A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability

  1. Emily A. Voigt
  2. Alana Gerhardt
  3. Derek Hanson
  4. Madeleine F. Jennewein
  5. Peter Battisti
  6. Sierra Reed
  7. Jasneet Singh
  8. Raodoh Mohamath
  9. Julie Bakken
  10. Samuel Beaver
  11. Christopher Press
  12. Patrick Soon-Shiong
  13. Christopher J. Paddon
  14. Christopher B. Fox
  15. Corey Casper

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Abstract

mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing stability, ease of manufacturing, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of bone marrow-resident antibody-secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability when stored lyophilized at room temperature for at least 6 months and at refrigerated temperatures for at least 10 months. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.

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  1. Version published to 10.1038/s41541-022-00549-y
  2. Rapid Reviews Infectious Diseases

    Cristian Smerdou

    Review 4: "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability"

    This preprint provides a proof-of-concept demonstration for a new COVID-19 vaccine that can be stored at room temperature. Reviewers noted that though the evidence generally justifies the main claim, more work is needed to use this as an alternative to existing licensed vaccines.

  3. Rapid Reviews Infectious Diseases

    Rushit N. Lodaya, Derek T. O'Hagan

    Review 3: "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability"

    This preprint provides a proof-of-concept demonstration for a new COVID-19 vaccine that can be stored at room temperature. Reviewers noted that though the evidence generally justifies the main claim, more work is needed to use this as an alternative to existing licensed vaccines.

  4. Rapid Reviews Infectious Diseases

    Hamid Mahmoudzadeh Niknam, Nastaran Sadat Savar

    Review 2: "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability"

    This preprint provides a proof-of-concept demonstration for a new COVID-19 vaccine that can be stored at room temperature. Reviewers noted that though the evidence generally justifies the main claim, more work is needed to use this as an alternative to existing licensed vaccines.

  5. Rapid Reviews Infectious Diseases

    Thomas Demoulins

    Review 1: "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability"

    This preprint provides a proof-of-concept demonstration for a new COVID-19 vaccine that can be stored at room temperature. Reviewers noted that though the evidence generally justifies the main claim, more work is needed to use this as an alternative to existing licensed vaccines.

  6. Rapid Reviews Infectious Diseases

    Strength of evidence

    Reviewers: Thomas Demoulins (University of Bern) | ๐Ÿ“˜๐Ÿ“˜๐Ÿ“˜๐Ÿ“˜๐Ÿ“˜
    Hamid Niknam, Nastaran Savar (Pasteur Institute of Iran) | ๐Ÿ“’๐Ÿ“’๐Ÿ“’โ—ป๏ธโ—ป๏ธ
    Rushit N. Lodaya, Derek T. O'Hagan (Rockville Center for Vaccines) | ๐Ÿ“’๐Ÿ“’๐Ÿ“’โ—ป๏ธโ—ป๏ธ
    Cristian Smerdou (Cima Universidad de Navarra) | ๐Ÿ“˜๐Ÿ“˜๐Ÿ“˜๐Ÿ“˜๐Ÿ“˜

  7. ScreenIT

    SciScore for 10.1101/2022.03.22.485230: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mouse studies: All animal work was done under the oversight of the Bloodworks Northwest Research Instituteโ€™s Institutional Animal Care and Use Committee (Seattle, WA).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Serum IgG, IgG1, and IgG2a titers by ELISA: SARS CoV-2 spike protein-binding IgG antibodies in mouse serum were measured by ELISA.
    IgG1
    suggested: None
    IgG
    suggested: None
    Plates were then washed, and spike protein-bound antibodies were detected using an Anti-Mouse IgG (Fc Specific)-Alkaline Phosphatase antibody (Sigma-Aldrich, #A2429) at a 1:4000 dilution in blocking buffer.
    Anti-Mouse IgG
    suggested: (Innovative Research Cat# HM4000, RRID:AB_1482268)
    Splenocytes were stained for viability with Zombie Green (BioLegend, San Diego, CA) in 50 ฮผL of PBS, and then Fc receptors were blocked with CD16/CD32 antibody (Invitrogen).
    CD16/CD32
    suggested: None
    IL-17A (Invitrogen, #88-7371-88), or IL-5 (BD Biosciences, #51-1805KZ) capture antibodies at a 1:200 dilution in ELISpot coating buffer (eBioscience, Waltham, MA).
    IL-17A
    suggested: (Thermo Fisher Scientific Cat# 88-7371-88, RRID:AB_2575104)
    IL-5
    suggested: (Novus Cat# NB 200-515, RRID:AB_535720)
    After a 3-hour incubation, plates were washed with PBS with 0.1% Tween 20, and secondary antibody (Goat Anti-Mouse IgG-HRP or IgA-HRP [SouthernBiotech, Birmingham, AL; #1030-05 and #1040-05]) was added at a 1:1000 dilution in PBS with 0.1% Tween and 5% FBS overnight at 4ยฐC.
    Anti-Mouse IgG-HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    After complexing, vaccine was lyophilized using a VirTis AdVantage 2.0 EL-85 (SP Industries, Warminster, PA) benchtop freeze dryer controlled by the microprocessor-based Wizard 2.0 software.
    EL-85
    suggested: RRID:CVCL_EL85)
    Briefly, HEK-293 cells (American Type Culture Collection, Manassas, VA; #CRL-3216) were plated at 4 x 105 cells/mL in 6-well plates 18-24 hours before the assay to achieve 50-70% confluency at assay start.
    HEK-293
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    All animal work was in compliance with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR Parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Animals, Eighth Edition.69 C57BL/6J mice obtained from The Jackson Laboratory (Harbor, ME) were used for all animal studies in this work.
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Software and Algorithms
    SentencesResources
    These sequences were then codon-optimized for mammalian (human) expression by Codex DNA (San Diego, CA) using a proprietary algorithm, synthesized by BioXp (Codex DNA), and inserted into AAHIโ€™s backbone saRNA expression vector by Gibson cloning.
    BioXp
    suggested: None
    All RNA samples were mixed with glyoxal loading dye (Invitrogen, Waltham, MA) 1:1 by volume, incubated at 50ยฐC for 20 minutes, loaded on a denatured 1% agarose gel in NorthernMax-Gly running buffer (Invitrogen) alongside Millennium RNA Markers (Thermo Fisher Scientific), and run at 120 V for 45 minutes before imaging on a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Spots were counted and data were analyzed using ImmunoSpot software (Cellular Technology Limited, Cleveland, OH)
    ImmunoSpot
    suggested: None
    All statistical analyses were conducted using Prism 9 (GraphPad Software, San Diego, CA).
    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.

  8. Version published to 10.1101/2022.03.22.485230v1 on bioRxiv