Scrutiny of human lung infection by SARS-CoV-2 and associated human immune responses in humanized mice

  1. Renren Sun
  2. Zongzheng Zhao
  3. Cong Fu
  4. Yixin Wang
  5. Zhendong Guo
  6. Chunmao Zhang
  7. Lina Liu
  8. Cheng Zhang
  9. Chang Shu
  10. Jin He
  11. Shucheng Hua
  12. Yuwei Gao
  13. Zheng Hu

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Abstract

There is an urgent need for animal models of COVID-19 to study immunopathogenesis and test therapeutic intervenes. In this study we showed that NSG mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by SARS-CoV-2, and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-seq examination identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, interferon stimulation, and pulmonary fibrosis between HL grafts from infected and control NSG-L mice. Furthermore, when infecting humanized mice with human immune system (HIS) and autologous HL grafts (HISL mice), the mice had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.

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  1. ScreenIT

    SciScore for 10.1101/2021.11.05.466755: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Discard human fetal samples of gestational age of 17-21 weeks were obtained with informed consent at the First Hospital of Jilin University.
    IACUC: Protocols involved in the use of human samples and animals were reviewed and approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University, and all experiments with SARS-CoV-2 (COVID-19) were performed in biosecurity level 3 laboratory according to the protocols approved by the Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Human immune cell reconstitution in humanized mice were determined by flow cytometry using following fluorochrome-conjugated antibodies: anti-human CD45, CD3, CD20, CD33, CD4,
    anti-human CD45
    suggested: (RayBiotech Cat# CS-11-0028, RRID:AB_1227892)
    CD3
    suggested: None
    CD20
    suggested: None
    CD33
    suggested: None
    ), CD20 (DAKO, L26), CD4 (ABclonal; ARC0328), or CD11c (Abcam; EP1347Y), ACE2 (Abcam; EPR4435(2)) antibodies, and the immunoreactivity was detected with UltraSensitiveTM Streptavidin-Peroxidase Kit (KIT-9710, Mai Xin, China) according to the manufacturer’s protocol.
    CD4
    suggested: None
    CD11c
    suggested: None
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 virus preparation and titer determination: SARS-CoV-2 (BetaCoV/Beijing/IME-BJ05-2020) was proliferated in Vero E6 cells, which are maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) with supplemented 2% fetal bovine serum (FBS; Gibco, Auckland, New Zealand).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice and human tissues: NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju (referred to as NSG) mice were purchased from Nanjing Biomedical Research Institute of Nanjing University.
    NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju
    suggested: None
    Software and Algorithms
    SentencesResources
    Examination was performed on a Cytek Aurora (Cytek Biosciences) and data were analyzed using the FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Reads were aligned with STAR RNA-seq aligner (Version 2.7.3a) using the UCSC/hg38 genome assembly and transcript annotation20.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Expression levels were calculated as Fragments per Kilobase of transcript per Million reads (FPKM) using Cufflinks software21.
    Cufflinks
    suggested: (Cufflinks, RRID:SCR_014597)
    Differential expression analysis was performed using Cuffdiff (version 2.2.1.
    Cuffdiff
    suggested: (Cuffdiff, RRID:SCR_001647)
    “Cluster Profiler” package (Release 3.16.1)22 and “DOSE” package (Release 3.14.0)23 in R software (version 4.0.2) was used for functional enrichment analysis, and GO biological processes terms at the significant level (q-value < 0.05) were employed.
    “Cluster
    suggested: None
    Profiler”
    suggested: None
    Statistical analysis: Data were analyzed using GraphPad Prism 8 software (San Diego, CA, USA) and presented as mean values ± SEM.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your 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: 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.

  2. Version published to 10.1101/2021.11.05.466755 on bioRxiv