Prokaryotic Production of Influenza Virus M2 Protein Fused to Leishmania Major HSP70 in Order to Prepare an Effective Flu Vaccine

Document Type : Original Article (s)

Authors

1 Department of Microbiology, School of Biological Science, Islamic Azad University, Qom Branch, Qom, Iran

2 Assistant Professor, Department of Virology, Influenza Research Lab, Pasteur Institute of Iran, Tehran, Iran

3 Assistant Professor, Department of Medical Virology and Immunology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran

4 Department of Virology, Influenza Research Lab, Pasteur Institute of Iran, Tehran, Iran

5 Lab Instructor, Influenza Research Lab, Pasteur Institute of Iran, Tehran, Iran

Abstract

Background: Influenza is a major cause of morbidity and mortality in the world. Permanent antigenic variation of influenza virus A causes a major concern to develop influenza vaccine. Now, some researchers are focusing on conserved antigens. The M2 protein is a proton-selective ion channel, integral in the viral envelope of the influenza virus A and allows the virus to enter and cause an infection in the host cells. This protein is conserved among all types of influenza virus A and an appropriate target for the development of influenza vaccine with broad-spectrum protection. In this study, Leishmania major heat shock protein-70 fused to M2 protein to prepare an effective vaccine against influenza virus A.Methods: Lm. HSP70 gene was cloned into BamHI and HindIII sites of pET28a vector. Influenza virus M2 gene was amplified via polymerase chain reaction (PCR) using specific primers and cloned into dephosphorylated linear pET28a vector upstream of Leishmania major HSP70 gene. The confirmed construct was transformed into Escherichia coli BL21 and protein expression was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG).Findings: The recombinant plasmids were confirmed via colony PCR, restriction enzyme analysis and sequencing. The result of sequencing revealed that the M2 gene was properly cloned into pET28a-HSP70 in the right frame to 6xhis tag. The protein expression was determined using Western blot analysis.Conclusion: Binding of HSP to the desired antigen induces increased level of immune responses. Hence, the chimer protein prepared in this study, could be an appropriate vaccine candidate to prevent influenza infection. The immunogenicity of this chimer protein with different formulation is going to evaluate in animal models. To investigate the effect of HSP on M2 immunogenicity, this chimer protein will be evaluated in future projects.

Keywords


  1. Rothberg MB, Haessler SD, Brown RB. Complications of viral influenza. Am J Med 2008; 121(4): 258-64.
  2. Nelson MI, Holmes EC. The evolution of epidemic influenza. Nat Rev Genet 2007; 8(3): 196-205.
  3. Zimmer G. RNA replicons - a new approach for influenza virus immunoprophylaxis. Viruses 2010; 2(2): 413-34.
  4. Cox RJ, Brokstad KA, Ogra P. Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol 2004; 59(1): 1-15.
  5. Gerdil C. The annual production cycle for influenza vaccine. Vaccine 2003; 21(16): 1776-9.
  6. Lamb RA, Lai CJ, Choppin PW. Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A 1981; 78(7): 4170-4.
  7. Wu F, Huang JH, Yuan XY, Huang WS, Chen YH. Characterization of immunity induced by M2e of influenza virus. Vaccine 2007; 25(52): 8868-73.
  8. Zebedee SL, Lamb RA. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol 1988; 62(8): 2762-72.
  9. Treanor JJ, Tierney EL, Zebedee SL, Lamb RA, Murphy BR. Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J Virol 1990; 64(3): 1375-7.
  10. Torok Z, Tsvetkova NM, Balogh G, Horvath I, Nagy E, Penzes Z, et al. Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase. Proc Natl Acad Sci U S A 2003; 100(6): 3131-6.
  11. Tamura Y, Saito K, Sato N. Heat shock protein inhibitor for molecular targeting therapy. Nihon Rinsho 2012; 70(Suppl 8): 135-9. (In Japanese).
  12. Rafati S, Gholami E, Hassani N, Ghaemimanesh F, Taslimi Y, Taheri T, et al. Leishmania major heat shock protein 70 (HSP70) is not protective in murine models of cutaneous leishmaniasis and stimulates strong humoral responses in cutaneous and visceral leishmaniasis patients. Vaccine 2007; 25(21): 4159-69.
  13. Esghaei M, Monavari SH, Tavassoti-Kheiri M, Shamsi-Shahrabadi M, Heydarchi B, Farahmand B, et al. Expression of the influenza M2 protein in three different eukaryotic cell lines. J Virol Methods 2012; 179(1): 161-5.
  14. Alavi-Esfahani MA, Fotouhi-Chahooki F, Saleh M, Tavakoli R, Farahmand B, Ghaemi A, et al. Over expression of influenza virus M2 protein in prokaryotic system. Iran J Virol 2012; 6(4): 13-9
  15. Fu TM, Freed DC, Horton MS, Fan J, Citron MP, Joyce JG, et al. Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus. Virology 2009; 385(1): 218-26.
  16. Slepushkin VA, Katz JM, Black RA, Gamble WC, Rota PA, Cox NJ. Protection of mice against influenza A virus challenge by vaccination with baculovirus-expressed M2 protein. Vaccine 1995; 13(15): 1399-402.
  17. Okuda K, Ihata A, Watabe S, Okada E, Yamakawa T, Hamajima K, et al. Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene. Vaccine 2001; 19(27): 3681-91.
  18. Zhao G, Du L, Xiao W, Sun S, Lin Y, Chen M, et al. Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant. Vaccine 2010; 28(44): 7233-40.
  19. Chen W, Lin Y, Liao C, Hsieh S. Modulatory effects of the human heat shock protein 70 on DNA vaccination. J Biomed Sci 2000; 7(5): 412-9.
  20. Li J, Li KN, Gao J, Cui JH, Liu YF, Yang SJ. Heat shock protein 70 fused to or complexed with hantavirus nucleocapsid protein significantly enhances specific humoral and cellular immune responses in C57BL/6 mice. Vaccine 2008; 26(25): 3175-87.
  21. Ebrahimi SM, Tebianian M, Toghyani H, Memarnejadian A, Attaran HR. Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris. Protein Expr Purif 2010; 70(1): 7-12.
  22. Ebrahimi SM, Tebianian M. Heterologous expression, purification and characterization of the influenza A virus M2e gene fused to Mycobacterium tuberculosis HSP70(359-610) in prokaryotic system as a fusion protein. Mol Biol Rep 2010; 37(6): 2877-83.
  23. Shaw A. Conserved proteins as potential universal vaccines. In: Rappuoli R, Giudice GD, editors. Influenza vaccines for the future. New York, NY: Springer; 2011. p. 313-25.
  24. Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci 2010; Chapter 5: Unit 5.24.1-29.