key: cord-1002551-4bdukagp
authors: Cao, Yunlong; Yisimayi, Ayijiang; Jian, Fanchong; Xiao, Tianhe; Song, Weiliang; Wang, Jing; Du, Shuo; Zhang, Zhiying; Liu, Pulan; Hao, Xiaohua; Li, Qianqian; Wang, Peng; An, Ran; Wang, Yao; Wang, Jing; Sun, Haiyan; Zhao, Lijuan; Zhang, Wen; Zhao, Dong; Zheng, Jiang; Yu, Lingling; Li, Can; Zhang, Na; Wang, Rui; Niu, Xiao; Yang, Sijie; Song, Xuetao; Zheng, Linlin; Li, Zhiqiang; Gu, Qingqing; Shao, Fei; Huang, Weijin; Wang, Youchun; Jin, Ronghua; Xiao, Junyu; Xie, Xiaoliang Sunney
title: Comprehensive Epitope Mapping of Broad Sarbecovirus Neutralizing Antibodies
date: 2022-02-07
journal: bioRxiv
DOI: 10.1101/2022.02.07.479349
sha: 7f749d768e098b9469090197f1de4da49a812a0e
doc_id: 1002551
cord_uid: 4bdukagp

Constantly emerging SARS-CoV-2 variants, such as Omicron BA.1, BA.1.1 and BA.2, pose a severe challenge to COVID-19 control1–10. Broad-spectrum antibody therapeutics and vaccines are needed for defending against future SARS-CoV-2 variants and sarbecovirus pandemics11–14; however, we have yet to gain a comprehensive understanding of the epitopes capable of inducing broad sarbecovirus neutralization. Here, we report the identification of 241 anti-RBD broad sarbecovirus neutralizing antibodies isolated from 44 SARS-CoV-2 vaccinated SARS convalescents. Neutralizing efficacy of these antibodies against D614G, SARS-CoV-1, Omicron variants (BA.1, BA.1.1, BA.2), RATG13 and Pangolin-GD is tested, and their binding capability to 21 sarbecovirus RBDs is measured. High-throughput yeast-display mutational screening was further applied to determine each antibody’s RBD escaping mutation profile, and unsupervised epitope clustering based on escaping mutation hotspots was performed7,15–18. A total of 6 clusters of broad sarbecovirus neutralizing antibodies with diverse breadth and epitopes were identified, namely Group E1 (S30919, BD55-3152 site), E3 (S2H9720 site), F1 (CR302221, S30422 site), F2 (DH104723, BD55-3500 site), F3 (ADG-224, BD55-3372 site) and B’ (S2K14625 site). Members of E1, F2 and F3 demonstrate the highest neutralization potency; yet, Omicron, especially BA.2, has evolved multiple mutations (G339D, N440K, T376A, D405N, R408S) to escape antibodies of these groups. Nevertheless, broad sarbecovirus neutralizing antibodies that survived Omicron would serve as favorable therapeutic candidates. Furthermore, structural analyses of selected drug candidates propose two non-competing antibody pairing strategies, E1-F2 and E1-F3, as broad-spectrum antibody cocktails. Together, our work provides a comprehensive epitope map of broad sarbecovirus neutralizing antibodies and offers critical instructions for designing broad-spectrum vaccines.

Group E1 antibodies display potent neutralizing activities against human infecting 117 sarbecoviruses, including both SARS-CoV-2 variants and SARS-CoV-1 variants (Fig. 118 1c, Extended Data Fig. 5b ). Their epitope is fully exposed regardless of the up and 119 down conformations of RBD and may involve a mixed protein and carbohydrate, 120 specifically the N-linked glycan on N343, as for S309 19 . Indeed this unique feature is 121 shared among Group E antibodies, evidenced by structural analyses of BD55-3152, 122 BD55-3546 and BD55-5840 in complex with spike proteins using cryo-electron 123 microscopy (cryo-EM) (Fig. 2a, Extended Data Fig. 6a-b) . Importantly, members of 124 Group E1 are generally sensitive to the changes of G339, E340, T345 and especially 125 R346, revealed by their escaping mutation profiles and structures (Fig. 2a, d) . Thus, 126 most E1 antibodies could not bind to clade 2 and 3 sarbecoviruses because of the 127 changes of RBD antigenic sites corresponding to G339, E340 and R346, as calculated 128 by multiple sequence alignment (MSA) (Fig. 1f, Fig. 2d ). Importantly, Omicron causes 129 considerable antibody escaping for Group E1 (Fig. 1d) ; however, a proportion of E1 Interestingly, despite the importance of R346 to E1 antibodies, the additional R346K 135 carried by BA.1.1 does not readily affect their efficacy (Fig. 1d) . This is foreseeable, as 136 Arg and Lys possess similar chemical properties. In fact, SARS-CoV-1 features a Lys 137 at the corresponding site (K333) (Fig. 2d) . The structure of BD55-3152 in complex with 138 the Omicron spike reveals that a CDRL2 Asp (D50) interacts with R346 (Fig. 2a) , 139 whereas the structure of BD55-3152 complexed with the SARS-CoV-1 spike shows 140 that the same Asp also coordinates K333 (Extended Data Fig. 8) . Nevertheless, most 141 E1 antibodies lost binding and neutralization ability towards clade 1a/1b sarbecoviruses 142 circulating among animals, including the bat coronavirus RaTG13 and the pangolin 143 coronavirus Pangolin-GD (Fig. 1e , Extended Data Fig. 5f-g) . This is largely because of 144 the Thr substitution in these sarbecoviruses (Fig. 2d) . In contrast to R346K, R346S/T 145 would greatly compromise the binding activities of E1 antibodies. 146 Group E2 antibodies are directed to the front chest of RBD. Previously, we solved cryo-148 EM structures of several neutralizing antibodies in this group, including BD-744 43 (Fig.   149 2b). These neutralizing antibodies target a relatively flat region partially exposed in the 150 down RBDs. The E2 antibodies are sensitive to mutations of R346, A348, A352, K356, 151 R357, L452, and I468, and these sites are largely not conserved in clade 1a/2/3 152 sarbecoviruses (Fig. 2d) . Therefore, the E2 Antibodies usually do not have broad Group F1, F2, and F3 antibodies cover a continuous surface on the backside of RBD 176 and can only bind to the up RBDs (Fig. 1b) . Members of the F1 group, such as 177 CR3022 21 and S304 22 , also require a wide open RBD to engage and do not directly 178 block ACE2, therefore displaying weak neutralizing activities in general (Fig. 1c) . 179 Escape hotspots for this group include SARS-CoV-2 residues 383-386, 390, and 391 180 (Fig. 3a, d) . Most of these sites are conserved in all sarbecoviruses, except an alanine 181 on 384 in clade 1a, which is also tolerated (Fig. 3d ). Due to their intrinsic weak The epitopes for group F2 antibodies are shifted upward compared with F1 (Fig. 3b) . 187 We solved the cryo-EM structure of two representative antibodies in this group, BD55- and ADG-2 24 (Fig. 1b) . Cryo-EM structures of BD55-4637 complexed with the 206 Omicron spike and BD55-3372 with the Delta spike reveal that F3 antibodies interact 207 with several ACE2-binding residues (Fig. 3c, Extended Data Fig. 6d) , and therefore 208 directly compete with ACE2. Major escape sites for this group of antibodies include 209 D405, R408, V503, G504, and Y508 (Fig. 3d) . Given the fact that D405, G504, and 210 Y508 are not conserved in clade 2 sarbecoviruses, F3 antibodies cannot bind to non-211 ACE2 utilizing clade 2 sarbecovirus (Extended Data Fig. 4e ), but showed good 212 neutralization breadth against clade 1a/b/2 sarbecovirus (Fig. 1e-f ). Similar to Group 213 E1, a proportion of F3 antibodies showed potent neutralization against Omicron BA.1, 214 despite the Y505H mutation carried by Omicron (Fig. 1d, 3d) . Moreover, several elite Notably, a unique subcluster of Group B antibodies also showed broad-spectrum 220 sarbecovirus neutralizing capability (Fig 1c) . Most Group B antibodies are SARS-CoV-221 2 specific since their major escaping mutations consist of E484 and F486, which are 222 not conserved in sarbecovirus clade (Extended Data Fig. 9) ; however, the rare sub-223 cluster B', featured by S2K146 25 , displayed skewed escaping mutation profiles toward 224 N487 and Y489, which are highly conserved in clade 1a/1b/3 (Extended Data Fig. 9 ), 225 making members of B' exhibits similar breadth as F3 antibodies. Sadly, most B' 226 antibodies failed to neutralize Omicron, except for S2K146 6 (Fig. 1d ).

The above analyses indicate that each epitope group's mutational escape hotspots are 229 closely related to the antibodies' sarbecovirus reactivity breadth. To further extrapolate 230 this observation, we simulated each antibody's sarbecovirus RBD binding spectrum 231 based on its escaping mutation profile and the corresponding MSA results of 232 sarbecovirus RBDs (Extended Data Fig. 10a-e) . Surprisingly, the simulated spectrum 233 well matches that obtained from ELISA (Extended Data Fig. 4a-e) , suggesting that the 234 antibody's breadth is mostly governed by the degree of conservation of its major 235 mutational escaping sites, and the escaping mutation profile of an antibody could be 236 used to predict its sarbecovirus binding spectrum.

Importantly, even broad sarbecovirus neutralizing antibodies are largely escaped by 239 Omicron (Fig. 1b) , which is consistent with that Omicron could greatly reduce the 240 neutralization efficacy of vaccinated SARS convalescents' plasma (Extended Data Fig.   241 1c). This supports the speculation that Omicron does not originate from zoonotic In addition, pairing non-competing antibodies into cocktails is a promising strategy to 262 reduce the chance of mutation-induced antibody evasion. As the epitopes of the E1 263 antibodies are located on the opposite sides of RBD compared to F2 and F3, we 264 envision that many E1 antibodies can function in combinations with an F2 or F3 265 counterpart. Indeed, several plausible strategies can be designed based on the structural 266 information presented in this study. For example, overlaying the targeting RBDs in the 267 BD55-5840 (E1) and BD55-3500 (F2) cryo-EM structures demonstrate that they can 268 readily bind to the Omicron RBD simultaneously (Fig. 4b) , whereas BD55-3546 (E1) 269 can bind concurrently with BD55-3372 (F3) on the Delta RBD (Fig. 4c) . Cryo-EM data collection, processing, and structure building 633 The samples for cryo-EM study were prepared essentially as previously described 51,53 .

All EM grids were evacuated for 2 min and glow-discharged for 30 s using a plasma Fig. 2 | Structural and escaping mutation analyses of group E1-E3 antibodies. a-c, High-resolution cryo-electron microscopy antibody structures of representative epitope group E1-E3 neutralizing antibodies. a, BD55-3152 (group E1) in complex of SARS-CoV-2 Omicron RBD complex. b, BD-744 (group E2) in complex of SARS-CoV-2 Beta RBD complex (PDB: 7EY0). c, S2H97 (group E3) in complex of SARS-CoV-2 RBD complex (PDB: 7M7W). Residues on the binding interface are marked. Residues highlighted in red indicate featuring escaping hotspots of the representative epitope groups. d, Averaged escape maps of antibodies in epitope group E1-E3, and corresponding multiple sequence alignment (MSA) of various sarbecovirus RBDs. Height of each amino acid in the escape maps represents its mutation escape score. Residues are colored corresponding to their chemical properties. Mutated sites in Omicron variants are marked in bold.

Omicron RBD against D614G pseudovirus and SARS pseudovirus for SARS convalescents before (red) and after (blue) the third dose vaccination (n=18) . Pearson's correlation coefficient is labeled. c, NT50 of plasma against different SARS-CoV-2 variants authentic virus, for SARS convalescents before and after the third dose vaccination (n=18). Statistical significance in a, c was determined by two-tailed Wilcoxon signed rank test(***p<0.001, **p<0.01, *p<0.05). NT50 values are displayed as geometric mean ± s. d. in the log10 scale. Residues on the binding interface are marked. 

IMGT standardized analysis of amino 650 acid sequences of variable, constant, and groove domains

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected 653

Hamsters and Provides a Potent Cocktail Pairing Strategy

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a 656 prerequisite conformational state for receptor binding

Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into 659 cocktail design against concerning variants

cryoSPARC: algorithms for rapid 662 unsupervised cryo-EM structure determination

UCSF Chimera--a visualization system for exploratory research and analysis

New tools for automated high-resolution cryo-EM structure determination in 667 RELION-3

Features and development of Coot

Macromolecular structure determination using X-rays, neutrons and 671 electrons: recent developments in Phenix

Structure visualization for researchers, educators, and 674 developers

are listed as inventors on the provisional patent applications of BD 679 series antibodies. X.S.X. and Y.C. are founders of Singlomics Biopharmaceuticals

Correspondence to Yunlong Cao or Ronghua Jin or Junyu Xiao or Xiaoliang Sunney

Request for materials described in this study should be directed to Yunlong Cao 685 and Xiaoliang Sunney Xie