Parasitology Research

, Volume 118, Issue 12, pp 3419–3427 | Cite as

Recombinant Opisthorchis viverrini tetraspanin expressed in Pichia pastoris as a potential vaccine candidate for opisthorchiasis

  • Luyen Thi Phung
  • Sujittra Chaiyadet
  • Nuttanan Hongsrichan
  • Javier Sotillo
  • Hang Dinh Thi Dieu
  • Canh Quang Tran
  • Paul J Brindley
  • Alex Loukas
  • Thewarach LahaEmail author
Helminthology - Original Paper


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.


Opisthorchis viverrini Tetraspanin Vaccination Pichia pastoris 


Funding information

This research was supported by Faculty of Medicine, Khon Kaen University, a project grant from the National Health and Medical Research Council of Australia (NHMRC), grant identification number APP1085309, and the National Cancer Institute, National Institute of Health, grant number 2R01CA164719-06A1. AL is supported by a senior principal research fellowship APP1117504.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

436_2019_6488_Fig5_ESM.png (1006 kb)
Supplemental Fig. 1

Recombinant form of Ov-LEL-TSP-2 expressed in P. pastoris. SDS-PAGE and Western blot analysis of samples from expression and purification of rOv-LEL-TSP-2 protein in P. pastoris. Protein marker (lane M), pre-induction supernatant (lane 1), methanol-induction supernatant (lane 2), flow through (lane 3), wash (lane 4), eluted protein (lane 5), purified protein in 20 mM HEPES (lane 6), western blot of rOv-LEL-TSP-2 probed with rabbit anti-Ov-TSP-2 serum (lane 7), western blot of 20 mM HEPES buffer probed with rabbit anti-Ov-TSP-2 serum-negative control (lane 8). The molecular weight of rOv-LEL-TSP-2 protein is approximately 12 kDa. (PNG 1006 kb)

436_2019_6488_MOESM1_ESM.tif (3.7 mb)
High Resolution (TIF 3770 kb)


  1. Boonmars T, Boonjaraspinyo S, Kaewsamut B (2009) Animal models for Opisthorchis viverrini infection. Parasitol Res 104(3):701–703. CrossRefPubMedGoogle Scholar
  2. Chaiyadet S, Sotillo J, Smout M, Cantacessi C, Jones MK, Johnson MS, Turnbull L, Whitchurch CB, Potriquet J, Laohaviroj M, Mulvenna J, Brindley PJ, Bethony JM, Laha T, Sripa B, Loukas A (2015) Carcinogenic liver fluke secretes extracellular vesicles that promote cholangiocytes to adopt a tumorigenic phenotype. J Infect Dis 212(10):1636–1645. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chaiyadet S, Krueajampa W, Hipkaeo W, Plosan Y, Piratae S, Sotillo J, Smout M, Sripa B, Brindley PJ, Loukas A, Laha T (2017a) Suppression of mRNAs encoding CD63 family tetraspanins from the carcinogenic liver fluke Opisthorchis viverrini results in distinct tegument phenotypes. Sci Rep 7(1):14342. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chaiyadet S, Smout M, Laha T, Sripa B, Loukas A, Sotillo J (2017b) Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells. Parasitol Int 66(4):494–502. CrossRefPubMedGoogle Scholar
  5. Chaiyadet S, Sotillo J, Krueajampa W, Thongsen S, Brindley PJ, Sripa B, Loukas A, Laha T (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 Negl Trop Dis 13(5):e0007450. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chavengkun W, Kompor P, Norkaew J et al (2016) Raw fish consuming behavior related to liver fluke infection among populations at risk of cholangiocarcinoma in Nakhon Ratchasima province, Thailand. Asian Pac J Cancer Prev 17(6):2761–2765PubMedGoogle Scholar
  7. Chen L, Chen Y, Zhang D, Hou M, Yang B, Zhang F, Zhang W, Luo X, Ji M, Wu G (2016) Protection and immunological study on two tetraspanin-derived vaccine candidates against schistosomiasis japonicum. Parasite Immunol 38(10):589–598. CrossRefPubMedGoogle Scholar
  8. Curti E, Kwityn C, Zhan B et al (2013) Expression at a 20L scale and purification of the extracellular domain of the Schistosoma mansoni TSP-2 recombinant protein: a vaccine candidate for human intestinal schistosomiasis. Hum Vaccin Immunother 9(11):2342–2350CrossRefGoogle Scholar
  9. Cwiklinski K, de la Torre-Escudero E, Trelis M, Bernal D, Dufresne PJ, Brennan GP, O'Neill S, Tort J, Paterson S, Marcilla A, Dalton JP, Robinson MW (2015) The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis. Mol Cell Proteomics 14(12):3258–3273. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dakshinamoorthy G, Munirathinam G, Stoicescu K, Reddy MV, Kalyanasundaram R (2013) Large extracellular loop of tetraspanin as a potential vaccine candidate for filariasis. PLoS One 8(10):e77394. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dang Z, Yagi K, Oku Y et al (2012) A pilot study on developing mucosal vaccine against alveolar echinococcosis (AE) using recombinant tetraspanin 3: vaccine efficacy and immunology. PLoS Negl Trop Dis 6(3):e1570. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dienz O, Eaton SM, Bond JP, Neveu W, Moquin D, Noubade R, Briso EM, Charland C, Leonard WJ, Ciliberto G, Teuscher C, Haynes L, Rincon M (2009) The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4+ T cells. J Exp Med 206(1):69–78. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Elkins DB, Haswell-Elkins M, Anderson RM (1986) The epidemiology and control of intestinal helminths in the Pulicat Lake region of Southern India. I. Study design and pre- and post-treatment observations on Ascaris lumbricoides infection. Trans R Soc Trop Med Hyg 80(5):774–792CrossRefGoogle Scholar
  14. Fantini MC, Dominitzki S, Rizzo A, Neurath MF, Becker C (2007) In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat Protoc 2(7):1789–1794. CrossRefPubMedGoogle Scholar
  15. Jittimanee J, Sermswan RW, Kaewraemruaen C et al (2012) Protective immunization of hamsters against Opisthorchis viverrini infection is associated with the reduction of TGF-beta expression. Acta Trop 122(2):189–195. CrossRefPubMedGoogle Scholar
  16. Khuntikeo N, Sithithaworn P, Loilom W et al (2016) Changing patterns of prevalence in Opisthorchis viverrini sensu lato infection in children and adolescents in northeast Thailand. Acta Trop 164:469–472. CrossRefPubMedGoogle Scholar
  17. Khuntikeo N, Titapun A, Loilome W, Yongvanit P, Thinkhamrop B, Chamadol N, Boonmars T, Nethanomsak T, Andrews RH, Petney TN, Sithithaworn P (2018) Current perspectives on opisthorchiasis control and cholangiocarcinoma detection in Southeast Asia. Front Med (Lausanne) 5:117. CrossRefGoogle Scholar
  18. Laha T, Pinlaor P, Mulvenna J, Sripa B, Sripa M, Smout MJ, Gasser RB, Brindley PJ, Loukas A (2007) Gene discovery for the carcinogenic human liver fluke, Opisthorchis viverrini. BMC Genomics 8:189. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. CrossRefGoogle Scholar
  20. Mulvenna J, Sripa B, Brindley PJ, Gorman J, Jones MK, Colgrave ML, Jones A, Nawaratna S, Laha T, Suttiprapa S, Smout MJ, Loukas A (2010) The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini. Proteomics 10(5):1063–1078. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Nowacki FC, Swain MT, Klychnikov OI, Niazi U, Ivens A, Quintana JF, Hensbergen PJ, Hokke CH, Buck AH, Hoffmann KF (2015) Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni. J Extracell Vesicles 4:28665. CrossRefPubMedGoogle Scholar
  22. Papatpremsiri A, Junpue P, Loukas A et al (2016) Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke. J Helminthol 90(1):39–47. CrossRefPubMedGoogle Scholar
  23. Pearson MS, Pickering DA, McSorley HJ, Bethony JM, Tribolet L, Dougall AM, Hotez PJ, Loukas A (2012) Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2. PLoS Negl Trop Dis 6(3):e1564. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Piratae S, Tesana S, Jones MK, Brindley PJ, Loukas A, Lovas E, Eursitthichai V, Sripa B, Thanasuwan S, Laha T (2012) Molecular characterization of a tetraspanin from the human liver fluke, Opisthorchis viverrini. PLoS Negl Trop Dis 6(12):e1939. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Saengsawang P, Promthet S, Bradshaw P (2016) Reinfection by Opisthorchis viverrini after treatment with praziquantel. Asian Pac J Cancer Prev 17(2):857–862CrossRefGoogle Scholar
  26. Sebina I, Fogg LG, James KR et al (2017) IL-6 promotes CD4(+) T-cell and B-cell activation during Plasmodium infection. Parasite Immunol 39(10). CrossRefGoogle Scholar
  27. Siegrist C-A (2018) Vaccine immunology. In: Plotkin SA, Orenstein WA, Offit PA, Edwards KM (eds) Plotkin’s caccines, 7th edn. Elsevier, Philadelphia, p 16–34.e7Google Scholar
  28. Sithithaworn P, Andrews RH, Nguyen VD, Wongsaroj T, Sinuon M, Odermatt P, Nawa Y, Liang S, Brindley PJ, Sripa B (2012) The current status of opisthorchiasis and clonorchiasis in the Mekong Basin. Parasitol Int 61(1):10–16. CrossRefPubMedGoogle Scholar
  29. Sotillo J, Pearson M, Potriquet J, Becker L, Pickering D, Mulvenna J, Loukas A (2016) Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates. Int J Parasitol 46(1):1–5. CrossRefPubMedGoogle Scholar
  30. Sripa B, Brindley PJ, Mulvenna J, Laha T, Smout MJ, Mairiang E, Bethony JM, Loukas A (2012a) The tumorigenic liver fluke Opisthorchis viverrini--multiple pathways to cancer. Trends Parasitol 28(10):395–407. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Sripa B, Thinkhamrop B, Mairiang E, Laha T, Kaewkes S, Sithithaworn P, Periago MV, Bhudhisawasdi V, Yonglitthipagon P, Mulvenna J, Brindley PJ, Loukas A, Bethony JM (2012b) Elevated plasma IL-6 associates with increased risk of advanced fibrosis and cholangiocarcinoma in individuals infected by Opisthorchis viverrini. PLoS Negl Trop Dis 6(5):e1654. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Tebeje BM, Harvie M, You H, Loukas A, McManus DP (2016) Schistosomiasis vaccines: where do we stand? Parasit Vectors 9(1):528. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Tran MH, Pearson MS, Bethony JM, Smyth DJ, Jones MK, Duke M, Don TA, McManus D, Correa-Oliveira R, Loukas A (2006) Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis. Nat Med 12(7):835–840. CrossRefPubMedGoogle Scholar
  34. Wang M, Jiang S, Wang Y (2016) Recent advances in the production of recombinant subunit vaccines in Pichia pastoris. Bioengineered 7(3):155–165. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Zhu L, Liu J, Dao J et al (2016) Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions. Sci Rep 6:25885. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Luyen Thi Phung
    • 1
    • 2
  • Sujittra Chaiyadet
    • 1
  • Nuttanan Hongsrichan
    • 1
  • Javier Sotillo
    • 3
    • 4
  • Hang Dinh Thi Dieu
    • 2
  • Canh Quang Tran
    • 2
  • Paul J Brindley
    • 5
  • Alex Loukas
    • 4
  • Thewarach Laha
    • 1
    Email author
  1. 1.Department of Parasitology, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand
  2. 2.Hai Duong Medical Technical UniversityHai Duong CityVietnam
  3. 3.Centro Nacional de MicrobiologíaInstituto de Salud Carlos IIIMadridSpain
  4. 4.Centre for Molecular Therapeutics, Australian Institute of Tropical Health and MedicineJames Cook UniversityCairnsAustralia
  5. 5.Department of Microbiology, Immunology and Tropical MedicineGeorge Washington UniversityWashingtonUSA

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