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Cited by 2,006SARS-CoV-2 and COVID-19 Researchvaccines and immunoinformatics approachesMonoclonal and Polyclonal Antibodies Research

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Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies

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Cao, Yunlong, Wang, Jing, Jian, Fanchong, Xiao, Tianhe, Song, Weiliang, Yisimayi, Ayijiang, Huang, Weijin, Li, Qianqian, Wang, Peng, An, Ran, Wang, Jing, Wang, Yao, Niu, Xiao, Yang, Sijie, Liang, Hui, Sun, Haiyan, Li, Tao, Yu, Yuanling, Cui, Qianqian, Liu, Shuo, Yang, Xiaodong, Du, Shuo, Zhang, Zhiying, Hao, Xiaohua, Shao, Fei, Jin, Ronghua, Wang, Xiangxi, Xiao, Junyu, Wang, Youchun, Xie, Xiaoliang Sunney·2022·Nature
DOI 10.1038/s41586-021-04385-3PMC PMC8866119

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Abstract

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening 1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A–F)—a grouping that is highly concordant with knowledge-based structural classifications 3–5 . Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A–D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309) 6 and group F (for example, CR3022) 7 , which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.

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Fig. 1 Omicron greatly reduces the neutralization potency of neutralizing antibodies of diverse epitopes. a, Schematic of MACS-based high-throughput yeast display mutation screening. mAb, monoclonal antibody. b, Representative antibody structures of each epitope group. c, t-distributed stochastic neighbour embedding (t-SNE) and unsupervised clustering of SARS-CoV-2 human neutralizing antibodies on the basis of each antibody escaping mutation profile. A total of six epitope groups (groups A–F) could be defined. d, Neutralization of the Omicron variant (spike-pseudotyped VSV) by 247 RBD neutralizing antibodies. Shades of red show the fold change in IC50 compared with D614G for each antibody. e, Neutralization of SARS-CoV-1 (spike-pseudotyped VSV) by 247 RBD neutralizing antibodies. Shades of red show the IC50 value (μg ml−1) of each antibody. All pseudovirus neutralization assays were conducted in biological duplicates or triplicates.
Fig. 1 Omicron greatly reduces the neutralization potency of neutralizing antibodies of diverse epitopes. a, Schematic of MACS-based high-throughput yeast display mutation screening. mAb, monoclonal antibody. b, Representative antibody structures of each epitope group. c, t-distributed stochastic neighbour embedding (t-SNE) and unsupervised clustering of SARS-CoV-2 human neutralizing antibodies on the basis of each antibody escaping mutation profile. A total of six epitope groups (groups A–F) could be defined. d, Neutralization of the Omicron variant (spike-pseudotyped VSV) by 247 RBD neutralizing antibodies. Shades of red show the fold change in IC50 compared with D614G for each antibody. e, Neutralization of SARS-CoV-1 (spike-pseudotyped VSV) by 247 RBD neutralizing antibodies. Shades of red show the IC50 value (μg ml−1) of each antibody. All pseudovirus neutralization assays were conducted in biological duplicates or triplicates.CC BY · PubMed Central
Fig. 2 The neutralizing abilities of group A–C antibodies are mostly abolished by Omicron. a–c, Escaping mutation profiles of representative neutralizing antibodies for group A (a), B (b) and C (c). For each site, the height of a letter indicates the detected mutation escape score of its corresponding residue. Sites mutated in Omicron are highlighted. d–f, Heat maps of site escape scores for neutralizing antibodies of epitope group A (d), B (e) and C (f). ACE2 interface residues are annotated with red blocks, and mutated sites in Omicron are marked in red. Annotations on the right side of heat maps represent the pseudovirus neutralizing IC50 fold change (FC) for Omicron and Beta compared to D614G. g–i, Representative structures of group A (g), group B (h) and group C (i) antibodies in complex with the RBD. Residues that are involved in important contacts are labelled. Omicron mutations are marked in blue. Antibody escaping mutations (Omicron) inferred from yeast display are labelled with squares.
Fig. 2 The neutralizing abilities of group A–C antibodies are mostly abolished by Omicron. a–c, Escaping mutation profiles of representative neutralizing antibodies for group A (a), B (b) and C (c). For each site, the height of a letter indicates the detected mutation escape score of its corresponding residue. Sites mutated in Omicron are highlighted. d–f, Heat maps of site escape scores for neutralizing antibodies of epitope group A (d), B (e) and C (f). ACE2 interface residues are annotated with red blocks, and mutated sites in Omicron are marked in red. Annotations on the right side of heat maps represent the pseudovirus neutralizing IC50 fold change (FC) for Omicron and Beta compared to D614G. g–i, Representative structures of group A (g), group B (h) and group C (i) antibodies in complex with the RBD. Residues that are involved in important contacts are labelled. Omicron mutations are marked in blue. Antibody escaping mutations (Omicron) inferred from yeast display are labelled with squares.CC BY · PubMed Central
Fig. 3 Most group D and E neutralizing antibodies are escaped by Omicron. a–c, Escaping mutation profiles of representative neutralizing antibodies for groups D (a), E (b) and F (c). For each site, the height of a letter indicates the detected mutation escape score of its corresponding residue. Sites mutated in Omicron are highlighted. d–f, Heat maps of site escape scores for neutralizing antibodies of epitope groups D (d), E (e) and F (f). ACE2 interface residues are annotated with red blocks, and mutated sites in Omicron are marked in red. Annotations on the right side of heat maps represent the pseudovirus neutralizing IC50 fold change (FC) for Omicron and Beta compared to D614G. g–j, Representative structures of group D (g), E (h) and F (i, j) antibodies in complex with the RBD. Residues that are involved in important contacts are labelled. Omicron mutations are marked in blue. Antibody escaping mutations (Omicron) inferred from yeast display are labelled with squares.
Fig. 3 Most group D and E neutralizing antibodies are escaped by Omicron. a–c, Escaping mutation profiles of representative neutralizing antibodies for groups D (a), E (b) and F (c). For each site, the height of a letter indicates the detected mutation escape score of its corresponding residue. Sites mutated in Omicron are highlighted. d–f, Heat maps of site escape scores for neutralizing antibodies of epitope groups D (d), E (e) and F (f). ACE2 interface residues are annotated with red blocks, and mutated sites in Omicron are marked in red. Annotations on the right side of heat maps represent the pseudovirus neutralizing IC50 fold change (FC) for Omicron and Beta compared to D614G. g–j, Representative structures of group D (g), E (h) and F (i, j) antibodies in complex with the RBD. Residues that are involved in important contacts are labelled. Omicron mutations are marked in blue. Antibody escaping mutations (Omicron) inferred from yeast display are labelled with squares.CC BY · PubMed Central
Fig. 4 Omicron escapes most neutralizing-antibody-based drugs. a, Neutralization of SARS-CoV-2 variants of concern (pseudotyped VSV) by nine neutralizing-antibody-based drugs. The pseudovirus neutralization assays for every VOC were performed in biological triplicates. The IC50 values shown are the average of three replicates shown in Extended Data Fig. 9. b, The sarbecovirus neutralization and binding capability (half-maximal effective concentration, EC50) of selected potent Omicron-neutralizing antibodies. The monoclonal antibody HG1K (IgG1 antibody against influenza A virus subtype H7N9) was used as the negative control.
Fig. 4 Omicron escapes most neutralizing-antibody-based drugs. a, Neutralization of SARS-CoV-2 variants of concern (pseudotyped VSV) by nine neutralizing-antibody-based drugs. The pseudovirus neutralization assays for every VOC were performed in biological triplicates. The IC50 values shown are the average of three replicates shown in Extended Data Fig. 9. b, The sarbecovirus neutralization and binding capability (half-maximal effective concentration, EC50) of selected potent Omicron-neutralizing antibodies. The monoclonal antibody HG1K (IgG1 antibody against influenza A virus subtype H7N9) was used as the negative control.CC BY · PubMed Central
Extended Data Fig. 1 Illustration of the SARS-CoV-2 spike protein with Omicron’s mutations. a, SARS-CoV-2 D614G spike protein structure overlayed with Omicron mutations. Omicron’s (BA.1) popular mutations are marked by red (for substitutions), blue (for insertions) and grey balls (for deletions). b, NTD-binding neutralizing antibodies shown together in complex with NTD. Substitutions and deletions of Omicron NTD are coloured blue and red, respectively.
Extended Data Fig. 1 Illustration of the SARS-CoV-2 spike protein with Omicron’s mutations. a, SARS-CoV-2 D614G spike protein structure overlayed with Omicron mutations. Omicron’s (BA.1) popular mutations are marked by red (for substitutions), blue (for insertions) and grey balls (for deletions). b, NTD-binding neutralizing antibodies shown together in complex with NTD. Substitutions and deletions of Omicron NTD are coloured blue and red, respectively.CC BY · PubMed Central
Extended Data Fig. 2 Comparison between FACS- and MACS-based deep mutational scanning. Deep mutational scanning maps with MACS-based (left) and FACS-based assays (right) of seven therapeutic neutralizing antibodies that have received emergency use authorization. Sites mutated in the Omicron variant are highlighted. Mutation amino acids of each site are shown by single letters. The heights represent mutation escape score, and colours represent chemical properties. FACS-based data were obtained from public datasets by Jesse Bloom.
Extended Data Fig. 2 Comparison between FACS- and MACS-based deep mutational scanning. Deep mutational scanning maps with MACS-based (left) and FACS-based assays (right) of seven therapeutic neutralizing antibodies that have received emergency use authorization. Sites mutated in the Omicron variant are highlighted. Mutation amino acids of each site are shown by single letters. The heights represent mutation escape score, and colours represent chemical properties. FACS-based data were obtained from public datasets by Jesse Bloom.CC BY · PubMed Central
Extended Data Fig. 3 Omicron neutralization IC50 fold-change distribution of 247 neutralizing antibodies of diverse epitopes. Fold-change of IC50 (VSV pseudovirus neutralization) compared to D614G by Beta and Omicron (BA.1) are shown for all 247 neutralizing antibodies tested. The effect of each RBD mutation of Omicron on antibody binding is inferred from yeast display mutation screening. Each antibody’s binding to Omicron RBD was validated through ELISA. All neutralization and ELISA assays were conducted in biological duplicates.
Extended Data Fig. 3 Omicron neutralization IC50 fold-change distribution of 247 neutralizing antibodies of diverse epitopes. Fold-change of IC50 (VSV pseudovirus neutralization) compared to D614G by Beta and Omicron (BA.1) are shown for all 247 neutralizing antibodies tested. The effect of each RBD mutation of Omicron on antibody binding is inferred from yeast display mutation screening. Each antibody’s binding to Omicron RBD was validated through ELISA. All neutralization and ELISA assays were conducted in biological duplicates.CC BY · PubMed Central
Extended Data Fig. 4 Heavy chain V/J segment recombination of neutralizing antibodies of each epitope group. a–f, Chord diagrams showing the heavy chain V segment and J segment recombination of epitope group A (a), B (b), C (c), D (d), E (e) and F (f). The width of the arc linking a V segment to a J segment indicates the antibody number of the corresponding recombination. The inner layer scatter plots show the V segment amino acid mutation rate, and black strips show the 25%~75% quantile of mutation rates.
Extended Data Fig. 4 Heavy chain V/J segment recombination of neutralizing antibodies of each epitope group. a–f, Chord diagrams showing the heavy chain V segment and J segment recombination of epitope group A (a), B (b), C (c), D (d), E (e) and F (f). The width of the arc linking a V segment to a J segment indicates the antibody number of the corresponding recombination. The inner layer scatter plots show the V segment amino acid mutation rate, and black strips show the 25%~75% quantile of mutation rates.CC BY · PubMed Central
Extended Data Fig. 5 Neutralization potency, heavy chain CDR3 length and mutation rate distribution for neutralizing antibodies of each epitope group. a, The length of H chain complementarity-determining region 3 (HCDR3) amino acid sequence for neutralizing antibodies in each epitope group (n = 66, 26, 57, 27, 39, 32 antibodies for epitope group A, B, C, D, E, F, respectively). HCDR3 lengths are displayed as mean ± s.d. b, The V segment amino acid mutation rate for neutralizing antibodies in each epitope group (n = 66, 26, 57, 27, 39, 32 antibodies for epitope group A, B, C, D, E, F, respectively). Mutation rates are calculated are displayed as mean ± s.d. c–e, The IC50 against D614G (c), Beta (d) and Omicron (e) variants for neutralizing antibodies in each epitope group (n = 66, 26, 57, 27, 39, 32 antibodies for epitope group A, B, C, D, E, F, respectively). IC50 values are displayed as mean ± s.d. in the log10 scale. Pseudovirus assays for each variant are biologically replicated twice. Dotted lines show the detection limit, which is from 0.0005 μg/mL to 10 μg/mL. IC50 geometric means are also labelled on the figure.
Extended Data Fig. 5 Neutralization potency, heavy chain CDR3 length and mutation rate distribution for neutralizing antibodies of each epitope group. a, The length of H chain complementarity-determining region 3 (HCDR3) amino acid sequence for neutralizing antibodies in each epitope group (n = 66, 26, 57, 27, 39, 32 antibodies for epitope group A, B, C, D, E, F, respectively). HCDR3 lengths are displayed as mean ± s.d. b, The V segment amino acid mutation rate for neutralizing antibodies in each epitope group (n = 66, 26, 57, 27, 39, 32 antibodies for epitope group A, B, C, D, E, F, respectively). Mutation rates are calculated are displayed as mean ± s.d. c–e, The IC50 against D614G (c), Beta (d) and Omicron (e) variants for neutralizing antibodies in each epitope group (n = 66, 26, 57, 27, 39, 32 antibodies for epitope group A, B, C, D, E, F, respectively). IC50 values are displayed as mean ± s.d. in the log10 scale. Pseudovirus assays for each variant are biologically replicated twice. Dotted lines show the detection limit, which is from 0.0005 μg/mL to 10 μg/mL. IC50 geometric means are also labelled on the figure.CC BY · PubMed Central
Extended Data Fig. 6 Escape hotspots of different epitope groups on the RBD surface. a–f, Aggregated site escape scores of antibodies for epitope group A–F, respectively. Epitope groups are distinguished by distinct colours, and the shades show normalized site escape scores. Escape hotspots of each epitope group are annotated by arrows.
Extended Data Fig. 6 Escape hotspots of different epitope groups on the RBD surface. a–f, Aggregated site escape scores of antibodies for epitope group A–F, respectively. Epitope groups are distinguished by distinct colours, and the shades show normalized site escape scores. Escape hotspots of each epitope group are annotated by arrows.CC BY · PubMed Central
Extended Data Fig. 7 Antibody-RBD interface distribution for neutralizing antibodies of each epitope group. a–f, Aggregated antibody-antigen interface of antibodies for epitope group A-F, respectively. Antibody-antigen interface was indicated from publicly available structures of neutralizing antibodies in complex with SARS-CoV-2 RBD. Different colours distinguish epitope groups, and the shade reflects group-specific site popularity to appear on the complex interface. Shared interface residues (Omicron) of each group are annotated.
Extended Data Fig. 7 Antibody-RBD interface distribution for neutralizing antibodies of each epitope group. a–f, Aggregated antibody-antigen interface of antibodies for epitope group A-F, respectively. Antibody-antigen interface was indicated from publicly available structures of neutralizing antibodies in complex with SARS-CoV-2 RBD. Different colours distinguish epitope groups, and the shade reflects group-specific site popularity to appear on the complex interface. Shared interface residues (Omicron) of each group are annotated.CC BY · PubMed Central
Extended Data Fig. 8 Comparison between mutation escape scores estimated from yeast display and neutralization of variants carrying corresponding mutations. a, K417N escape scores and corresponding K417N pseudovirus neutralizing IC50 fold change compared to D614G pseudovirus of antibodies within epitope group A. b, E484K/E484A escape scores and corresponding E484K pseudovirus neutralizing IC50 fold change compared to D614G pseudovirus of antibodies within epitope group C.
Extended Data Fig. 8 Comparison between mutation escape scores estimated from yeast display and neutralization of variants carrying corresponding mutations. a, K417N escape scores and corresponding K417N pseudovirus neutralizing IC50 fold change compared to D614G pseudovirus of antibodies within epitope group A. b, E484K/E484A escape scores and corresponding E484K pseudovirus neutralizing IC50 fold change compared to D614G pseudovirus of antibodies within epitope group C.CC BY · PubMed Central
Extended Data Fig. 9 Pseudovirus neutralization of neutralizing-antibody-based drugs against SARS-CoV-2 variants of concern. Pseudovirus (VSV-based) assays were performed using Huh-7 cells. Data are collected from three biological replicates and represented as mean ± s.d.
Extended Data Fig. 9 Pseudovirus neutralization of neutralizing-antibody-based drugs against SARS-CoV-2 variants of concern. Pseudovirus (VSV-based) assays were performed using Huh-7 cells. Data are collected from three biological replicates and represented as mean ± s.d.CC BY · PubMed Central
Extended Data Fig. 10 BLI response between neutralizing-antibody-based drugs and the RBD of SARS-CoV-2 wild type, Beta or Omicron strains. Antibodies were captured by Protein A sensor. The concentrations of RBD are shown in different colours. Dissociation constant (KD), association constant (ka), and dissociation rate constant (kd) are labelled. Neutralizing antibodies without binding are marked as ‘Escaped’.
Extended Data Fig. 10 BLI response between neutralizing-antibody-based drugs and the RBD of SARS-CoV-2 wild type, Beta or Omicron strains. Antibodies were captured by Protein A sensor. The concentrations of RBD are shown in different colours. Dissociation constant (KD), association constant (ka), and dissociation rate constant (kd) are labelled. Neutralizing antibodies without binding are marked as ‘Escaped’.CC BY · PubMed Central

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Authors
Cao, Y., Wang, J., Jian, F., Xiao, T., Song, W., Yisimayi, A., Huang, W., Li, Q., Wang, P., An, R., Wang, J., Wang, Y., Niu, X., Yang, S., Liang, H., Sun, H., Li, T., Yu, Y., Cui, Q., Liu, S., Yang, X., Du, S., Zhang, Z., Hao, X., Shao, F., Jin, R., Wang, X., Xiao, J., Wang, Y., & Xie, X. S.
Affiliations
  • Peking University (CN)
  • National Institutes for Food and Drug Control (CN)
  • Capital Medical University (CN)
  • Chinese Academy of Sciences (CN)
Corresponding author
  • Cao, Yunlong
  • Wang, Yao
  • Wang, Xiangxi
  • Xiao, Junyu
  • Wang, Youchun
  • Xie, Xiaoliang Sunney
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2021-12-23
Volume
602
Issue
7898
Pages
657-663
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-021-04385-3
PMC
PMC8866119
OpenAlex
W4220807057
Cited by
2,006
References
56
Citations by year
2026: 143 · 2025: 242 · 2024: 329 · 2023: 525 · 2022: 735 · 2021: 20
FWCI
127.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Peking University
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
Keywords
Omicron variant, SARS-CoV-2, receptor binding domain, neutralizing antibodies, antibody escape, K417N, vaccine design
MeSH terms
Angiotensin-Converting Enzyme 2, COVID-19, SARS-CoV-2, COVID-19 Vaccines, Antibodies, Monoclonal, Antibodies, Viral, Cells, Cultured, Convalescence, Humans, Immune Sera, Models, Molecular, Mutation, Neutralization Tests, Epitopes, B-Lymphocyte, Immune Evasion, Antibodies, Neutralizing, Spike Glycoprotein, Coronavirus

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