N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2

  1. Christopher M. Weiss
  2. Hongwei Liu
  3. Erin E. Ball
  4. Ashley R. Hoover
  5. Talia S. Wong
  6. Chun Fung Wong
  7. Samuel Lam
  8. Tomas Hode
  9. M. Kevin Keel
  10. Richard M. Levenson
  11. Wei R. Chen
  12. Lark L. Coffey

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Abstract

The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N- dihydro g alacto c hitosan (GC) is a novel mucoadhesive immunostimulatory polymer of β- 0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse.

IMPORTANCE

The ongoing COVID-19 pandemic necessitates new approaches to reduce disease caused by SARS-CoV-2. We tested the immunoadjuvant N-dihydro g alacto c hitosan (GC), used previously as an immunostimulant for tumor therapy and adjuvant for viral vaccines, as a potential COVID-19 countermeasure. When GC was administered before and after inoculation of a lethal dose of SARS-CoV-2 into the nose of humanized mice expressing an entry receptor for the virus, fewer mice showed weight loss and died compared to mice that received only the vehicle but no GC. GC-treated mice also had lower levels of infectious SARS-CoV-2 in their upper airway. These results suggest that GC may be a candidate to prevent or treat COVID-19.

Single Sentence Summary

The immunoadjuvant N-dihydrogalactochitosan diminishes SARS-CoV-2 disease in humanized ACE2 mice, representing a new countermeasure against COVID-19.

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  1. Version published to 10.1101/2021.08.10.455872v3 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.08.10.455872: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Ethics Statement: All mouse work was conducted on protocol #21868 approved by the institutional animal care and use committee (IACUC) at the University of California, Davis.
    Euthanasia Agents: Prior to euthanasia, whole blood was collected by submandibular vein puncture under isoflurane anesthesia.
    Sex as a biological variableMice: Equal numbers of male and female transgenic mice expressing the human ACE2 receptor on a K18 transgene in a C57Bl/6J background (B6.Cg-Tg(K18-ACE2)2Prlmn/J, referenced as ‘hACE2’) were purchased at 5 weeks of age from Jackson Laboratories (Sacramento, CA).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Virus: SARS-CoV-2/human/USA/CA-CZB-59×002/2020 (GenBank #MT394528), which was isolated from a patient in 2020 in Northern California and passaged once in Vero-E6 cells, was generously provided by Dr. Christopher Miller (University of California, Davis).
    Vero-E6
    suggested: None
    Antibody-virus dilution series were incubated for 1 hour at 37°C after which they were applied to confluent Vero CCL-81 cells in single-replicate and incubated for 1 hour at 5% CO2 and 37°C in a humidified incubator.
    Vero CCL-81
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Equal numbers of male and female transgenic mice expressing the human ACE2 receptor on a K18 transgene in a C57Bl/6J background (B6.Cg-Tg(K18-ACE2)2Prlmn/J, referenced as ‘hACE2’) were purchased at 5 weeks of age from Jackson Laboratories (Sacramento, CA).
    C57Bl/6J
    suggested: None
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Software and Algorithms
    SentencesResources
    For quantitative assessment of lung inflammation, digital images were captured and analyzed using ImageJ software (Fiji, NIH) to estimate the area of inflamed tissue that was visible to the naked eye at subgross magnification as a percentage of the total surface area of the lung section.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Statistics: All statistical tests were performed with GraphPad PRISM 9.0.2 (GraphPad Software).
    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:
    GC circumvents these limitations through a controlled and reproducible process of synthetically attaching galactose to the free-amino groups of the chitosan base, improving solubility (and thus bioavailability) while maintaining a physiological pH. GC has been previously used as a combination anti-tumor therapy due to its immunoadjuvant properties (38, 40), but its utility as a broad-acting antiviral compound has not previously been investigated. Immunoadjuvants stimulate non-specific innate immune responses through various mechanisms. Some use pattern-recognition receptors, including the Toll-like receptor (TLR) family of antigen detection complexes. While the mechanism of SARS-CoV-2 protection has not been determined for GC, chitosan, and similar polymers of N-acetylglucosamine, interact with TLR2, which serves as a rationale for inclusion of chitosan as a vaccine adjuvant. TLR2 signals through myeloid differentiation factor 88 (MyD88) to stimulate the nuclear factor kappa B (NF-κB) pathway and downstream inflammatory and anti-microbial cytokine responses (45, 46). TLR engagement of the canonical NF-κB pathway up-regulates both tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two potent pro-inflammatory effectors. IL-6, which is necessary for antiviral immunity against other virus families, has been identified as a target of dysregulation associated with hyperinflammatory responses in the lungs during SARS-family coronavirus infections. SARS-CoV-1 nucleocapsid ...

    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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  3. Version published to 10.1101/2021.08.10.455872v2 on bioRxiv
  4. Version published to 10.1101/2021.08.10.455872v1 on bioRxiv