This is the first study to determine the vaccine efficacy of Ov-EVs or the recombinant tetraspanins rOv-TSP-2 and rOv-TSP-3 against a challenge infection with metacercariae of O. viverrini in hamsters, and it supports the notion that targeting interactions between fluke EVs and their mammalian host cells holds promise for the discovery of novel vaccine candidates. Here, THBS2 is linked to infection.