Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine

  1. Alberto Grandi
  2. Michele Tomasi
  3. Cinzia Bertelli
  4. Teresa Vanzo
  5. Assunta Gagliardi
  6. Elena Caproni
  7. Silvia Tamburini
  8. Laura Fantappiè
  9. Gabriele Di Lascio
  10. Zeno Bisoffi
  11. Chiara Piubelli
  12. Maria Teresa Valenti
  13. Luca Dalle Carbonare
  14. Donato Zipeto
  15. Micol Ravà
  16. Valeria Fumagalli
  17. Pietro Di Lucia
  18. Davide Marotta
  19. Eleonora Sala
  20. Matteo Iannacone
  21. Peter Cherepanov
  22. Martino Bolognesi
  23. Massimo Pizzato
  24. Guido Grandi

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Abstract

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions.

Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

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  1. ScreenIT

    SciScore for 10.1101/2021.07.12.452027: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Animals showing such conditions were anesthetized and subsequently sacrificed in accordance with experimental protocols, which were reviewed and approved by the Animal Ethical Committee of The University of Trento and the Italian Ministry of Health.
    Euthanasia Agents: Cell Isolation and Flow Cytometry: Mice were euthanized by cervical dislocation.
    IRB: Samples had been collected and stored in the University of Verona biobank (Ethics Committee approval prot. N. 1538) and in Tropica Biobank of the IRCCS Sacro Cuore Don Calabria Hospital (Ethics Committee approval prot. N. 50950).
    Consent: All participants signed informed consent.
    Sex as a biological variableFive-week old CD1 female mice were immunized intraperitoneally (i.p.) on day 0, 14 and 28 with 10 μg of OMVs together with 2mg/ml Aluminium hydroxide.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Wells were washed three times with PBST and then incubated 45 min at room temperature with goat anti mouse alkaline phosphatase-conjugate antibodies at a final dilution of 1:2000 (SigmaAldrich).
    anti mouse alkaline phosphatase-conjugate
    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
    Briefly, Vero E6 cells were seeded at a density of 1.5 × 104 cells per well in flat-bottom 96-well tissue-culture plates.
    Vero E6
    suggested: None
    Preparation of viral pseudotypes and neutralization assays: SIV-based lentiviral particles pseudotyped with SARS-Cov-2 spike were produced in 10 cm plates prepared the day before transfection with 3 million HEK293T cells in 10 ml complete DMEM, supplemented with 10% FBS.
    HEK293T
    suggested: None
    Neutralization titres were tested on Huh-7 cells overexpressing ACE2.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Experimental Models: Organisms/Strains
    SentencesResources
    For virus challenge studies, B6.Cg-Tg(K18-ACE2)2Prlmn/J mice1 were purchased from The Jackson Laboratory.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    K18-hACE2 transgenic mice were immunized intraperitoneally with 10μl vaccine or PBS twice, 14 days apart.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Recombinant DNA
    SentencesResources
    Engineering BL21(DE3) E. coli strains with SARS-CoV-2 neutralizing epitopes: The pET21-FhuD2-RBM438-462, pET21-FhuD2-RBM467-509 and pET21-FhuD2-RBM438-509 plasmids carrying the Staphylococcus aureus Ferric hydroxamate receptor 2 (FhuD2) fused to one copy of RBM438-462, RBM467-509 and RBM438-509 SARS-CoV-2 epitopes respectively were assembled using the PIPE method as described in (ref. H. E. Klock, S. A. Lesley, The Polymerase Incomplete Primer
    pET21-FhuD2-RBM438-462
    suggested: None
    Briefly, pET21-FhuD2 plasmid was linearized by PCR, using FhuD2-v-R and pET-V-F primers (Table 1).
    pET21-FhuD2
    suggested: None
    pET-V-F
    suggested: None
    To confirm the correctness of the gene fusions, plasmids were sequenced (Eurofins, Ebersberg, Germany, EU) and E. coli BL21(DE3)Δ60 strain was transformed with pET21-FhuD2-RBM438-462, pET21-FhuD2-RBM467-509, pET21-FhuD2-RBM438-509 and pET21-FhuD2-RBM-BV438-509 plasmids and the derived recombinant strains were used for the production of engineered FhuD2-RBM438-462, FhuD2-RBM467-509, FhuD2-RBM438-509 and FhuD2-RBM-BV438-509 OMVs, respectively.
    pET21-FhuD2-RBM467-509
    suggested: None
    pET21-FhuD2-RBM438-509
    suggested: None
    pET21-FhuD2-RBM-BV438-509
    suggested: None
    OMV purification: OMVs from BL21(DE3)Δ60(pET-FhuD2-RBM438-509), BL21(DE3) Δ60(pET-FhuD2-RBM438-462), BL21(DE3)Δ60(pET-FhuD2-RBM467-509) and BL21(DE3)Δ60(pET-FhuD2-RBM-BV438-509), were purified in an EZ control bioreactor (Applikon Biotechnology, Schiedam, Netherlands) as previously described (Zanella et al., 2021).
    pET-FhuD2-RBM438-509
    suggested: None
    pET-FhuD2-RBM438-462
    suggested: None
    pET-FhuD2-RBM467-509
    suggested: None
    pET-FhuD2-RBM-BV438-509
    suggested: None
    Software and Algorithms
    SentencesResources
    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)
    Fitted sigmoidal curves and IC50 were obtained using Prism (GraphPad) with the least square variable slope method and using the dose-normalized response protocol.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Statistical analyses and software: Flow 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: 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.

    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.

  2. Version published to 10.1101/2021.07.12.452027v1 on bioRxiv