Defining the features and structure of neutralizing antibody targeting the silent face of the SARS-CoV-2 spike N-terminal domain

Abstract

Research on virus/receptor interactions has uncovered various mechanisms of antibody-mediated neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, understanding of neutralization by antibodies targeting the silent face, which recognize epitopes on glycan shields, remains limited, and their potential protective efficacy in vivo is not well understood. This study describes a silent face neutralizing antibody, 3711, which targets a non-supersite on the N-terminal domain (NTD) of the spike protein. Cryo-EM structure determination of the 3711 Fab in the spike complex reveals a novel neutralizing epitope shielded by glycans on the spike’s silent face. Antibody 3711 inhibits the interaction between the receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2) through steric hindrance and exhibits superior in vivo protective effects compared to other reported NTD-targeted monoclonal antibodies (mAbs). Competition assays and antibody repertoire analysis indicate the rarity of antibodies targeting the 3711-related epitope in SARS-CoV-2 convalescents, suggesting the infrequency of NTD silent face-targeted neutralizing antibodies during SARS-CoV-2 infection. As the first NTD silent face-targeted neutralizing antibody against SARS-CoV-2, the identification of mAb 3711, with its novel neutralizing mechanism, enhances our understanding of neutralizing epitopes on glycan shields and elucidates epitope-guided viral mutations that evade specific antibodies.

Keywords

glycan shield / neutralizing antibody / N-terminal domain / SARS-CoV-2 / silent face

References

[1] Ai J, Zhang H, Zhang Y, et al. Omicron variant showed lower neutralizing sensitivity than other SARS-CoV-2 variants to immune sera elicited by vaccines after boost. Emerg Microbes Infect. 2022; 11(1): 337-343.

[2] Cao Y, Wang J, Jian F, et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature. 2022; 602(7898): 657-663.

[3] Lan J, Ge J, Yu J, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020; 581(7807): 215-220.

[4] Ke Z, Oton J, Qu K, et al. Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature. 2020; 588(7838): 498-502.

[5] McCallum M, Czudnochowski N, Rosen LE, et al. Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement. Science. 2022; 375(6583): 864-868.

[6] Zhang Z, Liu W, Zhang S, et al. Two novel human coronavirus OC43 genotypes circulating in hospitalized children with pneumonia in China. Emerg Microbes Infect. 2022; 11(1): 168-171.

[7] Suryadevara N, Shrihari S, Gilchuk P, et al. Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein. Cell. 2021; 184(9): 2316-2331.e2315.

[8] McCallum M, De Marco A, Lempp FA, et al. N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell. 2021; 184(9): 2332-2347.e2316.

[9] Cerutti G, Guo Y, Zhou T, et al. Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single supersite. Cell Host Microbe. 2021; 29(5): 819-833.e817.

[10] Lok SM. An NTD supersite of attack. Cell Host Microbe. 2021; 29(5): 744-746.

[11] Chi X, Yan R, Zhang J, et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science. 2020; 369(6504): 650-655.

[12] Graham C, Seow J, Huettner I, et al. Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant. Immunity. 2021; 54(6): 1276-1289.e1276.

[13] Wang L, Zhou T, Zhang Y, et al. Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants. Science. 2021; 373(6556): eabh1766.

[14] Cerutti G, Guo Y, Wang P, et al. Neutralizing antibody 5–7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain. Cell Rep. 2021; 37(5): 109928.

[15] Rosa A, Pye VE, Graham C, et al. SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity. Sci Adv. 2021; 7(22): eabg7607.

[16] Li D, Edwards RJ, Manne K, et al. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies. Cell. 2021; 184(16): 4203-4219.

[17] Altomare CG, Adelsberg DC, Carreno JM, et al. Structure of a vaccine-induced, germline-encoded human antibody defines a neutralizing epitope on the SARS-CoV-2 spike N-terminal domain. mBio. 2022; 13(3): e0358021.

[18] Grant OC, Montgomery D, Ito K, Woods RJ. Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition. Sci Rep. 2020; 10(1): 14991.

[19] Watanabe Y, Allen JD, Wrapp D, McLellan JS, Crispin M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science. 2020; 369(6501): 330-333.

[20] Xu C, Wang Y, Liu C, et al. Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM. Sci Adv. 2021; 7(1): eabe5575.

[21] Shi R, Shan C, Duan X, et al. A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature. 2020; 584(7819): 120-124.

[22] Song G, Yuan M, Liu H, et al. Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift. bioRxiv. 2023.

[23] Corti D, Zhao J, Pedotti M, et al. Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus. Proc Natl Acad Sci USA. 2015; 112(33): 10473-10478.

[24] Zhou T, Zheng A, Baxa U, et al. A neutralizing antibody recognizing primarily N-linked glycan targets the silent face of the HIV envelope. Immunity. 2018; 48(3): 500-513.e506.

[25] Schoofs T, Barnes CO, Suh-Toma N, et al. Broad and potent neutralizing antibodies recognize the silent face of the HIV envelope. Immunity. 2019; 50(6): 1513-1529.e1519.

[26] Zhang Z, Zeng E, Zhang L, et al. Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo. Cell Discov. 2021; 7(1): 65.

[27] Sun X, Yi C, Zhu Y, et al. Neutralization mechanism of a human antibody with pan-coronavirus reactivity including SARS-CoV-2. Nat Microbiol. 2022; 7(7): 1063-1074.

[28] Hughes L, Gangavarapu K, Latif AA, et al. Outbreak.info genomic reports: scalable and dynamic surveillance of SARS-CoV-2 variants and mutations. Res Sq. 2022.

[29] Chen Q, Huang XY, Sun MX, et al. Transient acquisition of cross-species infectivity during the evolution of SARS-CoV-2. Natl Sci Rev. 2021; 8(11): nwab167.

[30] Sun J, Zhuang Z, Zheng J, et al. Generation of a broadly useful model for COVID-19 pathogenesis, vaccination, and treatment. Cell. 2020; 182(3): 734-743.e735.

[31] Yan Q, He P, Huang X, et al. Germline IGHV3-53-encoded RBD-targeting neutralizing antibodies are commonly present in the antibody repertoires of COVID-19 patients. Emerg Microbes Infect. 2021; 10(1): 1097-1111.

[32] Yan Q, Hou R, Huang X, et al. Shared IGHV1-69-encoded neutralizing antibodies contribute to the emergence of L452R substitution in SARS-CoV-2 variants. Emerg Microbes Infect. 2022: 1-41.

[33] Wang Y, Zhang L, Sang L, et al. Kinetics of viral load and antibody response in relation to COVID-19 severity. J Clin Invest. 2020; 130(10): 5235-5244.

[34] Shuai H, Chan JF, Yuen TT, et al. Emerging SARS-CoV-2 variants expand species tropism to murines. EBioMedicine. 2021; 73: 103643.

[35] Chi X, Xia L, Zhang G, et al. Comprehensive structural analysis reveals broad-spectrum neutralizing antibodies against SARS-CoV-2 Omicron variants. Cell Discov. 2023; 9(1): 37.

[36] Li Y, Shen Y, Zhang Y, Yan R. Structural basis for the enhanced infectivity and immune evasion of omicron subvariants. Viruses. 2023; 15(6): 1398.

[37] Lei J, Frank J. Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J Struct Biol. 2005; 150(1): 69-80.

[38] Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods. 2017; 14(4): 331-332.

[39] Grant T, Grigorieff N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife. 2015; 4: e06980.

[40] Zhang K. Gctf: real-time CTF determination and correction. J Struct Biol. 2016; 193(1): 1-12.

[41] Trabuco LG, Villa E, Mitra K, Frank J, Schulten K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure. 2008; 16(5): 673-683.

[42] Winn MD, Ballard CC, Cowtan KD, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011; 67(Pt 4): 235-242.

[43] Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 4): 486-501.

[44] Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods. 2017; 14(3): 290-296.

[45] Rosenthal PB, Henderson R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol. 2003; 333(4): 721-745.

[46] Chen S, McMullan G, Faruqi AR, et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy. 2013; 135: 24-35.

[47] Starr TN, Czudnochowski N, Liu Z, et al. SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature. 2021; 597(7874): 97-102.

[48] Wang Y, Zhang Z, Yang M, et al. Identification of a broad sarbecovirus neutralizing antibody targeting a conserved epitope on the receptor-binding domain. Cell Rep. 2024; 43(1): 113653.

[49] Bolotin DA, Poslavsky S, Mitrophanov I, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods. 2015; 12(5): 380-381.

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