MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

  1. Ruikang Liu
  2. Jeffrey L. Americo
  3. Catherine A. Cotter
  4. Patricia L. Earl
  5. Noam Erez
  6. Chen Peng
  7. Bernard Moss

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Abstract

Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals.

Significance

Vaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.30.424878: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Safety and Ethics: All experiments and procedures involving mice were approved under protocol LVD29E by the NIAID Animal Care and Use Committee according to standards set forth in the NIH guidelines, Ansaimal
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMice: Five-to six-week-old female BALB/cAnNTac and C57BL/6ANTac were obtained from Taconic Biosciences and B6.Cg-Tg(K18-ACE2)2Prlmn/J from Jackson Laboratories.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Titers of MVAs were determined in CEF by staining plaques with anti-VACV rabbit antibodies (36).
    anti-VACV
    suggested: (Creative Diagnostics Cat# DPAB-DC4864, RRID:AB_2501976)
    The membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS) for 1 h, washed with TBS with 0.1% Tween 20 (TBST), and then incubated at 4°C overnight with rabbit anti-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) or anti-FLAG M2 peroxidase antibody (Cat# A8592, MilliporeSigma) in 5% nonfat milk in TBST.
    anti-CoV-2
    suggested: None
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)
    The membrane that had been incubated with anti-RBD antibody was then incubated for 1 h with secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch).
    anti-RBD
    suggested: None
    For intracellular staining, cells were fixed with Cytofix/Cytoperm (BD Biosciences), permeabilized with Perm/Wash Buffer (BD Biosciences), and incubated with anti-CoV-2 Spike RBD mAb (SARS2-02) (13) followed by APC-conjugated goat anti-mouse IgG antibody (Cat# 405308, BioLegend).
    SARS2-02
    suggested: None
    anti-mouse IgG
    suggested: (BioLegend Cat# 405308, RRID:AB_315011)
    The binding of hACE2 to surface expressed S protein on infected HeLa cells was detected by incubating with 100 ng/106 cells of biotinylated human ACE2 protein (Cat# 10108-H08H-B, Sino Biological) followed by Alexa Fluor 647-conjugated anti-hACE2 antibody (Cat# FAB9332R, R&D Systems).
    anti-hACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Detection of S Protein by Flow Cytometry: HeLa cells were infected with 5 PFU per cell of rMVAs.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Detection of S-Binding Antibodies by ELISA: CoV-2 S protein produced in HEK293 cells was obtained from the NIAID Vaccine Research Center or Sino Biological and diluted in cold PBS to a concentration of 1 μg/ml.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    The TCID50 of the clarified culture medium was determined on Vero E6 cells after staining with crystal violet and scored by the Reed-Muench method.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Five-to six-week-old female BALB/cAnNTac and C57BL/6ANTac were obtained from Taconic Biosciences and B6.Cg-Tg(K18-ACE2)2Prlmn/J from Jackson Laboratories.
    BALB/cAnNTac
    suggested: None
    C57BL/6ANTac
    suggested: None
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Passive Serum Transfer: Serum for passive transfer was obtained from 20 BALB/c mice that were inoculated IM with rMVA S (WT) and 10 BALB/c mice with parental MVA at 0 and 3 weeks.
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    The stained cells were acquired on a FACSCalibur cytometer using Cell Quest software and analyzed with FlowJo (BD Biosciences).
    FACSCalibur
    suggested: None
    Stimulation and Staining of Lymphocytes: Splenocytes from individual mice or pooled from 3-5 mice were suspended at 1.5×107 cells/ml in RPMI (Quality Biological) supplemented with 10% heat-inactivated FBS, 10 U/ml penicillin, 10 μg/ml streptomycin, 2 mM L-glutamine, and 2 mM HEPES as previously described (61).
    Quality Biological
    suggested: None
    Approximately 100,000 events were acquired on a FACSCaliber cytometer using Cell Quest software and analyzed with FlowJo (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    NT50 were calculated using Prism (GraphPad Software) to plot dose-response curves, normalized using the average of the no virus wells as 100% neutralization, and the average of the no serum wells as 0%.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    After 18 h at 37°C, the cells were fixed in 2% paraformaldehyde and acquired with a FACSCalibur cytometer using Cell Quest software and analyzed with FlowJo.
    Cell Quest
    suggested: None
    The dilution of mouse serum that reduced the percentage of GFP-expressing cells by 50% (IC50) was determined by nonlinear regression using Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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 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.
    • 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 10.1101/2020.12.30.424878v1 on bioRxiv