A bacterial extracellular vesicle‐based intranasal vaccine against SARS‐CoV‐2 protects against disease and elicits neutralizing antibodies to wild‐type and Delta variants

  1. Linglei Jiang
  2. Tom A.P. Driedonks
  3. Wouter S.P. Jong
  4. Santosh Dhakal
  5. H. Bart van den Berg van Saparoea
  6. Ioannis Sitaras
  7. Ruifeng Zhou
  8. Christopher Caputo
  9. Kirsten Littlefield
  10. Maggie Lowman
  11. Mengfei Chen
  12. Gabriela Lima
  13. Olesia Gololobova
  14. Barbara Smith
  15. Vasiliki Mahairaki
  16. M. Riley Richardson
  17. Kathleen R. Mulka
  18. Andrew P. Lane
  19. Sabra L. Klein
  20. Andrew Pekosz
  21. Cory Brayton
  22. Joseph L. Mankowski
  23. Joen Luirink
  24. Jason S. Villano
  25. Kenneth W. Witwer

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Abstract

Several vaccines have been introduced to combat the coronavirus infectious disease‐2019 (COVID‐19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Current SARS‐CoV‐2 vaccines include mRNA‐containing lipid nanoparticles or adenoviral vectors that encode the SARS‐CoV‐2 Spike (S) protein of SARS‐CoV‐2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS‐CoV‐2 variants such as the Delta variant. Here, we present a novel, well‐characterized SARS‐CoV‐2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture‐derived Spike receptor‐binding domain (RBD). RBD‐conjugated outer membrane vesicles (RBD‐OMVs) were used to immunize the golden Syrian hamster ( Mesocricetus auratus ) model of COVID‐19. Intranasal immunization resulted in high titres of blood anti‐RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild‐type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD‐OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titres in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV‐based vaccine approaches.

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  1. Version published to 10.1002/jev2.12192
  2. Version published to 10.1101/2021.06.28.450181v4 on bioRxiv
  3. Version published to 10.1101/2021.06.28.450181v3 on bioRxiv
  4. Version published to 10.1101/2021.06.28.450181v2 on bioRxiv
  5. ScreenIT

    SciScore for 10.1101/2021.06.28.450181: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Study design, intranasal vaccination and virus challenge, and data/sample collection: All experimental procedures were approved by Johns Hopkins University Animal Care and Use Committee.
    Sex as a biological variable24 golden Syrian hamsters (Mesocricetus auratus, HsdHan®:AURA, 12 females, 12 males, 7-8 weeks old) were purchased from Envigo (Haslett, MI, USA) and were assigned to 3 immunization groups: 1) mock (vehicle) immunization, 2) unconjugated OMV (ctrl-OMV), and 3) RBD-OMV.
    Randomizationnot detected.
    BlindingPulmonary sections were examined by a pathologist who was blinded to the experimental groups.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blots were probed with primary antibodies: human anti-SARS-CoV-2 Spike (S-ECD/RBD) (cat# bcb03, Thermo Fisher, 1:1000, non-reducing conditions), anti-Salmonella typhimurium LPS (cat# ab8274, reducing conditions, 1:1000), and mouse anti-6xHis (ab18184, Abcam, 1:2000, reducing conditions) in 5% blocking buffer in PBS containing 0.1% v/v Tween 20 (PBS-T), incubating overnight at 4°C on a shaker.
    anti-SARS-CoV-2
    suggested: None
    anti-Salmonella
    suggested: (Abcam Cat# ab8274, RRID:AB_306423)
    anti-6xHis
    suggested: None
    Blots were washed 3x with PBS-T and incubated for 1 h at room temperature with appropriate secondary antibodies: mouse IgGk-BP-HRP (cat# sc-516102, SantaCruz) or goat anti-human-HRP (cat# 31410, Thermo Scientific), diluted 1:10,000 in 5% blocking buffer.
    anti-human-HRP
    suggested: None
    After a PBS rinse, grids were blocked in 1% BSA in PBS (30 min), followed by incubation with primary antibodies mouse anti-Spike (clone MM43, Sino Biological, 1:100) and mouse anti-S.
    anti-Spike
    suggested: (Sino Biological Cat# 40591-MM43, RRID:AB_2857934)
    anti-S
    suggested: None
    Single-particle interferometric reflectance imaging sensing (SP-IRIS): OMVs were pre-diluted 1:500 in PBS, followed by 1:1 dilution in incubation buffer (IB), and incubated at room temperature on ExoView R100 (NanoView Biosciences, Brighton, MA) custom virus chips printed with SARS-CoV2-Spike antibodies (clones D001, D003, MM43, Sino Biological),
    SARS-CoV2-Spike
    suggested: None
    Chips were then incubated for 1 h at RT with fluorescent antibodies against Spike (D001, CF555), (D003, CF647), (MM43, CF488) and LPS (CF647) diluted 1:1000 (final concentration of 500 ng/ml) in a 1:1 mixture of IB and blocking buffer.
    D001
    suggested: (Sino Biological Cat# 40150-D001-H, RRID:AB_2857930)
    CF555
    suggested: None
    D003
    suggested: (Sino Biological Cat# 40150-D003, RRID:AB_2827982)
    MM43
    suggested: (Sino Biological Cat# 40591-MM43, RRID:AB_2857934)
    CF647
    suggested: None
    Serology: Hamster antibody ELISA for RBD-specific IgG, IgA and IgM responses was performed as described previously (16).
    RBD-specific IgG
    suggested: None
    After washing plates 3x, HRP-conjugated secondary IgG (1:10000, Abcam, MA, USA), IgA (1:250, Brookwood Biomedical, AL, USA) or IgM (1:250, Brookwood Biomedical, AL, USA) antibodies were added.
    HRP-conjugated secondary IgG
    suggested: (Creative Diagnostics Cat# LAB-22, RRID:AB_2489589)
    IgA
    suggested: None
    IgM
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    On day 44, hamsters were challenged intranasally with 10^7 TCID50 of SARS-CoV-2 USA/Washington-1/2020, NR-52281 [BEI Resources, virus prepared as described previously (16)] diluted in 100 μl DMEM in an animal biosafety level 3 (ABSL3) facility.
    NR-52281
    suggested: None
    After a 1-h incubation at room temperature, the mixtures were added to VeroE6-TMPRSS2 cells in 96 well plates in sextuplet for 6 hours at 37°C.
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Briefly, tissue homogenates were 10-fold serially diluted in infection medium (Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2.5% fetal bovine serum, 1 mM glutamine, 1 mM sodium pyruvate, and penicillin (100 U/mL) and streptomycin (100 μg/mL) antibiotics), transferred in sextuplicate into 96-well plates containing confluent Vero-E6-TMPRSS2 cells (National Institute of Infectious Diseases,
    Vero-E6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Recombinant DNA
    SentencesResources
    Both constructs were synthesized by IDT (Coralville, IA, USA) and cloned into the pIRESpuro3 vector (cat# 631619, Takara Bio, USA) using EcoRI and BamHI restriction enzymes (R0101S and R0136S respectively, New England Biolabs) and a Rapid Ligation Kit (cat# K1423, Thermo Fisher, USA).
    pIRESpuro3
    suggested: None
    Production of OMV-RBD vaccine platform: OMVs were produced from S. typhimurium SL3261 ΔtolRA ΔmsbB cells harboring the expression plasmid pHbpD(Δd1)-SpyCatcher as described previously (14) and resuspended in PBS.
    pHbpD(Δd1)-SpyCatcher
    suggested: None
    Software and Algorithms
    SentencesResources
    Gels were scanned on a GS-800 calibrated densitometer (Bio-Rad), and the intensities of protein bands were determined using ImageJ (http://imagej.nih.gov/ij/).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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.

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

    About SciScore

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  6. Version published to 10.1101/2021.06.28.450181v1 on bioRxiv