding and hydrophobic contacts at the interface (H) rotated 180° along the y-axis relative to G. (I) Cryo-EM reconstruction for spike-Fab V6-2 complex. (J) Fab V6-2 binds the supersite I epitope on NTD. (K) Expanded view highlights interactions of V6-2 with NTD and illustrates hydrogen bonding and hydrophobic contacts at the interface (L) rotated 180° along the y-axis relative to K. Spike is colored grey, NTD light blue, V5-6 light chain yellow, V5-6 heavy chain indigo, V6-7 light chain green and V6-7 heavy chain purple, V6-2 light chain mustard, V6-2 heavy chain brown.
NTD lateral side binding antibodies recognize hydrophobic cavity
(A) Cryo-EM reconstruction for NTD-Fab V6-11 complex with spike trimer. As V6-11 dissociates the trimer, the focused map is shown in colors, and the putative trimer complex is shown in white (PDB:6VXX) to illustrate the incompatibility of the V6-11 complex with the trimeric spike. (B) Fab V6-11 targets the hydrophobic cavity of NTD lateral side. (C) Expanded view highlights interactions of V6-11 with NTD and illustrates hydrogen bonding and hydrophobic contacts at the interface (D) rotated 180° along the y-axis relative to C. (E) Cryo-EM reconstruction for spike-Fab V6-14 complex. (F) Fab V6-14 targets the hydrophobic cavity of NTD lateral side. (G) Expanded view highlights interactions of V6-14 with NTD and illustrates hydrogen bonding and hydrophobic contacts at the interface (H) rotated 180° along the y-axis relative to G. Spike is colored grey, NTD light blue, V6-11 light chain salmon, V6-11 heavy chain lavender, V6-14 light chain brown and V6-14 heavy chain green.
Functional characterization of SARS-CoV-2 spike-targeting antibodies
(A) Heatmap of relative binding to SARS-CoV-2 spike variants by flow cytometry. Percentage of spike positive cells binding each mAb was normalized such that the maximum % binding observed for each antibody across all variants is set to 100%. ACE2 and Flu-Ab binding to spike and variants are taken as positive and negative controls. Data are representative of 2 independent biological repeats. (B) Cell fusion assay-Quantification of syncytia formation of WA1 spike. Cell-cell fusion was represented as GFP positive area which was normalized to spike with no Ab. Shown are the means and standard deviation (SD) of three replicates. EK1C4 and Flu-Ab are positive and negative control for cell fusion inhibition respectively. Data are representative of 2 independent biological repeats.
Overview of cryo-EM data processing and local resolution analysis.
(A) Representative cryo-electron micrograph of the SARS-CoV-2 WA spike V3-9 complex. (B) Selected 2D class averages of the WA1 spike ectodomain bound to V3-9. (C) Local resolution maps and gold-standard Fourier shell correlation (FSC) curves for the global spike-Fab reconstruction generated using cryoSPARC v3.3.1. (D) and locally refined receptor-binding domain (RBD)-Fab complex. (E) Schematic representation of the overall cryo-EM data processing workflow.
Overview of cryo-EM data processing and local resolution analysis.
(A) Representative cryo-electron micrograph of the SARS-CoV-2 WA1 spike V6-4 complex. (B) Selected 2D class averages of the WA1 spike ectodomain bound to V6-4. (C) Local resolution maps and gold-standard Fourier shell correlation (FSC) curves for the global spike-Fab reconstruction generated using cryoSPARC v3.3.1. (D) and locally refined receptor-binding domain (RBD)-Fab complex. (E) Schematic representation of the overall cryo-EM data processing workflow.
Overview of cryo-EM data processing and local resolution analysis.
(A) Representative cryo-electron micrograph of the SARS-CoV-2 WA1 spike V5-6 complex. (B) Selected 2D class averages of the WA1 spike ectodomain bound to V5-6. (C) Local resolution maps and gold-standard Fourier shell correlation (FSC) curves for the global spike-Fab reconstruction generated using cryoSPARC v3.3.1. (D) and locally refined N-terminal domain (NTD)-Fab complex. (E) Schematic representation of the overall cryo-EM data processing workflow.
Overview of cryo-EM data processing and local resolution analysis.
(A) Representative cryo-electron micrograph of the SARS-CoV-2 WA1 spike V6-7 complex. (B) Selected 2D class averages of the WA1 spike ectodomain bound to V6-7. (C) Local resolution maps and gold-standard Fourier shell correlation (FSC) curves for the global spike-Fab reconstruction generated using cryoSPARC v3.3.1. (D) and locally refined N-terminal domain (NTD)-Fab complex. (E) Schematic representation of the overall cryo-EM data processing workflow.
Overview of cryo-EM data processing and local resolution analysis.
(A) Representative cryo-electron micrograph of the SARS-CoV-2 WA1 spike V6-2 complex. (B) Selected 2D class averages of the WA1 spike ectodomain bound to V6-2. (C) Local resolution maps and gold-standard Fourier shell correlation (FSC) curves for the global spike-Fab reconstruction generated using cryoSPARC v3.3.1. (D) and locally refined N-terminal domain (NTD)-Fab complex. (E) Schematic representation of the overall cryo-EM data processing workflow.
Overview of cryo-EM data processing and local resolution analysis.
(A) Representative cryo-electron micrograph of the SARS-CoV-2 WA1 spike V6-11 complex. (B) Selected 2D class averages of the WA1 spike ectodomain bound to V6-11. (C) Local resolution maps and gold-standard Fourier shell correlation (FSC) curves for the local spike-Fab reconstruction generated using cryoSPARC v3.3.1. (D) Schematic representation of the overall cryo-EM data processing workflow.
Glycans interaction with antibody.
(A) N122 glycan of spike interacting with CDRH2 and CDRL3. (B) N17 glycan of spike interacting with CDRL3. Glycans are shown as sticks in pink and antibody interacting residues are shown as sticks.
Analysis of heavy chain clashes with spike protomers
(A) The heavy chain of PVI.V6-11 exhibits steric clashes with the RBD of protomer B.(B) the heavy chain of PVI.V6-14 does not display any clashes with protomer B of the spike. PVI.V6-11 heavy chain is shown in lavender and PVI.V6-11 light chain is shown in salmon, PVI.V6-14 heavy chain is shown in green and PVI.V6-14 light chain is shown in orange. Protomer A, B and C of spike is shown in light blue, tan and gray respectively.
PVI.V6-11 competes with biliverdin.
Structural superposition of biliverdin bound NTD (PDB: 7B62) with PVI.V6-11 bound NTD (this study). Biliverdin is shown in green. PVI.V6-11 heavy chain is shown in lavender with CDRH3 loop highlighted in sticks. NTD is shown in light blue with interacting residues highlighted in sticks.
Multiple sequence alignment of RBD with epitope residues of RBD binders.
SARS-CoV-2 spike receptor-binding domain (RBD; amino acids 319-542) of WA1, B.1.1.7, P.1, B.1.617.2, BA.1, BQ.1.1, BA.5, BA.2.86, XBB.1.5, and JN.1. Epitope residues of PVI.V3-9 and PVI.V6-4 are indicated in heat map. Blue represents the heavy chain, yellow represents the light chain and white represents no interaction.
Multiple sequence alignment of NTD with epitope residues of NTD top binders.
SARS-CoV-2 spike N-terminal domain (NTD; amino acids 14-306) of WA1, B.1.1.7, P.1, B.1.617.2, BA.1, BQ.1.1, BA.5, BA.2.86, XBB.1.5, and JN.1. Epitope residues of Fab PVI.V5-6, PVI.V6-7 and PVI.V6-2 are indicated in heat map. Blue represents the heavy chain, yellow represents the light chain, green represents both heavy and light chain interaction and white represents no interaction.
Structural overlay of SARS-CoV-2 NTD variants reveals loop deviations and antibody clash.
(A) Superimposition of the NTD from the WA1 spike in complex with PVI.V5-6 onto NTD structures from BA.5 NTD with an RBD-bound antibody (PDB 8GTP), XBB.1.5 NTD (unliganded, PDB 8VKK), and XBB.1.5 in complex with an NTD-directed antibody (PDB 9CCJ) reveals significant conformational deviations in the N2, N3, and N5 loops. (B) Structural overlay shows that loop rearrangements in XBB.1.5 and BA.5 produce steric clashes with the PVI.V5-6 paratope. (C) Superimposition of the NTD from the WA1 spike in complex with PVI.V6-7 onto NTD structures from BA.5 NTD with an RBD-bound antibody (PDB), XBB.1.5 NTD (unliganded, PDB), and XBB.1.5 in complex with an NTD-directed antibody (PDB) reveals significant conformational deviations in the N2, N3, and N5 loops among these variants. (D) Structural overlay shows that loop rearrangements in XBB.1.5 and BA.5 produce steric clashes with the PVI.V6-7 paratope. (E) Superimposition of the NTD from the WA1 spike in complex with PVI.V6-7 onto NTD structures from BA.5 NTD with an RBD-bound antibody (PDB), XBB.1.5 NTD (unliganded, PDB), and XBB.1.5 in complex with an NTD-directed antibody (PDB) reveals significant conformational deviations in the N2, N3, and N5 loops among these variants. (F) Structural overlay shows that loop rearrangements in XBB.1.5 and BA.5 produce steric clashes with the PVI.V6-2 paratope.
Multiple sequence alignment of NTD with epitope residues of NTD lateral side binders.
SARS-CoV-2 spike N-terminal domain (NTD; amino acids 14-306) of WA1, B.1.1.7, P.1, B.1.617.2, BA.1, BQ.1.1, BA.5, BA.2.86, XBB.1.5, and JN.1. Epitope residues of Fab PVI.V6-11, and PVI.V6-14 are indicated in heat map. Blue represents the heavy chain, yellow represents the light chain, green represents both heavy and light chain interaction and white represents no interaction.
