Cost effective filamentous phage based immunization nanoparticles displaying a full-length hepatitis B virus surface antigen


Hepatitis B virus (HBV) is one of the major causes of chronic hepatitis, cirrhosis and liver cancer. In combating HBV infections, HBV diagnosis and vaccination are therefore critical. The hepatitis B virus surface antigen (HBsAg) is a key target molecule in developing vaccines and diagnostic systems. To date, although HBsAg has been expressed in bacteria, yeasts and mammalian cells, there are still limitations in the existing ones, which leave the necessity for searching new HBsAg production methods. In this study, a simple phage display-based method was developed to produce the purified full-length HBsAg molecules for further immunization studies. For this purpose, the HBsAg coding gene was cloned into a pCANTAB5E phagemid vector and expressed on the surface of M13 filamentous phages. The HBsAg-expressing phage nanosystem was then used as immunization agent in BALB/cJ mice. The ELISA results for sera obtained from mice immunized with HBsAg-displaying phage particles revealed an immune response against HBsAg. These results demonstrate the potential use of a full-length antigen to be displayed on phages as cost effective adjuvant-free immunization agents as an alternative to the highly purified and more expensive antigens conjugated with carrier molecules.

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Balcioglu, B. , Ozdemir-Bahadir, A. , Hinc, D. , Tamerler, C. and Erdag, B. (2014) Cost effective filamentous phage based immunization nanoparticles displaying a full-length hepatitis B virus surface antigen. Advances in Bioscience and Biotechnology, 5, 46-53. doi: 10.4236/abb.2014.51008.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Francois, G., Hallauer, J. and Van Damme, P. (2002) Hepatitis B vaccination: How to reach risk groups. Vaccine, 21, 1-4.
[2] Rantala, M. and van de Laar, M.J. (2008) Surveillance and epidemiology of hepatitis B and C in Europe—A review. Euro Surveill, 13.
[3] Szmuness, W., Stevens, C.E., Harley, E.J., Zang, E.A., Oleszko, W.R., William, D.C., Sadovsky, R., Morrison, J.M. and Kellner, A. (1980) Hepatitis B vaccine: Demonstration of efficacy in a controlled clinical trial in. The New England Journal of Medicine, 303, 833-841.
[4] Valenzuela, P., Medina, A. and Rutter, W. (1982) Synthesis and assembly of Hepatitis-B virus surface-antigen particles in yeast. Nature, 298, 347-350.
[5] Qin, S., Tang, H., Zhao, L., He, F., Lin, Y., Liu, L. and He, X. (2003) Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells. World Journal of Gastroenterology, 9, 1111-1113.
[6] Tan, G., Yusoff, K., Seow, H. and Tan, W. (2005) Antigenicity and immunogenicity of the immunodominant region of hepatitis B surface antigen displayed on bacteriophage T7. Journal of Medical Virology, 77, 475-480.
[7] Smith, G. (1985) Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science, 228, 1315-1317.
[8] Smith, G. and Petrenko, V. (1997) Phage display. Chemical Reviews, 97, 391-410.
[9] Vidova, B., Godany, A. and Sturdik, E. (2008) Phage display—A tool for detection and Prevention against pathogens. Nova Biotechnologica, 8, 23-33.
[10] Erdag, B., Balcioglu, K., Kumbasar, A., Celikbicak, O., Zeder-Lutz, G., Altschuh, D., Salih, B. and Baysal, K. (2007) Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity. Molecular Biotechnology, 35, 51-63.
[11] Prachasuphap, A., University, K., Kittigul, C., University, K., Sunthoranandh, P., University, K., Dhepakson, P., Center, M.B., Buddhirakkul, N., Center, M.B., Balachandra, K. and Medical Biotechnology Center, M.o.P.H. (2006) Construction of recombinant monoclonal antibody against hepatitis b surface antigen by phage display. Animal Cell Technology: Basic & Applied Aspects, 14, 227-232.
[12] Erdag, B., Balcioglu, B., Bahadir, A., Serhatli, M., Kacar, O., Bahar, A., Seker, U., Akgun, E., Ozkan, A., Kilic, T., Tamerler, C. and Baysal, K. (2011) Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2. Biotechnology and Applied Biochemistry, 58, 412-422.
[13] Tamerler, C., Khatayevich, D., Gungormus, M., Kacar, T., Oren, E.E., Hnilova, M. and Sarikaya, M. (2010) Molecular biomimetics: GEPI-based biological routes to technology. Biopolymers, 94, 78-94.
[14] Bayrovic, K., Erdag, B., Atalay, O.E. and Cirakoglu, B. (2001) Full resistance to tobacco mosaic virus infection conferred by the transgenic expression of a recombinant antibody in tobacco. Biotechnology & Biotechnology Equipment, 15, 21-27.
[15] Tang, K., Yusoff, K. and Tan, W. (2009) Display of Hepatitis B Virus PreS1 peptide on bacteriophage T7 and its potential in gene delivery into HepG2 cells. Journal of Virological Methods, 159, 194-199.
[16] Solomon, B. (2005) Generation of anti-beta-amyloid antibodies via phage display technology towards Alzheimer’s disease vaccination. Vaccine, 23, 2327-2330.
[17] Fang, J., Wang, G., Yang, Q., Song, J., Wang, Y. and Wang, L. (2005) The potential of phage display virions expressing malignant tumor specific antigen MAGE-A1 epitope in murine model. Vaccine, 23, 4860-4866.
[18] Kubar, A., Yapar, M., Ozyurt, M., Haznedaroglu, T. and Gun, H. (1988) Cloning of hepatitis B virus surface gene region to Escherichia coli. Flora, 6, 108-113.
[19] Sambrook, J., Fritsch, E.E. and Maniatis, T. (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Lab. Press, New York.
[20] Sorensen, H. and Mortensen, K. (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factories, 4.
[21] Lunin, V.G., Gol’dberg, E.Z., Grigor’ev, V.G., Mikhailov, M.M., Khudiakov, I.E., Skripkin, E.A., Smirnov, V.D., Naroditsky, B.S., Ketiladze, E.S. and Tikhonenko, T.I. (1983) Cloning and ex-pression of gene coding the hepatitis B virus surface antigen (HBsag) in Escherichia coli. Doklady Akademii Nauk Sssr, 268, 496-498.
[22] Guan, Z., Guo, B., Huo, Y., Guan, Z. and Wei, Y. (2010) Overview of expression of hepatitis B surface antigen in transgenic plants. Vaccine, 28, 7351-7362.
[23] Vianna, C.O., da Silva e Mouta Junior, S., de Oliveira da Silva, G., da Silva Freire, M. and de Moraes, M.T. (2003) Screening of CHO cell clones expressing histidine-tagged major S hepatitis B surface protein using a semi-quantitative PCR protocol. Journal of Virological Methods, 114, 171-174.
[24] Barbas, C.F.I., Burton, D.R., Scott, J.K. and Silverman, G.J. (2001) Phage display: A laboratory manual. Cold Spring Harbor Lab. Press, New York.
[25] Rakonjac, J., Bennett, N., Spagnuolo, J., Gagic, D. and Russel, M. (2011) Filamentous bacteriophage: Biology, phage display and nanotechnology applications. Current Issues in Molecular Biology, 13, 51-75.
[26] Irving, M.B., Pan, O. and Scott, J.K. (2001) Randompeptide libraries and antigen fragment libraries for epitope mapping and the development of vaccines and diagnostics. Current Opinion in Chemical Biology, 5, 314-324.
[27] Willis, A., Perham, R. and Wraith, D. (1993) Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage. Gene, 128, 79-83.
[28] Molenaar, T., Michon, I., de Haas, S., van Berkel, T., Kuiper, J. and Biessen, E. (2002) Uptake and processing of modified bacteriophage M13 in mice: Implications for phage display. Virology, 293, 182-191.
[29] Wang, L. and Yu, M. (2004) Epitope identification and discovery using phage display libraries: Applications in vaccine development and diagnostics. Current Drug Targets, 5, 1-15.
[30] Scala, G., Chen, X., Liu, W., Telles, J.N., Cohen, O.J., Vaccarezza, M., Igarashi, T. and Fauci, A.S. (1999) Selection of HIV-specific immunogenic epitopes by screening random peptide. Journal of Immunology, 162, 6155-6161.
[31] Puntoriero, G., Meola, A., Lahm, A., Zucchelli, S., Ercole, B.B., Tafi, R., Pezzanera, M., Mondelli, M.U., Cortese, R., Tramontano, A., Galfre, G. and Nicosia, A. (1998) Towards a solution for hepatitis C virus hypervariability: mimotopes of the. The EMBO Journal, 17, 3521-3533.
[32] Frenkel, D., Katz, O. and Solomon, B. (2000) Immunization against Alzheimer’s beta-amyloid plaques via EFRH phage. Proceedings of the National Academy of Sciences of the United Stated of America, 97, 11455-11459.
[33] Manoutcharian, K., Diaz-Orea, A., Gevorkian, G., Fragoso, G., Acero, G., Gonzalez, E., de Aluja, A., Villalobos, N., Gomez-Conde, E. and Sciutto, E. (2004) Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis. Veterinary Immunology and Immunopathology, 99, 11-24.
[34] Solomon, B. (2007) Active immunization against Alzheimer’s beta-amyloid peptide using phage display technology. Vaccine, 25, 3053-3056.
[35] Morales, J., Martinez, J., Manoutcharian, K., Hernandez, M., Fleury, A., Gevorkian, G., Acero, G., Blancas, A., Toledo, A., Cervantes, J., Maza, V., Quet, F., Bonnabau, H., de Aluja, A., Fragoso, G., Larralde, C. and Sciutto, E. (2008) Inexpensive anti-cysticercosis vaccine: S3Pvac expressed in heat inactivated M13 filamentous phage proves effective against naturally acquired Taenia solium porcine cysticercosis. Vaccine, 26, 2899-2905.
[36] Greenwood, J., Willis, A.E. and Perham, R.N. (1991) Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens. Journal of Molecular Biology, 220, 821-827.
[37] Mirano-Bascos, D., Tary-Lehmann, M. and Landry, S. (2008) Antigen structure influences helper T-cell epitope dominance in the human immune response to HIV envelope glycoprotein gp120. European Journal of Immunology, 38, 1231-1237.
[38] Chiang, C., Benencia, F. and Coukos, G. (2010) Whole tumor antigen vaccines. Seminars in Immunology, 22, 132-143.
[39] Kolodziej, P. and Young, R. (1991) Epitope tagging and protein surveillance. Methods in Enzymology, 194, 508-519.
[40] Ozdemir-Bahadir, A., Balcioglu, B., Uzyol, K., Hatipoglu, I., Sogut, I., Basalp, A. and Erdag, B. (2011) Phage Displayed HBV Core Antigen with Immunogenic Activity. Applied Biochemistry and Biotechnology, 165, 1437-1447.

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