REPORTS

Cite as: K. M. Hastie et al., Science
10.1126/science.abh2315 (2021).

Defining variant-resistant epitopes targeted by SARS-CoV-2
antibodies: A global consortium study
Kathryn M. Hastie1†, Haoyang Li1†, Daniel Bedinger2, Sharon L. Schendel1, S. Moses Dennison3, Kan Li3,
Vamseedhar Rayaprolu1, Xiaoying Yu1, Colin Mann1, Michelle Zandonatti1, Ruben Diaz Avalos1, Dawid Zyla1,
Tierra Buck1, Sean Hui1, Kelly Shaffer1, Chitra Hariharan1, Jieyun Yin1, Eduardo Olmedillas1, Adrian Enriquez1,
Diptiben Parekh1, Milite Abraha3, Elizabeth Feeney3, Gillian Q. Horn3, CoVIC-DB team1, Yoann Aldon4, Hanif
Ali5, Sanja Aracic6, Ronald R. Cobb7, Ross S. Federman8, Joseph M. Fernandez9, Jacob Glanville10, Robin Green8,
Gevorg Grigoryan8, Ana G. Lujan Hernandez11, David D. Ho12, Kuan-Ying A. Huang13, John Ingraham8, Weidong
Jiang14, Paul Kellam15,16, Cheolmin Kim17, Minsoo Kim17, Hyeong Mi Kim17, Chao Kong18, Shelly J. Krebs19, Fei
Lan9,20, Guojun Lang18, Sooyoung Lee17, Cheuk Lun Leung8, Junli Liu14, Yanan Lu9,21, Anna MacCamy22, Andrew
T. McGuire22, Anne L. Palser15, Terence H. Rabbitts5,23, Zahra Rikhtegaran Tehrani24, Mohammad M. Sajadi24,
Rogier W. Sanders4, Aaron K. Sato11, Liang Schweizer25, Jimin Seo17, Bingqing Shen25, Jonne J. Snitselaar4,
Leonidas Stamatatos22, Yongcong Tan18, Milan T. Tomic26, Marit J. van Gils4, Sawsan Youssef10, Jian Yu12, TomZ. Yuan11, Qian Zhang25, Bjoern Peters1,27, Georgia D. Tomaras3, Timothy Germann2, Erica Ollmann Saphire1,27*†These authors contributed equally to this work.
*Corresponding author. Email: erica@lji.org

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality
following severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Multiple mutations in
SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission andspillover/spillback events between humans and animals. To develop prevention and therapeutic strategies,
we formed an international consortium to map the epitope landscape on the SARS-CoV-2 Spike, defining
and structurally illustrating seven receptor-binding domain (RBD)-directed antibody communities with
distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal Spikemutations, individually and clustered together in variants, that impact antibody function among thecommunities. Key classes of RBD-targeted antibodies maintain neutralization activity against theseemerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment
cocktails and understanding how viral variants might affect antibody therapeutic efficacy.
Cell entry of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is mediated by its surface glycoprotein, Spike.
The S1 subunit of Spike contains the N-terminal domainFirst release: 23 September 2021

(NTD) and the receptor-binding domain (RBD), whichmediates recognition of the host cell receptor angiotensinconverting enzyme 2 (ACE2). The S2 subunit drives fusion

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1Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA. 2Carterra, 825 N. 300 W. Ste 309, Salt
Lake City, UT 84103, USA. 3Center for Human Systems Immunology, Departments of Surgery, Immunology, and Molecular Genetics and Microbiology and Duke Human
Vaccine Institute, Duke University, Durham, NC, 27701, USA. 4Department of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centers,
Location AMC, University of Amsterdam, Amsterdam Infection & Immunity Institute, 1105 AZ Amsterdam, the Netherlands. 5Quadrucept Bio Ltd., Cambridge CB23 6DW,
UK. 6Myrio Therapeutics Pty, Ltd., 1 Dalmore Drive, Scoresby, VIC 3179, Australia. 7National Resilience, Inc., 13200 NW Nano Ct., Alachua, FL 32615, USA. 8Generate
Biomedicines, Inc., 26 Landsdowne Street, Cambridge, MA 02139, USA. 9Activemotif, Inc., 1914 Palomar Oaks Way, Suite 150, Carlsbad, CA 92008, USA. 10Centivax, Inc.,
201 Gateway Blvd. Floor 1, South San Francisco, CA 94080, USA. 11Twist Bioscience, 681 Gateway Blvd., South San Francisco, CA 94080, USA. 12Aaron Diamond AIDS
Research Center, Columbia University Vagelos College of Physicians and Surgeons, 701 West 168th St. HHSC 1102, New York, NY 10032, USA. 13Division of Pediatric
Infectious Diseases, Department of Pediatrics, Chang Gung Memorial Hospital and Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan.
14Shanghai Henlius Biotech, Inc., 9/F, Innov Tower, Zone A, No. 1801 Hongmei Road, Xuhui District, Shanghai, China. 15Kymab, Ltd., The Bennet Building, Babraham
Research Campus, Cambridge CB22 3AT, UK. 16Department of Infectious Disease, Imperial College, London SW7 2AZ, UK. 17Celltrion, Inc., Department of Research andDevelopment, 23 Academy-ro Yeonsu-gu Incheon, Republic of Korea. 18Sanyou Biopharmaceuticals Co., Ltd., No. 188 Xinjunhuan Road, Building 6B-C, 3rd Floor, Minhang
District, Shanghai 201114, China. 19Emerging Infectious Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. 20Shanghai Key Laboratoryof Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan
University, Shanghai, China. 21AbCipher Biotechnology, 188 Xinjun Ring Road, Building 2, 4th Floor, Minhang District, Shanghai, 201114, China. 22Fred Hutchinson Cancer
Research Center, Vaccines and Infectious Diseases Division, Seattle, WA, USA. 23Institute of Cancer Research, Centre for Cancer Drug Discovery, London SM2 5NG, UK.
24Division of Clinical Care and Research, Institute of Human Virology, University of Maryland, Baltimore, MD 21201, USA. 25HiFiBiO, Inc., 237 Putnam Avenue, Cambridge, MA
02139, USA. 26National Resilience, Inc., 2061 Challenger Dr., Alameda, CA 94501, USA. 27Department of Medicine, University of California, San Diego, La Jolla, CA 92037,
USA.

First release: 23 September 2021

We first measured the affinity of 269 CoVIC mAbs forD614-Hexapro Spike ectodomain trimers and monomeric
RBD and NTD, and the ability of each of these mAbs to blockACE2-RBD binding (figs. S2 to S5, table S1, and covic.lji.org).
The panel, formed by candidates for therapeutic use, includes
NTD- or S2-directed antibodies, but is dominated by thosetargeting the RBD. In contrast to previous studies that classified mAbs using germline or structural information (10, 16),
the 186 RBD-reactive mAbs of CoVIC analyzed here were instead distinguished by a competition profile created by highthroughput surface plasmon resonance (HT-SPR). RBDdirected antibodies can be sorted into seven core “communities” (Fig. 1, fig. S6A, and table S2) that are broadly definedby the competition profiles of each mAb to one another. Communities can be further divided into finer clusters and bins
based on their discrete competition with other clusters
and/or their ability to compete with ACE2 (Fig. 1 and tables
S1 and S2).
To understand the position of each community relative tothe others, we next mapped the footprints by negative-stain
EM (NS-EM) for 25 example RBD-reactive mAbs chosen to
span the range of communities and key clusters (table S3). To
have a relatively agnostic view of antibody interactions with
Spike, mAbs were not chosen based on germline origin, CDR
feature or length, neutralization potency, particular antibody
origin (e.g., human, mouse or in silico) or format (e.g., IgG,
scFv-Fc, VHH-Fc, multivalent).
In parallel, we measured the neutralization activity of 41
RBD-directed mAbs (chosen to span the range of communities and key clusters) as well as a human ACE2-Fc formatbased therapeutic candidate (CoVIC-069) fusion format. Neutralization was measured against pseudoviruses displaying
the Spike protein bearing (a) the globally dominant G614 variation, (b) 15 single point mutations or deletions represented
in circulating strains, (c) constellations of mutations found infour VOCs [B.1.1.1 (alpha), B.1.351 (beta), P.1 (gamma), andB.1.617.2 (delta)] and one VOI [B.1.429 (epsilon)] and (d) two
pseudovariants carrying four mutations (termed 4xM, containing G261D, Y453F, F486L, and N501T) or five mutations(termed 5xM, carrying the 4xM mutations plus V367F) identified in human-mink spillover events (fig. S1).
The mAbs in RBD-1 through -3 target the receptor-binding
motif (RBM), compete with ACE2, and generally require the
RBD to be in the “up” conformation for binding (footprints defined in Fig. 2B, table S3, and covic.lji.org). Community RBD-1
contains hACE2-derived molecules and IgGs (e.g., CoVIC-259,
EMD-24335) that largely overlap with the RBM (Fig. 2B, fig.
S6B, and table S3). The footprint for RBD-2 mAbs is shiftedfrom the center of the ACE2 binding site toward the peak of
the RBM (Fig. 2B, fig. S6B, and table S3). RBD-2 is the largest
community and can be divided further into clusters and thenbins based on competition with other communities (Fig. 1).

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between virus and host cell membranes. Spike, particularly
the S1 subunit, is the primary target of neutralizing
antibodies against SARS-CoV-2 (1).
Since SARS-CoV-2 first emerged, recurrent mutations in
Spike arose during both human-to-human transmission (2–4)
and spillover/spillback events between humans and animals(5–8). Distinct Variants of Concern (VOCs) or Variants ofInterest (VOIs), including those first identified in the UK(alpha, B.1.1.7), South Africa (beta, B.1.351), Brazil (gamma,
P.1), India (delta, B.1.617.2) and California (epsilon, B.1.429)
carry several mutations associated with enhancement of human-to-human transmission (9). In particular, the receptorbinding motif (RBM) mutations K417, L452, E484 and N501
affect ACE2-Spike interactions (10). Variations at positions
N439 and S477 are frequently detected in patient samples (3,11, 12), whereas others such as V367F, Y453F and F486L areassociated with cross-species transmission (6, 8). The NTD isalso highly mutable and is especially prone to deletions:∆HV69-70 and ∆Y144 are both seen in B.1.1.7 and ∆HV69-70
is in the mink-associated Cluster V (6). ∆LAL242-244 appearsin B.1.351, and ∆FR157-158 is found in B.1.617.2 (9). The NTD
point mutations S13I and W152C alter disulfide bonding andconformation of the B.1.429 NTD (13) (fig. S1).
SARS-CoV-2 will continue to evolve. By understanding antibody footprints and the distinct ways by which antibodies
target Spike, we may deduce optimal combinations of mAbs
to prevent and treat infection by emerging variants and tominimize the risk of viral escape. We can also gauge the susceptibility of mapped antibodies to new mutations and predict whether newly identified mAbs might also be susceptible
to viral escape. Thus, we sought to define functionally important groups in an array of therapeutic candidates, and todissect how key mutations, both individually and combined
as in VOCs, affect antibody-mediated neutralization in a
pseudovirus neutralization assay.
The Coronavirus Immunotherapeutic Consortium(CoVIC) was formed to analyze candidate antibody therapeutics side-by-side in standardized assays (14) and now includes
over 350 monoclonal antibodies (mAbs) directed against the
SARS-CoV-2 Spike protein from 56 different partners acrossfour continents (15). The panel includes antibodies derived
from COVID-19 survivors, phage display, naïve libraries, insilico methods and other strategies, each elicited, evaluatedand selected using distinct criteria. The panel thus represents
a broader and deeper array of antibodies from which both
fundamental information and therapeutic cocktails can bederived. With the goals of FAIR (findable, accessible, interoperable, reusable) data analysis and management as well as
inclusion of otherwise inaccessible clinical candidates, candidate antibody therapeutics were blinded, and tested in multiple in vitro and in vivo assays with comparative data
uploaded into a publicly accessible database (covic.lji.org).

First release: 23 September 2021

and B.1.617.2 (delta) variants, which each contain only one or
two RBM-located mutations (N501Y, L452R or T478K/L452R
respectively). Curiously, the V367F mutation identified inmink populations enhances neutralization by some RBD-2
mAbs and in some cases this mutation can offset decreases inpotency resulting from other single point mutations. For example, CoVIC-040 has a 14- and 8-fold decrease in potency
against the F486L mutation and the F486L-containing 4xM
mink pseudovariant, respectively, but only a 4-fold decreaseagainst the 5xM mink pseudovariant, which contains V367F
in addition to the four mutations present in 4xM (Fig. 3, fig.
S10, and table S4). V367 is adjacent to an N-linked glycan at
position 343, which was recently implicated in providing agating mechanism for the RBD (21). Substitution of valinewith phenylalanine could alter the local environment of theN343 glycan moieties and enable the RBD to adopt a conformation more amenable to antibody interaction.
Antibodies in communities RBD-4 and RBD-5 bind to theouter face of the RBD and, like the Class 2 and Class 3 mAbs
previously defined in (16), can do so in either the “up” or“down” RBD conformation without steric hindrance (Fig. 2C,
figs. S6 and S12, and table S3). The footprints of these groups
largely overlap, but RBD-4 mAbs bind toward the outer edgeof the RBM and can block ACE2 (e.g., CoVIC-094, EMD24350; similar to antibody C002 (16)), whereas RBD-5 mAbs
bind away from the RBM, toward the “S309” site and do notblock ACE2 (e.g., CoVIC-134, EMD-24384; similar to antibody
REGN-10987 (17)) (10) (Figs. 1B and 2C, figs. S5 and S6B, andtables S1 to S3). Some RBD-4 and RBD-5 IgGs can crosslink
Spike trimers in solution (fig. S8C and table S3).
Interestingly, according to the five RBD-5 IgGs we imaged,
only those IgGs that show Spike-cross linking tendency havepotent neutralizing activity (Fig. 1B, fig. S13, and table S3). A
recent cryo-electron tomography study showed native Spiketrimers on the SARS-CoV-2 virion surface tilt at variable degrees relative to the viral envelope (22). This finding provides
a possibility for IgG-mediated Spike crosslinking on virions,and may contribute to the mechanism of neutralization of the
RBD-5 mAbs in the absence of ACE2 blocking (fig. S8D).
Most RBD-4 mAbs are impacted by E484K and/or L452R(represented in the B.1.429 variant) mutations (Fig. 3, fig. S10,
and table S4), and some are impacted by the N439K mutation, which is highly represented in sequences worldwide (3).
RBD-5 mAbs, however, show broad resistance to nearly all
mutations analyzed, with only two mAbs in this group showing moderate decreases in potency against V367F and N439K(Fig. 3, fig. S10, and table S4).
RBD-6 (e.g., CoVIC-250, EMD-24352) and RBD-7 (e.g.,
CoVIC-063, EMD-24353) antibodies bind to the inner face of
the RBD and access a previously described cryptic epitope(23, 24) (Fig. 2D, fig. S6B, and table S3). Like Class 4 antibodies described in (16), binding of Spike by RBD-6 and RBD-7

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Cluster 2a antibodies (e.g., CoVIC-252, EMD-24339) bind toward the inner face of the RBD and its binding area overlaps
highly with that of the therapeutic antibody REGN-10933(17). Antibodies in 2b.1 (e.g., CoVIC-010, EMD-24343; similar
to antibody COVA2-39 (18)) and 2b.2 (e.g., CoVIC-140, EMD24383; similar to antibody C144 (16)) bind toward the outerface of the RBD and mAbs in bin 2b.3 (e.g., CoVIC-002, EMD24345; similar to antibody S2E12 (19)) bind to the peak of the
RBD (Fig. 2B, fig. S7, and table S3). Lastly, RBD-3 mAbs binddown from the center of the ACE2 binding site toward the
RBD “mesa” (Fig. 2B and table S2; e.g., CoVIC-080, EMD24346; similar to antibody ADI-56046 (20)).
To simulate the authentic interactions between antibodies and Spike, intact IgGs were used for NS-EM structural
analysis whenever possible. RBD-1 IgGs tend to fully occupy
all three RBDs on one Spike and often crosslink two Spiketrimers, whereas most RBD-2 IgGs tend to bind bivalently toa single Spike trimer (figs. S8, A and B, and S9 and table S3).
RBD-3 IgGs can crosslink Spikes, and bivalent binding was
also observed in some cases (table S3 and fig. S9).
General epitope position, and particularly RBM epitopes,
is strongly associated with the propensity of particular Spikemutations to escape antibody-mediated neutralization (Fig.
3, fig. S10, and table S4). Neutralization by RBD-2a antibodies
is heavily impacted by the K417N mutation, but rarely by the
E484K mutation; those in RBD-2b are impacted by the E484K
mutation but less so by K417N. Similarly, RBD-2a antibodies
are resistant to the L452R mutation found in B.1.429 (epsilon)
and B.1.617.2 (delta), while only some RBD-2b antibodies are
sensitive to this mutation. Meanwhile, mAbs in RBD-3 are
impacted by both N501T/Y and E484K mutations (Fig. 3, figs.
S10 and S11, and table S4). In contrast to RBD-2 and -3, thesusceptibility of neutralization activity of antibodies in RBD1 to particular mutations is more variable (Figs. 2B and 3 andtables S3 and S4).
Regardless of the effect of particular single point mutations, nearly every RBD-1 or -2 mAb analyzed showed additive
decreases in potency against pseudovirus carrying constellations of multiple mutations in the RBM (Fig. 3, fig. S10, andtable S4). For B.1.351 and P.1, almost all RBD-1 and RBD-2
antibodies analyzed suffer a complete loss of neutralization
activity. For example, CoVIC-249 and CoVIC-010 show moderate or no change in IC50 against the single point mutations
K417N, E484K and N501Y, but CoVIC-249 loses all neutralization activity and CoVIC-010 potency falls by 1000-foldagainst B.1.351 (beta) and P.1 (gamma) which contain all
three mutations. Many RBD-2 antibodies also lose activity
against the 4xM mink pseudovariant that carries Y453F,
F486L and N501T mutations (Fig. 3, figs. S1B and S10, andtable S4).
In contrast, most RBD-1 and RBD-2 antibodies retain neutralization activity against B.1.1.7 (alpha), B.1.429 (epsilon)

First release: 23 September 2021

decreased or total loss of neutralization capacity for one ormore of the NTD-located deletions (∆69/70, ∆Y144, ∆157-158
and ∆242-244) found in circulating VOCs, regardless of theirbinding location on NTD (Fig. 4C, fig. S10, and table S4). All
NTD mAbs were impacted by P.1 (gamma), which lacks deletions and instead has several point mutations in the NTD. For
B.1.429 (epsilon), altered disulfide bonding in the NTD arising from the S13I and W152C mutations (13) also abrogatedmAb-mediated neutralization. Our results indicate that NTD
mutations decrease not only neutralization potency but also
the total fraction of virus neutralized (fig. S10).
Several therapeutic antibody cocktails comprising pairs ofdifferent mAbs against Spike are currently under investigation for post-exposure treatment of COVID-19 (16, 17, 31, 32).
However, the potency of some antibodies in these cocktails iscompromised by emerging SARS-CoV-2 variants (33, 34).
Meanwhile, exposure of virus to monoclonal or polyclonal
antibodies can promote antibody-resistant mutations in
Spike (34–37). Notably, SARS-CoV-2 variants that share critical mutations with B.1.1.7 (alpha) were isolated from an
immunocompromised COVID-19 patient who received threerounds of convalescent plasma treatment, indicating thateven a polyclonal therapeutic can drive evolution of resistantvirus strains in unresolved infections (38).
Potency, variant-resistance and the ability to co-bind areimportant considerations when selecting antibodies for therapeutic cocktails. The analysis of the 186 RBD-directed mAbs
presented here, each donated by different groups around theglobe and each selected in different ways, describes discrete
antibody communities, and functionally relevant sub-clusters
and/or bins. This analysis provides a competition grid, and aframework for cocktail selection. Notably, combining thisdata with neutralization potency and mutational analysis canguide selection of broadly protective therapeutic cocktails.
Overall, antibodies from community RBD-1 through RBD4 and those directed against the NTD are generally more potent than antibodies of other communities. The high potency
and non-overlapping epitopes of RBD- and NTD-directed
antibodies make them attractive as pairs for therapeutic
cocktails. However, members of each of these groups are also
highly susceptible to neutralization escape by mutations anddeletions found in emerging VOCs. Indeed, a CoVIC bispecific antibody targeting the RBD-1 and NTD-1 sites could
still neutralize single point mutations in the RBD (where the
NTD arm could compensate), but was ineffective against
B.1.351 (beta) and P.1 (gamma), which contain mutations thatsimultaneously escape both arms of the bispecific (fig. S14).
In contrast, RBD-5, -6 and -7 antibodies often have lower
potency but are more resistant to escape. Notably, theepitopes targeted by RBD-5, -6, and -7 antibodies have highsequence conservation among the Sarbecovirus subgenus of
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antibodies requires two RBDs to be in the “up” configuration(fig. S12). The representative IgGs in RBD-6 and RBD-7 eachshow stronger propensities to crosslink Spike trimers thanRBM-directed antibodies (fig. S9 and table S3). RBD-6 andRBD-7 antibodies primarily vary in their competition with
RBD-2a antibodies: the downward shift of the RBD-7 footprint
on the inner face of the RBD relative to the RBD-6 footprint
would allow simultaneous binding of RBD-2a antibodies with
RBD-7, but not RBD-6, antibodies (Figs. 1B and 2D, fig. S6B,
and table S2). This cryptic RBD-6/7 site is also recognized by
antibodies COVA1-16 (23) and CR3022 (24). Here, strategies ofsite recognition are further subdivided by competition subgroups, information useful for interpreting differences and antibody behavior and strategies for cocktail selection.
All RBD-6 and RBD-7a antibodies block ACE2, but antibodies in RBD-7b and 7c do not (Fig. 1B and tables S1 to S3).
The representatives from the RBD-7b and 7c clusters (CR3022
and CoVIC-021, respectively) demonstrate poor neutralization of pseudoviruses in our assay. The distinct difference inneutralization behavior between 7a and 7b/7c suggests thatat this cryptic epitope, competition with ACE2 is a determinant of neutralization (Fig. 1B and table S4) (25). Importantly,
due to their location away from the RBM, RBD-6 and RBD-7
antibodies are resistant to the mutations and variants analyzed (Fig. 3, fig. S10, and table S4).
Previous reports identified a “supersite” as the primarytarget for neutralizing antibodies directed against the NTD(26). In addition to RBD-directed antibodies, we also analyzed four CoVIC NTD-directed antibodies by NS-EM and inneutralization assays. Together these four antibodies,grouped as NTD-1 through NTD-3, encompass the approximate boundaries of the supersite. The two NTD-1 antibodies
bind from the top side of NTD to cover the NTD N terminus
and residue Y144 (Fig. 4, e.g., CoVIC-247, EMD-24355 and Table S3). The NTD-1 epitope overlaps with that of mAb 4A8(27) and other “supersite” binders (28, 29). The NTD-2 antibody (CoVIC-245, EMD-24360) approaches from the front
side of NTD and contacts Y144 as well as residues H69, V70,
W152 and G261, all of which are deleted or substituted inemerging variants (Fig. 4 and fig. S1). The NTD-2 footprint issimilar to the footprint of antibodies in the “antigenic site V''
group described in (26). The NTD-3 mAb (CoVIC-020, EMD24356) binds to the left side of the NTD, proximal to the RBDof the adjacent monomer and in contact with residue W152
(Fig. 4B). The NTD-3 mAb represents a novel epitope andbinding location of an anti-NTD antibody.
Unlike the RBD-directed antibodies, for which neutralization escape is strongly associated with antibody footprint, theNTD-directed antibodies are conformationally sensitive andaffected by mutations outside of the discrete footprint. Thisfinding is consistent with that for antibodies elicited by vaccines (30). Each of the four NTD mAbs analyzed exhibit acommunities might be achieved through engineering them as
multivalent formats, making them key members of a variantresistant cocktail that could also be suitable for treating otherSarbecovirus infections.
Taken together, the analysis presented here, made possible by broad participation of a few hundred therapeutic candidates in a global study, offers a detailed structural andcompetitive landscape of key antibody binding sites on Spike.
The results of this effort can be used to predict and interpret
effects of VOCs, and for strategic selection of durable therapeutics and cocktails against emerging variants.

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First release: 23 September 2021

ACKNOWLEDGMENTS
We are grateful for the multiple generous contributions of antibodies to the CoVIC
study, with special thanks to Matt Beasley, Shahrad Daraeikia, Vincent Dussupt,
Ben Kiefel, Sindy Liao, Chanjuan Liu, Letzibeth Mendez-Rivera, Pramila Rijal, Lisa
Schimanski, Pete Smith, Tiong Tan, Alain Townsend, Jack Wang, Run Yan, andLuo Yang. We thank the electron microscope facility of La Jolla Institute for
Immunology for the EM data collection. Funding: We gratefully acknowledge
philanthropic support of the Overton family for this urgent study (Coronavirus
Immunotherapeutic Consortium), COVID-19 Therapeutics Accelerator INV006133 (Coronavirus Immunotherapeutic Consortium), Bill and Melinda Gates
Foundation OPP1210938 (Coronavirus Immunotherapeutic Consortium), GHR
foundation (Coronavirus Immunotherapeutic Consortium), and NIH/NIAID grant
U19 AI142790-S1 (Coronavirus Immunotherapeutic Consortium). We also
acknowledge philanthropic support of Carolee Lee, FastGrants from Emergent
Ventures at the Mercatus Center, George Mason University for support ofessential instrumentation, and T.S. for support of the Saphire laboratory efforts
during the pandemic (EOS), Early Postdoc Mobility Fellowship of the Swiss
National Science Foundation P2EZP3_195680 (DZ), and Translating Duke Health
Immunology Initiative (GT). Author contributions: Conceptualization: KH, HL,
SS, EOS. Methodology: KH, SS, HL, DB, SD, KL, GT, EOS. Software: JI, GG, KL.
Validation: KH, HL, SMD, KL, DB, GT, EOS. Formal analysis: KH, HL, DB.
Investigation: KH, HL, SS, DB, DZ, VR, MZ, RDA, CM, TB, XY, SH, KS, CH, JY, EO,
AE, DB, SMD, DA, SD, KL, MA, GH, LF. Resources: HA, RF, JJF, JG, RG, GG, JI,
ALH, WJ, CK, MK, HMK, CK, FL, GL, SL, AL, JL, YA, ALP, PK, RC, MT, AM, ATM,
TR, ZR-T, MS, AS, LS, MJvG, TY, JS, BS, LS, YT, QZ. Data Curation: CoVIC-DB
team, BP, SS. Writing – original draft preparation: KH, HL, SS. Writing – review
and editing: EOS. Visualization: KH, HL, DB, DZ, KL, MS. Supervision: BP, GT, TG,
EOS. Project administration: SS, EOS. Funding acquisition: GT, TG, EOS. The
CoVIC-DB team includes Brendan Ha, Mari Kojima, Mahita Jarjapu, Randi Vita,
Anaїs Gambiez, Jason A. Greenbaum and James A. Overton under the directionof Bjoern Peters. We thank members of the Antibody Dynamics platform of the
Global Health-Vaccine Accelerator Platforms: Sarah Mudrak, Val Bekker, andKaren Makar for program management; David Beaumont and Mark Sampson fordata management; and Nathan Eisel, LaTonya Williams for technical expertise.
Competing interests: Authors declare that they have no competing interests.
Data and materials availability: EM maps in this study have been uploaded to
the EMDataResource. The EMDB access numbers are available in the main text
and table S3. Information concerning particular antibodies can be requested
through the Coronavirus Immunotherapeutics Consortium athttps://covic.lji.org. This work is licensed under a Creative Commons Attribution
4.0 International (CC BY 4.0) license, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is
properly cited. To view a copy of this license, visit
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SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abh2315
Materials and Methods
Figs. S1 to S15
Tables S1 to S4
References (40–56)
MDAR Reproducibility Checklist
24 February 2021; accepted 21 September 2021
Published online 23 September 2021
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Fig. 1 (previous page). The antigenic landscape of the SARS-CoV-2 receptor binding domain can be divided
into seven binding communities. (A) High-throughput SPR was used to determine the competitive relationshipbetween 186 RBD-directed mAbs. The dataset was analyzed by Carterra Epitope software to sort competition
profiles of clones into related clusters, which are represented as shared colored regions of the dendrogram.
The RBD epitope landscape can be broadly divided into seven communities containing mAbs that bind thereceptor-binding motif (RBD-1 through RBD-3), the outer face of the RBD (RBD-4 and RBD-5) or the inner faceof the RBD (RBD-6 and RBD-7). Communities can be further divided into smaller clusters (e.g., RBD-2a and-2b) and bins (e.g., RBD-2b.1, -2b.2 and -2b.3) based on their discrete competition with other clusters and/or
their ability to compete with ACE2 for Spike binding. Black bars indicate single clones that were used in further
analyses. Table S1 lists additional metrics for the indicated mAbs (i.e., ACE2 blocking, kinetic analyses andgermline information) and detailed information for the entire CoVIC panel is at covic.lji.org. (B) Binary heatmap matrix demonstrating the competition profile for the finer clusters and bins for the subset of single clones
indicated by black bars in panel (A). The matrix here contains representative examples. The complete
competition matrix for the study is in table S2. RBD-2 can be divided into clusters “a” and “b”, which havevarying ability to compete with mAbs in RBD-4 (e.g., RBD-2a mAbs do not compete while most RBD-2b mAbs
do). Cluster RBD-2b can be divided into three smaller bins that vary in their competition with both RBD-3 andRBD-4 mAbs: those in 2b.1, but not 2b.2 or 2b.3, compete with RBD-3 mAbs whereas mAbs in 2b.1 and 2b.2,
but not 2b.3, compete with RBD-4 mAbs. RBD-4 contains mAbs that do (RBD-4a) and do not (RBD-4b)
compete with ACE2. RBD-5 and RBD-7 have clusters of mAbs with lower neutralizing potency (i.e., RBD-5c andRBD-7b and RBD-7c) relative to the other cluster in the same community (i.e., RBD-5a and RBD-5b and RBD7a). Rows and columns indicate the immobilized mAb and injected analyte mAb, respectively. Table S2 shows
the complete matrix for competition between all 186 mAbs.

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Fig. 2 (next page). Negative stain EM analysis of representatives from each RBD-directed community.
(A) The location of important emerging mutations in RBD. The Spike trimer (adapted from PDB: 7A94 (39))
viewed from the top with one RBD “up” RBD, is shown, with individual Spike monomers colored white, grayand black. The RBM can be topologically divided into three sub-sections: the “Peak” that includes residues
F486, S477, T478 and E484, the “Valley” including residues Y453, K417 and L452 and the “Mesa” withresidue N501. Stars indicate residues on the central axis of RBD. The “Outer Face” (exposed in the RBD
down/closed conformation), and “Inner Face” (buried inside the trimer in the RBD down/closed
conformation) define the lateral faces of RBD and “Escarpment” (contains residues V367, N439 and glycan343). (B to D) NS-EM footprint of a representative antibody from each community mapped onto an RBD
monomer. The colored shading corresponds to the community colors in Fig. 1. The ACE2 binding site isoutlined with a dotted line. Side and top views of Spike trimers show the Fab approach angle and binding
stoichiometry for each representative. Table S3 shows NS-EM data for all 29 RBD-directed mAbs analyzed.

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Fig. 3. RBD-5, -6 and -7 antibodies retain neutralization activity against pseudovirus bearing mutations
singly or together in VOCs. Fold-change differences in potency for 38 RBD-directed antibodies and an
ACE2-Fc fusion (CoVIC-069) are shown in a heat map. In addition to VOCs, we also examined two
pseudoviruses bearing clusters of mink-associated mutations: 4xM (G261D, Y453F, F486L and N501T) and
5xM (G261D, Y453F, F486L, N501T and V367F). Fig. S1 lists mutations represented in each variant. Fig. S10shows neutralization curves for each virus-variant pair and table S4 lists fold-change values corresponding
to the heat map.

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Fig. 4. NS-EM and neutralization analysis of mAb targeting NTD. (A) Surface and cartoon (adapted from
PDB: 7A94 (39)) representation of the Spike NTD. The residue positions of mutations and deletions incirculating VOCs are indicated in three views of NTD. Fig. S1 lists mutations represented in each variant.
(B) Footprints for three NTD-targeted antibodies with the NTD “supersite” (26) indicated as a dotted line.
The NTD-directed antibodies shown here define the approximate boundaries of the neutralizing epitope
landscape. Additional NS-EM data are in table S3. (C) Fold-change in potency of pseudovirus neutralization
experiments for each antibody-variant pair.

Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global
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Horn, , Yoann Aldon, Hanif Ali, Sanja Aracic, Ronald R. Cobb, Ross S. Federman, Joseph M. Fernandez, Jacob Glanville,
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Cheuk Lun Leung, Junli Liu, Yanan Lu, Anna MacCamy, Andrew T. McGuire, Anne L. Palser, Terence H. Rabbitts, Zahra
Rikhtegaran Tehrani, Mohammad M. Sajadi, Rogier W. Sanders, Aaron K. Sato, Liang Schweizer, Jimin Seo, Bingqing Shen,
Jonne J. Snitselaar, Leonidas Stamatatos, Yongcong Tan, Milan T. Tomic, Marit J. van Gils, Sawsan Youssef, Jian Yu, Tom Z.
Yuan, Qian Zhang, Bjoern Peters, Georgia D. Tomaras, Timothy Germann, and Erica Ollmann Saphire

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