CORONAVIRUS

Structural basis for neutralization of SARS-CoV-2 andSARS-CoV by a potent therapeutic antibodyZhe Lv1,2*, Yong-Qiang Deng3*, Qing Ye3*, Lei Cao1*, Chun-Yun Sun4*, Changfa Fan5*, Weijin Huang6,Shihui Sun3, Yao Sun1, Ling Zhu1, Qi Chen3, Nan Wang1,2, Jianhui Nie6, Zhen Cui1,2, Dandan Zhu1,Neil Shaw1, Xiao-Feng Li3, Qianqian Li6, Liangzhi Xie4,7,8†, Youchun Wang6†, Zihe Rao1†,Cheng-Feng Qin3†, Xiangxi Wang1,2†

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. There are noapproved vaccines or therapeutics for treating COVID-19. Here we report a humanized monoclonalantibody, H014, that efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as wellas authentic SARS-CoV-2 at nanomolar concentrations by engaging the spike (S) receptor bindingdomain (RBD). H014 administration reduced SARS-CoV-2 titers in infected lungs and preventedpulmonary pathology in a human angiotensin-converting enzyme 2 mouse model. Cryo–electronmicroscopy characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragmentunveiled a previously uncharacterized conformational epitope, which was only accessible whenthe RBD was in an open conformation. Biochemical, cellular, virological, and structural studiesdemonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitopeanalysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncovered broadcross-protective epitopes. Our results highlight a key role for antibody-based therapeuticinterventions in the treatment of COVID-19.

The coronavirus disease 2019 (COVID-19)pandemic has afflicted >7 million peoplein more than 200 countries and regions,resulting in 400,000 deaths as of 8 June,according to the World Health Organi-

zation. The etiological agent of this pandemicis the newly emerging coronavirus, severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2), which, together with the closely relatedSARS-CoV, belongs to the lineage B of thegenus Betacoronavirus in the Coronaviridaefamily (1). Sharing an amino acid sequenceidentity of ~80% in the envelope-locatedspike (S) glycoprotein, both SARS-CoV-2 andSARS-CoV use human angiotensin-convertingenzyme 2 (hACE2) to enter host cells. Cel-lular entry is achieved by the homotrimericS-mediated virus-receptor engagement through

the receptor-binding domain (RBD) followedby virus-host membrane fusion (1, 2). Abroga-tion of this crucial role played by the S proteinin the establishment of an infection is themaingoal of neutralizing antibodies and the focusof therapeutic interventions as well as vaccinedesign (3–6). Several previously characterizedSARS-CoV neutralizing antibodies (NAbs) weredemonstrated to exhibit very limited neutral-ization activities against SARS-CoV-2 (7–9).Among these, CR3022, a weakly neutralizingantibody against SARS-CoV, is tight binding,but non-neutralizing for SARS-CoV-2, indica-tive of possible conformational differences inthe neutralizing epitopes (9). More recentstudies have reported two SARS-CoV neutral-izing antibodies, 47D11 and S309, that havebeen shown to neutralize SARS-CoV-2 as well(10, 11), suggesting that broad cross-neutralizingepitopes exist within lineage B. Convalescentplasma containingSARS-CoV-2NAbshave beenshown to confer clear protection in COVID-19patients (12, 13), yet gaps in our knowledgeconcerning the immunogenic features and keyepitopes of SARS-CoV-2 have hampered thedevelopment of effective immunotherapeuticsagainst the virus.The RBDs of SARS-CoV and SARS-CoV-2

have an amino acid sequence identity of around75%, raising the possibility that RBD-targetingcross-neutralizing NAbs could possibly be iden-tified. Using a phage display technique, weconstructed an antibody library that wasgenerated from RNAs extracted from periph-eral lymphocytes of mice immunized withrecombinant SARS-CoV RBD. SARS-CoV-2RBD was used as the target for screening

the phage antibody library for potential hits.Antibodies that showed tight binding forSARS-CoV-2 RBD were further propagated aschimeric antibodies and tested for neutraliz-ing activities with a vesicular stomatitis virus(VSV)–based pseudotyping system (fig. S1) (14).Among the antibodies tested, clone 014, whichshowed potent neutralizing activity againstSARS-CoV-2 pseudovirus, was humanized andnamed H014. To evaluate the binding affin-ities, we monitored real-time association anddissociation of H014 binding to either SARS-CoV-2RBDor SARS-CoVRBDusing theOCTETsystem (Fortebio). Both H014 immunoglobulinG (IgG) and Fab fragments exhibited tight bind-ing to both RBDs with comparable bindingaffinities at subnanomolar concentrations forSARS-CoV-2 RBD and SARS-CoV RBD, respec-tively (Fig. 1A and fig. S2). Pseudovirus neu-tralization assays revealed thatH014 has potentneutralizing activities: a 50% neutralizingconcentration value (IC50) of 3 nM and 1 nMagainst SARS-CoV-2 and SARS-CoV pseudo-viruses, respectively (Fig. 1B). Plaque-reductionneutralization test (PRNT) conducted againstan authentic SARS-CoV-2 strain (BetaCoV/Beijing/AMMS01/2020) verified the neutral-izing activities with an IC50 of 38 nM, 10-foldlower than those observed in the pseudotyp-ing system (Fig. 1C). We next sought to assessin vivo protection efficacy of H014 in our pre-viously established hACE2 humanizedmousemodel that was sensitized to SARS-CoV-2 in-fection (15). In this model, as a result of hACE2expression on lung cells, SARS-CoV-2 gainsentry into the lungs and replicates as in a hu-mandisease, exhibiting lung pathology at 5 dayspost-infection (dpi). hACE2-humanized micewere treated by intraperitoneal injection ofH014 at 50 mg per kilogram of body weighteither 4 hours after (one dose, therapeutic) or12 hours before and 4 hours after (two doses,prophylactic plus therapeutic) intranasal in-fectionwith 5 × 105 plaque-forming units (PFU)of SARS-CoV-2 (BetaCoV/Beijing /AMMS01/2020). All challenged animals were sacrificedat day 5. Whereas the viral loads in the lungsof the phosphate-buffered saline (PBS) group(control) increased rapidly to ~107 RNA copies/gat day 5 (Fig. 1D), in the prophylactic andprophylactic plus therapeutic groups, H014treatment resulted in a ~10-fold and 100-foldreduction of viral titers in the lungs at day 5,respectively (Fig. 1D). Lung pathology analysisshowed that SARS-CoV-2 caused mild inter-stitial pneumonia characterized by inflamma-tory cell infiltration, alveolar septal thickening,and distinctive vascular system injury uponPBS treatment. By contrast, no obvious lesionsof alveolar epithelial cells or focal hemorrhagewere observed in the lung sections from micethat received H014 treatment (Fig. 1E), indic-ative of a potential therapeutic role forH014 intreating COVID-19.

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1CAS Key Laboratory of Infection and Immunity, NationalLaboratory of Macromolecules, Institute of Biophysics,Chinese Academy of Sciences, Beijing 100101, China.2University of Chinese Academy of Sciences, Beijing 100049,China. 3State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology, Academy ofMilitary Medical Sciences, Beijing, China. 4Beijing Proteinand Antibody R&D Engineering Center, Sinocelltech Ltd.,Beijing 100176, China. 5Division of Animal Model Research,Institute for Laboratory Animal Resources, NationalInstitutes for Food and Drug Control (NIFDC), Beijing102629, China. 6Division of HIV/AIDS and Sex-TransmittedVirus Vaccines, Institute for Biological Product Control,National Institutes for Food and Drug Control (NIFDC),Beijing 102629, China. 7Beijing Antibody Research KeyLaboratory, Sino Biological Inc., Building 9, Jing Dong BeiTechnology Park, No.18 Ke Chuang 10th St, BDA, Beijing,100176, China. 8Cell Culture Engineering Center, ChineseAcademy of Medical Sciences & Peking Union MedicalCollege, Beijing 100005, China.*These authors contributed equally to this work.†Corresponding author. Email: xiangxi@ibp.ac.cn (X.W.); qincf@bmi.ac.cn (C.-F.Q.); wangyc@nifdc.org.cn (Y.W.); liangzhi@yahoo.com (L.X.); raozh@tsinghua.edu.cn (Z.R.)

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The overall structure of SARS-CoV-2 S trimerresembles those of SARS-CoV and other corona-viruses. Each monomer of the S protein iscomposed of two functional subunits. The S1subunit binds the host cell receptor, whereasthe S2 subunit mediates fusion of the viralmembranewith the host cell membrane (2, 16).The four domainswithin S1 includeN-terminaldomain (NTD), RBD, and two subdomains (SD1and SD2), the latter of which are positionedadjacent to the S1 and S2 cleavage site. Hinge-like movements of the RBD give rise to twodistinct conformational states referred to as“close” and “open,” where close correspondsto the receptor-inaccessible state and opencorresponds to the receptor-accessible state,which is reported to be metastable (17–20).Cryo–electron microscopy (cryo-EM) charac-terization of the stabilized SARS-CoV-2 Sectodomain in complex with the H014 Fabfragment revealed that the complex adoptsthree distinct conformational states, corre-sponding to one RBD open + two RBDs closed(state 1), two RBDs open + one RBD closed(state 2), and all three open RBDs (state 3) (Fig.2A). The structure of the completely closed(state 4) SARS-CoV-2 S trimer without anyFab bound was also observed during three-

dimensional (3D) classification of the cryo-EMdata, albeit in the presence of excessive Fab,suggesting that the binding sites of H014 areexposed through protein “breathing” followedby a stochastic RBD movement (Fig. 2A). Wedetermined asymmetric cryo-EM reconstruc-tions of the four states at 3.4 to 3.6 Å (figs. S3to S6 and table S1). However, the electron po-tential maps for the binding interface betweenRBD and H014 are relatively weak, owing toconformational heterogeneity. To solve thisproblem, we performed focusing classificationand refinement by using a “block-based” re-construction approach to further improve thelocal resolution up to 3.9 Å, enabling reliableanalysis of the interaction mode (figs. S5 andS6). Detailed analysis of the interactions be-tween H014 and S was done using the bindinginterface structure.H014 recognizes a conformational epitope

on one side of the open RBD, only involvingprotein–protein contacts, distinct from thereceptor-binding motif (RBM) (Fig. 2B). TheH014 paratope constitutes all six complementary-determining region (CDR) loops (CDRL1 to -3and CDRH1 to -3) and the unusual heavy-chain framework (HF-R, residues 58 to 65)that forges tight interactions with the RBD,

resulting in a buried area of ~1000 Å2 (Fig.2C). Variable domains of the light chain andheavy chain contribute ~32 and 68% of theburied surface area, respectively, through hy-drophobic and hydrophilic contacts. The H014epitope is composed of 21 residues, primarilylocated in the a2-b2-h2 (residues 368 to 386),h3 (residues 405 to 408 and 411 to 413), a4(residue 439), and h4 (residues 503) regions,which construct a cavity on one side of theRBD (Fig. 2, B and D, and fig. S7). The 12-residue-long CDRH3 inserts into this cavity,and the hydrophobic residue (YDPYYVM)–enriched CDRH3 contacts the h3 and edge ofthe five-stranded b-sheet (b2) region of theRBD (Fig. 2D). Tight binding between the RBDand H014 is primarily due to extensive hydro-phobic interactions contributed by two patches:one formed by F54 from CDRH2; Y101 fromCDRH3; and A411, P412, and Y508 of the RBDand the other composed of Y49 from CDRL2;P103, Y104, and Y105 from CDRH3; and V407,V503, and Y508 of the RBD (Fig. 2E and tableS2). Additionally, hydrophilic contacts fromCDRH1 and HF-R further enhance the RBD-H014 interactions, leading to an extremelyhigh binding affinity at subnanomolar con-centration at temperatures of 25° or 37°C (Fig.

Lv et al., Science 369, 1505–1509 (2020) 18 September 2020 2 of 5

Fig. 1. H014 is a lineage B cross-neutralizing antibody of therapeuticvalue. (A) Affinity analysis of the bindingof H014 to RBD of SARS-CoV-2 andSARS-CoV. Biotinylated RBD proteins of(upper) SARS-CoV-2 or (lower) SARS-CoV were loaded on Octet SA sensor andtested for real-time association anddissociation of the H014 antibody. Globalfit curves are shown as black dottedlines. The vertical dashed lines indicatethe transition between association anddisassociation phases. (B) Neutralizingactivity of H014 against SARS-CoV-2 andSARS-CoV pseudoviruses (PSV). Serialdilutions of H014 were added to test itsneutralizing activity against (upper)SARS-CoV-2 and (lower) SARS-CoV PSV.Neutralizing activities are representedas mean ± SD. Experiments wereperformed in triplicate. (C) In vitroneutralization activity of H014 againstSARS-CoV-2 by PRNT in Vero cells.Neutralizing activities are representedas mean ± SD. Experiments were per-formed in duplicate. (D) Groups of hACE2mice challenged with SARS-CoV-2 weretreated intraperitoneally with H014 in twoindependent experimental settings: (i) asingle dose at 4 hours after infection(therapeutic, T); and (ii) two doses at12 hours before and 4 hours after challenge (prophylactic plus therapeutic, P+T). Virus titers in the lungs were measured 5 days post-infection (dpi) and are presentedas RNA copies per gram of lung tissue. n = 7, 3, and 3, respectively; (n: number of independent experiments of each group). *P < 0.05, **P < 0.01. Red dashedline represents limit of detection. (E) Histopathological analysis of lung samples at 5 dpi. Scale bars, 100 mm.

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2E and fig. S8). Residues that constitute theepitope aremostly conserved,with three single-sitemutants (R408I, N439K, andV503F) in thisregion among currently circulating SARS-CoV-2 strains reported (fig. S9). In addition, anumber of SARS-CoV-2 isolates bear a com-monmutation, V367F, in the RBD (21), whichlies adjacent to themajor epitopepatcha2-b2-h2.Constructs of the recombinant RBD harbor-ing point mutations of the above-mentionedresidues and other reported substitutions ex-hibited an indistinguishable binding affinityfor H014 (fig. S10), suggesting that H014 mayexhibit broad neutralization activities againstSARS-CoV-2 strains currently circulating world-wide. Of the 21 residues in the H014 epitope, 17(81%) are identical between SARS-CoV-2 andSARS-CoV (fig. S9), which explains the cross-reactivity and comparable binding affinities.To investigate whether H014 interferes with

the binding of RBDs of SARS-CoV-2 or SARS-CoV to ACE2, we performed competitive bind-ing assays at both protein and cellular levels.The enzyme-linked immunosorbent assay(ELISA) indicated that H014 competed withrecombinant ACE2 for binding to RBDs ofSARS-CoV-2 and SARS-CoV with EC50 valuesof 0.7 and 5 nM, respectively (Fig. 3A). Ad-ditionally, H014 efficiently blocked both theattachment of SARS-CoV-2 RBD to ACE2-expressing 293T cells and the binding ofrecombinant ACE2 to SARS-CoV-2 S expressing

293T cells (Fig. 3B). To verify its potential fullocclusion on trimeric S, we conducted two setsof surface plasmon resonance (SPR) assays byexposing the trimeric S first toH014 and then toACE2 or vice versa. As expected, binding ofH014 completely blocked the attachment ofACE2 to trimeric S. Moreover, ACE2 could bedisplaced from trimeric S and replaced by H014(Fig. 3C). To further verify these results in a cell-based viral infection model, we performed realtime reverse transcription–quantitative poly-merase chain reaction (RT-PCR) analysis toquantify the amount of virus remaining onthe host cell surface, which was exposed toantibodies before or after attachment of virusto cells at 4°C. H014 efficiently preventedattachment of SARS-CoV-2 to the cell surfacein a dose-dependent manner, and the viralparticles that had already bound to the cellsurface could be partially stripped by H014(Fig. 3D). Superimposition of the structureof the H014-SARS-CoV-2 trimeric S complexover the ACE2–SARS-CoV-2 RBD complexstructure revealed clashes between the ACE2and H014, arising from an overlap of the re-gions belonging to the binding sites locatedat the apical helix (h4) of the RBD (Fig. 3E).This observation differs substantially fromthose of most known SARS RBD-targetedantibody complexes, where the antibodiesdirectly recognize the RBM (22–25). Thus,the ability of H014 to prevent SARS-CoV-2

from attaching to host cells can be attributedto steric clashes with ACE2.Similar to the RBM, theH014 epitope is only

accessible in the open state, indicative of a roleakin to that of the RBM—involving dynamicinterferences in interactions with host cells.Our structures, together with previously re-ported coronavirus S structures, not includinghuman coronavirus HKU1 (20), have observedthe breathing of the S1 subunit, which me-diates the transition between “closed” and“open” conformation (Fig. 4A) (2, 9, 20). Incontrast to the hinge-like movement of theRBD observed in most structures, the confor-mational transition from “closed” to “open”observed in our structures mainly involves twosteps of rotations: (i) counterclockwise move-ment of SD1 by ~25° encircling the hinge point(at residue 320); (ii) counterclockwise rotationby ~60° of the RBD itself (Fig. 4B). Theseconformational rotations of the SD1 and RBDat proximal points relay an amplified altera-tion at the distal end, leading to opening upof adequate space for the binding of H014 orACE2 (Fig. 4B). Ambiguously, the special con-formational transition observed in our com-plex structures results from the engagementof H014 or a synergistic movement of the SD1and RBD.Humoral immunity is essential for protec-

tion against coronavirus infections, and passiveimmunization of convalescent plasma has been

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Fig. 2. Cryo-EM structures of the SARS-CoV-2S trimer in complex with H014. (A) Orthogonal viewsof SARS-CoV-2 S trimer with three RBDs in theclosed state (left), one RBD in the open state andcomplexed with one H014 Fab (middle), two RBDs inthe open state and each complexed with one H014Fab. NTD, N-terminal domain. All structures arepresented as molecular surfaces with different colorsfor each S monomer (cyan, violet, and yellow), andthe H014 Fab light (hot pink) and heavy (purple-blue)chains. (B) Cartoon representations of the structureof SARS-CoV-2 RBD in complex with H014 Fab withthe same color scheme as in (A). Residues thatconstitute the H014 epitope and the RBM are shownas spheres and colored in green and blue, respec-tively. The overlapped residues between the H014epitope and the RBM are shown in red. (C andD) Interactions between the H014 and SARS-CoV-2RBD. The CDRs of the H014 that interact withSARS-CoV-2 RBD are displayed as thick tubes overthe cyan surface of the RBD (C). The H014 epitope isshown as a cartoon representation over the surfaceof the RBD (D). (E) Details of the interactionsbetween the H014 and SARS-CoV-2 RBD. Someresidues involved in the formation of hydrophobicpatches and hydrogen bonds are shown as sticksand labeled. Color scheme is the same as in (A).Abbreviations for the amino acid residues: A, Ala;D, Asp; F, Phe; G, Gly; K, Lys; L, Leu; N, Asn; P, Pro;Q, Gln; S, Ser; T, Thr; V, Val; and Y, Tyr.

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demonstrated to be effective in treating SARSand COVID-19 (12, 26). A number of SARS-CoV–specific neutralizing antibodies—includingm396, 80R, and F26G19—have been previouslyreported to engage primarily the RBM core(residues 484 to 492 in SARS-CoV) providedthat the RBD is in an open conformation (Fig.4, C and D) (24, 27, 28). Unexpectedly, none ofthese antibodies has so far demonstrated im-pressive neutralizing activity against SARS-CoV-2 (7, 8). S230, a SARS-CoV neutralizingantibody that functionally mimics receptorattachment and promotes membrane fusion,despite requiring an open state of the RBD aswell, recognizes a small patch at the top of theRBM (Fig. 4, C and D) (17). Recently, two otherantibodies (CR3022 and S309) originatingfrom the memory B cells of SARS survivorsin 2003, demonstrated strong binding toSARS-CoV-2 (9, 10). However, CR3022 failed toneutralize SARS-CoV-2 in vitro, which couldbe attributed to the binding of CR3022 to acryptic epitope, which is distal from the RBDand is only accessible when at least two RBDsare in the open state. Furthermore, CR3022does not compete with ACE2 for binding tothe RBD (Fig. 4, C and D) (9). By contrast,S309, a more recently reported neutralizingmonoclonal antibody (mAb) against both SARS-CoV and SARS-CoV-2 recognizes a conservedglycan-containing epitope accessible in both theopen and closed states of SARS-CoV and SARS-CoV-2 RBDs (10). The neutralization mecha-nism of S309 does not depend on directblocking of receptor binding, but it inducesantibody-dependent cell cytotoxicity (ADCC)and antibody-dependent cellular phagocyto-sis (ADCP). We reported a potent therapeuticantibody, H014, which can cross-neutralizeSARS-CoV-2 andSARS-CoV infections by block-ing attachment of the virus to the host cell (Fig.3). Distinct from other antibodies, H014 bindsone full side of the RBD, spanning from theproximal h2 to the distal edge of the RBM, andpossesses partially overlapping epitopes withSARS-CoV–specific antibodies such as m392,80R, F26G19, CR3022, and S309 (Fig. 4, C andD). Although both SARS-CoV and SARS-CoV-2use ACE2 to enter host cells, high sequencevariations in theRBMand local conformationalrearrangements at the distal end of the RBMfrom the two viruses have been observed (8).These probably lead to the failure of some anti-bodies, especially those targeting theRBMofSARS-CoV, in cross-binding and cross-neutralizingSARS-CoV-2. Three cross-reactive antibodies(CR3022, S309, andH014) recognizemore con-served epitopes located beyond the RBM, amongwhich the CR3022 epitope is most distantfrom the RBM (Fig. 4, C and D), suggesting apossible reason for its inability to neutralizeSARS-CoV-2. H014 binds more conserved epi-topes, someofwhich are in close proximity to theRBM, rendering it effective in cross-neutralizing

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Fig. 3. Mechanism of neutralization of H014. (A) Competitive binding assays by ELISA. RecombinantSARS-CoV-2 (upper) or SARS-CoV (lower) RBD protein was coated on 96-well plates; recombinant ACE2 andserial dilutions of H014 were then added for competitive binding to SARS-CoV-2 or SARS-CoV RBD. Valuesare the mean ± SD. Experiments were performed in triplicate. (B) Blocking of SARS-CoV-2 RBD binding to293T-ACE2 cells by H014 (upper). Recombinant SARS-CoV-2 RBD protein and serially diluted H014 wereincubated with ACE2-expressing 293T cells (293T-ACE2) and tested for binding of H014 to 293T-ACE2 cells.Competitive binding of H014 and ACE2 to SARS-CoV-2-S cells (lower). Recombinant ACE2 and seriallydiluted H014 were incubated with 293T cells expressing SARS-CoV-2 spike protein (SARS-CoV-2-S) andtested for binding of H014 to SARS-CoV-2-S cells. Bovine serum albumin was used as a negative control(NC). Values are mean ± SD. Experiments were performed in triplicate. (C) BIAcore SPR kinetics ofcompetitive binding of H014 and ACE2 to SARS-CoV-2 S trimer. For both panels, SARS-CoV-2 S trimerwas loaded onto the sensor. In the upper panel, H014 was first injected, followed by ACE2, whereas in thelower panel, ACE2 was injected first and then H014. The control groups are depicted by black curves.(D) Amount of virus on the cell surface, as detected by RT-PCR. Preattachment mode: Incubate SARS-CoV-2and H014 first, then add the mixture into cells (left); postattachment mode: Uncubate SARS-CoV-2 and cellsfirst, then add H014 into virus-cell mixtures (right). High concentrations of H014 prevented attachment ofSARS-CoV-2 to the cell surface when SARS-CoV-2 was exposed to H014 before cell attachment. Valuesrepresent mean ± SD. Experiments were performed in duplicates. (E) Clashes between H014 Fab and ACE2upon binding to SARS-CoV-2 S. H014 and ACE2 are represented as the molecular surface; SARS-CoV-2 Strimer is shown in ribbon representation. Inset is a zoomed-in view of the interactions of the RBD, H014, andACE2 and the clashed region (oval ellipse) between H014 and ACE2. The H014 Fab light and heavy chains,ACE2, and RBD, are presented as cartoons. The epitope, RBM, and the overlapped binding region of ACE2and H014 on RBD are highlighted in green, blue, and red, respectively.

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lineage B viruses. Additionally, antibodies re-cognizing more conserved patches beyondthe RBM would function synergistically withantibodies targeting the RBM to enhance neu-tralization activities, which could mitigate therisk of viral escape. The molecular features ofH014 epitopes unveiled in this study facilitatethe discovery of broad cross-neutralizing epi-topes within lineage B and pose interestingtargets for structure-based rational vaccinedesign. Our studies also highlight the promiseof antibody-based therapeutic interventionsfor the treatment of COVID-19.

REFERENCES AND NOTES

1. P. Zhou et al., Nature 579, 270–273 (2020).2. A. C. Walls et al., Cell 181, 281–292.e6 (2020).

3. Q. Gao et al., Science 369, 77–81 (2020).4. D. Corti et al., Proc. Natl. Acad. Sci. U.S.A. 112, 10473–10478

(2015).5. J. Pallesen et al., Proc. Natl. Acad. Sci. U.S.A. 114,

E7348–E7357 (2017).6. A. C. Walls et al., Nat. Struct. Mol. Biol. 23, 899–905

(2016).7. X. Tian et al., Emerg. Microbes Infect. 9, 382–385 (2020).8. J. Lan et al., Nature 581, 215–220 (2020).9. M. Yuan et al., Science 368, 630–633 (2020).10. D. Pinto et al., bioRxiv 2020.04.07.023903 [Preprint].

10 April 2020. https://doi.org/10.1101/2020.04.07.023903.11. C. Wang et al., Nat. Commun. 11, 2251 (2020).12. A. Casadevall, L. A. Pirofski, J. Clin. Invest. 130, 1545–1548

(2020).13. C. Shen et al., JAMA 323, 1582 (2020).14. J. Nie et al., Emerg. Microbes Infect. 9, 680–686 (2020).15. S. H. Sun et al., Cell Host Microbe 28, 124–133.e4 (2020).16. D. Wrapp et al., Science 367, 1260–1263 (2020).17. A. C. Walls et al., Cell 176, 1026–1039.e15 (2019).18. M. Gui et al., Cell Res. 27, 119–129 (2017).19. Y. Yuan et al., Nat. Commun. 8, 15092 (2017).

20. R. N. Kirchdoerfer et al., Nature 531, 118–121 (2016).21. Z. Z. Junxian Ou et al., bioRxiv 2020.03.15.991844

[Preprint]. 23 March 2020. https://doi.org/10.1101/2020.03.15.991844.

22. J. ter Meulen et al., PLOS Med. 3, e237 (2006).23. J. Sui et al., Proc. Natl. Acad. Sci. U.S.A. 101, 2536–2541 (2004).24. E. N. van den Brink et al., J. Virol. 79, 1635–1644 (2005).25. J. D. Berry et al., J. Virol. Methods 120, 87–96 (2004).26. A. C. Cunningham, H. P. Goh, D. Koh, Crit. Care 24, 91 (2020).27. P. Prabakaran et al., J. Biol. Chem. 281, 15829–15836

(2006).28. W. C. Hwang et al., J. Biol. Chem. 281, 34610–34616 (2006).

ACKNOWLEDGMENTS

We thank X. Huang, B. Zhu, and G. Ji for cryo-EM data collectionand the Center for Biological imaging (CBI) at the Institute ofBiophysics for EM work. This study was supported by the NationalKey Research and Development Program (2020YFA0707500,2018YFA0900801), the Strategic Priority Research Program(XDB29010000), National Science and Technology MajorProjects of Infectious Disease funds (grants 2017ZX103304402),Beijing Municipal Science and Technology Project (Z201100005420017),and Zhejiang Provincial Basic Public Welfare Research Project(LED20C010001). X.W. was supported by the Ten Thousand TalentProgram and the NSFS Innovative Research Group (no. 81921005).C.-F.Q. was supported by the National Science Fund forDistinguished Young Scholar (no. 81925025) and the InnovativeResearch Group (no. 81621005) from the NSFC, and the InnovationFund for Medical Sciences (no. 2019-I2M-5-049) from the ChineseAcademy of Medical Sciences. L.X. and C.-Y.S. are inventorson patent application (202010219867.1) submitted by SinocelltechLtd. that covers the intellectual property of H014. Authorcontributions: Z.L., Y.-Q.D., C.-Y.S., Q.Y., L.C., C.F., W.H., Y.S.,Q.C., S.S., N.W., D.Z., J.N., Z.C., N.S., and X.-F.L. performedexperiments; X.W., C.-F.Q., Z.R., L.X., and Y.W. designed the study;all authors analyzed data; and X.W., L.Z., C.-F.Q., and Z.R. wrotethe manuscript. Competing interests: All authors have nocompeting interests. Data and materials availability: Cryo-EMdensity maps of the apo SARS-CoV-2 S trimer, SARS-CoV-2S trimer in complex with one Fab, SARS-CoV-2 S trimer in complexwith two Fabs, SARS-CoV-2 S trimer in complex with three Fabs,and binding interface have been deposited at the ElectronMicroscopy Data Bank with accession codes EMD-30325, EMD-30326, EMD-30332, EMD-30333, and EMD-30331, and relatedatomic models have been deposited in Protein Data Bankunder accession codes 7CAB, 7CAC, 7CAI, 7CAK, and 7CAH,respectively. H014 is available from Sinocelltech Group under amaterial transfer agreement with Sinocelltech. This work islicensed under a Creative Commons Attribution 4.0 International(CC BY 4.0) license, which permits unrestricted use,distribution, and reproduction in any medium, provided theoriginal work is properly cited. To view a copy of this license,visit https://creativecommons.org/licenses/by/4.0/. This licensedoes not apply to figures/photos/artwork or other contentincluded in the article that is credited to a third party;obtain authorization from the rights holder before usingsuch material.”

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/369/6510/1505/suppl/DC1Materials and MethodsFigs. S1 to S10Tables S1 and S2References (29–41)

View/request a protocol for this paper from Bio-protocol.

3 May 2020; accepted 16 July 2020Published online 23 July 202010.1126/science.abc5881

Lv et al., Science 369, 1505–1509 (2020) 18 September 2020 5 of 5

Fig. 4. Breathing of the S1 subunit and epitopes of neutralizing antibodies. (A) H014 can only interactwith the “open” RBD, whereas the “closed” RBD is inaccessible to H014. The open RBD and RBD-boundH014 are depicted in lighter colors corresponding to the protein chain that they belong to. Color scheme isthe same as in Fig. 2A. (B) Structural rearrangements of the S1 subunit of SARS-CoV-2 transition fromthe closed state to the open state. SD1, subdomain 1; SD2, subdomain 2; RBD’, RBD (closed state) fromadjacent monomer. SD1, SD2, NTD, RBD, and RBD’ are colored in pale green, light orange, cyan, blueand yellow, respectively. The red dot indicates the hinge point. The angles between the RBD and SD1 arelabeled. (C) Epitope location analysis of neutralizing antibodies on SARS-CoV and SARS-CoV-2 S trimers.The S trimer structures with one RBD open and two RBD closed from SARS-CoV and SARS-CoV-2were used to show individual epitope information, which is highlighted in green. The accessible andinaccessible states are encircled and marked by green ticks and red crosses. (D) Footprints of theseven mAbs on RBDs of SARS-CoV (left) and SARS-CoV-2 (right). RBDs are rendered as molecularsurfaces in light blue. Footprints of different mAbs are highlighted in different colors as labeled in thegraph. Epitopes recognized by the indicated antibodies are labeled in blue (non-overlapped), yellow(overlapped once), and red (overlapped twice).

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antibodyStructural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic

Zihe Rao, Cheng-Feng Qin and Xiangxi WangChen, Nan Wang, Jianhui Nie, Zhen Cui, Dandan Zhu, Neil Shaw, Xiao-Feng Li, Qianqian Li, Liangzhi Xie, Youchun Wang, Zhe Lv, Yong-Qiang Deng, Qing Ye, Lei Cao, Chun-Yun Sun, Changfa Fan, Weijin Huang, Shihui Sun, Yao Sun, Ling Zhu, Qi

originally published online July 23, 2020DOI: 10.1126/science.abc5881 (6510), 1505-1509.369Science 

, this issue p. 1505Sciencereceptor-binding domain of the spike and blocking its attachment to the host receptor.together with biochemical, cellular, and virological studies, showed that the antibody acts by binding to the

electron microscopy structure,−monoclonal antibody that protected against SARS-CoV-2 in a mouse model. The cryo report a humanized et al.neutralize the virus hold promise as therapeutics and could inform vaccine design. Lv

makes antibodies, many of which target the spike protein, a key player in host cell entry. Antibodies that potently In response to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the immune system

A steric block to SARS-CoV-2

ARTICLE TOOLS http://science.sciencemag.org/content/369/6510/1505

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REFERENCES

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