Structural basis for broad coronavirus neutralization

Abstract

Three highly pathogenic β-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARS-CoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight β-coronavirus spike glycoproteins, including all five human-infecting β-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryo-electron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with β-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.

Figure 1.. Identification and characterization of a cross-reactive and broadly neutralizing coronavirus mAb

(A) Mouse immunization and B6 mAb selection scheme. MERS-CoV and SARS-CoV S₁ subunits fused to human Fc and the respiratory syncytial virus fusion glycoprotein (RSV F) ectodomain trimer fused to a foldon and a his-tag were used as decoys during selection. (B-E) Binding of MERS-CoV S (B), OC43 S (C), SARS-CoV S (D) and SARS-CoV-2 S (E) ectodomain trimers to the B6 mAb immobilized at the surface of biolayer interferometry biosensors. Data were analyzed with the ForteBio software, and global fits are shown as dashed lines. The vertical dotted lines correspond to the transition between the association and dissociation phases. Approximate apparent equilibrium dissociation constants (K_D, app) are reported due to the binding avidity resulting from the trimeric nature of S glycoproteins. (F-H) B6-mediated neutralization of VSV particles pseudotyped with MERS-CoV S (F), OC43 S (G) and HKU4 S (H). Data were evaluated using a non-linear sigmoidal regression model with variable Hill slope. Fit is shown as dashed lines and experiments were performed in triplicate with at least two independent mAb and pseudotyped virus preparations.

Figure 2.. B6 targets a linear epitope in the coronavirus S₂ fusion machinery.

(A-B) Molecular surface representation of a composite model of the B6-bound MERS-CoV S cryoEM structure and of the B6-bound MERS-CoV S stem helix peptide crystal structure shown from the side (A) and viewed from the viral membrane (B). MERS-CoV S protomers are colored pink, cyan and gold and the B6 Fab heavy and light chains are colored purple and magenta, respectively. The composite model was generated by docking the crystal structure of B6 bound to the MERS-CoV stem helix in the cryoEM map. (C) Identification of a conserved 15 residue sequence spanning the stem helix. Residue numbering for MERS-CoV S and SARS-CoV-2 S are indicated on top and bottom of the alignment, respectively. (D) Binding of 0.1 μM B6 mAb or (E) 1 μM B6 Fab to biotinylated coronavirus S stem helix peptides immobilized at the surface of biolayer interferometry biosensors.

Figure 3.. Molecular basis for the broad B6 cross-reactivity with a conserved coronavirus stem helix peptide.

(A) Crystal structure of the B6 Fab (surface rendering) in complex with the MERS-CoV S stem helix peptide. (B-C) Crystal structures of the B6 Fab bound to the MERS-CoV S (B) or HKU4 S (C) stem helix reveal a conserved network of interactions except for the substitution of D1236_MERS-CoV with E1237_HKU4 which preserves the salt bridge triad formed with CDRH3 residue R104 and CDRL1 residue H33. (D-F) Crystal structures of the B6 Fab bound to the MERS-CoV S (D), OC43 S (E) or SARS-CoV/SARS-CoV-2 S (F) stem helix showcasing the conservation of the paratope/epitope interface except for the conservative substitution of F1238_MERS-CoV with W1240_OC43 or Y1137_SARS-CoV/Y1155_SARS-CoV-2. The B6 heavy and light chains are colored purple and magenta, respectively, and only selected regions are shown in panels (B-F) for clarity. The coronavirus S stem helix peptides are rendered in ribbon representation and colored gold with interacting side chains shown in stick representation.

Figure 4.. B6 binding disrupts the stem helix bundle and sterically inhibits membrane fusion.

(A) CryoEM map of prefusion SARS-CoV-2 S (EMD-21452) filtered at 6 Å resolution to emphasize the intact trimeric stem helix bundle. (B) CryoEM map of the MERS-CoV S–B6 complex showing a disrupted stem helix bundle. (C) Model of B6-induced S stem movement obtained through comparison of the apo SARS-CoV-2 S and B6-bound MERS-CoV S structures. (D-F) Proposed mechanism of inhibition mediated by the B6 mAb. B6 binds to the hydrophobic core (red) of the stem helix bundle and disrupts its quaternary structure (D-E). The B6 disrupted state likely prevents S₂ subunit refolding from the pre- to the post-fusion state and blocks viral entry (F).