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Research article

Clinical Microbiology and Biomedical Sciences

Insect Cell Surface Expression of Hemagglutinin (HA) of Egyptian H5N1 Avian Influenza Virus Under Transcriptional Control of Whispovirus Immediate Early-1 Promoter

Mohamed Rasheed Gadalla 1, Ayman Hany El-Deeb 1, Mohamed Maged Emara 1, 2 and Hussein Aly Hussein 1*

1Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt, 2Qatar Biomedical Research Institute, Qatar Foundation, Education City, 5825, Doha, Qatar

Received: May 12, 2014; Accepted: August 6, 2014

J. Microbiol. Biotechnol. 2014; 24(12): 1719-1727

Published December 28, 2014 https://doi.org/10.4014/jmb.1405.05028

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

Keywords

Avian influenza, Hemagglutinin, Baculovirus, Whispovirus immediate early-1, Promoter

References

  1. Chen CY, Liu HJ, Tsai CP, Chung CY, Shih YS, Chang PC, et al. 2010. Baculovirus as an avian influenza vaccine vector:differential immune responses elicited by different vector forms. Vaccine 28: 7644-7651.
    Pubmed CrossRef
  2. Choi JG, Kim MC, Kang HM, Kim KI, Lee KJ, Park CK, et al. 2013. Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens. Vet. Microbiol. 162: 623-630.
    Pubmed CrossRef
  3. Cornelissen LA, de Vries RP, de Boer-Luijtze EA, Rigter A, Rottier PJ, de Haan CA. 2010. A single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus H5N1. PLoS One 5: e10645.
    Pubmed CrossRef
  4. Cox MM. 2012. Recombinant protein vaccines produced in insect cells. Vaccine 30: 1759-1766.
    Pubmed CrossRef
  5. Crawford AM, Miller LK. 1988. Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 62:2773-2781.
    Pubmed
  6. Gao H, Wang Y, Li N, Peng WP, Sun Y, Tong GZ, Qiu HJ. 2007. Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells. J. Biotechnol. 131: 138-143.
    Pubmed CrossRef
  7. He F, Ho Y, Yu L, Kwang J. 2008. WSSV ie1 promoter is more efficient than CMV promoter to express H5 hemagglutinin from influenza virus in baculovirus as a chicken vaccine. BMC Microbiol. 8: 238.
    Pubmed CrossRef
  8. He F, Madhan S, Kwang J. 2009. Baculovirus vector as a delivery vehicle for influenza vaccines. Expert Rev. Vaccines 8: 455-467.
    Pubmed CrossRef
  9. Hitchman RB, Possee RD, King LA. 2009. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat. Biotechnol. 3: 46-54.
    Pubmed CrossRef
  10. Hu YC, Luo YL, Ji WT, Chulu JL, Chang PC, Shieh H, et al. 2006. Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system. J. Virol. Methods 135: 43-48.
    Pubmed CrossRef
  11. Hu YC, Yao K, Wu TY. 2008. Baculovirus as an expression and/or delivery vehicle for vaccine antigens. Expert Rev. Vaccines 7: 363-371.
    Pubmed CrossRef
  12. Killian ML. 2008. Hemagglutination assay for the avian influenza virus. Methods Mol. Biol. 436: 47-52.
    Pubmed
  13. Kretzschmar E, Kuroda K, Klenk HD. 1995. Expression of the hemagglutinin protein of influenza virus. Analysis of protein modifications. Methods Mol. Biol. 39: 317-336.
    Pubmed
  14. Kuroda K, Hauser C, Rott R, Klenk HD, Doerfler W. 1986. Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO J. 5: 1359-1365.
    Pubmed
  15. Lee C W, S enne DA, S uarez DL. 2 004. Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (differentiating infected from vaccinated animals) strategy for the control of avian influenza. Vaccine 22: 3175-3181.
    Pubmed CrossRef
  16. Lin YJ, Deng MC, Wu SH, Chen YL, Cheng HC, Chang CY, et al. 2008. Baculovirus-derived hemagglutinin vaccine protects chickens from lethal homologous virus H5N1 challenge. J. Vet. Med. Sci. 70: 1147-1152.
    Pubmed CrossRef
  17. Liu G, Zhang F, Shi J, Tian G, Chen H, Yu K, Meng Q. 2013. A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge. Vaccine 31: 5398-5404.
    Pubmed CrossRef
  18. Liu WJ, Chang YS, Wang CH, Kou GH, Lo CF. 2005. Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp. Virology 334: 327-341.
    Pubmed CrossRef
  19. Liu YK, Chu CC, Wu TY. 2006. Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells. Acta Pharmacol. Sin. 27: 321-327.
    Pubmed CrossRef
  20. Lu L, Yu L, Kwang J. 2007. Baculovirus surface-displayed hemagglutinin of H5N1 influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity. Biochem. Biophys. Res. Commun. 358: 404-409.
    Pubmed CrossRef
  21. Madhan S, Prabakaran M, Kwang J. 2010. Baculovirus as vaccine vectors. Curr. Gene Ther. 10: 201-213.
    Pubmed CrossRef
  22. Mueller M, Renzullo S, Brooks R, Ruggli N, Hofmann MA. 2010. Antigenic characterization of recombinant hemagglutinin proteins derived from different avian influenza virus subtypes. PLoS One 5: e9097.
    Pubmed CrossRef
  23. O'Reilly DR. 1997. Use of baculovirus expression vectors. Methods Mol. Biol. 62: 235-246.
    Pubmed
  24. Palomares LA, Estrada-Mondaca S, Ramirez OT. 2004. Production of recombinant proteins: challenges and solutions. Methods Mol. Biol. 267: 15-52.
    Pubmed
  25. Pedersen JC. 2008. Hemagglutination-inhibition test for avian influenza virus subtype identification and the detection and quantitation of serum antibodies to the avian influenza virus. Methods Mol. Biol. 436: 53-66.
    Pubmed
  26. Possee RD. 1986. Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. Virus Res. 5: 43-59.
    CrossRef
  27. Prabakaran M, Madhan S, Prabhu N, Qiang J, and Kwang J. 2010. Gastrointestinal delivery of baculovirus displaying influenza virus hemagglutinin protects mice against heterologous H5N1 infection. J. Virol. 84: 3201-3209.
    Pubmed CrossRef
  28. Treanor J. 2009. Recombinant proteins produced in insect cells. Curr. Top Microbiol. Immunol. 333: 211-225.
    Pubmed CrossRef
  29. Treanor JJ, Wilkinson BE, 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: 1732-1737.
    CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2014; 24(12): 1719-1727

Published online December 28, 2014 https://doi.org/10.4014/jmb.1405.05028

Copyright © The Korean Society for Microbiology and Biotechnology.

Insect Cell Surface Expression of Hemagglutinin (HA) of Egyptian H5N1 Avian Influenza Virus Under Transcriptional Control of Whispovirus Immediate Early-1 Promoter

Mohamed Rasheed Gadalla 1, Ayman Hany El-Deeb 1, Mohamed Maged Emara 1, 2 and Hussein Aly Hussein 1*

1Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt, 2Qatar Biomedical Research Institute, Qatar Foundation, Education City, 5825, Doha, Qatar

Received: May 12, 2014; Accepted: August 6, 2014

Abstract

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate
surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus
(AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1
promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully
cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into
DH10Bac competent cells, and the recombinant bacmid was generated via site-specific
transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9)
insect cells to construct the recombinant baculovirus and to induce expression of the HA
protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed
hemadsorption activity. Hemagglutination activity was also detected in both extra- and
intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by
reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was
observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE
analysis of the expressed protein revealed the presence of a visually distinguishable band of
~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis
confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of
H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The
expressed protein was displayed on the plasma membrane of infected insect cells under the
control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

Keywords: Avian influenza, Hemagglutinin, Baculovirus, Whispovirus immediate early-1, Promoter

References

  1. Chen CY, Liu HJ, Tsai CP, Chung CY, Shih YS, Chang PC, et al. 2010. Baculovirus as an avian influenza vaccine vector:differential immune responses elicited by different vector forms. Vaccine 28: 7644-7651.
    Pubmed CrossRef
  2. Choi JG, Kim MC, Kang HM, Kim KI, Lee KJ, Park CK, et al. 2013. Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens. Vet. Microbiol. 162: 623-630.
    Pubmed CrossRef
  3. Cornelissen LA, de Vries RP, de Boer-Luijtze EA, Rigter A, Rottier PJ, de Haan CA. 2010. A single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus H5N1. PLoS One 5: e10645.
    Pubmed CrossRef
  4. Cox MM. 2012. Recombinant protein vaccines produced in insect cells. Vaccine 30: 1759-1766.
    Pubmed CrossRef
  5. Crawford AM, Miller LK. 1988. Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 62:2773-2781.
    Pubmed
  6. Gao H, Wang Y, Li N, Peng WP, Sun Y, Tong GZ, Qiu HJ. 2007. Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells. J. Biotechnol. 131: 138-143.
    Pubmed CrossRef
  7. He F, Ho Y, Yu L, Kwang J. 2008. WSSV ie1 promoter is more efficient than CMV promoter to express H5 hemagglutinin from influenza virus in baculovirus as a chicken vaccine. BMC Microbiol. 8: 238.
    Pubmed CrossRef
  8. He F, Madhan S, Kwang J. 2009. Baculovirus vector as a delivery vehicle for influenza vaccines. Expert Rev. Vaccines 8: 455-467.
    Pubmed CrossRef
  9. Hitchman RB, Possee RD, King LA. 2009. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat. Biotechnol. 3: 46-54.
    Pubmed CrossRef
  10. Hu YC, Luo YL, Ji WT, Chulu JL, Chang PC, Shieh H, et al. 2006. Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system. J. Virol. Methods 135: 43-48.
    Pubmed CrossRef
  11. Hu YC, Yao K, Wu TY. 2008. Baculovirus as an expression and/or delivery vehicle for vaccine antigens. Expert Rev. Vaccines 7: 363-371.
    Pubmed CrossRef
  12. Killian ML. 2008. Hemagglutination assay for the avian influenza virus. Methods Mol. Biol. 436: 47-52.
    Pubmed
  13. Kretzschmar E, Kuroda K, Klenk HD. 1995. Expression of the hemagglutinin protein of influenza virus. Analysis of protein modifications. Methods Mol. Biol. 39: 317-336.
    Pubmed
  14. Kuroda K, Hauser C, Rott R, Klenk HD, Doerfler W. 1986. Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO J. 5: 1359-1365.
    Pubmed
  15. Lee C W, S enne DA, S uarez DL. 2 004. Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (differentiating infected from vaccinated animals) strategy for the control of avian influenza. Vaccine 22: 3175-3181.
    Pubmed CrossRef
  16. Lin YJ, Deng MC, Wu SH, Chen YL, Cheng HC, Chang CY, et al. 2008. Baculovirus-derived hemagglutinin vaccine protects chickens from lethal homologous virus H5N1 challenge. J. Vet. Med. Sci. 70: 1147-1152.
    Pubmed CrossRef
  17. Liu G, Zhang F, Shi J, Tian G, Chen H, Yu K, Meng Q. 2013. A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge. Vaccine 31: 5398-5404.
    Pubmed CrossRef
  18. Liu WJ, Chang YS, Wang CH, Kou GH, Lo CF. 2005. Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp. Virology 334: 327-341.
    Pubmed CrossRef
  19. Liu YK, Chu CC, Wu TY. 2006. Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells. Acta Pharmacol. Sin. 27: 321-327.
    Pubmed CrossRef
  20. Lu L, Yu L, Kwang J. 2007. Baculovirus surface-displayed hemagglutinin of H5N1 influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity. Biochem. Biophys. Res. Commun. 358: 404-409.
    Pubmed CrossRef
  21. Madhan S, Prabakaran M, Kwang J. 2010. Baculovirus as vaccine vectors. Curr. Gene Ther. 10: 201-213.
    Pubmed CrossRef
  22. Mueller M, Renzullo S, Brooks R, Ruggli N, Hofmann MA. 2010. Antigenic characterization of recombinant hemagglutinin proteins derived from different avian influenza virus subtypes. PLoS One 5: e9097.
    Pubmed CrossRef
  23. O'Reilly DR. 1997. Use of baculovirus expression vectors. Methods Mol. Biol. 62: 235-246.
    Pubmed
  24. Palomares LA, Estrada-Mondaca S, Ramirez OT. 2004. Production of recombinant proteins: challenges and solutions. Methods Mol. Biol. 267: 15-52.
    Pubmed
  25. Pedersen JC. 2008. Hemagglutination-inhibition test for avian influenza virus subtype identification and the detection and quantitation of serum antibodies to the avian influenza virus. Methods Mol. Biol. 436: 53-66.
    Pubmed
  26. Possee RD. 1986. Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. Virus Res. 5: 43-59.
    CrossRef
  27. Prabakaran M, Madhan S, Prabhu N, Qiang J, and Kwang J. 2010. Gastrointestinal delivery of baculovirus displaying influenza virus hemagglutinin protects mice against heterologous H5N1 infection. J. Virol. 84: 3201-3209.
    Pubmed CrossRef
  28. Treanor J. 2009. Recombinant proteins produced in insect cells. Curr. Top Microbiol. Immunol. 333: 211-225.
    Pubmed CrossRef
  29. Treanor JJ, Wilkinson BE, 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: 1732-1737.
    CrossRef