title: Study of immunogenicity of recombinant proteins based on hemagglutinin and neuraminidase conservative epitopes of influenza A virus.
abstract: BACKGROUND
  Recombinant hemagglutinin (rHA) and neurominidase (rNA) developed in our investigation are amino acid sequence consensus variants of H1N1 2009 subtype influenza virus strain, also including immunogenic epitopes typical for other influenza virus subtypes (H3N1 and H5N1). Substitutions were made: typical for Russian virus isolates (in HA - S220T, NA - D248N) and in active centers of molecules - R118L, R293L, R368L; C92S, C417S to increase recombinant proteins stability in E. coli. The aim of the present work was to study immunogenicity of the obtained rHA and rNA.
  
  
  METHODS
  Fragments aa 83-469 of NA and aa 61-287 of HA were chosen because they include the main B-cell epitopes and are the minimal structures for correct folding of target proteins. The designed nucleotide sequences were synthesized and purified and the expression of rNA and rNA were analyzed. For immunization and virus challenge we used influenza viruses A/California/04/2009 (H1N1), A/PR/8/34 (H1N1), A/Perth/16/2009 (H3N2), A/Chicken/Kurgan/05/2005 R.G. (H5N1), and B/Florida/04/2006. Specific IgG levels were determined by ELISA in 96-well ELISA plates. Significant differences of survival in mouse groups were analyzed by Mantel-Cox (log-rank) and Gehan-Breslow-Wilcoxon tests.
  
  
  RESULTS
  The obtained results demonstrate the high immunogenicity and ability of indicated proteins mixture to provide similar cross-protection against influenza viruses of the H1N1 subtype.
  
  
  CONCLUSIONS
  The data obtained suggest efficient pluripotent vaccine creation based on HA and NA conservative regions.
authors: LLC Pharma Gene, Saint-Petersburg, Russia. hiv-1111@mail.ru
authors: Ilya
authors: I
authors: Dukhovlinov
authors: Ruslan
authors: R
authors: Al-Shekhadat
authors: Ekaterina
authors: E
authors: Fedorova
authors: Ludmila
authors: L
authors: Stepanova
authors: Marina
authors: M
authors: Potapchuk
authors: Irina
authors: I
authors: Repko
authors: Olga
authors: O
authors: Rusova
authors: Anton
authors: A
authors: Orlov
authors: Ludmila
authors: L
authors: Tsybalova
authors: Oleg
authors: O
authors: Kiselev
mesh_terms: D000818:Animals; D001835:Body Weight; D000939:Epitopes / Q000276:immunology*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology* / Q000302:isolation & purification; D007114:Immunization; D007074:Immunoglobulin G / Q000097:blood; D009980:Influenza A virus / Q000276:immunology*; D051379:Mice; D009439:Neuraminidase / Q000276:immunology* / Q000302:isolation & purification; D009976:Orthomyxoviridae Infections / Q000097:blood / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology* / Q000302:isolation & purification; D016019:Survival Analysis
chemical_list: D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D009439:Neuraminidase
references: Mol Cell Biol. 1985 Sep;5(9):2181-9
references: 3016520
references: Intervirology. 2007;50(1):58-62
references: 17164559
references: J Virol. 2005 Mar;79(5):2814-22
references: 15709000
references: Hum Immunol. 2010 May;71(5):468-74
references: 20156506
references: Science. 2009 Apr 10;324(5924):246-51
references: 19251591
references: Clin Vaccine Immunol. 2009 Jun;16(6):824-9
references: 19386796
references: Nat Struct Mol Biol. 2009 Mar;16(3):265-73
references: 19234466
references: Immunol Lett. 2008 Jan 29;115(2):144-52
references: 18160106
references: Prog Clin Biol Res. 1980;47:367-75
references: 7010377
references: Gene. 1992 Jan 2;110(1):89-94
references: 1544581
references: Annu Rev Biochem. 2000;69:531-69
references: 10966468
references: Nucleic Acids Res. 2004 Apr 15;32(7):e59
references: 15087491
references: Vaccine. 2009 Oct 23;27(45):6330-9
references: 19840669
references: PLoS Pathog. 2009 Mar;5(3):e1000350
references: 19300497
references: Vaccine. 2006 Mar 15;24(12):2176-85
references: 16310896
references: Vaccine. 2002 Jun 21;20(21-22):2772-80
references: 12034104
references: Arch Med Res. 2009 Nov;40(8):643-54
references: 20304251
references: J Clin Microbiol. 1986 Jul;24(1):157-60
references: 3722363
references: Vaccine. 2009 Oct 19;27(44):6234-8
references: 19686692
references: Science. 2010 Aug 27;329(5995):1060-4
references: 20647428
references: Proc Natl Acad Sci U S A. 1981 Sep;78(9):5376-80
references: 6170982
references: Vaccine. 2009 Mar 26;27(15):2099-107
references: 19356612
references: Biochem Biophys Res Commun. 2010 Mar 19;393(4):614-8
references: 20152806
references: BMC Struct Biol. 2009 Apr 07;9:21
references: 19351406
references: Vaccine. 2003 Feb 14;21(9-10):932-9
references: 12547605
references: BioDrugs. 2006;20(6):335-40
references: 17176120
references: Microbiol Rev. 1992 Mar;56(1):152-79
references: 1579108
references: PLoS One. 2010 Feb 17;5(2):e9268
references: 20174650
references: Vaccine. 2010 Oct 4;28(42):6852-7
references: 20723626