Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry

Figures

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  Figure 1.. Identification of the S2K146 cross-reactive and broadly neutralizing sarbecovirus mAb. A) Binding of site I-targeting S2E12, site IV-targeting S309, or site II-targeting S2X259 recombinant IgG1 (second phase) following association of S2K146 mAb (first phase) to His-tagged SARS-CoV-2 RBD immobilized on anti-His sensors, as measured by biolayer interferometry. B) Cross-reactivity of S2K146 (upper panel) and S2E12 (lower panel) with 22 RBDs from sarbecovirus clades (in brackets) and SARS-CoV-2 variants analyzed by ELISA. EC50 of at least two independent experiments are shown. C) Flow cytometry analysis of S2K146 cross-reactivity with a panel of 12 S glycoproteins representative of sarbecovirus clades 1a and 1b transiently expressed on the surface of mammalian cells. D) S2K146 cross-reactivity with sarbecovirus RBDs displayed at the surface of yeast. E) S2K146- and S2E12-mediated neutralization of replication-competent SARS-CoV-2 (USA-WA1/2020) and SARS-CoV-2 VOC viruses. F) S2K146 and S2K146-UCAmediated neutralization of VSV pseudotypes harboring SARS-CoV-2 S or SARS-CoV S.

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  Figure 2.. The S2K146 broadly neutralizing mAb recognizes RBD antigenic site I. A) CryoEM structure of the prefusion SARS-CoV-2 S ectodomain trimer with three S2K146 Fab fragments bound to two open RBDs and one partially closed RBD viewed along two orthogonal orientations. SARS-CoV-2 S protomers are colored cyan, pink and gold. S2K146 heavy chain and light variable domains are colored purple and magenta. Glycans are rendered as blue spheres. B) Ribbon diagram of the S2K146-bound SARS-CoV-2 RBD. C) Zoomed-in view of the contacts formed between S2K146 and the SARS-CoV-2 RBD. Selected epitope residues are shown as sticks and electrostatic interactions are indicated with dashed lines. D) S2K146 epitope residues shown as sticks and colored purple (labeled) if they are involved in ACE2 binding or grey otherwise (unlabeled). E) The side chains of the ten S2K146 epitope residues conserved between the SARS-CoV-2 (purple) and SARS-CoV (PDB 2AJF (29), green) RBDs are shown as sticks. F) The side chains of the six S2K146 epitope residues conservatively substituted between the SARS-CoV-2 (purple) and SARS-CoV (green) RBDs are shown as sticks.

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  Figure 3.. S2K146 is resilient to a broad spectrum of escape mutations. A) Molecular surface representation of the SARS-CoV-2 RBD with the S2K146 epitope colored purple and the ACE2 footprint indicated as a black outline. B) Mapping of RBD mutations reducing S2K146 binding using DMS of the yeast-displayed SARS-CoV-2 RBD. Sites of strong escape (purple underlines in Supplemental Figure S5.A) are shown in logo plot. Letters are colored according to how mutations affect the RBD affinity for ACE2 as measured via yeast display as measured in Starr et al. (24). C) Molecular surface representation of the SARS-CoV-2 RBD with the S2E12 epitope colored grey and the ACE2 footprint indicated as a black outline. The N343 glycan is rendered as blue spheres in A and C. D) Mapping of RBD mutations reducing S2E12 binding using DMS of the yeast-displayed SARS-CoV-2 RBD. Sites of strong escape (purple underlines in Supplemental Figure S5A) are shown in logo plot, as measured previously in Starr et. al(14)). E) Zoomed-in view of the Y489H S2K146 neutralization escape mutation. The S2K146 heavy and light chain variable domains are shown as transparent purple and magenta surfaces with ribbons, respectively. F)…

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  Figure 4.. S2K146 blocks receptor attachment, triggers premature S refolding and protects against SARS-CoV-2 challenge therapeutically. A) Superimposition of the S2K146-bound (purple) and ACE2-bound (green, PDB: 6M0J (36)) SARS-CoV-2 RBD structures showing steric overlap. B) Pre-incubation of serial dilutions of S2K146 with SARS-CoV-2 RBD prevents binding to immobilized human ACE2 (hACE2) ectodomain in ELISA. C) S2K146-mediated S1-shedding from cell-surface expressed SARS-CoV-2 S as determined by flow cytometry. S2E12 mAb was used as positive control whereas S2M11 was used as a negative control. D) Cropped electron micrographs of negatively stained SARS-CoV-2 S trimer before (left, prefusion state) or after (right, postfusion state) incubation with S2K146. One representative micrograph for each dataset is shown out of 93 (SARS-CoV-2 S alone) and of 225 (SARS-CoV-2 S with S2K146) micrographs. E-F) Quantification of viral RNA (E) and replicating virus titers (TCID50), (F) in the lung of Syrian hamster 4 days post-intranasal infection with SARSCoV-2 Beta VOC following therapeutic administration of S2K146 mAb at three different doses: 10-5-1mg/Kg (n=6/5 animal for each group). S2E12 mAb was…