Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

  1. Rinke Bos
  2. Lucy Rutten
  3. Joan E. M. van der Lubbe
  4. Mark J. G. Bakkers
  5. Gijs Hardenberg
  6. Frank Wegmann
  7. David Zuijdgeest
  8. Adriaan H. de Wilde
  9. Annemart Koornneef
  10. Annemiek Verwilligen
  11. Danielle van Manen
  12. Ted Kwaks
  13. Ronald Vogels
  14. Tim J. Dalebout
  15. Sebenzile K. Myeni
  16. Marjolein Kikkert
  17. Eric J. Snijder
  18. Zhenfeng Li
  19. Dan H. Barouch
  20. Jort Vellinga
  21. Johannes P. M. Langedijk
  22. Roland C. Zahn
  23. Jerome Custers
  24. Hanneke Schuitemaker

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Abstract

Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

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  1. Version published to 10.1038/s41541-020-00243-x
  2. ScreenIT

    SciScore for 10.1101/2020.07.30.227470: (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 variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For CR3022, CR3015 (van den Brink et al., 2005), CR3046 and S309 (Pinto et al., 2020) the heavy and light chain were cloned into a single IgG1 expression vector to express a fully human IgG1 antibody.
    CR3015
    suggested: (Fitzgerald Industries International Cat# 10C-CR3015M1, RRID:AB_1288272)
    CR3046
    suggested: (Fitzgerald Industries International Cat# 20C-CR3046R, RRID:AB_1288447)
    S309
    suggested: None
    human IgG1
    suggested: None
    Subsequently, cells were incubated in 50 μl/well blocking buffer containing primary antibodies ACE2-Fc (5 µg/mL, 1 µg/mL and 0.2 µg/mL)(1 µg/mL for radar plot), S309 (1 µg/mL), SAD-S35 (1 µg/mL), CR3015 (5 µg/mL), CR3022 (5 µg/mL), CR3046 (5 µg/mL), and convalescent serum (1:400) for 1 hr at 4 °C.
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    After blocking, the cells were incubated with 50 μl/well of secondary antibodies HRP conjugated Mouse Anti Human IgG (Jackson, 1:2500) or HRP Conjugated goat anti mouse IgG (Jackson, 1:2500) then incubated 40 min at 4 °C.
    Anti Human IgG
    suggested: None
    anti mouse IgG
    suggested: None
    Cells were washed twice with FACS buffer and stained with goat anti-Human IgG Alexa Fluor 647 (Invitrogen) or goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen) secondary antibody for 30 min in FACS buffer.
    anti-Human IgG
    suggested: None
    anti-Mouse IgG
    suggested: None
    After incubation, the membrane was washed three times with TBST for 5 min and subsequently incubated for 1 hr with 1:10,000 IRDye 800CW-conjugated goat-anti-human secondary antibody (Li-COR) in TBST-5% Blocker.
    secondary
    suggested: None
    For IgG1 and IgG2a ELISAs, a similar protocol was followed as described above, but respectively using Goat anti-Mouse IgG1-HRP and Goat anti-Mouse IgG2a-HRP as secondary antibodies.
    anti-Mouse IgG1-HRP
    suggested: None
    anti-Mouse IgG2a-HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Flow cytometry: MRC-5 cells (0.4×106 cells/well) were seeded in 6-well plates and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes at 5000 vp/cell for 48 hrs.
    MRC-5
    suggested: None
    BioLayer Interferometry (BLI): Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2.
    Expi293F
    suggested: RRID:CVCL_D615)
    Acceptor HEK293 cells were transfected in 6-well plates (Corning) with ACE2, TMPRSS2 and the PEP86 subunit, or just the PEP86 subunit (‘No spike’) as negative control.
    HEK293
    suggested: None
    Vero-E6 cells were seeded at 12,000 cells/well in 96-well tissue culture plates 1 day prior to infection.
    Vero-E6
    suggested: None
    After 30 min incubation, the mixture was inoculated onto susceptible Vero E6 cells in MW96 well plates.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals: Female BALB/c or C57BL6 mice (specific pathogen-free), aged 8-12 weeks at the start of the study were purchased from Charles River laboratories (Sulzfeld, Germany).
    BALB/c
    suggested: None
    C57BL6
    suggested: None
    Software and Algorithms
    SentencesResources
    Data processing for the different proteins was performed using Biopharma finder 3.1 (Thermo Scientific).
    Biopharma
    suggested: (TransCelerate BioPharma, RRID:SCR_003728)
    Overlays between brightfield and GFP channels were made in ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc. Cary, NC, US) and R version 3.6.1 (2019-07-05).
    SAS Institute
    suggested: (Statistical Analysis System, RRID:SCR_008567)

    Results from OddPub: Thank you for sharing your data.

    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
    NCT04436276Active, not recruitingA Study of Ad26.COV2.S in Adults (COVID-19)

    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

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  3. Version published to 10.1101/2020.07.30.227470v1 on bioRxiv