Extracellular vesicles from adult Fasciola gigantica provide clues for new vaccine targets against fascioliasis

Abstract Background: Extracellular vesicles (EVs) released by helminths are important in parasite-host communication. However, little is known about the character and contents of Fasciola gigantica EVs. A better understanding of EVs released by F. gigantica will help reveal the mechanism of F. gigantica-host interaction and aid in the search for new vaccine candidates for fascioliasis control. Methods: We used ultracentrifugation to purify two different populations of EVs (15K EVs and 100K EVs) from adult F. gigantica culture media. The morphology and size of the purified EVs were determined by transmission electron microscopy (TEM) and ZETASIZER NANO ZSP. With the aim of identifying diagnostic markers or potential vaccine candidates, proteins within the isolated 100K EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS). Then, mice were vaccinated with ESPs, 15K EVs, 100K EVs, and rFg-HSP70 combined with alum adjuvant followed by challenge infection with F. gigantica metacercariae. Fluke recovery and antibody levels were used to evaluate vaccine protection. Results: TEM analysis and Nanoparticle tracking analysis indicated successful isolation of two subpopulations of EVs (15K EVs and 100K EVs) from adult F. gigantica culture supernatants using differential centrifugation. A total of 755 proteins were identified in the 100K EVs. Exosome biogenesis or vesicle trafficking proteins, ESCRT pathways proteins,as well as exosome markers, heat shock proteins and 14-3-3, were identified in the 100K EVs. These results indicate that the isolated 100K EVs were exosome-like vesicles. The functions of the identified proteins may be associated with immune regulation, immune evasion, and virulence. Mice immunized with F. gigantica ESP, 15K EVs, 100K EVs, and rFg-HSP70 exhibited a fluke burden reduction of 67.90%, 60.38%, 37.73%, and 56.6%, respectively, compared with the adjuvant control group. The vaccination of mice with F. gigantica 100K EVs, 15K EVs, ESP, and rFg-HSP70 induced significant production of specific IgG, IgG1, and IgG2a in sera. Conclusion: This study suggests that proteins within the exosome-like vesicles of F. gigantica have immunomodulatory, immune evasion, and virulence functions. This knowledge may lead to new strategies for immunotherapy, vaccination, and the diagnosis of fascioliasis.