Fig 2: Design and purification of RBD-nanoparticleA. Computer-assisted modeling of RBD-nanoparticle based on previously solved structures of H. pylori ferritin (PDB: 3EGM) and SARS-CoV-2 RBD (PDB: 7JMP). RBD forms radial projections on threefold axis point of fully assembled nanoparticle.B. Coomassie staining of purified ferritin-nanoparticle and RBD-nanoparticle following SDS-PAGE.C. Size exclusion chromatography peaks of the concentrated supernatants from HEK293T transfected with plasmids encoding secreted ferritin-nanoparticle or RBD-nanoparticles. The supernatants were concentrated with 100 kDa MWCO and 500 kDa MWCO filters on TFF system and loaded to Superdex 200 Increase 10/300 GL and HiPrep 16/60 Sephacryl S-500 HR gel filtration columns on Bio-rad NGC chromatography system, respecitvely.

Fig 3: Molecular design and biochemical and antigenic characterization of ferritin nanoparticles (FT) and DBV Gn Head-ferritin (GnH-FT) nanoparticles(A) Schematic representation of GnH-FT based on previously solved structures and domains of DBV Gn and Gc. The construct was transfected to HEK293T cells to collect the cell supernatant at 72 hours after transfection for purification. (SP: signal peptide, TM: transmembrane domain) (B, C) Size exclusion chromatograms to purify FT (B) and GnH-FT (C) using Superdex 200. Increase 10/300 GL and Superose6 Increase 10/300 GL columns, respectively, on Bio-Rad NGC chromatography system. Fractions corresponding to the colored arrows were separately collected for further analyses.(D, E) Fractions from size exclusion chromatograms of FT and GnH-FT were further analyzed by gradient (7% - 20%) SDS-PAGE and Coomassie Brilliant blue staining. Fractions corresponding to the black and red arrows from FT and GnH-FT purifications were loaded to SDS-PAGE gel without boiling (“NB”) and with boiling (“B”) to characterize head-mediated disassembly of the 24-mer nanoparticle. Intact FT nanoparticle and GnH-FT nanoparticle each has expected molecular weight of approximately 432 kDa and 1,560 kDa. Dissembled FT and GnH-FT monomer each has expected molecular weight of 18 kDa and 65 kDa.(F) Western-blot analysis of different fractions collected from GnH-FT size exclusion chromatogram. In house-generated mouse monoclonal antibody recognizing DBV Gn Head region was used to detect GnH-FT subunit monomers.