Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

  1. Asha Ann Philip
  2. John Thomas Patton

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Abstract

Among vaccines administered to children are those targeting rotavirus, a segmented double-stranded RNA virus that represents a major cause of severe gastroenteritis. To explore the feasibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding cassettes for NSP3, a translational 2A stop-restart signal, and a FLAG-tagged portion of the SARS-CoV-2 spike (S) protein: S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended RBD (ExRBD), or S2 core (CR) domain. Generation of rSA11 containing the S1 coding sequence required a sequence insertion of 2.2 kbp, the largest such insertion yet introduced into the rotavirus genome. Immunoblotting showed that rSA11 viruses containing the smaller NTD, RBD, ExRBD, and CR coding sequences expressed S-protein products of expected size, with ExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, the rSA11 virus containing the full-length S coding sequence (rSA11/NSP3-fS1) failed to express its expected 80 kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of immunogenic portions of the SARS-CoV-2 S protein, including NTD, RBD, ExRBD, and CR, that have sizes smaller than the S1 fragment.

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  1. Version published to 10.3390/vaccines9050449
  2. ScreenIT

    SciScore for 10.1101/2021.02.18.431835: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    , rabbit monoclonal PCNA [13110S, Cell Signaling Technology (CST), 1:1000] antibody or rabbit anti-RBD (ProSci 9087; 1:200
    PCNA [ 13110S , Cell Signaling Technology ( CST)
    suggested: None
    anti-RBD
    suggested: None
    Primary antibodies were detected using 1:10,000 dilutions of horseradish peroxidase (HRP)-conjugated secondary antibodies: horse anti-mouse IgG (CST),
    anti-mouse IgG
    suggested: None
    Blots were probed with FLAG antibody (1:2000) to detect fRBD and fExRBD and NSP2 antibody (1:2000).
    FLAG
    suggested: None
    NSP2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To summarize, BHK-T7 cells were transfected with SA11 pT7 plasmids and pCMV-NP868R using Mirus TransIT-LT1 transfection reagent.
    BHK-T7
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    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.

    About SciScore

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  3. Version published to 10.1101/2021.02.18.431835v1 on bioRxiv