GlyPerA™ mouth spray maintains epithelial integrity upon SARS-CoV-2 infection of NHBE cultures

To monitor, if single administration of the GlyPerA™ solution, which can be used as mouth- or nose spray, protects respiratory tissues from SARS-CoV-2-mediated destruction over time, NHBE cultures were kept in culture for 2 days following treatment and infection. The solution was either added pure (GlyPerA pure) or diluted 1/100 (GlyPerA dil.) 5 min prior infection with BA.1 or BA.2 at an MOI 0.005. The diluted solution was further added simultaneous to infection (GlyPerA dil. SIM Omicron) or 45 min post infection (GlyPerA dil. POST Omicron) with Omicron. Analysis of transepithelial electrical resistance (TEER; Fig. 1a), an indicator for tissue integrity, was performed on 1 dpi and 2 dpi. These analyses revealed that upon Omicron infection, TEER values slightly dropped 1 dpi (left) and significantly decreased 2 dpi (right) compared to GlyPerA-treated UI controls (UI; Fig. 1a). This drop in TEER was also consistent with tissue destruction on 2 dpi analyzed by immunofluorescence analyses (Fig. 1a, lower right; Additional file 1: Fig. S1, right) and LDH from basolateral supernatants as cytotoxicity measure (Additional file 1: Fig. S1, left). While in GlyPerA-treated UI controls, an intact ciliated layer (red) with mucus-producing goblet cells (orange) was observed, Omicron-infected tissues illustrated porous, infected (green) tissue structures (Fig. 1a, lower right). Nuclei were stained using Hoechst (Fig. 1a, lower right, blue). Moreover, occludin staining revealed fully intact tight junctions in GlyPerA-treated UI controls, while these were lost in Omicron-infected tissue models on 2 dpi (Additional file 1: Fig. S1, occludin in blue, right). GlyPerA treatment significantly rescued epithelial integrity on 2 dpi (right) independent on the concentration of GlyPerA (pure vs. diluted) and time of administration (pre, sim, post) in infected cultures (Fig. 1a). TEER values of GlyPerA-treated and Omicron-infected tissues were in the range of the mock-treated, uninfected control (Fig. 1a). Thus, tissue integrity was greatly rescued in infection experiments on both days analyzed post infection, if GlyPerA™ solution was administered prior, simultaneous to and post SARS-CoV-2 exposure only once.

Fig. 1

Disruption of epithelial integrity by Omicron BA.1 or BA.2 can be avoided by GlyPerA™ treatment. a Pseudostratified epithelia were infected by apical addition of SARS-CoV-2 BA.1 and BA.2 with or without GlyPerA™ treatment and incubated for 48 h. GlyPerA™ solution was added pure or diluted (1/100) 5 min prior infection (GlyPerA pure PRE Omicron, GlyPerA dil. PRE Omicron), and the diluted solution in addition simultaneous with (GlyPerA dil. SIM Omicron) or 45 min post (GlyPerA dil. POST Omicron) infection. TEER was measured on 1 dpi (left) and 2 dpi (right) using a EVOM volt-ohm-meter. TEER in Ω/cm2 was determined for all conditions (UI, Omicron, GlyPerA pure PRE Omicron, GlyPerA dil. PRE Omicron, GlyPerA dil. SIM Omicron, GlyPerA dil. POST Omicron) and plotted on a bar graph. Bars represent the mean + SD from 3 independent pseudostratified epithelia measured in triplicates. Statistical significance was calculated using One-way ANOVA with Tukey’s multiple comparisons test. To further validate the drop in TEER, immunofluorescence analyses were performed to stain for the tissue integrity. For this, 2 dpi tissue models were stained using Hoechst (nuclei, blue), acetylated tubulin (cilia, red), MUC5AC (mucus-producing cells, orange) and virus (SARS-CoV-2, green). b Viral RNA was measured from UI, Omicron, GlyPerA pure PRE Omicron, GlyPerA dil. PRE Omicron, GlyPerA dil. SIM Omicron, GlyPerA dil. POST Omicron cultures of pseudostratified epithelia on 3 dpi. The experiment was repeated at least 3 times and statistically significantl differences were determined by one-way ANOVA with Tukey´s multiple comparisons test. All values are means ± SD. In a and b *P < 0.05, **P < 0.01, ***P < 0.001

GlyPer™ addition significantly lowers SARS-CoV-2 viral loads compared to infected controls independent on the time of administration

In accordance to TEER, absolute quantification of viral load from 3 dpi subnatants of differently treated cells revealed protection from infection by applying the GlyPer™ solution (Fig. 1b). The protection from viral infection was independent on whether the GlyPer™ formulation was added shortly prior infection (GlyPerA pure PRE and GlyPerA dil. PRE Omicron), simultaneously (GlyPerA dil. SIM Omicron) or 45 min post infection with BA.1 or BA.2 (GlyPerA dil. POST Omicron) (Fig. 1b). Viral copy numbers in GlyPerA-treated and infected subnatants were slightly higher than background levels of uninfected (UI) and significantly lower compared to Omicron-infected (Omicron) cultures (Fig. 1b). Here, we demonstrated that single application of GlyPerA™ solution blocked SARS-CoV-2 infection of HAE cultures by significantly reducing virus release independent on the time of administration.

GlyPerA™ protects from SARS-CoV-2 infection and intracellular (IC) C3 activation in primary HAE cells

Lastly, we monitored primary NHBE cell infection and inflammation after applying SARS-CoV-2 variants of concern (VOCs) Omicron BA.1 and BA.2 in absence and presence of GlyPerA™ solution by imaging analyses. In these experiments, a multiplicity of infection (MOI) of 0.005 was used to determine morphogenesis, inflammation (IC C3 activation) and cytopathic effects of SARS-CoV-2 infection in human airway epithelial cells.

GlyPerA™ solution was carefully added onto the apical side of fully differentiated, pseudostratified epithelia cultured at air–liquid interphase as described by us [3,4,5, 8, 9] to realistically mimic the distribution within the oral or nasal cavity. Epithelia were incubated for 5 min with diluted GlyPerA™ solution prior applying clinical isolates derived from BA.1- or BA.2-infected individuals or the solution was added simultaneously with the viral preparations. The clinical specimen were anonymized before use and the study has been approved by the ethics committee of the Medical University of Innsbruck (approval no. ECS1166/2020). Uninfected tissues treated with GlyPerA™ solution alone (UI) served as control in all imaging experiments. After 3 days post infection (3 dpi), tissue models were fixed and stained for immunofluorescence (IF) analyses using Alexa594-labeled antibodies against the SARS-CoV-2 spike 1 (S1) and nucleocapsid (N) proteins (Fig. 2a and Additional file 2: Videos S1a and Additional file 3: S1b, orange, SARS-CoV-2 (N/S)) to detect virus, Hoechst for nuclei (Fig. 2a and Additional file 2: Videos S1a and Additional file 3: Video S1b, blue), acetylated tubulin for cilia (Fig. 2a and Additional file 2: Video S1a and Additional file 3: Video S1b, red), and complement component C3 as marker for innate immune activation of NHBE cells (Fig. 2a and Videos S1a and Additional file 3: Video S1b, green).

Fig. 2

GlyPerA™ shields primary HAE cells from SARS-CoV-2 Omicron BA.1 and BA.2 infection and innate immune activation. Visualization of virus binding (SARS-CoV-2 S1/N, orange) and complement (C3-FITC, green) in SARS-CoV-2 infected 3D pseudostratified epithelia. Pseudostratified epithelia were apically treated with GlyPerA™ prior exposure to SARS-CoV-2. On 3 dpi, filters were fixed, stained for höchst (blue), SARS-CoV-2 S1/N (orange), complement C3 (green) and acetylated tubulin (red) and then analysed by HCS. a XYZ-stacks of uninfected (UI, panel 1), BA.2-infected (panel 2), GlyPerA™-pre-treated and BA.2-infected (panel 3) HAE cultures were analyzed using the Operetta CLS HCS and the 63XWATER objective. Cells were stained using C3-FITC (green) as indicator for innate immune activation, SARS-CoV-2-S1/N-Alexa594 (orange) for virus detection, höchst for imaging nuclei (blue) and acetylated tubulin for staining cilia (red). High IC C3 mobilization was monitored in BA.2-infected cultures, while no virus and low C3 signals were detected in UI (panel 1) and GlyPerA™/BA.2- (panel 3) cultures. Scale bars represent 50 µm (XY) and 20 µm (YZ, XZ), respectively. Three independent experiments were performed. b Numbers of nuclei per condition (UI, Omicron, GlyPerA dil. PRE Omicron, GlyPerA dil. SIM Omicron), viral signals and c areas of C3 production were absolutely quantified from at least three different areas using the Harmony™ 4.9 software. Statistical significances were analyzed with GraphPad Prism software using One-way ANOVA and Tukey’s post test. In b and c **P < 0.01, ***P < 0.001, ****P < 0.0001

We recently found that infection in primary airway epithelial cells was escorted by massive intracellular (IC) C3 mobilization and secretion of the anaphylatoxin C3a from HAE cells [4]. Thus, we used IC C3 as marker for innate immune activation during infection of NHBE cells with BA.1 and BA.2 in absence and presence of GlyPerA™.

Z-stack analyses of Omicron (BA.1 or BA.2)-infected NHBE cells treated with GlyPerA™ revealed an intact, pseudostratified epithelium with no detectable virus or virus-infected cells (Fig. 2a, right panels, and Video S1b). In contrast, epithelia infected with Omicron BA.1 or BA.2 illustrated extensive destruction of epithelia with virus-infected, inflamed cells (Fig. 2a, middle panels, and Video S1a, green, orange). GlyPerA-mock treated UI cells served as controls for intact epithelia; these showed the most intact cilia layer as well as the lowest IC C3 activation (Fig. 2a, left panel).

When absolutely quantifying nuclei in more than 1500 cells per condition, we found that Omicron infection significantly decreased the nuclei count compared to UI on 3 dpi (Fig. 2b, left). GlyPerA-treatment prior to or simultaneous with BA.1 or BA.2 significantly rescued cell numbers compared to infected cultures, but were decreased in comparison to UI (Fig. 2b, left). Moreover, GlyPerA treatment significantly rescued from infection compared to infected tissues independent on the time of addition (pre, sim), when viral particles were counted (Fig. 2b, right). These analyses are in accordance to absolutely quantified viral copy numbers using RT-PCR (Fig. 1b).

Lastly, Omicron infection was associated with concomitant activation of IC C3 (green) (Fig. 2a, middle panels, and Additional file 2: Video S1a), while the IC C3 mobilization was significantly decreased, when tissue samples were pretreated with GlyPerA solution (Fig. 2a, right panels, Fig. 2c, Additional file 3: Video S1b) or when the solution was added simultaneously (Fig. 2c).

Upon quantifying C3 signals from more than 1000 cells/condition in UI, Omicron- and GlyPerA/Omicron-exposed, pseudostratified respiratory epithelia, we found highly significant differences in percentages of C3+ areas (Fig. 2c) These analyses demonstrate that infection going along with tissue destruction and intracellular C3 mobilization on 3 dpi induced in NHBE cultures upon SARS-CoV-2 interactions can be avoided by pre-treatment with or simultaneous addition of GlyPerA™ solution to epithelia.