key: cord-0684702-54617ubj
authors: McCallum, Matthew; Walls, Alexandra C.; Sprouse, Kaitlin R.; Bowen, John E.; Rosen, Laura; Dang, Ha V.; deMarco, Anna; Franko, Nicholas; Tilles, Sasha W; Logue, Jennifer; Miranda, Marcos C.; Ahlrichs, Margaret; Carter, Lauren; Snell, Gyorgy; Pizzuto, Matteo Samuele; Chu, Helen Y.; Van Voorhis, Wesley C.; Corti, Davide; Veesler, David
title: Molecular basis of immune evasion by the delta and kappa SARS-CoV-2 variants
date: 2021-08-12
journal: bioRxiv
DOI: 10.1101/2021.08.11.455956
sha: 33823c7cfe46f19e5bd54f31c0cdb4e99ef4a0e9
doc_id: 684702
cord_uid: 54617ubj

Worldwide SARS-CoV-2 transmission leads to the recurrent emergence of variants, such as the recently described B.1.617.1 (kappa), B.1.617.2 (delta) and B.1.617.2+ (delta+). The B.1.617.2 (delta) variant of concern is causing a new wave of infections in many countries, mostly affecting unvaccinated individuals, and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing the impact of individual mutations on immune evasion. Mutations in the B.1.617.1 (kappa) and B.1.617.2 (delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including an unexpected remodeling of the B.1.617.2 (delta) N-terminal domain. The binding affinity of the B.1.617.1 (kappa) and B.1.617.2 (delta) receptor-binding domain for ACE2 is comparable to the ancestral virus whereas B.1.617.2+ (delta+) exhibits markedly reduced affinity. We describe a previously uncharacterized class of N-terminal domain-directed human neutralizing monoclonal antibodies cross-reacting with several variants of concern, revealing a possible target for vaccine development.

B.1.617.2, and B.1.617.2+ S (GMTs 270, 410, and 94), respectively, compared to G614 S (GMT: 850) (Fig. 1B, Fig S1-S2) . The average neutralization potency of the Janssen Ad26.COV2.Selicited plasma was reduced 5-, 4-, and ≥6-fold for B.1.617.1, B.1.617.2, and B.1.617.2+ S (GMTs 9.8, 12, and 8.4), respectively, compared to D614G S (GMT: 47) (Fig. 1C, Fig S1-S2) . These data demonstrate that all three variants lead to reductions in neutralization potency from vaccineelicited Abs with B.1.617.2+ causing the greatest decrease, on par with what was observed for the B.1.351 (beta) variant of concern (1, 3). Furthermore, polyclonal Abs from half of the Janssenvaccinated individuals evaluated completely lost their ability to neutralize one or multiple variants in our assay, likely as a result of the moderate GMTs against G614 S pseudovirus (41). Table S1 ). Data are an average of n = 2 replicates and are representative of at least two independent assays. Dashed line indicates the limit of detection for the assay. Means (shown as thick black horizontal lines) were compared against G614 by twoway ANOVA (Dunnet's test); *, p<0.05; ***, p<0.001; ****, p<0.0001. (D-F) Incidence of variants of concern and variants of interest as a proportion of viruses sequenced in the world (D), India (E), and the USA (F) deposited to GISAID (analyzed using outbreak.info) from December 1, 2020 to July 4, 2021. B. (Fig 1D-F) Table  S1 ). The high incidence of B.1.617.2 is consistent with recent studies showing that the B.1.617.2 variant has enhanced transmissibility, replication kinetics and viral loads in oropharyngeal and nose-throat swabs of infected individuals relative to the ancestral virus and other variants (37, 44) .

We determined electron cryomicroscopy (cryoEM) structures of the B.1.617.1 and B.1.617.2 S glycoproteins to gain insights into their reduced antibody sensitivity and provide a structural framework to understand the role of shared and distinct mutations. The B.1.617.1 S structure -harboring five of the HexaPro stabilizing mutations (45), a native furin cleavage site, and engineered in the closed conformation with S383C/D985C mutations (46) -was determined in complex with S309 (an RBD-targeted neutralizing mAb (15) ) and S2L20 (an NTD-targeted non-neutralizing mAb (23, 47) ) at 2.4 Å resolution (Fig 2A, Fig S3A-C, Table S3 ). Local classification and refinements of S309 bound to the B.1.617.1 RBD were used to account for conformational dynamics and improve local resolution of this region to 3.3 Å (Fig 2A,C and Fig  S3A) . The B.1.617.2 S structure -harboring some of the HexaPro (45) and VFLIP (48) stabilizing mutations and a native furin cleavage site -was obtained in complex with S2M11 (an RBDtargeted neutralizing mAb (19) ) and S2L20 at 2.4 Å resolution (Fig 2B, Fig S3D-F, Table S3 ). . SARS-CoV-2 S protomers are colored pink, cyan, and gold, whereas the S2L20 Fab heavy and light chains are colored dark and light green, respectively. The S309 Fab heavy and light chains are colored dark and light orange, respectively (A). The S2M11 Fab heavy and light chains are colored dark and light gray, respectively (B). Only the Fab variable domains are resolved and therefore modeled in the map. N-linked glycans are rendered as dark blue spheres. (C) Zoomed in view of the S309-bound B. The RBD is the main target of serum neutralizing activity in convalescent and vaccinated individuals and comprises several antigenic sites recognized by neutralizing Abs with a range of neutralization potencies and breadth (15) (16) (17) (18) (49) (50) (51) . The B.1.617.1 S and B.1.617.2 S structures resolve the complete RBD and provide high-resolution blueprints of the residue substitutions found in these two variants (Fig 2C-D) . In both structures, the L452R side chain, which is part of antigenic site Ib (18) , is oriented identically (and modeled in the same rotameric configuration) to what we previously observed in the B.1.427/B.1.429 S structure (47) (Fig 2E-F) . The L452R mutation reduces neutralization mediated by some clinical mAbs, such as bamlanivimab (LY-CoV555) and regdanvimab (CT-P59), due to steric alteration of this antigenic site that is incompatible with binding (Fig 2E-F) (47) . The B.1.617.1 E484Q RBD mutation is located within the receptor-binding motif, at the boundary between antigenic sites Ia/Ib (18) , and could affect neutralization from mAbs recognizing both subsites. However, the E484Q mutation is conservative as it substitutes the side chain carboxylic group with an amide group through replacement of an oxygen with a nitrogen atom. Residue 484 is involved in the epitopes recognized by bamlanivimab (LY-CoV555) (20, 52) and casirivimab (REG10933) (53, 54) , and both mAbs have dampened binding or neutralization potency against E484Q-harboring mutants likely due to disruption of electrostatic interactions (52, 54) . The B.1.617.2 T478K RBD mutation is also located at the boundary between antigenic sites Ia/Ib but does not affect binding to mAbs that have received an emergency use authorization in the US (52, 55, 56) (Fig 2D) .

E484Q, L452R, and T478K mutations are part of antigenic site I which we previously showed to be immunodominant (16) (17) (18) . Both the B.1.351 (beta) and P.1 (gamma) variants of concern harbor the E484K mutation which is associated with a reduction of vaccine-elicited neutralizing Ab titers and mAb neutralization (38, 41, 57) . The E484Q mutation has also been reported to reduce serum neutralizing Ab titers alone or in combination with the L452R mutation (41, 54, 58) . The effect of the T478K mutation on polyclonal Ab responses has not been characterized as thoroughly as the L452R and E484Q substitutions. The recurrent emergence of mutants carrying residue substitutions at position 452 and 484 underscores the apparent hyperfocusing of neutralizing antibody responses at these antigenic subsites and provides a possible explanation for their acquisition in multiple SARS-CoV-2 lineages.

We set out to assess the impact of the RBD mutations present in the B.1.617.1, B.1.617.2, and B.1.617.2+ on binding affinity for the ACE2 receptor to assess potential impact on a component of viral transmissibility. The B.1.617.1 (L452R/E484Q) and B.1.617.2 (L452R/T478K) RBDs recognized immobilized ACE2 with roughly similar efficiency to the wildtype RBD, as observed by ELISA (Fig 2G, Fig S4, Table S4 ). ACE2 binding to the B.1.617.2+ (K417N/L452R/T478K) and B.1.1.7 (N501Y) RBDs was respectively much weaker and much tighter than for any other variants evaluated in our assay, in agreement with the enhanced affinity conferred by N501Y (4, 59) (Fig 2G, Table S4 ). We confirmed these results using surface plasmon resonance (Fig 2H, Fig S4 Table  S3 . and biolayer interferometry (Fig 2I, Fig S4X, Table S4 ) binding analysis of the monomeric human ACE2 ectodomain to immobilized RBDs, indicating that the B.1.617.1 and B.1.617.2 RBDs interact with ACE2 with roughly comparable affinity to the wildtype RBD whereas the B.1.617.2+ RBD is severely attenuated. These data concur with evaluation of the effect of individual residue mutations on ACE2 binding using deep-mutational scanning of yeast-displayed RBDs (59) , the positioning of the R452 and K478 side chains away from the ACE2-binding interface, and the conservative nature of the E484Q substitution (Fig 2J-K) . Finally, these data point to the key contribution of the salt bridge formed between the K417SARS-CoV-2 and D30ACE2 side chains for receptor engagement (Fig 2J-K) , in agreement with previous studies (60, 61) . Since none of the B.1.617.1 and B.1.617.2 RBD mutations increase ACE2 binding markedly and all of them reside in the most immunogenic antigenic site, we propose that they emerged mainly as a result of antibody-mediated selective pressure to reduce immune recognition.

Although multiple antigenic sites are present at the surface of the NTD, a single supersite of vulnerability is known to be targeted by neutralizing Abs elicited upon infection and vaccination (23) (24) (25) . This antigenic supersite (designated site i) comprises the NTD N-terminus (residues 14-20), a β-hairpin (residues 140-158), and a loop (residues 245-264). To improve the cryoEM map resolution of the B.1.617.1 and B.1.617.2 NTDs bound by S2L20 and visualize the structural changes associated with their respective constellation of mutations, we used focused 3D classification and local refinement. Two of the three B.1.617.1 NTD mutations, G142D and E154K, map to the supersite β-hairpin (Fig 3A, Table S1 ) and we have previously shown that G142D abrogates binding of 3 out of 5 NTD-specific neutralizing mAbs tested (23) . The B.1.617.1 T95I substitution occurs outside the antigenic supersite and is unlikely to contribute to immune evasion significantly (Fig 3A) . All of the B.1.617.2 mutations are found within the antigenic supersite: T19R, G142D, E156G, and 157-158del (Fig 3B) . The T19R substitution abrogates the glycosylation sequon at position N17, as supported by the lack of a resolved glycan at this position in the cryoEM map. We previously showed that T19A, which also removes the N17 glycosylation sequon, decreased binding to 4 out 5 NTD neutralizing mAbs tested (23) . Residues 156-158 participate in the supersite β-hairpin and their mutation/deletion in the B.1.617.2 NTD lead to striking structural remodeling: residues 151-159 adopt an alpha-helical conformation whereas this segment is β-stranded in the absence of this mutation/deletion (Fig 3B) . Based on these findings, we hypothesized that B.1.617.1 and B.1.617.2 variant S glycoproteins would escape recognition by most neutralizing NTD Abs (Fig 3C & 3D) . (Fig 4A,  Fig S4) . Out of 11 neutralizing mAbs tested, we observed a 10-fold or greater reduction in binding for 8, 10, 10, 10, 3, and 11 mAbs to B. (Fig 4A) . Collectively, these data indicate that NTD-specific neutralizing Abs exert a selective pressure participating in evolution of the antigenic supersite leading to neutralization escape in numerous variants (62) (63) (64) 4A) . To provide a structural framework for understanding the S2X303 binding breadth, we characterized its Fab fragment bound to B.1.617.1 S using cryoEM (Fig 4B) . Focused classification and local refinement of the S2X303-bound NTD yielded a map at 3.5 Å resolution revealing the unique recognition mode of this mAb (Fig 4C, Fig S3G-I, Table S3 ).

S2X303 recognizes the NTD with an angle of approach almost orthogonal relative to several previously described NTD-specific neutralizing mAbs and its epitope is only partially overlapping with the NTD antigenic supersite (23) . Specifically, the S2X303 complementary determining region 3-dominated paratope exclusively contacts residues 123-125 (along with the glycan at position N122), that are part of the NTD galectin-like distal β-sheet, and residues 144-154 within the supersite β-hairpin (Fig 4D-E) . As a result, S2X303 defines a new class of NTDspecific mAbs that differs from S2X333 (23), a canonical ultrapotent NTD-specific mAb, and from P008_056 (65), which interferes with biliverdin binding, with respect to their footprints and angles of approach (Fig 4E) . Moreover, S2X303 exhibits unprecedented cross-reactivity compared to all other mAbs and can bind to a large number of variants (Fig 4A) . enhance cleavage at the S1/S2 boundary and cell-cell fusion (37, 67, 68) . Since the S1/S2 cleavage site is key for transmission and pathogenicity (69, 70) , it appears likely that this mutation contributes to the success of these lineages. Furthermore, the L452R mutation, which augments stability/expression (59) , increases viral replication kinetics relative to the ancestral virus (71) . The enhanced ability of B.1.1.7 to antagonize host innate immunity through upregulation of orf6 and orf9b was suggested to have participated in the success of this variant (72) . It is possible that B.1.617.2 evolved a similar strategy to reach global domination warranting further studies to uncover the contribution of innate immune antagonism to the continued emergence of variants with greater transmissibility.

We demonstrate here that S309, the parent mAb of sotrovimab which has received an emergency use authorization from the FDA, is unaffected by antigenic drift observed in variants of concern and interest due to recognition of a conserved RBD epitope (12, 15, 56) . The recent discovery of multiple additional conserved antigenic sites recognized by RBD-specific mAbs with (near) pan-sarbecovirus neutralizing activity (16, 17, 21, (73) (74) (75) and the anticipated continued emergence of SARS-CoV-2 variants motivate the clinical development and deployment of some of these mAbs for prophylaxis with at-risk patients and for treatments of unvaccinated individuals or breakthrough infections. Moreover, next-generation vaccine candidates have recently been described to elicit broad sarbecovirus immunity (41, 76, 77) , holding the promise to be resilient to the emergence of SARS-CoV-2 variants and of new zoonotic sarbecoviruses. Looking forward, the discovery of broadly neutralizing mAbs targeting the fusion machinery makes tangible the development of a universal β-coronavirus vaccine (27) (28) (29) (30) 78) . 

Average daily prevalence for B. 2) were obtained from GISAID (using outbreak.info) and plotted using GraphPad PRISM software (version 9.2.0).

Blood samples were collected from participants who had received both doses of the Pfizer's BNT162b2 vaccine or Moderna's mRNA-1273 vaccine and were 7-30 days post second dose. Individuals were enrolled in the UWARN: COVID-19 in WA study at the University of Washington in Seattle, WA. This study was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350). The J&J/Janssen Ad26.COV2.S vaccine samples were collected as part of the HAARVI study and was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00000959). Baseline sociodemographic and clinical data for these individuals are summarized in Table S2 .

Recombinant mAbs were expressed in ExpiCHO cells at 37 °C and 8% CO2. Cells were transfected using ExpiFectamine. Transfected cells were supplemented 1 day after transfection with ExpiCHO Feed and ExpiFectamine CHO Enhancer. Cell culture supernatant was collected eight days after transfection and filtered through a 0.2 μm filter. Recombinant antibodies were affinity purified on an ÄKTA xpress FPLC device using 5 mL HiTrapTM MabSelectTM PrismA columns followed by buffer exchange to Histidine buffer (20 mM Histidine, 8% sucrose, pH 6) using HiPrep 26/10 desalting columns.

The WT, B.1.1.7, B.1.617.1, B.1.617.2, and B.1.617.2+ SARS-CoV-2 RBD construct were synthesized by GenScript into pCMVR with an N-terminal mu-phosphatase signal peptide, and a C-terminal octa-histidine tag (GHHHHHHHH) and an avi tag (GLNDIFEAQKIEWHE)). The boundaries of the construct are N-328-RFPN-331 and 528-KKST-531.

The hACE2 construct was previously synthesized by Twist into pTwist+CMV(residues 1-615 with a C-terminal avi tag-10xHis-GGG-tag, and N-terminal signal peptide) (59) .

The SARS-CoV-2 S ectodomain with hexapro mutations (45), S383C/D985C mutations (46) , the native furin cleavage site (RRAR), and B.1.617.1 spike mutations (T95I, G142D, E154K, L452R, E484Q, D614G, P681R and Q1071H) was synthesised by GenScript into pCMV with a C-terminal foldon and avi tag followed by an octa-histidine tag. The SARS-CoV-2 S ectodomain with VFLIP mutations (48) , the native furin cleavage site (RRAR), and B.1.617.2 spike mutations (T19R, G142D, E156G, T478K, and D950N substitutions and a deletion of residues 157 and 158) was synthesised by GenScript into pCMV with a C-terminal avi tag followed by an octa-histidine tag. The SARS-CoV-2 S ectodomain with hexapro mutations and P.1 spike mutations (L18F,  T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F) was synthesised by GenScript into pCMVR with foldon, a C-terminal avi tag followed by an octahistidine tag. The SARS-CoV-2 S ectodomain with the 2P mutations and B.1.1.7 spike mutations (del69/70, del144/145, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H) was synthesised by GenScript into pCMV with a C-terminal foldon and avi tag followed by an octahistidine tag. The SARS-CoV-2 S ectodomain with the 2P mutations and B.1.351 spike mutations (D80A, D215G, 242-244del, R246I, K417N, E484K, N501Y , D614G, and A701V) was synthesised by GenScript into pCMV with a C-terminal foldon and avi tag followed by an octahistidine tag.

hACE2 and the biotinylated RBDs were produced in 25 mL cultures of Expi293F Cells (ThermoFisher Scientific) grown in suspension using Expi293 Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 5% CO2 incubator rotating at 130 rpm. Cells grown to a density of 3 million cells per mL were transfected using the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) and cultivated for four days. Proteins were purified from clarified supernatants using a nickel HisTrap HP affinity column (Cytiva) and washed with ten column volumes of 20 mM imidazole, 25 mM sodium phosphate pH 8.0, and 300 mM NaCl before elution with a gradient of 500 mM imidazole. Proteins were buffer exchanged into 20 mM sodium phosphate pH 8 and 100 mM NaCl and concentrated using 30 kDa or 10 kDa centrifugal filters (Amicon Ultra, MilliporeSigma) for hACE2 and the biotinylated RBDs, respectively, before being flash frozen.

The SARS-CoV-2 S ectodomains were produced in 100 mL cultures of Expi293F Cells (ThermoFisher Scientific) grown in suspension using Expi293 Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. Cells grown to a density of 2.5 million cells per mL were transfected using the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) and cultivated for four days at which point the supernatant was harvested. S ectodomains were purified from clarified supernatants using a Cobalt affinity column (Cytiva, HiTrap TALON crude), washing with 20 column volumes of 20 mM Tris-HCl pH 8.0 and 150 mM NaCl and eluted with a gradient of 600 mM imidazole. The S ectodomain was then concentrated using a 100 kDa centrifugal filter (Amicon Ultra 0.5 mL centrifugal filters, MilliporeSigma), residual imidazole was washed away by consecutive dilutions in the centrifugal filter unit with 20 mM Tris-HCl pH 8.0 and 150 mM NaCl, and finally concentrated to 2 mg/mL and flash frozen.

SARS-CoV-2 D614G, B.1.617.1 (mutations T95I, G142D, E154K, L452R, E484Q, D614G, P681R and Q1071H), B.1.617.2 (mutations T19, G142D, E156G, 157-158del, T478K, D950N) and B.1.617.2+ (B.1.617.2 mutations plus K417N) pseudotypes were prepared similarly as previously described (47) . Briefly, HEK-293T cells seeded in poly-D-lysine coated 100 mm dishes at ~75 % confluency were washed five times with Opti-MEM and co-transfected with Lipofectamine 2000 (Life Technologies) with 24 µg of the respective S glycoprotein plasmids. After 5 h at 37°C, media supplemented with 20% FBS and 2% PenStrep was added. After 20 hours, cells were washed five times with DMEM and cells were transduced with VSVΔG-luc (39) and incubated at 37°C. After 2 h, infected cells were washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background. After 18-24 h, the supernatant was harvested and clarified by low-speed centrifugation at 2,500 g for 10 min. The supernatant was then filtered (0.45 μm) and concentrated 10 times using a 30 kDa cut off membrane. The pseudotypes were then aliquoted and frozen at -80 °C.

To At this stage, excess DMEM was removed from the cells and 40 µL from each well (containing sera and pseudovirus) was transferred to the 96-well plate seeded with HEK-293T cells expressing hACE2 and incubated at 37°C for 2 h. After 2 h, an additional 40 µL of DMEM supplemented with 20% FBS and 2% PenStrep was added to the cells.

After 20 h, 40 µL of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark for 5 min. The plates were immediately read on a Biotek plate reader. Measurements were done in duplicate with biological replicates. Relative luciferase units were plotted and normalized in Prism (GraphPad): cells alone without pseudovirus was defined as 0 % infection, and cells with virus only (no sera) was defined as 100 % infection. Prism (GraphPad) nonlinear regression with "[inhibitor] versus normalized response with a variable slope" was used to determine IC50 values from curve fits. Means of these duplicates were compared against G614 by two-way ANOVA (Dunnet's test) using GraphPad PRISM software (version 9.2.0).

Undiluted pseudoviruses were added to 4X SDS-PAGE loading buffer. Samples were run on a 4%-15% gradient Tris-Glycine Gel (BioRad) and transferred to PVDF membranes. An anti-S2 SARS-CoV-2 S polyclonal primary antibody (1:1,500 dilution,Invitrogen PA5-114534) and an Alexa Fluor 680-conjugated goat anti-rabbit secondary antibody (1:20,000 dilution, Jackson Laboratory 111-625-144) were used for Western-blotting. A LI-COR processor was used to develop images.

For ELISA experiments with NTD-targeted mAbs, 384-well Maxisorp plates (ThermoFisher Scientific 464718) were coated overnight at 4°C with 2 μg/mL of S glycoprotein in 20mM HEPES pH 8 and 150mM NaCl. Plates were slapped dry and blocked with Blocker Casein in TBS (ThermoFisher Scientific 37532) for one hour at 37°C. Plates were slapped dry and mAbs were serially diluted 1:5 in TBST with an initial concentration of 50 µg/ml. Plates were left for one hour at 37°C and washed 4x with TBST, then 1:5000 Goat anti-Human (ThermoFisher Scientific A18817) was added. Plates were left for one hour at 37°C and washed 4x with TBST, and then TMB Microwell Peroxidase (Seracare 5120-0083) was added. The reaction was quenched after 4 minutes with 1 N HCl and the A450 of each well was read using a BioTek plate reader.

For ELISA experiments with RBDs, 384-well Maxisorp plates (ThermoFisher Scientific 464718) were coated overnight at 4°C with 4 µg/mL of hACE2-His in 20mM Sodium Phosphate pH 8 and 100mM NaCl. Plates were slapped dry and blocked with Blocker Casein in TBS (ThermoFisher Scientific 37532) for one hour at 37°C. Plates were slapped dry and wild-type RBD, B.1.1.7 RBD, B.1.351 RBD, B.1.617.1 RBD, B.1.617.2 RBD, and B.1.617.2+ RBD were serially diluted 1:3 in TBST with an initial concentration of 1542nM. Plates were left for one hour at 37°C, then washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 2 μg/mL S309 mAb (15) . Plates were left for one hour at 37°C and washed 4x with TBST, then 1:5000 Goat anti-Human (ThermoFisher Scientific A18817) was added. TMB Microwell Peroxidase (Seracare 5120-0083) was added after another hour at 37°C and 4x wash with TBST. The reaction was quenched after 1-2 minutes with 1 N HCl and the A450 of each well was read using a BioTek plate reader. mg/ml S309 Fab and 2.2 μL of 67 mg/mL S2L20 Fab in 150 mM NaCl and 20 mM Tris-HCl pH 8 for 30 min at 37°C. Alternatively, 50 μL of 2 mg/mL SARS-CoV-2 S B.1.617.2 ectodomain was incubated with 34 µl 2.9 mg/ml S2M11 Fab in 150 mM NaCl and 20 mM Tris-HCl pH 8 for 10 min at 37°C, and then 2.2 μL of 67 mg/mL S2L20 Fab was added for 20 min at 37°C. Alternatively, 50 μL of 2 mg/mL SARS-CoV-2 S B.1.617.1 ectodomain was incubated with 3.6 µl 28 mg/ml S2X303 Fab in 150 mM NaCl and 20 mM Tris-HCl pH 8 for 30 min at 37°C.

Unbound Fab was then washed away with six consecutive dilutions in 400 μL of 20 mM Tris-HCl pH 8.0 and 150 mM NaCl over a 100 kDa centrifugal filter (Amicon Ultra 0.5 mL centrifugal filters, MilliporeSigma). The complex was concentrated to 3.6 mg/mL and 3 μL was immediately applied onto a freshly glow discharged 2.0/2.0 UltraFoil grid (200 mesh), plunge frozen using a vitrobot MarkIV (ThermoFisher Scientific) using a blot force of −1 and 6.5 s blot time at 100% humidity and 23°C.

Data were acquired using the Leginon software (79) to control a FEI Titan Krios transmission electron microscope equipped with a Gatan K3 direct detector and operated at 300 kV with a Gatan Quantum GIF energy filter. The dose rate was adjusted to 3.75 counts/superresolution pixel/s, and each movie was acquired in 75 frames of 40 ms with a pixel size of 0.843 Å and a defocus range comprised between −0.2 and −2.0 μm.

Movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking and extraction (with a downsampled pixel size of 1.686 Å and box size of 256 pixels 2 ) were carried out using Warp (80) . Reference-free 2D classification was performed using cryoSPARC (81) to select well-defined particle images. 3D classification with 50 iterations each (angular sampling 7.5 ̊ for 25 iterations and 1.8 ̊ with local search for 25 iterations) were carried out using Relion without imposing symmetry. 3D refinements were carried out using non-uniform refinement in cryoSPARC (82) before particle images were subjected to Bayesian polishing using Relion (83) during which particles were re-extracted with a box size of 512 Å at a pixel size of 0.843 Å. Next, 86 optics groups were defined based on the beamtilt angle used for data collection. Another round of non-uniform refinement in cryoSPARC was then performed concurrently with global and per-particle defocus refinement. For focused classification, particles were symmetry-expanded in Relion (84, 85) , and the particles were 3D classified in Relion without alignment using a mask that encompasses part of the NTD and the S2L20 VH/VL region, or the RBD and the S309 VH/VL region. Particles in well-formed 3D classes were then used for local refinement in cryoSPARC. Reported resolutions are based on the gold-standard Fourier shell correlation of 0.143 criterion and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution (86, 87) .

UCSF Chimera (88) and Coot (89) were used to fit atomic models of S2M11, S2L20, S309, and SARS-CoV-2 S (PDB 7LXY, 7N8I, 7R6W) into the cryo-EM maps. The model was then refined and rebuilt into the map using Coot (89) , Rosetta (90, 91) , Phenix (92) , and ISOLDE (93) . Model validation and analysis used MolProbity (94), EMringer (95), Phenix (92) and Privateer (96) . Figures were generated using UCSF ChimeraX (97) .

His-avi-tagged wildtype, B.1.1.7 (N501Y) B.1.617.1 (L452R, E484Q), B.1.617.2 (L452R, T478K), or B.1.617.2+ (K417N, L452R, T478K) RBD were biotinylated and immobilized at 5 ng/μL in undiluted 10X kinetics buffer (Pall) to SA sensors that were pre-hydrated in water for at least 10 minutes and then equilibrated into 10X Kinetics Buffer (Pall). The RBDs were loaded to a level of 1nm total shift. The loaded tips were then dipped into a dilution series of monomeric ACE2-his in 10X Kinetics Buffer (Pall) starting at 1000 or 5000 nM for 300 seconds prior to 300 seconds dissociation in 10X Kinetics buffer for kinetics determination. The data were baseline subtracted and the plots fitted using the Pall FortéBio/Sartorius analysis software (v.12.0). Data were plotted in Graphpad Prism (v.9.0.2). These experiments were done side-by-side with two separate RBD protein preparations and two separate ACE2 preparations and a representative experiment is shown.

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AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells

Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN. bioRxiv

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

Broad sarbecovirus neutralization by a human monoclonal antibody

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

The neutralizing antibody

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single supersite

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

de van der Schueren, Others, Structural basis for broad coronavirus neutralization

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes. bioRxivorg (2021)

Correlates of protection against SARS-CoV-2 in rhesus macaques

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra

The Indian SARS-CoV-2 Genomics Consortium (INSACOG), The CITIID-NIHR BioResource COVID-19 Collaboration

Nussenzweig, mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

Acquisition of complement resistance through incorporation of CD55/decay-accelerating factor into viral particles bearing baculovirus GP64

Spike Protein for Neutralization Assays

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines. bioRxiv (2021)

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals

Virological and serological kinetics of SARS-CoV-2 Delta variant vaccine-breakthrough infections: a multi-center cohort study

Viral infection and

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation

Structure-based design of a highly stable, covalently-linked SARS-CoV-2 spike trimer with improved structural properties and immunogenicity

Antibodies elicited by mRNA-1273 vaccination bind more broadly to the receptor binding domain than do those from SARS-CoV-2 infection

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Potently neutralizing and protective human antibodies against SARS-CoV-2

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

The Spike Proteins of SARS-CoV-2 B.1.617 and B.1.618 Variants Identified in India Provide Partial Resistance to Vaccine-elicited and Therapeutic Monoclonal Antibodies

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19

The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2. bioRxiv (2021)

Serum Neutralizing Activity Elicited by mRNA-1273 Vaccine -Preliminary Report

The Genotype to Phenotype Japan (G2P-Japan) Consortium, SARS-CoV-2 B.1.617 mutations L452 and E484Q are not synergistic for antibody evasion

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape

Persistence and Evolution of SARS-CoV-2 in an Immunocompromised Host

Case Study: Prolonged infectious SARS-CoV-2 shedding from an asymptomatic immunocompromised cancer patient

SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity

Infection and Vaccine-Induced Neutralizing-Antibody Responses to the SARS-CoV-2 B.1.617 Variants

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Others, SARS-CoV-2 spike P681R mutation, a hallmark of the Delta variant, enhances viral fusogenicity and pathogenicity. bioRxiv

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody

A broadly neutralizing antibody protects against SARS-CoV, pre-emergent bat CoVs, and SARS-CoV-2 variants in mice. bioRxivorg

Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice

Automated molecular microscopy: the new Leginon system

Real-time cryo-electron microscopy data preprocessing with Warp

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction

A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis

New tools for automated high-resolution cryo-EM structure determination in RELION-3

RELION: implementation of a Bayesian approach to cryo-EM structure determination

Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy

High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy

UCSF Chimera--a visualization system for exploratory research and analysis

Features and development of Coot

Automatically Fixing Errors in Glycoprotein Structures with Rosetta

Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

ISOLDE: a physically realistic environment for model building into lowresolution electron-density maps

MolProbity: all-atom structure validation for macromolecular crystallography

EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy

Privateer: software for the conformational validation of carbohydrate structures

Meeting modern challenges in visualization and analysis

Measurements were performed using a Biacore T200 instrument. The Cytiva Biotin CAPture Kit, Series S, was used for surface capture of biotinylated RBDs. Running buffer was HBS-EP+ pH 7.4 (Cytiva) and measurements were performed at 25 C. Experiments were performed with a 3-fold dilution series of monomeric hACE2: 200 nM, 67 nM, 22 nM, 7.4 nM. Association was 300 s and dissociation was 450 s. Data were double reference-subtracted and fit to a 1:1 binding model using Biacore Evaluation software.