Article

Memory B cell repertoire for recognition of evolving

SARS-CoV-2 spike
Graphical abstract Authors
Pei Tong, Avneesh Gautam,
Ian W. Windsor, ..., Goran Bajic,
Stephen C. Harrison,
Duane R. Wesemann

Correspondence
[email protected]

In brief
Unbiased charting of memory B cell
receptor-encoded antibodies from
COVID-19 convalescent subjects
identifies seven major antibody
competition groups recognizing epitopic
regions with group assignment
correlating with cross-CoV-reactivity
breadth and neutralization potency.
SARS-CoV-2 variants tend to escape
antibodies from groups with the most
potent neutralizers, but many retain
affinity, showing that redundant
components of a primary immune
Highlights response establish durable protection
d Seven major epitopic regions of SARS-CoV-2 spike are from evolving pathogens.
consistently targeted by human Abs

d Ab group assignment correlates with CoV binding breadth

and neutralization potency

d SARS-CoV-2 variants tend to escape Abs from the groups

with most potent neutralizers

d Intra-group Ab binding redundancy confers robustness

against emerging variants

Tong et al., 2021, Cell 184, 4969–4980

September 16, 2021 ª 2021 Elsevier Inc.
https://doi.org/10.1016/j.cell.2021.07.025 ll
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Article
Memory B cell repertoire for recognition
of evolving SARS-CoV-2 spike
Pei Tong,1,14 Avneesh Gautam,1,14 Ian W. Windsor,2,5,9,14 Meghan Travers,1 Yuezhou Chen,1 Nicholas Garcia,1
Noah B. Whiteman,1 Lindsay G.A. McKay,3,4 Nadia Storm,3,4 Lauren E. Malsick,3,4 Anna N. Honko,3,4 Felipe J.N. Lelis,1
Shaghayegh Habibi,1 Simon Jenni,2 Yongfei Cai,5 Linda J. Rennick,6,7 W. Paul Duprex,6,7 Kevin R. McCarthy,6,7
Christy L. Lavine,8 Teng Zuo,1 Junrui Lin,1 Adam Zuiani,1 Jared Feldman,9 Elizabeth A. MacDonald,2 Blake M. Hauser,9
Anthony Griffths,3,4 Michael S. Seaman,8 Aaron G. Schmidt,9,10,11 Bing Chen,5,11 Donna Neuberg,12 Goran Bajic,2,5,15
Stephen C. Harrison,2,5,11,13 and Duane R. Wesemann1,11,16,*
1Department of Medicine, Division of Allergy and Immunology, Division of Genetics, Brigham and Women’s Hospital, Harvard Medical School,

Boston, MA 02115, USA

2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
3Department of Microbiology, Boston University School of Medicine, Boston, MA 02115, USA
4National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA 02115, USA
5Laboratory of Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
6The Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
7The Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
8Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
9Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
10Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA
11Massachusetts Consortium on Pathogen Readiness, Boston, MA 02115, USA
12Department of Data Science, Dana-Farber Cancer Institute, Boston, MA 02215, USA
13Howard Hughes Medical Institute, Boston, MA 02115, USA
14These authors contributed equally
15Present address: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
16Lead contact

*Correspondence: [email protected]
https://doi.org/10.1016/j.cell.2021.07.025

SUMMARY

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but
with unknown reach from original infection to antigenically drifted variants. We charted memory B cell recep-
tor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found
seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion
of published and newly determined structures of antibody-S complexes identified corresponding epitopic re-
gions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and conver-
gent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many
members of the groups associated with the most potent neutralizing activity, some antibodies in each of
those groups retained affinity—suggesting that otherwise redundant components of a primary immune
response are important for durable protection from evolving pathogens. Our results furnish a global atlas
of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robust-
ness against emerging variants.

INTRODUCTION immunity in humans. We focus on B cells, because antibodies,

a key part of the immune defense, are sufficient to protect
Coronavirus (CoV) disease 2019 (COVID-19), caused by the se- against SARS-CoV-2 infection in animal models (Robbiani
vere acute respiratory syndrome (SARS) CoV-2, has become a et al., 2020; Schäfer et al., 2021).
pandemic of historic effect. Although vaccines have been devel- Antibodies are both soluble effector molecules and the anti-
oped in record time, new variants continue to emerge and gen-receptor component of the B cell receptor (BCR). BCRs
threaten to evade immune responses. We need to understand evolve enhanced pathogen binding through immunoglobulin
how SARS-CoV-2 is recognized and remembered by the (Ig) gene somatic hypermutation (SHM) and selection in
immune system to illuminate requirements for broad protective lymphoid tissue germinal centers (GCs), leading to antibody

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affinity maturation (Victora and Nussenzweig, 2012) and genera- most potent neutralizers, while the S2-1 group had the greatest
tion of both antibody-secreting plasma cells (PCs) and memory recognition breadth across CoVs. The emerging SARS-CoV-2
B cells. Higher avidity interactions encourage terminal differenti- variants, particularly B.1.351, escaped binding by many RBD-2
ation of B cells into PCs; memory B cells frequently have lower and NTD-1 antibodies. Nonetheless, because mutations in those
avidity but more cross-reactive specificities (Akkaya et al., 2020). variants had varying effects on the affinity of antibodies within
Both PC-derived secreted antibody and memory B cells sup- a competition group, we conclude that the presence in an indi-
ply immune memory to prevent repeat infection, but with non- vidual of otherwise redundant, memory B cells for a given epit-
redundant roles. Secreted antibodies can prophylactically opic region can confer recognition breadth for dynamically
thwart pathogen invasion with fixed recognition capability, while mutating S.
memory B cells harbor expanded pathogen recognition capacity
and can differentiate quickly into PCs to contribute dynamically RESULTS
to the secreted antibody repertoire (Akkaya et al., 2020). More-
over, memory B cells retain plasticity to adapt to viral variants Anti-S antibody general subunit reactivity and breadth
through GC re-entry and SHM-mediated evolution (Turner across individuals
et al., 2020). To identify the general pattern of SARS-CoV-2 S recognition by
The viral spike (S) glycoprotein binds ACE2 on host cells and memory B cells in convalescent subjects, we sorted single
mediates viral fusion with the host (Li et al., 2003). Its fusogenic CD19+ CD27+ IgG+ B cells recognizing soluble prefusion-stabi-
activity depends on a furin-mediated cleavage, resulting in lized S trimer (Figures 1A, S1A, and S1B) from 19 individuals
N-terminal S1 and C-terminal S2 fragments (Xia et al., 2020) 24 (median) days (range 13–63 days) following recovery from
and on a subsequent cleavage of S2 mediated either by cathep- COVID-19 (Data S1). We also sorted S-reactive B cells that did
sins or by a serine protease, TMPRSS2 (Hoffmann et al., 2020). not bind RBD from three individuals because less is known about
The S glycoprotein is the principal neutralizing antibody target these antibodies. S-reactive B cells made up 0.2% (0.07%–
and the focus of most vaccines. Secreted SARS-CoV-2 S anti- 0.4%) of the total B cell population (Figure 1A, left panel), with
bodies available in serum can decline with time (Chen et al., RBD-binding cells representing about a quarter of S-reactive
2020; Dan et al., 2021) and lose reactivity to emerging variants IgG+ B cells (Figure 1A, right panel). We cloned and expressed
(Garcia-Beltran et al., 2021; Wang et al., 2021a). Antibodies Ig-heavy (H) and -light (L) chains from individual, sorted memory
cloned from memory B cells target the S glycoprotein in both B cells into human IgG1 and k or l vectors. We detected IgG in
distinct and redundant ways—complementary and competitive 255 of the culture supernatants, which we used to screen for
recognition, respectively (Brouwer et al., 2020; Dejnirattisai binding to SARS-CoV-2 S (Figures 1B, 1C, and S1C–S1E). Of
et al., 2021; Graham et al., 2021; Hansen et al., 2020; Liu et al., the 255 IgGs, 217 bound SARS-CoV-2 S and/or RBD, as
2020; Wang et al., 2021b). Many of these antibodies have been assayed by flow cytometry (157 from the S+ sorting with an
identified and characterized; their positions within the distribu- additional 1 that bound to RBD but not S, and 59 from
tion of practical, complementary recognition of SARS-CoV-2 S S+/RBD sorting) (Figure 1C); 166 of the 217 bound recombinant
in the human memory B cell repertoire have not. Moreover, the SARS-CoV-2 S, as assayed by ELISA (116 from S+ sorting and
recognition reach of memory B cells induced by one SARS- 50 from S+RBD sorting).
CoV-2 strain toward evolving stains across the major epitopic re- We estimated, by ELISA and, where possible, yeast display of
gions is not yet clear. the subdomains (Figures S1D and S1E), the proportion of mAbs
We present here an unbiased global assessment of the distri- that bound to RBD, NTD, and S2. Recombinant and yeast-dis-
bution of memory B cell-encoded antibodies among cooperative played S2 protein could have any of several conformations,
and competitive recognition clusters on the SARS-CoV-2 S and all or parts of the polypeptide chain might be disordered;
glycoprotein and examine features that direct their collaborative mAbs that bound S2 on ELISA plates might therefore tend
robustness against emerging SARS-CoV-2 variants. In a to recognize linear epitopes or even the S2 post-fusion
comprehensive competition analysis of 152 monoclonal anti- conformation. Indeed, most of the S2-binding mAbs had rela-
bodies (mAbs) from 19 subjects for binding with trimeric S ecto- tively low ELISA-determined affinities for intact, recombinant,
domain, we have identified seven recurrently targeted competi- prefusion S, although a few bound more tightly to S expression
tion groups—three for antibodies with epitopes on the receptor- on the surface of 293T cells (Figure S1F). Of the 157 S-reactive
binding domain (RBD), two for epitopes on the N-terminal mAbs sorted with SARS-CoV-2-stabilized S trimer, a total of
domain (NTD), and two for S2 epitopes. We show that these 36 (23%) were RBD specific as assayed by ELISA, by yeast
groups represent the major practical antibody footprints, with display, or both (Figure 1B). We detected 16 (10%) mAbs that
rare antibodies outside them. We map the clusters onto S by bound the NTD and 49 (31%) that bound S2 (Figure 1C). Eleven
including previously characterized antibodies and new, cryo- of the 49 S2 binders bound cell-surface-expressed, but not
genic electron microscopy (cryo-EM)-determined structures. Ig ELISA-based, SARS-CoV-2 S.
repertoire analysis indicates both divergent and convergent We also assessed mAbs by ELISA for cross-reactivity to other
clones with the competition groups. CoV S glycoproteins. Those of SARS, MERS, and common cold
Antibodies mapped to groups we named based on the S b-CoVs HKU1 and OC43 have sequences with 75.8%, 28.6%,
domain (i.e., RBD, NTD, and S2) followed by a number corre- 25.1%, and 25.5% amino-acid identity, respectively, with
sponding to the abundance, within our population, of mAbs SARS-CoV-2 S; the more distantly related common cold
that bind the designated domain. RBD-2 and NTD-1 were the a-CoVs, NL63 and 229E, just 18.3% and 20.2%. Of the 157 S

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Figure 1. SARS-CoV-2 spike-specific mAb binding profiles

(A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19+ cells. Left: IgG+CD27+ cells from 18 donors and the subset of
those that expressed S-binding BCRs. Right: cells from three donors expressing S-binding BCRs and sorted to recover principally those that did not bind RBD.
(B) Summary of all productive mAbs (recombinant human IgG1) screened by ELISA (with recombinant S ectodomain trimer) and cell-surface expression assays
(both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown next to each chart. To
the right of the charts for the two sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding mAbs for those subjects from
whom at least ten paired-chain BCR sequences were recovered.
(C) Binding to a panel of S proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA and by association with S expressed on the
surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for S+ sorted (left) and S+RBD sorted cells (right). Left panel, 157 clones bound
to S and an additional one bound to only RBD but not S. Pink indicates ELISA screens. Blue indicates cell-based screens. Each short section of a row represents
an antibody. The rows labeled VH mutation and VL mutation are heatmaps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated.
(D) Dot plots of VH and VL mutation counts in mAbs that bound RBD, NTD, S2, and a ‘‘broad CoV group’’ that included MERS, HKU1, and OC43.
***p < 0.001, ****p < 0.0001; nonparametric Kruskal-Wallis multiple comparison. Horizontal lines show mean ± SEM. See also Figure S1.

ectodomain-sorted mAbs, 47 (29.9%) bound to SARS-CoV S up to 100-fold differences in affinity. We also included 15 pub-
and 8 to other b-CoV S glycoproteins. These 8 cross-reactive lished mAbs with known structures as references (Figure S2A).
mAbs have higher mutation levels than do RBD, NTD, and the We identified seven major clusters of competing mAbs (Fig-
other S2-binding mAbs from our cohort (Figures 1C and 1D). Af- ure S2A). The three RBD clusters overlapped to varying extents,
finities of mAbs derived from the two individuals that donated as expected for sites on a relatively small domain. Asymmetric
twice to this study (G32 and C41) did not differ between time competition (one mAb blocks binding of another, but the second
points (spaced 31 and 33 days apart, respectively; the two does not block the first) occurred when one had much higher af-
time points were combined in the analysis) (Figure S1G). Among finity than the other—e.g., S309 (Pinto et al., 2020), which binds
the 59 S-binding mAbs cloned from the S+RBD sorted memory more tightly than do most of the RBD-1 mAbs we isolated. The
B cells, ELISA detected 23 (39%) mAbs that bound NTD (11 of clusters define relatively broad epitopic regions, as the footprints
which also bound NTD on yeast), and 14 (23.7%) that bound of two mAbs within a cluster might not overlap with each other
S2, of which 7 (11.9%) cross-reacted with SARS-CoV S. One but both might overlap with the footprint of a third (e.g.,
mAb bound RBD (Figure 1C). REGN10933 and REGN10987, both of which competed with
C12A2, although they have completely distinct footprints at
Unbiased global competition analysis defines seven S either end of the RBD receptor-binding motif (RBM). Some
epitopic regions crosstalk between clusters is also evident (e.g., C93D9, which
We used a competition ELISA to determine pairwise overlaps of bound the RBD, blocked both RBD-2 and NTD-1 mAbs). The
the 105 mAbs in our panel for which we could detect signal at published 4-8, 4A8, and COVA1-22 mAbs (Brouwer et al.,
1 mg/mL. By adding a biotinylated version of each mAb together 2020; Chi et al., 2020; Liu et al., 2020), which have been shown
with excess of each of the other mAbs individually into ELISA to bind the NTD, compete with each other and map to NTD-1.
plates pre-coated with pre-fusion-stabilized SARS-CoV-2 S NTD-2 mAbs cluster distinctly from NTD-1, indicating minimal
(Wrapp et al., 2020), we could detect competition of mAbs with spatial overlap of these two NTD regions. One NTD-1 mAb

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Figure 2. Seven recurrently targeted major epitopic regions

(A) Cross competition matrix for 73 S+ mAbs with affinity sufficient for detection by ELISA. Blocking mAbs (columns) added at 100 mg/mL; detection antibodies
(rows) at 1 mg/mL. Intensity of color shows strength of blocking, from 0 signal (complete blocking) to 70% full signal (top gradient at right of panel: orange).
Hierarchical clustering of mAbs by cross competition into seven groups (plus a singleton labeled S2-3), enclosed in square boxes, with designations shown and in
colors from dark blue (NTD-1) to dark red (S2-3). Green arrows on the left designate mAbs newly reported here. The lower parts of the panel show competition of
blocking mAbs with soluble, human ACE2 (second gradient at right: dark red); log10 (IC50) (IC50 unit mg/mL) in pseudovirus (614D S) neutralization assay (third
gradient at right: violet); area under the curve for ELISA binding (ELISA AUC: brown); and binding (ELISA) to recombinant domains and heterologous S (green).
(B) Competition in cell-based assay for 36 mAbs with binding in ELISA format too weak for reliable blocking measurement (rows). Blocking mAbs (columns)
selected from each of the seven clusters in the ELISA assay. Strength of blocking shown as intensity of orange color, as in (A).
(C) Distribution of a mAbs from two individual subjects (expressed as percent of sequence pairs recovered from that subject) into the seven principal clusters, plus
a non-assigned (unknown) category (unk) and S2-3. Top row shows mAb distribution from all subjects and the bottom row shows the distribution of all other
individuals minus C12 and G32.
See also Figure S2.

(C81H11) competed strongly with mAbs from S2-1, and a sec- bound MERS S. Thus, S2-1 mAbs appear to recognize a region
ond could be assigned on the basis of competition either to of S2 conserved among SARS-CoV-2, SARS-CoV, MERS,
NTD-1 or to S2-1, suggesting structural adjacency of at least HKU1, and OC43, as shown previously for S2 mAbs (Ng et al.,
some sites in these two clusters (Figure S2A). Several segments 2020; Song et al., 2020). Isolation of a single mAb (C12B3) that
of S2 are in contact with either RBD or NTD, some with differen- bound S2 but did not map to any of the seven major clusters sug-
tial exposure depending on whether the RBD is ‘‘up’’ or ‘‘down.’’ gests that the immune system may target additional regions of
Inclusion of common mAbs shared among studies reveals S2 but that those responses are subdominant.
how epitopic regions determined here directly by analysis of
the competition matrix relate to other classifications, particularly Memory B cells dominant across individuals in natural
for RBD. For instance, RBD-2 roughly corresponds to recently infection
described class 1 and class 2 (Barnes et al., 2020), RBS-A, B, We probed the relative distribution of epitopes recognized by
and C mAbs (Yuan et al., 2021), and RBD-A (Rogers et al., 2020). SARS-CoV-2-specific memory B cells in the population repre-
Thirty-six mAbs in the ELISA competition analysis cross-re- sented by our cohort by ELISA-based and cell-surface-based
acted with SARS-CoV. These mapped mostly to the RBD-1 (11 assays. We clustered all the S+ mAbs (Figure 2) and S+RBD-
mAbs) and S2-1 (17 mAbs) clusters. Four mAbs that mapped mAbs from a separate sorting step (Figures S2B and S2C). In
to S2-1 (C15C3, C7A4, C7A9, and G32Q1) also bound the com- the former set, comprising 73 mAbs that bound strongly enough
mon cold b-CoVs, and two of these (C7A9 and C15C3) also for the ELISA competition assay, the order of epitopic region

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Figure 3. Spatial distribution of the major
RBD and NTD epitopic regions
Surface regions of the SARS-CoV-2 S trimer con-
tacted by mAbs in four of the seven principal clus-
ters, according to the color scheme shown, with a
representative Fab for all except RBD-3. The
C81C10 Fab defines an epitope just outside the
margin of NTD-1, but it does not compete with any
antibodies in RBD-2. The RBD-2 Fv shown is that of
C121 (PDB ID: 7K8X: Barnes et al., 2020), which fits
most closely, of the many published RBD-2 anti-
bodies, into our low-resolution map for C12A2. Left:
views normal to and along threefold axis of the
closed, all-RBD-down conformation; right: similar
views of the one-RBD-up conformation. C121
(RBD-2) can bind both RBD down and RBD up;
G32R7 (RBD-1) binds only the ‘‘up’’ conformation of
the RBD. The epitopes of the several published
RBD-3 antibodies are partly occluded in both
closed and open conformations of the RBD; none
are shown here as cartoons. A cartoon of the poly-
peptide chain of a single subunit (dark red) is shown
within the surface contour for an S trimer (gray).

Spatial distribution of the major RBD

and NTD epitopic regions
We included in the competition assays
mAbs for which published structures
show their interaction with S. By cryo-
EM, we determined structures of Fab frag-
ments of six mAbs from the RBD-1, RBD-
2, and NTD-1 clusters bound with S ecto-
frequencies was RBD-1 (27.4%), S2-1 (19.2%), NTD-1 (17.8%), domain, to fill gaps in the representation of mAbs from those
RBD-2 (15.1%), and NTD-2 (8.2%) (Figure 2A). There were 36 clusters in published work. Two of those reconstructions are at
more mAbs that had insufficient affinity for ELISA competition relatively high resolution (those of Fab C12C9 in NTD-1, and
but that bound cell surface SARS-CoV-2 S (Figures 2B and Fab G32R7 in RBD-1), three (C12A2 and C93D9 in RBD-2, and
S3A). We mapped ELISA-insufficient mAbs to the seven clusters C81C10 at the periphery of NTD-1) at intermediate resolution,
using the cell-based assay. We used 20 mAbs (2 RBD-3, 4 RBD- and one (C12C11 in NTD-1) at much lower resolution.
2, 4 RBD-1, 2 NTD-2, 4 NTD-1, 2 S2-1, and 2 S2-2) from the RBD-1
ELISA-mapped competition clusters as blocking mAbs, a non- The complex with Fab G32R7 (Figure 3) has three RBDs in the
COVID-19-related blocking antibody as negative control, and ‘‘up’’ configuration, each bound with a Fab. The epitope is part
self-blocking as positive control (Figures 2B and S3A). We of the RBD surface that faces outward in the ‘‘down’’ configura-
used an additional 118 mAbs (distributed across the seven clus- tion of the domain, but interference of the bound Fab with the
ters) to map the 13 mAbs that failed to be mapped clearly with NTD of the anticlockwise neighboring subunit (as viewed from
the initial 20 (Figure S3B). above the S) would prevent binding to a down-oriented RBD.
Cell-based competition showed that mAbs with affinities too The connection of the RBD allows a range of orientations for
low to test by ELISA mapped primarily to S2 and to the NTD (Fig- the up configuration, and association with the G32R7 Fab does
ures 2B and S2C). Analysis of ELISA-mapped mAbs that were not fix the orientation of the RBD, blurring density in a 3D recon-
also mapped with the cell-based competition assay showed struction of the intact S. Local refinement of an RBD-Fab subpar-
that the two assays were consistent (Figure S3C) and suggested ticle then yielded a map with well-defined features at the
that cell-surface binding extended the dynamic range of the interface (Figure 3; Data S3 and S4). RBD contacts are all with
ELISA competition assay to include less tightly binding mAbs, the H-chain variable domain (VH), principally CDRH2, framework
justifying use of the combined competition results in subsequent residues in the C, D, and E strands, and CDRH3. The unusually
analyses. This combined approach showed that frequencies of long CDRH3 (24 residues) also interacts with three glycans—
cluster-targeting mAbs from the two individuals that contributed one on RBD Asn343 and the others on NTD Asn122 and
the most clones (C12 and G32) were largely similar to the aggre- Asn165. Although VH approaches the NTD closely enough to
gate of all others (Figure 2C). In addition, competition analysis of interact with the glycans, we could identify just one likely
purified polyclonal IgGs indicated that samples from two blood additional contact with an NTD side-chain (Phe157). S309, an
draws one month apart had similar principal targets (Figure S3D). RBD-1 neutralizing antibody isolated from a convalescent

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Figure 4. Distribution of pseudovirus neutralization potency in each competition cluster

Both IC50 (A and C) and IC80 (B and D) shown for infection in two different cell lines. (A) and (B) pair: 293FT cells expressing hACE2 and TMPRSS2. (C) and (D) pair:
TZM.bl cells expressing hACE2. Color gradient indicates frequency of the clones in each cluster that have the neutralization potency shown by the vertical scale.
See also Figure S4.

SARS-CoV donor that also neutralizes SARS-CoV-2 (Pinto et al., NTD-2

2020), has contacts with the RBD that do not overlap those of We have no structures of NTD-2 mAbs bound with S, but from
G32R7, but the L chain of the latter would collide with it. non-competition with NTD-1, insensitivity to NTD loop deletions
RBD-2 and RBD-3 (see below), and exposure of NTD surfaces on the trimer, we
Most potently neutralizing mAbs cluster in RBD-2; many pub- suggest that the NTD-2 epitopes may be on one of the lateral
lished structures show modes of antibody binding within this faces of the NTD.
group (Barnes et al., 2020; Piccoli et al., 2020). Their epitopes
include various parts of the ACE2 binding site (i.e., the RBM) Neutralizing function maps largely to RBD-2 and NTD-1
at one apex of the domain. For the mAbs characterized clusters
here, low-resolution structure of Fab C12A2 showed that its Using two different pseudovirus assays, we determined neutral-
epitope was essentially identical to that of published antibody ization by mAbs from each of the seven clusters and found
2-4 (Data S5) (Liu et al., 2020). The same IGVH encodes the H neutralizing mAbs in five of the seven clusters (RBD-1, -2, and
chains of both mAbs, and the L-chain genes are closely -3, and NTD-1 and NTD-2) (Figure 4). The most potent were in
related (overall amino-acid sequence identity). They contact RBD-2, as expected from their co-clustering with known strong
the slight concavity in the center of the RBM, the site for neutralizers such as REGN10987 and REGN10933, which are
most of the neutralizing mAbs represented by structures in used as a mAb drug cocktail (Baum et al., 2020), and from
the PDB. The probably immunosubdominant RBD-3 class in- many previous reports (Brouwer et al., 2020; Ju et al., 2020;
cludes several antibodies for which published structures are Liu et al., 2020). Among Abs from that cluster, 52%–58%
available; we included CR3022, an antibody originally isolated neutralized with IC50<0.1 mg/mL and 29%–35% with
from a SARS-CoV convalescent subject that cross-reacts with IC80<0.1 mg/mL (Figure 4). Alternate SARS-CoV-2 S pseudo-
SARS-CoV-2 (ter Meulen et al., 2006). Its epitope on the RBD typed virus neutralization protocols showed similar results (Fig-
is nearly opposite that of G32R7 (Figure 3), in an epitopic ure 4). The strongest of the RBD-1 mAbs had IC50 in the range
region partly occluded in the down configuration of the RBD of 1 mg/mL; those in RBD-3 were, in general, much weaker.
previously referred to as a cryptic supersite (Piccoli There is strong correlation between pseudotyped and authentic
et al., 2020). virus (Figure S4). NTD-1 mAbs appeared more sensitive to the
NTD-1 neutralization assay used, but a few, such as C12C9, ap-
NTD-1 cluster includes mAbs with neutralizing activity, including proached or exceeded the strongest in RBD-1 (Figure S4A).
C12C9 and C12C11. The latter, judging from the low-resolution NTD-2 mAbs were, in general, less potent than NTD-1 mAbs,
map (Data S5), appears to have a footprint that coincides with and none of the S2 mAbs neutralized infection, with the possible
that of the published 4A8 antibody (PDB ID: 7C2L; Chi et al., exception of very weak neutralization by G32C5 (IC50 of
2020). Like the G32R7 complex, the C12C9 complex also 22 mg/mL) (Figure S4C).
required local subparticle refinement to yield a map interpretable
at the level of side-chain contacts at the Fab-NTD interface. Its High diversity and some sequence convergence in
footprint overlaps that of 4A8, but it is displaced slightly toward competition clusters
the threefold axis of the S trimer. Both mAbs have principal con- Variable region exons of IgH and IgL genes are each assembled
tacts in two NTD surface loops, residues 140–160 and 245–260. by V(D)J recombination from a diversity of gene segments.
The C81C10 mAb, which we have grouped in NTD-1 but which Preference of VH gene segment usage frequencies differed
competes with only two of the most weakly binding members among the seven mAb clusters (Figure 5A). Enrichment for
of that cluster, appears to represent a distinct and potentially VH3-53, previously reported to be associated with SARS-
subdominant subset. An 8-Å resolution structure (Data S5) CoV-2 S (Yuan et al., 2020), was exclusively within the RBD-2
bound with S trimer shows that its epitope is at the bottom of group. VH3 family mAbs are particularly abundant in all
the NTD, well displaced from the epitopes of C12C9, C12C11, the clusters. VH3-30 and VH3-30-3, which have average fre-
and 4A8. quency in the general human repertoire of 5.4% and 1.3%,

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Figure 5. High diversity and some sequence convergence in competition clusters

(A) IgH VH gene segments of the 167 mAbs characterized by binding SARS-CoV-2 S in either ELISA or cell-surface expression. Inner ring indicates VH family; the
outer ring indicates specific VHs. PBMC repertoire is from 350 million reads of deep sequencing (Briney et al., 2019). S binders include 167 clones in Data S2. *p <
0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Bonferroni correction. Red asterisks: comparing to S binders; black asterisks: comparing to a non-selected B cell
repertoire from PBMCs.
(B) Maps of pairwise distances of CDRH3 (lower left triangle) and CDRL3 (upper right triangle) for the NTD-2 and S2-1 cluster mAbs from (A). Antibodies in both
clusters arranged by VH usage. Clones converging on identical VH/VL and closest distance of CDRL3 from the same cluster are shown. Pairwise distances
analyzed by MEGA X. Intensity of color shows the distance from 0 (identical) to 1 (no identity). Sequence alignment for the mAbs from the indicated clusters with
identical VH and VL and similar CDR3s. Differences in CDR3s from the reference sequence (bold) are in red; dashes indicate missing amino acids; dots represent
identical amino acids.
(C) Summary of convergent sequences of anti-SARS-CoV-2 S and RBD antibodies from independent datasets. Ig sequences derived from binding to DIII of Zika
virus E protein, and HA of influenza virus H1N1 were used as control datasets. Convergent sequences had identical VH and VL and >50% identity in CDRH3
and CDRL3.
(D) Representative convergent clones from different individuals and independent datasets from (C).
See also Figure S5.

respectively, account between them for over 30% of the mAbs chains of ‘‘natural antibodies,’’ also associates strongly with
in RBD-1, and for 16 of the 19 mAbs in S2-2. The VH1 and VH4 polyreactivity. VH and VL somatic mutation levels were gener-
families are co-dominant with VH3 in NTD-1 and NTD-2, ally, but not significantly, greater in S2-1 (Figure S5A)
respectively (Figure 5A). VH1-69-encoded mAbs are enriched IgH and IgL variable regions each have three complementary
in S2-1, which contains most of the cross-reactive mAbs to determining regions (CDRs), which are the principal contact sites
other CoVs. VH1-69-encoded mAbs are frequently observed for antigen. CDRs 1 and 2 for H and L chain are encoded within
in antiviral responses to influenza virus, HCV, and HIV-1 the VH and VL gene segments, respectively. Diverse, non-tem-
(Chen et al., 2019), and previous work reported that SARS- plated sequences produced by VDJH junctions encode CDRH3
CoV-2 S-specific mAbs isolated from SARS-CoV-infected pa- regions, which have dominant roles in most Ab-antigen interac-
tients also showed an enriched VH1-69 gene segment usage tions. CDRL3 is also diverse due to VJL junctional heterogeneity,
(Wec et al., 2020). VH1-69, which is well represented in H but it has fewer non-templated sequence additions. Intracluster

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Figure 6. Influence of mutations found in var-
iants of concern on binding and neutraliza-
tion by human mAbs
(A) Positions of mutations in amino acid sequences
of B.1.1.7, B.1.351 and P.1. Left panel: RBD (gray
backbone cartoon) and Fvs representing each of the
three RBD clusters (green, yellow, and orange
backbone cartoons for RBD-1, -2, and -3, respec-
tively). Red spheres show side chains at positions of
RBD mutations N501Y (found in all three variants of
concern), E484K, and K417N/T (found in B.1.351
and P.1). Right panel: NTD (gray backbone cartoon)
and Fvs representing the NTD-1 cluster (blue
backbone cartoon) and the C81C10 non-neutral-
izing antibody (cyan backbone cartoon). Spheres
show side chains at positions of mutations in B.1.1.7
(yellow), B.1.351 (orange), and P.1 (red). One NTD
substitution, L18F (brown), is in both B.1.351 and
P.1. Although D242-244 is a deletion within a b
strand, its effect will be to reconfigure the 248–260
loop (orange asterisk), as residues 245–247 will shift
into the positions of the deleted residues in the
strand. See also Figure S6.
(B) Heatmap showing binding of 119 mAbs to
Nextstrain cluster 20A.EU1 (A222V), Danish mink
variant (D69-70 and Y453F), B.1.1.7 (D69-70, D144,
N501Y, A570D, P681H, T716I, S982A, and
D1118H), B.1.351 (L18F, D80A, D215G, D242-244,
K417N, E484K, N501Y, and A701V), and P.1 (L18F,
T20N, P26S, D138Y, R190S, K417T, E484K, N501Y,
H655Y, T1027I, and D1176F) (top), and NTD dele-
tion variants (bottom). Variants include D614G.
Relative binding intensities of the tested mAbs for
each variant are shown in shades of blue.
(C) Heatmap showing neutralization potency of 119
mAbs to D614G, B.1.1.7, B.1.351, and P.1. Log10
transformed IC50 shown in shades of dark red. IC50,
mg/mL.
See also Figure S6.

mAb CDR3 sequence comparisons showed little sequence sim- Finally, we note that antibody C93D9 represents a striking
ilarity (Figure S5B). NTD-2 contained a subcluster of identical example of structural convergence (Figures S5C and S5D). All
CDRL3 sequences that were associated with the same VH but two of the 20 mAbs from the literature shown in Figure S5E
and VL segments from two different individuals (C81 and have the same VH and a non-random selection of VL but diver-
C12) (Figure 5B). S2-1 had a small subcluster of CDRH3 and gent CDRH3 sequences and lengths. Nonetheless, all 20, as
CDRL3 sequence similarities from five different study partici- well as C93D9, bind the RBM in almost identical poses—consis-
pants (C83, C102, C163, C12, and C53) (Figure 5B). These tent with germline-encoded CDRs as the principal binding con-
data indicate substantial intracluster CDR3 diversity with rare tacts (Yuan et al., 2020).
instances of CDR3 sequence similarity between different
individuals. Escape from RBD-2 and NTD-1 mAbs by viral variants
We also asked whether we could find evidence of sequence Emergence of SARS CoV-2 variants that enhance transmissi-
convergence with other COVID-19 datasets for which paired bility, such as the variant B.1.1.7 (Rambaut et al., 2020), and in
IgH and IgL sequence data are available. Convergent sequence some cases reduce the neutralization titers of convalescent
criteria of (1) same VH and VL, and (2) no less than 50% CDRH3 sera, such as the variant B.1.351 (Tegally et al., 2021), indicates
and CDRL3 identity (Croote et al., 2018), revealed rare se- more rapid evolution of the virus than expected from the error-
quences very similar to representatives from RBD-1, RBD-2, correcting properties of CoV RNA-dependent RNA polymerases.
and NTD-1 in independent datasets for SARS-CoV-2 (Kreer In the case of B.1.351 in particular, the clusters of three substitu-
et al., 2020; Liu et al., 2020; Robbiani et al., 2020), but not for tions and one deletion in the NTD and three substitutions in the
mAbs against Zika (Robbiani et al., 2017) or influenza (Wrammert RBD concentrate at contacts of the most potent of the many
et al., 2011) viruses (Figures 5C and 5D). We also found conver- well-characterized neutralizing mAbs (Figure 6A). Moreover,
gent pairs within our own dataset representing both S2-1 and recurrent deletions in loops of the NTD appear to accelerate
S2-2 (Figure 5D). SARS CoV-2 antigenic evolution (McCarthy et al., 2021).

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We examined the effects of naturally occurring mutant S pro- the pandemic. Immune responses in these SARS-CoV-2-naive
tein on binding of mAbs in each competition group (Figures 6B donors were to early and relatively homogeneous variants circu-
and 6C). B.1.1.7 had lower affinity for various mAbs in the lating well before emergence of the B.1.1.7 and B.1.351 strains
RBD-1 and NTD-1 cluster. None of the RBD-1 mAbs lost binding first reported in December 2020, and probably before the spread
completely, and testing a variant with just the deletion at position in New England of the D614G variant that did not substantially
144 in the NTD showed that this single mutation caused loss of alter antigenicity (Muik et al., 2021; Xie et al., 2021). This set of
binding by nearly two-thirds of the mAbs in the NTD-1 cluster. BCR sequences and corresponding mAbs thus represents
Mutations in B.1.351 S had more pronounced effects, particu- responses to a relatively homogeneous infectious virus and pro-
larly on mAbs in the RBD-2 and NTD-1 clusters, as expected vides a valuable tool for examining the degree to which these an-
from the positions of the sequence changes. In addition to the tibodies retain recognition of emerging variants and for studying
N501Y substitution also present in the variant B.1.1.7, an the extent to which loss of neutralizing titer correlates with loss of
E484K mutation lies at the center of the epitope for many of longer-term protection.
the most potent RBD-2 neutralizing mAbs (Figure 6A). About Finding that the same regions of S are targeted across multiple
one-third of the RBD-2 mAbs retained modest to high affinity, individuals may confer pressure for viral escape. This may be a
but the variant S failed to bind any of the NTD-1 cluster downside of what appears to be a consistent and robust repre-
(excluding C81C10—a peripheral NTD-1 member as discussed sentation of germline antibodies against neutralizing SARS-
above), with the marginal exception of 4A8. The P.1 variant CoV-2 targets in the human antibody repertoire. The fact that
escaped RBD-2 mAbs in a manner mirroring B.1.351, but with mAbs that lose reactivity to emerging variants tend to be mem-
much less escape from NTD-1 mAbs (Figures 6B and S6A). bers of the most potent neutralizing mAb clusters is consistent
mAbs in S2-related groups were not affected by B.1.1.7, with in vivo protection by these antibody groups.
B.1.351, or P.1, despite the presence of some mutations within The competition clusters we have identified are roughly anal-
S2. One exception is the singleton S2-3 mAb C12B3, an S2 ogous to genetic complementation groups. Competition can
binder that loses binding to B.1.1.7 (Figure 6B). Loss of neutral- result from overlapping binding footprints or non-overlapping
ization potency tracked with loss of binding (Figures 6B, 6C, but neighboring footprints that lead to mutual exclusion of IgGs
S6B, and S6C). bound at the two adjacent epitopes. Competition can also result
Differential effects on antibodies with overlapping but still from stabilization by one antibody of a conformation (e.g., the
distinct epitopes illustrate the potential importance of a redun- up-down conformational isomerism of the RBD) that excludes
dant, polyclonal response. Although C12C9, C12C11, and 4A8 or lowers affinity of the other. Any of these mechanisms may
all contact the 140–160 loop (Figure 3) and all are sensitive to contribute to the clusters we have mapped, but the outcome in
the recurrent multi-position deletions at D141-144 and D243- all cases is an apparent redundancy of binding capacity in a
244, only the latter two are sensitive to the recurrent single-posi- broadly polyclonal response that may nonetheless impart recog-
tion deletions at 144 or 146 (Figures 6B and S6A). Moreover, nition breadth toward an evolving pathogen within a single
although they are in the same convergent structural class whose individual.
members bind the RBM in nearly identical poses (Figures S5C Complementary recognition of non-overlapping viral targets
and S5D), CC12.1 (Yuan et al., 2021) nearly fails to recognize by non-competing antibodies in the repertoire can reduce the
B.1.351 (7%) while C93D9 retains some marginal affinity likelihood of viral escape (Keeffe et al., 2018). Our data suggest
(29%) (Figure 6B). Thus, apparently redundant memory B cell an additional mechanism for preventing viral escape: competing
clones can have non-redundant functional roles. antibodies may help retain recognition of a rapidly evolving anti-
gen by their differential sensitivity to specific mutations. The po-
DISCUSSION tential dynamic reach of otherwise redundant mAb recognition,
illustrated by selective retention of affinity for variants by some
Our results illustrate the landscape of memory B cell coverage of antibodies within a cluster but not by others, may give selective
the SARS-CoV-2 S glycoprotein in convalescent donors. Unlike advantage to immune mechanisms that yield multiple competing
the terminally differentiated plasma cells that determine the pro- antibodies to critical epitopes, as those that retain adequate af-
file of serum antibodies, memory B cells clonally expand upon finity can then re-activate, expand, and potentially undergo
re-exposure to antigen, some differentiating into fresh anti- further affinity maturation. The presence of some antibodies
body-secreting cells and others re-entering GCs to undergo that retain cross-strain neutralizing activity suggests that protec-
further SHM-mediated diversification and affinity maturation. tion from variants may depend upon robustness of B cell re-
These outcomes offer a layer of flexibility for adaptation to drifted sponses to parent S. Escape from neutralization is a likely feature
or related viral strains, if available secreted antibodies fail to pre- of variant emergence. It will be important to determine the de-
vent initial infection. Loss of protection against overt or severe gree to which antibodies with retained affinity for immunodomi-
disease is not an inevitable consequence of a waning serum anti- nant neutralizing S protein targets influence protection from
body titer. This atlas of B cell memory therefore maps, systemat- variant-driven clinical disease.
ically, a crucial component of the long-term immune response to
SARS-CoV-2 infection. Limitations of study
The donors for this study experienced COVID-19 symptom The participants in this study suffered from mild COVID-19.
onset between March 3 and April 1, 2020, and blood draws While this represents the majority of cases, it will be important
analyzed here were between April 2 and May 13, 2020, early in for future studies to examine the degree to which antigen

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recognition may differ in cases of more severe disease. In addi- and Prevention), and SARS-CoV-2, Isolate hCoV-19/South Africa/KRISP-
tion, our study only includes adults. Inclusion of children in future K005325/2020, NR-54009 (contributed by Alex Sigal and Tulio de Oliveira).
The SARS-CoV-2 S model in the graphical abstract is from PBD (ID:6XR8).
studies will illuminate how SARS-CoV-2 S recognition may differ
in this age group.
AUTHOR CONTRIBUTIONS

STAR+METHODS D.R.W. designed the study. P.T. and A. Gautam conducted ELISA and FACS
experiments. I.W.W., S.J., and G.B. conducted cryo-EM experiments. P.T.,
M.T., A. Gautam, Y. Chen, A.Z., T.Z., and J.L. performed single-cell sorting
Detailed methods are provided in the online version of this paper
and antibody cloning. P.T. and J.F. prepared yeast expression constructs.
and include the following: P.T., K.R.M., W.P.D., L.J.R., and Y. Cai prepared S variant expression con-
structs. P.T., A. Gautam, I.W.W., G.B., N.G., N.B.W., D.N., S.C.H., and
d KEY RESOURCES TABLE D.R.W. analyzed data. S.H., M.T., F.J.N.L., and A. Gautam recruited patients
d RESOURCE AVAILABILITY and processed samples. P.T., C.L.L., M.T., and M.S.S. performed pseudovirus
B Lead contact neutralization assays and analysis. L.G.A.M., A.N.H., L.E.M., N.S., and A. Grif-
B Materials availability fiths performed authentic virus neutralization assays and analysis. M.T.,
B Data and code availability I.W.W., E.A.M., G.B., J.F., B.M.H., A.G.S., Y. Cai, and B.C. contributed recom-
binant protein. N.G., N.B.W., and P.T. prepared the first draft. S.C.H. and
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
D.R.W. supervised research and wrote the paper.
B Human study participants
d METHOD DETAILS DECLARATION OF INTERESTS
B SARS-CoV-2 S-specific single B cell sorting
B Antibody cloning and production The authors declare no competing interests.
B Monoclonal antibody screening with ELISA
B Cell surface binding assays Received: March 23, 2021
B ELISA-based antibody competition Revised: June 14, 2021
Accepted: July 15, 2021
B Cell-based antibody competition
Published: July 23, 2021
B Antibody binding to S variants
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Goat anti-human IgG-alkaline Southern Biotech Cat# 2040-04; RRID:AB_2795643
phosphatase (AP)
Streptavidin-alkaline phosphatase BD Biosciences Cat# 554065, RRID:AB_10053566
Human CD19 MicroBeads Miltenyi Biotec Cat# 130-050-301, RRID:AB_2848166
Anti-flag-APC BioLegend Cat# 637307; RRID:AB_2561496
Anti-flag-PE BioLegend Cat# 637310; RRID:AB_2563148
Anti-His-PE BioLegend Cat# 362603, RRID:AB_2563634
Anti-human-IgG-PerCP-Cy5.5 BioLegend Cat# 410710; RRID:AB_2565788
Anti-human-CD27-APC-Cy7 BioLegend Cat# 356424; RRID:AB_2566773
Anti-human-CD19-BV510 BioLegend Cat# 302242; RRID:AB_2561668
Anti-human-IgD-FITC BioLegend Cat# 348206; RRID:AB_10612567
Anti-human-IgM-BV605 BioLegend Cat# 314524; RRID:AB_2562374
Anti-human-IgG- Alexa Fluor 647 Thermo Fisher Scientific Cat# A-21445, RRID:AB_2535862
anti-c-Myc IgY Thermo Fisher Scientific Cat# A-21281, RRID:AB_2535826
DyLight 649 Streptavidin BioLegend Cat#405224
goat anti-chicken IgG Alexa Fluor 488 Thermo Fisher Scientific Cat# A-11039, RRID:AB_142924
Bacterial and virus strains
E.coli TOP10 Thermo Fisher Scientific Cat# C404003
E.coli Stbl3 Thermo Fisher Scientific Cat# C737303
SARS-CoV-2, isolate USA-WA1/2020 University of Texas Medical Branch N/A
SARS-CoV-2, Isolate hCoV-19/USA/ BEI Resources NR-54011
CA_CDC_5574/2020
SARS-CoV-2, Isolate hCoV-19/South Africa/ BEI Resources NR-54009
KRISP-K005325/2020
Biological samples
COVID-19 convalescent PBMC This paper N/A
Pre-COVID19 PBMC (collected before This paper N/A
10/01/2019)
Chemicals, peptides, and recombinant proteins
SARS-CoV-2 S (sorting) GeneScript Cat# Z03481
SARS-CoV-2 S (ELISA) Obtained from the lab of Dr. Bing Chen N/A
SARS-CoV-2 RBD Obtained from the lab of Dr. Aaron Schmidt N/A
SARS-CoV-2 S2 Sino Biological Cat# 40590-V08B
SARS-CoV-2 NTD Sino Biological Cat# 40591-V49H
SARS-CoV RBD Obtained from the lab of Dr. Aaron Schmidt N/A
MERS RBD Obtained from the lab of Dr. Aaron Schmidt N/A
HKU1 RBD Obtained from the lab of Dr. Aaron Schmidt N/A
SARS-CoV S Sino Biological Cat# 40634-V08B
MERS S Sino Biological Cat# 40069-V08B
HKU1 S Sino Biological Cat# 40606-V08B
OC43 S Sino Biological Cat# 40607-V08B
NL63 S Sino Biological Cat# 40604-V08B
229E S Sino Biological Cat# 40605-V08B
Human ACE2 Obtained from the lab of Dr. Bing Chen N/A
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Bovine serum albumin (BSA) Sigma Cat# A2153
Triton X-100 Fisher Scientific Cat# 9002-93-1
10x phosphate buffered saline (PBS) Boston BioProducts Cat# BM-220-10XS
Glycine Sigma-Aldrich Cat# G7126
Zinc chloride Sigma-Aldrich Cat# 793523
Magnesium chloride Sigma-Aldrich Cat# M0250
Tween-20 Sigma-Aldrich Cat# P1379
Ficoll-Paque PLUS Cytiva Cat# 17144003
4’,6-diamidino-2-phenylindole, Thermo Fisher Scientific Cat# D1306
dihydrochloride (DAPI)
Dithiothreitol (DTT) Thermo Fisher Scientific Cat# R0861
RNaseOUT Thermo Fisher Scientific Cat# 10777-019
Random hexamer primer Thermo Fisher Scientific Cat# FERSO142
10 mM dNTPs Promega Cat# U1515
IGEPAL CA-630 Sigma-Aldrich Cat# I8896
SuperScript III reverse transcriptase Thermo Fisher Scientific Cat# 18080085
HotStarTaq DNA polymerase QIAGEN Cat# 203205
Protein A agarose beads Thermo Fisher Scientific Cat# 20334
Papain resin Thermo Fisher Scientific Cat# 20341
Talon cobalt resin Takara bio Cat# 635503
Strep-Tactin Sepharose resin IBA Life Sciences Cat# 2-1201-010
4-20 % Tris-Glycine SDS-PAGE Thermo Fisher Scientific Cat# XP04200BOX
1M Tris-HCl (pH8.0) Boston BioProducts Cat# BBT-80-500
Gentian Violet RICCA Chemicals Cat# 3233-4
Deposited data
Primary data This paper Mendeley Data: https://doi.org/
10.17632/tfvsfg47c7.1
Heavy and light chain sequences This paper GenBank:
MW718328-MW718631
Atomic structure This paper PDB:
7N62 and 7N64
Cryo-EM structure This paper EMDB:
EMD-24192 to EMD-24194 and
EMD-24196 to EMD-24198
Critical commercial assays
Fab preparation kit Thermo Fisher Scientific Cat# 44985
Lipofectamine 3000 transfection reagent Thermo Fisher Scientific Cat# L3000008
ExpiFectamine Kit Thermo Fisher Scientific Cat# A14525
ONE-Glo luciferase assay system Promega Cat# E6120
EZ-Link Sulfo-NHS-LC-Biotin Thermo Fisher Scientific CAT# 21338
Experimental models: Cell lines
HEK293T ATCC Cat# CRL-3216
VERO C1008 (E6) BEI Resources Cat# NR-596
EBY100 S. cerevisiae Strain Thermo Fisher Scientific Cat# C83900
ACE2/TMPRSS2-expressing HEK293T Obtained from the lab of Dr. Marc Johnson N/A
Expi293F Thermo Fisher Scientific Cat# A14527
ACE2-expressing TZM.bl Obtained from the lab of N/A
Dr. Michael S. Seaman
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Oligonucleotides
Primer set for Ig PCR (Tiller et al., 2008) IDT DNA N/A
Recombinant DNA
pCHA vector Obtained from the lab of Dr. K. Dane Wittrup N/A
pmaxGFP Lonza Cat#: VDC-1040
Plasmid encoding 2p Obtained from the lab of Dr. Jason McLellan N/A
Plasmid encoding hexapro Obtained from the lab of Dr. Jason McLellan N/A
HDM-SARS2-spike-delta21-D614G Addgene Cat# 158762
HDM-SARS2-spike-D69-70-delta21-D614G Obtained from the lab of Dr. W. Paul Duprex N/A
HDM-SARS2-spike-D141-144-delta21-D614G Obtained from the lab of Dr. W. Paul Duprex N/A
HDM-SARS2-spike-D144-delta21-D614G Obtained from the lab of Dr. W. Paul Duprex N/A
HDM-SARS2-spike-D146-delta21-D614G Obtained from the lab of Dr. W. Paul Duprex N/A
HDM-SARS2-spike-D210-delta21-D614G Obtained from the lab of Dr. W. Paul Duprex N/A
HDM-SARS2-spike-D243-244-delta21-D614G Obtained from the lab of Dr. W. Paul Duprex N/A
HDM-SARS2-spike-A222V-delta21-D614G This paper N/A
HDM-SARS2-spike-D 69-70 Y453F- This paper N/A
delta21-D614G
HDM-SARS2-spike-B.1.1.7-delta21-D614G This paper N/A
(Pseudovirus)
HDM-SARS2-spike-B.1.351-delta21-D614G This paper N/A
(Pseudovirus)
HDM-SARS2-spike-P.1-delta21-D614G This paper N/A
(Pseudovirus)
Plasmid encoding B.1.1.7 (Flow cytometry) Obtained from the lab of Dr. Bing Chen N/A
Plasmid encoding B.1.351 (Flow cytometry) Obtained from the lab of Dr. Bing Chen N/A
Plasmid encoding P.1 (Flow cytometry) Obtained from the lab of Dr. Bing Chen N/A
HDM-SARS2-Spike-delta21 Addgene Cat#155130
pLenti CMV Puro LUC (w168-1) Addgene Cat#17477
psPAX2 Addgene Cat#12260
Software and algorithms
GraphPad Prism 8 and 9 GraphPad Software https://www.graphpad.com/scientific-
software/prism/
IgBLAST NCBI https://www.ncbi.nlm.nih.gov/igblast/
Clustal Omega EMBL-EBI https://www.ebi.ac.uk/Tools/msa/
clustalo/
R Free Software Foundation/GNU R v4.0.0
Bioconductor software in R Bioconductor https://www.bioconductor.org/
MEGAX Molecular Evolutionary Genetics Analysis https://www.megasoftware.net/
FlowJo 9.9.6 and 10.7.1 BD https://www.flowjo.com/
UCSF Chimera UCSF https://www.cgl.ucsf.edu/chimera/

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Duane R.
Wesemann. [email protected]

Materials availability
Reagents and materials presented in this study are available upon request, in some cases after completion of a material transfer
agreement.

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Data and code availability

The atomic models have been deposited in the RCSB Protein Data Bank (PDB) under the accession numbers 7N62 (for C12C9) and
7N64 (for G32R7), and the electron microscopy maps have been deposited in the Electron Microscopy Data Bank (EMDB) under the
accession numbers EMD-24192 to EMD-24194 (for C12C9, G32R7, C12C11) and EMD-24196 to EMD-24198 (for C93D9, C81C10
and C12A2). Sequences of the monoclonal antibodies characterized are deposited in GenBank, accession numbers: MW718328 -
MW718631.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Human study participants

The study and protocol were approved by Partners Institutional Review Board. Volunteers aged 18 and older with a history of COVID-
19 were enrolled between March and May 2020. COVID-19 was diagnosed by a healthcare professional based on symptoms and a
positive nasopharyngeal swab RT-PCR test except for G32, who was diagnosed by an antibody test. Participants self-reported data
for body-mass index (BMI), symptom onset and recovery dates and self-rated the severity of their COVID-19 symptoms on a 1-10
scale, with 1 describing very mild symptoms and 10 describing very severe symptoms. Blood samples were collected at least 2 weeks
after symptom resolution. Symptom duration is the time between symptom onset and recovery dates. Detailed information about the
cohort is in Data S1. BMI was calculated as participant’s weight in kilograms divided by the square of their height in meters. Blood
samples were processed within 4 h of sample collection. PBMCs and plasma samples were isolated by density gradient centrifuga-
tion with Ficoll-Paque PLUS (GE Healthcare) and stored at 80 C until use.

METHOD DETAILS

SARS-CoV-2 S-specific single B cell sorting

B cells, enriched from PBMCs with human CD19 MicroBeads (Miltenyi), were incubated with 2 mg/ ml flag-tagged S protein or mixture
of flag-tagged S protein (Genscript, Cat. Z03481) and His-tagged RBD (Chen et al., 2020) on ice for 30 min. Cells were then washed
with 2% fetal bovine serum (FBS) (Hyclone) in PBS and stained with mixture of anti-human IgG (Percpcy5.5; Biolegend Cat. 410710),
anti-human IgD (FITC; Biolegend Cat. 348205), anti-human IgM (Bv605; Biolegend Cat. 314524), anti-CD27 (APCcy7; Biolegend Cat.
356404). A mixture of PE- and APC-conjugated anti-flag antibodies (Biolegend Cat. 637309 and 637307) was also added for gating
S-specific double positive cells, or a mixture of PE-conjugated anti-His (Biolegend Cat. 362603) and APC-conjugated anti-flag, for
S positive and RBD negative cells. Memory B cells were gated on DAPI-CD19+IgM-IgD-IgG+CD27+. Individual S double positive or
S+RBD- cells were sorted with a FACSAria Fusion (BD Biosciences) into each well of 96-well microplates containing 4 ml/well of lysis
buffer (0.5X PBS, 10 mM dithiothreitol, and 4U RNaseOUT). Lysed cells were immediately frozen and stored at 80 C until use.

Antibody cloning and production

Cloning and expression of mAbs from single, SARS-CoV-2 S-specific B cells were performed as described previously (Chen et al.,
2020). In brief, mRNA from lysed B cells was reverse transcribed with SuperScript III (ThermoFisher) and random hexamers. Two
rounds of PCR were performed to amplify heavy and light chain transcripts. Amplified products from the second round PCR were
detected by agarose gel and further verified by Sanger sequencing. Sequences were analyzed with IgBlast (https://www.ncbi.
nlm.nih.gov/igblast/), and sequence confirmed PCR products were then amplified with gene specific primers containing restriction
enzyme sites for cloning into human IgG1, k and l expression vectors (gifts from Michel C. Nussenzweig, Rockefeller University). For
small scale antibody production, paired heavy and light chains were co-transfected into HEK293T cells (ATCC, Cat. CRL-3216) in 6-
well plates with Lipofectamine 3000 (ThermoFisher, Cat. L3000015) following manufacturer’s instructions. Cells were cultured in
DMEM supplemented with 10% FBS and incubated at 37 C with 5% CO2. The medium was replaced with fresh medium at 12 h
post-transfection and the supernatant harvested after 48 h. Cell debris were removed by centrifugation at 2000 g for 10 min and
the cleared supernatant stored at 4 C for further use.
For large scale antibody production, paired heavy chain and light chain were co-transfected in Expi293F cells (ThermoFisher, Cat.
A14527) with ExpiFectamine (ThermoFisher, Cat. A14525) in 250 mL Erlenmeyer flasks following manufacturer’s instructions. Cells
were cultured in Expi293 expression medium at 37 C and 8% CO2 with shaking at 125 RPM. On day 7, cells were removed by centri-
fuging at 2000 RPM for 10 min. Clear supernatants were incubated overnight at 4 C with protein A agarose beads (ThermoFisher, Cat.
20334), followed by washing with PBS, elution with 0.1 M Glycine (pH 2.7) and neutralization with 1 M Tris-HCl (pH 8.0). Purified an-
tibodies were dialyzed against PBS for further use.

Monoclonal antibody screening with ELISA

SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described (Chen et al., 2020). SARS-CoV-2 S2
and NTD proteins were purchased from Sino biological (PA, USA). ELISA was carried out as described (Chen et al., 2020). Briefly,
MaxiSorp 96-well ELISA plates (ThermoFisher) were coated with 50 ng/well of the antigen in PBS at 4 C overnight. Plates were
blocked with 150 mL of 4% BSA in PBS for at least 2 h. Supernatant of antibody-expressing cells was diluted 10-fold for the first
well and 4-fold serial diluted for subsequent wells, applied to the plates, and incubated at 4 C overnight. Plates were washed 4 times

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with PBS supplemented with 0.05% Tween-20 (PBST). Anti-human IgG-alkaline phosphatase (Southern Biotech, Birmingham, AL) at
a final concentration of 1 mg/mL in 1% BSA and 0.05% Tween-20 was added at 50 ml/well and incubated for 1 h at room temperature.
Plates were washed three times with PBST. Developing solution (0.1 M lycine, pH 10.4, with 1 mM MgCl2 and 1 mM ZnCl2, containing
alkaline phosphatase substrate p-nitrophenyl phosphate (Sigma-Aldrich) at final concentration of 1.6 mg/mL) was then added to the
plates at 100 ml/well, and incubated for 2 h at room temperature. Absorbance was measured at 405 nm by microplate reader (Biotek
Synergy H1).
For median effective concentration (EC50) and area under curve (AUC) analysis of antibody binding by ELISA, 5-fold serial dilutions
were produced from an initial concentration of 10 mg/mL of purified antibody in the ELISA procedure described above. EC50 and AUC
were calculated with GraphPad Prism 9.

Cell surface binding assays

Antibodies were tested for binding to surface-expressed SARS-CoV-2 spike, RBD, and NTD on HEK293 T cells (spike) and on yeast
(RBD, NTD). For yeast expression assays, RBD (aa 319-529) and NTD (aa 17-286) were cloned into a pCHA vector (gift of K. Dane
Wittrup, Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA). Plasmids were chemically transformed into yeast cell
line EBY100 to display RBD or NTD as previously described (Chen et al., 2020). Briefly, single clones were cultured in SDCAA selec-
tion medium for 48 h at 30 C and 250 RPM. Cells were pelleted and resuspended in SGCAA medium to an absorbance of 0.5-1 at
600 nm, cultured at 20 C with shaking at 250 RPM for another 48 h to induce expression. RBD and NTD were detected by anti-c-Myc
IgY antibody (ThermoFisher, Cat. A21281). Yeast expressing RBD or NTD were incubated with antibody supernatant and anti-c-Myc
IgY on ice for 30 min and then washed with PBS with 2% FBS twice. Cells were then stained with goat anti-chicken IgG (Alexa Fluor
488, ThermoFisher, A11039) and goat anti-human IgG (Alexa Fluro 647, ThermoFisher, A21445) on ice for 15 min, followed by
washing twice with PBS supplemented with 2% FBS (FACS buffer). Cells were resuspended in FACS buffer and detected by flow
cytometry (BD Canto II). Data were analyzed by FlowJo 10.7.1. For analysis of antibody binding to SARS-CoV-2 S on HEK293T cells,
a plasmid containing Wuhan-Hu-1 S (HDM-SARS2-spike-delta21, Addgene, Cat. 155130) was co-transfected with pmaxGFP
(Lonza) in HEK293T cells using Lipofectamine 3000. Fresh medium was added at 24 h, and cells were harvested at 48 h post-trans-
fection in PBS with 2 mM EDTA. Cells were stained with antibody supernatant on ice for 1 h, washed twice with FACS buffer, and
stained with goat anti-human IgG (Alexa Fluro 647 ThermoFisher, A21445) and DAPi (to distinguish dead and live cells). After washing
twice with FACS buffer, cells were resuspended in FACS buffer and detected by flow cytometry (BD Canto II). S+ cells were identified
by gating on DAPi-GFP+. Data were analyzed in FlowJo 10.7.1. For median effective concentration (EC50) of antibody binding to
SARS-CoV-2 S in the cell-based assay, eight three-fold serial dilutions of purified antibody were produced starting from 10 mg/
mL, followed by flow cytometric binding analysis as above. EC50 was calculated with GraphPad Prism 9.

ELISA-based antibody competition

The competition assay was performed as described (Liu et al., 2020). Briefly, detection antibodies were biotinylated with EZ-Link
Sulfo-NHS-LC-Biotin (ThermoFisher) according to manufacturer’s protocol. 50 ng/well of SARS-CoV-2 S protein were coated on
ELISA plates at 4 C overnight. Plates were blocked with 150 mL of 4% BSA in PBS for 2 h. 30 mL of 2 mg/mL biotinylated antibody
were mixed with 30 mL of 200 mg/mL blocking antibody and added to ELISA plates. For purified polyclonal IgG competition, bio-
tinylated polyclonal IgGs at saturated concentration were used to compete with 100 mg/mL blocking antibody. For antibody compe-
tition with hACE2 (aa18-615), 100 ng/well hACE2 were coated on ELISA plates at 4 C overnight. Plates were blocked with 4% BSA,
and a mixture of 30 mL of 2 mg/mL Twin-Strep-tag HexaPro S (Hsieh et al., 2020) and 30 mL of 200 mg/mL blocking antibody was then
added. Plates were incubated for 2 h at 37 C and washed 4 times with PBST. 50 ml/well of streptavidin-alkaline phosphatase (BD
Biosciences, Cat. 554065) was added to the wells using a dilution of 1:1000 dilution of the stock solution according to the manufac-
turer’s instructions, and incubated for 1 h at room temperature. Plates were washed 5 times with PBST and developed at room tem-
perature for 2 h. Absorbance was measured at 405 nm by microplate reader (Biotek Synergy H1). The detection signal was calculated
by (OD value of mixture antibodies-OD value of PBS)/ (OD value of biotinylated antibody alone-OD value of PBS) x100%. Negative
values were treated as 100% competition.

Cell-based antibody competition

S (HDM-SARS2-spike-delta21, Addgene, Cat. 155130) and GFP (pmaxGFP) were co-expressed in HEK293T cells. 30 mL of 2 mg/mL
biotinylated antibody were mixed with 30 mL of 200 mg/mL blocking antibody and added to cells. After 1 h incubation on ice, followed
by washing twice with FACS buffer, cells were stained with 50 mL of 1:1000 diluted DyLight 649 Streptavidin (BioLegend, Cat. 405224)
and DAPi. After washing twice with FACS buffer, cells were resuspended in FACS buffer and detected by flow cytometry (BD Canto
II). S+ cells were gated on DAPi-GFP+. Data were analyzed by FlowJo 10.7.1. The detection signal was calculated by (MFI of mixture
antibodies-MFI of PBS)/ (MFI of biotinylated antibody alone-MFI of PBS) x100%. Negative values were treated as 100% competition.

Antibody binding to S variants

Variants included: Wuhan-Hu-1-D614G S (HDM-SARS2-spike-delta21-D614G, Addgene, Cat. 158762), recurring NTD deletions as
described (McCarthy et al., 2021) (D69-70, D141-144, D144, D146, D210, D243-244), Nextstrain cluster 20A.EU1 (A222V), Danish
mink variant (D 69-70 and Y453F), UK B.1.1.7 (D69-70, D144, N501Y, A570D, P681H, T716I, S982A, D1118H), SA B.1.351 (L18F,

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D80A, D215G, D242-244, K417N, E484K, N501Y, A701V) and P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y,
T1027I, D1176F). We note that plasmids with D144 and D145 have the same coding sequence due to the presence of tyrosine at both
sites. All variants contain D614G. For binding, 10 mg/mL of antibody were incubated with cells, with goat anti-human IgG as second-
ary antibody for detection by flow cytometry (BD Canto II). S+ cells were gated on DAPi-GFP+. Data were analyzed by FlowJo 10.7.1.
Binding for each mAb was first normalized (‘‘normalized IgG MFI’’) by dividing the MFI for that mAb by the MFI for GFP. The normal-
ized MFI for binding the D614G spike was used as a reference (normalized D614G spike IgG MFI). The relative binding intensities of
the tested mAbs for each variant, calculated as the ratio of the normalized variant IgG MFI and the normalized D614G spike IgG MFI.
Relative binding signal > 1 was treated as no loss of binding and set to 1.

Pseudovirus production and neutralization assay

Pseudovirus particles were produced as described (Chen et al., 2020). HEK293T cells were co-transfected with spike envelope
plasmid (HDM-SARS2-spike-delta21, Addgene, Cat. 155130) or plasmids encoding variant spike (HDM-SARS2-spike-delta21-
D614G, HDM-SARS2-spike-B.1.1.7-delta21-D614G, HDM-SARS2-spike-B.1.351-delta21-D614G, HDM-SARS2-spike-P.1-
delta21-D614G) package plasmid (psPAX2, Addgene, Cat. 12260) and backbone plasmid (pLenti CMV Puro LUC, Addgene, Cat.
17477) with Lipofectamine 3000. Medium was replaced with fresh medium at 24 h, and supernatants were harvested at 48 h
post-transfection and clarified by centrifugation at 300 g for 10 min before aliquoting and storing at 80 C. SARS-Cov-2 pseudovirus
neutralization assay was performed as described (Johnson et al., 2020), with target cell line 293FT expressing human ACE2 and
serine protease TMPRSS2 (provided by Marc C. Johnson, University of Missouri) or TZM.bl expressing human ACE2. Cells at
1.8 3 104 cell/well were seeded in 96-well plates 16 h in advance. Serial diluted mAb was mixed with pseudovirus and incubated
for 1 h at 37 C before adding to cells. Cells infected without mAb were scored as 100% infection; cells cultured without pseudovirus
or mAb as blank controls. After 48 h incubation at 37 C with 5% CO2, cells were processed with luminescent regent (ONE-GloTM,
Promega) according to manufacturer’s instructions, and luminescence (RLU) was measured with a microplate reader (Biotek
Synergy H1). Inhibition was calculated by 100-(RLU of mAb-RLU of blank)/ (RLU of pseudovirus -RLU of blank) x100%. Values for
half inhibition (IC50) and 80% inhibition (IC80) were calculated with GraphPad Prism 9.

Authentic virus propagation and neutralization assay

SARS-CoV-2 propagation: All work with infectious SARS-CoV-2 was performed under Biosafety Level-4 conditions at the National
Emerging Infectious Diseases Laboratories (NEIDL). Passage 4 SARS-CoV-2 USA-WA1/2020 was received from the University of
Texas Medical Branch. SARS-CoV-2, isolate USA/CA_CDC_5574/2020 (from the B1.1.7 lineage) was isolated from a nasopharyn-
geal swab on December 29, 2020 in San Diego County, California, USA. Passage 3 master stock material was received at NEIDL
from BEI Resources (Cat. NR-54011, Lot: 70041598). SARS-CoV-2, isolate hCoV-19/South Africa/KRISP-K005325/2020 (also
referred to as 501Y.V2.HV and 501Y.V2.HV001) was isolated from an oropharyngeal swab from a 40-year-old human male in Ugu
district, KwaZulu-Natal, South Africa on November 16, 2020. Passage 4 master stock material was received at NEIDL from BEI Re-
sources (Cat. NR-54009, Lot: 70041942). Viruses were amplified in T225 flasks of VeroE6 cells infected at an approximate MOI of
0.001 plaque forming units (PFU)/cell in DMEM + 2% HI-FBS. Infected cells were observed daily for progression of cytopathic effect
(CPE). Stock supernatant was harvested and clarified by centrifugation at 5,250 RCF at 4 C for 10 min and heat inactivated FBS con-
centration (GIBCO) was increased to a final concentration of 10% prior to cryopreservation at 80 C. Stocks were characterized to
include negative sterility testing using trypic soy broth and thioglycolate medium with dextrose (negative for growth out to 14 days)
and analysis for mycoplasma species from DNA using the MycoSEQ detection system (ThermoFisher), which is able to detect > 90
mycoplasma species if present (none identified). Endotoxin levels were determined using the Lonza QCL-1000 endpoint chromo-
genic LAL assay and were 0.164 EU/mL for NSU-V014 and 0.156 EU/mL for NSU-V015.
Cell Culture: NR-596 VeroE6 cells (BEI Resources) were maintained in Dulbecco’s modified Eagle medium (DMEM) (GIBCO) with
10% heat inactivated FBS (GIBCO), GlutaMAX (GIBCO), non-essential amino acids (GIBCO) and sodium pyruvate (GIBCO). One day
prior to the assay, VeroE6 cells were seeded at a density of 8.0 3 105 cells per well of a 6-well plate (Falcon Polystyrene Microplates,
Cat. 353934) in 2 mL media.
Viral neutralization reduction assays: An Avicel plaque reduction assay was used to quantify plaques. Antibody samples were seri-
ally diluted in Dulbecco’s Phosphate Buffered Saline (DPBS)(GIBCO) using two-fold dilutions. Dilutions were prepared in triplicate per
antibody and plated in triplicate. Each dilution was incubated at 37 C and 5% CO2 for 1 h with 1000 PFU/mL of SARS-CoV-2. Con-
trols included DPBS as a negative control and 1000 PFU/mL SARS-CoV-2 incubated with DPBS. The maintenance medium was
removed from each plate and 200 mL of each inoculum dilution was added to confluent monolayers of NR-596 Vero E6 cells (including
a positive and mock negative control) in triplicate and incubated for 1 h at 37 C/5% CO2 with gentle rocking every 10-15 min to pre-
vent monolayer drying. The overlay was prepared by mixing by inversion Avicel 591 overlay (DuPont Nutrition & Biosciences, Wil-
mington, DE) and 2X Modified Eagle Medium (Temin’s modification, GIBCO) supplemented with 2X antibiotic-antimycotic (GIBCO),
2X GlutaMAX (GIBCO) and 10% FBS (GIBCO) in a 1:1 ratio. After 1 h, 2 mL of overlay was added to each well and the plates was
incubated for 48 h at 37 C/5% CO2. 6-well plates were then fixed using 10% neutral buffered formalin prior to removal from BSL-
4 space. The fixed plates were then stained with 0.2% aqueous Gentian Violet (RICCA Chemicals, Arlington, TX) in 10% neutral buff-
ered formalin for 30 min, followed by rinsing and plaque counting. The half maximal inhibitory concentrations (IC50) were calculated
using GraphPad Prism 8.

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Fab preparation
Fab fragments were expressed with a His-tag heavy chain expression vector and co-transfected with a light chain vector in Expi293F
cells. Fab fragments were also produced by papain digestion with a Fab preparation kit (ThermoFisher, Cat. 44985) according to
manufacturer’s protocol. In brief, 0.5-1.0 mg of IgG1 antibodies were mixed with 125 mL papain resin (ThermoFisher, Cat. 20341)
for 5 h in the digestion buffer provided, containing 20 mM cysteine, pH 7.4. Undigested antibody and Fc fragments were removed
by incubating digested products with a protein A column overnight at 4 C, then collecting the Fab-containing flow-through. Fabs
were analyzed by 4%–20% Tris-Glycine SDS-PAGE (ThermoFisher, Cat. XP04200BOX). C12C9 and C12A2 Fabs were prepared
by expressing Fabs with a 3C-cleavable histag that was removed using 3C protease (Pierce) following Talon resin purification, as
described (Schmidt et al., 2015).

Statistical analysis
Competition clusters were processed in two steps. The mAbs were first grouped based on binding to SARS-CoV-2 subdomains
(RBD, NTD, S2). These mAbs, together with ungrouped mAbs, were then clustered based on competition in ELISA or in the cell-based
assay, taking reduction of signal by 30% as the competition threshold.
In order to determine the presence of epitope dependent, VH-segment preferential usage, we used resampling to bootstrap
p values. For each cluster with size n, we resampled n VH segments from the observed VH segments in our dataset m times with
replacement. P values were generated by counting the number of resampled clusters for which the frequency of a VH-segment
matched or exceeded the frequency observed in the 167 S binders in Data S2, dividing the quantity of these instances by the number
of trials m, and performing a Bonferroni correction (e.g., multiplying the p value by the number of unique VH-segments) (Nielsen et al.,
2020). For these data, the 7 clusters range from 5 to 39 members, and 1 million resampled clusters were generated for each cluster.
We also used the same methods to compare the VH-segment composition of each cluster to the VH-segment composition of the
general human PBMC repertoire, exchanging the weights of the unique VH-segments with their representation in the averaged gen-
eral repertoire from 10 healthy controls (Briney et al., 2019).
Public clones were screened from previously reported clones with a total of 616 SARS-CoV-2 related clones (Chi et al., 2020; Kreer
et al., 2020; Liu et al., 2020; Robbiani et al., 2020). We also screened 133 Zika (Robbiani et al., 2017) and 98 Flu (H1N1) (Wrammert
et al., 2011) related clones. Public clones converged on identical VH and VL alleles, with at least 50% identity in CDRL3 and 50%
identity in CDRH3.

Other analysis
Phylogenetic analysis of CoV spikes was done comparing pairwise identity using Clustal Omega. GenBank accession numbers used
are the following: SARS (MN985325.1), MERS (JX869059.2), HKU1 (Q0ZME7.1), OC43 (AAT84362.1), NL63 (AAS58177.1), and 229E
(AAK32191.1). MEGA X was used for pairwise distance analysis of CDRH3 and CDRL3s. For CDR3s that exceeded the MEGA X
range, we manually set them to the maximum value of 3.

Protein Expression and purification for cryo-EM

Plasmids encoding stabilized variants 2P (Wrapp et al., 2020) and hexapro (Hsieh et al., 2020) of SARS-CoV2 S protein were gifts from
Jason McLellan (University of Texas, Austin). Spike proteins for electron microscopy were expressed in Expi293F cells grown in
Expi293 medium after transfection with spike-encoded plasmid DNA using the Expifectamine 293 transfection kit (ThermoFisher,
Waltham, MA). Cells were grown for 6 days before subjecting conditioned media to affinity chromatography following centrifugation
and 0.2 mm filtration. The 2P variant of spike was applied first to a Talon cobalt resin (Takara Bio) and eluted with 200 mM imidazole
followed by purification over a S200 size exclusion chromatography column (Cytiva). Alternatively, the hexapro variant of spike was
applied to a Streptactin resin (IBA Life Sciences), eluted with 2.5 mM desthiobiotin, and used without further purification.

Cryo-EM grid preparation

Grids were glow discharged (PELCO easiGlow) for 30 s at 15 mA and prepared with a Gatan Cryoplunge 3 by applying 3.5 mL of sam-
ple and blotting for 4.0 s in the chamber maintained at a humidity between 86% and 90%. Protein complexes were formed with spike
and a 3-fold excess of Fab one h before freezing and applied with without further purification.
Preliminary studies on 2P S protein complexes with C12C11 were performed with 1.2 mg/mL total protein and C-flat 1.2-1.3 400 Cu
mesh grids (Protochips). Structures of C12A2 and C12C9 bound with 2P S protein were determined using Quantifoil 1.2-1.3 400 mesh
Cu grids and 0.5 mg/mL total protein, the former with 0.1% w/v octyl b-D-glucopyranoside to reduce orientation bias. Structures of
C93D9, G32R7, and C81C10 with hexapro S protein were prepared with thick C-flat 1.2-1.3 400 Cu mesh grids and 1.2 mg/mL total
protein.

Cryo-EM image recording

Images for C12C11 complexes were recorded on a Talos Arctica microscope operated at 200 keV with a Gatan K3 direct electron
detector. Images for C12A2, C12C9, and G32R7 complexes were recorded on a Titan Krios microscope operated at 300 keV with a
Gatan BioQuantum GIF/K3 direct electron detector. Images for C93D9 and C81C10 were recorded on an FEI Technai F20

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microscope operated at 200 keV with a Gatan K2 Summit direct electron detector. Automated recording was with Serial EM (Mas-
tronarde, 2005) in all cases. Specifications and statistics for images from each of these complexes are in Table S1.

Cryo-EM image analysis and 3D reconstruction

Image analysis for all structures was carried out in RELION (Scheres, 2012). Beam-induced motion correction of micrograph movies
was performed with UCSF MotionCor2 (Zheng et al., 2017) followed by contrast transfer function estimation with CTFFIND-4.1
(Rohou and Grigorieff, 2015), both as implemented in RELION. Particles were picked from motion corrected micrographs using
crYOLO (Wagner et al., 2019). A general model was used to pick particles from datasets collected on the Talos Arctica and Titan
Krios; a specific model was trained to pick particles from F20 micrographs.
Extracted particles were downsampled and subjected to 2D classification, two rounds for the Titan Krios datasets. Initial models
were prepared, and the best of three was used as a reference for 3D classification with C3 symmetry imposed. For the Talos Actica
and F20 datasets, all particles from reasonable classes were combined and subjected to 3D autorefinement and sharpening, yielding
final reconstructions at 8-11Å resolution. The Titan Krios datasets for C12C9 and G32R7 required additional rounds of 3D classifi-
cation and 3D autorefinement to converge to final C3-symmetric, full particle reconstructions of 3.0 and 3.6 Å nominal resolutions
for C12C9 and G32R7, respectively, but with much lower resolution for the Fab-bound domains. We therefore carried out local refine-
ment as follows. Particle stacks from the final C3-symmetric, full particle maps were symmetry expanded and back projected to
create a new C3-expanded reconstruction. Models of the most similar heavy and light chains were extracted from the protein
data bank and combined to create initial models for the Fabs of C12C9 (heavy: 5ggu, light: 6ghg) and G32R7 (heavy: 4qf1, light:
7byr). Fab models and the NTD (residues 14-290 of 7c2l) were docked into the reconstructed maps; the RBD from 7bz5 was also
docked into the G32R7 map. These docked PDB models were used to prepare initial masks with a sphere radius of 8 Å using
NCSMASK and a soft edge of 5 pixels, added with relion_mask_create. The soft mask was then used to make a background-
subtracted, subparticle stack as implemented in RELION. Fab-occupied and well-resolved subparticles were identified with 3D
classification without alignment. Further rounds of 3D classification with and without alignment were carried out along with 3D
autorefinement to obtain final sharpened maps with resolutions of 4.0 Å for both C12C9 and G32R7 Fabs. Detailed descriptions
of the particle processing are in Data S3 and statistics, including model refinement, are in Table S1. Fourier shell correlations are
in Data S4 and S5.

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Supplemental figures

Figure S1. Sorting strategy for SARS-CoV-2-specific memory B cells and characterization of monoclonal antibodies, related to Figure 1
(A) Representative flow cytometry plots showing CD19+, CD27+, SARS-CoV-2 S-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic
control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD IgM-IgG+CD27+ and finally on S (B) Representative flow
cytometry plots showing S-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the S gate. (C)
Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK293T cells. Cells were gated on DAPI GFP+ population. (D) Representative flow
plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (E) Representative flow plot of mAb supernatant
(legend continued on next page)
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bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Figure 1C for the screening color scheme. (F) Bar graph of Log10(EC50) of
antibodies targeting RBD, NTD and S2 using ELISA and cell-based assay. EC50 (mg/mL), RBD (n = 23), NTD clusters (n = 15) and S2 (n = 15). ***p < 0.001, ****p <
0.0001; Paired nonparametric Wilcoxon test. Data are mean values ± SEM (G) Dot plot of Log10(EC50) of antibodies in the indicated bins by cell-based assay.
Antibodies are from subjects G32 and C41, sorted with S. Each dot represents one monoclonal antibody. EC50 (mg/mL), 13-39 days (n = 13), 40-63 days (n = 8). No
significance; nonparametric Mann-Whitney test. Data are mean values ± SEM.
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Figure S2. Competition mapping including antibodies from S+RBD sort and antibodies with published structures, related to Figure 2
(A) Cross competition matrix by ELISA-based competition. Including antibodies from cells gated as S+RBD increased representation of NTD and S2 clusters.
Color and shading scheme, groups defined by hierarchical clustering, and recombinant protein binding as in Fig 2A. Arrows designate antibodies described in the
text, including those reported here (green) and those from published work by others (blue). (B) Cross competition matrix for mAbs from S+RBD sort by ELISA-
based competition. (C) Competition in cell-based assay, for antibodies with binding in ELISA format too weak for reliable blocking measurement. See Figure 2B
for procedures, heat-map color scheme, etc.
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Figure S3. Cell-based competition assay and comparison with ELISA, related to Figure 2
(A) Representative flows plot for competition, by 20 blocking antibodies representing each of the seven principal clusters (from ELISA: Figure S2A), for binding
cell-surface expressed S protein by 3 biotinylated antibodies. A non-COVID-19 related antibody and a self-blocking antibody were used as negative and positive
controls. (B) Heatmap of 19 mAbs with hierarchical clustering from cell-based competition assay with 118 blocking antibodies. See Figure 2B for procedures,
heat-map color scheme, etc. (C) Heatmap of 17 mAbs with hierarchical clustering from ELISA-based (left panel) and cell-surface (right panel) cross-competition.
See Figure 2B for procedures, heat-map color scheme, etc. (D) Heatmap of polyclonal IgG with hierarchical clustering from ELISA-based competition with
12 blocking antibodies and a non-COVID-19 control antibody. Intensity of color shows binding intensity of detection polyclonal antibodies, from 0 signal
(complete blocking) to 100% full signal.
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Figure S4. Neutralization profiles for monoclonal antibodies of seven clusters, related to Figure 4
(A) Authentic virus (WA1) neutralization profiles of 9 antibodies. (B) Pseudovirus neutralization profiles in two cell lines for antibodies from NTD-1 cluster. Left
panel: neutralization profiles in 293FT cells co-expressing hACE2 and TMPRSS2 as target cells (n = 39). Right panel: neutralization profiles in TZM.bl cells
expressing hACE2 as target cells (n = 13). (C) Pseudovirus neutralization profiles in 293FT cells co-expressing hACE2 and TMPRSS2 for antibodies from RBD-1
(n = 22), RBD-2 (n = 23), RBD-3 (n = 5), NTD-2 (n = 16), S2-1 (n = 32) and S2-2 (n = 19) clusters. Data are mean values ± SD for authentic virus assays and
pseudovirus assays using 293FT/hACE2/TMPRSS2 cells. Data are mean values ± SEM for assays using TZM.bl/hACE2 cells.
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Figure S5. Diversity of antibody sequences and convergent C93D9 class of antibodies, related to Figure 5
(A) V(D)J and VJ mutation levels in each of the 7 principal competition groups. Mutations in VH and VL (excluding CDR3) counted by IgBLAST. (B) Maps of pairwise
distances of CDRH3 (lower left triangle) and CDRL3 (upper right triangle) for the RBD-1, RBD-2, RBD-3, NTD-1 and S2-2 cluster antibodies related to Figure 5B.
(C) Two views of 20 Fab structures, listed in (E), bound with SARS-CoV-2 RBD. Structures all superposed on the RBD; heavy-and light-chains of each Fab in a
distinct color. The figure includes only the RBD from 6YZ5 (not one of the 20), with the RBM in light orange and the rest of the chain in gray. (D) View as in the right-
hand panel in (C), but showing only the Fab from 7B3O (the closest in sequence to C93D9), with CDRs labeled. The most intimate contacts with RBM residues are
from CDRH1, CDRH2 and CDRL1, many with residues constrained in potential variability by ACE2 interaction. (E) Maps of pairwise distances of CDRH3 (lower left
triangle) and CDRL3 (upper right triangle) for the 21 C93D9 class antibodies in (C) and (D). Pairwise distances analyzed by MEGA X. Intensity of color shows the
distance, from 0 (identical) to 1 (no identity). The VH and VL genes encoding the antibodies are shown in the indicated groups. Differences in CDR3s from the
reference sequences (bold) are in red; dashes indicate missing amino acids; dots represent identical amino acids. IGHV3-66 and IGHV3-53 are very similar VH
gene segments, differing by only one encoded amino-acid residue.
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Figure S6. Representative flow plots for mAb binding and neutralization of indicated variants, related to Figure 6
Flow plots for binding of 7 mAbs to Nextstrain cluster 20A.EU1 (A222V), Danish mink variant (D69-70 and Y453F), B.1.1.7 (D69-70, D144, N501Y, A570D, P681H,
T716I, S982A, D1118H), B.1.351 (L18F, D80A, D215G, D241-243, K417N, E484K, N501Y, A701V), P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y,
H655Y, T1027I, D1176F) and NTD deletion variants. All variants contain the D614G mutation. Plasmids with variant S co-expressed with pmaxGFP in HEK293T
cells. Cells were gated on DAPi GFP+. mAb C81E2 was used as positive control, and PBS, as negative control. (B) Authentic B.1.1.7 virus neutralization profiles
for 6 antibodies. (C) Authentic B.1.351 virus neutralization profiles for 6 antibodies. Data are mean values ± SD for authentic virus assays.