Intranasal Administration of ACIS KEPTIDE™ Prevents SARS-CoV2-Induced Acute Toxicity in K18-hACE2 Humanized Mouse Model of COVID-19: A Mechanistic Insight for the Prophylactic Role of KEPTIDE™ in COVID-19

  1. Gunnar Gottschalk
  2. James F Keating
  3. Kris Kesler
  4. Konstance Knox
  5. Avik Roy

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Abstract

Previously, we have demonstrated that ACIS KEPTIDE™, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE™ attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*10 6 ) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 μg/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities. In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo , significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice.

Highlights

  • ACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 & 8). Intranasal inoculation of SARS-CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6) and kidney tubular cells (Fig.8). ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8).

  • ACIS KEPTIDE™ completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8).

  • ACIS KEPTIDE™ improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult.

  • Intranasal administration of 0.05% Beta-propiolactone (βPL)-inactivated SARS-CoV2 (1 *10 6 ) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation ( Supplemental videos ) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality.

  • The peptide backbone of ACIS KEPTIDE™ provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8). Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8) of SARS-CoV2-treated K18-hACE2 mice.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.13.378257: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Animals are provided standard mouse chow and water ad libitum and closely monitored for health and overall well-being daily by the investigator and supervised by a certified veterinarian in accordance with standard animal care guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableData collection, audiovisual recordings, and statistical analysis: Six to eight weeks old K18-hACE2 mice (n=8; 4 males and 4 females) were included to explore the toxic effect of KEPTIDE™ treatment.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The rehydrated slides were blocked with 2% horse-serum followed by incubation with primary antibodies (Mouse anti-S-glycoprotein antibody, 1:200 dilution, Millipore, Cat #
    anti-S-glycoprotein
    suggested: None
    ; Rabbit anti-ACE2 antibody, 1:200 dilution, Abcam, Cat # ab272690) for 2 hrs.
    anti-ACE2
    suggested: None
    Next day, cells were thoroughly washed with PBST, incubated with Rabbit-anti ACE2 antibody (1:200 dilution) for 2 hrs, washed with PBST (X 3), incubated with 2° antibody [Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L); 1:200 dilution] for 1 hr, washed, and then mounted with fluoromount media.
    Rabbit-anti ACE2
    suggested: None
    Anti-Rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To generate virus stocks, Vero E6 cells were cultured in complete DMEM (+ 2% FCS), inoculated with virus at a MOI of 0.05.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Acquisition, justification, treatment, and disposal of K18-hACE2 mice: Six weeks-old K18-hACE2 mice (B6. Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from Jackson laboratories (Stock #034860).
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    B6. Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: None
    Software and Algorithms
    SentencesResources
    Coppe Healthcare solutions has retained the approval, license and CDC-certification to handle SARS-CoV2 virus.
    Coppe Healthcare
    suggested: None
    Slides were thoroughly dried under air-blower at 70-80°C and imaged in a LOMO phase-contrast light microscope with ToupView 3.7 software.
    ToupView
    suggested: (ToupView , RRID:SCR_017998)
    Data were plotted in GraphPad Prism 8 software as XY scatter plots with X axis of “days” and Y axis of health variables.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Quantitative estimation of ACE-2-ir cells in bronchiolar epithelia and Mean color intensity of CE-2 expression in kidney tubular cells were performed in ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Data were analyzed with a one-way ANOVA in a GraphPad Prism 8 software considering the treatment as a single factor.
    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: 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.

    About SciScore

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  2. Version published to 10.1101/2020.11.13.378257v1 on bioRxiv