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Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals

Stéphanie Thébault, Nathalie Lejal, Alexis Dogliani, Amélie Donchet, Agathe Urvoas, Marie Valerio-Lepiniec, Muriel Lavie, Cécile Baronti, Franck Touret, Bruno da Costa, Clara Bourgon, Audrey Fraysse, Audrey Saint-Albin-Deliot, Jessica Morel, Bernard Klonjkowski, Xavier de Lamballerie, Jean Dubuisson, Alain Roussel, Philippe Minard, Sophie Le Poder, Nicolas Meunier, Bernard Delmas
doi: https://doi.org/10.1101/2022.05.10.491295
Stéphanie Thébault
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Nathalie Lejal
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Alexis Dogliani
2Centre National de la Recherche Scientifique, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Marseille, France
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Amélie Donchet
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Agathe Urvoas
3Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette cedex, France
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Marie Valerio-Lepiniec
3Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette cedex, France
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Muriel Lavie
4Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Lille, France
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Cécile Baronti
5Unité des Virus Émergents (UVE), Aix Marseille Université, IRD 190, INSERM 1207, Marseille, France
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Franck Touret
5Unité des Virus Émergents (UVE), Aix Marseille Université, IRD 190, INSERM 1207, Marseille, France
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Bruno da Costa
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Clara Bourgon
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Audrey Fraysse
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Audrey Saint-Albin-Deliot
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Jessica Morel
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Bernard Klonjkowski
6UMR Virologie, INRAE-ENVA-ANSES, École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, 94704 Paris, France
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Xavier de Lamballerie
5Unité des Virus Émergents (UVE), Aix Marseille Université, IRD 190, INSERM 1207, Marseille, France
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Jean Dubuisson
4Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Lille, France
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Alain Roussel
2Centre National de la Recherche Scientifique, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Marseille, France
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Philippe Minard
3Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette cedex, France
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Sophie Le Poder
6UMR Virologie, INRAE-ENVA-ANSES, École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, 94704 Paris, France
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Nicolas Meunier
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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Bernard Delmas
1Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350, Jouy-en-Josas, France
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  • For correspondence: bernard.delmas@inrae.fr
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Abstract

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

Author Summary The entry of SARS-CoV-2 in permissive cells is mediated by the binding of its spike to angiotensin-converting enzyme 2 (ACE2) on the cell surface. To select ligands able to block this interaction, we screened a library of phages encoding artificial proteins (named αReps) for binding to its receptor binding domain (RBD). Two of them were able to bind the RBD with high affinity and block efficiently the virus entry in cultured cells. Assembled αReps through covalent or non-covalent linkages blocked virus entry at lower concentration than their precursors (with around 20-fold activity increase for a trimeric αRep). These αReps derivates neutralize efficiently SARS-CoV-2 β, γ, δ and Omicron virus variants. Instillation of an αRep dimer in the nasal cavity effectively reduced virus replication in the hamster model of SARS-CoV-2 and pathogenicity.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 11, 2022.
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Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals
Stéphanie Thébault, Nathalie Lejal, Alexis Dogliani, Amélie Donchet, Agathe Urvoas, Marie Valerio-Lepiniec, Muriel Lavie, Cécile Baronti, Franck Touret, Bruno da Costa, Clara Bourgon, Audrey Fraysse, Audrey Saint-Albin-Deliot, Jessica Morel, Bernard Klonjkowski, Xavier de Lamballerie, Jean Dubuisson, Alain Roussel, Philippe Minard, Sophie Le Poder, Nicolas Meunier, Bernard Delmas
bioRxiv 2022.05.10.491295; doi: https://doi.org/10.1101/2022.05.10.491295
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Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals
Stéphanie Thébault, Nathalie Lejal, Alexis Dogliani, Amélie Donchet, Agathe Urvoas, Marie Valerio-Lepiniec, Muriel Lavie, Cécile Baronti, Franck Touret, Bruno da Costa, Clara Bourgon, Audrey Fraysse, Audrey Saint-Albin-Deliot, Jessica Morel, Bernard Klonjkowski, Xavier de Lamballerie, Jean Dubuisson, Alain Roussel, Philippe Minard, Sophie Le Poder, Nicolas Meunier, Bernard Delmas
bioRxiv 2022.05.10.491295; doi: https://doi.org/10.1101/2022.05.10.491295

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