Spike protein multiorgan tropism suppressed by antibodies targeting SARS-CoV-2

  1. Molly Brady
  2. Conor McQuaid
  3. Alexander Solorzano
  4. Angelique Johnson
  5. Abigail Combs
  6. Chethana Venkatraman
  7. Akib Rahman
  8. Hannah Leyva
  9. Wing-Chi Edmund Kwok
  10. Ronald W. Wood
  11. Rashid Deane

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Abstract

While there is SARS-CoV-2 multiorgan tropism in severely infected COVID-19 patients, it’s unclear if this occurs in healthy young individuals. In addition, for antibodies that target the spike protein (SP), it’s unclear if these reduce SARS-CoV-2/SP multiorgan tropism equally. We used fluorescently labeled SP-NIRF to study viral behavior, using an in vivo dynamic imaging system and ex in vivo tissue analysis, in young mice. We found a SP body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. SP uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or CSF. Thus, the brain vascular barriers were effective in restricting the entry of SP into brain parenchyma in young healthy mice. While both anti-ACE2 and anti-SP antibodies suppressed SP biodistribution and organ uptake, anti-SP antibody was more effective. By extension, our data support the efficacy of these antibodies on SARS-CoV-2 multiorgan tropism, which could determine COVID-19 organ-specific outcomes.

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  1. Version published to 10.1038/s42003-021-02856-x
  2. ScreenIT

    SciScore for 10.1101/2021.07.30.454520: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal studies were performed in accordance with the National Institute of Health guidelines and using protocols approved by the University Committee on Animal Resources.
    Euthanasia Agents: Tissue Preparation for fluorescence imaging: After the duration of the experiment, mice were transcardially perfused using cold phosphate buffered saline (PBS) and paraformaldehyde (PFA) (4% in PBS, pH: 7.3).
    Sex as a biological variableMice: C57BL/6J (2-3 months old; Jackson Laboratory; Bar Harbor, ME, USA), male mice were used.
    RandomizationA training image was created from the (ROIs) to train a three-way random trees pixel classifier to account for variation, and distinguish between positive fluorescence and negative background.
    BlindingThe experimenter was blinded to the experiment design, and unaware of the tracers, T1 and T2 used in these studies.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-SARS-CoV-2 spike protein antibody (SARS-CoV-2 Spike Protein Monoclonal Antibody (cat# MA5-36087; immunogen SARS-CoV-2 spike protein that interact with HeLa cell expressed SARS-CoV-2) and anti ACE2 antibody (ACE2 Recombinant Rabbit Monoclonal Antibody (Cat# MA532307; interact with mouse ACE2) were obtained from Life Technologies Corporation.
    Anti-SARS-CoV-2 spike protein
    suggested: (Thermo Fisher Scientific Cat# MA5-36087, RRID:AB_2866698)
    anti ACE2
    suggested: None
    The groups of mice were as follows: young male mice (2-3 months old), T1 (anti-ACE2 antibody; young male mice (2-3 months old)) and T2 (anti-spike protein antibody; young male mice (2-3 months old)).
    T1
    suggested: (US Biological Cat# C2256-23M, RRID:AB_2291331)
    anti-ACE2
    suggested: (Biotrend Cat# ACE23-M, RRID:AB_2223312)
    T2 ( anti-spike protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Materials: SARS-CoV-2 Spike proteins (recombinant SARS-CoV-2 Spike Protein (S-RBD; cat# RP-87678, HEK293 cell expressed and binds ACE2) and ovalbumin Alexa Fluor-488 conjugate (Cat# 034781; molecular weight 45 kDa) were obtained from Life Technologies Corporation, Carlsbad CA, USA.
    HEK293
    suggested: None
    Anti-SARS-CoV-2 spike protein antibody (SARS-CoV-2 Spike Protein Monoclonal Antibody (cat# MA5-36087; immunogen SARS-CoV-2 spike protein that interact with HeLa cell expressed SARS-CoV-2) and anti ACE2 antibody (ACE2 Recombinant Rabbit Monoclonal Antibody (Cat# MA532307; interact with mouse ACE2) were obtained from Life Technologies Corporation.
    HeLa
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: C57BL/6J (2-3 months old; Jackson Laboratory; Bar Harbor, ME, USA), male mice were used.
    C57BL/6J
    suggested: None
    Software and Algorithms
    SentencesResources
    SP was labeled separately with Alexa Fluor 555 using a kit (Microscale protein labeling kit; ThermoFisher Scientific; Waltham, MA, USA) and by following the manufacturer instructions.
    ThermoFisher Scientific
    suggested: None
    Imaging settings and recordings were accomplished through a custom-built LabVIEW program (National Instruments, Austin, TX, USA).
    LabVIEW
    suggested: (LabView , RRID:SCR_014325)
    Using ImageJ software (National Institutes of Health, Bethesda, MD, USA), regions of interest (ROIs) were identified, and the ROI fluorescence intensity recorded at the same settings, as reported [39].
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Peak intensity (Imax) was identified as the average of 10 seconds at the peak identified using GraphPad Prism software (Version 9.1.0 (216)).
    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:
    Limitations of the present study: We used the RBD of the SP as a surrogate of the virus (SARS-CoV-2) to study its biodistribution and elimination, since RBD plays a major role for viral entry into host cells. However, the distribution pattern might be different for the actual virus, and the molecular weight of SP would influence filtration at the kidneys and convective flow in peripheral organs, such as muscles. Thus, a lower molecular weight RBD (< albumin) likely represent a maximum biodistribution. The biodistribution of SP was not systematically evaluated in all organs/cells, such blood cells, pancreas and others. Therefore, it is possible that an organ not studied may also play a role in SP uptake. However, these other organs may not be as significant, since there was no significant SP- NIRF level in the rest of the mouse. Thus, we focused on organs with significant signal. The interaction of the whole virus with host cells may be different. Nevertheless, our studies and other that use the SP would provide critical data to better and safely model the virus behavior in a host before using the virus in studies. In the current study the efficacy of anti-SP and anti-ACE2 antibodies was assessed and thus, intravenous injection of SP was used. However, the main route of viral entry into the body in inhalation of droplets. In terrestrial animals, injection of fluid into the nose is not physiological. The SP would need to specially formulate into an aerosol for inhalation studie...

    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 10.1101/2021.07.30.454520 on bioRxiv