Chaiyadet, Sujittra, Sotillo, Javier, Krueajampa, Watchara, Thongsen, Sophita, Brindley, Paul J., Sripa, Banchob, Loukas, Alex, and Laha, Thewarach (2019) Vaccination of hamsters with Opisthorchis viverrini extracellular vesicles and vesicle-derived recombinant tetraspanins induces antibodies that block vesicle uptake by cholangiocytes and reduce parasite burden after challenge infection. PLoS Neglected Tropical Diseases, 13 (5). e0007450.

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Abstract

Background: The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model.

Methodology/Principal: findings EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection.

Conclusion/Significance: Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge.

Author summary: Cholangiocarcinoma (CCA) is a significant public health problem in countries throughout Southeast Asia. In these areas CCA has a strong association with chronic infection with the food-borne liver fluke Opisthorchis viverrini. Current control of the infection relies on chemotherapy and health education, however these approaches are not sustainable in isolation. Hence, there is an urgent need for a vaccine against this neglected tropical disease. A vaccine against O. viverrini would confer anti-cancer protection in similar fashion to the acclaimed vaccine for human papillomavirus and cervical cancer. Toward this goal, secreted extracellular vesicles (EVs) of O. viverrini and recombinant proteins from the surface of EVs were generated and tested as vaccines in a hamster challenge model. Vaccination of hamsters with EVs and recombinant proteins induced production of antibodies in serum and bile, and those antibodies blocked uptake of EVs by primary bile duct cells in vitro. Challenge of vaccinated hamsters with infective stage flukes markedly reduced adult fluke recovery compared to the adjuvant control group. This is the first report of successful vaccination of hamsters with O. viverrini EVs and recombinant vesicle surface proteins, and provides proof-of-concept for development of subunit vaccines for this carcinogenic infection.

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