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

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.

A molecular assembly system for presentation of antigens on the surface of HBc virus-like particles.

Hepatitis B virus-like particles, icosahedral structures formed by multiple core protein dimers, are promising immune-enhancing vaccine carriers for foreign antigens. Insertions into the surface-exposed immunodominant loop are especially immunogenic. However, the need to conserve the particulate structure to ensure high immunogenicity imposes restraints on the nature of the heterologous sequence that can be inserted. We propose a new approach to constructing HBc particles linked to the target epitopes that relies on non-covalent interactions between the epitope and pre-assembled unmodified HBc particles. Interaction was enabled by fusion of the epitope to the GSLLGRMKGA peptide, binding to the spike tips. This peptide may be used as a "binding tag" allowing in vitro construction of HBc particles carrying the target peptide. Such virus-like particles carrying multiple copies of the extracellular domain of the M2 protein of different influenza strains appeared to be highly immunogenic and protected immunised mice against a lethal influenza challenge.