Utility of edible plant-derived exosome-like nanovesicles as a novel delivery platform for vaccine antigen delivery

Plant-derived exosome-like nanovesicles (PENVs) contain various biomolecules that can be used as delivery cargoes, such as small compounds, small interfering RNAs, DNAs, and recombinant proteins. Edible PENVs are nontoxic, useful for oral administration, and easily prepared in high amounts from a variety of plants, vegetables, and fruits. In this study, we evaluated whether PENVs can be used as delivery cargoes for recombinant vaccine antigens. Thus, we isolated PENVs from grapefruits and mandarin oranges by differential centrifugation and characterized their sizes and morphologies. Our results showed lipid bilayer morphologies of grapefruit-derived nanovesicles (GNVs) with the average size of 46 ± 8.5 nm and mandarin orange-derived NVs (MNVs) with the average size of 227 ± 6.4 nm. However, exposure to GNVs and MNVs did not cause cytotoxicity in Vero monkey kidneys or in MDCK canine kidney cells. To evaluate their utility as carriers of mRNA or recombinant proteins, GNVs and MNVs were loaded with GFP mRNA, Alexa Fluor 647-labeled heat shock protein 70 (AF-HSP70), or recombinant hepatitis B surface antigen (HBsAg). Our results showed that mRNA-GFP, human HSP70 protein, and HBsAg were efficiently loaded into both GNVs and MNVs. Interestingly, the oral administration of HBsAg-loaded GNVs to mice resulted in significantly higher levels of serum IgG than in the experimental control group (HBsAg without NVs). Therefore, our results report, for the first time, that edible PENVs loaded with HBsAg administered to mice via oral vaccination were effective in vaccine development.