Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus) polymerase activity by the minigenome method with a fluorescent protein.

INTRODUCTION: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. OBJECTIVE: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. MATERIALS AND METHODS: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. RESULTS: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. CONCLUSION: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

[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.

[MECHANISMS OF ATTENUATION OF COLD-ADAPTED STRAIN A/KRASNO- DAR/101/35/59 (H2N2)].

AIM: Study of mechanisms of attenuation of cold-adapted (ca) influenza virus strain A/ Krasnodar/101/35/59 (H2N2), associated with disruption of NS1 protein functions. MATERIALS AND METHODS: Study of interferonogenic activity of ca strain A/Krasnodar/101/35/59 (H2N2), its parent variant A/Krasnodar/101/59 (H2N2), virulent strain A/WSN/33 (H1N1) and a number of single gene and multiple gene reassortants between these strains, obtained using reverse genetics, was carried out. Study of dynamics of IFNß gene expression was carried out by using a methodical approach of RT-PCR in real time mode. RESULTS: Inclusion of PB-1 gene of ca strain A/ Krasnodar/101/35/59 (H2N2) with reversion to wild type into genome composition of virulent strain A/WSN/33 (H1N1) does not result in a sharp change of interferonogenic activity of the reassortant. At the same time, similar inclusion of PB-1 gene of ca strain resulted in an incredible growth of interferonogenic activity of the reassortant. On the other hand, inclusion of NP-gene of wild type strain A/Krasnodar/101/59 (H2N2) into genome composition of the wild type strain A/WSN/33 did not differ by effect on interferonogenicity of the reassortant from inclusion of NP-gene of ca strain. CONCLUSION: Both constellations of genes of parent variants and mutations localized in these genes could affect formation of attenuation phenotype of reassortants. The data obtained allow to assume possible mechanisms of attenuation of ca strains, associated with disruption.of NS gene function.

[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.

[Activating effect of a germanium-organic compound on immunocompetent cells during intranasal immunization of mice with a live influenza vaccine].

AIM: Detailed characteristic of results of intranasal immunization of mice with one of two variants of vaccinating influenza virus, particularly in combination with a low molecular weight germanium-organic compound (LMW-GOC). An additional aim is evaluation of effect of LMW-GOC on the parameters of immune system in case of intranasal administration of the preparation without the addition of vaccinating virus. MATERIALS AND METHODS: The study was carried out in female CBA mice (18-20 g, 6 animals per group). Intranasal immunization was carried out by 2 different variants of B/Victoria influenza virus--once or twice with a 2 week interval. Cells for study were obtained from spleen and nasal- and bronchial-associated lymphoid tissue (NALT/ BALT) 24 hours and 7 days after intranasal administration of the preparations. The main method of the study--determination of the level of expression of various markers oflymphocytes in comparison with the level of the same markers in the cells of control group animals by using flow cytometry method. The mean parameters obtained were determined by using program package WinMDI 2.8. RESULTS: The main results were the increase of level of expression of various lymphocyte markers obtained from mice after intranasal administration of the vaccines and their combination with LMW-GOC or LMW-GOC only without the participation of the vaccines. A significant increase of the expression of TLR9 marker compared with other parameters was noted. Administration to mice of wild B/Victoria strain notably more frequently conditioned the decrease of expression of some parameters compared with administration of the cold adapted strain. Effect of LMW-GOC without the vaccine also conditioned the increase of levels of markers however a combination of the preparations with the vaccine was more effective. CONCLUSION: The increase of level of expression of a number of lymphocyte markers may serve as a sign of successful intranasal vaccination against influenza. LMW-GOC preparation increases immune stimulating effect of intranasally administered vaccines and in none of the cases weakens the stimulating result of effect of the vaccines, and in many cases increases it. LMW-GOC may be studied as a main or additional adjuvant for intranasal application of 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.

[Study of immunogenicity and protective efficacy of a novel inactivated vaccine with chitosan against influenza A/H1N1/2009].

AIM: Study of immunogenicity and protective efficacy of a novel inactivated vaccine with chitosan against influenza A/H1N1/2009. MATERIALS AND METHODS: Influenza virus A/California/7/2009 (H1N1) strain was used in the study. Mice were immunized twice (21 day interval) with experimental samples of inactivated influenza vaccine: No. 1--without the addition of chitosan, No. 2--with addition of chitosan. The blood was obtained 21 days after the first and 10 days after the second immunization with the vaccines and was treated with RDE. Antibody levels were evaluated in HI reaction. RESULTS: HI reaction method showed that antibody titers induced after immunization of vaccine No. 2 were higher than those induced after immunization with vaccine No. 1. Evaluation of protective efficacy of the vaccines against an experimental form of influenza infection in mice showed that after immunization with vaccine that does not contain chitosan the level of virus accumulation does not differ from the control statistically significantly (p < or = 0.05), at the same time the level of virus accumulation in the lungs of infected animals immunized with chitosan containing vaccine significantly (significantly with 95% probability) decreased by an average 3.01g when compared with control. CONCLUSION: Comparative analysis of immunogenicity and protective efficacy of experimental samples of inactivated influenza vaccine against influenza A/H 1N1/2009 showed that the vaccine with the addition of chitosan stimulates the formation of a higher immune response and promotes a more significant suppression of influenza A infectious agent reproduction in the lung target-organ.

[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.

[A study of the interaction of boraadamantane with liposomes].

Changes in the surface potential of liposomes obtained from dipalmitoyl phospatidylcholine during their interaction with the new antiviral preparation boraadmantane have been studied. It has been concluded that the saturation of the lipid bilayer of liposomes by boraadmantane occurs at concentrations of the preparation above 10 microg/ml.

[Antiviral activity of panavir in experimental Measles virus infection in cell cultures].

Antiviral activity of Panavir was studied in a model of experimental infection due to Morbillivirus in subcultures of cells Vero and B-16. It was shown that at multiplicity of the infection (0.001-0.0001 TCD50/cell) Panavir inhibited reproduction of the virus in concentrations of 12-100 mcg/0.2 ml.

[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.

[Influence of chitosan on immunophenotype and functional activity of murine mononuclear leukocytes after immunization with inactivated influenza vaccine].

In the overwhelming majority of countries inactivated vaccines, which form mainly humoral immunity, are used for prevention of influenza. The objective of the study was to assess the combined effect of inactivated influenza vaccine and chitozan on cellular immunity in CBA line mice. Intramuscular administration of 2 doses (with 4 week interval) of inactivated influenza vaccine and chitozan resulted in increased cytotoxic activity of splenic NK cells against NK-sensitive cell line K562 as well as in increased proliferative activity of mononuclear leukocytes, and numbers of CD3 T-lymphocytes, NKT cells, B-lymphocytes in animals' spleens. Combination of inactivated influenza vaccine with chitozan modulated the number MHC II-expressing cells by eliminating the increased reactivity of immune system cells as well as increased the number of MHC I-expressing cells. This point on the activation of cellular properties, which recognize intracellular pathogens, and thus on activation of both humoral and cellular factors of immune response. It can be proposed that inclusion of chitozan in the vaccine allows to modulate switching of the immune response from Th-2 to Th-1 type.

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.