key: cord-1031461-60gscdyu
authors: Du, Shuo; Cao, Yunlong; Zhu, Qinyu; Wang, Guopeng; Du, Xiaoxia; He, Runsheng; Xu, Hua; Zheng, Yinghui; Wang, Bo; Bai, Yali; Ji, Chenggong; Yisimayi, Ayijiang; Wang, Qisheng; Gao, Ning; Xie, X. Sunney; Su, Xiao-dong; Xiao, Junyu
title: Structures of potent and convergent neutralizing antibodies bound to the SARS-CoV-2 spike unveil a unique epitope responsible for exceptional potency
date: 2020-07-09
journal: bioRxiv
DOI: 10.1101/2020.07.09.195263
sha: b25e03f639f3d5939589836371606d0f28daa8d5
doc_id: 1031461
cord_uid: 60gscdyu

Understanding the mechanism of neutralizing antibodies (NAbs) against SARS-CoV-2 is critical for effective vaccines and therapeutics development. We recently reported an exceptionally potent NAb, BD-368-2, and revealed the existence of VH3-53/VH3-66 convergent NAbs in COVID-19. Here we report the 3.5-Å cryo-EM structure of BD-368-2’s Fabs in complex with a mutation-induced prefusion-state-stabilized spike trimer. Unlike VH3-53/VH3-66 NAbs, BD-368-2 fully blocks ACE2 binding by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their “up” and “down” positions. BD-368-2 also triggers fusogenic-like structural rearrangements of the spike trimer, which could impede viral entry. Moreover, BD-368-2 completely avoids the common epitope of VH3-53/VH3-66 NAbs, evidenced by multiple crystal structures of their Fabs in tripartite complexes with RBD, suggesting a new way of pairing potent NAbs to prevent neutralization escape. Together, these results rationalize a unique epitope that leads to exceptional neutralization potency, and provide guidance for NAb therapeutics and vaccine designs against SARS-CoV-2.

Coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 38 (SARS-CoV-2), has become a global pandemic (Callaway et al., 2020 ). An important structural protein of 629. In CDRH2, Tyr58 not only mediates packing interactions as described above for Phe58 in BD-604 but 137 also forms hydrogen bonds with Thr415 S ( Figure S4B ). In CDRH3, Tyr102 is present, together with two 138 additional tyrosine residues, Tyr99 and Tyr103 ( Figure S4A ). Tyr102 is slightly pushed away by Tyr103 to we first attempted to obtain the crystal structure of BD-368-2 Fab in complex with RBD. However, this RBD together with the Fabs of several VH3-53/VH3-66 antibodies. We subsequently determined the crystal 165 structures of three tripartite complexes consisting of the Fabs of these antibodies and RBD: BD-and RBD are highly similar to those seen in the binary complexes described above ( Figure S5C -E). Five loop in the VH domain, and light chain CDRL1 and CDRL2 ( Figure 5A ). The remaining two CDRs, especially CDRH2, do not directly contact RBD, suggesting that the interaction between BD-368-2 and RBD 175 could be further enhanced by structure-based protein engineering. Gly26, Phe27, and Ala28 in CDRH1 176 cradle Tyr449 S ( Figure 5B ). Tyr32 in CDRH1 and Arg102 in CDRH3 together form robust packing with present between the heavy chain CDRs and RBD, involving heavy chain residues Arg100, Tyr105, Asp106, and RBD residues Gly482 S , Glu484 S ( Figure 5B ). Ser75 and Asn77 in the DE loop of the VH domain form 180 hydrogen bonds with Arg346 S and Asn450 S ( Figure 5C ). The light chain of BD-368-2 Fab mainly plays a 181 supportive role in stabilizing the conformation of the heavy chain residues. Direct interactions between the 182 light chain and RBD are seen between Asn33 in CDRL1 and Asn481 S , which form reciprocal hydrogen 183 bonds between their respective main chain and side chain groups ( Figure 5D ). Asn33, Tyr35, Tyr37, and

Leu55 together create a pocket to accommodate Val483 S .

The epitope of BD-368-2 does not significantly overlap with the binding site of ACE2 on RBD. 

Cryo-EM structure of BD-368-2 in complex with the prefusion-stabilized S trimer

To further investigate the molecular mechanism by which BD-368-2 neutralizes SARS-CoV-2, we set to 197 characterizing its interaction with the S trimer using cryo-EM. In the beginning, we used the 2P variant of Figure 6A) . We subsequently determined a cryo-EM structure of BD-368-2 Fab in complex with 211 S-6P at an overall resolution of 3.5 Å ( Figure S6 , Table S3 ).

S-6P exhibits an asymmetric conformation as previously observed (Cao et al., 2020; Wrapp et al., 2020), sandwiched between the NTD and RBD of the adjacent RBD-"up" protomer. The VH domain of this Fab is 217 close to a glycan attached to Asn165 in the NTD of the RBD-"up" protomer, whereas the VL domain 218 appears to contact the "up" RBD directly. In a way, it seems that besides its own RBD target, this Fab is also 219 exploiting the adjacent S protomer to gain further avidity. Together, our structural analyses suggest that BD-220 368-2 can bind to the RBDs regardless of their "up" and "down" positions to achieve full occupancy of the S 221 trimer. Consistently, BD-368-2 IgG exhibits markedly increased binding affinities for the S trimer compared 222 to its Fab, likely because of the multivalent interactions ( Figure 6D ).

Here we performed a systematic structural analysis of the SARS-CoV-2 NAbs. Our results shed light on their 

We further show that the epitopes of the VH3-53/VH3-66 NAbs and BD-368-2 have no overlaps, and can 238 engage one RBD simultaneously. These results provide a foundation for combination therapy. In fact, BD-239 368-2 may further potentiate the activity of the VH3-53/66 antibodies, since it can induce rapid structural 240 changes of the S trimer, which may lead to the exposure of the RBDs that were originally in the "down" 

Besides the VH3-53/VH3-66 antibodies, further structural analyses suggest that BD-368-2 appears to be able 252 to bind RBD together with two other antibodies: CR3022 and S309 (Figure 7 ). Like CR3022, S309 was also These antibodies each have a unique epitope and displays a distinct binding pose. Among them, BD-368-2 255 binds to the RBD regardless of its "up" and "down" state, blocks the engagement of ACE2, and causes 256 drastic conformational changes of the S trimer. All these effects likely contribute to its potent neutralizing 257 activity. S309 recognizes a glycan-containing epitope and can also bind both the "up" and "down" RBDs 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the There are restrictions on the availability of antibodies due to limited stock and continued consumption. We 279 are glad to share remaining antibodies with reasonable compensation for processing and shipping upon completion of a Material/Data Transfer Agreement for non-commercial usage.

purposes but not their disclosure to third parties, are needed to obtain the sequences by contacting the Data 286 Access Committee.

In Vitro expression of the antibodies and ELISA quantification

All antibody sequences in this manuscript were generated in the previous study (Cao et al., 2020) . The 292 antibodies were in vitro expressed using HEK293 cells, and the binding specificities were quantity by ELISA 293 against the Spike protein and the RBD protein, as described previously. An antibody is defined as ELISA-294 positive when the OD450 is saturated using 1 μg/mL RBD/S protein.

Surface plasmon resonance

The dissociation coefficients for the binding between BD-368-2 and the S trimers were measured using a 

The pseudovirus neutralization assays were performed using Huh-7 cell lines, as described previously (Cao 

Protein expression and purification

The SARS-CoV-2 RBD (residues 319-541) with an N-terminal His6 tag was cloned into a modified pFastBac 317 vector (Invitrogen) that encodes a gp67 signal peptide. Bacmids were generated using the Bac-to-Bac system 

For data collection, the crystals were transferred to a solution containing the crystallization solution 358 supplemented with 20% ethylene glycol or 20% glycerol before they were flash-cooled in liquid nitrogen.

Diffraction data were collected at the Shanghai Synchrotron Radiation Facility (beamline BL17U) and the 360 National Facility for Protein Science Shanghai (beamline BL19U). The data were processed using HKL2000 indicates that the antibody's IC50 is higher than 1 μg/mL. The detailed characteristics of the antibodies shown 575 here are listed in Table S1 .

(B) Characteristics of the potent VH3-53/VH3-66 convergent NAbs selected based on VDJ sequences. KD 577 targeting RBD was measured by using surface plasmon resonance (SPR) with a 1:1 binding model. See also Figure S1 and Figure S2 . 

(C) BD-629 Fab is modeled onto one of the "down" RBD in the same S trimer structure. The "down" RBD is 607 not available for the interaction with BD-629, due to the steric hindrance imposed by an adjacent protomer. 

RBD and BD-629 Fab are shown using the same color scheme as in Figure 2 . 

The binary complex is shown in white. 

The Figures S6. Workflow for the 3D reconstruction of the cryo-EM structure of the S-6P trimer in 695 complex with three BD-368-2 Fabs

A representative raw image collected using a Titan Krios 300 kV equipped with a K2 detector

Gold standard Fourier shell correlation (FSC) curve with the estimated resolution. 700 (E) Eulerian angle distribution of the particles used in the final 3D reconstruction

Local resolution estimation of the final density map analyzed by ResMap

We thank the staff of the Shanghai Synchrotron Radiation Facility (beamline BL17U) and the National