Advanced Biomedical Research

Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine

Authors

1 Department of Biology, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran

2 Department of Genetics, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran

4 Department Of Rabies, Pasteur Institute of Iran, Tehran, Iran

5 Department Of Rabies, Pasteur Institute of Iran; Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

6 Department Of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran

Abstract

Background: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study.
Materials and Methods: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein.
Results: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain.
Conclusion: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models.

Keywords

1.
Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect 2011;17:107-15.  Back to cited text no. 1
    
2.
Roohvand F, Kossari N. Advances in hepatitis C virus vaccines, Part one: Advances in basic knowledge for hepatitis C virus vaccine design. Expert Opin Ther Pat 2011;21:1811-30.  Back to cited text no. 2
    
3.
Roohvand F, Kossari N. Advances in hepatitis C virus vaccines, part two: Advances in hepatitis C virus vaccine formulations and modalities. Expert Opin Ther Pat 2012;22:391-415.  Back to cited text no. 3
    
4.
Ishii S, Koziel MJ. Immune responses during acute and chronic infection with hepatitis C virus. Clin Immunol 2008;128:133-47.  Back to cited text no. 4
    
5.
Fynan EF, Webster RG, Fuller DH, Haynes JR, Santoro JC, Robinson HL. DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci U S A 1993;90:11478-82.  Back to cited text no. 5
    
6.
Cui Z. DNA vaccine. Adv Genet 2005;54:257-89.  Back to cited text no. 6
    
7.
Memarnejadian A, Roohvand F, Arashkia A, Berjisian F, Aghasadeghi MR. Designing, constructing and immunogenic evaluation of polyepitope DNA constructs by the application of hepatitis C virus immunodominant epitopes in BALB/c mice. Yakhteh Med J2009;11:122-33.  Back to cited text no. 7
    
8.
Buteau C, Markovic SN, Celis E. Challenges in the development of effective peptide vaccines for cancer. Mayo Clin Proc 2002;77:339-49.  Back to cited text no. 8
    
9.
Memarnejadian A, Roohvand F, Arashkia A, Rafati S, Shokrgozar MA. Polytope DNA vaccine development against hepatitis C virus: A streamlined approach from in silico design to in vitro and primary in vivo analyses in BALB/c mice. Protein Pept Lett 2009;16:842-50.  Back to cited text no. 9
    
10.
Arashkia A, Roohvand F, Memarnejadian A, Aghasadeghi MR, Rafati S. Construction of HCV-polytope vaccine candidates harbouring immune-enhancer sequences and primary evaluation of their immunogenicity in BALB/c mice. Virus Genes 2010;40:44-52.  Back to cited text no. 10
    
11.
Im EJ, Hong JP, Roshorm Y, Bridgeman A, Létourneau S, Liljeström P, et al. Protective efficacy of serially up-ranked subdominant CD8+T cell epitopes against virus challenges. PLoS Pathog 2011;7:e1002041.  Back to cited text no. 11
    
12.
Zhang Y, Ma CJ, Ni L, Zhang CL, Wu XY, Kumaraguru U, et al. Cross-talk between programmed death-1 and suppressor of cytokine signaling-1 in inhibition of IL-12 production by monocytes/macrophages in hepatitis C virus infection. J Immunol 2011;186:3093-103.  Back to cited text no. 12
    
13.
Behr MA, Small PM. A historical and molecular phylogeny of BCG strains. Vaccine 1999;17:915-22.  Back to cited text no. 13
    
14.
Klade CS, Wedemeyer H, Berg T, Hinrichsen H, Cholewinska G, Zeuzem S, et al. Therapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41. Gastroenterology 2008;134:1385-95.  Back to cited text no. 14
    
15.
Yutani S, Komatsu N, Shichijo S, Yoshida K, Takedatsu H, Itou M, et al. Phase I clinical study of a peptide vaccination for hepatitis C virus-infected patients with different human leukocyte antigen-class I-A alleles. Cancer Sci 2009;100:1935-42.  Back to cited text no. 15
    
16.
Warren WN, Brian JL, Sujata VM, Philip JT. Potential DNA vaccine integration into host cell genome: Merck research laboratories west point. Pennsyluania1948;6:30-9.  Back to cited text no. 16
    
17.
Halliday J, Klenerman P, Barnes E. Vaccination for hepatitis C virus: Closing in on an evasive target. Expert Rev Vaccines 2011;10:659-72.  Back to cited text no. 17
    
18.
Kaneko T, Moriyama T, Udaka K, Hiroishi K, Kita H, Okamoto H, et al. Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope. Eur J Immunol 1997;27:1782-7.  Back to cited text no. 18
    
19.
Sallberg MM, Frelin L, Diepolder H, Jung MC, Mathiesen I, Fons M, et al. First clinical trial of therapeutic vaccination using naked DNA delivered by in vivo electroporation shows antiviral effects in patients with chronic Hepatitis C. J Hepatol 2009:50:S18-9.  Back to cited text no. 19
    
20.
Alvarez-Lajonchere L, Shoukry NH, Gra B, Amador-Cañizares Y, Helle F, Bédard N, et al. Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial. J Viral Hepat 2009;16:156-67.  Back to cited text no. 20
    
21.
Sette A, Newman M, Livingston B, McKinney D, Sidney J, Ishioka G, et al. Optimizing vaccine design for cellular processing, MHC binding and TCR recognition. Tissue Antigens 2002;59:443-51.  Back to cited text no. 21
    
22.
Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, et al. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA 1996;93:1759-63.  Back to cited text no. 22
    
23.
Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol 1994;68:1494-500.  Back to cited text no. 23
    
24.
Choo QL, Kuo G, Ralston R, Weiner A, Chien D, Van Nest G, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91:1294-8.  Back to cited text no. 24
    
25.
Lagging LM, Meyer K, Hoft D, Houghton M, Belshe RB, Ray R. Immune responses to plasmid DNA encoding the hepatitis C virus core protein. J Virol 1995;69:5859-63.  Back to cited text no. 25
    
26.
Major ME, Vitvitski L, Mink MA, Schleef M, Whalen RG, Trépo C, et al. DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid. J Virol 1995;69:5798-805.  Back to cited text no. 26
    
27.
Tokushige K, Wakita T, Pachuk C, Moradpour D, Weiner DB, Zurawski VR Jr, et al. Expression and immune response to hepatitis C virus core DNA-based vaccine constructs. Hepatology 1996;24:14-20.  Back to cited text no. 27
    
28.
Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 2001;194:1395-406.  Back to cited text no. 28
    
29.
Shi L, Liu S, Fan GX, Sheng L, Ren HX, Yuan YK. Effective induction of type 1 cytotoxic T cell responses in mice with DNA vaccine encoding two hepatitis C virus cytotoxic T lymphocyte epitopes. Viral Immunol 2006;19:702-11.  Back to cited text no. 29
    
30.
Memarnejadian A, Roohvand F. Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine. Cell Immunol 2010;261:93-8.  Back to cited text no. 30
    
31.
Keating GM, Noble S. Recombinant hepatitis B vaccine (Engerix-B): A review of its immunogenicity and protective efficacy against hepatitis B. Drugs 2003;63:1021-51.  Back to cited text no. 31
    
32.
Patient R, Hourioux C, Vaudin P, Pagès JC, Roingeard P. Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: Implications for the design of new vaccine strategies. N Biotechnol 2009;25:226-34.  Back to cited text no. 32
    
33.
Novagen, Inc. pET system manual. 8th ed. 1991. United States and Canada. TB055 8th Edition 02/99. Available from: http://research.fhcrc.org/content/dam/stripe/hahn/methods/biochem/pet.pdf.   Back to cited text no. 33
    
34.
Novagen, Inc. pET system manual. 11th ed. 2005. United States and Canada. TB055 11th Edition 01/06. Available from: http://kirschner.med.harvard.edu/files/protocols/Novagen_petsystem.pdf.   Back to cited text no. 34
    
35.
Giacalone MJ, Gentile AM, Lovitt BT, Berkley NL, Gunderson CW, Surber MW. Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques 2006;40:355-64.  Back to cited text no. 35
    
36.
Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 1986;189:113-30.  Back to cited text no. 36
[PUBMED]    
37.
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 1990;185:60-89.  Back to cited text no. 37
[PUBMED]    
38.
Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 2005;41:207-34.  Back to cited text no. 38
    
39.
Suter-Crazzolara C, Unsicker K. Improved expression of toxic proteins in E. coli. Biotechniques 1995;19:202-4.  Back to cited text no. 39
    
40.
Sørensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 2005;115:113-28.  Back to cited text no. 40
    
41.
Miyada CG, Stoltzfus L, Wilcox G. Regulation of the araC gene of Escherichia coli: Catabolite repression, autoregulation, and effect on araBAD expression. Proc Natl Acad Sci U S A 1984;81:4120-4.  Back to cited text no. 41
[PUBMED]    
42.
Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 1991;219:37-44.  Back to cited text no. 42
    
43.
Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci 2010;24:1-29.  Back to cited text no. 43
    
44.
Ring GM, O'Connell MA, Keegan LP. Purification and assay of recombinant ADAR proteins expressed in the yeast Pichia pastoris or in Escherichia coli. Methods Mol Biol 2004;265:219-38.  Back to cited text no. 44
    
45.
Saïda F, Uzan M, Odaert B, Bontems F. Expression of highly toxic genes in E. coli: Special strategies and genetic tools. Curr Protein Pept Sci 2006;7:47-56.  Back to cited text no. 45
    
46.
Narayanan A, Ridilla M, Yernool DA. Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions. Protein Sci 2011;20:51-61.  Back to cited text no. 46
    
Advanced Biomedical Research
Volume 2015, october
October 2015