Preferences help
enabled [disable] Abstract
Number of results
2014 | 61 | 3 | 561-572
Article title

Influenza virus hemagglutinin as a vaccine antigen produced in bacteria

Title variants
Languages of publication
Recombinant subunit vaccines based on hemagglutinin proteins produced in bacteria (bacterial HAs) are promising candidates for enhancing the supply of vaccines against influenza, especially for a pandemic. Over 20 years after the failure to obtain the antigen with native HA characteristics in the early 1980’s, there are increasing data on successful production of HA proteins in bacteria. The vast majority of bacterial HAs have been based on the HA1 subunit of HA expressed separately or as a component of conjugate vaccines, but those based on the ectodomain and the HA2 subunit have also been reported. The most of HAs have been efficiently expressed as insoluble aggregates called inclusion bodies. Refolded and purified proteins were extensively studied for structure, the ability to bind to sialic acid-containing receptors, antigenicity, immunogenicity and efficacy. The results from these studies contradict the view that glycosylation determines the correct structure of the hemagglutinin, as they proved that bacterial HAs can be valuable vaccine antigens when appropriate folding and purification methods are applied to rationally designed proteins. The best evidence for success in bacterial production of protective HA is that vaccines based on proprietary Toll-like Receptor (VaxInnate) and bacteriophage Qβ-VLPs (Cytos Biotechnology) technologies have been advanced to clinical studies.

Physical description
  • Institute of Biotechnology and Antibiotics, Warsaw, Poland
  • Aguilar-Yáñez JM, Portillo-Lara R, Mendoza-Ochoa GI, García-Echauri SA, López-Pacheco F, Bulnes-Abundis D, Salgado-Gallegos J, Lara-Mayorga IM, Webb-Vargas Y, León-Angel FO, Rivero-Aranda RE, Oropeza-Almazán Y, Ruiz-Palacios GM, Zertuche-Guerra MI, DuBois RM, White SW, Schultz-Cherry S, Russell CJ, Alvarez MM (2010) An influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli. PLoS One 5: e11694.
  • Atsmon J, Kate-Ilovitz E, Shaikevich D, Singer Y, Volokhov I, Haim KY, Ben-Yedidia T (2012) Safety and immunogenicity of multimeric-001 - a novel universal influenza vaccine. J Clin Immunol 32: 595-603.
  • Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22: 1399-1408.
  • Barbey-Martin C, Gigant B, Bizebard T, Calder LJ, Wharton SA, Skehel JJ, Knossow M (2002) An antibody that prevents the hemagglutinin low pH fusogenic transition. Virology 294: 70-74.
  • Biesova Z, Miller MA, Schneerson R, Shiloach J, Green KY, Robbins JB, Keith JM (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.
  • Bizebard T, Gigant B, Rigolet P, Rasmussen B, Diat O, Bösecke P, Wharton SA, Skehel JJ, Knossow M (1995) Structure of influenza virus haemagglutinin complexed with a neutralizing antibody. Nature 376: 92-94.
  • Bommakanti G, Citron MP, Hepler RW, Callahan C, Heidecker GJ, Najar TA, Lu X, Joyce JG, Shiver JW, Casimiro DR, ter Meulen J, Liang X, Varadarajan R (2010) Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci USA 107: 13701-13706.
  • Bommakanti G, Lu X, Citron MP, Najar TA, Heidecker GJ, ter Meulen J, Varadarajan R, Liang X (2012) Design of Escherichia coli-expressed stalk domain immunogens of H1N1 hemagglutinin that protect mice from lethal challenge. J Virol 86: 13434-13444.
  • Bosch BJ, Bodewes R, de Vries RP, Kreijtz JH, Bartelink W, van Amerongen G, Rimmelzwaan GF, de Haan CA, Osterhaus AD, Rottier PJ (2010) Recombinant soluble, multimeric HA and NA exhibit distinctive types of protection against pandemic swine-origin 2009 A(H1N1) influenza virus infection in ferrets. J Virol 84: 10366-10374.
  • Bright RA, Ross TM, Subbarao K, Robinson HL, Katz JM (2003) Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine. Virology 308: 270-278.
  • Chen J, Lee KH, Steinhauer DA, Stevens DJ, Skehel JJ, Wiley DC (1998) Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation. Cell 95: 409-417.
  • Chen J, Skehel JJ, Wiley DC (1999) N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil. Proc Natl Acad Sci USA 96: 8967-8972.
  • Chen J, Wharton SA, Weissenhorn W, Calder LJ, Hughson FM, Skehel JJ, Wiley DC (1995) A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation. Proc Natl Acad Sci USA 92: 12205-12209.
  • Chiu FF, Venkatesan N, Wu CR, Chou AH, Chen HW, Lian SP, Liu SJ, Huang CC, Lian WC, Chong P, Leng CH (2009) Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus. Biochem Biophys Res Commun 383: 27-31.
  • 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.
  • Davis AR, Bos T, Ueda M, Nayak DP, Dowbenko D, Compans RW (1983) Immune response to human influenza virus hemagglutinin expressed in Escherichia coli. Gene 21: 273-284.
  • Davis AR, Nayak DP, Ueda M, Hiti AL, Dowbenko D, Kleid DG (1981) Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli. Proc Natl Acad Sci USA 78: 5376-5380.
  • de Vries RP, Smit CH, de Bruin E, Rigter A, de Vries E, Cornelissen LA, Eggink D, Chung NP, Moore JP, Sanders RW, Hokke CH, Koopmans M, Rottier PJ, de Haan CA (2012) Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin. J Virol 86: 11735-11744.
  • DuBois RM, Aguilar-Yañez JM, Mendoza-Ochoa GI, Oropeza-Almazán Y, Schultz-Cherry S, Alvarez MM, White SW, Russell CJ (2011) The receptor-binding domain of influenza virus hemagglutinin produced in Escherichia coli folds into its native, immunogenic structure. J Virol 85: 865-872.
  • Dukhovlinov I, Al-Shekhadat R, Fedorova E, Stepanova L, Potapchuk M, Repko I, Rusova O, Orlov A, Tsybalova L, Kiselev O (2013) Study of immunogenicity of recombinant proteins based on hemagglutinin and neuraminidase conservative epitopes of influenza A virus. Med Sci Monit Basic Res 19: 221-227.
  • Dumon-Seignovert L, Cariot G, Vuillard L (2004) The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expr Purif 37: 203-206.
  • Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, Throsby M, Goudsmit J, Wilson IA (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324: 246-251.
  • Farahmand B, Khodabandeh M, Mahboudi F, Fotouhi F, Saleh M, Barkhordari F, Tabatabaian M, Nasab FP, Kheiri MT (2012) Influenza virus hemagglutinin: a model for protein N-glycosylation in recombinant Escherichia coli. Intervirology 55: 219-224.
  • Fleury D, Barrère B, Bizebard T, Daniels RS, Skehel JJ, Knossow M (1999) A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site. Nat Struct Biol 6: 530-534.
  • Ha Y, Stevens DJ, Skehel JJ, Wiley DC (2002) H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. EMBO J 21: 865-875.
  • Hong M, Lee PS, Hoffman RM, Zhu X, Krause JC, Laursen NS, Yoon SI, Song L, Tussey L, Crowe JE Jr, Ward AB, Wilson IA (2013) Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site. J Virol 87: 12471-12480.
  • Horthongkham N, Srihtrakul T, Athipanyasilp N, Siritantikorn S, Kantakamalakul W, Poovorawan Y, Sutthent R (2007) Specific antibody response of mice after immunization with COS-7 cell derived avian influenza virus (H5N1) recombinant proteins. J Immune Based Ther Vaccines 5: 10.
  • Jegerlehner A, Zabel F, Langer A, Dietmeier K, Jennings GT, Saudan P, Bachmann MF (2013) Bacterially produced recombinant influenza vaccines based on virus-like particles. PLoS One 8: e78947.
  • Jeon SH, Arnon R (2002) Immunization with influenza virus hemagglutinin globular region containing the receptor-binding pocket. Viral Immunol 15: 165-176.
  • Johansson BE, Brett IC (2007) Changing perspective on immunization against influenza. Vaccine 25: 3062-3065.
  • Johansson BE, Bucher DJ, Kilbourne ED (1989) Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol 63: 1239-1246.
  • Khurana S, Chearwae W, Castellino F, Manischewitz J, King LR, Honorkiewicz A, Rock MT, Edwards KM, Del Giudice G, Rappuoli R, Golding H (2010a) Vaccines with MF59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian H5N1 influenza virus. Sci Transl Med 2: 15ra5.
  • Khurana S, Larkin C, Verma S, Joshi MB, Fontana J, Steven AC, King LR, Manischewitz J, McCormick W, Gupta RK, Golding 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, Suguitan AL Jr, Rivera Y, Simmons CP, Lanzavecchia A, Sallusto F, Manischewitz J, King LR, Subbarao K, Golding H (2009) Antigenic fingerprinting of H5N1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets. PLoS Med 6: e1000049.
  • Khurana S, Verma S, Verma N, Crevar CJ, Carter DM, Manischewitz J, King LR, Ross TM, Golding H (2010b) Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus. PLoS One 5: e11548.
  • Khurana S, Verma S, Verma N, Crevar CJ, Carter DM, Manischewitz J, King LR, Ross TM, Golding H (2011b) Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomerization, hemagglutination, and cross-protective immunity in ferrets. J Virol 85: 1246-1256.
  • Kim CS, Epand RF, Leikina E, Epand RM, Chernomordik LV (2011) The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion. J Biol Chem 286: 13226-13234.
  • Krystal M, Elliott RM, Benz EW Jr, Young JF, Palese P (1982) Evolution of influenza A and B viruses: conservation of structural features in the hemagglutinin genes. Proc Natl Acad Sci USA 79: 4800-4804.
  • Lamb RA, Choppin PW (1983) The gene structure and replication of influenza virus. Annu Rev Biochem 52: 467-506.
  • Li H, Ding J, Chen YH (2003) Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus. Immunobiology 207: 305-313.
  • Liu G, Song L, Reiserova L, Trivedi U, Li H, Liu X, Noah D, Hou F, Weaver B, Tussey L (2012) Flagellin-HA vaccines protect ferrets and mice against H5N1 highly pathogenic avian influenza virus (HPAIV) infections. Vaccine 30: 6833-6838.
  • Liu G, Tarbet B, Song L, Reiserova L, Weaver B, Chen Y, Li H, Hou F, Liu X, Parent J, Umlauf S, Shaw A, Tussey L (2011) Immunogenicity and efficacy of flagellin-fused vaccine candidates targeting 2009 pandemic H1N1 influenza in mice. PLoS One 6: e20928.
  • Loeffen WL, de Vries RP, Stockhofe N, van Zoelen-Bos D, Maas R, Koch G, Moormann RJ, Rottier PJ, de Haan CA (2011) Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus. Vaccine 29: 1545-1550.
  • McMurry JA, Johansson BE, De Groot AS (2008) A call to cellular & humoral arms: enlisting cognate T cell help to develop broad-spectrum vaccines against influenza A. Hum Vaccin 4: 148-157.
  • Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260: 289-298.
  • Nayak DP, Davis AR, McQueen NL, Bos TJ, Jabbar MA, Sivasubramanian N, Lionelli G (1985) Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cDNAs in prokaryotic and eukaryotic cells. Vaccine 3 (3 Suppl): 165-171.
  • Okuno Y, Isegawa Y, Sasao F, Ueda S (1993) A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol 67: 2552-2558.
  • Osterholm MT, Kelly NS, Manske JM, Ballering KS, Leighton TR, Moore KA (2012) The compelling need for game-changing influenza vaccines: an analysis of the influenza vaccine enterprise and recommendations for the future. University of Minnesota Center for Infectious Disease Research and Policy.
  • Park MH, Chang J (2012) Immunogenicity and protective efficacy of a dual subunit vaccine against respiratory syncytial virus and influenza virus. Immune Netw 12: 261-268.
  • Pica N, Palese P (2013) Toward a universal influenza virus vaccine: prospects and challenges. Annu Rev Med 64: 189-202.
  • Roberts PC, Garten W, Klenk HD (1993) Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin. J Virol 67: 3048-3060.
  • Rudolph W, Ben Yedidia T (2011) A universal influenza vaccine: where are we in the pursuit of this 'Holy Grail'? Hum Vaccin 7: 10-11.
  • Saelens X, Vanlandschoot P, Martinet W, Maras M, Neirynck S, Contreras R, Fiers W, Jou WM (1999) Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin. Eur J Biochem 260: 166-175.
  • Sánchez-Arreola PB, López-Uriarte S, Marichal-Gallardo PA, González-Vázquez JC, Pérez-Chavarría R, Soto-Vázquez P, López-Pacheco F, Ramírez-Medrano A, Rocha-Pizaña MR, Alvarez MM (2013) A baseline process for the production, recovery, and purification of bacterial influenza vaccine candidates. Biotechnol Prog 29: 896-908.
  • Shen S, Mahadevappa G, Oh HL, Wee BY, Choi YW, Hwang LA, Lim SG, Hong W, Lal SK, 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.
  • Shih AC, Hsiao TC, Ho MS, Li WH (2007) Simultaneous amino acid substitutions at antigenic sites drive influenza A hemagglutinin evolution. Proc Natl Acad Sci USA 104: 6283-6288.
  • Skehel JJ, Cross K, Steinhauer D, Wiley DC (2001) Influenza fusion peptides. Biochem Soc Trans 29: 623-626.
  • Skehel JJ, Stevens DJ, Daniels RS, Douglas AR, Knossow M, Wilson IA, Wiley DC (1984) A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci USA 81: 1779-1783.
  • Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69: 531-569.
  • Skibinski DA, Hanson BJ, Lin Y, von Messling V, Jegerlehner A, Tee JB, Chye de H, Wong SK, Ng AA, Lee HY, Au B, Lee BT, Santoso L, Poidinger M, Fairhurst AM, Matter A, Bachmann MF, Saudan P, Connolly JE (2013) Enhanced neutralizing antibody titers and Th1 polarization from a novel Escherichia coli derived pandemic influenza vaccine. PLoS One 8: e76571.
  • Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, Jacobs A, Liu G, Huang Y, Desai P, Maksymiuk G, Takahashi V, Umlauf S, Reiserova L, Bell R, Li H, Zhang Y, McDonald WF, Powell TJ, Tussey L (2008) Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs. PLoS One 3: e2257.
  • Song L, Zhang Y, Yun NE, Poussard AL, Smith JN, Smith JK, Borisevich V, Linde JJ, Zacks MA, Li H, Kavita U, Reiserova L, Liu X, Dumuren K, Balasubramanian B, Weaver B, Parent J, Umlauf S, Liu G, Huleatt J, Tussey L, Paessler S (2009) Superior efficacy of a recombinant flagellin: H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin. Vaccine 27: 5875-5884.
  • Steel J, Lowen AC, Wang TT, Yondola M, Gao Q, Haye K, García-Sastre A, Palese P (2010) Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio 1: e00018-10.
  • Suarez DL (2005) Overview of avian influenza DIVA test strategies. Biologicals 33: 221-226.
  • Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen LM, Santelli E, Stec B, Cadwell G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox NJ, Bankston LA, Donis RO, Liddington RC, Marasco WA (2009) Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273.
  • Sylte MJ, Suarez DL (2009) Influenza neuraminidase as a vaccine antigen. Curr Top Microbiol Immunol 333: 227-241.
  • Taylor DN, Treanor JJ, Sheldon EA, Johnson C, Umlauf S, Song L, Kavita U, Liu G, Tussey L, Ozer K, Hofstaetter T, Shaw A (2012) Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin fusion vaccine with improved safety and immune response. Vaccine 30: 5761-5769.
  • Taylor DN, Treanor JJ, Strout C, Johnson C, Fitzgerald T, Kavita U, Ozer K, Tussey L, Shaw A (2011) Induction of a potent immune response in the elderly using the TLR-5 agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125, STF2.HA1 SI). Vaccine 29: 4897-4902.
  • Treanor JJ, Taylor DN, Tussey L, Hay C, Nolan C, Fitzgerald T, Liu G, Kavita U, Song L, Dark I, Shaw A (2010) Safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125) in healthy young adults. Vaccine 28: 8268-8274.
  • Verma S, Dimitrova M, Munjal A, Fontana J, Crevar CJ, Carter DM, Ross TM, Khurana S, Golding 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.
  • Wacker M, Linton D, Hitchen PG, Nita-Lazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW, Aebi M (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298: 1790-1793.
  • Wang CC, Chen JR, Tseng YC, Hsu CH, Hung YF, Chen SW, Chen CM, Khoo KH, Cheng TJ, Cheng YS, Jan JT, Wu CY, Ma C, Wong CH (2009) Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc Natl Acad Sci USA 106: 18137-18142.
  • Wei CJ, Xu L, Kong WP, Shi W, Canis K, Stevens J, Yang ZY, Dell A, Haslam SM, Wilson IA, Nabel GJ (2008) Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol 82: 6200-6208.
  • Weldon WC, Wang BZ, Martin MP, Koutsonanos DG, Skountzou I, Compans RW (2010) Enhanced immunogenicity of stabilized trimeric soluble influenza hemagglutinin. PLoS One 5: e12466.
  • Wiley DC, Skehel JJ (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem 56: 365-394.
  • Wiley DC, Wilson IA, Skehel JJ (1981) Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289: 373-378.
  • Wilson IA, Cox NJ (1990) Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8: 737-771.
  • Wilson IA, Skehel JJ, Wiley DC (1981) Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289: 366-373.
  • Xie QM, Ji J, Du LQ, Cao YC, Wei L, Xue CY, Qin JP, Ma JY, Bi YZ (2009) Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine. Poult Sci 88: 1608-1615.
  • Xuan C, Shi Y, Qi J, Zhang W, Xiao H, Gao GF (2011) Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines. Protein Cell 2: 997-1005.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.