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Inter-phylum transfer of molecular information is exquisitely exemplified in the uptake of parasite extracellular vesicles (EVs) by their target mammalian host tissues. The oriental liver fluke, Opisthorchis viverrini is the major cause of bile duct cancer in people in Southeast Asia. A major mechanism by which O. viverrini promotes cancer is through the secretion of excretory/secretory products which contain extracellular vesicles (Ov EVs). Ov EVs contain microRNAs that are predicted to impact various mammalian cell proliferation pathways, and are internalized by cholangiocytes that line the bile ducts. Upon uptake, Ov EVs drive relentless proliferation of cholangiocytes and promote a tumorigenic environment, but the underlying mechanisms of this process are unknown. Moreover, purification and characterization methods for helminth EVs in general are ill defined. We therefore compared different purification methods for Ov EVs and characterized the sub-vesicular compartment proteomes. Two CD63-like tetraspanins (Ov -TSP-2 and TSP-3) are abundant on the surface of Ov EVs, and could serve as biomarkers for these parasite vesicles. Anti-TSP-2 and -TSP-3 IgG, as well as different endocytosis pathway inhibitors significantly reduced Ov EV uptake and subsequent proliferation of cholangiocytes in vitro. Silencing of Ov-tsp-2 and tsp- 3 gene expression in adult flukes using RNA interference resulted in substantial reductions in Ov EV secretion, and those vesicles that were secreted were deficient in their respective TSP proteins. Our findings shed light on the importance of tetraspanins in fluke EV biogenesis and/or stability, and provide a conceivable mechanism for the efficacy of anti-tetraspanin subunit vaccines against a range of parasitic helminth infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The human liver fluke, Opisthorchis viverrini infects several million people in Thailand. Chronic opisthorchiasis combined with nitrosamine ingested with food frequently leads bile duct cancer, cholangiocarcinoma (CCA). Repeated infection by O. viverrini increases hepatobiliary disease as well as increases the risk of CCA. Praziquantel (PZQ) is the drug of choice for treatment of opisthorchiasis. Whereas PZQ is considered safe to use, recent reports have cautioned that repeated use might influence the risk of liver fluke infectioninduced CCA. Therefore, the efficacy of repeated treatment with PZQ warranted evaluation. Here the efficacy of PZQ treatment was investigated in hamsters repeatedly infected with O. viverrini and in hamsters both infected with O. viverrini and exposed to the carcinogen dimethylnitrosamine (DMN). Experimental hamsters were infected with O. viverrini and treated two times with 300 mg/kg of PZQ. Other hamsters were infected with O. viverrini as well as provided with drinking water that contained 12.5 ppm DMN, following by two treatments with PZQ. As controls, uninfected hamsters, uninfected hamsters treated with PZQ, and uninfected hamsters treated with both PZQ with DMN were included. Histopathology of hepatobiliary tissues was examined, specifically for infiltration of white blood cells, proliferation of cholangiocytes and fibrosis. The pathology including inflammation, bile duct cell proliferation and liver fibrosis decreased significantly by one week following treatment with PZQ. Following repeated infection with O. viverrini and exposure to DMN, and treatment with PZQ, disease was less marked compared to hamsters infected twice with O. viverrini but without treatment with PZQ. Severe disease manifested in hamsters with repeated liver fluke infection without PZQ treatment. Differences in pathology were not evident among the control hamsters – noninfected hamsters, uninfected hamster receiving PZQ only and uninfected hamsters treated with both PZQ with DMN. These findings confirmed the efficacy of PZQ for treatment of opisthorchiasis. [ABSTRACT FROM AUTHOR]