SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy

  1. Alexander M. Horspool
  2. Chengjin Ye
  3. Ting Y. Wong
  4. Brynnan P. Russ
  5. Katherine S. Lee
  6. Michael T. Winters
  7. Justin R. Bevere
  8. Theodore Kieffer
  9. Ivan Martinez
  10. Julien Sourimant
  11. Alexander Greninger
  12. Richard K. Plemper
  13. James Denvir
  14. Holly A. Cyphert
  15. Jordi Torrelles
  16. Luis Martinez-Sobrido
  17. F. Heath Damron

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Abstract

SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

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    SciScore for 10.1101/2021.05.05.442784: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: All ABSL-3 animal experiments were conducted under West Virginia University (WVU
    Euthanasia Agents: Euthanasia and necropsy of SARS-CoV-2 infected mice: Euthanasia was conducted by administering 200 µL of pentobarbital (Patterson Veterinary 07-805-9296, 390 mg/kg diluted in 0.9% sterile NaCl) and cardiac puncture.
    Sex as a biological variableMale and female (Figures 2-3) or male (Figures 4-7) eight-week-old B6.Cg-Tg(K18-hACE2)2Prlmn/J mice (Jackson Laboratory 034860) were anesthetized with a single intraperitoneal dose of ketamine (Patterson Veterinary 07-803-6637, 80 mg/kg) + xylazine (Patterson Veterinary 07-808-1947, 8.3 mg/kg) and the 50µL infectious dose was administered with a pipette intranasally, 25µL per nare.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Secondary antibody (100µL 1:500 anti-human IgG HRP, Invitrogen 31410) was added and plates were incubated for 10 minutes at room temperature shaking at 60rpm.
    anti-human IgG
    suggested: None
    After blocking, 100µL were transferred into 2µL of antibody cocktail including 0.5µg of: hamster anti-mouse CD3e BV510: BD Biosciences 563024
    anti-mouse CD3e
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For viral growth kinetics, Vero E6 cells (6-well plate, 106 cells/well, triplicates) were infected (MOI 0.01) with SARS-CoV-2 WA-1, B.1.1.7 or B.1.351.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Male and female (Figures 2-3) or male (Figures 4-7) eight-week-old B6.Cg-Tg(K18-hACE2)2Prlmn/J mice (Jackson Laboratory 034860) were anesthetized with a single intraperitoneal dose of ketamine (Patterson Veterinary 07-803-6637, 80 mg/kg) + xylazine (Patterson Veterinary 07-808-1947, 8.3 mg/kg) and the 50µL infectious dose was administered with a pipette intranasally, 25µL per nare.
    B6.Cg-Tg(K18-hACE2)2Prlmn/J
    suggested: None
    Software and Algorithms
    SentencesResources
    Raw reads were quality filtered using Trimmomatic v0.39 (Bolger et al., 2014) and mapped to a SARS-CoV-2 reference genome (Genbank Accession No. MN985325) with Bowtie2 v2.4.1 (Langmead and Salzberg, 2012).
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    We genotyped each sample for low frequency VoCs with LoFreq* v2.1.3.1 (Wilm et al., 2012) and filtered sites with allele frequencies less than 20%.
    LoFreq*
    suggested: None
    SARS-CoV-2 viral RNA from stocks used for K18-hACE2 transgenic mice infection was deep sequenced and reads were aligned to the MN908947.3 reference genome using BWA version 0.7.17 (Li and Durbin, 2009) and trimmed for base-calling quality using iVar version 1.3.1 (Grubaugh et al., 2019) with default parameters.
    BWA
    suggested: (BWA, RRID:SCR_010910)
    iVar
    suggested: None
    Coverage was computed using samtools mpileup version 1.11 (Li et al., 2009).
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    LAVA constructs a candidate reference genome from early passage virus using bwa (Li and Durbin, 2009), removes PCR duplicates with Picard, calls variants with VarScan (Koboldt et al., 2009, 2012), and converts these changes into amino acid changes with Annovar (Wang et al., 2010).
    Picard
    suggested: (Picard, RRID:SCR_006525)
    VarScan
    suggested: (VARSCAN, RRID:SCR_006849)
    Annovar
    suggested: (ANNOVAR, RRID:SCR_012821)
    1.351 is accession number MZ065365 and SRA BioProject PRJNA726258.
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    CT values and copy numbers were calculated and analyzed in Microsoft Excel and GraphPad Prism v9.0.0.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Statistical analyses: All statistical tests were performed on groups with n > 3 in GraphPad Prism v9.0.0.
    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: We detected the following sentences addressing limitations in the study:
    It is important to point out the caveat that we did not investigate antibody dependent enhancement of infection. The observed differences in our mouse challenge studies may be attributable to the alterations in the viral B.1.351 S RBD region of the S protein, which may increase affinity for the hACE2 receptor (Bozdaganyan et al., 2021; Laffeber et al., 2021; Ozono et al., 2021; Shah et al., 2020; Tian et al., 2021) and could result in increased infectivity and pathogenicity. Although this yet speculative, sequencing results in Supplementary Figure 2-3 illustrate that many other mutations exist within the B.1.351 SARS-CoV-2 genome. Mutations within other viral proteins, particularly those involved in viral replication and genome packaging may provide additional advantages for this VoC during infection. It is currently unknown what impact the B.1.351 ORF7a deletion we observed has on pathogenesis, but other studies have suggested limited impacts of similar ORF7a mutations in cell culture (Chiem et al., 2020; Narayanan et al., 2008; Sims et al., 2005). Dissecting the consequences of these B.1.351 non-S mutations will be useful in determining the mechanisms behind the increased disease severity observed here and may help better inform containment of these VoCs, and others emerging in the future. Several additional observations were made in this study, including discovering significant differences in cytokine production of B.1.351 SARS-CoV-2 VoC infected mice. IL-27 is an indicato...

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.05.05.442784v1 on bioRxiv