Broad neutralization of SARS-CoV-2 variants by circular mRNA producing VFLIP-X spike in mice

  1. Chotiwat Seephetdee
  2. Kanit Bhukhai
  3. Nattawut Buasri
  4. Puttipatch Leelukkanaveera
  5. Pat Lerdwattanasombat
  6. Suwimon Manopwisedjaroen
  7. Nut Phueakphud
  8. Sakonwan Kuhaudomlarp
  9. Eduardo Olmedillas
  10. Erica Ollmann Saphire
  11. Arunee Thitithanyanont
  12. Suradej Hongeng
  13. Patompon Wongtrakoongate

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Abstract

Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X elicited neutralizing antibodies for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in T H 1 and T H 2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA confers humoral and cellular immune responses, as well as neutralizing activity against broad SARS-CoV-2 variants.

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  1. Version published to 10.1101/2022.03.17.484759v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.03.17.484759: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mouse immunization: Mouse experiments were performed under the Animal Ethics approved by Faculty of Science, Mahidol University (MUSC63-016-524).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blotted proteins were detected with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) and a donkey anti-rabbit IgG-HRP antibody (sc-2077, Santa Cruz Biotechnology).
    anti-rabbit IgG-HRP
    suggested: (Santa Cruz Biotechnology Cat# sc-2077, RRID:AB_631745)
    sc-2077
    suggested: None
    Flow cytometry: To detect surface protein expression, transfected HEK293T cells were stained with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) and an Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, Invitrogen).
    A11034
    suggested: None
    Cells were then blocked with 20% FBS in PBS and incubated with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) followed by an Alexa Fluor 594-conjugated goat anti-rabbit IgG (A11037, Invitrogen).
    anti-rabbit IgG
    suggested: None
    A11037
    suggested: (Thermo Fisher Scientific Cat# A-11037, RRID:AB_2534095)
    1.1.529 S 15-mer (overlapping by 11 amino acids) peptide pool (GenScript) in plates pre-coated with anti-IFN-γ or anti-IL-4 antibodies.
    anti-IFN-γ
    suggested: None
    anti-IL-4
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T-hACE2 cells were cultured with 1 μg/ml puromycin to maintain stable expression of hACE2.
    HEK293T-hACE2
    suggested: RRID:CVCL_A7UK)
    Immunofluorescence assay: Transfected HEK293T cells were fixed with 4% paraformaldehyde (PFA).
    HEK293T
    suggested: RRID:CVCL_HA71)
    Briefly, serially diluted heat-inactivated sera (starting with a dilution of 1:20) were pre-incubated with equal volumes of 100 TCID50 of SARS-CoV-2 for one hour at 37°C. 100 ml of the virus-serum mixtures were added to pre-seeded Vero E6/TMPRSS2 cell monolayers (1×104 cells/well) in duplicate on a 96-well microtiter plate.
    Vero E6/TMPRSS2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    In vivo bioluminescence imaging: For detection of in vivo expression of FLuc-encoding circRNA-LNP, 7-week-old female BALB/c mice were inoculated with 1 μg of the circRNA-LNP via intramuscular route.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Recombinant DNA
    SentencesResources
    Briefly, the SARS-CoV-2 pseudovirus were produced by co-transfection of plasmids pHAGE-CMV-Luc2-IRES-ZsGreen-W, HDM-Hgpm2, HDM-tat1b, pRC-CMV-Rev1b, and SARS-CoV-2 spike expressing plasmid into HEK293T cells using jetPRIME transfection reagent.
    pHAGE-CMV-Luc2-IRES-ZsGreen-W
    suggested: RRID:Addgene_164432)
    pRC-CMV-Rev1b
    suggested: RRID:Addgene_164443)
    Software and Algorithms
    SentencesResources
    An unrooted phylogenetic tree comparing amino acid sequences derived from wildtype spike, SARS-CoV-2 spike variants of concern (VOCs) and variants of interest (VOIs), and wildtype-X spike (wildtype spike with the six rationally substituted amino acids) was constructed by maximum likelihood using IQ-TREE software.
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)
    Cells were then acquired on a BD Accuri C6 plus and analyzed by FlowJo software version 10.6.2.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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

    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 10.1101/2022.03.17.484759v1 on bioRxiv