Single dose immunization with a COVID-19 DNA vaccine encoding a chimeric homodimeric protein targeting receptor binding domain (RBD) to antigen-presenting cells induces rapid, strong and long-lasting neutralizing IgG, Th1 dominated CD4 + T cells and strong CD8 + T cell responses in mice

  1. Gunnstein Norheim
  2. Elisabeth Stubsrud
  3. Lise Madelene Skullerud
  4. Branislava Stankovic
  5. Stalin Chellappa
  6. Louise Bjerkan
  7. Katarzyna Kuczkowska
  8. Elisabeth Müller
  9. Monika Sekelja
  10. Agnete B. Fredriksen

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Abstract

The pandemic caused by the SARS-CoV-2 virus in 2020 has led to a global public health emergency, and non-pharmaceutical interventions required to limit the viral spread are severely affecting health and economies across the world. A vaccine providing rapid and persistent protection across populations is urgently needed to prevent disease and transmission. We here describe the development of novel COVID-19 DNA plasmid vaccines encoding homodimers consisting of a targeting unit that binds chemokine receptors on antigen-presenting cells (human MIP-1α /LD78β), a dimerization unit (derived from the hinge and C H 3 exons of human IgG3), and an antigenic unit (Spike or the receptor-binding domain (RBD) from SARS-CoV-2). The candidate encoding the longest RBD variant (VB2060) demonstrated high secretion of a functional protein and induced rapid and dose-dependent RBD IgG antibody responses that persisted up to at least 3 months after a single dose of the vaccine in mice. Neutralizing antibody (nAb) titers against the live virus were detected from day 7 after one dose. All tested dose regimens reached titers that were higher or comparable to those seen in sera from human convalescent COVID-19 patients from day 28. T cell responses were detected already at day 7, and were subsequently characterized to be multifunctional CD8 + and Th1 dominated CD4 + T cells. Responses remained at sustained high levels until at least 3 months after a single vaccination, being further strongly boosted by a second vaccination at day 89. These findings, together with the simplicity and scalability of plasmid DNA manufacturing, safety data on the vaccine platform in clinical trials, low cost of goods, data indicating potential long term storage at +2° to 8°C and simple administration, suggests the VB2060 candidate is a promising second generation candidate to prevent COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.08.416875: (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 variableImmunization of animals: 6-week-old, female BALB/c mice were obtained from Janvier Labs (France).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For primary detection antibody, either biotinylated anti-human MIP-1α (R&D Systems) or SARS-CoV-2/2019-nCoV Spike/RBD Antibody (1:1000) (Sino Biological) was used.
    anti-human MIP-1α
    suggested: None
    SARS-CoV-2/2019-nCoV
    suggested: None
    Streptavidin-HRP (1:3000) or anti-rabbit IgG-HRP (1:5000) was added as secondary detection antibody.
    Streptavidin-HRP
    suggested: (Cell Signaling Technology Cat# 3999, RRID:AB_10830897)
    anti-rabbit IgG-HRP
    suggested: None
    Plates were washed 3x and incubated with a 1:50,000 dilution of anti-mouse total IgG-HRP antibody (Southern Biotech) and incubated for 1h at 37°C.
    anti-mouse total IgG-HRP
    suggested: None
    Microplaques were detected using a SARS-CoV-2 antibody specific for the SARS-CoV-2 RBD Spike protein and a rabbit HRP conjugate, infected foci were detected using TrueBlueTM substrate.
    SARS-CoV-2
    suggested: None
    Cells were further stained with the extracellular antibodies (anti-CD3, anti-CD4, anti-CD8 and γδTCR), fixed and permeabilized, and stained for detection of cytokines (anti-TNFα, anti-IFN-γ, anti-IL-4, anti-IL-17 antibodies) and a transcription factor (anti-FoxP3 antibody).
    anti-CD3
    suggested: (Thermo Fisher Scientific Cat# 8822-6853-41, RRID:AB_2575278)
    anti-CD4,
    suggested: None
    anti-CD8
    suggested: None
    anti-TNFα
    suggested: None
    anti-IFN-γ
    suggested: (Bio-Rad Cat# M6000007NY, RRID:AB_2784537)
    anti-IL-4
    suggested: None
    anti-IL-17
    suggested: None
    anti-FoxP3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    An ELISA was performed to verify the amount of VB10.COV2 protein produced by the HEK293 cells and secreted into the cell supernatant.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    The virus/serum mixtures were then transferred to washed Vero E6 (ECACC 85020206) cell monolayers in 96-well flat-bottomed plates, allowed to adsorb at 37°C for a further hour, before removal of the virus inoculum and replacement with overlay (1% w/v CMC in complete media).
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Immunization of animals: 6-week-old, female BALB/c mice were obtained from Janvier Labs (France).
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    These positive cells were further analyzed using Boolean gating algorithm in FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Statistical analyzes of antibody responses in sera to compare groups were performed by two-tailed Mann-Whitney test was performed (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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT02529930CompletedAn Exploratory Safety and Immunogenicity Study of HPV16+ Imm…
    NCT04405349RecruitingInvestigating the Combination of VB10.16 and Atezolizumab in…
    NCT03548467Active, not recruitingA Study to Evaluate Safety and Efficacy of Multiple Dosing W…

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 40. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2020.12.08.416875v1 on bioRxiv