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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 human liver fluke Opisthorchis viverrini is a food-borne trematode that causes hepatobiliary disease in humans throughout Southeast Asia. People become infected by consuming raw or undercooked fish containing metacercariae. Development of a vaccine to prevent or minimize pathology would decrease the risk of severe morbidity, including the development of bile duct cancer.Methods: We produced an oral vaccine based on recombinant Bacillus subtilis spores expressing the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (Ov-TSP-2), a protein that is abundant on the surface of O. viverrini secreted extracellular vesicles (EVs). Recombinant spores expressing Ov-TSP-2-LEL were orally administered to hamsters prior to challenge infection with O. viverrini metacercariae.Results: Vaccinated hamsters generated serum IgG as well as bile IgG and IgA responses to Ov-TSP-2-LEL, and serum IgG from vaccinated hamsters blocked the uptake of fluke EVs by a human bile duct epithelial cell line. Vaccinated hamsters had 56% reductions in both adult flukes and fecal eggs compared to the control group.Conclusions: These findings indicate that oral vaccination of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL is efficacious at reducing infection intensity and could form the basis of a vaccine for control of carcinogenic liver fluke infection in humans. [ABSTRACT FROM AUTHOR]

Opisthorchiasis affects millions of people in Southeast Asia and has been strongly associated with bile duct cancer. Current strategic control approaches such as chemotherapy and health education are not sustainable, and a prophylactic vaccine would be a major advance in the prevention of the disease. Tetraspanins are transmembrane proteins previously described as potential vaccine candidates for other helminth infections and are also found in the membranes of the tegument and extracellular vesicles of O. viverrini. Here, we investigated the potential of a recombinant protein encoding for the large extracellular loop of O. viverrini tetraspanin-2 (rOv-LEL-TSP-2) in a hamster vaccination model. Hamsters were vaccinated with 50 and 100 μg of rOv-LEL-TSP-2 produced from Pichia pastoris yeast combined with alum CpG adjuvant via the intraperitoneal route. The number of worms recovered from hamsters vaccinated with rOv-LEL-TSP-2 was significantly reduced compared to adjuvant control groups. Fecal egg output was also significantly reduced in vaccinated animals, and the average length of worms recovered from vaccinated animals was significantly shorter than that of the control group. Vaccinated animals showed significantly increased levels of anti-rOv-TSP-2 IgG in the sera after three immunizations, as well as increased levels of several T helper type 1 cytokines in the spleen including IFN-γ and IL-6 but not the Th2/regulatory cytokines IL-4 or IL-10. These results suggest that rOv-TSP-2 could be a potential vaccine against opisthorchiasis and warrants further exploration. [ABSTRACT FROM AUTHOR]

Background: The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens. Methodology/Principal Findings: The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Conclusions/Significance: This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed. Author Summary: Liver fluke infection is a fish borne disease that afflicts millions of residents in Thailand and Laos. Infection results from eating undercooked freshwater fish contaminated with larvae of the worm Opisthorchis viverrini. Infection can lead to cancer of the bile ducts (cholangiocarcinoma). Indeed, O. viverrini is designated as a Group 1 carcinogen by the World Health Organization, i.e. a definitive cause for cancer. Proteins produced at the surface and/or released from this parasite play pivotal roles in maintaining the infection and disease. These proteins are valid targets for development of vaccines and new drugs. Tetraspanins are prominent in the tegument (the surface covering) of parasites closely related to O. viverrini where they are exposed to immune responses. Similar molecules on the surface of O. viverrini may be vital for the parasite's survival and may make effective vaccines. Here the gene coding for O. viverrini tetraspanin-1 (Ov-TSP-1) was investigated. We used electron microscopy to show that Ov-TSP-1 is expressed in the tegument. We then silenced expression of the gene encoding Ov-TSP-1 and showed that this resulted in malformation of the tegument, highlighting the importance of this molecule for parasite development and its potential as a vaccine target. [ABSTRACT FROM AUTHOR]