SARS-CoV-2 spike RBD and nucleocapsid encoding DNA vaccine elicits T cell and neutralising antibody responses that cross react with variants

  1. VA Brentville
  2. M Vankemmelbeke
  3. RL Metheringham
  4. P Symonds
  5. KW Cook
  6. RA Urbanowicz
  7. T Tsoleridis
  8. CM Coleman
  9. K-C Chang
  10. A Skinner
  11. E Dubinina
  12. I Daniels
  13. S Shah
  14. M Argonza
  15. J Delgado
  16. V Dwivedi
  17. V Kulkarni
  18. JE Dixon
  19. AG Pockley
  20. SE Adams
  21. SJ Paston
  22. JM Daly
  23. JK Ball
  24. LG Durrant

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Abstract

Although the efficacy of vaccines targeting SARS-CoV-2 is apparent now that the approved mRNA and adenovirus vector vaccines are in widespread use, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. We have therefore designed a DNA vaccine encoding both the SARS-CoV-2 spike receptorbinding domain (‘RBD’) and nucleocapsid proteins, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4 + Th1 and CD8 + T-cell responses to both proteins in mice and rats, with responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD in mice that efficiently neutralise in pseudotype and live virus neutralisation assays and show cross reactivity with spike proteins from the variants B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). The vaccine also showed good protection in a viral challenge model in ACE2 receptor transgenic mice. This DNA platform can be easily adapted to target variant proteins and we show that a vaccine variant encoding the Beta variant sequence stimulates cross-reactive humoral and T cell responses. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for rapidly targeting emerging SARS-CoV-2 variants.

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  1. Version published to 10.1101/2021.06.18.448932v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.06.18.448932: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody binding to S1 antigen was detected using a three-step approach consisting of anti-mouse Fc biotin (Sigma Aldrich B7401) followed by streptavidin-HRPO (Life Technologies SA1007) and finally TMB (3,3’, 5, 5’-tetramethylbenzidine) Core+ reagent (Bio-Rad BUF062C) for development.
    S1
    suggested: None
    anti-mouse
    suggested: (Sigma-Aldrich Cat# B7401, RRID:AB_258619)
    All assays included a standard curve of mouse neutralising S1 antibody and mouse anti-N antibody (Sino Biological).
    anti-N
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, 1.5 × 106 HEK293T cells were seeded overnight in a 10 cm diameter Primaria-coated dish (Corning).
    HEK293T
    suggested: None
    For infectivity and neutralisation testing of SARS-CoV-2 pseudoparticles, 1 × 105 VeroE6 cells were seeded on white 96-well tissue culture plates (Corning) and incubated overnight at 37°C.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    HLA-A2.1+/+ HLADP4+/+ hCD4+/+ (HHDII/DP4; EM:02221, European Mouse Mutant Archive), HLA-A2/DR1 (HHDII/DR1; Pasteur Institute), HLA-A2 (HHDII; Pasteur Institute) transgenic mice or BALB/c and C57Bl/6 (Charles River) aged 7–12 weeks were used.
    HLA-A2.1+/+ HLADP4+/+ hCD4+/+
    suggested: None
    detected: (IMSR Cat# EM_02221, RRID:IMSR_EM:02221)
    BALB/c
    suggested: None
    C57Bl/6
    suggested: None
    Recombinant DNA
    SentencesResources
    DNA plasmids: The backbone of the vaccine plasmids was derived from the FDA regulatory compliant vector backbone of pVAX1 (Invitrogen) for use in humans.
    pVAX1
    suggested: RRID:Addgene_137910)
    The sequences of both chains within each expression cassette of the pVaxDC vectors were confirmed by the dideoxy chain termination method [30].
    pVaxDC
    suggested: None
    The plasmid pCMV3-2019-nCoV-Spike (S1+S2)-long encodes full-length spike from SARS-CoV-2 amino acid 1–1273 (GenBank accession number YP_009724390 /QHD43416.1) was obtained from Sino Biological (catalogue number VG40589-UT).
    pCMV3-2019-nCoV-Spike
    suggested: None
    This contained codon optimised cDNA for expression of the protein in mammalian cells inserted into the KpnI/XbaI sites of the mammalian expression vector pCMV3-untagged under control of the high-level expression mammalian human enhanced cytomegalovirus (CMV) immediate early promoter.
    pCMV3-untagged
    suggested: None
    Transfections were performed with 2 µg each of the murine leukemia virus (MLV) Gag-Pol packaging vector (phCMV-5349), luciferase encoding reporter plasmid (pTG126) and plasmid encoding Wuhan strain SARS-CoV-2 spike using 24 µL polyethylenimine (Polysciences) in Optimem (Gibco).
    pTG126
    suggested: None
    Software and Algorithms
    SentencesResources
    Neutralising activities were reported as reciprocal serum dilution levels corresponding to 50% inhibitory dilution (ID50) values and were calculated by nonlinear regression (GraphPad Prism version 9.1.2), using lower and upper bounds (0% and 100% inhibition) as constraints to assist curve fitting.
    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: We detected the following sentences addressing limitations in the study:
    DNA vectors also lend themselves well to use in multiple homologous prime boost immunisation regimes, which is often not compatible with other viral vector-based vaccination strategies due to limitations of anti-vector immunity but may be vital to combat rapidly emerging virus variants. Data from our cancer vaccine platform demonstrates the stability of the DNA vector used in this study and its safety in human subjects [27]. The bivalent DNA SARS-CoV-2 vaccine stimulates strong humoral and cellular immunity that can be easily adapted to emerging virus variants. Furthermore, the responses elicited by both original Lineage A and B.1.351 (Beta) variant vaccines show cross-reactivity with multiple viral variants and support the potential clinical application of these vaccines.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    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: 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.06.18.448932v1 on bioRxiv