Comparison of Mucosal and Intramuscular Immunization against SARS-CoV-2 with Replication-Defective and Replicating Single-cycle Adenovirus Vaccines

  1. Haley E. Mudrick
  2. Erin B. McGlinch
  3. Brian J. Parrett
  4. Jack R. Hemsath
  5. Mary E. Barry
  6. Jeffrey D. Rubin
  7. Chisom Uzendu
  8. Michael J. Hansen
  9. Courtney L. Erskine
  10. Virginia P. VanKeulen
  11. Aleksandra Drelich
  12. Chien-Te Kent Tseng
  13. Shane Massey
  14. Madiha Fida
  15. Gina A. Suh
  16. Tobias Peikert
  17. Matthew S. Block
  18. Gloria R. Olivier
  19. Michael A. Barry

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Abstract

SARS-CoV-2 enters the body at mucosal surfaces, such as the nose and lungs. These events involve a small number of virions at these mucosal barriers and are therefore a strategic point to stop a COVID-19 infection before it starts. Despite this, most vaccines against COVID-19 are being injected into the muscle where they will not generate the highest levels of mucosal protection. The vaccines that are approved for use in humans are all replication-defective (RD) mRNA, DNA, or adenovirus (Ad) vaccines that do not amplify antigen transgenes. We developed single cycle adenovirus (SC-Ad) vectors that replicate antigen genes up to 10,000-fold in human cells, but that are disabled from producing infectious Ad particles. We show here that SC-Ad expressing the full-length SARS-CoV-2 spike protein produces 100-fold more spike protein than a matched RD-Ad-Spike vector. When Ad-permissive hamsters were immunized with these vaccines by intranasal (IN) or intramuscular (IM) routes, SC-Ad produced significantly stronger antibody responses as compared to RD-Ad against the spike protein that rose over 14 weeks after one immunization. Single IN or IM immunizations generated significant antibody responses in serum and in bronchoalveolar lavages (BALs). IN priming, but not IM priming, generated HLA-restricted CD8 T cell responses in BALs. SC-Ad-Spike generated antibodies that retain binding to spike receptor binding domains (RBDs) with mutations from new viral variants. These data suggest empowering the genomes of gene-based vaccines with the ability to amplify antigen genes can increase potency. This may be particularly advantageous when applying mucosal vaccines to combat mucosal pathogens like SARS-CoV-2.

One Sentence Summary

Arming adenovirus vaccines with the ability to replicate vaccine antigen genes may increase potency for systemic, or more importantly, mucosal immunization against mucosal pathogens.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.20.440651: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: This study was conducted in Mayo Clinic’s AAALAC (Association for the Assessment and Accreditation of Laboratory Animal Care)- accredited facilities and were approved by the Institutional Animal Care and Use Committee (
    Euthanasia Agents: Mice were euthanized via CO2 gas, then sterilized with 70% ethanol.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody ELISAs: Binding IgG and IgA antibody responses in mouse serum, hamster serum, and bronchoalveolar lavage fluid were measured by ELISA against spike S1 protein and SARS-CoV-2 receptor binding domain variants.
    IgA
    suggested: None
    For hamster samples, the secondary antibodies used were: Peroxidase Conjugated Affinity Purified Anti-Golden Syrian Hamster IgG (H&L) Goat (Rockland Inc.), and Rabbit Anti-Hamster IgA (Brookwood Biomedical).
    Anti-Golden Syrian Hamster IgG
    suggested: None
    Anti-Hamster IgA
    suggested: None
    For mouse samples, the secondary antibodies used were: GOXMO HRP HIGH XADS (Invitrogen), and HRP-Goat Anti-Mouse IgA (Invitrogen).
    Anti-Mouse IgA
    suggested: None
    After 24 h, cells were transferred to nitrocellulose plates, coated with anti-IFN-γ antibody, and incubated for 24 more hours.
    anti-IFN-γ
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Western Blotting: Human A549 lung cells were infected with RD- or SC-Ad spike at the indicated multiplicities of infection (MOI) and harvested 24 hours later.
    A549
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals: BALB/c mice were purchased from Charles River Laboratories.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Recombinant DNA
    SentencesResources
    This full-length sequence was inserted into the shuttle plasmid pAd6-NdePfl-CMV-MCS-3X-LZL.
    pAd6-NdePfl-CMV-MCS-3X-LZL
    suggested: None
    This sequence was recombined into pAd6-ΔE1-ΔE3 and pAd6-ΔIIIa-ΔE3 by red recombination as in (20–22) to generate RD-Ad6-spike and SC-Ad-Spike, respectively.
    pAd6-ΔE1-ΔE3
    suggested: None
    pAd6-ΔIIIa-ΔE3
    suggested: None
    Software and Algorithms
    SentencesResources
    Single-cycle Adenovirus Expressing Wild-type SARS-CoV-2 spike: A codon-optimized cDNA encoding the original wild-type spike protein from severe acute respiratory syndrome coronavirus 2 isolate 2019-nCoV_HKU-SZ-002a_2020, accession number MN938384.1 was synthesized by Genewiz.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Statistical Analysis: Prism 9 Graphical software was used for all statistical analyses.
    Statistical Analysis: Prism
    suggested: None

    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

    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 10.1101/2021.04.20.440651v1 on bioRxiv