In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice

  1. Narayanappa Amruta
  2. Elizabeth B. Engler-Chiurazzi
  3. Isabel C. Murray-Brown
  4. Timothy E. Gressett
  5. Ifechukwude J. Biose
  6. Wesley H. Chastain
  7. Gregory Bix

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin α5β1, and human ACE2 to facilitate viral entry into host cells in vitro . We previously found that inhibition of integrin α5β1 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro . In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo . We discovered that treatment with single- or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 hours after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence and improved lung histology in a majority of mice 72 hours post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin α5 and αv (an α5-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C–X–C motif chemokine ligand 10 ( Cxcl10 ), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin α5β1 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.08.443275: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: The Institutional Animal Care and Use Committee of Tulane University reviewed and approved all procedures for sample handling, inactivation, and removal from a BSL3 containment (permit number 3430 (#5)).
    Euthanasia Agents: After 3 days post infection (dpi), the mice were euthanized by CO2 asphyxiation followed by cervical dislocation and lungs were harvested for histology, immunofluorescence and qRT-PCR analysis.
    Sex as a biological variableMice and Ethics Statement: Male heterozygous K18-hACE c57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J, 10-weeks-old) were obtained from The Jackson Laboratory.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibody (Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig) 1:1000, NR-10361) incubation was achieved at room temperature for 1 h.
    Anti-SARS Coronavirus ( antiserum , Guinea Pig )
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice and Ethics Statement: Male heterozygous K18-hACE c57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J, 10-weeks-old) were obtained from The Jackson Laboratory.
    K18-hACE c57BL/6J
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistics: Statistical tests were performed with GraphPad Prism, 8.4.3 version (GraphPad Software, San Diego, CA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    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:
    A limitation of this study is that it employed a limited number of K18 hACE2 mice, a function of the logistical difficulty of doing live virus BSL-3 studies. We also dichotomized ATN-161-treated animals into ‘responder’ and ‘non-responder’ groups such that mice were considered non-responders if they displayed Genomic N values > 2×109, Sgm-N values > 1×105 and viral immunohistology staining counts >0.7 values observed in the SARS-CoV-2 group in the SARS-CoV-2 + saline group. ATN-161 responders had significantly lower genomic lung viral loads than SARS-CoV-2-infected animals. Accordingly, this bimodal distribution of responders may be due to our use of heterozygous (HT) K18-hACE2 mice, as the K18-hACE2 homozygous mouse model completely replaces mACE2 expression with hACE2 under the mAce2 promoter [49]. Thus, it might be possible that our use of HT K18-hACE2 mice in this study produced variable expression patterns of mACE2 vs hACE2, reducing the efficacy of ATN-161 in mice that expressed higher levels of mACE2 relative to hACE2. ATN-161 interference with mACE2-α5β1 interactions would presumably have minimal to no effect on SARS-CoV-2 infection as compared to impacting hACE2-α5β1 interactions. ATN-161 reduced Sgm-N viral load among pooled ATN-161-treated mice. When we re-analyzed the data by comparing lung viral load in the responder/non-responder groups to that of the SARS-CoV-2 + Saline-treated mice, we found that ATN-161 responders had significantly lower Sgm-N lung viral load...

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

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  2. Version published to 10.1101/2021.05.08.443275 on bioRxiv