Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system

Document Type : Full paper (Original article)


1 Ph.D. Student in Plant Biotechnology, Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

3 Influenza Research Lab, Pasteur Institute of Iran, Tehran, Iran

4 Razi Vaccine and Serum Research Institute, Mashhad, Iran


The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. In this study, bacterial system has been developed for expression and purification of properly folded HA1 antigen as a rapid response to emerging pandemic strains. Here, a recombinant H5N1 (A/Indonesia/05/05) hemagglutinin globular domain, the synthesized HA1 (1-320 amino acids), was amplified and cloned into pET-28a bacterial expression vector. Then, his-tagged HA1 protein was expressed in Escherichia coli BL21 under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA chromatography. Migration size of protein was detected at 40 KDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight. Since most antigenic sites are in the HA1 domain of HA, using this domain of influenza virus as antigen is of great importance in vaccine development. The ability of the antibody stimulation against HA1 expressed in bacterial cells is also examined using enzyme-linked immunosorbent assay (ELISA) analysis. Upon immunization of rabbits, oligomeric HA1 elicited potent neutralizing antibodies and high levels of serum antibody binding to HA1. Our findings suggest that HA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.


Biesova, Z; Miller, MA; Schneerson, R; Shiloach, J; Green, KY; Robbins, JB and Keith, JM (2009). Preparation, characterization, and immunogenicity in mice of a re-combinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus. Vaccine. 27: 6234-6238.
Bradford, MM (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.
Chiu, FF; Venkatesan, N; Wu, CR; Chou, AH; Chen, HW; Lian, SP; Liu, SJ; Huang, CC; Lian, WC; Chong, P and Leng, CH (2009). Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus. Biochem. Biophys. Res. Commun., 383: 27-31.
Dilillo, DJ; Tan, GS; Palese, P and Ravetch, JV (2014). Broadly neutralizing hemagglutinin stalk-specific anti-bodies require Fcgamma R interactions for protection against influenza virus in vivo. Nature Med., 20: 143-151.
Janknecht, R; de Martynoff, G; Lou, J; Hipskind, RA; Nordheim, A and Stunnenberg, HG (1991). Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc. Natl. Acad. Sci. U. S. A., 88: 8972-8976.
Johansson, BE and Brett, IC (2007). Changing perspective on immunization against influenza. Vaccine. 25: 3062-3065.
Kanekiyo, M; Wei, CJ; Yassine, HM; McTamney, PM; Boyington, JC; Whittle, JR; Rao, SS; Kong, WP; Wang, L and Nabel, GJ (2013). Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature. 499: 102-106.
Khurana, S; Larkinb, C; Vermaa, S; Joshib, MB; Fontanac, J; Stevenc, AC; Kinga, LR; Manischewitza, J; McCormickb, W; Guptab, RK and Goldinga, H (2011a). Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines. Vaccine. 29: 5657-5665.
Khurana, S; Verma, S; Verma, N; Crevar, CJ; Carter, DM; Manischewitz, J; King, LR; Ross, TM and Golding, H (2010). Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza
virus. PLoS One. 5: e11548. doi: 10.1371/journal.pone. 0011548.
Khurana, S; Verma, S; Verma, N; Crevar, CJ; Carter, DM; Manischewitz, J; King, LR; Ross, TM and Golding, H (2011b). Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomeriza-tion, hemagglutination, and crossprotective immunity in ferrets. J. Virol., 85: 1246-1256.
Lin, YJ; Deng, MC; Wu, SH; Chen, YL; Cheng, HC; Chang, CY; Lee, MS; Chien, MS and Huang, CC (2008). Baculovirus derived hemagglutinin vaccine protects chickens from lethal homologous virus H5N1 challenge. J. Vet. Med. Sci., 70: 1147-1152.
Olsen, B; Munster, VJ; Wallensten, A; Waldenstrom, J; Osterhaus, ADME and Fouchier, RAM (2006). Global patterns of influenza A virus in wild birds. Science. 312: 384-388.
Shen, S; Mahadevappa, G; Oh, HL; Wee, BY; Choi, YW; Hwang, LA; Lim, SG; Hong, W; Lal, SK and Tan, YJ (2008). Comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza A (H5N1) virus expressed in insect cells and bacteria. J. Med. Virol., 80: 1972-1983.
Shoji, Y; Bi, H; Musiychuk, K; Rhee, A; Horsey, A; Roy, G; Green, B; Shamloul, M; Farrance, CE; Taggart, B; Mytle, N; Ugulava, N; Rabindran, S; Mett, V; Chichester, JA and Yusibov, V (2009). Plant-derived hemagglutinin protects ferrets against challenge infection with the A/Indonesia/05/05 strain of avian influenza. Vaccine. 27: 1087-1992.
Shoji, Y; Chichester, JA; Jones, M; Manceva, SD and Damon, E (2011). Plant based rapid production of recombinant subunit hemagglutinin vaccine targeting H1N1 and H5N1 influenza. Hum. Vaccines. 7: 41-50.
Stevens, J; Blixt, O; Tumpey, TM; Taubenberger, JK; Paulson, JC and Wilson, IA (2006). Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science. 312: 404-410.
Swayne, DE and Suarez, DL (2000). Highly pathogenic avian influenza. Rev. Sci. Tech., 19: 463-482.
Tonegawa, K; Nobusawa, E; Nakajima, K; Kato, T; Kutsuna, T and Kuroda, K (2003). Analysis of epitope recognition of antibodies induced by DNA immunization against hemagglutinin protein of influenza A virus. Vaccine. 21: 3118-3125.
Treanor, JJ; Campbell, JD; Zangwill, KM; Rowe, T and Wolff, M (2006). Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N. Engl. J. Med., 354: 1343-1351.
Tsai, HJ; Chi, LA and Yu, AL (2012). Monoclonal antibodies targeting the synthetic peptide corresponding to the polybasic cleavage site on H5N1 influenza hemagglutinin. J. Biomed. Sci., 19: 37-44.
Verma, S; Dimitrova, M; Munjal, A; Fontana, J; Crevar, CJ; Carter, DM; Ross, TM; Khurana, S and Goldinga, H (2012). Oligomeric recombinant H5 HA1 vaccine produced in bacteria protects ferrets from homologous and heterologous wild-type H5N1 influenza challenge and controls viral loads better than subunit H5N1 vaccine by eliciting high-affinity antibodies. J. Virol., 86: 12283-12293.
Wei, C; Nurul, T; Wahida, AG and Shaharum, S (2014). Construction and heterologous expression of a truncated haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli. Trop. Biomed., 31: 1-10.