Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance
2022; Springer Nature; Volume: 23; Issue: 4 Linguagem: Inglês
10.15252/embr.202154199
ISSN1469-3178
AutoresJustin D. Walter, Melanie Scherer, Cedric A. J. Hutter, Alisa A. Garaeva, Iwan Zimmermann, Marianne Wyss, Jan Rheinberger, Yelena Ruedin, Jennifer C. Earp, Pascal Egloff, Michèle Sorgenfrei, Lea M. Hürlimann, Imre Gonda, Gianmarco Meier, Sille Remm, Sujani Thavarasah, Geert van Geest, Rémy Bruggmann, Gert Zimmer, Dirk Jan Slotboom, Cristina Paulino, Philippe Plattet, Markus A. Seeger,
Tópico(s)SARS-CoV-2 detection and testing
ResumoArticle7 March 2022Open Access Transparent process Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance Justin D Walter Justin D Walter orcid.org/0000-0002-1492-3055 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Conceptualization, Supervision, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Melanie Scherer Melanie Scherer orcid.org/0000-0001-5554-9664 Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Cedric A J Hutter Cedric A J Hutter orcid.org/0000-0002-8920-3343 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Software, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Alisa A Garaeva Alisa A Garaeva orcid.org/0000-0002-7394-8981 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Software, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Iwan Zimmermann Iwan Zimmermann orcid.org/0000-0003-3476-4749 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Linkster Therapeutics AG, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Marianne Wyss Marianne Wyss orcid.org/0000-0002-2292-3282 Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Investigation Search for more papers by this author Jan Rheinberger Jan Rheinberger orcid.org/0000-0002-9901-2065 Department of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Resources, Software, Investigation Search for more papers by this author Yelena Ruedin Yelena Ruedin orcid.org/0000-0002-6852-6119 Institute of Virology and Immunology, Bern & Mittelhäusern, Switzerland Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Investigation Search for more papers by this author Jennifer C Earp Jennifer C Earp orcid.org/0000-0003-3944-4533 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Pascal Egloff Pascal Egloff orcid.org/0000-0001-8948-3704 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Linkster Therapeutics AG, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Michèle Sorgenfrei Michèle Sorgenfrei orcid.org/0000-0001-8480-197X Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Lea M Hürlimann Lea M Hürlimann orcid.org/0000-0001-9907-7830 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Imre Gonda Imre Gonda orcid.org/0000-0001-9951-181X Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Gianmarco Meier Gianmarco Meier orcid.org/0000-0002-4455-430X Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Sille Remm Sille Remm orcid.org/0000-0001-5204-4556 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Sujani Thavarasah Sujani Thavarasah orcid.org/0000-0002-3957-2298 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Geert van Geest Geert van Geest orcid.org/0000-0002-1561-078X Interfaculty Bioinformatics Unit and Swiss, Institute of Bioinformatics, University of Bern, Bern, Switzerland Contribution: Resources, Software Search for more papers by this author Rémy Bruggmann Rémy Bruggmann orcid.org/0000-0001-5629-6363 Interfaculty Bioinformatics Unit and Swiss, Institute of Bioinformatics, University of Bern, Bern, Switzerland Contribution: Resources, Software Search for more papers by this author Gert Zimmer Gert Zimmer orcid.org/0000-0002-2708-2507 Institute of Virology and Immunology, Bern & Mittelhäusern, Switzerland Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Supervision, Investigation, Methodology, Writing - review & editing Search for more papers by this author Dirk J Slotboom Dirk J Slotboom orcid.org/0000-0002-5804-9689 Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Supervision, Writing - review & editing Search for more papers by this author Cristina Paulino Corresponding Author Cristina Paulino [email protected] orcid.org/0000-0001-7017-109X Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Department of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Supervision, Funding acquisition, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Philippe Plattet Corresponding Author Philippe Plattet [email protected] orcid.org/0000-0003-3313-2598 Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Markus A Seeger Corresponding Author Markus A Seeger [email protected] orcid.org/0000-0003-1761-8571 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Justin D Walter Justin D Walter orcid.org/0000-0002-1492-3055 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Conceptualization, Supervision, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Melanie Scherer Melanie Scherer orcid.org/0000-0001-5554-9664 Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Cedric A J Hutter Cedric A J Hutter orcid.org/0000-0002-8920-3343 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Software, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Alisa A Garaeva Alisa A Garaeva orcid.org/0000-0002-7394-8981 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Software, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Iwan Zimmermann Iwan Zimmermann orcid.org/0000-0003-3476-4749 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Linkster Therapeutics AG, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Marianne Wyss Marianne Wyss orcid.org/0000-0002-2292-3282 Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Investigation Search for more papers by this author Jan Rheinberger Jan Rheinberger orcid.org/0000-0002-9901-2065 Department of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Resources, Software, Investigation Search for more papers by this author Yelena Ruedin Yelena Ruedin orcid.org/0000-0002-6852-6119 Institute of Virology and Immunology, Bern & Mittelhäusern, Switzerland Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Investigation Search for more papers by this author Jennifer C Earp Jennifer C Earp orcid.org/0000-0003-3944-4533 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Pascal Egloff Pascal Egloff orcid.org/0000-0001-8948-3704 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Linkster Therapeutics AG, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Michèle Sorgenfrei Michèle Sorgenfrei orcid.org/0000-0001-8480-197X Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Lea M Hürlimann Lea M Hürlimann orcid.org/0000-0001-9907-7830 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Imre Gonda Imre Gonda orcid.org/0000-0001-9951-181X Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Gianmarco Meier Gianmarco Meier orcid.org/0000-0002-4455-430X Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Sille Remm Sille Remm orcid.org/0000-0001-5204-4556 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Sujani Thavarasah Sujani Thavarasah orcid.org/0000-0002-3957-2298 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Investigation Search for more papers by this author Geert van Geest Geert van Geest orcid.org/0000-0002-1561-078X Interfaculty Bioinformatics Unit and Swiss, Institute of Bioinformatics, University of Bern, Bern, Switzerland Contribution: Resources, Software Search for more papers by this author Rémy Bruggmann Rémy Bruggmann orcid.org/0000-0001-5629-6363 Interfaculty Bioinformatics Unit and Swiss, Institute of Bioinformatics, University of Bern, Bern, Switzerland Contribution: Resources, Software Search for more papers by this author Gert Zimmer Gert Zimmer orcid.org/0000-0002-2708-2507 Institute of Virology and Immunology, Bern & Mittelhäusern, Switzerland Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Supervision, Investigation, Methodology, Writing - review & editing Search for more papers by this author Dirk J Slotboom Dirk J Slotboom orcid.org/0000-0002-5804-9689 Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Supervision, Writing - review & editing Search for more papers by this author Cristina Paulino Corresponding Author Cristina Paulino [email protected] orcid.org/0000-0001-7017-109X Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Department of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Contribution: Supervision, Funding acquisition, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Philippe Plattet Corresponding Author Philippe Plattet [email protected] orcid.org/0000-0003-3313-2598 Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Markus A Seeger Corresponding Author Markus A Seeger [email protected] orcid.org/0000-0003-1761-8571 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Contribution: Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Justin D Walter1,†, Melanie Scherer2,†, Cedric A J Hutter1,†, Alisa A Garaeva1,3,†, Iwan Zimmermann1,4, Marianne Wyss2, Jan Rheinberger5, Yelena Ruedin6,7, Jennifer C Earp1, Pascal Egloff1,4, Michèle Sorgenfrei1, Lea M Hürlimann1, Imre Gonda1, Gianmarco Meier1, Sille Remm1, Sujani Thavarasah1, Geert van Geest8, Rémy Bruggmann8, Gert Zimmer6,7, Dirk J Slotboom3, Cristina Paulino *,3,5, Philippe Plattet *,2 and Markus A Seeger *,1 1Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland 2Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland 3Department of Membrane Enzymology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands 4Linkster Therapeutics AG, Zurich, Switzerland 5Department of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands 6Institute of Virology and Immunology, Bern & Mittelhäusern, Switzerland 7Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland 8Interfaculty Bioinformatics Unit and Swiss, Institute of Bioinformatics, University of Bern, Bern, Switzerland † These authors contributed equally to this work *Corresponding author. Tel: +31 50 363 34 02; E-mail: [email protected] *Corresponding author. Tel: +41 31 631 23 70; E-mail: [email protected] *Corresponding author. Tel: +41 44 634 53 96; E-mail: [email protected] EMBO Reports (2022)23:e54199https://doi.org/10.15252/embr.202154199 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants. Synopsis Sybodies Sb#15 and Sb#68 inhibit SARS-CoV-2 infectivity by targeting non-overlapping epitopes on the spike glycoprotein. Covalent sybody fusion and valency engineering enhances neutralization potency against variants and impedes emergence of escape mutants. Two synthetic nanobodies were in vitro selected against the SARS-CoV-2 receptor-binding domain (RBD). Sb#15 and Sb#68 neutralize viral infection by blocking ACE2 association with the SARS-CoV-2 spike protein. Sb#15 and Sb#68 bind to distinct epitopes and promote a unique up/out conformation of the RBD. Sybody fusions increase neutralization potency and attenuate the emergence of novel escape mutants. Introduction The spike glycoprotein is the most prominent surface-exposed entity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and possesses the vital molecular machinery required for recognition and fusion with host membranes (Wrapp et al, 2020b). To date, most authorized vaccines against coronavirus disease 2019 (COVID-19) rely on exposure of patients solely to the spike protein (Poland et al, 2020). Similarly, the spike protein is the exclusive target of currently approved monoclonal antibody therapies for COVID-19 (Taylor et al, 2021). Unfortunately, recent months have seen the emergence and rapid spread of mutant viral strains conferring amino acid changes in the spike protein, which can attenuate neutralization by many convalescent, vaccine-induced, and monoclonal antibodies (Garcia-Beltran et al, 2021; Harvey et al, 2021). Therefore, from a public health perspective, it is imperative to pursue the development of therapeutic strategies that can withstand the continued emergence of SARS-CoV-2 escape mutants. The spike protein mutations that cause increased virulence and immune evasion are predominantly found in the receptor-binding domain (RBD) (Piccoli et al, 2020; Harvey et al, 2021), which is specifically responsible for host recognition via interaction with human angiotensin-converting enzyme 2 (ACE2) (Benton et al, 2020). The RBD harbors two hotspots for antibody recognition. One of these epitopes overlaps with the ACE2 binding interface and is evolutionarily unique in SARS-CoV-2; the second so-called “cryptic” epitope is found in a peripheral region that is conserved among RBDs from several characterized coronaviruses (Yuan et al, 2020). While individually targeting either epitope with antibodies quickly results in the emergence of escape mutants (Weisblum et al, 2020; Greaney et al, 2021), there is growing evidence that simultaneous engagement of both epitopes via polyvalent antibodies may mitigate viral escape (De Gasparo et al, 2021; Koenig et al, 2021). Here, we present two synthetic single-domain antibodies (sybodies), designated Sb#15 and Sb#68, that recognize non-overlapping epitopes on the RBD. Sybodies offer several advantages over conventional antibodies such as the potential for rapid development, low-cost production in prokaryotic expression systems, and facile engineering (Zimmermann et al, 2018; Jovčevska & Muyldermans, 2020). Cryogenic electron microscopy (cryo-EM) revealed that Sb#15 binds within the ACE2 interface, whereas Sb#68 engages the adjacent conserved cryptic epitope. Structural analysis also demonstrated that the dual presence of Sb#15 and Sb#68 resulted in the adoption of a novel RBD conformation that we termed up/out. Fusion of Sb#15 and Sb#68 yielded a bispecific construct, termed GS4, that displayed enhanced avidity and neutralization potency relative to the separate sybodies. Exposure of SARS-CoV-2 to the individual sybodies in vitro resulted in the rapid emergence of escape mutants, including a Q493R-RBD variant (within the ACE2 epitope) that has recently been observed in COVID-19 patients treated with a monoclonal antibody (Focosi et al, 2021), as well as a novel P384H-RBD mutation in the cryptic epitope. In contrast, no escape mutants were detected upon treatment with GS4. Finally, we found that additional valency engineering via covalent trimerization of GS4, giving a construct we termed Tripod-GS4r, resulted in further enhancement of viral neutralization potential against the B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) SARS-CoV-2 variants of concern (VOCs). Overall, our study demonstrates favorable prospects for such multivalent sybodies to be a valuable therapeutic tool vis-à-vis future SARS-CoV-2 variants or comparable forthcoming viral pandemics. Results Identification of a sybody pair that (i) simultaneously bind to the spike RBD, (ii) compete with ACE2 interaction and (iii) efficiently neutralize viruses We sought to engineer a pair of synthetic nanobodies (sybodies), which may mitigate viral escape due to the simultaneous binding of discrete non-overlapping epitopes of the SARS-CoV-2 spike (S) glycoprotein. Using our established sybody generation workflow (Zimmermann et al, 2018, 2020), we conducted a selection campaign against the isolated receptor-binding domain (RBD) of the spike protein. Upon screening single sybody clones with ELISA and grating-coupled interferometry (GCI), we identified six sybodies exhibiting affinities against the RBD ranging from 24 to 178 nM (Figs 1A and EV1A, Appendix Table S1). In this study, we focus on sybodies Sb#15 and Sb#68 (Figs 1A and EV1B) that can simultaneously bind the immobilized spike protein (Fig 1B) and exhibit affinities of 12 and 9 nM, respectively, when probing against the entire spike protein stabilized by two prolines (S-2P) (Fig 1A). Figure 1. Sybodies Sb#15 and Sb#68 bind non-overlapping epitopes on the spike protein, and inhibit ACE2 binding Affinity determination of Sb#15 and Sb#68 against the immobilized spike protein (S-2P) using GCI. The data were fitted using a heterogeneous ligand model. Left, GCI epitope-binning experiment showing Sb#15 (blue), Sb#68 (red), and their combination (black) against immobilized spike protein (S-2P). Since both sybodies were present at saturating concentrations, the increased amplitude is indicative of simultaneous binding. Right, ELISA experiment confirming dual-binding of Sb#15 and Sb#68. Myc-tagged Sb#15 was immobilized on an anti-myc antibody-coated ELISA plate, followed by exposure of biotinylated RBD, which was premixed with tag-less sybodies (indicated on the x-axis). Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Kinetic characterization of sybodies by GCI RBD-vYFP and ECD were immobilized as indicated and the six top sybodies were injected at increasing concentrations ranging from 1.37 nM to 1 μM. Data were fitted using a Langmuir 1:1 model. In-depth affinity characterization of Sb#15 and Sb#68. RBD-vYFP and S-6P were immobilized as indicated and Sb#15 and Sb#68 were injected at concentrations ranging from 1.95 to 250 nM for Sb#15 and 3.9 to 500 nM for Sb#68. For RBD, data were fitted using a Langmuir 1:1 model. For S-6P, the data were fitted with the heterogeneous ligand model, because the 1:1 model was clearly not appropriate to describe the experimental data. Corresponding data for S-2P is shown in main Fig 1A. Download figure Download PowerPoint To investigate whether Sb#15 and/or Sb#68 could block the interaction between the spike protein and ACE2, we performed an ACE2 competition experiment using GCI. To this end, spike protein was coated on a GCI chip and Sb#15 (200 nM), Sb#68 (200 nM) as well as a non-randomized convex sybody control (Sb#0, 200 nM) were injected alone or together with ACE2 (100 nM) to monitor binding (Fig 1C). Indeed, Sb#0 did not bind when injected alone and consequently did not disturb ACE2 binding when co-injected. Conversely, both Sb#15 and Sb#68 were found to dominate over ACE2 in the association phase during co-injection, and the resulting curves are highly similar to what was observed when these two sybodies were injected alone. This experiment demonstrated that Sb#15 and Sb#68 compete with ACE2 for access to its binding site on the spike protein. Having established that Sb#15 and Sb#68 could bind the spike protein and block ACE2 association in vitro, we next asked whether these sybodies (as well as the additional candidate sybodies Sb#16 and Sb#45 Appendix Fig S1, Appendix Table S1) could inhibit the SARS-CoV-2 fusogenic machinery in viral neutralization assays. To varying extents, all assayed sybodies neutralized vesicular stomatitis viruses (VSV) that were pseudotyped with SARS-CoV-2 spike protein (Zettl et al, 2020), with estimated IC50 values of 2.3 µg/ml (147 nM) and 2.3 µg/ml (138 nM), for Sb#15 and Sb#68, respectively (Fig 2A and B, Table 1). As a positive control for our VSV neutralization assay, we used the previously characterized RBD-binding sybody MR3 (Li et al, 2021), which in our assay displayed an IC50 value of 0.4 µg/ml, equivalent to the reported value (Li et al, 2021). Since Sb#15 and Sb#68 can bind simultaneously to full-length spike protein, we mixed Sb#15 and Sb#68 together to investigate potential additive or synergistic neutralizing activity of these two independent sybodies. Indeed, consistent with the binding assays, the simultaneous presence of both sybodies resulted in slightly improved neutralization profiles with IC50 values reaching 1.7 µg/ml (53 nM), suggesting an additive effect. In addition to the individual sybodies, we also explored potential avidity effects of sybodies genetically fused to human IgG1 Fc domains. The respective homodimeric sybody-Fc constructs exhibited VSV pseudotype IC50 values of 1.2 µg/ml (16 nM) and 3.9 µg/ml (50 nM) for Sb#15 and Sb#68, respectively (Fig 2C, Table 1). This improvement in VSV neutralization potency suggests that the bivalent arrangement of the Fc fusion constructs resulted in a discernible avidity effect. For neutralization of live SARS-CoV-2 we employed a classical virus neutralization assay and confirmed that both sybodies successfully inhibited cell entry by infectious SARS-CoV-2, with ND50 values of 8.8 µg/ml (561 nM) for Sb#15 and 6.3 µg/ml (377 nM) for Sb#68 (Table 1). The approximately 3- to 6-fold discrepancy in neutralization efficacies, measured using either live SARS-CoV-2 virus or pseudotyped VSV, may reflect slight differences in viral physiology (variation of incorporated spikes per viral particle) or could owe to the different assay methods (luciferase emission versus plaque reduction determination). Collectively, these data highlight the successful discovery of a pair of sybodies (Sb#15 and Sb#68) that bind simultaneously to the spike RBD, compete with ACE2 interaction, and neutralize viral infection in vitro. Figure 2. Neutralization of viral entry using pseudotyped VSVs Neutralization assays using VSVΔG pseudotyped with wild-type SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. Error bars correspond to standard deviation of three biological replicates. Sb#15, Sb#68, or an equimolar mixture of both sybodies. Sb#14, Sb#16, Sb#45, or, as a positive control, the previously described sybody MR3. Neutralization by bivalent sybody-Fc fusions. Download figure Download PowerPoint Table 1. Summary of neutralization assay results. Binders SARS-CoV-2 pseudovirus Live SARS-CoV-2 IC50 (µg/ml) IC50 (nM) ND50 (µg/ml) ND50 (nM) Sb#14 2.8 178.3 nn nn Sb#15 2.3 146.5 8.8 561 Sb#16 20 1250 nn nn Sb#45 15 910 nd nd Sb#68 2.3 137.7 6.3 377 Sb#15+Sb#68 1.7 52.5 nd nd GS4 0.02 0.7 0.08 2.6 MR3 0.4 24 2.3 140 Tripod-GS4r 0.01 0.08 0.08 0.6 Sb#14-Fc 2.9 37.8 nd nd Sb#15-Fc 1.2 15.5 nd nd Sb#16-Fc 0.6 7.8 nd nd Sb#45-Fc 1.6 20.3 nd nd Sb#68-Fc 3.9 49.6 nd nd nn, non-neutralizing; nd, not determined. Structural basis of Sb#15 and Sb#68 neutralizing activity To gain structural insights into how Sb#15 and Sb#68 recognize the RBD and neutralize viruses, we performed single-particle cryo-EM analysis of purified sybody-spike protein complexes. Three cryo-EM datasets were collected, allowing a glimpse of the spike protein either simultaneously bound to both sybodies, or associated to Sb#15 or Sb#68 alone (Fig 3, Appendix Table S2). The highest resolution was obtained for the spike protein in complex with both sybodies (Figs 3 and EV2, Appendix Fig S1A–K), whereas structures with the individual sybodies were determined based on fewer particles and mainly served to unambiguously assign the binding epitopes of Sb#15 (Appendix Fig S2A–G, Fig EV3A and B) and Sb#68 (Appendix Fig S3A–K, Fig EV4). Analysis of the spike/Sb#15/Sb#68 particles after 3D classification revealed that the spike protein adopts two distinct conformations (Appendix Fig S1F and G). The first conformation (30% of particles) has a three-fold symmetry, with three RBDs in the up conformation (3up) and two sybodies bound to each of the RBDs, confirming that Sb#15 and Sb#68 bind simultaneously (Figs 3A and EV2A, Appendix Fig S1F and G).
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