COVID- e Vax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19

  1. Antonella Conforti
  2. Emanuele Marra
  3. Fabio Palombo
  4. Giuseppe Roscilli
  5. Micol Ravà
  6. Valeria Fumagalli
  7. Alessia Muzi
  8. Mariano Maffei
  9. Laura Luberto
  10. Lucia Lione
  11. Erika Salvatori
  12. Mirco Compagnone
  13. Eleonora Pinto
  14. Emiliano Pavoni
  15. Federica Bucci
  16. Grazia Vitagliano
  17. Daniela Stoppoloni
  18. Maria Lucrezia Pacello
  19. Manuela Cappelletti
  20. Fabiana Fosca Ferrara
  21. Emanuela D’Acunto
  22. Valerio Chiarini
  23. Roberto Arriga
  24. Abraham Nyska
  25. Pietro Di Lucia
  26. Davide Marotta
  27. Elisa Bono
  28. Leonardo Giustini
  29. Eleonora Sala
  30. Chiara Perucchini
  31. Jemma Paterson
  32. Kathryn Ann Ryan
  33. Amy-Rose Challis
  34. Giulia Matusali
  35. Francesca Colavita
  36. Gianfranco Caselli
  37. Elena Criscuolo
  38. Nicola Clementi
  39. Nicasio Mancini
  40. Rüdiger Groß
  41. Alina Seidel
  42. Lukas Wettstein
  43. Jan Münch
  44. Lorena Donnici
  45. Matteo Conti
  46. Raffaele De Francesco
  47. Mirela Kuka
  48. Gennaro Ciliberto
  49. Concetta Castilletti
  50. Maria Rosaria Capobianchi
  51. Giuseppe Ippolito
  52. Luca G. Guidotti
  53. Lucio Rovati
  54. Matteo Iannacone
  55. Luigi Aurisicchio

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Abstract

The COVID-19 pandemic caused by the β-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID- e Vax – a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD – induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID- e Vax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID- e Vax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

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  1. Version published to 10.1101/2021.06.14.448343v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.06.14.448343: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces.
    Field Sample Permit: All experimental work was conducted under the authority of a UK Home Office approved project license that had been subject to local ethical review at PHE Porton Down by the Animal Welfare and Ethical Review Body (AWERB) as required by the Home Office Animals (Scientific Procedures) Act 1986 Vaccination: Mice.
    Sex as a biological variableSixteen, 7-week-old female Sprague-Dawley rats, with a body weight range of 140-155 grams, were purchased from Envigo (Italy).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Pseudoparticles were harvested after 16-24 hours: the supernatant was centrifuged at 1200 rpm 5 min to pellet debris, the supernatant of anti-VSV-G expressing hybridoma cells (Anti-VSV-G antibody (I1, produced from CRL-2700 mouse hybridoma cells, ATCC) added to virus stocks at 1:10 (v/v) to block residual VSV-G containing particles.
    Anti-VSV-G
    suggested: None
    I1
    suggested: None
    Then, slides were stained with Alexa Fluor 568 Goat Anti-Rabbit antibody for 2h RT.
    Anti-Rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Transient expression of recombinant SARS-CoV-2 Spike proteins and Western Blotting: HEK293 cells were transiently transfected with SARS-CoV-2-S fragments expression vectors using Lipofectamine 2000 Transfection reagent (Thermo Fisher Scientific).
    HEK293
    suggested: None
    In brief, HEK293T cells were seeded and the next day, transfected with 44 μg plasmid encoding SARS-CoV-2 Spike (pCG1-SARS-2-S, Wuhan Hu-1) using Transit LT-1 (Mirus).
    HEK293T
    suggested: None
    In EDA, Vero E6 cells were seeded into 96 wells plates and infected at 95% of confluency with base 10 dilutions of virus stock.
    Vero E6
    suggested: None
    For neutralization experiments with SARS-CoV-2 VSV-based pseudoparticles, Caco-2 cells were seeded in 96-well plates in 180 μl medium at 10,000 cells/well.
    Caco-2
    suggested: None
    HEK293TN-hACE2 were generated by transduction of HEK293TN with a lentiviral vector engineered to stably express hACE2 (described elsewhere, paper submitted).
    HEK293TN-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals: BALB/c (H-2d) and C57Bl/6 mice (H-2b) were purchased from Envigo (Italy).
    C57Bl/6
    suggested: None
    B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from The Jackson Laboratory.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Sixteen, 7-week-old female Sprague-Dawley rats, with a body weight range of 140-155 grams, were purchased from Envigo (Italy).
    Sprague-Dawley
    suggested: None
    K18-hACE2 mice were immunized with 10 ug of COVID-eVax or saline solution twice 21 days apart intra-muscularly followed by electroporation as described above.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    In order to identify immunodominant RBD epitopes (here highlighted in yellow), Elispot assay was performed by stimulating splenocytes from RBD vaccinated Balb/c mice for 20h with RBD peptide pools.
    Balb/c
    suggested: None
    Recombinant DNA
    SentencesResources
    For the construction of RBD, N/R and S1 constructs, the cDNA corresponding to each region was amplified via PCR by using sequence-specific primers and directionally cloned into the linearized pTK1A-TPA vector by enzymatic restriction PacI/NotI.
    pTK1A-TPA
    suggested: None
    FL expression vector was generated by In-fusion Cloning System (Takara), amplifying the cDNA by using specific primers overlapping both the synthetic gene and the acceptor empty vector pTK1A.
    pTK1A
    suggested: None
    In brief, HEK293T cells were seeded and the next day, transfected with 44 μg plasmid encoding SARS-CoV-2 Spike (pCG1-SARS-2-S, Wuhan Hu-1) using Transit LT-1 (Mirus).
    pCG1-SARS-2-S
    suggested: None
    The next day, medium was removed, 15 ml fresh medium and then VSV(Fluc_eGFP)-VSV-G added to deliver the defective viral reporter genome (ΔVSV-G, kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) (33).
    ΔVSV-G
    suggested: None
    The following day, 32 μg of reporter plasmid pLenti CMV-GFP-TAV2A-LUC Hygro, 12.5 μg of pMDLg/pRRE (Addgene #12251), 6.25 μg of pRSV-Rev (Addgene #12253), and 9 μg pcDNA3.1_ Spike_del19 were cotransfected following a calcium phosphate transfection.
    pLenti CMV-GFP-TAV2A-LUC
    suggested: None
    pMDLg/pRRE
    suggested: RRID:Addgene_12251)
    pRSV-Rev
    suggested: RRID:Addgene_12253)
    pcDNA3.1_ Spike_del19 (Addgene #155297) was generated by deletion of last 19aa of Spike starting from pcDNA3.1-SARS2-Spike (a gift from Fang Li, Addgene plasmid # 145032). pLenti CMV-GFP-TAV2A-LUC Hygro was generated from pLenti CMV GFP Hygro (Addgene #17446) by addition of T2A-Luciferase by PCR cloning.
    pcDNA3.1_ Spike_del19
    suggested: RRID:Addgene_155297)
    pcDNA3.1-SARS2-Spike
    suggested: RRID:Addgene_145032)
    pLenti CMV
    suggested: RRID:Addgene_140746)
    Software and Algorithms
    SentencesResources
    The serum dilution inhibiting 50% of the CPE (IC50) was calculated using Prism 7 (GraphPad Software, San Diego, CA, USA) as described (36).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Brightfield images were acquired through an Aperio Scanscope System CS2 microscope and an ImageScope program (Leica Biosystem) following the manufacturer’s instructions.
    ImageScope
    suggested: (ImageScope, RRID:SCR_014311)
    Flow cytometry data were collected using FlowJo Version 10.5.3 (Treestar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analyses were performed with GraphPad Prism software version 8 (GraphPad).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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
    NCT04642638Active, not recruitingSafety, Immunogenicity, and Efficacy of INO-4800 for COVID-1…

    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 10.1101/2021.06.14.448343v1 on bioRxiv