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.