Sequence changes in the live attenuated, cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus.

Nucleotide sequences were determined for the RNA segments coding for proteins other than the hemagglutinin and neuraminidase of the A/Leningrad/134/57 (H2N2) wild-type (A/Len/wt) virus and its two cold-adapted (ca) and attenuated variants, A/Leningrad/134/17/57 (A/Len/17/ca) and A/Leningrad/134/47/57 (A/Len/47/ca) that are used in the U.S.S.R. in the preparation of reassortant live attenuated vaccines. Ten nucleotide differences were detected between the sequences of the A/Len/wt and A/Len/17/ca viruses; of these, eight were deduced to encode amino acid (aa) substitutions. One aa substitution each was predicted for the PB2, M1, M2, and NS2 proteins, whereas two aa substitutions each were predicted for the PB1, and PA proteins of the A/Len/17/ca virus. Four additional nucleotide changes were found in the genome of the A/Len/47/ca virus; three of these were detected to code for one additional aa substitution each for the PB2, PB1, and NP proteins.     

Phenotypic and genetic analyses of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2)

For the past three decades the cold-adapted (ca) and temperature sensitive (ts) master donor strain, A/Leningrad/134/17/57 (H2N2) has been successfully used as the basis for the live attenuated reassortant influenza A vaccine. This donor strain was developed from A/Leningrad/134/57 (H2N2) wild-type (wt) virus following 17 passages in eggs at 25 degrees C. Our detailed investigation has revealed that the A/Leningrad/134/17/57 (Len/17) master donor stock is a mixed population comprised of numerous variants of the ca/ts Len/17 influenza virus. We have identified these variants to exhibit a broad range in their temperature sensitive phenotype when assayed on Madin-Darby canine kidney (MDCK) cells at 37 degrees C. A selection of these variant clones has been fully characterized by sequencing in order to understand the variability in the ts phenotype. This study has also addressed the feasibility of using cell culture technology for the propagation and subsequent manufacturing of the cold-adapted influenza vaccine (CAIV), particularly with respect to retaining the defined mutations that contribute toward the ca/ts phenotype.     

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.     

Immunogenic and isotype-specific responses to Russian and US cold-adapted influenza a vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57, and A/Ann Arbor/6/60 (H2N2) in mice

The immunogenicity of the Russian cold-adapted (ca) donor stains, A/Leningrad (Len)/134/17/57 and A/Leningrad/134/47/57, and the US strain A/Ann Arbor (AA)/6/60-ca, were compared in BALB/c mice with their respective wild-type parental viruses. Each ca donor strain was less immunogenic than its wild-type parent. The vaccinating dose, when administered twice, which prevented multiplication of a standard challenge of parental wild-type virus in 50% of mice (the 50% protective dose or PD(50)), was shown for A/Len/134/17/57-ca, A/Len/134/47/57-ca, and A/AA/6/60-ca to be 10(3.77), 10(4.32), and 10(4.70), respectively. These findings were extended by measuring the number of antibody secreting cells induced in the lungs and mediastinal lymph nodes of mice infected with the same ca donors using an ELISPOT assay. When each donor strain was administered twice at a dose of 100 PD(50) over a 3-week interval, the overall immunoglobulin isotype antibody secreting cell profiles were shown to be similar. However, A/Len/134/17/57-ca and A/Len/134/47/57-ca induced significantly higher total immunoglobulin responses in the lungs than A/AA/6/60-ca (P < 0.05). A/Len/134/17/57-ca also induced a significantly greater IgA response in the lungs than A/AA/6/60-ca (P < 0.05). These results suggest that A/Len/134/17/57-ca is a superior immunogen to A/Len/134/47/57-ca which in turn is more immunogenic than A/AA/6/60-ca.     

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.     

Mutations in the genes coding for hemagglutinin and neuraminidase in cold-adapted variants of the influenza virus A/Leningrad/134/57 (H2N2)

An analysis of ts-mutations in the genomes of native and cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus based on the use of fowl plague virus ts mutants was carried out. The recombination test was done by the conventional method in chick embryo fibroblast culture (genes PB2, PB1, PA, NP, NA, M and NS) or cell systems permissive for reproduction of human influenza virus (gene HA). The cold-adapted strain A/Len/17 used for preparation of live influenza vaccine (LIV) for adults was shown to have 4 ts mutations: three in "internal" genes (PB2, NP, and M) and one in gene 4 coding for hemagglutinin (HA). The more attenuated cold-adapted donor A/Len/47 for preparation of similar LIV for children acquired three additional ts mutations: two (PB1 and NS) in "internal" genes and one in gene 6 coding for neuraminidase (NA). The accumulation of ts mutations in the genome of cold-adapter strains was found to be accompanied by a decrease in their pneumotropicity for mice as well as their detectability in different organs of these animals.     

Analysis of mutations in the genome of cold-adapted strains of influenza A virus using extended modification of polymerase chain restriction method

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.     

The growth of attenuated influenza vaccine donor strains in continuous cell lines

The growth of the Russian live attenuated influenza vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57 and B/USSR/60/69 was studied in cells of the VERO and Madin-Darby canine kidney (MDCK) lines as six-well cultures and cell factories infected at different multiplicities of infection. Yields for A/Leningrad/134/17/57 and A/Leningrad/134/47/57 were comparable in either cell line over a range of multiplicities but were about 10-fold lower than in the allantoic fluids of infected chicken embryos. For both A/Leningrad/134/47/57 and B/USSR/60/69, yields from the MDCK line were about 10-fold higher than for the VERO line. For B/USSR/60/69, yields in eggs were approximately 100-fold higher than those obtained in the MDCK line. A feature of the growth of B/USSR/60/69 was its reduced capacity to produce infectious progeny in either cell line at multiplicities of infection of 2.0 or 1.0 pfu/cell. Inhibition was due probably due to the presence of defective-interfering particles and was not detected with A/Leningrad/134/17/57 or A/Leningrad/134/47/57 in cultures of either line infected at the same multiplicities. Yields for both A/Leningrad/134/47/57 and B/USSR/60/69 in cells of the MDCK line were comparable when grown in six-well cultures or cell factories.     

Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants

Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 x 10(10) pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.     

Production of early cytokines after infection with A/Leningrad/134/57 (H2N2) wide-type virus and cold-adapted attenuated vaccine viruses

The production of proinflammatory cytokines was studied following experimental infection of BALB/c mice with influenza viruses that differed in virulence. The generation of TNF-alpha, IL-6, IL-12, and IFN-gamma was investigated in the lung homogenates in the early periods after intranasal infection of mice with A/Leningrad/134/57 (H2N2) wild-type virus and cold-adapted attenuated vaccine viruses: A/Leningrad/134/17157 (H2N2) and A/Leningrad/134/47/57 (H2N2). Wild-type virus induced substantially higher levels of proinflammatory cytokines: TNF-alpha, IL-6, IL-12, and IFN-gamma. After infection with the cold-adapted viruses, the levels of the cytokines were reduced as compared to those induced by the wild-type virus. The A/Leningrad/134/47/57 virus was marked by a noticeable production of IL-6 and IFN-gamma in the murine lung, but it was less than with wild-type virus infection. At the same time, the more attenuated strain A/Leningrad/134/47/57 induced TNF-alpha and IFN-gamma in the quantities similar to those in the control animals. Thus, a response of proinflammatory cytokines in early infection in the murine lung depended on the level of viral replication in the lower respiratory tract and on the attenuation of influenza virus strains.     

Attenuated ts-recombinants of influenza A/USSR/77 (H1N1) virus obtained by crossing with the cold-adapted donor A/Leningrad/134/57 (H2N2) virus

Conditions of obtaining attenuated influenza virus recombinants by crossing of a cold-adapted donor with A (H1N1) influenza virus that reappeared in 1977 were studied. Temperature-sensitive recombinants suitable for intranasal immunization of adults with low titres of anti-haemagglutinin and anti-neuraminidase antibodies, and possessing sufficiently high immunogenicity were obtained by crossing of native parent strains and cross-reactivation techniques. It was confirmed that the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus variant is a prospective donor of attenuation for obtaining recombinants--candidates for live influenza vaccine strains.     

B-cell and cytotoxic lymphocyte immune response to virulent,attenuated and reassortant influenza viruses

The quantitative estimation of the production of the virus specific IgA-, IgM-, IgG1, IgG2a, IgG2b and IgG3-antibody secreting B-cells (ASC) and extent of the immune response of the cytotoxic T-lymphocytes was carried out in mice after primary and secondary immunization with the parental cold adapted (ca) virulent epidemic wild-type (wt) master strain viruses and their reassortant variant (RV) with the incorporated in their genom different genes from ca master strain. The chick embryo derived ca master strain virus A/Len/134/47/57 (H2N2) reduced or eliminated the viral potency to induce the primary B-cellular response. Reassortant incorporation of wt derived HA and NA genes into the ca virus genome restored the virus immunogenic activity concerning the ASC production, but at the lower level than parental virulent virus. Reassortance of the viruses according to generally accepted genom formula 6/2 was associated with decrement of the functional activity of the cytotoxic T-lymphocytes memory and of the primary immune response especially. The data obtain demonstrate the necessity for the control of the immunogenicity of the reassortant viruses at the cloning stage.     

The effect of amplifying reproduction of influenza virus in mouse lungs during simultaneous infection with two cold-adapted strains

Reproduction of cold-adapted (ca) strains of influenza virus in the lungs of white mice after separate and combined inoculation and the properties of isolates derived from the infected animals were studied. It was shown that after combined inoculation with ca and ts strains A/Leningrad/134/17/57 (H2N2) and A/PR/8/59/1 (H1N1) ca recombinants could develop loosing some ts mutations and possessing (unlike the master strains) pneumo-virulence for mice. All the pneumo-virulent reassortants inherited hemagglutinin from the ca A/PR/8/59/1 strain and PB1 protein from the ca A/Leningrad/134/17/57 strain. The results indicate that it is unsafe to construct live recombinant divaccines by combining the recombinants produced from different donors of attenuation.     

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.     

Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model

Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.     

Characterization of Reverse Genetics-Derived Cold-Adapted Master Donor Virus A/Leningrad/134/17/57 (H2N2) and Reassortants with H5N1 Surface Genes in a Mouse Model

Live attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV), we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parental wt viruses compared to the ca master donor viruses as well as the H5N1 reassortants. All ca viruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of the ca MDV. Mice inoculated twice 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologous wt H5N1 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV viruses warrant further development into a vaccine for human use.     

Genome composition analysis of 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 vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6: 2 reassortant viruses containing the gene segments of the surface antigens haemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6: 2 reassortant viruses are 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, sboth selection and creening needs to be both rapid and unambiguous. The screening of reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets is described to select both influenza A and influenza B 6: 2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine, unambigously.     

Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the PB2, PB1, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.     

Cell-based assay for the determination of temperature sensitive and cold adapted phenotypes of influenza viruses

The determination of temperature sensitive (ts) and cold adapted (ca) phenotype for influenza A and B strains has been conducted traditionally using embryonated chicken eggs. As attempts are made to move away from the use of eggs in the manufacturing process of influenza vaccines, it will become useful to develop cell-based assays to support cell culture-based vaccine production. In this study, MDCK cells have been evaluated as a tool for determining the ts and ca phenotypes associated with live attenuated influenza viruses. Direct comparisons were made of these phenotypes carried out in eggs. Reassortants made from the Russian live attenuated influenza donor strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 were prepared entirely in MDCK cells and their phenotypes evaluated using the MDCK cell-based assay. It is concluded that MDCK cells are more sensitive than eggs for the measurement of ts and ca phenotype of influenza viruses (particularly for influenza A) and they provide an alternative means for screening candidate reassortants prior to determining their genome composition.     

Location of ts defects in the genome of cold-adapted recombinant influenza A virus vaccine strains

The ts phenotype and location of ts mutations were studied in the genome of parent viruses and those obtained by recombination of cold-adapted strains A/Leningrad/134/17/57 or A/Leningrad/134/47/57 with epidemic H1N1 and H3N2 influenza A virus strains. The epidemic H1N1 and H3N2 strains under study possessed a ts phenotype and contained ts mutations in one or two genes. The ts phenotype was lost following three clonings at 40 degrees C, suggesting that influenza virus strains isolated from humans may be heterogeneous and contain virions either carrying or not carrying the ts mutations in their genomes. Two cold-adapted strains possessing a distinct ts phenotype contained ts mutations in three (A/Leningrad/134/17/57 virus after 17 passages at 25 degrees C) or in five (A/Leningrad/134/47/57 variant after 30 additional passages at 25 degrees C) genes coding for non-glycosylated proteins. When compared with cold-adapted donor strains, the recombinants had either the same set or additional ts mutations. However, no ts mutation was detected in a gene which had been inherited from the donor strain. It is suggested that, in addition to the analysis of the genome composition, in cold-adapted recombinant influenza virus strains recommended as vaccine candidates it is necessary to control the number of genes containing ts mutations.