Introduction of Two Prolines and Removal of the Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike-Based SARS-CoV-2 Vaccine in the Mouse Model

  1. Fatima Amanat
  2. Shirin Strohmeier
  3. Raveen Rathnasinghe
  4. Michael Schotsaert
  5. Lynda Coughlan
  6. Adolfo García-Sastre
  7. Florian Krammer

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Abstract

A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.

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  1. Version published to 10.1128/mbio.02648-20
  2. ScreenIT

    SciScore for 10.1101/2020.09.16.300970: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Mouse vaccinations and challenge: All animal procedures were performed by adhering to the Institutional Animal Care and Use Committee (IACUC) guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableSix to eight week old, female, BALB/c mice (Jackson Laboratories) were immunized intramuscularly with 3 μg of recombinant protein per mouse with an adjuvant, AddaVax (Invivogen) in a volume of 50 ul.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Next, primary antibody was prepared in PBS containing 1% non-fat milk using anti-hexahistidine antibody (Takara Bio, catalog #631212) at a dilution of 1:3000.
    anti-hexahistidine
    suggested: None
    The secondary solution was prepared with 1% non-fat milk in PBS-T using anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-Aldrich) at a dilution of 1:3,000.
    anti-mouse IgG ( whole molecule)–alkaline phosphatase ( AP )
    suggested: None
    The next day, cells were stained with an anti-nuceloprotein antibody (ThermoFisher; Catalog # PA5-81794) according to our published protocol (20).
    anti-nuceloprotein
    suggested: None
    IHC staining was performed using an anti-SARS-CoV nucleoprotein antibody (Novus Biologicals cat.
    anti-SARS-CoV nucleoprotein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    High titer Ad-hACE2 stocks were amplified in TRex™-293 cells, purified by CsCl ultracentrifugation and infectious titers determined by tissue-culture infectious dose-50 (TCID50), adjusting for plaque forming unit (PFU
    TRex™-293
    suggested: RRID:CVCL_D585)
    The RBD protein was produced in 293F cells and purified using Ni-NTA resin and this procedure has been described in detail earlier (19).
    293F
    suggested: RRID:CVCL_D615)
    Briefly, Vero.E6 cells were seeded at a density of 20,000 cells per well in a 96-well cell culture plate.
    Vero.E6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Six to eight week old, female, BALB/c mice (Jackson Laboratories) were immunized intramuscularly with 3 μg of recombinant protein per mouse with an adjuvant, AddaVax (Invivogen) in a volume of 50 ul.
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    Data was analyzed using GraphPad Prism 7 and are under the curve (AUC) values were measured and graphed (18).
    GraphPad Prism
    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 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

    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.

  3. Version published to 10.1101/2020.09.16.300970 on bioRxiv