Preclinical study of DNA vaccines targeting SARS-CoV-2

  1. Hiroki Hayashi
  2. Jiao Sun
  3. Yuka Yanagida
  4. Takako Otera
  5. Ritsuko Kubota-Kotetsu
  6. Tatsuo Shioda
  7. Chikako Ono
  8. Yoshiharu Matsuura
  9. Hisashi Arase
  10. Shota Yoshida
  11. Ryo Nakamaru
  12. Ryoko Ide
  13. Akiko Tenma
  14. Sotaro Kawabata
  15. Takako Ehara
  16. Makoto Sakaguchi
  17. Hideki Tomioka
  18. Munehisa Shimamura
  19. Sachiko Okamoto
  20. Yasunori Amaishi
  21. Hideto Chono
  22. Junichi Mineno
  23. Takano Komatsuno
  24. Yoshimi Saito
  25. Hiromi Rakugi
  26. Ryuichi Morishita
  27. Hironori Nakagami

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Abstract

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.21.347799: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All experiments were approved by the Ethical Committee for Animal Experiments of the Osaka University Graduate School of Medicine.
    Consent: The use of patients sera with written informed consent was approved by an Ethics Committee of Research Instituted for Microbial Diseases Osaka University (approval number 31-14-1).
    IRB: The use of patients sera with written informed consent was approved by an Ethics Committee of Research Instituted for Microbial Diseases Osaka University (approval number 31-14-1).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAnimal protocol: Seven-week-old male Sprague-Dawley (SD) rats were purchased from Clea Japan Inc. (Tokyo, Japan), and housed with free access to food and water in a temperature and light cycle-controlled facility.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After the membrane was blocked with PBS containing 5 % skim milk for 1 hour at room temperature (RT), the membrane was incubated with an antibody against SARS-CoV-2 Spike (GNX135356, GeneTex, Inc.) at 4 °C overnight.
    antibody against SARS-CoV-2 Spike ( GNX135356
    suggested: None
    Sandwich ELISA for detection of secreted spike protein: A diluted capture antibody for the spike protein (GTX632604, GeneTex) was coated on a 96-well plate and incubated at 4 °C overnight.
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    Ninety-six-well PVDF membrane-bottomed plates (Merck Millipore) were coated with an anti-rat IFNγ capture antibody or IL4 capture antibody and incubated at 4 °C overnight.
    anti-rat IFNγ
    suggested: None
    The washed plates were incubated with a biotinylated polyclonal antibody specific for rat IFNγ or IL4 for 2 hours at 4 °C.
    IL4
    suggested: None
    After washing with PBS-T, plate was incubated with anti-his antibody conjugated with HRP (abcam) for 2 h at RT.
    anti-his
    suggested: None
    As first antibody, 1μg/ml of anti-SARS-CoV/SARS-CoV-2 nucleoprotein monoclonal antibody, HM1054 (EastCoast Bio) was used.
    anti-SARS-CoV/SARS-CoV-2 nucleoprotein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, DNA transfection, western blotting, and immunostaining: Human embryonic kidney 293 (HEK293) cells were cultured in DMEM containing 10 % FBS plus penicillin/streptomycin and grown at 37 °C in a humidified 5 % CO2 incubator.
    HEK293
    suggested: None
    Pseudoviruses were incubated with a series of dilutions of inactivated rat serum for 1 hour at 37 °C, and then added to Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    VeroE6/TMPRSS2 cells (4×104 cells/well) were seeded on 96-well plates and incubated at 37° for 24 hours.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animal protocol: Seven-week-old male Sprague-Dawley (SD) rats were purchased from Clea Japan Inc. (Tokyo, Japan), and housed with free access to food and water in a temperature and light cycle-controlled facility.
    Sprague-Dawley
    suggested: RRID:RGD_70508)
    Software and Algorithms
    SentencesResources
    The value of the half-maximal antibody titer of each sample was calculated from the highest absorbance in the dilution range by using Prism 6 software.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Signals were detected with a ChemiDoc Touch Imaging System (Bio-Rad) and analyzed with Image Lab software version 6.0.1 (
    Image Lab
    suggested: (Image Lab Software, RRID:SCR_014210)
    One-way ANOVA followed by Tukey’s post hoc test was used to assess significant differences in each experiment using Prism 6 software (GraphPad Software).
    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 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

    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 10.1101/2020.10.21.347799 on bioRxiv