Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

  1. Flavia Chiuppesi
  2. Marcela d’Alincourt Salazar
  3. Heidi Contreras
  4. Vu H Nguyen
  5. Joy Martinez
  6. Soojin Park
  7. Jenny Nguyen
  8. Mindy Kha
  9. Angelina Iniguez
  10. Qiao Zhou
  11. Teodora Kaltcheva
  12. Roman Levytskyy
  13. Nancy D Ebelt
  14. Tae Hyuk Kang
  15. Xiwei Wu
  16. Thomas Rogers
  17. Edwin R Manuel
  18. Yuriy Shostak
  19. Don J Diamond
  20. Felix Wussow

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Abstract

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.01.183236: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Mouse immunization: The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute of City of Hope (COH) approved protocol 20013 assigned for this study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological)
    anti-SARS-CoV-1
    suggested: None
    anti-SARS-CoV1 NP
    suggested: None
    Anti-rabbit polyclonal antibody conjugated with horseradish peroxidase (Sigma-Aldrich) was used as a secondary antibody and protein bands were visualized with chemiluminescent substrate (ThermoFisher).
    Anti-rabbit
    suggested: None
    Anti-SARS-CoV-1 S1 mouse (40150-R007, Sino Biological) and S2 rabbit (GTX632604, GeneTex) monoclonal antibodies, anti-SARS-CoV-1 N rabbit monoclonal antibody (40143-R001, Sino Biological), and anti-vaccinia rabbit polyclonal antibody (9503-2057, Bio Rad) were used in dilution 1:2,000.
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    anti-SARS-CoV-1 N
    suggested: None
    anti-vaccinia
    suggested: None
    One hour later anti-mouse or anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (A11001, A21206; Invitrogen) were added to the cells at a dilution of 1:4,000.
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)
    After washing the plate, 1:3,000 dilution of HRP-conjugated anti-mouse IgG secondary antibody (W402B, Promega) was added and incubated for one additional hour.
    anti-mouse IgG
    suggested: None
    The assay was performed as described above except for the secondary antibodies (1:2,000. goat Anti-Mouse IgG2a cross absorbed HRP antibody, Southern biotech, 1083-05; Goat anti-Mouse IgG1 cross absorbed HRP antibody, Thermo Fisher, A10551).
    Anti-Mouse IgG2a
    suggested: (Thermo Fisher Scientific Cat# A10551, RRID:AB_2534048)
    anti-Mouse IgG1
    suggested: (Thermo Fisher Scientific Cat# A10551, RRID:AB_2534048)
    A10551
    suggested: None
    The IgG2a/IgG1 ratio was calculated by dividing the absorbance read in the well incubated with the IgG2a secondary antibody divided the absorbance for the same sample incubated with the IgG1 antibody.
    IgG2a
    suggested: None
    IgG1
    suggested: None
    Cross-adsorbed goat anti-human IgG (H+L) secondary antibody (A18811, Invitrogen) was used at a dilution of 1:3,000.
    anti-human IgG
    suggested: (Thermo Fisher Scientific Cat# A18811, RRID:AB_2535588)
    A18811
    suggested: None
    Anti-mouse CD28 and CD49d antibodies (1μg/ml; BioLegend) were added as co-stimulation.
    Anti-mouse CD28
    suggested: None
    CD49d
    suggested: None
    In experiments testing double recombinants SARS-CoV2 vectors IL-2 antibody was not included and PE-CF594 anti-mouse IL-4 (clone 11B11, 562450, BD) and BV421 rat anti mouse IL-10 (clone JES5-16E3, 563276, BD) were added.
    IL-2
    suggested: None
    anti-mouse IL-4
    suggested: (Thermo Fisher Scientific Cat# 88-7771-88, RRID:AB_476398)
    BV421 rat anti mouse IL-10
    suggested: (BioLegend Cat# 505022, RRID:AB_2563240)
    anti
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and Viruses: BHK-21 (CCL-10), A549 (CCL-185), HeLa (CCL-2), 293T (
    BHK-21
    suggested: None
    HeLa
    suggested: None
    CRL-1573), 143B (CRL-8303), MRC-5 (CCL-171), HEK293/17 (CRL11268), THP-1 (TIB-202), ARPE-19 (CRL-2302) were purchased from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations.
    THP-1
    suggested: None
    HEK293T/ACE2 were a kind gift of Pamela J.
    HEK293T/ACE2
    suggested: None
    Fully infected BHK cell monolayers were usually visible at 8-12 days post transfection/infection. sMVA virus from infected BHK cell monolayers was prepared by conventional freeze/thaw method and passaged once on BHK cells before producing ultra-purified virus stocks on CEF. sMVA or recombinant sMVA-CoV-2 vectors were reconstituted either with FPV HP1.441 (sMVA hp, sMVA-N/S,
    BHK
    suggested: None
    Host cell range: sMVA and wtMVA host cell range using various human cell lines (HeLa, 293T, MRC-5, A549, and 143B) BHK cells, and CEF was determined as follows.
    293T
    suggested: None
    MRC-5
    suggested: None
    A549
    suggested: None
    MVA neutralization assay: ARPE-19 cells were seeded in 96 well plates (1.5×104 cells/well).
    ARPE-19
    suggested: None
    HEK293T cells overexpressing ACE-2 receptor were seeded the day before transduction at a density of 2×105 cells per well in a 96-well plate in complete DMEM (47).
    HEK293T
    suggested: None
    For the ADE assay, THP1 cells were seeded at a confluency of 2×106 cells/mL in a 96 well plate and co-incubated for 48 hours with serum samples diluted at 1:5,000 or 1:50,000 in the presence of SARS-CoV-2-Spike pseudotyped or VSV-G luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen.
    THP1
    suggested: None
    Briefly, HeLa-ACE2 cells were seeded in 12 μL complete DMEM at a density of 2×103 cells per well.
    HeLa-ACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    6 weeks old C57BL/6 (C57BL/6J, 000664) or Balb/c (BALB/cJ, 000651) were purchased from the Jackson laboratories.
    C57BL/6
    suggested: None
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Balb/c
    suggested: RRID:IMSR_JAX:000651)
    BALB/cJ
    suggested: RRID:IMSR_JAX:000651)
    Software and Algorithms
    SentencesResources
    De novo assembly was done using either canu v1.7.1 or wtdbg2 v2.5.
    canu
    suggested: (Canu, RRID:SCR_015880)
    Images were acquired and processed using Zen software (Zeiss)
    Zen
    suggested: None
    Pictures from each well were processed using Image-Pro Premier (Media Cybernetics) and the fluorescent area corresponding to the area covered by MVA-Venus infected cells was calculated.
    Image-Pro Premier
    suggested: (Image-Pro Premier , RRID:SCR_016497)
    Analysis was performed using FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistics: Statistical evaluation was pursued using GraphPad Prism (v8.3.0).
    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: 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

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  2. Version published to 10.1101/2020.07.01.183236v1 on bioRxiv