Annals of Laboratory Medicine

Korean Society for Laboratory Medicine (대한진단검사의학회)
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Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

  • Taherkhani, Reza (Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Scien) ;
  • Makvandi, Manoochehr (Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Scien) ;
  • Farshadpour, Fatemeh (Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Scien)
  • Received : 2013.08.12
  • Accepted : 2013.10.02
  • Published : 2014.03.02
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Abstract

Background: Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. Methods: Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by $Ni^{2+}$-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. Results: High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P =0.625). Conclusions: The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.

Keywords

References

  1. Srivastava R, Aggarwal R, Jameel S, Puri P, Gupta VK, Ramesh VS, et al. Cellular immune responses in acute hepatitis E virus infection to the viral open reading frame 2 protein. Viral Immunol 2007;20:56-65. https://doi.org/10.1089/vim.2006.0053
  2. Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, et al. Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010;376:895-902. https://doi.org/10.1016/S0140-6736(10)61030-6
  3. Aggarwal R, Shukla R, Jameel S, Agrawal S, Puri P, Gupta VK, et al. T-cell epitope mapping of ORF2 and ORF3 proteins of human hepatitis E virus. J Viral Hepat 2007;14:283-92. https://doi.org/10.1111/j.1365-2893.2006.00796.x
  4. Yamashita T, Mori Y, Miyazaki N, Cheng RH, Yoshimura M, Unno H, et al. Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure. Proc Natl Acad Sci USA 2009; 106:12986-91. https://doi.org/10.1073/pnas.0903699106
  5. Uchida T. Cloning and sequencing of cDNA of the hepatitis E virus genome--application to diagnosis. Nihon Rinsho 1993;51:352-6.
  6. Zhou YX, Lee MY, Ng JM, Chye ML, Yip WK, Zee SY, et al. A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice. World J Gastroenterol 2006;12:306-12.
  7. Xing L, Wang JC, Li TC, Yasutomi Y, Lara J, Khudyakov Y, et al. Spatial configuration of hepatitis E virus antigenic domain. J Virol 2011;85: 1117-24. https://doi.org/10.1128/JVI.00657-10
  8. Zhang J, Liu CB, Li RC, Li YM, Zheng YJ, Li YP, et al. Randomized-controlled phase II clinical trial of a bacterially expressed recombinant hepatitis E vaccine. Vaccine 2009;27:1869-74. https://doi.org/10.1016/j.vaccine.2008.12.061
  9. Emerson SU and Purcell RH. Recombinant vaccines for hepatitis E. Trends Mol Med 2001;7:462-6. https://doi.org/10.1016/S1471-4914(01)02106-2
  10. Jiménez de Oya N, Galindo I, Gironés O, Duizer E, Escribano JM, Saiz JC. Serological immunoassay for detection of hepatitis E virus on the basis of genotype 3 open reading frame 2 recombinant proteins produced in Trichoplusia ni larvae. J Clin Microbiol 2009;47:3276-82. https://doi.org/10.1128/JCM.00750-09
  11. Sehgal D, Malik PS, Jameel S. Purification and diagnostic utility of a recombinant hepatitis E virus capsid protein expressed in insect larvae. Protein Expr Purif 2003;27:27-34. https://doi.org/10.1016/S1046-5928(02)00574-0
  12. Naik S, Aggarwal R, Naik SR, Dwivedi S, Talwar S, Tyagi SK, et al. Evidence for activation of cellular immune responses in patients with acute hepatitis E. Indian J Gastroenterol 2002;21:149-52.
  13. Zhou YH, Purcell RH, Emerson SU. A truncated ORF2 protein contains the most immunogenic site on ORF2: antibody responses to non-vaccine sequences following challenge of vaccinated and non-vaccinated macaques with hepatitis E virus. Vaccine 2005;23:3157-65. https://doi.org/10.1016/j.vaccine.2004.12.020
  14. Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH. Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol 2006;87:697-704. https://doi.org/10.1099/vir.0.81545-0
  15. Tsarev SA, Tsareva TS, Emerson SU, Govindarajan S, Shapiro M, Gerin JL, et al. Successful passive and active immunization of cynomolgus monkeys against hepatitis E. Proc Natl Acad Sci U S A 1994;91:10198-202. https://doi.org/10.1073/pnas.91.21.10198
  16. Zhang M, Emerson SU, Nguyen H, Engle RE, Govindarajan S, Gerin JL, et al. Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells. Vaccine 2001;20:853-7. https://doi.org/10.1016/S0264-410X(01)00399-1
  17. Saiz JC, Gonzalez MJ, Borca MV, Sobrino F, Moore DM. Identification of neutralizing antigenic sites on VP1 and VP2 of type A5 foot-and-mouth disease virus, defined by neutralization-resistant variants. J Virol 1991; 65:2518-24.
  18. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24: 1596-9. https://doi.org/10.1093/molbev/msm092
  19. Yang EC, Chi BR, Li X, Liu Y, Gao P, Jia P, et al. Expression and characterization of a recombinant truncated capsid protein of hepatitis E virus in Pichia pastoris. Chem Res Chin Univ 2010;26:235-9.
  20. Gholipour A, Moosavian M, Galehdari H, Makvandi M, Memari HR, Alvandi A. Cloning and periplasmic expression of peptidoglycan-associated lipoprotein (PAL) protein of Legionella pneumophila in Escherichia coli. Jundishapur J Microbiol 2010;3:1-9.
  21. Hao B-C, Lan X, Xing X-Y, Xiang H-T, Wen F-Q, Hu Y-Y, et al. Cloning and prokaryotic expression of cDNAs from hepatitis E virus structural gene of the SW189 strain. Thai J Vet Med 2012;42:179-84.
  22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. https://doi.org/10.1016/0003-2697(76)90527-3
  23. Jacobs D, Vercammen M, Saman E. Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain. Clin Diagn Lab Immunol 1999;6:24-9.
  24. Liu XS, Wang Y, Zhang Y, Fang T, Pan L, Lü J, et al. Cloning, codon-optimized expression and homology modeling of structural protein VP1 from foot and mouth disease virus. Afr J Microbiol Res 2011;5:486-95.
  25. Wang X, Li X, Zhang Z, Shen X, Zhong F. Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli. Protein Expr Purif 2010;72:101-6. https://doi.org/10.1016/j.pep.2010.02.011
  26. Garmory HS, Brown KA, Titball RW. DNA vaccines: improving expression of antigens. Genet Vaccines Ther 2003;1:2. https://doi.org/10.1186/1479-0556-1-2
  27. Ramakrishna L, Anand KK, Mohankumar KM, Ranga U. Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol 2004;78:9174-89. https://doi.org/10.1128/JVI.78.17.9174-9189.2004
  28. Ruan B, Zumbika E, Wang SY, Chen Y, Ma YL, Chen YG, et al. Cloning and expression of cDNAs from hepatitis E virus structural gene. Hepatobiliary Pancreat Dis Int 2003;2:387-90.