Fig. 3. Design and characterization of the flexible OMV-based antigen display platform-antigen presentation on BMDCs and enrichment of antigens and adjuvants in lymph nodes.

a Schematic illustration of ClyA-Catcher (CC) OMVs system for antigen display. SpyCatcher (SpC) and SnoopCatcher (SnC) were expressed as fusion proteins with ClyA (ClyA-Catcher, CC) on the OMVs surface. SpyTag (SpT) or SnoopTag (SnT)-labeled antigens bind to CC OMVs through isopeptide bond formation between the tag and catcher. b TEM and DLS analysis of CC OMVs. Scale bar, 100 nm. c, d The conjugation of SpT-HA (c) or SnT-HA (d) to ClyA-Catchers on the CC OMVs surface was verified by western blot analysis using an anti-HA antibody. e The simultaneous display of two Catcher/Tag pairs by the same OMVs. SpT-Cys- and SnT-Cys labeled with 5 (white arrow) and 10 nm (red arrow) gold nanoparticles, respectively, were used to identify SpC and SnC on CC OMVs. Scale bar, 20 nm. f Confocal microscopy images of antigen presentation by BMDCs incubated with the indicated formulations for 12 h. The cell nuclei were stained blue (DAPI), and MHCI-OVA complexes were stained green (PE-anti-mouse H-2Kb bound to SIINFEKL) (n = 3). BF bright field. Scale bar, 50 µm. g Confocal microscopy images of antigen uptake by BMDCs after incubation with the indicated OMVs formulations for 12 h. The cell nuclei were stained blue (DAPI), and the antigen was labeled with Cy5.5 (red) (n = 3). Scale bar, 50 µm. h, i Lymph node accumulation of OMVs in vivo (n = 3). Various organs and the inguinal draining lymph nodes of mice were collected 12 h after s.c. immunization with the indicated OMVs formulations to examine the accumulation of Cy5.5 fluorescence (h). Frozen sections of the lymph nodes were prepared and examined by fluorescence microscopy (i). Cell nuclei were stained blue (DAPI), and the antigen was labeled with Cy5.5 (red). Scale bar, 1 mm. Source data are provided as a Source Data file.