key: cord-0784122-gxkxdkdq
authors: Wang, Zijun; Muecksch, Frauke; Cho, Alice; Gaebler, Christian; Hoffmann, Hans-Heinrich; Ramos, Victor; Zong, Shuai; Cipolla, Melissa; Johnson, Briana; Schmidt, Fabian; DaSilva, Justin; Bednarski, Eva; Ben Tanfous, Tarek; Raspe, Raphael; Yao, Kaihui; Lee, Yu E.; Chen, Teresia; Turroja, Martina; Milard, Katrina G.; Dizon, Juan; Kaczynska, Anna; Gazumyan, Anna; Oliveira, Thiago Y.; Rice, Charles M.; Caskey, Marina; Bieniasz, Paul D.; Hatziioannou, Theodora; Barnes, Christopher O.; Nussenzweig, Michel C.
title: Analysis Of Memory B Cells Identifies Conserved Neutralizing Epitopes On The N-Terminal Domain Of Variant SARS-Cov-2 Spike Proteins
date: 2022-04-07
journal: Immunity
DOI: 10.1016/j.immuni.2022.04.003
sha: 8b69d44fe89c97e7d8ef66a601165a8703d1ab74
doc_id: 784122
cord_uid: gxkxdkdq

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for antibodies. Here, we use NTD-specific probes to capture anti-NTD memory B cells in a longitudinal cohort of infected individuals, some of whom were vaccinated. We found 6 complementation groups of neutralizing antibodies. 58% targeted epitopes outside the NTD supersite, 58% neutralized either Gamma or Omicron, and 14% were broad neutralizers that also neutralized Omicron. Structural characterization revealed that broadly active antibodies targeted three epitopes outside the NTD supersite including a class that recognized both the NTD and SD2 domain. Rapid recruitment of memory B cells producing these antibodies into the plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants, including Omicron.

Several independent studies purified anti-SARS-CoV-2 specific B cells from infected or 43 vaccinated individuals using soluble spike (S) protein as a bait. In all cases the neutralizing 44 antibodies obtained by this method targeted the RBD most frequently and were generally more 45 this site are also mutated in the S protein of PMS20, a synthetic construct that is highly antibody 72 resistant and chimeric proteins build from WT and PMS20 proteins show that NTD-specific 73 antibodies are an important component of the neutralizing activity in convalescent and vaccine 74 recipient plasma (Schmidt et al., 2021b) . NTD supersite mutations are therefore likely to 75 contribute to the poor plasma neutralizing activity against the Omicron variant in individuals that 76 received 2 doses of currently available vaccines or convalescent individuals exposed to pre- To focus on the development and evolution of the human antibody response to NTD, we studied 85 a cohort of SARS-CoV-2 convalescent and/or mRNA vaccinated individuals using the isolated 86 NTD domain as a probe to capture memory B cells producing antibodies specific to this domain. 87

We isolated 275 mAbs across 6 participants, of which 103(37.5%) neutralized at least one of 88 three strains, Wuhan-Hu-1, Gamma, or PMS20. Among the 43 neutralizing antibodies that were 89 further characterized, we found 6 complementation groups based on competition binding 90 experiments. Three of the broad neutralizers were characterized structurally. C1520 and C1791 91 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to 92 previously described antibodies allowing for greater potency and cross-reactivity with 7 different 93 variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously 94 J o u r n a l P r e -p r o o f al., 2020; Wang et al., 2021b)), some anti-NTD antibodies were very potent with IC50 values as 210 low as 0.17 nanograms per milliliter ( Figure 4A ). Of the 103 neutralizing anti-NTD antibodies 211 with demonstrable neutralizing activity 14 were specific for Wuhan-Hu-1, 20 were limited to 212

Gamma, and 13 were PMS20-specific ( Figure 4B ). The remaining 56 antibodies neutralized 2 or 213 more viruses ( Figure 4B ). Antibodies targeting the NTD supersite are enriched in VH1-24, VH3-214 30 and VH3-33 and these 3 VH genes account for 59 of the 103 antibodies tested (Figures 3D, 215 4B and S6E, Table S4 ). Notably, despite its mutations in the NTD supersite, 24 antibodies 216 neutralized PMS20 and 6 neutralized all 3 viruses suggesting that some of these antibodies might 217 bind to epitopes outside of the supersite. 218

To document the neutralizing breadth of the 6 broadest antibodies, we tested them against 220 viruses pseudotyped with SARS-CoV-2 Alpha, Beta, Delta, Iota and Omicron and SARS-CoV S 221 proteins ( Figure 4C ). Although none of the antibodies neutralized SARS-CoV, 4 of the 6 222 antibodies neutralized all strains tested albeit at relatively high neutralizing concentrations. 223

However, pseudovirus neutralization was incomplete even at very high antibody concentrations 224 for 2 of the more potent antibodies tested ( Figure 4D ). 225

To determine whether intact virus neutralization resembles pseudovirus neutralization we 227 performed microneutralization experiments using authentic SARS-CoV-2-WA1/2020 (Robbiani 228 et al., 2020) and -Beta. In contrast to the pseudovirus, the two antibodies that showed the most 229 incomplete neutralization profiles against pseudovirus, C1520 and C1565, reached complete 230 neutralization and were exquisitely potent with IC50s in the low nanogram per milliliter range 231 against both strains ( Figures 4C and 4E ). We conclude that some naturally arising memory anti-232 J o u r n a l P r e -p r o o f NTD antibodies produced in response to Wuhan-Hu-1 infection and immunization are 233 insensitive to the mutations found in Omicron and other variants of concern. 234 235

To determine whether our collection of anti-NTD neutralizing antibodies target overlapping 237 epitopes we performed biolayer interferometry competition experiments ( Figure 5 ). Among the 238 43 antibodies with the highest neutralizing activity tested there were 6 discernible 239 complementation groups. Groups I and II were overlapping and highly enriched in VH3-30, 240

VH3-33 and VH1-24 respectively which accounted for nearly 90% of the antibodies in these 2 241 groups ( Figure 5B ). These antibodies neutralized either or both Wuhan-Hu-1 and Gamma, but 242 none neutralized PMS20 or omicron that are mutated in the NTD supersite. Thus, these 2 groups Voss et al., 2021). Group III and IV are also overlapping but in contrast, groups III and IV only 3 247 of the 19 neutralize Wuhan-Hu-1 and those that do are broad. Notably, the remaining antibodies 248 fail to measurably neutralize Wuhan-Hu-1 but neutralize PMS20 and/or Omicron ( Figure 5B and 249 Table S4 ). Unlike other VH1-24 encoded antibodies that fall into complementation group II, 250 C1704 and C1557 in group III only neutralize PMS20 but no other strains, suggesting the 251 mechanism of their neutralization might be unusual. Group V contains 4 members, 3 of which 252 are broad. The final group VI contains 2 members, C1621 only neutralizes Wuhan-Hu-1, and 253 C1554 neutralized broadly but not potently. The broadest neutralizing anti-NTD antibodies 254 appear to recognize sites outside of the supersite and are not dominated by VH1-24 and VH3-255 J o u r n a l P r e -p r o o f 30/3-33. Altogether 16 out of the 43 anti-NTD neutralizing antibodies tested neutralized PMS20 256 and/or Omicron but not Wuhan-Hu-1. Thus, the B cell memory compartment produced in 257 response to infection with Wuhan-Hu-1 contains antibodies that bind to this strain with high 258 affinity, but do not neutralize it and instead neutralize PMS20 and/or Omicron. 259 260 Anti-NTD antibodies define neutralizing epitopes outside of the supersite 261

To delineate the structural basis for broad-recognition of NTD-directed antibodies, we 262 determined structures of WT SARS-CoV-2 S 6P (Hsieh et al., 2020) bound to Fab fragments of 263 C1717 (group III), C1520 (group IV), and C1791 (group V) using single-particle cryo-electron 264 microscopy (cryo-EM) ( Figure S7 and Table S5 ). Global refinements yielded maps at 2.8Å 265 (C1520-S), 3.5Å (C1717-S), and 4.5Å (C1791-S) resolutions, revealing Fab fragments bound to 266 NTD epitopes on all three protomers within a trimer irrespective of 'up'/'down' RBD 267 conformations for all three Fab-S complexes. C1520 and C1791 Fabs recognize epitopes on 268 opposite faces of the NTD, with binding poses orthogonal to the site i antigenic supersite and 269 distinct from C1717 pose, consistent with BLI mapping data ( Figures 5B and S7J) . A 4.5Å 270 resolution structure of antibody C1791 (VH3-23*01/VK1-17*01) bound to a S trimer revealed a 271 glycopeptidic NTD epitope wedged between the N61NTD-and N234NTD-glycans, engaging 272 several N-terminal regions including the N1-, N2-and b8-b9 hairpin loops ( Figure S7K ). The 273 binding pose of C1791 is similar to the cross-reactive antibody S2L20, which was shown to 274 maintain binding against single NTD mutations and several VOCs but was non- McCallum et al., 2021b). The C1520 epitope comprises residues along the supersite beta-hairpin 280 (residues 152-158), the b8-strand (residue 97-102), N4-loop (residues 178-188), and N-linked 281 glycans at positions N122 and N149 ( Figure 6D ). Targeting of the NTD epitope was driven 282 primarily by heavy chain contacts (the buried surface area (BSA) of NTD epitope on the C1520 283 HC represented ~915Å 2 of ~1150Å 2 total BSA), mediated by the 20-residue long CDRH3 that 284 contributed 55% of the antibody paratope ( Figure 6D 20-residue long CDRH3 of C1520 displaces the supersite beta-hairpin and N4 loops, which acts 289 as a gate for the hydrophobic pocket ( Figure 6F ), in a manner similar to antibody P008-056 290 ( Figure 6G ). This contrasts Ab5-7 that directly buries the tip of its CDRH3 into the hydrophobic 291 pocket, and S2X303 that maintains a closed gate and partially-overlaps with the supersite beta-292 hairpin ( Figure 6G ). Thus, C1520's increased cross-reactivity and neutralization breadth relative 293 to Ab5-7 and S2X303 is likely mediated by displacement of the supersite beta-hairpin and N4-294 loops, which harbor escape mutations found in several SARS-CoV-2 VOCs and can undergo 295 structural remodeling to escape antibody pressure ( Figure 6F and N603SD2-glycans and is positioned in close proximity (<12 Å) to the S2-fusion peptide 304 region ( Figure 7A and 7B). All six CDR loops contribute to an epitope that spans both the NTD 305 and SD2 regions (residues 600-606) with a spike epitope BSA of ~1325 Å 2 ( Figure 7B ). CDRH1 306 and CDRH3 loops mediate extensive hydrogen bond and van der Waals contacts with the C-307 terminus of the NTD (residues 286-303) and SD2 loop (residues 600-606), whereas CDRH2 308 engages N-terminal regions (residues 27-32, 57-60) and NTD loop residues 210-218 ( Figure 7C Figure 7F ). We speculate that the stabilization of the N282NTD-glycan against the 316 adjacent protomer and the proximity of the C1717 LC to the S2 fusion machinery could 317 potentially contribute to C1717's neutralization mechanism by preventing access to the S2´ 318 cleavage site or destabilization of S1. Such a mechanism could explain the lack of Delta VOC 319 neutralization, as this variant has been shown to have enhanced cell-cell fusion activity relative 320 humans, rapid development of neutralizing antibodies to SARS-CoV-2 is associated with better 328 clinical outcomes (Khoury et al., 2021) . Although there was initial concern that antibodies might 329 enhance disease and some NTD antibodies were found to enhance infection in vitro, they were • All scripts and the data used to process antibody sequences are publicly available on 599 Zenodo (DOI: 10.5281/zenodo.6380908). 600

• Any additional information required to reanalyze the data reported in this paper is 601 available from the lead contact upon request. Table S1 . Briefly, 500 µL of serial 10-fold virus dilutions in Opti-MEM were used to infect 4x10 5 cells 731 seeded the day prior into wells of a 6-well plate. After 1.5 h adsorption, the virus inoculum was 732 removed, and cells were overlayed with DMEM containing 10% FBS with 1.2% microcrystalline 733 cellulose (Avicel). Cells were incubated for 4 days at 33 C, followed by fixation with 7% 734 formaldehyde and crystal violet staining for plaque enumeration. All SARS-CoV-2 experiments 735 were performed in a biosafety level 3 laboratory. 736

To confirm virus identity and evaluate for unwanted mutations that were acquired during the 737 amplification process, RNA from virus stocks was purified using TRIzol Reagent (ThermoFisher 738

Scientific, catalog no. 15596026). Brief, 200 L of each virus stock was added to 800 L TRIzol 739 J o u r n a l P r e -p r o o f Reagent, followed by 200 L chloroform, which was then centrifuged at 12,000 g x 5 min. The 740 upper aqueous phase was moved to a new tube, mixed with an equal volume of isopropanol, and 741 then added to a RNeasy Mini Kit column (QIAGEN, catalog no. 74014) to be further purified 742

following the manufacturer's instructions. Viral stocks were subsequently confirmed via next 743 generation sequencing using libraries for Illumina MiSeq. 744 745

The day prior to infection VeroE6UNC cells were seeded at 1x10 4 cells/well into 96-well plates. 747

Antibodies were serially diluted (4-fold) in cell culture medium, consisting of medium 199 (Lonza, 748

Inc.) supplemented with 1% bovine serum albumin (BSA) and 1x penicillin/streptomycin. Next, Antibodies were identified and sequenced as described previously (Robbiani et al., 2020) . In 793 brief, RNA from single cells was reverse-transcribed (SuperScript III Reverse Transcriptase, 794

Invitrogen, 18080-044) and the cDNA stored at −20 °C or used for subsequent amplification of 795 the variable IGH, IGL and IGK genes by nested PCR and Sanger sequencing. Sequence analysis 796 was performed using MacVector. Amplicons from the first PCR reaction were used as templates 797

for sequence-and ligation-independent cloning into antibody expression vectors. Recombinant 798 monoclonal antibodies and Fabs were produced and purified as previously described (Robbiani 799 et al., 2020) . 

PHENIX: a comprehensive Python-based 936 system for macromolecular structure solution

A serological assay to detect 939 SARS-CoV-2 seroconversion in humans

SARS-CoV-2 mRNA vaccination induces 942 functionally diverse antibodies to NTD, RBD, and S2

SARS-CoV-2 neutralizing antibody 945 structures inform therapeutic strategies

Structures of Human

Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of 949 Antibodies

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid 952 mutational escape seen with individual antibodies

High resolution single particle 954 refinement in EMAN2

Potent neutralizing antibodies from 957 COVID-19 patients define multiple targets of vulnerability

Heavily mutated Omicron variant puts scientists on alert

Broadly neutralizing antibodies overcome SARS-CoV-961 2 Omicron antigenic shift

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies

Neutralizing antibody 5-7 defines a distinct site of vulnerability in 966 SARS-CoV-2 spike N-terminal domain

Potent SARS-CoV-2 neutralizing antibodies directed against spike N-969 terminal domain target a single supersite

MolProbity: all-atom structure validation for 972 macromolecular crystallography

A neutralizing human antibody binds to the N-terminal domain of the Spike protein 975 of SARS-CoV-2

Anti-SARS-CoV-2 receptor-binding domain antibody 978 evolution after mRNA vaccination

SARS-CoV-2 Omicron-B.1.1.529 leads to 981 widespread escape from neutralizing antibody responses

A Public Database of Memory and 984 Naive B-Cell Receptor Sequences

Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19. 987

Low-dose in vivo protection and 990 neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

Germinal Center and Extrafollicular B Cell Responses in 993 Vaccination

Features and development of Coot

Genomics and epidemiology of the P.1 998 SARS-CoV-2 lineage in Manaus

A pipeline approach to single-particle 1000 processing in RELION

Evolution of antibody immunity to SARS-1003 CoV-2

Visualizing density maps with UCSF Chimera. 1005

UCSF ChimeraX: Meeting modern challenges in visualization and analysis

Mapping mutations to the SARS-CoV-2 RBD that escape 1011 binding by different classes of antibodies

Targets of T Cell Responses to SARS-CoV-1014

Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

mRNA booster immunization elicits potent neutralizing serum activity 1018 against the SARS-CoV-2 Omicron variant

Rep: A Database of 1020

Curated Antibody Repertoires for Exploring Antibody Diversity and Predicting Antibody 1021 Prevalence

Early Treatment for Covid-19 with SARS-CoV-2 Neutralizing 1024

Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire 1027 sequencing data

Amino acid side-chain partition energies and distribution of residues in soluble 1029 proteins

Studies in humanized mice and convalescent humans yield a SARS-1032

CoV-2 antibody cocktail

A Combination of Receptor-Binding 1035 Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS

A SARS-CoV-2 Infection Model in Mice 1039 Demonstrates Protection by Neutralizing Antibodies

Structure-based design of prefusion-stabilized SARS-1042 CoV-2 spikes

Neutralizing antibody levels are highly 1045 predictive of immune protection from symptomatic SARS-CoV-2 infection

Longitudinal Isolation of Potent Near-1049

Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients

Inference of macromolecular assemblies from crystalline 1051 state

A simple method for displaying the hydropathic character of 1053 a protein

In vitro and in vivo functions of SARS-CoV-2 infection-1056 enhancing and neutralizing antibodies

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

An infectivity-enhancing site on the SARS-CoV-2 spike protein 1062 targeted by antibodies

Automated electron microscope tomography using robust prediction 1064 of specimen movements

N-terminal domain antigenic mapping reveals a site of 1067 vulnerability for SARS-CoV-2

Molecular basis of immune evasion by the Delta 1070 and Kappa SARS-CoV-2 variants

Correlates of protection against SARS-CoV-2 in rhesus 1073 macaques

Affinity maturation of SARS-CoV-2 1076 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations

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

Considerable escape of SARS-CoV-2 1083 Omicron to antibody neutralization

Reduced sensitivity of SARS-CoV-2 variant 1086 Delta to antibody neutralization

cryoSPARC: algorithms for 1088 rapid unsupervised cryo-EM structure determination

Convergent antibody responses to SARS-CoV-2 in 1091 convalescent individuals

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection 1094 from disease in a small animal model

SARS-CoV-2 can recruit a heme metabolite to evade antibody 1097 immunity

Variant Neutralization in Serum from Vaccinated and Convalescent Persons

Plasma Neutralization of the SARS-CoV-2 1102 Omicron Variant

Measuring SARS-CoV-2 1105 neutralizing antibody activity using pseudotyped and chimeric viruses

High genetic barrier to SARS-CoV-2 1108 polyclonal neutralizing antibody escape

A vaccine-induced public antibody protects against 1111 SARS-CoV-2 and emerging variants

High frequency of shared clonotypes in human B cell 1114 receptor repertoires

Neutralizing and protective human 1117 monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Neutralizing and protective human 1121 monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

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Germinal centers

Prevalent, protective, and convergent IgG 1141 recognition of SARS-CoV-2 non-RBD spike epitopes

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mRNA vaccine-elicited antibodies to 1151 SARS-CoV-2 and circulating variants

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Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 1165 Delta variant

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Rapid isolation and profiling of a diverse panel of 1171 human monoclonal antibodies targeting the SARS-CoV-2 spike protein

alone, (4) Blocking: sensors immersed 5 min with IgG isotype control at 10 µg/mL. (5) Baseline: 808 sensors immersed 30 sec in buffer alone, (6) Antigen association: sensors immersed 5 min with 809 NTD at 10 µg/mL. Expression and purification of stabilized SARS-CoV-2 6P ectodomain was conducted as 823 previously described (Barnes et al., 2020a) . Briefly, constructs encoding the SARS-CoV-2 S 824 ectodomain (residues 16-1206 with 6P stabilizing mutations (Hsieh et al., 2020) , a mutated furin 825 cleavage site, and C-terminal foldon trimerization motif followed by hexa-His tag) were used to 826 transiently transfect Expi293F cells (Gibco). Four days after transfection, supernatants were 827 harvested and S 6P proteins were purified by nickel affinity and size-exclusion chromatography. 828Peak fractions from size-exclusion chromatography were identified by SDS-PAGE, and fractions 829 J o u r n a l P r e -p r o o f corresponding to spike trimers were pooled and stored at 4˚C. Fabs and IgGs were expressed, 830 purified, and stored as previously described (Barnes et al., 2020a) . 831 832

Purified Fabs were mixed with SARS-CoV-2 S 6P trimer at a 1.1:1 molar ratio of Fab-to-834 protomer for 30 minutes at room temperature. Fab-S complexes were concentrated to 3-4 mg/mL 835 prior to deposition on a freshly glow-discharged 300 mesh, 1.2/1.3 Quantifoil grid (Electron 836Microscopy Sciences). Immediately prior to deposition of 3 µL of complex onto grid, fluorinated 837 octyl-maltoside (Anatrace) was added to the sample to a final concentration of 0.02% w/v. 838Samples were vitrified in 100% liquid ethane using a Mark IV Vitrobot (Thermo Fisher) after 839 blotting at 22˚C and 100% humidity for 3s with Whatman No. 1 filter paper. 840 841 Cryo-EM data collection and processing 842Single-particle cryo-EM data were collected on a Titan Krios transmission electron microscope 843 (Thermo Fisher) equipped with a Gatan K3 direct detector, operating at 300 kV and controlled 844 using SerialEM automated data collection software (Mastronarde, 2005) . A total dose of ~60 e -845 /Å 2 was accumulated on each movie comprising 40 frames with a pixel size of 0.515 Å (C1520-S 846 dataset) or 0.852 Å (C1717-S and C1791-S) and a defocus range of -1.0 and -2.5 µm. Further 847 data collection parameters are summarized in Table S5 . 848 849 Movie frame alignment, CTF estimation, particle-picking and extraction were carried out using 850 cryoSPARC v3.1 (Punjani et al., 2017 ). Reference-free particle picking and extraction were 851 performed on dose-weighted micrographs curated to remove images with poor CTF fits or signs 852 J o u r n a l P r e -p r o o f of crystalline ice. A subset of 4x-downsampled particles were used to generate ab initio models, 853 which were then used for heterogeneous refinement of the entire dataset in cryoSPARC. Particles 854 belonging to classes that resembled Fab-S structures were extracted, downsampled x2 and 855 subjected to 2D classification to select well-defined particle images. 3D classifications (k=6, 856 tau_fudge=4) were carried out using Relion v3.1.1(Fernandez-Leiro and Scheres, 2017) without 857 imposing symmetry and a soft mask. Particles corresponding to selected classes were re-858 extracted without binning and 3D refinements were carried out using non-uniform refinement in 859 cryoSPARC. Particle stacks were split into individual exposure groups based on the beamtilt 860 angle used for data collection and subjected to per particle CTF refinement and aberration 861 corrections. Another round of non-uniform refinement in cryoSPARC was then performed. For 862 focused classification and local refinements of the Fab VHVL-NTD interface, particles were 3D 863 classified in Relion without alignment using a mask that encompassed the Fab-NTD region. 864Particles in good 3D classes were then used for local refinement in cryoSPARC. Details of 865 overall resolution and locally-refined resolutions according to the gold-standard Fourier shell 866 correlation of 0.143 criterion (Bell et al., 2016) can be found in Table S5 . 867 868 Cryo-EM structure modeling, refinement, and analyses 869Coordinates for initial complexes were generated by docking individual chains from reference 870 structures (see Table S5 ) into cryo-EM density using UCSF Chimera (Goddard et al., 2007; 871 Pettersen et al., 2004) . Initial models for Fabs were generated from coordinates from PDB 6RCO 872 (for C1717 Fab) or PDB 7RKS (for C1520). Models were refined using one round of rigid body der Waals interactions between atoms were assigned as interactions that were <4.0Å. Hydrogen 884 bond and van der Waals interaction assignments are tentative due to resolution limitations. Spike 885 epitope residues were defined as residues containing atom(s) within 4Å of a Fab atom for the 886 C1520-S and C1717-S complexes, and defined as spike C atom within 7Å of a Fab C atom 887 for the C1791-S complex. 888 889

Antibody sequences were trimmed based on quality and annotated using Igblastn v.1.14. with 891 IMGT domain delineation system. Annotation was performed systematically using Change-O 892 toolkit v.0.4.540 (Gupta et al., 2015) . Heavy and light chains derived from the same cell were 893 paired, and clonotypes were assigned based on their V and J genes using in-house R and Perl 894 scripts ( Figure S3 ). All scripts and the data used to process antibody sequences are publicly 895 available on GitHub (https://github.com/stratust/igpipeline/tree/igpipeline2_timepoint_v2). The table highlights the reagents, genetically modified organisms and strains, cell lines, software, instrumentation, and source data essential to reproduce results presented in the manuscript. Depending on the nature of the study, this may include standard laboratory materials (i.e., food chow for metabolism studies, support material for catalysis studies), but the table is not meant to be a comprehensive list of all materials and resources used (e.g., essential chemicals such as standard solvents, SDS, sucrose, or standard culture media do not need to be listed in the table). Items in the table must also be reported in the method details section within the context of their use. To maximize readability, the number of oligonucleotides and RNA sequences that may be listed in the table is restricted to no more than 10 each. If there are more than 10 oligonucleotides or RNA sequences to report, please provide this information as a supplementary document and reference the file (e.g., See Table S1 for XX) in the key resources table.

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