Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B

  1. Jun Liu
  2. Patrick Budylowski
  3. Reuben Samson
  4. Bryan D. Griffin
  5. Giorgi Babuadze
  6. Bhavisha Rathod
  7. Karen Colwill
  8. Jumai A. Abioye
  9. Jordan A. Schwartz
  10. Ryan Law
  11. Lily Yip
  12. Sang Kyun Ahn
  13. Serena Chau
  14. Maedeh Naghibosadat
  15. Yuko Arita
  16. Queenie Hu
  17. Feng Yun Yue
  18. Arinjay Banerjee
  19. W. Rod Hardy
  20. Karen Mossman
  21. Samira Mubareka
  22. Robert A. Kozak
  23. Michael S. Pollanen
  24. Natalia Martin Orozco
  25. Anne-Claude Gingras
  26. Eric G. Marcusson
  27. Mario A. Ostrowski

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Abstract

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle–formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.

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  1. Version published to 10.1126/sciadv.abj9815
  2. ScreenIT

    SciScore for 10.1101/2021.05.11.443286: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Ethics: All animal work was approved by the Animal Care Committees of The University of Toronto.
    IACUC: Ethics: All animal work was approved by the Animal Care Committees of The University of Toronto.
    Consent: For studies involving human samples, written informed consent was obtained from convalescent COVID-19 patients, and samples were obtained and used according to a research ethics board (REB) approved protocol (St.
    IRB: For studies involving human samples, written informed consent was obtained from convalescent COVID-19 patients, and samples were obtained and used according to a research ethics board (REB) approved protocol (St.
    Sex as a biological variableMouse vaccination: Female C57BL/6 mice of 6-to 8-week old were vaccinated intramuscularly twice with a 3-week interval.
    RandomizationAll animals were randomly assigned to different treatment groups.
    BlindingThe performers measuring SARS-CoV-2 neutralization by mice sera and SARS-CoV-2 virus in mouse tissues and the pathologist examining animal pathology were blinded to the sample groupings.
    Power AnalysisThe sample size of mice was determined by power analysis assuming 60% protection efficacy.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Wells were then washed 3 times with 200µL PBST before incubation for 1 hour with secondary antibodies (HRP-labeled Goat anti-mouse IgG1/IgG2b/IgG2c purchased from SouthernBiotech or HRP-labeled Goat anti-mouse IgG Fcγ purchased from Jackson ImmunoResearch, West Grove, PA) in 1% w/v milk powder in PBS-T.
    anti-mouse IgG1/IgG2b/IgG2c
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Following a 0.2 µm filtration process, the LNPs were subjected to quality tests including RNA concentration, encapsulation efficiency, particle size, pH and osmolality. mRNA transfection of HEK293T cells and detection of the expressed immunogens: S mRNA, Sfurinmut mRNA, and RBD mRNA were transfected into HEK293T cells using Lipofectamine® MessengerMAX™ transfection reagent (Thermo Fisher Scientific, Mississauga, ON, Canada) according to the manufacturer’s protocol.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Serial dilutions of the sera were mixed with 100 TCID50 SARS-CoV-2 virus (isolate SARS-CoV-2-SB2-P3 PB Clone 1, passage 3(40)) in serum free DMEM, incubated at 37°C for 1 hour, and then added onto the VeroE6 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    For the neutralization assay, diluted mouse sera (1:40 from stock sera) were serially diluted (from 2.5 to 4-fold dilutions over 7 dilutions to encompass a complete neutralization curve per sample) and incubated with diluted pseudovirus at a 1:1 ratio for 1 hour at 37°C before being transferred to plated HEK293T-ACE2/TMPRSS2 cells and incubated for an additional 48 hours at 37°C and 5% CO2.
    HEK293T-ACE2/TMPRSS2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse vaccination: Female C57BL/6 mice of 6-to 8-week old were vaccinated intramuscularly twice with a 3-week interval.
    C57BL/6
    suggested: None
    In some experiments, both male and female BALB/c mice of 6-to 8-week old were used.
    BALB/c
    suggested: None
    Recombinant DNA
    SentencesResources
    An E gene DNA standard (pUC57-2019-nCoV-PC:E, GenScript, Piscataway, NJ) was also run at the same time for conversion of Ct value to genomic copies, by using the Rotor-Gene Q software (QIAGEN).
    pUC57-2019-nCoV-PC:E
    suggested: None
    Software and Algorithms
    SentencesResources
    FlowJo (BD) was used to analyze the flow cytometry data.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    OD values of each PTX-COVID19-B vaccinated mouse serum minus average of OD values of 4 tdTomato control mouse sera at the same dilution were used to calculate EC50 titer using the 4-parameter logistic regression analysis in GraphPad Prism 8 (GraphPad Software, La Jolla, CA)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All statistical analysis was performed by using GraphPad Prism 8.
    GraphPad Prism
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04765436RecruitingPTX-COVID19-B, an mRNA Humoral Vaccine, is Intended for Prev…

    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.
    • Thank you for including a protocol registration statement.

    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.

  3. Version published to 10.1101/2021.05.11.443286v1 on bioRxiv