Extracellular Vesicles of Giardia duodenalis: Unravelling Their Virulence Factors and Potential to Induce Protection Against Experimental Giardiasis.

Extracellular vesicles (EVs) are key mediators of intercellular communication, enabling trans-kingdom exchange of proteins, nucleic acids, lipids, and metabolites. In Giardia duodenalis, EVs contribute to parasite survival, host-pathogen interactions, and immune modulation. In this study, EVs were isolated from trophozoites grown in two serum-free media of distinct nutritional complexity (TYI-33 and DMEM) and characterized to define their molecular composition, pathogenic potential, and immunogenic properties. Nanoparticle tracking analysis showed vesicles predominantly 51-150 nm in diameter. Proteomic profiling identified 227 proteins in TYI-33 EVs and 97 in DMEM EVs, including molecules involved in metabolism, antioxidant defence, and virulence. Among these, giardipains 1-3, enolase, fructose-bisphosphate aldolase, ornithine carbamoyltransferase, and arginine deiminase were detected. TYI-33 EVs induced disruption of epithelial junctions and apoptotic-like changes in IEC-6 monolayers, consistent with contact-independent cytopathic activity. To assess their immunoprotective capacity, gerbils were intranasally immunized with TYI-33 EVs and challenged with G. duodenalis. Immunized animals showed significant reductions in intestinal trophozoite burden and faecal cyst shedding at 7, 14, and 21 days post-infection, accompanied by increased serum IL-17F and IL-22. Collectively, these findings indicate that Giardia EVs, while carrying virulence-associated factors, also possess strong immunogenic properties, supporting their potential as a novel mucosal vaccine platform against giardiasis.