Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2143–2154 | Cite as

Immunological features and efficacy of the recombinant subunit vaccine LTB-EMY162 against Echinococcus multilocularis metacestode

Biotechnological products and process engineering


Alveolar echinococcosis is a zoonotic disease caused by the infection of the larval stage Echinococcus multilocularis with worldwide distribution especially in the northwest China. It is important to develop a well-tolerated immunoprophylaxis against E. multilocularis for alveolar echinococcosis control. In this study, a prokaryotic expression system for recombinant immunogen LTB-EMY162 was established, and the immunological features, sensitized lymphocyte, IL-4/IFN-γ secreted, prophylactic effect, and therapeutic effect were also evaluated. Arctic Express (DE3) system, Ni2+-charged and molecular sieve chromatography were used to obtain a high-purity 29 kDa protein. The ELISA and lymphocyte proliferation assay showed that LTB-EMY162 induced high-titer specific IgG against EMY162 and E. multilocularis protoscoleces protein in BALB/c mice and promoted sensitized T lymphocyte cell proliferation, and LTB-EMY162 stimulated Th cell to secrete IL-4 and IFN-γ and induced a Th1/Th2 mixed type immunological response. We also found that LTB-EMY162 significantly inhibited the cysts formation by challenging with 1000 E. multilocularis protoscoleces. The growth of protoscoleces and cysts were also significantly decreased by treating with LTB-EMY162 in 1000 protoscoleces intraperitoneal injection therapeutic mice model. In conclusion, we have constructed a subunit vaccine LTB-EMY162 which has prevention and therapeutic effect against E. multilocularis infection.


Echinococcus multilocularis Alveolar echinococcosis Subunit vaccine EMY162 



We thank all of the staffs for their helpful suggestions and advice during this project. We would like to thank the editor for the helpful comments on the manuscript.


This work was funded by National Natural Science Foundation of China (No.81360300, Tang F; No. 32571231, Ge RL), the Province Natural Science Foundation of Qinghai (No. 2015-ZJ-745), the 123Talent Project of Qinghai University, the Key Project of Qinghai Provincial Health and Family Planning Commission (No. 2016-wjzd-04), the National Basic Research Program of China (No. 2012CB518200, L-RG), and the Science and technology project of Qinghai Agriculture and Animal Husbandry Bureau (NMSY-2017-09). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All experiments were conducted with adherence to the NIH Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Ethical and Experimental Committee of Qinghai University (QHDX-2014-07). This article does not contain any studies with human participants performed by any of the authors.


  1. Batçık OE, A Ö KO, Ekşi MŞ, Bilge T (2016) Cerebral alveolar echinococcosis concomitant with liver and lung lesions in a young adult patient: case report and literature review. Turkiye Parazitol Derg:169–171Google Scholar
  2. Boubaker G, Hemphill A, Huber CO, Spiliotis M, Babba H, Gottstein B (2015) Prevention and immunotherapy of secondary murine alveolar echinococcosis employing recombinant EmP29 antigen. PLoS Negl Trop Dis 9(6):e0003795. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Capron M, Capron A, Torpier G, Bazin H, Bout D, Joseph M (1978) Eosinophil-dependent cytotoxicity in rat schistosomiasis. Involvement of IgG2a antibody and role of mast cells. Eur J Immunol 8(2):127–133. CrossRefPubMedGoogle Scholar
  4. Clements DA, Gilbert GL (1990) Immunisation for the prevention of Haemophilus influenzae type b infections: a review. Aust NZ J Med 20(6):828–834. CrossRefGoogle Scholar
  5. Dang Z, Yagi K, Oku Y, Kouguchi H, Kajino K, Watanabe J, Matsumoto J, Nakao R, Wakaguri H, Toyoda A (2009) Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis. Vaccine 27(52):7339–7345. CrossRefPubMedGoogle Scholar
  6. Emery I, Liance M, Leclerc C (1997) Secondary Echinococcus multilocularis infection in A/J mice: delayed metacestode development is associated with Th1 cytokine production. Parasite Immunol 19(11):493–503CrossRefPubMedGoogle Scholar
  7. Giraudoux P, Raoul F, Afonso E, Ziadinov I, Yang Y, Li LI, Tiaoying LI, Quéré J, Feng X, Wang Q (2013) Transmission ecosystems of Echinococcus multilocularis in China and Central Asia. Parasitology 140(13 (control of cestode zoonoses in Asia: role of basic and applied science)):1655–1666CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gottstein B, Müller N, Jr SJC, Vogel M, Tanner I, Seebeck T (1990) Humoral and cellular immune response in mice and dogs induced by a recombinant Echinococcus multilocularis antigen produced by a Salmonella typhimurium vaccine strain. Parasite Immunol 12(2):163–174. CrossRefPubMedGoogle Scholar
  9. Guo L, Yin R, Liu K, Lv X, Li Y, Duan X, Chu Y, Xi T, Xing Y (2014) Immunological features and efficacy of a multi-epitope vaccine CTB-UE against H. pylori in BALB/c mice model. Appl Microbiol Biotechnol 98(8):3495–3507CrossRefPubMedGoogle Scholar
  10. Hemphill A, Muller J (2009) Alveolar and cystic echinococcosis: towards novel chemotherapeutical treatment options. J Helminthol 83(2):99–111. CrossRefPubMedGoogle Scholar
  11. Ishizuka EK, Ferreira MJ, Grund LZ, Coutinho EMM, Komegae EN, Cassado AA, Bortoluci KR, Lopes-Ferreira M, Lima C (2012) Role of interplay between IL-4 and IFN-γ in the in regulating M1 macrophage polarization induced by Nattectin. Int Immunopharmacol 14(4):513–522. CrossRefPubMedGoogle Scholar
  12. Joliat GR, Melloul E, Petermann D, Demartines N, Gillet M, Uldry E, Halkic N (2015) Outcomes after liver resection for hepatic alveolar echinococcosis: a single-center cohort study. World J Surg 39(10):2529CrossRefPubMedGoogle Scholar
  13. Kamishima T, Harabayashi T, Ishikawa S, Kubota KC, Nonomura K, Omatsu T, Onodera Y, Shirato H, Terae S (2009) Alveolar hydatid disease of the adrenal gland: computed tomography and magnetic resonance imaging findings. Jpn J Radiol 27(5):225–228. CrossRefPubMedGoogle Scholar
  14. Katoh Y, Kouguchi H, Matsumoto J, Goto A, Suzuki T, Oku Y, Yagi K (2008) Characterization of em Y162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis. Biochim Biophys Acta 1780(1):1–6CrossRefPubMedGoogle Scholar
  15. Khusmith S, Panitchakorn J, Krudsood S, Wilairatana P, Looareesuwan S (2001) IgE elevation and anti-plasmodium falciparum IgE antibodies: association of high level with malaria resistance. Southeast Asian J Trop Med Public Health 32(4):696–701PubMedGoogle Scholar
  16. King CL, Ottesen EA, Nutman TB (1990) Cytokine regulation of antigen-driven immunoglobulin production in filarial parasite infections in humans. J Clin Investig 85(6):1810–1815. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kizaki T, Kobayashi S, Ogasawara K, Day NK, Good RA, Onoé K (1991) Immune suppression induced by protoscoleces of Echinococcus multilocularis in mice. Evidence for the presence of CD8dull suppressor cells in spleens of mice intraperitoneally infected with E. multilocularis. J Immunol 147(5):1659PubMedGoogle Scholar
  18. Klaver AC, Patrias LM, Coffey MP, Finke JM, Loeffler DA (2010) Measurement of anti-Abeta1-42 antibodies in intravenous immunoglobulin with indirect ELISA: the problem of nonspecific binding. J Neurosci Methods 187(2):263–269. CrossRefPubMedGoogle Scholar
  19. Kouguchi H, Matsumoto J, Katoh Y, Oku Y, Suzuki T, Yagi K (2007) The vaccination potential of EMY162 antigen against Echinococcus multilocularis infection. Biochem Biophys Res Commun 363(4):915–920. CrossRefPubMedGoogle Scholar
  20. Kouguchi H, Matsumoto J, Nakao R, Yamano K, Oku Y, Yagi K (2013) Characterization of a surface glycoprotein from Echinococcus multilocularis and its mucosal vaccine potential in dogs. PLoS One 8(7):e69821. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Le G, Li X, Feng T, He Y, Xing Y, Deng X, Tao X (2012) Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from Helicobacter pylori urease a subunit. Appl Microbiol Biotechnol 93(5):1937–1945CrossRefGoogle Scholar
  22. Moro P, Schantz PM (2009) Echinococcosis: a review. Int J Infect Dis 13(2):125–133. CrossRefPubMedGoogle Scholar
  23. Nail LC, Reimundes ER, Galluzzo CW, Dan L, Ibrahim YL, Lobrinus JA, Chappuis F (2017) Disseminated alveolar echinococcosis resembling metastatic malignancy: a case report. J Med Case Rep 11(1):113. CrossRefGoogle Scholar
  24. Nunnari G, Pinzone MR, Gruttadauria S, Celesia BM, Madeddu G, Malaguarnera G, Pavone P, Cappellani A, Cacopardo B (2012) Hepatic echinococcosis: clinical and therapeutic aspects. World J Gastroenterol 18(13):1448–1458. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Ricardblum S, Bressonhadni S, Vuitton DA, Ville G, Grimaud JA (1992) Hydroxypyridinium collagen cross-links in human liver fibrosis: study of alveolar echinococcosis. Hepatology 15(4):599–602CrossRefGoogle Scholar
  26. Sá-Nunes A, Faccioli L, Sforcin J (2003) Propolis: lymphocyte proliferation and IFN-γ production. J Ethnopharmacol 87(1):93–97CrossRefPubMedGoogle Scholar
  27. Siles-Lucas M, Merli M, Mackenstedt U, Gottstein B (2003) The Echinococcus multilocularis 14-3-3 protein protects mice against primary but not secondary alveolar echinococcosis. Vaccine 21(5–6):431–439. CrossRefPubMedGoogle Scholar
  28. Torgerson PR, Keller K, Magnotta M, Ragland N (2010) The global burden of alveolar echinococcosis. PLoS Negl Trop Dis 4(6):e722. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Turcanu V, Hirst TR, Williams NA (2002) Modulation of human monocytes by Escherichia coli heat-labile enterotoxin B-subunit; altered cytokine production and its functional consequences. Immunology 106(3):316–325. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Verweij WR, De HL, Holtrop M, Agsteribbe E, Brands R, van Scharrenburg GJ, Wilschut J (1998) Mucosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen. Vaccine 16(20):2069–2076. CrossRefPubMedGoogle Scholar
  31. Vuitton DA (2003) The ambiguous role of immunity in echinococcosis: protection of the host or of the parasite? Acta Trop 85(2):119–132. CrossRefPubMedGoogle Scholar
  32. Wang J, Zhang C, Wei X, Oleg B, Lv G, Lu X, Georges M, Vuitton DA, Hao W, Lin R (2013) TGF-βand TGF-β/Smad signaling in the interactions between Echinococcus multilocularis and its hosts. PLoS One 8(2):e55379. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Wang H, Zhang F, Ma X, Ma H, Zhu Y, Liu X, Zhu M, Wen H, Ding J (2014) Prokaryotic expression and identification of B- and T-cell combined epitopes of Em95 antigen of Echinococcus multilocularis. Int J Clin Exp Pathol 7(8):5117–5122PubMedPubMedCentralGoogle Scholar
  34. Yang YR, Vuitton DA, Jones MK, Craig PS, Mcmanus DP (2005) Brain metastasis of alveolar echinococcosis in a hyperendemic focus of Echinococcus multilocularis infection. Trans R Soc Trop Med Hyg 99(12):937–941. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Research Center for High Altitude MedicineQinghai UniversityXiningChina
  2. 2.Qinghai-Utah Joint Research Key Lab for High Altitude MedicineXiningChina
  3. 3.Qinghai Key Laboratory of Science and Technology for High Altitude MedicineXiningChina
  4. 4.Qinghai Key Laboratory for EchinococcosisXiningChina
  5. 5.Ningxia Key Laboratory of Clinical and Pathogenic MicrobiologyGeneral Hospital of Ningxia Medical UniversityYinchuanChina
  6. 6.Ningxia Key Laboratory of Cerebrocranial Diseases, School of Laboratory MedicineNingxia Medical UniversityYinchuanChina
  7. 7.Research Center for High Altitude ZoonosisQinghai UniversityXiningChina

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