Structural and biochemical rationale for enhanced spike protein fitness in delta and kappa SARS-CoV-2 variants.

Biological materials availability

All unique biological materials described in this manuscript are made available upon reasonable request from the corresponding author and following the completion of a materials transfer agreement. Pseudovirus neutralization assay SARS-CoV-2 S protein Delta and Kappa genes were synthesised and inserted into pcDNA3.1 (GeneArt Gene Synthesis, Thermo Fisher Scientific). The production of the SARS-CoV-2 wild-type (D614G) S protein was described previously 23 . Variant pseudotyped retroviral particles were produced in HEK293T cells as described previously 46 . Briefly, a third-generation lentiviral packaging system was utilised in combination with plasmids encoding the full-length SARS-CoV-2 spike, along with a transfer plasmid encoding luciferase and GFP as a dual reporter gene. Pseudoviruses were harvested 60 h after transfection, filtered with a 0.45 µm PES filter, and frozen. For neutralization assays, HEK293T-ACE2-TMPRSS2 cells 47 (BEI Resources cat# NR-55293) were seeded in 96- or 384-well plates at 50,000 or 20,000 cells respectively. The next day, pseudovirus preparations normalised for viral capsid p24 levels (Lenti-X™ GoStix™ Plus) were incubated with dilutions of the indicated antibodies or sera (obtained under the approval of the UBC Clinical Research Ethics Board), or media alone for 1 h at 37 °C prior to addition to cells and incubation for 48 h. Cells were then lysed and luciferase activity assessed using the ONE-Glo™ EX Luciferase Assay System (Promega) according to the manufacturer’s specifications. Detection of relative luciferase units was carried out using a Varioskan Lux plate reader (Thermo Fisher). Percent neutralisation was calculated relative to signals obtained in the presence of virus alone for each experiment. The kits, reagents and DNA described in this section are summarised in Supplementary Tables 2 and 6 .

Expression and purification of recombinant spike protein constructs

The wild-type SARS-CoV-2 S HexaPro expression plasmid was previously described 45 and was a gift from Jason McLellan (Addgene plasmid #154754; http://n2t.net/addgene:154754 ; RRID:Addgene_154754). The VoC RBD mutations were introduced by site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs). Successful cloning was confirmed by Sanger sequencing (Genewiz, Inc.). The following primers were used (5′–3′): B.1.617.1 E484A HexaPro Forward: TAATGGCGTGgcgGGCTTCAACTGCTACTTCCCAC Reverse: CAAGGGGTGCTGCCGGCC B.1.617.1 E484I HexaPro Forward: TAATGGCGTGatcGGCTTCAACTGCTACTTCCCACTG Reverse: CAAGGGGTGCTGCCGGCC Expi293F cells (Thermo Fisher, Cat# A14527, Supplementary Table 5 ) were grown in suspension culture using Expi293 Expression Medium (Thermo Fisher, Cat# A1435102) at 37 °C, 8% CO 2 . Cells were transiently transfected at a density of 3 × 10 6 cells/mL using linear polyethylenimine (Polysciences Cat# 23966-1). The media was supplemented 24 h after transfection with 2.2 mM valproic acid, and expression was carried out for 3–5 days at 37 °C, 8% CO 2 . The supernatant was harvested by centrifugation and filtered through a 0.22-μM filter prior to loading onto a 5 mL HisTrap excel column (Cytiva). The column was washed for 20 CVs with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl), 5 CVs of wash buffer supplemented with 20 mM imidazole and the protein eluted with elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions containing the protein were pooled and concentrated (Amicon Ultra 100 kDa cut off, Millipore Sigma) for gel filtration. Gel filtration was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer (20 mM Tris pH 8.0, 150 mM NaCl). Peak fractions corresponding to soluble protein were pooled and concentrated to 4.5–5.5 mg/mL (Amicon Ultra 100 kDa cut off, Millipore Sigma). Protein samples were flash-frozen in liquid nitrogen and stored at −80 °C. Antibody production VH-FC ab8, IgG ab1, Fab S309, and Fab S2M11 were produced as previously described 17 , 18 . Plasmids encoding the light and heavy chains for Fab 4A8 and Fab 4–8 were synthesised (GeneArt Gene Synthesis, Thermo Fischer Scientific). Heavy chains were designed to incorporate a C terminal 6x histidine tag. Expi293 cells were transfected at a density of 3 × 10 6 cells/mL using linear polyethylenimine (Polysciences Cat# 23966-1). In all, 24-h following transfection, media was supplemented with 2.2 mM valproic acid, and expression was carried out for 3–5 days at 37 °C, 8% CO 2 . The supernatant was harvested by centrifugation and filtered through a 0.22-μM filter prior to loading onto a 5 mL HisTrap excel column (Cytiva). The column was washed for 20 CVs with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl), 5 CVs of wash buffer supplemented with 20 mM imidazole. The protein was eluted with elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions containing the protein were pooled and concentrated (Amicon Ultra 10 kDa cut off, Millipore Sigma) for gel filtration. Gel filtration was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer (20 mM Tris pH 8.0, 150 mM NaCl). Peak fractions corresponding to soluble protein were pooled and concentrated to 8–20 mg/mL (Amicon Ultra 10 kDa cut off, Millipore Sigma). Protein samples were stored at 4 °C until use. The antibodies described in this section are summarised in Supplementary Table 3 .

Electron microscopy sample preparation and data collection

For cryo-EM, S protein samples were prepared at 2.25 mg/mL, with and without the addition of ACE2 (Supplementary Table 4 ) at 0.5 mg/mL (1:1.25S protein trimer:ACE2 molar ratio). Vitrified samples of all S protein samples were prepared by first glow discharging Quantifoil R1.2/1.3 Cu mesh 200 holey carbon grids for 30 s using a Pelco easiGlow glow discharge unit (Ted Pella) and then applying 1.8 µL of protein suspension to the surface of the grid at a temperature of 10 °C and a humidity level of >98%. Grids were blotted (12 s, blot force −10) and plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). All cryo-EM samples were imaged using a 300 kV Titan Krios G4 transmission electron microscope (Thermo Fisher Scientific) equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at ×155,000 magnification (calibrated pixel size of 0.5 Å per physical pixel) over a defocus range of −0.5 µm to −3 µm with a total dose of 40 e − /Å 2 using EPU automated acquisition software. For negative stain, S protein samples were prepared at 0.1 mg/mL. Grids of Cu mesh 300 with continuous ultra-thin carbon film (CF300-Cu-UL, Electron Microscopy Sciences) were glow discharged for 15 s using the Pelco easiGlow. Samples were allowed to adsorb for 30 s before blotting away excess liquid, followed by a brief wash with MilliQ H 2 O. Grids were stained by three successive applications of 2% (w/v) uranyl formate (20 s, 20 s, 60 s). Negative stain grids were imaged using a 200 kV Glacios transmission electron microscope (Thermo Fisher Scientific) equipped with a Falcon3 camera operated in linear mode. Micrographs were collected using EPU at ×92,000 magnification (physical pixel size 1.6 Å) over a defocus range of −1 µm to −2 µm with a total accumulated dose of 120 e − /Å 2 . The software and algorithms described in this section are summarised in Supplementary Table 7 .

Image processing

The detailed workflow for the data processing is summarised in Supplementary Figs. 4 – 10 . In general, all data processing was performed in cryoSPARC v.3.2 48 unless stated otherwise. Motion correction in patch mode (EER upsampling factor 1, EER number of fractions 40), CTF estimation in patch mode, reference-free particle picking, and particle extraction were performed on-the-fly in cryoSPARC. After preprocessing, particles were subjected to 2D classification and/or 3D heterogeneous classification. The final 3D refinement included per particle CTF estimation and aberration correction. For B.1.617.1 spike proteins, focused refinements were performed with a soft mask covering all six RBDs. For the complexes of spike protein ectodomain and human ACE2, focused refinements were performed with a soft mask covering a single RBD and its bound ACE2. Global resolution and focused resolution were determined according to the gold-standard FSC 49 . The software and algorithms described in this section are summarised in Supplementary Table 7 .

Model building and refinement

For models of spike protein ectodomain alone, the SARS-CoV-2 HexaPro S trimer with N501Y mutation (PDB code 7MJG) was docked into the cryo-EM density map using UCSF Chimera v.1.15 50 . Then, mutation and manual adjustment were done with COOT v.0.9.3 51 , followed by iterative rounds of refinement in COOT and Phenix v.1.19 52 . Glycans were added at N-linked glycosylation sites in COOT. For models of complex of spike protein ectodomain and human ACE2, the RBD-ACE2 subcomplex was built using published coordinates (PDB code 7MJN) as the initial model, followed by refinement against focused refinement maps. The obtained model was then docked into global refinement maps together with the other individual domains of the spike protein. Model validation was performed using MolProbity 53 . Figures were prepared using UCSF Chimera, UCSF ChimeraX v.1.1.1 54 and PyMOL (v.2.2 Schrodinger, LLC). The software and algorithms described in this section are summarised in Supplementary Table 7 .

Biolayer interferometry S protein-ACE2 binding assay

The kinetics of SARS-CoV-2 trimers and human ACE2 binding were analysed with the biolayer interferometer BLItz (ForteBio, Menlo Park, CA). Protein-A biosensors (ForteBio: 18–5010) were coated with ACE2-mFc (40 µg/mL) for 2 min and incubated in DPBS (pH = 7.4) to establish baselines. Concentrations of 125, 250, 500, and 1000 nM spike trimers were used for association for 2 min followed by dissociation in DPBS for 5 min. The association ( k on ) and dissociation ( k off ) rates were derived from the fitting of sensorgrams and used to calculate the binding equilibrium constant (K D ). Enzyme-linked immunosorbent assay (ELISA) For ELISA, 100 µL of wild-type (D614G), Kappa or Delta SARS-CoV-2 S proteins were coated onto 96-well MaxiSorp™ plates at 2 µg/mL in PBS overnight at 4 °C. All washing steps were performed three times with PBS + 0.05% Tween 20 (PBS-T). After washing, wells were incubated with blocking buffer (PBS-T + 1% casein) for 1 h at room temperature. After washing, wells were incubated with dilutions of primary antibodies in PBS-T + 0.5% BSA buffer for 1 h at room temperature. After washing, wells were incubated with goat anti-human IgG (Jackson ImmunoResearch) at a 1: 8000 dilution in PBS-T + 1% casein buffer for 1 h at room temperature. After washing, the substrate solution (Pierce™ 1-Step™) was used for colour development according to the manufacturer’s specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific). Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Biological materials availability

All unique biological materials described in this manuscript are made available upon reasonable request from the corresponding author and following the completion of a materials transfer agreement.

Supplementary information Supplementary Information Reporting Summary