A broadly neutralizing biparatopic Nanobody protects mice from lethal challenge with SARS-CoV-2 variants of concern

  1. Teresa R. Wagner
  2. Daniel Schnepf
  3. Julius Beer
  4. Karin Klingel
  5. Natalia Ruetalo
  6. Philipp D. Kaiser
  7. Daniel Junker
  8. Martina Sauter
  9. Bjoern Traenkle
  10. Desiree I. Frecot
  11. Matthias Becker
  12. Nicole Schneiderhan-Marra
  13. Annette Ohnemus
  14. Martin Schwemmle
  15. Michael Schindler
  16. Ulrich Rothbauer

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Abstract

The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailed in vitro characterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infection in vivo , human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses and in situ hybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.08.455562: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All experiments were in compliance with the German animal protection law and approved by the animal welfare committee of the Regierungspraesidium Freiburg (permit G-20/91).
    Euthanasia Agents: Infected ketamine/ xylazine-anesthetized mice were prepared for histological analyses by transcardial perfusion with 15 ml of 4% formaldehyde solution and stored in 4 % formaldehyde at 4 °C until organs were processed further.
    Sex as a biological variableHemizygous 8-14 week-old animals of both sexes were used in accordance with the guidelines of the Federation for Laboratory Animal Science Associations and the National Animal Welfare Body.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    48 h post-infection, cells were fixed with 80% acetone, and immune fluorescence (IF) staining was performed using an anti-SARS-CoV-2 nucleocapsid antibody (rabbit) and a goat anti-rabbit Alexa594 conjugated secondary antibody.
    anti-SARS-CoV-2 nucleocapsid antibody
    suggested: None
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Protein expression and purification: Confirmed constructs were expressed in Expi293 cells.
    Expi293
    suggested: RRID:CVCL_D615)
    Virus neutralization assay (VNT): Caco-2 (Human Colorectal adenocarcinoma,
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    Swabs were collected in OptiMEM containing 0.3% BSA and titers determined by plaque assay using Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    In vivo infection experiments: Transgenic (K18-hACE2)2Prlmn mice were purchased from The Jackson Laboratory and bred and kept under specific-pathogen-free conditions in the animal facilities of the University Medical Center Freiburg.
    K18-hACE2)2Prlmn
    suggested: None
    Recombinant DNA
    SentencesResources
    DNA coding for bipNbs were cloned into pCDNA3.4 expression vector seamlessly downstream of comprising N-terminal signal peptide (MGWTLVFLFLLSVTAGVHS) for secretory pathway using type IIS restriction enzyme Esp3I and EcoRI site.
    pCDNA3.4
    suggested: RRID:Addgene_131198)
    The expression plasmid pCAGGS encoding the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (amino acids 319-541) was kindly provided by F. Krammer. RBDs of SARS-CoV-2 variants of concern (VOCs) B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma), A.1.617.2 (Delta), B.1.429 (Epsilon), P3 (Theta), B.1.617.1 (Kappa) and A.23.1 were generated by PCR amplification of fragments from WT or cognate DNA template and subsequent fusion PCR by overlap extension to introduce described mutations.
    pCAGGS
    suggested: RRID:Addgene_18926)
    Software and Algorithms
    SentencesResources
    ThermControl v2.0.4 the melting (Tm) and aggregation (TAgg/ Tturbidity) temperature was determined.
    ThermControl
    suggested: None
    Infection rates were calculated as the ratio of DAPI-positive over Alexa594-positive cells, which were automatically counted by the Gen5 software (BioTek).
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    Inhibitory concentration 50 (IC50) was calculated as the half-maximal inhibitory dose using 4-parameter nonlinear regression (GraphPad Prism).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.08.08.455562v1 on bioRxiv