Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps

Abstract

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Figure 1.. Cryo-EM reconstruction of WT-fl-Spike with computationally designed, CVD293 or linker variant of the affinity matured variant, CVD432

a-b. Cryo-EM reconstructions of WT-fl-Spike with CVD293 or CVD432 showing the heterogeneity in distribution of all RBD down, 1-RBD- or 2-RBD-up states and variable ACE2 occupancy. Also shown is schematic of the primary structure of CVD293 or CVD432 and the engineered mutations, colored by domain.

Figure 2.. Cryo-EM reconstruction of WT-Spike-RBD with engineered ACE2 Fc-fusions reveal contributions from hydrophobic interactions at RBD-ACE2 interface

a. WT-Spike-RBD/CVD293 and WT-Spike-RBD/CVD432 models colored by estimated per residue Q-score ranging from 0 (red) to 0.7 (purple). The color bar shows corresponding estimated resolution in Å for each Q-score. Expected Q-score for 3.5 Å map is 0.49 and expected Q-score for 3.36 Å map is 0.52. b-c. Cryo-EM reconstructions of WT-Spike-RBD with either CVD293 or CVD432 show favorable π–π stacking interactions between WT-Spike-RBD residue Y489 and engineered ACE2 residue F31. Additionally, there are also hydrophobic interactions between WT-Spike-RBD residue L455 and CVD293 residue I34 are also seen. Hydrogen bond interactions between WT-Spike-RBD residue Q493 and CVD293 or CVD432 residue Q35 are not apparent in the cryo-EM consensus model. d. The Rosetta lowest energy model for CVD293 is overlaid with the cryo-EM model. Both models show hydrophobic and hydrogen bond interactions between CVD293 and WT-Spike-RBD residues that contribute to improved interface energy (REU) compared to the ACE2-WT Spike RBD interaction.

Figure 3.. Multi-model pipeline improves confidence of molecular interactions at the interface residues in cryo-EM derived models of WT-Spike-RBD with engineered ACE2 Fc-fusions

a. Multi-model pipeline with average Rosetta interface energy and average per-residue side-chain RMSD metrics for interface residue rotamer positions. b. Average per-residue side chain RMSD for interface helix residues of CVD293 and CVD432 c-d. Superposition of critical interface residues of the top 80 selected cryo-EM based models for CVD293 and CVD432. e. Average Rosetta interface energy for CVD293 design model, CVD293 cryo-EM based models, CVD432 design model and CVD432 cryo-EM based models.

Figure 4.. Binding to Omicron- and Delta-RBD and neutralization of Omicron- and Delta-SARS-CoV-2 VOCs by CVD293 and CVD432

a-b. Left panel - Predictions based on Rosetta interface energy calculations suggest that Omicron-RBD binds CVD293 and CVD432 with high affinity. Residue pair interactions of RBD residues with CVD293/CVD432 residue F31 (yellow), with residue I34/S34 (blue) and with residue Q35 (red) are shown. Right panel - Zoomed in view of the interface of the models Omicron-RBD/CVD293 and Omicron-RBD/CVD432. Wheat-colored residues indicate RBD interactions ACE2 K/F31. Blue residues indicate RBD interactions with ACE2 H/I/S34. Magenta residues indicate RBD interactions with more than one engineered ACE2 residue c. Biolayer interferometry measurements for CVD293 or CVD432 interactions with Omicron- or Delta-RBD. d. CVD293 and CVD432 potently neutralize vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV-2 Omicron- and Delta-Spike. Error bars represent standard deviation over all technical replicates from two biological replicates.