Genetic Basis of Attenuation of Cold-Adapted Influenza Strain B/Leningrad/14/17/55 - Backup Master Donor Virus for Influenza Type B Live Attenuated Vaccines.

The reassortant vaccine strain of live attenuated influenza vaccine inherits temperature sensitivity and areactogenicity from cold-adapted attenuated master donor virus. In Russia, B/ USSR/60/69 master donor virus (B60) is currently in use for the preparation of live attenuated type B influenza vaccine candidates. Trivalent live attenuated influenza vaccine based on A/ Leningrad/134/17/57 and B60 are licensed for the use in Russia for single dose vaccination of adults and children over 3 years. B/Leningrad/14/17/55 (B14) cold-adapted virus is a backup master donor virus for live attenuated type B influenza vaccine. According to our preliminary estimates, it is more attenuated than B60, which can allow expanding applicability of this vaccine for children under 3 years of age. In this paper, the role of B14 genes in its attenuation was assessed. Representative collection of reassortants of B14 with epidemic influenza B viruses was obtained, a phenotypic analysis of reassortants was performed, and their pathogenicity for animals was assessed. The leading role of PB2 and PA genes in attenuation of B14 master donor virus was proven.

Experimental Study of Genetic Constellation of Cold-Adapted Master Donor Viruses for Live Attenuated Influenza Vaccine Type B.

We studied the constellation of genes encoding polymerase complex proteins of master donor viruses for Russian live attenuated influenza vaccine type B. Reassortants of the reserve attenuation donor B/Leningrad/14/17/55 with B/USSR/60/69 master donor virus currently used for manufacturing seasonal influenza vaccine were prepared and examined. Most reassortants obtained by the classical reassortment method inherited all genes from the B/Leningrad/14/17/55 virus except the gene encoding PB1 subunit of the polymerase complex. One reassortant was selected for further evaluation of the role of PB1 gene. Greater attenuation of the strain for experimental animals (mice) in comparison with the original strains was demonstrated. This indicates high degree of constellation of genes of cold-adapted master donor viruses and the important compensating role of amino acid substitutions in the PB1 protein of B/Leningrad/14/17/55 donor virus in preventing viral hyperattenuation.

Composition of the Stabilizer and Conditions of Lyophilization for Preserving Infectious Activity of Influenza Virus.

For stabilization of vaccine preparations, they are lyophilized. The composition of the protective medium is an important parameter affecting the quality of the vaccine after drying. In view of the risk of spreading prion diseases, the use of media containing animal proteins is not recommended. In this study, protective media containing no animal proteins and lyophilization regimen were determined. The optimum lyophilization regimen consisted of three stages: freezing at -70°C, main stage at -35°C, and drying at 24°C. Protective medium containing 4% trehalose or protective medium with 10% sucrose and 5% soy peptone ensured highest stability of the lyophilized vaccine preparation in temperature range of 4-24°C. This can help to overcome possible break in the cold chain, which is important during transporting or storage of vaccine preparations.

Development of Pyrosequencing Assay for Evaluation of Genetic Stability of Vaccine Strains of Live Attenuated Influenza Type B Vaccine.

We developed a pyrosequencing protocol for monitoring of stability of attenuating mutations in the genome of vaccine reassortants based on master donor virus of Russian live attenuated influenza vaccine B/USSR/60/69. The developed protocol allows rapid and accurate assessment of mutations and can be used for analysis of genetic stability of reassortants during vaccine strain development and manufacturing, as well as genetic stability of vaccine isolates of influenza B virus during pre-clinical and clinical trials.

[Codon optimization of hemagglutinin as a promising tool for improving of the influenza vaccines immunogenicity].

Modification of the codon bias of sequences is a promising tool of the gene expression control. The theoretical basis of the codon optimization is reviewed, data on experiments in changing the viral gene codon bias for purposes of vaccine development are discussed. Research into the field of the influenza vaccine immunogenicity improvement with codon optimization method is reviewed. Prospects of the use of the codon optimization technique for influenza vaccine development are considered.

[Contribution of neuraminidase of influenza viruses to the sensitivity to the serum inhibitors and reassortment efficiency].

The live attenuated influenza vaccine (LAIV) consists of reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from the circulating wild-type (WT) parental and the 6 internal protein-encoding gene segments from the cold-adapted attenuated master donor viruses (genome composition 6:2). In this study, we describe the obstacles to developing LAIV vaccine strains depending on the phenotypic peculiarities of the WT viruses used for reassortment. The genomic composition analysis of 849 reassortants revealed that over 80% of the reassortants based on the inhibitor-resistant WT viruses inherited WT NA as compared to 26% of reassortants based on the inhibitor-sensitive WT viruses. In addition, the highest percentage of the vaccine genotype reassortants was achieved when WT parental viruses were resistant to the non-specific serum inhibitors. We demonstrate that NA may play a role in the influenza virus sensitivity to the non-specific serum inhibitors. Replacing NA of the inhibitor-sensitive WT virus with the NA of the inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to the non-specific inhibitors.

[Peculiarity of reassortment of current wild type influenza viruses with master donor viruses for live influenza vaccine].

The live attenuated influenza vaccine (LAIV) currently licensed in Russia consists of the reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments from the circulating wild-type viruses and the six internal protein-encoding gene segments from cold-adapted master donor viruses (MDV) A/Leningrad/134/17/57 (H2N2) or B/USSR/60/69. Presently, only classical reassortment technique is approved for the generation of Russian LAIV strains. In this work, we describe the obstacles to the development of LAIV 6:2 vaccine strains depending on the phenotypic properties of the wild-type viruses used for reassortment. It was demonstrated that the highest percentage of 6:2 vaccine reassortants could be achieved when wild-type parental viruses were resistant to non-specific gamma-inhibitors. It was shown that it was impossible to generate 6:2 vaccine reassortants possessing six internal genes of the AILeningrad113417/57 (H2N2) master donor virus and avian HA and NA genes from H5N1-PR8 viruses using classical reassortment technique. It was suggested that strong constellation effects between the gene segments of the parental viruses could affect the virus gene reassortment. A strong interaction between the genome segments encoding neuraminidase of avian origin and PB2 gene of PR8 virus was observed. When the PB2 gene was inherited from cold-adapted master donor virus, the neuraminidase was also found to be of MDV origin.

[Genome composition analysis of the reassortant influenza viruses used in seasonal and pandemic live attenuated influenza vaccine].

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.

[Restriction analysis of genome composition of live influenza vaccine].

Live attenuated cold-adapted influenza vaccine (LIV) has been used in Russia for over 50 years and proved to be safe and effective. Currently, Russian reassortant LAIV is based on influenza AILeningrad/134/17/57 (H2N2) and B/USSR/60/69 Master Donor Viruses (MDVs) which are cold-adapted (ca), temperature-sensitive (ts), and attenuated (att), respectively. The MDVs are used to generate attenuated reassortant vaccine viruses containing the surface antigens of current wild type (wt) influenza A (HINI) and A (H3N2) viruses and wt influenza B virus. The ca/ts/att phenotype of these viruses limits replication in the upper respiratory tract. Reassortment typically yields numerous viruses with different genome constellations, rapid screening is needed to select proper vaccine viruses. In this study, screening of reassortant vaccine strains for live attenuated influenza vaccine generated from currently circulating influenza A and B viruses by RFLP assay is described.

[Leading role of genes coding polymerase complex in attenuation of domestic donor viruses for A and B live influenza vaccine].

AIM: To identify the genes that are responsible for attenuation of donor viruses for live influenza vaccine. MATERIALS AND METHODS. Analysis of phenotypical properties of reassortants of wild type A and B influenza viruses with A/Leningrad/134/17/57 (H2N2) (A17) and B/USSR/60/69 (B60) master donor viruses was performed by comparison of their capability to grow at different temperatures in chicken eggs or/and MDCK cells. RESULTS: Ts phenotype of 178 reassortants of A17 with current non-ts influenza A wild type viruses and 33 reassortants of B60 with current non-ts influenza B wild type viruses were evaluated. Reassortants inherited two polymerase genes PB2 and PA or PB 1 from A17 regularly demonstrated ts phenotype. The polymerase PA and PB2 gene segments of B60 independently controlled manifestation of ts phenotype of B60 based reassortants. The other nonpolymerase genes played no role in manifestation of ts phenotype of reassortants A17 and B60 viruses. CONCLUSION: The molecular basis for the development ts phenotype of both A and B influenza vaccine reassortant viruses determined by polymerase genes complex.

[Live influenza vaccine: laboratory markers of attenuation].

Screening for candidate reassortants is an important step in the development of live influenza vaccine (LIV). The temperature-sensitive (ts) and cold-adapted (ca) phenotypes of vaccine strains are generally determined, by employing chicken embryos, and used as ts and ca attenuation markers. However, it is difficult to use the egg-determined ts phenotypes of vaccine candidate reassortants as an attenuation marker due to a wide circulation of natural ts epidemic influenza viruses. This study used two new alternative ts and ca attenuation markers in MDCK cells. The MDCK cell line was shown to be able to differentiate cold-adapted influenza viruses from any epidemic strains whereas they were undistinguishable when using eggs. The reduced ability of influenza type A vaccine viruses to grow in the MDCK cell culture at temperatures above 37 degrees C can be successfully used as a "cell-culture" ts marker. The similar marker for influenza B viruses may serve their reduced activity in the MDCK cells at 38 degrees C. The high reproductive activity of cold-adapted viruses in the MDCK cells at 26 degrees C was shown to be a suitable ca attenuation marker. The presented attenuation markers may be included into the standard scheme of primary screening of ts reassortant candidates for commercial live influenza vaccine as additional selection factors and may be used as basic markers in the design of culture vaccine.

[Growth characteristics of single gene mutants of A/Leningrad/134/57(H2N2) influenza virus and cold-adapted mutant A/Leningrad/134/17/57(H2N2)].

The authors examined a role of some mutated A/Leningrad/134/17/57(H2N2) virus genes in the realization of growth characteristics. The latter of single gene reassortants (SGRs) (PB2, PB1, PA, M, and NS), epidemic virus and attenuation donor were assessed by infecting MDCK cells and hen embryos at a low inoculation index. Viral replication in the hen embryos and cultured tissue was compared at 34 degrees C. The viruses and reassortants tested showed a high growth capacity in the hen embryos (9.5-10.5 Ig TCID50). The growth curves of viruses were studied on the cultured MDCK cells at a low inoculation index indicated that Len/17 and the single gene reassortants M and NS had the highest growth capacity. At the same time the growth of both PB1 and PB2 SGRs was less extensive. The reproduction of PB2 SGR was 100-1000 times less than that of other viruses tested. M, NS, and PA gene mutations did not affect viral growth in hen embryos and cultured tissue while PB2 gene mutation and its constellations with other genes caused a reduction in viral growth in the cultured tissue.

[Efficacy of production of reassortant of epidemic strains and cold-adapted influenza viruses in chicken embryo and MDCK cells].

Reassortant strains for modern live influenza vaccines are prepared using growing chicken embryos. It is very important to switch manufacture of influenza vaccines from chicken embryos to cell cultures, especially due to the threat of future pandemic, when there will be need of big quantities of vaccine for immunization of all age groups. Efficacy of production of reassortant strains with 6:2 vaccine formulation of genome (6 internal genes from the donor of attenuation and 2 genes coding external antigens--hemagglutinin and neuraminidase--from epidemic strain) in MDCK cell culture, using standard techniques employed for production of the vaccine in chicken embryos, was studied. It was shown that yield frequency of aforementioned reassortants of influenza A viruses did not exceed 5.7% whereas in chicken embryos vaccine 6:2 reassortants were isolated with frequency of 4%. For influenza B viruses, yield of 6:2 reassortants in growing chicken embryos exceeded 67% whereas in MDCK cell culture we were unable to produce clones with required genome composition. Thus, existing method while effective for production of vaccine reassortants in chicken embryos is low effective for isolation of 6:2 reassortants in MDCK cell culture. Fundamentally new techniques are needed for production of reassortant strains for live influenza vaccine in cell culture.

[Genetic stability of cold-adapted influenza viruses].

The stability of cold adaptation, temperature-sensitivity, and marker mutations that are typical of attenuated influenza A and B viruses--master donor strains and their based reassortant vaccine strains was studied. After 5 sequential passages in chick embryos (CE) at resolving temperatures of 32 and 37 degrees C, the master donor strains and vaccine viruses retained their adaptability and temperature sensitive phenotype. Passage at the temperatures maximally permissible for viral reproduction (39 and 38 degrees C for influenza A and B viruses, respectively, aborted infection just during the second passage. After a series of passages at all study temperatures), there was neither loss or nor substitution of the marker mutations typical of the cold-adapted and temperature-sensitive phenotype of attenuated viruses. The study supports the high genetic stability of attenuated cold-adapted influenza A and B viruses during CE passage not only at the optimum, but also at elevated incubation temperatures.

[Phenotyte of epidemic influenza B virus strains isolated in different years].

The reproducing ability at elevated temperatures (non-ts phenotype) was examined for 38 influenza B virus strains isolated in different years in different countries. Out of the 7 strains isolated in 1940 to 1973, only one showed temperature-sensitivity of reproduction (a ts phenotype). In 1984 to 1988, the proportion of temperature-sensitive strains increased up to 55% (6 of 11). Since the late 1990s, the majority (90%) of the study influenza B viruses demonstrated a pronounced ts phenotype. Influenza B virus strains were also examined for their resistance to serum inhibitors. Prior to the divergence of influenza B viruses into two lines: B/Jamagata and B/Victoria, the epidemic viruses exhibited a high resistance to nonspecific inhibitors of normal equine serum. This property was also preserved in all study B/Victoria strains; however, 83% of the B/Jamagata viruses were inhibitor-sensitive. The present study has demonstrated the heterogenicity of epidemic influenza B viruses in temperature- and inhibitor-sensitivity.

[Genetic and phenotypic analysis of heterogeneous population of a cold-adapted donor of the A/Leningrad/134/17/57 (H2N2) attenuation and of the donor-based reassortant influenza vaccine strains].

Cold-adapted (CA) temperature sensitive and attenuated virus A/Leningrad/134/17/57 (H2N2) (Len/17) has been recently used in Russia as a donor of internal genes in the preparation of reassortant vaccine strains of CA live influenza vaccine (LIV) for all age groups. The Len/17 population was found to be heterogeneous and to be made up of clones, which differ by combinations of mutations in internal genes. Around 50% of the Len/17 population had clones with all 8 coding mutations in internal genes. The others were made up of clones with mutation combinations, which were different from the original Len/17. The PCR restriction method was used to analyze 5 clones of Len/17 and 8 LIV vaccine strains. There were no Ala-86-Thr mutation in the M2 protein in 4 clones and 3 vaccine strains. The PB-1 gene of 4 clones and 3 vaccine strains had a mutation encoding Met-317-IIe more typical of a more attenuated virus A/Leningrad/134/47/57 (H2N2) (Len/47). The NP protein of a clone had a mutation Leu-341-IIe also typical of Len/47. However, neither the absence of mutation in the M2 gene nor an extra mutation in the PB1 gene affected the attenuation extent of reassortant CALIV.

Generation of influenza transfectants using purified recombinant nucleocapsid protein.

Affinity-purified type A influenza virus nucleocapsid protein expressed by a recombinant baculovirus vector was used in in vitro RNA transcription reactions to create RNP complexes containing a synthetic influenza A virus NS gene. When used in transfection assays, the baculovirus-expressed NP was shown to be biologically active allowing the efficient isolation of transfectant viruses containing the artificially-introduced NS gene. The results demonstrate that NP is the only virion protein necessary in the reconstituted RNP complexes used for transfection thus eliminating the need for purified RNP complexes containing active polymerase.

[Immune response to live influenza vaccine]. / Immunnyi otvet na zhivuiu groppoznuiu vaktsinu.

Priority data on the induction, by using a Russian live cold-adapted reassortant influenza vaccine (LIV), of the cellular and humoral immunity with regard for attenuation and genetic reassortment of vaccine stains as well as with regard for the age of vaccinated persons and the production of Th1 (IFNY, IL-2) and Th2 (IL-4) cytokine markers in vitro are presented. It was demonstrated in vivo that a pathogenic virus of the A group by far more actively induced the lymphocyte apoptosis as compared with attenuated genetically reassorted stains. Unlike the influenza pathogenic virus, the genetically attenuated and reassorted strain did not produce any negative effects on the induction of cellular immunity. A comparative study of the LIV immunogenic properties in vaccinated persons showed an advantage of LIV over inactivated influenza vaccine (IIV) in stimulating the cellular and local immunity in the elderly. Unlike IIV, LIV induced an active and balanced immune response developing due to Th1 and Th2 activation. LIV was found to stimulate well enough the production of IFN and IL-2 in both young and old persons.

[Effect of remantadine on the development of toxicoses caused by different strains of influenza virus]. / Vliianie remantadina na razvitie toksikoza, vyzvannogo razlichnymi shtammami virusa grippa.

The effect of remantadine on the development of viral toxicosis caused in mice by original and remantadine-resistant strains of influenza virus was studied. A definite reduction of the antitoxic effectiveness of remantadine was demonstrated in passages of toxigenic influenza A virus strain in its presence. Prophylactic administration of remantadine protected mice from the development of toxic pulmonary edema and death caused by inoculation of a massive dose of influenza B virus. Passages of influenza B virus in the presence of remantadine produced a strain causing toxicosis in mice the course of which was not affected by remantadine any longer.

[Analysis of mutations in the genome of cold-adapted strains of influenza A virus using extended modification of polymerase chain restriction method]. / Analiz mutatsii v genome kholodoadaptirovannykh shtammov virusa grippa A s ispol'zovaniem rasshirennoi modifikatsii PTSP-restriktsionnogo metoda.

Cold-adapted influenza viruses A/Leningrad/13 4/17/5 7 (H2N2) (Len/17) and A/Leningrad/I 34/4 7/57 (H2N2) (Len/47) are used in Russia to prepare live reassortant cold-adapted influenza vaccines (LIV) for adults and children, respectively. Comparison between the nucleotide sequences of the Len/17 strain and the initial wild-type strain A/Leningrad/13 4/5 7 (H2N2) revealed ten nucleotide substitutions (eight of them encoding). Four additional substitutions (three encoding) were found in the genome of the Len/47 virus. Gene segment restriction site (PCR-restriction) analysis was used for identification of the genotype of reassortant influenza viruses. Conventional methods of PCR-restriction analysis detect only five encoding nucleotides substitutions in the internal genes of the Len/17 and seven substitutions in the internal genes of the Len/47 virus. An extended modification of the PCR-restriction method detect all encoding mutations in the internal genes of the Len/17 and Len/47 viruses (eight and eleven encoding substitutions, respectively). This method is advantageous for genome composition analysis of reassortant influenza vaccine strains and for investigating the genetic stability of LIV during replication in vaccines.