Simultaneous and sequential multi-species coronavirus vaccination

  1. Lei Peng
  2. Zhenhao Fang
  3. Paul A. Renauer
  4. Andrew McNamara
  5. Jonathan J. Park
  6. Qianqian Lin
  7. Xiaoyu Zhou
  8. Matthew B. Dong
  9. Biqing Zhu
  10. Hongyu Zhao
  11. Craig B. Wilen
  12. Sidi Chen

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Abstract

Although successful COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging for development of multi-species coronavirus vaccines. Here we developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 (Delta variant), SARS-CoV and MERS-CoV, and test how multiplexing of these LNP-mRNAs can induce effective immune responses in animal models. A triplex scheme of LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV, with a relatively weaker MERS-CoV response in this setting. Single cell RNA-seq profiled the global systemic immune repertoires and the respective transcriptome signatures of multiplexed vaccinated animals, which revealed a systemic increase in activated B cells, as well as differential gene expression signatures across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated the feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.

One sentence summary

Multiplexed mRNA vaccination in simultaneous and sequential modes provide broad and potent immunity against pathogenic coronavirus species.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.07.491038: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Single cell RNA-seq: PBMCs were collected from mRNA-LNP vaccinated and control mice were collected as described above for mouse immunization and sample collection, and normalized to 1000 cells/μL.
    Sex as a biological variableMouse immunization: 6-8 weeks old female C57BL/6Ncr (B6) mice were purchased from Charles River and used for vaccine immunogenicity study.
    RandomizationAll experiments utilize randomized littermate controls.
    BlindingReplication, randomization, blinding and reagent validations: Replicate experiments have been performed for all key data shown in this study.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: All cell lines tested negative for mycoplasma. Figs.

    Table 2: Resources

    Antibodies
    SentencesResources
    Thereafter, cells were washed twice and incubated with PE–anti-human FC antibody (Biolegend, 410708) in MACS buffer for 30 min on ice.
    PE–anti-human FC
    suggested: None
    Anti-mouse secondary antibody (Fisher, Cat# A-10677) was diluted to 1:2500 in blocking buffer and incubated at room temperature for one hour.
    Anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A-10677, RRID:AB_2534060)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T (ThermoFisher), Huh-7 and 293T-hACE2 (Dr Bieniasz’ lab) cell lines were cultured in complete growth medium, Dulbecco’s modified Eagle’s medium (DMEM; ThermoFisher) supplemented with 10% Fetal bovine serum (FBS, Hyclone), 1% penicillin-streptomycin (Gibco) (D10 media for short).
    HEK293T
    suggested: None
    Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of corresponding spike plasmids, in the presence of 198 µl PEI.
    293T
    suggested: None
    The SARS-CoV and SARS-CoV-2 pseudovirus neutralization assays were performed on 293T-hACE2 cell, while the MERS-CoV neutralization assay was performed on Huh-7 cells.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    One day before infection, 293T-hACE2 cells were plated in a 96 well plate with 0.01 x106 cells per well.
    293T-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse immunization: 6-8 weeks old female C57BL/6Ncr (B6) mice were purchased from Charles River and used for vaccine immunogenicity study.
    C57BL/6Ncr
    suggested: RRID:MGI:2160593)
    Recombinant DNA
    SentencesResources
    The spike sequences were cloned by Gibson Assembly (NEB) into pcDNA3.1 plasmid for the mRNA transcription and pseudovirus assay.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    1.617.2 variant S protein (Delta variant-Δ19), SARS-CoV S protein (SARS-CoV-Δ19) and MERS S protein (MERS-CoV-Δ16) were generated based on the pSARS-CoV-2-Δ19.
    pSARS-CoV-2-Δ19
    suggested: None
    Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of corresponding spike plasmids, in the presence of 198 µl PEI.
    pCCNanoLuc2AEGFP
    suggested: None
    Software and Algorithms
    SentencesResources
    Coronavirus spike sequence alignment: The spike sequence used to produce the LNP-mRNA vaccines were aligned using Clustal Omega 43 and visualized in Jalview 44.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Jalview
    suggested: (Jalview, RRID:SCR_006459)
    Cell culture: HEK293T (ThermoFisher), Huh-7 and 293T-hACE2 (Dr Bieniasz’ lab) cell lines were cultured in complete growth medium, Dulbecco’s modified Eagle’s medium (DMEM; ThermoFisher) supplemented with 10% Fetal bovine serum (FBS, Hyclone), 1% penicillin-streptomycin (Gibco) (D10 media for short).
    ThermoFisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
    Analysis was performed using FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The 50% inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism (GraphPad Software Inc.).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Differential expression was performed using the edgeR analysis pipeline and quasi-likelihood (QL) F tests 53, 54.
    edgeR
    suggested: (edgeR, RRID:SCR_012802)
    Schematic illustrations: Schematic illustrations were created with Affinity Designer or BioRender.
    BioRender
    suggested: (Biorender, RRID:SCR_018361)
    Genomic sequencing raw data are deposited to Gene Expression Omnibus (GEO) with a pending accession code.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

    Results from OddPub: Thank you for sharing your code and data.

    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
    NCT04796896RecruitingA Study to Evaluate Safety and Effectiveness of mRNA-1273 CO…

    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 10.1101/2022.05.07.491038v1 on bioRxiv