ColdZyme Maintains Integrity in SARS-CoV-2-Infected Airway Epithelia

Abstract

SARS-CoV-2 infection causing the COVID-19 pandemic calls for immediate interventions to avoid viral transmission, disease progression, and subsequent excessive inflammation and tissue destruction. Primary normal human bronchial epithelial cells are among the first targets of SARS-CoV-2 infection. Here, we show that ColdZyme medical device mouth spray efficiently protected against virus entry, excessive inflammation, and tissue damage. Applying ColdZyme to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI) completely blocked binding of SARS-CoV-2 and increased local complement activation mediated by the virus as well as productive infection of the tissue model. While SARS-CoV-2 infection resulted in exaggerated intracellular complement activation immediately following infection and a drop in transepithelial resistance, these parameters were bypassed by single pretreatment of the tissues with ColdZyme mouth spray. Crucially, our study highlights the importance of testing already evaluated and safe drugs such as ColdZyme mouth spray for maintaining epithelial integrity and hindering SARS-CoV-2 entry within standardized three-dimensional (3D) in vitro models mimicking the in vivo human airway epithelium.IMPORTANCE Although our understanding of COVID-19 continuously progresses, essential questions regarding prophylaxis and treatment remain open. A hallmark of severe SARS-CoV-2 infection is a hitherto-undescribed mechanism leading to excessive inflammation and tissue destruction associated with enhanced pathogenicity and mortality. To tackle the problem at the source, transfer of SARS-CoV-2, subsequent binding, infection, and inflammatory responses have to be avoided. In this study, we used fully differentiated, mucus-producing, and ciliated human airway epithelial cultures to test the efficacy of ColdZyme medical device mouth spray in terms of protection from SARS-CoV-2 infection. Importantly, we found that pretreatment of the in vitro airway cultures using ColdZyme mouth spray resulted in significantly shielding the epithelial integrity, hindering virus binding and infection, and blocking excessive intrinsic complement activation within the airway cultures. Our in vitro data suggest that ColdZyme mouth spray may have an impact in prevention of COVID-19.

Keywords: ColdZyme; SARS-CoV-2; airway epithelia; anaphylatoxins; antiviral response.

FIG 1

ColdZyme mouth spray protects primary human airway epithelial (HAE) cells from SARS-CoV-2 binding and innate immune activation. Visualization of virus binding (SARS-CoV-2-S1/N, red) and complement (C3-FITC, green) in SARS-CoV-2-infected 3D pseudostratified epithelia. Multilayered epithelia were apically treated with solvent control or ColdZyme mouth spray prior to exposure to SARS-CoV-2 (MOI 0.1). After 2 h, filters were fixed; stained for Hoechst (blue), SARS-CoV-2-S1/N (red), complement C3 (green), and WGA (orange); and then analyzed by HCS. (a) Overview on the whole Transwell filter of solvent (left)- and ColdZyme (right)-pretreated and infected HAE cultures using ×5 magnification. One representative filter and one detail out of the filter are illustrated. Scale bars represent 2 mm or 1 mm as indicated. (b) Z-stacks of six fields of uninfected (UI, left), SARS-CoV-2-exposed (middle), and ColdZyme/SARS-CoV-2 (right)-exposed cells were analyzed using the Operetta CLS HCS and the 63× water objective. Cells were stained using C3-FITC (green) as indicator for innate immune activation, SARS-CoV-2-S1/N-Alexa 594 (red) for virus detection, Hoechst stain for imaging nuclei (blue), and WGA for staining lectins (orange). Massive IC C3 mobilization and SARS-CoV-2 binding/uptake were monitored in SARS-CoV-2-exposed cultures (middle), while no virus and low C3 signals were detected in UI (left) and ColdZyme/SARS-CoV-2-exposed (right) cultures. Scale bars represent 100 μm, and three independent experiments were performed. (c) Z-stacks of several representative single fields of SARS-CoV-2-exposed regions under the different conditions (ColdZyme/UI, left; SARS-CoV-2, middle; ColdZyme/SARS-CoV-2, right) are shown. Scale bars represent 50 μm, and three independent experiments were performed. (d) More than 2,500 cells (upper left) were analyzed for their expression of C3 (upper right) and SARS-CoV-2 (lower left), where up to 50% of the analyzed SARS-CoV-2-infected cells were stained positive for C3 (upper right) or virus (lower left), while only background signals were detected in UI or ColdZyme/SARS-CoV-2-exposed cells. Too, significantly higher levels of SARS-CoV-2/C3 double-positive cells were analyzed in the infected cultures compared to UI or treated ones (lower right). Statistical significances were analyzed on >2,500 cells with GraphPad Prism software using one-way ANOVA and Tukey’s posttest.

FIG 2

Disruption of epithelial integrity is contingent on extensive C3 mobilization and C3a secretion by SARS-CoV-2 and can be avoided by ColdZyme pretreatment. (a) Multilayered epithelia were infected by apical addition of SARS-CoV-2 (MOI 0.1) with or without ColdZyme pretreatment and incubated for 72 h. TEER was measured using an EVOM voltohmmeter. TEER in Ω/cm² was determined for all conditions (UI, ColdZyme/UI, SARS-CoV-2, ColdZyme/SARS-CoV-2) and plotted on a bar graph. Bars represent the mean + SD from 3 to 6 independent pseudostratified epithelia. Statistical significance was calculated using one-way ANOVA with Tukey’s multiple-comparison test. (b) Image analyses of ColdZyme/UI, ColdZyme/SARS-CoV-2, and SARS-CoV-2 primary HAE cells. Filters were differentially treated according to the labeling, fixed after 3 days, and stained using C3-FITC (green), SARS-CoV-2-S1 (red), phalloidin (orange), and Hoechst stain (blue). Image analyses revealed intact epithelial tissue structures (orange) and nuclei (blue) in ColdZyme/UI (upper panel) and ColdZyme/SARS-CoV-2-infected (middle panel) HAE, while tissue integrity was disrupted in SARS-CoV-2-infected cultures, which also illustrated high C3 and SARS-CoV-2-S1 staining (red and green, lower panel). Under each condition, one representative Z-stack (upper left), xyz stack (upper middle), 3D analyses of all stainings (lower left) and virus/C3 stainings (lower middle), and two imaged fields (right) are depicted. Infection experiments were performed three times independently. Scale bars represent 50 μm or 100 μm as indicated. (c) C3a level determination in SARS-CoV-2-infected or ColdZyme-pretreated/SARS-CoV-2-infected pseudostratified epithelia. Multilayered epithelia were pretreated or not with ColdZyme prior to infection with SARS-CoV-2 (MOI 0.1) for 72 h. Uninfected (UI) and ColdZyme/UI cultures served as controls. Basolateral supernatants were harvested, and C3a levels were determined using a BD OptEIA human C3a ELISA kit. C3a levels in ng/ml were determined for all UI and infected epithelia and plotted on a bar graph. Dots represent values from independent experiments. Statistical significance was calculated using one-way ANOVA with Tukey’s multiple-comparison test. (d) Spot analyses were performed on UI, ColdZyme/UI, SARS-CoV-2-infected, and ColdZyme/SARS-CoV-2-infected HAE cultures. SARS-CoV-2 spots (Alexa 594) were counted on an average of 1,200 cells (Hoechst, nuclear count; Alexa 647, cytoplasm) using the RMS spot analyses (Harmony software; Operetta CLS, Perkin-Elmer). Due to background spots in the UI and ColdZyme/UI cultures probably due to autofluorescence of dead cells in the 3D cultures, all conditions were normalized to UI. One out of three representative experiments was set as 1.0, and all other samples were normalized to this. The experiment was repeated at least 3 times, and one-way ANOVA with Tukey’s multiple-comparison test was used to calculate statistical significance. ns, not significant.

FIG 3

Increased infection of nasal epithelial cells using SARS-CoV-2 was impeded by pretreatment with ColdZyme. Visualization of SARS-CoV-2-infected pseudostratified nasal epithelia pretreated with ColdZyme mouth spray. Multilayered epithelia were pretreated for 30 min with ColdZyme sprayed apically and infected by apical addition of SARS-CoV-2 (MOI 0.1). d3pI cells were fixed and stained for Hoechst (blue), SARS-CoV-2-S1 (red), complement C3 (green), and phalloidin (orange) and then analyzed by HCS. The upper panel illustrates SARS-CoV-2-infected cultures, and the lower panel illustrates ColdZyme/SARS-CoV-2-infected cultures. Under each condition, one representative Z-stack (upper left), xyz stack (upper right), and 3D analyses of all stainings (lower left) and virus/C3 stainings (lower right) are depicted. Stainings were repeated thrice. Scale bars represent 50 μm.