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