SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains

  1. Peter Radvak
  2. Hyung-Joon Kwon
  3. Martina Kosikova
  4. Uriel Ortega-Rodriguez
  5. Ruoxuan Xiang
  6. Je-Nie Phue
  7. Rong-Fong Shen
  8. James Rozzelle
  9. Neeraj Kapoor
  10. Taylor Rabara
  11. Jeff Fairman
  12. Hang Xie

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Abstract

SARS-CoV-2 variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta) show increased transmissibility and enhanced antibody neutralization resistance. Here we demonstrate in K18-hACE2 transgenic mice that B.1.1.7 and B.1.351 are 100-fold more lethal than the original SARS-CoV-2 bearing 614D. B.1.1.7 and B.1.351 cause more severe organ lesions in K18-hACE2 mice than early SARS-CoV-2 strains bearing 614D or 614G, with B.1.1.7 and B.1.351 infection resulting in distinct tissue-specific cytokine signatures, significant D-dimer depositions in vital organs and less pulmonary hypoxia signaling before death. However, K18-hACE2 mice with prior infection of early SARS-CoV-2 strains or intramuscular immunization of viral spike or receptor binding domain are resistant to the lethal reinfection of B.1.1.7 or B.1.351, despite having reduced neutralization titers against these VOC than early strains. Our results thus distinguish pathogenic patterns in K18-hACE2 mice caused by B.1.1.7 and B.1.351 infection from those induced by early SARS-CoV-2 strains, and help inform potential medical interventions for combating COVID-19.

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  1. Version published to 10.1038/s41467-021-26803-w
  2. ScreenIT

    SciScore for 10.1101/2021.06.05.447221: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All procedures were performed according to the animal study protocols approved by the FDA White Oak Animal Program Animal Care and Use Committee.
    Sex as a biological variableMice: Hemizygous B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE) female mice (JAX Stock No. 034860) and noncarrier c57BL/6J male mice (JAX Stock No. 000664) were mated to maintain live colonies at the ABSL2 facility of FDA White Oak Vivarium
    RandomizationAt the age of 8-10 weeks, K18-hACE mice of both sexes were randomly assigned to experimental groups at approximately 1:1 ratio and were transferred to the ABSL3 facility for infection.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Only hACE2 (+) offspring as confirmed by genotyping (Transnetyx) were retained and were used for experiments.

    Table 2: Resources

    Antibodies
    SentencesResources
    After incubation at 37 °C, 5% CO2 for two days, virus-infected cells were detected using an in-house rabbit polyclonal antibody specific for SARS-CoV-2 N/M/E followed by horseradish peroxidase-conjugated goat anti-mouse IgG (Invitrogen).
    anti-mouse IgG
    suggested: None
    Bound antibodies were detected using peroxidase-conjugated goat anti-mouse IgM heavy chain (Southern Biotech) or IgG (H+L) antibodies (Invitrogen) followed by One-step TMB substrate (ThermoFisher).
    peroxidase-conjugated goat anti-mouse IgM heavy chain ( Southern Biotech )
    suggested: None
    IgG ( H+L ) antibodies ( Invitrogen ) followed by One-step TMB substrate ( ThermoFisher)
    suggested: None
    Virus-infected cells were detected using in-house raised rabbit anti-S polyclonal antibody along with peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen).
    anti-S
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The virus-serum mixtures were then added to Vero E6 cells (2X104 cells/well) pre-seeded in 96-well tissue culture plates and were incubated at 37°C, 5% CO2 for 2 days.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Hemizygous B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE) female mice (JAX Stock No. 034860) and noncarrier c57BL/6J male mice (JAX Stock No. 000664) were mated to maintain live colonies at the ABSL2 facility of FDA White Oak Vivarium
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    At the age of 8-10 weeks, K18-hACE mice of both sexes were randomly assigned to experimental groups at approximately 1:1 ratio and were transferred to the ABSL3 facility for infection.
    K18-hACE
    suggested: None
    In separated experiments, K18-hACE2 mice of both sexes were primed and boosted at 3 weeks intervals with S (10 µg/dose) or RBD (20 µg/dose) emulsified with AddaS03™ (InvivoGen) via intramuscular route.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Recombinant DNA
    SentencesResources
    Recombinant proteins: The DNA sequences encoding the full-length S (residues 1-1213) and RBD (residues 319-541) of WA (GenBank: MN985325.1) with a T4 trimerization domain and a 6xHis tag at the C-terminus were codon-optimized and were subcloned into pcDNA5/FRT mammalian expression vector (ThermoFisher).
    pcDNA5/FRT
    suggested: RRID:Addgene_31983)
    Threshold cycle (Ct) values were calculated using MxPro qPCR software (Agilent) and the N gene copies in individual tissues were interpolated from a standard curve constructed by serial dilutions of a pCC1-CoV2-F7 plasmid expressing SARS-CoV-2 N57.
    pCC1-CoV2-F7
    suggested: None
    The copies of expressed hACE2 were interpolated from a standard curve created by serial dilutions of a pSBbi-bla ACE2 plasmid expressing hACE2 (kindly provided by J Yewdell, NIAID/NIH).
    pSBbi-bla ACE2
    suggested: None
    hACE2
    suggested: RRID:Addgene_1786)
    Software and Algorithms
    SentencesResources
    Heatmap was generated using Prism 8.4.3 (GraphPad, San Diego, CA)
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Tissue sections were visualized using a Leica Aperio AT2 slide scanner with Aperio ImageScope DX clinical viewing software (Histoserv).
    ImageScope
    suggested: (ImageScope, RRID:SCR_014311)
    R version 4.0.3 (https://www.r-project.org/) was used to perform all the above analyses.
    https://www.r-project.org/
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)

    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.

  3. Version published to 10.1101/2021.06.05.447221 on bioRxiv