Site-specific mutants as candidates for live influenza vaccines were resulted from directly introducing into the genome of the pathogenic influenza virus A/WSN/33 (H1N1) strain ts mutations derived from the genes encoding the polymerase complex proteins from some cold-adapted strains serving as attenuation donor. Here we present the data of a comparative study examining immune system arms in mice immunized intranasally with influenza virus mutants and classical cold-adapted reassortant obtained by crossing cold-adapted strain Donor A/Krasnodar/101/35/59 (H2N2) with strain A/WSN/33 (H1N1) bearing surface antigens (hemagglutinin and neuraminidase) similar to mutants. Immunophenotyping mononuclear leukocytes from immunized mice indicated at moderate suppressive effect after using site-specific mutant and the HA reassortant viruses on some immune cell subsets. All viruses in immunized mice resulted in activation of certain lymphocyte subsets including MHC II-positive cells, CD45+/CD19+ B lymphocytes and natural killer cells (CD16/32+/CD3–). Timescale and magnitude of activation markedly differed for each cell subsets. Mice immunized with mutants M26 and U2 peaked with count of CD16/32+/CD3– expressing cells on day 2 after the second immunization compared with control (p 0.05) that may suggest about an important role for NK cells in activating immune response. In contrast, no significant changes were observed during the study in percentage of CD4+/CD25+/Fox P3 regulatory T cells, CD4+ T helpers and CD8+ cytotoxic cells, except for a sharply decreased count of activated CD4+/CD25+ cells (4-fold) on day 7 after immunization with mutant virus M26. Moreover, mutants U2 and M26 more moderately increased percentage of TLR2- and TLR4-positive cells. The viruses studied ambiguously affected count of TLR9-expressing cells in immunized animals. All viruses increased phagocytic activity in monocytes, but not neutrophils. Despite the moderate activation of innate and adaptive immunity arms, site-specific mutants more profoundly affected humoral reactions inducing increased antibody titers, so that immunogenicity of mutant viruses was higher than that of the cold-adapted reassortant. Thus, the findings hold a promise of using site-specific mutants as live influenza vaccines.

Aim. Study of changes in the phenotypic characteristics of the virulent A/WSN/33 (H1N1) strain of influenza A virus under the influence of the inclusion of site-specific mutations in the PB1-gene of this strain.Materials and methods. Using a two-step polymerase reaction in the PB1 gene of A/ WSN/33 (H1N1) strain were included ts mutations taken from the genome of attenuated CA donors-strains: A/Ann Arbor/6/60 (H2N2), A/Leningrad 134/17/57 (H2N2) and A/ Krasnodar/101/35/59. Ts-phenotype, att-phenotype, immunogenicity, as well as weight loss in mice infected with these mutants were studied in the obtained site-specific mutants.Results. It was shown that the inclusion of ts mutations from the genome of CA donorsstrains of attenuation in the PB1 gene of the virulent A/WSN/33 (H1N1) strain leads to a change in the phenotypic characteristics of this strain to different degrees.Discussion. Analysis of the genome of CA strains- donors of attenuation of influenza virus indicates the crucial importance of the presence of functional defects in the PB1– protein for the formation of the attenuation phenotype of the virus.Conclusion. The technology of site-specific mutagenesis canbe successfully used to modify the PB1 gene of a virulent influenza A virus strain in order to construct a new generation of live influenza vaccines.

Aim. Study of biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene. Materials and methods. Site-specific mutants of A/WSN/33 of influenza virus, having in PB2-gene ts-mutations from genome of cold-adapted (CA) master-strains: A/Ann Arbor/6/60 (H2N2); A/Leningrad/134/17/57 (H2N2); A/Krasnodar/101/35/59 (H2N2) were obtained with help of reverse genetics methods. The ts-phenotype, att-phenotype, immunogenicity and protective efficacy in homologous and heterologous control infections were studied in the obtained site-specific mutants. Results. It was shown that the inclusion in the PB2-gene of the virulent A/WSN/33 strain as single mutations and a combination of mutations from the genomes of CA donor-strains leads to a change in the ts-phenotype and att-phenotype of the mutants obtained. These mutants had high protective efficacy in homologous and heterologous control infection. Conclusion. The results obtained allow us to consider the site-specific mutants of influenza virus as possible candidates for live influenza vaccines.

Aim. Study of biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene. Materials and methods. Site-specific mutants of A/WSN/33 of influenza virus, having in PB2-gene ts-mutations from genome of cold-adapted (CA) master-strains: A/Ann Arbor/6/60 (H2N2); A/Leningrad/134/17/57 (H2N2); A/Krasnodar/101/35/59 (H2N2) were obtained with help of reverse genetics methods. The ts-phenotype, att-phenotype, immunogenicity and protective efficacy in homologous and heterologous control infections were studied in the obtained site-specific mutants. Results. It was shown that the inclusion in the PB2-gene of the virulent A/WSN/33 strain as single mutations and a combination of mutations from the genomes of CA donor-strains leads to a change in the ts-phenotype and att-phenotype of the mutants obtained. These mutants had high protective efficacy in homologous and heterologous control infection. Conclusion. The results obtained allow us to consider the site-specific mutants of influenza virus as possible candidates for live influenza vaccines. Цель. Изучение биологических свойств аттенуированных вариантов штамма А/WSN/33(Н1N1) вируса гриппа А, полученных с помощью сайт-специфического мутагенеза РВ2-гена. Материалы и методы. С помощью методов обратной генетики получены сайт-специфические мутанты штамма А/WSN/33, имеющие в РВ2-гене ts-мутации из генома холодоадаптированных (ХА) штаммов-доноров аттенуации: А/Энн Арбор/6/60(Н2N2), А/Ленинград/134/17/57(Н2N2), А/Краснодар/101/35/59 (Н2N2). У полученных сайт-специфических мутантов исследован ts-фенотип, att-фенотип, иммуногенность и защитная эффективность при гомологичном и гетерологичном контрольном заражении. Результаты. Показано, что включение в РВ2-ген вирулентного штамма А/WSN/33 как единичных ts-мутаций, так и комбинации ts-мутаций из генома известных ХА штаммов-доноров аттенуации ведет к изменению ts- и att-фенотипа полученных сайт-специфических мутантов. Наблюдалось падение способности к размножению при повышенной температуре и снижение вирулентности для мышей при интраназальном заражении. Полученные мутанты имели высокую защитную эффективность при гомологическом и гетерологическом контрольном заражении. Заключение. Полученные данные позволяют сделать вывод, что некоторые сайт-специфические мутанты не уступают по защитной эффективности как при гомологичном, так и гетерологичном контрольном заражении ХА реассортантным вакцинным вариантам. Результаты работы дают основания рассматривать некоторые из этих мутантов как возможные кандидаты в живые гриппозные вакцины.

The hemostatic and immunostimulating activity and cytotoxicity were determined for a number of chitosans differing in molecular weight (from 3 to 510 kDa) and degree of acetylation (from 1 to 25 mol%) that were used as adjuvants in inactivated poliomyelitic, influenza, and live influenza vaccines. It has been shown that the hemostatic activity of chitosan increased sharply with an increase in its molecular weight. In oligochitosan with a molecular weight of <16 kDa, it was smaller by a factor of 15–100 than in chitosan with a molecular weight of 20–510 kDa. The level of increase in the immunogenicity of vaccines containing oligochitosan as adjuvants was not lower than that for the vaccine including high-molecular chitosan. However, the immunostimulatory activity of oligochitosan depended on the degree of acetylation, reaching a maximum value at 6 mol%. It was shown that all oligochitosans and chitosans with a molecular mass below ~50 kDa showed almost no cytotoxicity at a concentration of ≤2.5 mg/mL, which enable their use as adjuvants for inactivated and live vaccines at the optimal ratio of molecular weight to the degree of acetylation.

Relevance. Live influenza vaccines are highly effective and currently used in the Russian Federation and the United States. Goal. To investigate especial features of immune response of mice, immunized by different types of live influenza vaccines and infected later by virulent influenza strain. Materials and methods. Mice were immunized by two types of live influenza vaccines candidate: cold-adapted (CA) reassortant, which inherited the 6 «internal» genes from the CA donor A/Krasnodar/101/35/59 (H2N2) and 2 genes encoding the surface proteins HA and NA from the virulent strain A/WSN/33 (H1N1) and site-specific mutants on the basis of A/WSN/33 strain, in the genome of which has been included mutations from genes of CA strains of influenza virus, encoding proteins of the polymerase complex. Immunized mice were then infected by a virulent A/WSN/33 strain of influenza virus. Results. It was shown that CA reassortant RKr35/WSN/33 and site-specific mutants Tr. № 5 and № 8 in contrast to the virulent A/WSN/33 strain were characterized by pronounced ts-phenotype and att-phenotype. Both types of live vaccines after two-time intranasal immunization induced in mice a relatively low level of humoral antibodies (log2 4,5 ± 1,2 - log2 6,0 ± 0,7). Despite the low level of induction of humoral response both types of live vaccines had similar marked protective efficiency. However, animals immunized with CA reassortant was characterized by a significant weight loss after infection with a virulent influenza strain (25%), while the infection of animals, immunized with site-specific mutants, by a similar dose of virulent flu strain has very little impact on their weight characteristics (8 - 13%). Conclusion. The data obtained indicate that antibodies to surface proteins of the virion, induced in the process of immunization, slightly inhibited the initial replication of the virulent strain and suggest that these differences in the symptoms of the infection depend on the characteristics of activated T-cell immunity. This circumstance could have a significant impact on metabolic processes in organism of infected animals.

Addition of chitosan as an adjuvant to subunit vaccine from the swine origin influenza virus A/California/7/09 (H1N1) increases vaccine immunogenicity by 8-16 times and significantly enhances its protective potency. Single immunization with chitosan adjuvanted vaccine induced similar antibody titers as two immunizations with unadjuvanted vaccine. Chitosan stabilized the immunogenicity of subunit vaccine when stored at 4o C. The antigenic specificity of the A/California/7/09 (H1N1) virus strain did not resemble substantially that of the human influenza strains A/Brisbane/59/07 (H1N1) and A/Solomon Isles/3/06 (H1N1), which are among the 2008/2009 and 2007/2008 seasonal influenza vaccines, respectively, as well as that of the human influenza H1N1 virus strains that circulated about 30 years ago.

Optimal conditions are determined for growing cold-adapted reassortant strains of a live influenza vaccine in MDCK cell line cultivated in a fermenter with a serum-free medium and microcarriers. The studied MDCK cell line meet all national and WHO requirements for the finite cell lines used for the production of biological preparations. CA reassortant vaccine strains grown in such conditions which fully preserve its mutations and the mutations lead to amino acid substitution in all genome segments of the studied CA reassortants. Under optimal cultivation conditions, the output of a monovalent live CA influenza vaccine in a 10-l fermenter may reach 100,000 doses.

All rimantadine-resistant variants of influenza virus prepared by consecutive passages in the presence of rimantadine had increased virion transcriptase activity as compared to the original strains. The increased virion transcriptase activity of rimantadine-resistant strains was unrelated to the possible role of M1 protein, since RNPs isolated from the virions of these variants also revealed higher transcriptase activity as compared to RNPs isolated from rimantadine-sensitive virus. The study of rimantadine-resistant recombinant X-4 which inherited from the resistant fowl plague virus (FPV) only the gene 7 coding for M proteins provided additional evidence for the suggestion that the increased virion transcriptase activity of rimantadine-resistant influenza virus variants is coincidental rather than directly associated with such resistance.

Features of the genome and antigenic specificity of hemagglutinin of some influenza A (H1N1) virus strains circulating in the epidemic period of 1982-1983 were studied comparatively. Analysis of the genome of the isolates under study in comparison with that of the reference A/England/333/80 strain and with each other has established changes not only in the genes coding for hemagglutinin and neuraminidase but also most of the genes coding for unglycolysed proteins. The antigenic specificity of hemagglutinin of the isolates under study examined with rat antisera and monoclonal antibodies was found to be quite dissimilar. Hemagglutinin of the A/Dunedin/27/83 strain induced antibodies capable of reacting predominantly with the homologous strain whereas antibodies to hemagglutinin of the A/Leningrad/16/16/82 and A/Chile/1/83 strains had a wide spectrum of antigenic specificity and neutralized well the hemagglutinin of different variants of influenza virus of H1N1 serotype circulating at that time. Among the 1982-1983 isolates studied, the A/Leningrad/16/16/82 strain was selected which, by its hemagglutinin properties, is optimal for preparation of inactivated vaccine, as was confirmed by the study of an experimental batch of such vaccine in volunteers.

Development of immunological status of children after oral administration of a live influenza vaccine was followed using different tests of the cellular (blast transformation assay, identification of T, B and nil lymphocytes) and humoral (haemagglutination inhibition test, neuraminidase-inhibition test, neutralization test) immune response. Direct correlation was observed between the increase of neutralizing antibody and the lymphocyte stimulation indices in blast transformation assay (BTA). In vaccinated children the reactivity of lymphocytes was reduced and the amount of T lymphocytes decreased. The influenza vaccine has been shown to possess weak sensitizing properties.

For some years the Moscow Research Institute of Virus Preparations carried out studies concerned with the elaboration of conditions for production and control of mumps vaccine from the Leningrad-3 (L-3) strain under experimental production conditions. Twenty nine batches of the vaccine were prepared, tested and studied in extensive field trials. The paper presents the optimal conditions for the preparation of the live mumps vaccine from the L-3 strain in Japanese quail embryo cell cultures. In monkey experiments the L-3 strain was shown to have no neurovirulence. The system of biological control for the mumps vaccine permits to release the preparation meeting the current requirements for safety of live tissue culture parenteral vaccines. The data on the stability of the mumps vaccine from the L-3 strain are presented.