[INCLUSION OF SITE-SPECIFIC MUTATIONS INTO CONSERVATIVE SEG- MENTS OF PA-GENE RESULTS IN ATTENUATION OF VIRULENT INFLUENZA VIRUS STRAIN A/WSN/33].

AIM: Study the possibility of obtaining attenuated variants of influenza virus by including specially selected site-specific mutations into a conservative sequence of PA-gene (terminal segment of COOH-domain of the PA-gene) of a virulent strain. MATERIALS AND METHODS: A/ WSN/33 - a virulent strain of influenza virus was used in the study. Inclusion of site-specif- ic mutations into PA-gene of the A/WSN/33 virulent strain was carried out using a two-step mutation PCR. Cloning was carried out using GoldenGate reaction. 8-plasmid transfection system based on pHW2000 vector was used. Transformation was carried out in rubidium competent bacterial cells of DH5(α strain. Transfection was done using Lipofectamine LTX (Invitrogen) reagentin a 293T and MDCK cells' co-culture. RESULTS: Transfectants with F658A substitution in the COOH-domain of the PA-gene were shown to acquire ts-phenotype and sharply reduce the ability to reproduce in mice lungs. Introduction of F658A substitution into COOH-domain of the PA-gene in combination with introduction of ts-mutations from ca influenzavirus strains into the genome ofthe virulent strain resulted in obtaining transfectants that have phenotypic characteristics typical for live influenza vaccine candidates. CONCLUSION: The ability to obtain attenuated variants of influenza viruses by introducing spe- cially selected site-specific mutations into conservative sequence of the PA-gene is shown.

[COLD-ADAPTED A/KRASNODAR/101/35/59 (H2N2) STRAIN--A PROMISING STRAIN-DONOR OF ATTENUATION FOR PROCURATION OF LIVE INFLUENZA VACCINES].

AIM: Study of ts, ca, att phenotype, immunogenicity and protective effectiveness of reassortants obtained by a way of recombination of a new influenza cold-adapted (ca) strain donor of attenuation A/Krasnodar/101/35/59 (H2N2) and virulent strain of influenza virus. MATERIALS AND METHODS: Viruses were used: ca strain A/Krasnodar/101/35.59 (H2N2), virulent strains: A/Kumamoto/102/02 (H3N2) and A/Bern/07/95. For determination of ts and ca phenotype, titration of viruses in chicken embryos was carried out simultaneously at optimal, decreased and increased temperature. Protective effect of immunization was evaluated during intranasal infection of mice with a virulent strain of influenza virus. RESULTS: All the obtained reassortants possessed 6 internal genes from strain-donor of attenuation and 2 genes, coding HA and NA-proteins from virulent strains. Ca reassortants were characterized by ts and ca phenotype, had antigenic specificity and good immunogenicity, had high protective effectiveness. CONCLUSION: The data obtained indicate on the perspectiveness of ca strain A/Krasnodar/101/35/59 (H2N2)as a donor of attenuation for live influenza vaccines.

[New cold-adapted donor strains for live influenza vaccine].

Cold-adapted (CA) strains A/Krasnodar/35 and B/Victoria/63 were isolated using passages of A/Krasnodar/101/59 and B/Victoria/2/87 wild type strains at low temperatures. The resulting CA strains possessed TS and CA phenotypes and had a reduced ability to reproduce in mouse lungs and nasal turbinates. They displayed a high protective efficacy in experiments on mice. The two CA strains reproduced well in chick embryos and MDCK cell line without change of TS and CA markers. The CA A/Krasnodar/35 strain during passages at low temperature acquired 13 mutations in the 6 internal genes, 8 of those mutations led to amino acid changes. The CA B/Victoria/63 strain acquired 8 mutations in the internal genes, 6 of which led to amino acid changes. The intranasal vaccination of mice with the CA A/Krasnodar/35 strain led to a transitory suppression of various lymphocyte subpopulations, as well as to an increase in the number of some other cell types. The CA strains in question may be used in the future as attenuation donors for live influenza vaccines.

[Increasing the immunogenicity of inactivated chitosan adjuvanted vaccine from A/California/7/09 (H1N1) strain and analyzing the antigenic specificity of this influenza virus strain].

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 4 degrees 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.

[Investigation of antiviral activity of adamantan boron derivaties on pandemic influenza virus models].

Comparative investigation of the virus-inhibiting activity of some boron-containing compounds showed that products BG 12 and BG 4 had the highest inhibitory effect on pandemic viruses. The minimum inhibitory concentration (MIC) of the products was 0.1 mcg/ml. The use of liposomes loaded with BG 12 molecules in the optimal concentration (0.1 mcg/ml) resulted in inhibition of the avian plague virus growth in the MDCK cells. Possible design of efficient drugs for antiviral protection based on the complexes liposomes--boron-containing compounds is discussed.

[Enhancing the immunogenicity of inactivated polio vaccines with chitosan used as an adjuvant].

Addition of chitosan to inactivated trivalent polio vaccine or inactivated preparations of attenuated poliomyelitis viruses (Sabin strains) significantly increases immunogenicity of these inactivated poliomyelitis virus preparations. High neutralizing antibody titers are detected after two immunizations of mice and a single immunization of rats, as well as when the antigen dose was reduced by 4 times. Addition of chitosan as an adjuvant significantly induces cellular immunity.

[Increase of immunogenicity of cold-adapted influenza vaccine by using adjuvant].

AIM: To assess increase of protective efficacy of live cold-adapted (ca) influenza vaccine after addition of adjuvant chitozan. MATERIALS AND METHODS: Used viruses: ca donor of attenuation A/Krasnodar/101/35/59 (H2N2) and epidemic strain A/Krasnodar/101/59 (H2N2); as an adjuvant--derivative of chitozan and microparticles of chitozan. Experiments were performed in outbred mice. Protective effect of immunization was measured by intranasal challenge by virulent strain of virus. Immune response was assessed by ELISA and indirect hemagglutination inhibition assay. RESULTS: During intranasal immunization of mice with intact CA donor of attenuation A/Krasnodar/101/35/59 (H2N2) addition of 1% solution of chitozan glutamate to vaccine material resulted in increased serum IgG in immunized mice and protective effect of immunization. Addition of adjuvant to ca donor strain did not influence on its ts-characteristic. It was shown that inactivated with ultraviolet radiation ca donor strain in combination with chitozan did not protect against infection caused by virulent strain A/Krasnodar/101/59, whereas the same doses of intact ca donor strain with chitozan were protective. Chitozan did not enhance replication of donor strain in upper respiratory tract of mice. CONCLUSION: Obtained data demonstrate that chitozan as a mucous-adhesive adjuvant could increase efficacy of live ca influenza vaccine.

[Chitosan as an adjuvant for inactivated vaccines against avian influenza viruses].

AIM: To study chitozan as an adjuvant for inactivated vaccines against A/H5 influenza viruses. MATERIALS AND METHODS: Avian A/H5 influenza viruses were grown on chicken embryos or on MDCK cell line; viruses-containing fluid was inactivated with formalin. Mice were vaccinated intramuscularly with inactivated avian influenza virus mixed with chitozan and then levels of hemagglutination-inhibiting and neutralizing antibodies as well as protective efficacy against both homologous and drifted strains of avian influenza viruses A/H5 were measured. RESULTS: Addition of chitozan to inactivated preparations of A/H5 avian influenza viruses for immunization of mice significantly increased levels of hemagglutination-inhibiting and neutralizing antibodies to both homologous and drifted variants of A/H5 influenza viruses, including those containing neuraminidase from other subtype as well as strains isolated 10 - 20 years earlier than virus used for vaccination. Chitozan significantly improved protective efficacy of inactivated avian influenza vaccines against infection with both homologous and drifted variant of the virus. Vaccination with inactivated avian influenza viruses A/H5 and chitozan induced high levels of antibodies even after single immunization as well as after administration of 8-fold reduced dose of preparation. CONCLUSION: Chitozan is a perspective adjuvant for inactivated vaccines against avian influenza viruses, which could significantly improve immune response and protective efficacy against both homologous and drifted variants of avian influenza viruses A/H5.

[Chitosan as an adjuvant for parenteral inactivated influenza vaccines].

Addition of 0.5% chitosan derivative to parenteral inactivated influenza vaccines increased antibody titers in the single immunization of mice by 4-5 times while double immunization showed 6-to-10-fold increases as compared with immunization without chitosan. Moreover, chitosan-containing vaccines induced the generation of antibodies to the drift variants of influenza virus. When the mice were given inactivated influenza virus A/H5N2 vaccine containing chitosan, immunogenicity and protective efficacy were much higher than when they received a vaccine containing no chitosan.

Molecular mechanisms of reversion to the ts+ (non-temperature-sensitive) phenotype of influenza A cold-adapted (ca) virus strains.

A ts+ ca- (non-temperature-sensitive, non-cold-adapted) revertant of the A/Leningrad/134/47/57 ca strain influenza virus [A/Leningrad/134/47/ts+18/1957(H2N2)], obtained in our previous study, lost phenotypic manifestation of ts mutations by the PB2, NP and NS genes, although, according to sequencing data, it acquired only two true reversions of a mutation in the PB2 and PB1 genes. Direct sequencing showed the appearance of 27 additional mutations (13 coding) in the genes encoding the PB2, PB1, PA, NP, M and NS proteins of the revertant, along with the above-mentioned two true reversions. We conjecture that some of these mutations suppressed phenotypic manifestation of ts mutations in the NS and NP genes.

[Molecular mechanisms of reversions to the ts+ -phenotype of cold-adapted influenza virus A strains--attenuation donors for live influenza reassortant vaccines].

A ts+ revertant of cold-adapted (ca) strain A/Leningrad/134/47/57--the attenuation donor for live influenza reassortant vaccines--was obtained by passages of the ca strain in chick embryos at nonpermissive temperatures. The ts+ revertant acquired the ability to grow in chick embryos at 40 degrees C and lost the capacity to reproduce there at 25 degrees C. A complementation-recombination test using the fowl plague virus (FPV0 ts-mutants showed the loss of the ts-phenotype in the RNA-segments of ts+ revertants' genome coding for PB2, NP, and NS (NS2) proteins. However, PCR-restriction analysis revealed a true reversion in RNA-segment coding for PB2 protein only. All the investigated mutations in the ts+ revertant genome were preserved. This phenomenon could be explained by the appearance of intragenic and extragenic suppression mutations in the ts+ revertant genome. The data of the complementation-recombination test suggest that reversion of ts-phenotype occurs more frequently due to extra- or intragenic suppression rather than as a result of a true mutation loss. Estimation of the genetic stability of vaccine ca strains of influenza virus should be based on the combined use of PCR-restriction and complementation tests.

Development of cell culture (MDCK) live cold-adapted (CA) attenuated influenza vaccine.

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.

[Further development (MDCK) of live cold-adapted influenza vaccine: cultivation of vaccine strains in production fermenters].

Optimal conditions were developed for cultivating the cold-adapted reassortant live influenza vaccine (CARLIV) in MDCK cells, which were in their turn cultivated in fermenters with serum-free medium and microcarrier. The use of MDCK cells meets all national and WHO requirements to continuous cells used in the production of biological preparations. CARLIV cultivated under such conditions well preserve their ts-mutations and mutation, which entail substitutions of amino acids, in all CARLIV genome segments. Provided the cultivation conditions are optimal, the output of multivalent CARLIV in a 101 fermenter can reach 100000 doses.

[Development of a live culture of cold-adapted reassortant influenza vaccines]. / Razrabotka kul'tural'noi zhivoi kholodoadaptirovannoi reassortantnoi grippozonoi vaktsiny.

Optimal conditions for the cultivation of the MDCK cell lines in the laboratory spinner or by using the Eagle-MEM with or without fetal serum were worked out. The cold-adapted reassortant vaccine strains of virus influenza A/H1N1, A/H3N2 and B are well replicated in the MDCK cells both in a monolayer and in the spinner by using the serum-free medium. A maximum virus titer depends on a multiplicity of infection used in a fetal medium and on the addition of trypsin. Under the optimal conditions, the titer of the studied cold-adapted reassortants, while using a serum-free medium, reaches as much as 10(9.0)-10(9.5) EID50/ml.

Virion transcriptase activity of rimantadine-sensitive and rimantadine-resistant variants of human influenza virus.

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.

[Genomic and antigenic analysis of strains of influenza A virus (H1N1) isolated in 1982-1983: approaches to the choice of the optimal vaccine strain]. / Genomnyi i antigennyi analiz shtammov virusa grippa A (H1N1) 1982-1983 gg. vydeleniia: podkhody k vyboru optimal'nogo vaktsinnogo shtamma.

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.

Characteristics of cell-mediated immunity after oral administration of a live influenza vaccine.

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.