Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient as a source of immune escape variants

  1. Sebastian Weigang
  2. Jonas Fuchs
  3. Gert Zimmer
  4. Daniel Schnepf
  5. Lisa Kern
  6. Julius Beer
  7. Hendrik Luxenburger
  8. Jakob Ankerhold
  9. Valeria Falcone
  10. Janine Kemming
  11. Maike Hofmann
  12. Robert Thimme
  13. Christoph Neumann-Haefelin
  14. Svenja Ulferts
  15. Robert Grosse
  16. Daniel Hornuss
  17. Yakup Tanriver
  18. Siegbert Rieg
  19. Dirk Wagner
  20. Daniela Huzly
  21. Martin Schwemmle
  22. Marcus Panning
  23. Georg Kochs

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Abstract

The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient’s escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants.

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  1. Version published to 10.1038/s41467-021-26602-3
  2. ScreenIT

    SciScore for 10.1101/2021.04.30.21256244: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Written informed consent was obtained from all participants and the study was conducted according to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany: No. F-2020-09-03-160428 and no. 322/20) and the Declaration of Helsinki (1975).
    IRB: Written informed consent was obtained from all participants and the study was conducted according to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany: No. F-2020-09-03-160428 and no. 322/20) and the Declaration of Helsinki (1975).
    IACUC: All experiments were in compliance with the German animal protection law and approved by the animal welfare committee of the Regierungspraesidium Freiburg (permit G-20/91).
    Sex as a biological variableHemizygous 8-12-week-old animals of both sexes were used in accordance with the guidelines of the Federation for Laboratory Animal Science Associations and the National Animal Welfare Body.
    RandomizationPhylogenetic and variant analysis: All available sequences from Germany deposited in GISAID (http://gisaid.org/) between February and April 2020 were downloaded (as of 11th of February 2021) and 250 sequences randomly subsampled excluding sequences already deposited by the Virology in Freiburg (Extended data table 3).
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Fixed and permeabilized cells were incubated with dilutions of the post-infectious mouse sera and SARS-CoV-2-specific antibodies were detected by fluorescence-labeled secondary anti-mouse IgG antiserum.
    SARS-CoV-2-specific
    suggested: None
    anti-mouse IgG
    suggested: None
    Detection of the primary antibodies was performed with fluorescent-labeled (Li-COR) secondary antibodies.
    Li-COR) secondary antibodies.
    suggested: None
    SARS-CoV-2 N- and spike-specific primary antibodies and AF568-labeled goat-anti-rabbit (Invitrogen, #A11011, 1:400) secondary antibody as well as AF488-labeled Phalloidin (Hypermol, #8813-01, 1:250) were used for staining.
    SARS-CoV-2
    suggested: None
    N-
    suggested: (UC Davis/NIH NeuroMab Facility Cat# N400/24, RRID:AB_2877580)
    Cells were incubated in medium containing the monoclonal mAb I1 antibody (ATCC) directed against VSV-G.
    VSV-G
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virus titers were determined by plaque assay on VeroE6 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    VeroE6 or human bronchial epithelium Calu-3 cells (ATCC-HTB-55) in 6-well plates, 1 x 106 cells, were infected with a moi of 0.001 for 1.5h.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    BHK-21 cells were transfected with the pCAGGS-S plasmids and later inoculated with 5 ffu/cell of VSV*ΔG(FLuc), coding for firefly luciferase, as described33.
    BHK-21
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Infection of K18-hACE2 transgenic mice: Transgenic (K18-hACE2)2Prlmn mice18 were purchased from The Jackson Laboratory and bred locally.
    K18-hACE2)2Prlmn
    suggested: None
    Recombinant DNA
    SentencesResources
    Virus pseudotype VSV*ΔG(FLuc) neutralization assay: cDNAs encoding the S protein were prepared from oropharyngeal swab samples of the COVID-19 patient obtained at days d14 and d105 and were cloned into the eukaryotic expression vector pCAGGS32.
    pCAGGS32
    suggested: None
    Single and double spike mutations were introduced into the pCAGGS-S(d14) construct.
    pCAGGS-S(d14
    suggested: None
    BHK-21 cells were transfected with the pCAGGS-S plasmids and later inoculated with 5 ffu/cell of VSV*ΔG(FLuc), coding for firefly luciferase, as described33.
    pCAGGS-S
    suggested: None
    Software and Algorithms
    SentencesResources
    Fluorescence images were generated using a LSM800 confocal laser-scanning microscope (Zeiss) equipped with a 63X, 1.4 NA oil objective and Airyscan detector and processed with Zen blue software (Zeiss) and ImageJ/Fiji.
    ImageJ/Fiji
    suggested: (BioVoxxel Toolbox, RRID:SCR_015825)
    The de-multiplexed raw reads were subjected to a custom Galaxy pipeline, which is based on bioinformatics pipelines on usegalaxy.eu (doi: 10.5281/zenodo.3685264)35.
    Galaxy
    suggested: (Galaxy, RRID:SCR_006281)
    The raw reads were pre-processed with fastp (https://www.biorxiv.org/content/10.1101/274100v2) and mapped to the SARS-CoV-2 Wuhan-Hu-1 reference genome (Genbank: NC_045512) using BWA-MEM (https://academic.oup.com/bioinformatics/article/25/14/1754/225615).
    BWA-MEM
    suggested: (Sniffles, RRID:SCR_017619)
    Variants (SNPs and INDELs) were called with the ultrasensitive variant caller LoFreq (https://academic.oup.com/nar/article/40/22/11189/1152727), demanding a minimum base quality of 30 and a coverage of at least 5-fold.
    LoFreq
    suggested: (LoFreq, RRID:SCR_013054)
    The effects of the mutations were automatically annotated in the vcf files with SnpEff (https://www.tandfonline.com/doi/full/10.4161/fly.19695).
    SnpEff
    suggested: (SnpEff, RRID:SCR_005191)
    For the phylogenetic analysis, the sequences were first aligned with MAFFT (v7.45)36 and a tree was constructed with IQ-Tree (v2.1.2)37.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    IQ-Tree
    suggested: (IQ-TREE, RRID:SCR_017254)
    To visualize the phylogenetic tree a custom R script was written utilizing the ggtree (v2.2.4) (https://academic.oup.com/mbe/article-abstract/35/12/3041/5142656), treeio (v1.12.0) (https://academic.oup.com/mbe/article-abstract/37/2/599/5601621) and ggplot2 (v3.3.3) packages (https://link.springer.com/chapter/10.1007/978-3-319-24277-4_12).
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    An in-house R script was also used to plot the variant frequencies that were detected by LoFreq as a heatmap (pheatmap package v1.0.12).
    pheatmap
    suggested: (pheatmap, RRID:SCR_016418)

    Results from OddPub: Thank you for sharing your code.

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

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  3. Version published to 10.1101/2021.04.30.21256244 on medRxiv