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

  1. Stéphanie Thébault
  2. Nathalie Lejal
  3. Alexis Dogliani
  4. Amélie Donchet
  5. Agathe Urvoas
  6. Marie Valerio-Lepiniec
  7. Muriel Lavie
  8. Cécile Baronti
  9. Franck Touret
  10. Bruno Da Costa
  11. Clara Bourgon
  12. Audrey Fraysse
  13. Audrey Saint-Albin-Deliot
  14. Jessica Morel
  15. Bernard Klonjkowski
  16. Xavier de Lamballerie
  17. Jean Dubuisson
  18. Alain Roussel
  19. Philippe Minard
  20. Sophie Le Poder
  21. Nicolas Meunier
  22. Bernard Delmas

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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 EC 50 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 EC 50 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.

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  1. Version published to 10.1371/journal.ppat.1010799
  2. ScreenIT

    SciScore for 10.1101/2022.05.10.491295: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsField Sample Permit: The study was carried out following a protocol approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A-16) and authorized by the French ministry of Research under the number APAFIS#25384-2020041515287655 v6 in accordance with the French and European regulations.
    IRB: The study was carried out following a protocol approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A-16) and authorized by the French ministry of Research under the number APAFIS#25384-2020041515287655 v6 in accordance with the French and European regulations.
    Sex as a biological variableGolden Syrian hamster infections and assessment of αREPs antiviral activity: Hamster infections: Thirty-two specific-pathogen-free (SPF) 8 weeks-old Golden Syrian hamsters (Mesocricetus auratus, males, provided by Janvier-Labs, Le Genest-Saint-Isle, France) housed under BSL-III conditions were kept according to the standards of French law for animal experimentation.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The sections were then incubated overnight in PBS with 0.2% BSA and 0.05% Tween-20 with primary antibodies directed against SARS-CoV-2 Nucleocapsid Protein (1:500; mouse monoclonal, # ZMS1075, Merck);
    SARS-CoV-2 Nucleocapsid Protein ( 1:500
    suggested: None
    Fluorescence staining was performed using goat anti-rabbit Alexa-Fluor-488 (1:800; Molecular Probes – A32731; Invitrogen, Cergy Pontoise, France) and donkey anti-mouse Alexa-Fluor 555 (1:800; Molecular Probes – A32773; Invitrogen, Cergy Pontoise, France) secondary antibodies.
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# A32731, RRID:AB_2633280)
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A32773, RRID:AB_2762848)
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, HEK-293TT cells (106 cells per P6 well) were transfected with plasmids encoding GAG-POL, F-LUC and SARS-CoV-2 spikes.
    HEK-293TT
    suggested: None
    Viral stocks were prepared by propagation in Vero E6 cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% (v/v
    Vero E6
    suggested: RRID:CVCL_XD71)
    This mixture was added to Vero-E6 cells (CRL-1586, ATCC) seeded in a 96-well plate one day before.
    Vero-E6
    suggested: None
    To prepare the virus working stocks, a 25cm2 culture flask of confluent VeroE6 TMPRSS2 cells growing with MEM medium with 2.5% FCS was inoculated at a multiplicity of infection (MOI) of 0.001.
    VeroE6 TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    EC50 and EC90 determination: One day prior to infection, 5×104 VeroE6/TMPRSS2 cells per well were seeded in 100 µL assay medium (containing 2.5% FCS) in 96 well culture plates.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    SARS CoV-2 Beta (SA lineage B 1.351) was isolated in France in 2021, The strain is available through EVA GLOBAL: UVE/SARS-CoV-2/2021/FR/1299-ex SA (lineage B 1.351) at https://www.european-virus-archive.com/virus/sars-cov-2-uvesars-cov-22021fr1299-ex-sa-lineage-b-1351. Sars-Cov-2 Gamma (SARS-CoV-2/2021/JP/TY7-503 lineage P.1, ex Brazil) was isolated in Japan in January 2021.
    Sars-Cov-2 Gamma
    suggested: None
    Recombinant DNA
    SentencesResources
    Production of the receptor binding domain (RBD) of the SARS-CoV-2 spike: The RBD (223 amino acids starting at position 319 of the spike sequence) coding sequence was cloned in frame behind a sequence encoding a signal peptide and in front of a His-tag coding sequence in the eukaryotic pYD11 expression plasmid.
    pYD11
    suggested: None
    αReps expression and purification: The αRep genes encoding the RBD binders were subcloned in the bacterial expression vector pQE81 and resulting plasmids used for transforming Rosetta cells.
    pQE81
    suggested: None
    Software and Algorithms
    SentencesResources
    The sections were then incubated overnight in PBS with 0.2% BSA and 0.05% Tween-20 with primary antibodies directed against SARS-CoV-2 Nucleocapsid Protein (1:500; mouse monoclonal, # ZMS1075, Merck);
    Merck)
    suggested: None
    Images were quantified using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2012) to threshold specific staining of SARS-CoV-2 in the dorso-medial area of the nasal cavity.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    All data obtained were analyzed using GraphPad Prism 8 software (Graphpad software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2022.05.10.491295v1 on bioRxiv