Fig. 5: The CCD/RBD-HR vaccine targets nasal epithelial cells.

a Representative immunofluorescent images of nasal mucosa harvested from BALB/c mice (n = 3) immunized with PBS or RBD-HR (green) with or without CCD intranasally 24 h before. Blue: DAPI. Scale bars: 100 μm. b Left: BALB/c mice (n = 3) were immunized with PBS or fluorescently labeled RBD-HR with or without CCD intranasally. 24 h later, the nasal mucosa was harvested, and the uptake of RBD-HR by primary NECs (EpCAM⁺) was assessed with FCM. Right: RBD-HR uptake by isolated primary NECs ex vivo after incubation with RBD-HR with or without CCD for 30 min. c Maturation of mouse NECs 24 h after immunization as determined by the expression of CD40, CD80, CD86, and MHC II using FCM. n = 3 mice per group. d A mouse NEC cell line was stimulated with PBS, RBD-HR, and CCD/RBD-HR for 24 h, and the expression of maturation markers was examined. e Mouse NECs were isolated and incubated with PBS or fluorescently labeled RBD-HR (green) with or without CCD for 24 h. Confocal microscopy was used to image the localization of RBD-HR (green) and MHC II (red) on primary NECs. Blue: DAPI. Scale bars: 10 μm. Similar results were obtained in three independent experiments. f Concentration of IL-6 in the supernatant of the stimulated NEC cell line at 24 h. g, h Mouse primary NECs were isolated and stimulated with PBS, RBD-HR, or CCD/RBD-HR in the presence or absence of anti-MHC II antibodies for 24 h, followed by co-culture with splenic T cells. 48 h later, fractions of CD4⁺CD44⁺ and CD4⁺CD69⁺ T cells were assessed with FCM. Data were displayed with floating bars in (b–d) and (f–h). The middle line indicates the median and the box shows the data range. Data were presented as mean values ± SEM of three (n = 3) in b, g, and four (n = 4) independent experiments in (d, f, h). P values were calculated with One-way ANOVA followed by Dunnett’s multiple comparisons tests. Source data are provided as a Source Data file.