Journal of Genetics

, 98:53 | Cite as

Variability of the EG95 antigen-coding gene of Echinococcus granulosus in animal and human origin: implications for vaccine development

  • V. Sreevatsava
  • Sumanta De
  • S. Bandyopadhyay
  • P. Chaudhury
  • A. K. Bera
  • Ramachandran Muthiyan
  • Arun Kumar De
  • P. Perumal
  • Jai Sunder
  • Gayatri Chakraborty
  • D. BhattacharyaEmail author
Research Article


In the present study, the genetic variability of the EG95 protein-coding gene in several animal and human isolates of Echinococcus granulosus was investigated. A total of 24 isolates collected from cattle, buffalo, sheep, goat, dog and man were amplified by Eg95-coding gene-specific primers. From the generated sequence information, a conceptual amino acid sequence was deduced. Phylogenetically, the Eg95 coding gene belongs to the Eg95-1/Eg95-2/Eg95-3/Eg95-4 cluster. Further confirmation on the maximum composite likelihood analysis revealed that the overall transition/transversion bias was 2.913. This finding indicated that there is bias towards transitional and transversional substitution. Using artificial neural networks, a B-cell epitope was predicted on primary sequence information. Stretches of amino acid residues varied between animal and human isolates when hydrophobicity was considered. Flexibility also varied between larval and adult stages of the organism. This observation is important to develop vaccines. However, cytotoxic T-lymphocyte epitopes on primary sequence data remained constant in all isolates. In this study, agretope identification started with hydrophobic amino acids. Amino acids with the same physico-chemical properties were present in the middle. The conformational propensity of the Eg95-coding gene of 156 amino acid residues had \(\upalpha \)-turns and \(\upbeta \)-turns, and \(\upalpha \)-amphipathic regions up to 129, 138–156 and 151–155 residues, respectively. The results indicated potential T-cell antigenic sites. The overall Tajima’s D value was negative (−2.404165), indicative of negative selection pressure.


Eg95 gene epitope mapping selection pressure Echinococcus granulosus 


  1. Alvarez-Rojas C. A., Gauci C. G., Nolan M. J., Harandi M. F. and Lightowlers M. W. 2012 Characterization of the Eg95 gene family in the G6 genotype of Echinococcus granulosus. Mol. Biochem. Parasitol. 183, 115–121.CrossRefGoogle Scholar
  2. Arbabi M., Pirestani M., Delavari M., Hooshyar H., Abdoli A. and Sarvi S. 2017 Molecular and morphological characterizations of Echinococcus granulosus from human and animal isolates in Kashan, Markazi Province, Iran. Iran J. Parasitol. 12, 177–187.PubMedGoogle Scholar
  3. Arend A. C., Zaha A., Ayala F. J. and Haag K. L. 2004 The Echinococcus granulosus antigen B shows a high degree of genetic variability. Exp. Parasitol. 108, 76–80.CrossRefGoogle Scholar
  4. Barnes T. S., Hinds L. A., Jenkins D. J., Coleman G. T., Colebrook A. L., Kyngdon C. T. et al. 2009 Efficacy of the EG95 hydatid vaccine in a macropodid host, the tammar wallaby. Parasitology 136, 461–468.CrossRefGoogle Scholar
  5. Bhasin M. and Raghava G. P. 2004 Prediction of CTL epitopes using QM, SVM and ANN techniques. Vaccine 22, 3195–3204.CrossRefGoogle Scholar
  6. Boubaker G., Gottstein B., Hemphill A., Babba H. and Spiliotis M. 2014 Echinococcus P29 antigen: molecular characterization and implication on post-surgery follow-up of CE patients infected with different species of the Echinococcus granulosus complex. PLoS One 9, e98357.CrossRefGoogle Scholar
  7. Brunak S. and Buus S. 2000 Identifying cytotoxic T cell epitopes from genomic and proteomic informat ion: ‘The human MHC project’. Rev. Immunogenet. 2, 477.PubMedGoogle Scholar
  8. Chow C., Gauci C. G., Cowman A. F. and Lightowlers M. W. 2001 A gene family expressing a host-protective antigen of Echinococcus granulosus. Mol. Biochem. Parasitol. 118, 83–88.CrossRefGoogle Scholar
  9. Craig P. S., McManus D. P., Lightowlers M. W., Chabalgoity J. A., Garcia H. H., Gavidia C. M. et al. 2007 Prevention and control of cystic echinococcosis. Lancet Infect. Dis. 7, 385–394.CrossRefGoogle Scholar
  10. Dudek N. L., Perlmutter P., Aguilar M. I., Croft N. P. and Purcell A. W. 2010 Epitope discovery and their use in peptide based vaccines. Curr. Pharm. Des. 16, 3149–3157.CrossRefGoogle Scholar
  11. Eckert J. and Thompson R. C. A. 1997 Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans. Acta Trop. 64, 19–34.CrossRefGoogle Scholar
  12. Gauci C., Merli M., Muller V., Chow C., Yagi K., Mackenstedt U. et al. 2002 Molecular cloning of a vaccine antigen against infection with the larval stage of Echinococcus multilocularis. Infect. Immun. 70, 3969–3972.CrossRefGoogle Scholar
  13. Gauci C. G., Alvarez-Rojas C. A., Chow C. and Lightowlers M. W. 2018 Limitations of the Echinococcus granulosus genome sequence assemblies for analysis of the gene family encoding the EG95 vaccine antigen. Parasitology 145, 807–813.CrossRefGoogle Scholar
  14. Goldsby R. A., Kindt T. J. and Osborne B. A. 2000 Kuby immunology, 4th edition, pp. 63–81, 173–198. W. H. Freeman, New York.Google Scholar
  15. Haag K. L., Gottstein B. and Ayala F. J. 2009 The EG95 antigen of Echinococcus spp. contains positively selected amino acids, which may influence host specificity and vaccine efficiency. PLoS One 4, e5362.CrossRefGoogle Scholar
  16. Hemphill A. and Kern P. 2008 Special issue: experimental studies in echinococcosis. Exp. Parasitol. 119, 437–438.CrossRefGoogle Scholar
  17. Huang F., Dang Z., Suzuki Y., Horiuchi T., Yagi K., Kouguchi H. et al. 2016 Analysis on gene expression profile in oncospheres and early stage metacestodes from Echinococcus multilocularis. PLoS Negl. Trop. Dis. 10, e0004634.CrossRefGoogle Scholar
  18. Jenkins D. J. and Macpherson C. N. 2003 Transmission ecology of Echinococcus in wild-life in Australia and Africa. Parasitology 127, S63–S72.CrossRefGoogle Scholar
  19. Jones D. T. 1999 Protein secondary structure prediction based on position specific scoring matrices. J. Mol. Biol. 292, 195–202.CrossRefGoogle Scholar
  20. Kamenetzky L., Muzulin P. M., Guttierrez A. M., Angel S. O., Zaha A., Guarnera E. A. et al. 2005 High polymorphism in genes encoding antigen B from human infections strains of Echinococcus granulosus. Parasitology 131, 805–815.CrossRefGoogle Scholar
  21. Kennedy D. A. and Read A. F. 2017 Why does drug resistance readily evolve but vaccine resistance does not? Proc. Biol. Sci. 284, 20162562.CrossRefGoogle Scholar
  22. Larrieu E., Mujica G., Gauci C. G., Vizcaychipi K., Seleiman M., Herrero E. et al. 2015 Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: second study of impact. PLoS Negl. Trop. Dis. 9, e0004134.CrossRefGoogle Scholar
  23. Leinikki P., Lehtinen M., Hyoty H., Parkkonen P., Kantanen M. L. and Hakulinen J. 1993 Synthetic peptides as diagnostic tools in virology. Adv. Virus Res. 42, 149–186.CrossRefGoogle Scholar
  24. Li Y., Liu X., Zhu Y., Zhou X., Cao C., Hu X. et al. 2013 Bioinformatic prediction of epitopes in the Emy162 antigen of Echinococcus multilocularis. Exp. Ther. Med. 6, 335–340.CrossRefGoogle Scholar
  25. Lightowlers M. W. and Heath D. D. 2004 Immunity and vaccine control of Echinnococuss granulosus infection in animal intermediate hosts. Parassitologia 46, 27–31.PubMedGoogle Scholar
  26. Lightowlers M. W., Jensen O. and Fernandez E. 1999 Vaccination trails in Australia and Argentina confirm the effectiveness of the EG95 hydatid vaccine in sheep. Int. J. Parasitol. 29, 531–534.CrossRefGoogle Scholar
  27. Lightowlers M. W., Lawrence S. B., Gauci C. G., Young J., Ralston M. J., Maas D. et al. 1996 Vaccination against hydatidosis using a defined recombinant antigen. Parasite Immunol. 18, 457–462.CrossRefGoogle Scholar
  28. Lightowlers M. W., Gauci C. G., Chow C., Drew D. R., Gauci S. M., Jenkins D. J. et al. 2003 Molecular and genetic characterization of the host-protective oncospheres antigens of taeniid cestode parasites. Int. J. Parasitol. 33, 1207–1217.CrossRefGoogle Scholar
  29. Nono J. K., Pletinckx K., Lutz M. B. and Brehm K. 2012 Excretory/secretory-products of Echinococcus multilocularis Larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro. PLoS Negl Trop Dis. 6, e1516.CrossRefGoogle Scholar
  30. Nunnari G., Pinzone M. R., Gruttadauria S., Celesia B. M., Madeddu G., Malaguarnera G. et al. 2012 Hepatic echinococcosis: clinical and therapeutic aspects. World J. Gastroenterol. 18, 1448–1458.CrossRefGoogle Scholar
  31. Pan D., Bera A. K., Bandyopadhyay S., Das S. K., Bandyopadhyay S., Bhattacharyya S. et al. 2009 Relative expression of antigen B coding gene of bubaline isolates of Echinococcus granulosus in fertile and sterile cysts. J. Helminthol. 28, 1–4.Google Scholar
  32. Pan D., Bera A. K., Das S. K., Bandyopadhyay S., Manna B. and Bhattacharya D. 2010a Polymorphism and natural selection of antigen B1 of Echinococcus granulosus isolated from different host assemblages in India. Mol. Biol. Rep. 37, 1477–1482.CrossRefGoogle Scholar
  33. Pan D., Bera A. K, De S., Bandyopadhyay S., Das S. K., Manna B. et al. 2010b Relative expression of the 14-3-3 gene in different morphotypes of cysts of Echinococcus granulosus isolated from the India buffalo. J. Helminthol. 15, 1–4.Google Scholar
  34. Pellequer J. L., Westhof E. and Van Regenmortel M. H. 1991 Predicting location of continuous epitopes in proteins from their primary structures. Methods Enzymol. 203, 176–201.CrossRefGoogle Scholar
  35. Regenmortel M. H. V. V. 2006 Immunoinformatics may lead to a reappraisal of the nature of B cell epitopes and of the feasibility of synthetic peptide vaccines. J. Mol. Recognit. 19, 183–187.CrossRefGoogle Scholar
  36. Rosenzvit M. C., Camica F., Kamenentzky L., Muzulin P. M. and Gutierrez A. M 2006 Identification and intra-specific variability analysis of secreted membrane-bound proteins from Echinococcus granulosus. Parasitol Int. 55, 63–67.CrossRefGoogle Scholar
  37. Saha S. and Raghava G. P. 2006 Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65, 40–48.CrossRefGoogle Scholar
  38. Singh H. and Raghava G. P. 2001 Propred: prediction of HLA-DR binding sites. Bioinformatics 7, 1236–1237.CrossRefGoogle Scholar
  39. Spotin A., Mahami-Oskouei M., Harandi M. F., Baratchian M., Bordbar A., Ahmadpour E. et al. 2017 Genetic variability of Echinococcus granulosus complex in various geographical populations of Iran inferred by mitochondrial DNA sequences. Acta Trop. 165, 10–16.CrossRefGoogle Scholar
  40. Spouge J. L., Guy H. R., Cornette J. L., Margalit H., Cease K., Berzofsky J. A. et al. 1987 Strong conformational propensities enhance T cell antigenicity. J. Immunol. 138, 204–212.PubMedGoogle Scholar
  41. Tamura K., Dudley J., Nei M. and Kumar S. 2007 MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.CrossRefGoogle Scholar
  42. Thompson R. C. A., Lymbery A. J. and Constantine C. C. 1995 Variation in Echinococcus: towards a taxonomic revision of the genus. Adv. Parasitol. 35, 145–175.CrossRefGoogle Scholar
  43. Thompson R. C. A. and McManus D. P. 2001 Aetiology: parasites and life-cycles. In WHO/OIE manual on echinococcosis in humans and animals: A public health problem of global concern (ed. J. Eckert, M. A. Gemmel, F. X. Meslin, Z. S. Pawlowski), pp. 1–9. World Organization for Animal Health, Paris.Google Scholar
  44. Torgerson P. R. and Heath D. D. 2003 Transmission dynamics and control options for Echinococcus granulosus. Parasitology 127, 43–58.CrossRefGoogle Scholar
  45. WHO (2018) Echinococcosis. (accessed on 14-01-2019).Google Scholar
  46. WHO-Informal Working Group on Echinococcosis 2003 PAIR: Puncture, aspiration, injection, Re-aspiration. An option for the treatment of cystic echinococcosis. WHO, Geneva.Google Scholar
  47. Woollard D. J., Heath D. D. and Lightowlers M. W. 2000 Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens. Parasitology 121, 145–153.CrossRefGoogle Scholar
  48. Zhang J. 2008 Positive selection, not negative selection, in the pseudogenization of rcsA in Yersinia pestis. Proc. Natl. Acad. Sci. USA 105, E69; author reply E70.Google Scholar
  49. Zhang W., Lij Y. H., Zhang Z., Turson G., Aili H., Wang J. et al. 2003 Echinococcus granulosus from Xinjiang, PR China cDNAs encoding the EG95 vaccine antigen are expressed in different life cycle stages and are conserved in the oncosphere. Am. J. Trop. Med. Hyg. 68, 40–43.CrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2019

Authors and Affiliations

  • V. Sreevatsava
    • 1
  • Sumanta De
    • 1
  • S. Bandyopadhyay
    • 1
  • P. Chaudhury
    • 2
  • A. K. Bera
    • 1
  • Ramachandran Muthiyan
    • 3
  • Arun Kumar De
    • 3
  • P. Perumal
    • 3
  • Jai Sunder
    • 3
  • Gayatri Chakraborty
    • 3
  • D. Bhattacharya
    • 3
    Email author
  1. 1.Eastern Regional StationIndian Veterinary Research InstituteKolkataIndia
  2. 2.Indian Veterinary Research InstituteBareillyIndia
  3. 3.ICAR-Central Island Agricultural Research InstitutePort BlairIndia

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