key: cord-0831974-iamwpct3 authors: Claireaux, Mathieu; Caniels, Tom G; de Gast, Marlon; Han, Julianna; Guerra, Denise; Kerster, Gius; van Schaik, Barbera DC; Jongejan, Aldo; Schriek, Angela I.; Grobben, Marloes; Brouwer, Philip JM; van der Straten, Karlijn; Aldon, Yoann; Capella-Pujol, Joan; Snitselaar, Jonne L; Olijhoek, Wouter; Aartse, Aafke; Brinkkemper, Mitch; Bontjer, Ilja; Burger, Judith A; Poniman, Meliawati; Bijl, Tom PL; Torres, Jonathan L; Copps, Jeffrey; Martin, Isabel Cuella; de Taeye, Steven W; de Bree, Godelieve J; Ward, Andrew B; Sliepen, Kwinten; van Kampen, Antoine HC; Moerland, Perry D; Sanders, Rogier W; van Gils, Marit J title: A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike date: 2021-12-03 journal: bioRxiv DOI: 10.1101/2021.12.01.470767 sha: b3bedd4fddeb2ba57820fa1157c859eebc8cb1ba doc_id: 831974 cord_uid: iamwpct3 Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that ∼82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (∼0.05%) and unswitched B cells (∼0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 49 2019 and its increased spread as a result of novel viral variants has posed considerable danger to global 50 health. Multiple vaccines are now in use that confer high levels of protection. As of November 25th 2021, 51 54% of the world's population has received at least one dose of a coronavirus disease 2019 52 vaccine, illustrating the rapid development and distribution of vaccines (Ritchie et al. 2020) . A major goal 53 of these vaccines is to induce neutralizing antibodies (NAbs) and memory B cells that protect against 54 subsequent infection. Most licensed vaccines aim to induce immunity against SARS-CoV-2 S, a trimeric glycoprotein on 56 the surface of the virion, that is the only known target for NAbs. It consists of an apical S1 subunit 57 encompassing an N-terminal domain (NTD) and the receptor-binding domain (RBD) that is responsible for 58 binding to the ACE2 receptor; and a membrane-proximal S2 subunit which is responsible for fusion of viral 59 and cellular membranes. Coronavirus (CoV) S proteins can suffer from instability and deteriorate into non-60 native forms leading to altered exposure of antibody epitopes (Hsieh et al. 2020; Berger and Schaffitzel 61 2020; Juraszek et al. 2021) . Therefore, some of the approved vaccines, including those from J&J/Janssen, 62 Moderna and Pfizer/BioNTech, but not those from Oxford/AstraZeneca and several others, contain 63 modifications to stabilize the S proteins. These changes include the removal of the furin cleavage site 64 between S1 and S2 and two proline substitutions (K986P/V987P) in S2, resulting in a prefusion stabilized 65 S trimer termed S-2P (Sadoff et al. 2021; Baden et al. 2021; Polack et al. 2020) . Although S-2P is more 66 stable than wild-type S (S-WT), subsequent studies revealed that even S-2P suffers from instability issues 67 and displays open conformations (Juraszek et al. 2021; Henderson et al. 2020; Hsieh et al. 2020) . Several 68 additional stabilization strategies have been described, including the introduction of an additional four 69 prolines (F817P/A892P/A899P/A942P), resulting in S-6P (HexaPro S), which shows considerably 70 4 increased stability and resistance to heat/freeze cycles compared to S-2P and S-WT (Hsieh et al. 2020; 71 Henderson et al. 2020; McCallum et al. 2020; Juraszek et al. 2021 ). Early in the pandemic many groups identified and isolated potent NAbs from COVID-19 patients 73 that defined important epitopes on S, which have led to emergency use authorization of several monoclonal 74 antibody (MAb) therapies for COVID-19 (Taylor et al. 2021) . Most of the potently neutralizing MAbs target 75 the immunodominant RBD and NTD on the apex of S, whereas MAbs against the S2 domain, while 76 generally less potent, tend to be more broadly reactive across sarbecoviruses and endemic human CoVs 77 (HCoVs) (Shiakolas et al. 2021; Amanat, Thapa, et al. 2021) . B cells that arise during SARS-CoV-2 infection 78 target both S1 and S2 domains, but their ontogeny is often unclear. S1 NAbs are usually poorly cross-79 reactive with different SARS-CoV-2 variants or other HCoVs and have not undergone extensive somatic 80 hypermutation (SHM), suggesting that they originate from de novo activation of naive B cells (Kreer et al. 81 2020; Brouwer et al. 2020; Zost et al. 2020; Rogers et al. 2020; Robbiani et al. 2020) . In contrast, a subset 82 of S2 NAbs cross-bind and cross-neutralize endemic HCoVs, suggesting that they arise from pre-existing 83 memory B cells, formed during infection with an endemic HCoV and reactivated upon SARS-CoV-2 84 infection (Song et al. 2021) . Thus, it is conceivable that in SARS-CoV-2 infection and similarly, vaccination, 85 the humoral immune response is driven by both de novo activation of naive B cells and reactivation of 86 memory B cells. Although the NAb response to SARS-CoV-2 infection and vaccination has been studied 88 extensively, non-neutralizing MAbs (non-NAbs) have not been a focal point. While understudied, non-NAbs 89 make up a substantial portion of the antibody repertoire after infection and the majority of vaccine-induced 90 anti-S Abs are also non-neutralizing (Amanat, Thapa, et al. 2021; Dugan et al. 2021; Sakharkar et al. 2021 ). Non-NAbs can contribute to immunity through effector mechanisms such as antibody-dependent cellular 92 cytotoxicity (ADCC) and phagocytosis (ADCP) (Ullah et al. 2021; Chan et al. 2021; Winkler et al. 2021; 93 5 Schäfer et al. 2021; Shiakolas et al. 2021) , while high levels of pro-inflammatory antibodies may contribute 94 to severe disease (Chakraborty et al. 2021; Larsen et al. 2021) . Non-NAbs elicited by infection target S as 95 well as other viral proteins such as nucleoprotein (N) and ORF8 (Dugan et al. 2021 properties and epitopes of non-NAbs as well as their B cell origins, is therefore highly relevant for both 104 understanding humoral immunity against SARS-CoV-2 and improving vaccines. Here, we characterized 105 the human baseline B cell repertoire that recognizes SARS-CoV-2 S prior to any antigenic SARS-CoV-2 S 106 encounter through infection or vaccination. We found that an unusually high proportion (~0.1%) of naive B 107 cells is able to recognize SARS-CoV2 S and that these naive B cells show a highly enriched usage of heavy 108 chain immunoglobulin V gene (IGHV)1-69 and kappa chain immunoglobulin V gene (IGKV)3-11 in their B 109 cell receptor (BCR). A subset of these naive B cells exhibits polyreactivity and can be activated upon SARS- Recent studies on sera and memory B cells from recovered COVID-19 patients suggest that a fraction of 119 the antibody response against SARS-CoV-2 S could stem from pre-existing cross-reactive memory B cells 120 induced by prior infection with endemic HCoVs (Ng et al. 2020; Song et al. 2021; Grobben et al. 2021; Tong 121 et al. 2021) . Therefore, we interrogated the baseline SARS-CoV-2 S-reactive B cell repertoire from ten 122 unexposed and unvaccinated healthy donors (HD01-10), sampled in 2019 and early 2020, before the first 123 official case report of SARS-CoV-2 infection in the Netherlands. We developed a novel combinatorial B cell 124 staining approach with labelled antigenic probes, allowing for the simultaneous identification of B cells that 125 are reactive to six different pathogens in a single sample (Table S1 ). SARS-CoV-2 S-reactive B cells were 126 compared to those recognizing pre-encountered antigens, including influenza A virus hemagglutinin 127 (H1N1pdm09 HA), RSV fusion protein (RSV F) and tetanus toxoid (TT), as well as those against 128 unencountered antigens HIV-1 envelope glycoprotein (Env) and hepatitis C virus (HCV) envelope 129 glycoprotein (E1E2; Fig. 1A , Fig. S1A -E). B cells specific for SARS-CoV-2 were present at high frequency 130 in unexposed individuals (median 0.086% of total B cells), which was ~10-30-fold higher compared to the 131 frequency of B cells reactive with other unencountered antigens HIV-1 Env and HCV E1E2 (median 0.007% 132 and 0.0025%, respectively), but in a similar range to the frequency of B cells reactive with previously 133 encountered antigens H1N1 HA (0.066%), RSV F (0.042%) and TT (0.12%, Fig. 1B ). Next, we analyzed the naive and memory subsets of these antigen-specific B cells, which can be 135 subdivided into four populations based on their surface expression of IgD and CD27: naive (IgD + /CD27 -), 136 classical memory (IgD -/CD27 + ), CD27memory (IgD -/CD27 -) and unswitched memory (IgD + /CD27 + ) (Fig. 1A lower panel, C-D and Fig. S2A , B). There were virtually no naive B cells specific to HIV-1 Env and HCV 7 E1E2 (~0.001%) (Fig. 1C ). In contrast, the proportion of B cells that recognize SARS-CoV-2 S was 139 significantly higher (0.11%) in the naive compartment than those specific against any other probe tested 140 ( Fig. 1C) . This indicates that a de novo B cell response stemming from the naive compartment likely plays 141 a role in SARS-CoV-2 infection. In the classical memory compartment, B cells specific against recurring seasonal infections (H1N1 HA and 143 RSV F; 0.084% and 0.095%, respectively) or those elicited against a vaccine component (TT; 0.17%) were 144 significantly more predominant than those against SARS-CoV-2 S (0.049%) (Fig. 1D) , as expected. However, the SARS-CoV-2 S-reactive memory B cell frequency was significantly higher than that of HIV-1 146 Env and HCV E1E2 (Fig. 1D , 0.0088% and 0.0094%, respectively). We further confirmed the presence of 147 pre-existing SARS-CoV-2 S-reactive humoral immunity by observing significantly higher plasma Ig levels 148 against SARS-CoV-2 S compared to SARS-CoV S, MERS-CoV S and HIV-1 Env-specific Igs ( Fig. S2E -F). The majority of the SARS-CoV-2 S-binding classical memory B cells were IgG+. In contrast, memory B 150 cells reactive with the vaccine antigen TT were frequently IgM+, while H1N1 HA and RSV F reactive B cells 151 were almost exclusively IgM-, probably reflecting recurrent antigenic stimulation (Fig. 1E, right panel; Fig. 152 S2C). Similar results were obtained for the unswitched memory compartment, with SARS-CoV-2 having a 153 higher S-reactive B cell frequency (0.042%) than that of HIV-1 Env (0.0083%) and HCV E1E2 (0.0018%), 154 comparable to RSV F (0.034%), but lower than to H1N1 HA (0.076%) and TT (0.26%) (Fig. S2B) In eight out of ten supernatants from unexposed individuals, the secretion of specific antibodies against 172 SARS-CoV-2 S was similar to that against endemic HCoVs HKU1-CoV S and OC43-CoV S ( Fig. 1F -H). Taken together, while the majority of SARS-CoV-2 S-reactive B cells belong to the naive compartment, the 174 memory B cell compartment of the majority of HDs has the capacity to produce SARS-CoV-2 S-reactive 175 antibodies despite not having previously encountered SARS-CoV-2. One to two donors appear to show 176 some response to HIV-1 Env and HCV E1E2 (Fig. 1G-H) , while they were confirmed HIV-1 and HCV-naive, 177 an observation that requires further study but may relate to rare cross-reactivity of virus-reactive antibodies 178 (Williams, Han, and Haynes 2018 Next, we analyzed whether specific HC/LC pairings were preferred in SARS-CoV-2 S-reactive 272 naive B cells in unexposed individuals. Indeed, we observed significant enrichment of multiple HC/LC 273 pairings, in particular IGHV1-69/IGKV3-11, which was found in 164 out of 1755 total naive HC/LC pairs in 274 S-reactive B cells (9.3%), corresponding to 8.5% on average for the nine donors included (Fig. 3F ). These 275 13 results match our earlier observations (Fig. 2D) . Moreover, IGHV1-69 was used in seven out of the ten 276 highest-frequency HC/LC pairings (IGHV1-69 paired with IGKV3-11, IGKV3-20, IGKV3-15, IGKV1-39, 277 IGKV4-1, IGKV1-5, IGKV2-28) and four of these pairings were significantly overrepresented in SARS-CoV- To characterize the epitopes of the IGHV1-69/IGKV3-11 clonotype, we studied six non-clonal IGHV1-305 69/IGKV3-11 MAbs isolated from two COVID-19 patients in an earlier study (Brouwer et al. 2020) , which 306 all showed high identity to germline V genes and bear an average-length CDRH3 ( However, several studies have shown that even S-2P is unstable and can deteriorate into aberrant 349 conformations, albeit less efficiently so than S-WT (Hsieh et al. 2020; Juraszek et al. 2021) . Indeed, we 350 found that COVA1-07, COVA2-14 and COVA2-17 bound efficiently to S-2P as measured by bio-layer 351 interferometry (BLI). In fact, the binding kinetics to S-2P were similar to those of S2, revealing that the non-352 neutralizing S2 epitope is well exposed on S-2P. Next, we tested a more stabilized S trimer that incorporates (Fig. 4G) . Overall, the frequency of S-2P-reactive B cells was 1.6-fold higher than that of S-363 6P-reactive cells (Fig. 4G and Fig. 4H left panel) . Strikingly, the loss of reactivity toward S-6P is explained (Table S1) Luminex assays were performed as described previously (Grobben et al. 2021) . In short, expressed 759 glycoproteins were covalently coupled to Luminex Magplex beads using a ratio of 75 µg protein to 12.5 760 million beads for SARS-CoV-2 S and equimolarly for all other proteins. Next, 1:10.000-diluted plasma or 761 undiluted stimulated B cell supernatant was added to the protein-coated beads overnight at 4°C. The 762 following day, beads were washed with TBS/0.05% Tween using a magnetic separator and resuspended 763 in 1,3 µg/mL goat anti-human IgG-PE and incubated for 2 hours at room temperature (Southern Biotech). After washing, the beads were resuspended in Magpix drive fluid (Luminex). The plates were read on a 765 Magpix (Luminex). Specific binding was attributed to median fluorescence intensity (MFI) levels >3x above 766 background. Influenza A Virus Hemagglutinin Trimer Head and Stem Proteins Identify and Quantify Different Hemagglutinin-Specific B Cell Subsets in 991 Thrombocytopenic Purpura with Possible Association with AstraZeneca-Oxford COVID-19 Vaccine Introduction of Two Prolines and Removal of the 997 Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike-Based SARS-CoV-2 Vaccine in the Mouse Model SARS-CoV-2 mRNA Vaccination Induces Functionally 1001 Diverse Antibodies to NTD, RBD, and S2 Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine SARS-CoV-2 Neutralizing Antibody Structures 1007 Inform Therapeutic Strategies An Anti-SARS-CoV-2 Non-Neutralizing Antibody with Fc-Effector 1010 Function Defines a New NTD Epitope and Delays Neuroinvasion and Death in K18-hACE2 Mice The SARS-CoV-2 Spike Protein: Balancing Stability and 1013 Infectivity Silencing of HIV-1 1015 with RNA Interference: A Multiple shRNA Approach Commonality despite 1018 Exceptional Diversity in the Baseline Human Antibody Repertoire Two-Component Spike Nanoparticle 1021 Vaccine Protects Macaques from SARS-CoV-2 Infection Potent Neutralizing Antibodies Patients Define Multiple Targets of Vulnerability First Report of a de Novo iTTP Episode Associated with an mRNA-Based Anti Emerging SARS-CoV-2 Variants of Concern Evade 1030 Humoral Immune Responses from Infection and Vaccination Proinflammatory IgG Fc Structures in Patients with 1034 Severe COVID-19 The Fc-Mediated Effector Functions of a Potent SARS-CoV-2 1037 Isolated from an Early Convalescent COVID-19 Patient, Are Essential 1038 for the Optimal Therapeutic Efficacy of the Antibody Neutralizing Antibodies: Genetics, Structures, and Relevance to Rational Vaccine Design Immune Correlates of Protection by mRNA-1273 Vaccine 1044 against SARS-CoV-2 in Nonhuman Primates Large-Scale Sequence and Structural Comparisons of 1047 Human Naive and Antigen-Experienced Antibody Repertoires Von Willebrand Factor Multimer Formation Contributes to 1051 Generation of Inhibitory Autoantibodies to ADAMTS13 in 1054 Profiling B Cell Immunodominance after SARS-CoV-2 Infection Reveals 1057 Antibody Evolution to Non-Neutralizing Viral Targets Evidence for Antibody as a Protective Correlate 1060 for COVID-19 Vaccines Infection Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring 1066 Comprehensive Database for Human and Mouse Immunoglobulin and T Cell Receptor Genes Fab and Fc Contribute to Maximal Protection against SARS-CoV-2 1072 Following NVX-CoV2373 Subunit Vaccine with Matrix-M Vaccination Cross-Reactive Antibodies 1076 after SARS-CoV-2 Infection and Vaccination Polyreactive Broadly Neutralizing B Cells Are Selected 1080 to Provide Defense against Pandemic Threat Influenza Viruses Parallel Detection of Antigen-Specific T-Cell Responses 1083 by Multidimensional Encoding of MHC Multimers Integrated Analysis of Multimodal Single-Cell Data Controlling the SARS-CoV-2 Spike Glycoprotein 1089 Structure-Based Design of Prefusion-Stabilized SARS-CoV-2 1092 A Systematic Review of Antibody Mediated 1095 Immunity to Coronaviruses: Kinetics, Correlates of Protection, and Association with Severity Breadth and Function of Antibody Response to Acute SARS-CoV-2 1099 Infection in Humans Stabilizing the Closed 1102 SARS-CoV-2 Spike Trimer Structures and Distributions of SARS-CoV-2 Spike Proteins on Intact Virions CoV-2 Vaccines in Nonhuman Primates and Humans VarScan: Variant Detection in 1111 Massively Parallel Sequencing of Individual and Pooled Samples Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-1114 Neutralizing Antibodies from COVID-19 Patients Appion: An Integrated, Database-Driven Pipeline to Facilitate EM Image 1117 Processing Afucosylated IgG Characterizes Enveloped Viral Responses 1120 and Correlates with COVID-19 Severity Géraldine 1123 IMGT®, the International ImMunoGeneTics Information System® 25 Years on Free SARS-CoV-2 Spike 1126 Protein S1 Particles May Play a Role in the Pathogenesis of COVID-19 Infection Fast and Accurate Long-Read Alignment with Burrows-Wheeler 1129 and 1000 Genome Project Data Processing Subgroup Sequence Alignment/Map Format and SAMtools SARS-CoV-2-Reactive Mucosal B Cells in the Upper Respiratory Tract of 1135 Uninfected Individuals Acquired Thrombotic Thrombocytopenic Purpura: A Rare 1138 Disease Associated with BNT162b2 Vaccine The ADAMTS13-von Willebrand Factor Axis Structure-Guided Covalent Stabilization of Coronavirus Spike Glycoprotein Trimers in the Closed 1145 MULTI-Seq: Sample Multiplexing for Single-Cell 1148 RNA Sequencing Using Lipid-Tagged Indices HIV Antibodies. Antigen Modification Regulates 1151 Competition of Broad and Narrow Neutralizing HIV Antibodies Structure-Based Design of a Fusion Glycoprotein 1154 Vaccine for Respiratory Syncytial Virus Preexisting and de Novo Humoral Immunity to SARS-CoV-2 in Humans UCSF Chimera--a Visualization System for 1160 Exploratory Research and Analysis Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine cryoSPARC: Algorithms 1165 for Rapid Unsupervised Cryo-EM Structure Determination CoV-AbDab: 1167 The Coronavirus Antibody Database Pandemic (COVID-19) Convergent Antibody Responses to SARS-CoV-2 in Convalescent 1173 Individuals Isolation of Potent SARS-CoV-2 Neutralizing Antibodies and Protection from 1176 Disease in a Small Animal Model SARS-CoV-2 Can Recruit a Heme Metabolite to Evade Antibody Immunity Thrombocytopenic Purpura after First Vaccination Dose of BNT162b2 mRNA COVID-19 Vaccine Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-1186 19 Prolonged Evolution of the Human B Cell Response to 1189 SARS-CoV-2 Infection Virus Vaccines: Proteins Prefer Prolines Antibody Potency, Effector Function, and Combinations in 1194 Protection and Therapy for SARS-CoV-2 Infection in Vivo Cross-Reactive Coronavirus Antibodies with Diverse Epitope 1198 Specificities and Fc Effector Functions Relapse of Thrombotic Thrombocytopenic Purpura after COVID-19 Vaccine Structure and Immunogenicity of a Stabilized HIV-1 Envelope 1206 Trimer Based on a Group-M Consensus Sequence Cross-Reactive Serum and Memory B-Cell Responses to Spike Protein in 1209 SARS-CoV-2 and Endemic Coronavirus Infection Automated Molecular Microscopy: The New 1212 Immunogenicity of Stabilized 1215 HIV-1 Envelope Trimers with Reduced Exposure of Non-Neutralizing Epitopes Neutralizing Monoclonal Antibodies for Treatment of COVID-19 Memory B Cell Repertoire for Recognition of Evolving SARS-CoV-2 Spike Live Imaging of SARS-CoV-2 Infection in Mice Reveals 1225 Neutralizing Antibodies Require Fc Function for Optimal Efficacy TiltPicker: Software Tools to Facilitate Particle Selection in Single Particle Electron Microscopy Cross-Reactivity of HIV Vaccine 1231 Responses and the Microbiome Human Neutralizing Antibodies against SARS-CoV-2 Require Intact Fc Effector 1234 Functions for Optimal Therapeutic Protection Broad Auto-Reactive IgM Responses Are Common in Critically Ill Patients Including Those with COVID-19 An Alternative Binding Mode of IGHV3-53 Antibodies to the SARS-1240 CoV-2 Receptor Binding Domain DNA Vaccine Protection against SARS-CoV-2 in 1244 New Tools for Automated High-Resolution Cryo-EM Structure 1247 Determination in RELION-3 Potently Neutralizing and Protective Human Antibodies against 1250 SARS-CoV-2 penicillin/streptomycin (100U/mL and 100 μg/mL, respectively) were transfected with purified DNA as 792 described previously (Brouwer et al. 2020 Polyreactivity ELISAs were performed as described elsewhere (Guthmiller et al. 2020) . High-binding half-829 area 96-well plates (Costar) were coated with 2 μg/mL Salmonella enterica serovar Typhimurium flagellin 830 (Invitrogen), 10 μg/mL calf thymus double-stranded DNA (ThermoFisher), 5 μg/mL human insulin (Sigma-831 51 Aldrich) and 10 μg/mL Escherichia coli lipopolysaccharide (Sigma-Aldrich) in PBS and stored at RT 832 overnight. Separate plates were coated with 10 μg/mL bovine cardiolipin (Sigma-Aldrich) in 99% ethanol 833 and allowed to air-dry overnight. The following day, plates were washed with demineralized water and 834 blocked with PBS/0.05% Tween/1mM ethylenediaminetetraacetic acid (EDTA) for 1 h. MAbs were serially 835 diluted five-fold (starting concentration 10 μg/mL) and binding to the plates was allowed for 2 h at RT. After RESEDA (https://bitbucket.org/barbera/reseda/) was used to further analyze the BCR repertoire. Sequences assembled by CellRanger were aligned to the IMGT gene database (Giudicelli, Chaume, and 913 Lefranc 2005) with BWA (default settings) (Li and Durbin 2010) . Variants were called with samtools mpileup 914 (Li et al. 2009 ) and VarScan (Koboldt et al. 2009 ) with minimum coverage set to 1 read to avoid missing 915 54 mutations from antibody parts covered by a single sequence read. Consequently, any differences with 916 respect to the IMGT sequences were considered as SHM. The CDR3 sequences were determined by 917 translating the nucleotide sequences to peptide sequences and searching for conserved motifs in the V and 918 J genes. The information from RESEDA was merged with the results obtained with Seurat. Clustering and BCR repertoire analysis 921 In total, 1965 paired heavy/light chain BCRs were recovered, which could be assigned reliably to a single 922 HD using the HTOs. K-means clustering with three determinants (IgD CLR/CD27 CLR/total number of 923 mutations in HC/LC V region) was used to cluster B cells. The optimal number of clusters (k = 2) was 924 determined using the average silhouette method and was used to assign a phenotype (memory/naive). MAbs in molar excess were complexed with S2 for 30 min at RT. Immune complexes were diluted to ~20 933 μg/mL and deposited on glow-discharged, carbon-coated 400 mesh copper grids (Electron Microscopy 934 Sciences). Sample was blotted from the grid with filter paper followed by two successive additions of 2% 935 w/v uranyl formate stain with blotting. Grids were imaged on a Tecnai T20 (FEI) electron microscope with 936 a CMOS 4k camera (TVIPS) at 200 kV, 62,000x nominal magnification, and 1.77Å/pixel. Micrographs 937 were collected using Leginon, particles were picked using Difference of Gaussians picker, and particles 938 were cleaned through successive rounds of reference-free 2D classification in Relion 3.0 (Suloway et al. 939 2005; Voss et al. 2009; Lander et al. 2009; Zivanov et al. 2018) . Particles were also processed in 940 CryoSPARC2 and reconstructed using Ab Initio (Punjani et al. 2017) .