Applied Biochemistry and Biotechnology

, Volume 172, Issue 7, pp 3635–3645 | Cite as

Expression of Avian Influenza Virus (H5N1) Hemagglutinin and Matrix Protein 1 in Pichia pastoris and Evaluation of their Immunogenicity in Mice

  • M. Subathra
  • P. Santhakumar
  • Sureddi Satyam Naidu
  • M. Lakshmi Narasu
  • T. M. A. Senthilkumar
  • Sunil K. Lal


The conventional avian influenza vaccines rely on development of neutralizing antibodies against the HA and NA antigens. However, these antigens are highly variable, and hence there is a need for better vaccine candidates which would offer broader protection in animals. The M1 of avian influenza is another major structural protein that has conserved epitopes that are reported to induce CD8+ T cells and can contribute to protection against morbidity and mortality from influenza. Hence in an effort to study the immune response of rM1 either alone or in combination with rHA, the hemagglutinin (HA) and matrix protein (M1) of A/Hatay/2004/H5N1 strain of avian influenza were expressed in Pichia pastoris as his-tagged proteins and purified through Ni-NTA chromatography. The His-tag was removed using TEV protease cleavage site and the immunogenicity of purified rHA and rM1 either alone or in combination was determined in mice. One group of mice was immunized with 5 μg of purified rHA, the other group was immunized with rM1, and a third group of mice were immunized with 5 μg of rHA and rM1. All the animals were boosted twice, once on 28 days postimmunization (dpi) and the second on 42 dpi. The immune response was evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) assay. The group of mice immunized with rHA and rM1 together showed significantly higher immune response against rHA and rM1 than mice immunized with either HA or M1 antigens. The addition of rM1 with rHA resulted in increased HI titer in animals immunized with both the antigens. These results suggest that the HA and M1 expressed in P. pastoris can be utilized in combination for the development of faster and cost-effective vaccines for circulating and newer strains of avian influenza and would aid in combating the disease in a pandemic situation, in which production time matters greatly.


Matrix protein1 Hemagglutinin Highly pathogenic avian influenza virus H5N1 Immunogenicity 



This study was supported by the funding from the Department of Science and Technology (DST), New Delhi, India. The authors also thank Dr. V.A. Srinivasan (Former Director) and Dr. Dev Chandran, (Former Scientist), To the Indian Immunologicals Ltd, Hyderabad, for providing valuable technical support during this study.


  1. 1.
    Lambkin, R., Novelli, P., Oxford, J., & Gelder, C. (2004). Human genetics and responses to influenza vaccination: clinical implication. American Journal of Pharmacogenomics, 4, 293–298.CrossRefGoogle Scholar
  2. 2.
    Johnson, M. P., Meitin, C. A., Bender, B. S., & Small, P. A., Jr. (1993). Recombinant vaccinia immunization in the presence of passively administered antibody. Vaccine, 11(6), 665–669.CrossRefGoogle Scholar
  3. 3.
    Heiny, A. T., Miotto, O., Srinivasan, K. N., Khan, A. M., Zhang, G. L., Brusic, V., et al. (2007). Evolutionarily conserved protein sequences of influenza A viruses, avian and human, as vaccine targets. PLoS ONE, 2, e1190.CrossRefGoogle Scholar
  4. 4.
    Berkhoff, E. G. M., de Wit, E., Geelhoed-Mieras, M. M., Boon, A. C. M., Symons, J., Fouchier, R. A. M., et al. (2006). Fitness costs limit escape from cytotoxic T lymphocytes by influenza A viruses. Vaccine, 24, 6594–6596.CrossRefGoogle Scholar
  5. 5.
    Bui, H.-H., Peters, B., Assarsson, E., Mbawuike, I., & Sette, A. (2007). Ab and T cell epitopes of influenza A virus, knowledge and opportunities. Proceedings of the National Academy of Sciences of the United States of America, 104, 246–251.CrossRefGoogle Scholar
  6. 6.
    Lee, L. Y., Ha, L. A., Simmons, C., de Jong, M. D., Chau, N. V., Schumacher, R., et al. (2008). Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) inhealthy individuals. Journal of Clinical Investigation, 118, 3478–3490.Google Scholar
  7. 7.
    Treanor, J. J., Campbell, J. D., Zangwill, K. M., Rowe, T., & Wolff, M. (2006). Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. New England Journal of Medicine, 354(13), 1343–1351.CrossRefGoogle Scholar
  8. 8.
    Horimoto, T., & Kawaoka, Y. (2006). Strategies for developing vaccines against H5N1 influenza A viruses. Trends in Molecular Medicine, 12(11), 506–514.CrossRefGoogle Scholar
  9. 9.
    Stephenson, I., Nicholson, K. G., Wood, J. M., Zambon, M. C., & Katz, J. M. (2004). Confronting the avian influenza threat: vaccine development for a potential pandemic. Lancet Infectious Diseases, 4(8), 499–509.CrossRefGoogle Scholar
  10. 10.
    Wood, J. M., & Robertson, J. S. (2004). From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza. Nature Reviews Microbiology, 2(10), 842–847.CrossRefGoogle Scholar
  11. 11.
    Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C. A., & Gingeras, T. R. (1987). Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Research, 15(9), 3859–3876.CrossRefGoogle Scholar
  12. 12.
    Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K., & Romanos, M. A. (1991). High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology (N Y), 9(5), 455–460.CrossRefGoogle Scholar
  13. 13.
    Louisirirotchanakul, S., Lerdsamran, H., Wiriyarat, W., Sangsiriwut, K., Chaichoune, K., Pooruk, P., et al. (2007). Erythrocyte binding preference of avian influenza H5N1 viruses. Journal of Clinical Microbiology, 45, 2284–2286.CrossRefGoogle Scholar
  14. 14.
    Wiriyarat, W., Lerdsamran, H., Pooruk, P., Webster, R. G., Louisirirotchanakul, S., Ratanakorn, P., et al. (2010). Erythrocyte binding preference of 16 subtypes of low pathogenic avian influenza and 2009 pandemic influenza A (H1N1) viruses. Veterinary Microbiology, 146, 346–349.CrossRefGoogle Scholar
  15. 15.
    Biesova, Z., Miller, M. A., Schneerson, R., Shiloach, J., Green, K. Y., Robbins, J. B., et al. (2009). Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus. Vaccine, 27, 6234–6238.CrossRefGoogle Scholar
  16. 16.
    Chen, Z., Yoshikawa, T., Kadowaki, S., Hagiwara, Y., Matsuo, K., Asanuma, H., et al. (1999). Protection and antibody responses in different strains of mouse immunized with plasmid DNAs encoding influenza virus haemagglutinin, neuraminidase and nucleoprotein. Journal of General Virology, 80(Pt 10), 2559–2564.Google Scholar
  17. 17.
    Deroo, T., Jou, W. M., & Fiers, W. (1996). Recombinant neuraminidase vaccine protects against lethal influenza. Vaccine, 14(6), 561–569.CrossRefGoogle Scholar
  18. 18.
    Keitel, W. A., Cate, T. R., Atmar, R. L., Turner, C. S., Nino, D., Dukes, C. M., et al. (1996). Increasing doses of purified influenza virus hemagglutinin and subvirion vaccines enhance antibody responses in the elderly. Clinical and Diagnostic Laboratory Immunology, 3(5), 507–510.Google Scholar
  19. 19.
    Robinson, H. L., Hunt, L. A., & Webster, R. G. (1993). Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine, 11(9), 957–960.CrossRefGoogle Scholar
  20. 20.
    Fang, F., Cai, X. Q., Chang, H. Y., Wang, H. D., Yang, Z. D., & Chen, Z. (2008). Protection abilities of influenza B virus DNA vaccines expressing hemagglutinin, neuraminidase, or both in mice. Acta Virologica, 52(2), 107–112.Google Scholar
  21. 21.
    Okuda, K., Ihata, A., Watabe, S., Okada, E., Yamakawa, T., Hamajima, K., et al. (2001). Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene. Vaccine, 19(27), 3681–3691.CrossRefGoogle Scholar
  22. 22.
    Park, K. S., Seo, Y. B., Lee, J. Y., Im, S. J., Seo, S. H., Song, M. S., et al. (2011). Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e. Vaccine, 29(33), 5481–5487.CrossRefGoogle Scholar
  23. 23.
    Johansson, B. E. (1999). Immunization with influenza A virus hemagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine. Vaccine, 17(15–16), 2073–2080.CrossRefGoogle Scholar
  24. 24.
    Jones, T., Allard, F., Cyr, S. L., Tran, S. P., Plante, M., Gauthier, J., et al. (2003). A nasal Proteosome influenza vaccine containing baculovirus-derived hemagglutinin induces protective mucosal and systemic immunity. Vaccine, 21(25–26), 3706–3712.CrossRefGoogle Scholar
  25. 25.
    Crawford, J., Wilkinson, B., Vosnesensky, A., Smith, G., Garcia, M., Stone, H., et al. (1999). Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes. Vaccine, 17, 2265–2274.CrossRefGoogle Scholar
  26. 26.
    Sugiura, T., Sugita, S., Imagawa, H., Kanaya, T., Ishiyama, S., Saeki, N., et al. (2001). Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system. Journal of Virological Methods, 98(1), 1–8.CrossRefGoogle Scholar
  27. 27.
    Powers, D. C., Smith, G. E., Anderson, E. L., Kennedy, D. J., Hackett, C. S., Wilkinson, B. E., et al. (1995). Influenza A virus vaccines containing purified recombinant H3 hemagglutinin are well tolerated and induce protective immune responses in healthy adults. Journal of Infectious Diseases, 171(6), 1595–1599.CrossRefGoogle Scholar
  28. 28.
    Treanor, J. J., Wilkinson, B. E., Masseoud, F., Hu-Primmer, J., Battaglia, R., O'Brien, D., et al. (2001). Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans. Vaccine, 19(13–14), 1732–1737.CrossRefGoogle Scholar
  29. 29.
    Vanlandschoot, P., Maertens, G., Jou, W. M., & Fiers, W. (1993). Recombinant secreted haemagglutinin protects mice against a lethal challenge of influenza virus. Vaccine, 11(12), 1185–1187.CrossRefGoogle Scholar
  30. 30.
    Mahmood, K., Bright, R. A., Mytle, N., Carter, D. M., Crevar, C. J., Achenbach, J. E., et al. (2008). H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses. Vaccine, 26(42), 5393–5399.CrossRefGoogle Scholar
  31. 31.
    Bright, R. A., Carter, D. M., Daniluk, S., Toapanta, F. R., Ahmad, A., Gavrilov, V., et al. (2007). Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin. Vaccine, 25(19), 3871–3878.CrossRefGoogle Scholar
  32. 32.
    Fouchier, R. A., Munster, V., Wallensten, A., Bestebroer, T. M., Herfst, S., Smith, D., et al. (2005). Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology, 79(5), 2814–2822.CrossRefGoogle Scholar
  33. 33.
    Shi, W., Lei, F., Zhu, C., Sievers, F., & Higgins, D. G. (2010). A complete analysis of HA and NA genes of influenza A viruses. PLoS One, 5(12), e14454.CrossRefGoogle Scholar
  34. 34.
    Rao, S., Kong, W. P., Wei, C. J., Yang, Z. Y., Nason, M., Styles, D., et al. (2008). Multivalent HA DNA vaccination protects against highly pathogenic H5N1 avian influenza infection in chickens and mice. PLoS One, 3(6), e2432.CrossRefGoogle Scholar
  35. 35.
    Quan, F. S., Huang, C., Compans, R. W., & Kang, S. M. (2007). Virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus. Journal of Virology, 81(7), 3514–3524.CrossRefGoogle Scholar
  36. 36.
    Wang, L., Suarez, D. L., Pantin-Jackwood, M., Mibayashi, M., Garcia-Sastre, A., Saif, Y. M., et al. (2008). Characterization of influenza virus variants with different sizes of the non-structural (NS) genes and their potential as a live influenza vaccine in poultry. Vaccine, 26(29–30), 3580–3586.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • M. Subathra
    • 1
  • P. Santhakumar
    • 2
  • Sureddi Satyam Naidu
    • 2
  • M. Lakshmi Narasu
    • 1
  • T. M. A. Senthilkumar
    • 3
  • Sunil K. Lal
    • 4
  1. 1.Centre for BiotechnologyJawaharlal Nehru Technological UniversityHyderabadIndia
  2. 2.Indian Immunologicals LtdHyderabadIndia
  3. 3.Department of Animal BiotechnologyMadras Veterinary CollegeChennaiIndia
  4. 4.Virology GroupInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia

Personalised recommendations