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Study of immunogenicity of recombinant proteins based on hemagglutinin and neuraminidase conservative epitopes of Influenza A virus

Ilya Dukhovlinov, Ruslan Al-Shekhadat, Ekaterina Fedorova, Ludmila Stepanova, Marina Potapchuk, Irina Repko, Olga Rusova, Anton Orlov, Ludmila Tsybalova, Oleg Kiselev

Med Sci Monit Basic Res 2013; 19:221-227

DOI: 10.12659/MSMBR.884002

Available online: 2013-08-23

Published: 2013-08-23


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.
Material and 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.

Keywords: Neuraminidase - isolation & purification, Orthomyxoviridae Infections - virology, Mice, Influenza A virus - immunology, Immunoglobulin G - blood, Immunization, Hemagglutinin Glycoproteins, Influenza Virus - isolation & purification, Epitopes - immunology, Body Weight, Animals, Plasmids - metabolism, Recombinant Proteins - isolation & purification, Survival Analysis