[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.

[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.

Human airway epithelial cells present antigen to influenza virus-specific CD8+ CTL inefficiently after incubation with viral protein together with ISCOMATRIX.

In the present paper, an in vitro model was established in which the interaction between influenza virus-specific CD8+ T cells and human airway epithelial cells can be studied. To this end, the human lung epithelial cell line A549 was transduced with the HLA-A*0201 gene. This MHC class I allele is involved in the presentation of the immunodominant M158-66 cytotoxic T lymphocyte (CTL) epitope of the influenza A virus matrix protein. The A549-HLA-A2 cells and a CD8+ T cell clone specific for the M158-66 epitope were used to evaluate ISCOMATRIX (IMX), which is considered a potential mucosal adjuvant for influenza vaccines, for its capacity to activate virus-specific CTL after incubation with epithelial cells. It was found that virus infected epithelial cells activated virus-specific CTL efficiently. However, incubation of epithelial cells with ISCOMATRIX and recombinant M1 protein activated CD8+ T cells inefficiently, unlike the incubation of C1R cells expressing a HLA-A2 trans gene or HLA-A2+ B-lymphoblastoid cells with these reagents. It was concluded that this lack of antigen presentation by epithelial cells indicate that these cells are not subject to killing by virus-specific CTL upon instillation with ISCOMATRIX-based vaccines, which may be a favorable property of mucosal vaccines.

Sequence variation in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) contribute to the control of viral infections by recognizing peptides of viral proteins presented by MHC class I molecules on infected cells. Some viruses have developed strategies to evade recognition by CTL. One of these strategies involves antigenic variation in CTL epitopes as described for viruses chronically infecting their host like EBV, HIV, HBV and HCV. Here we show three examples of variation in CTL epitopes in the influenza virus nucleoprotein (NP) associated with escape from CTL immunity. The first two involve a mutation at position 384 of the NP, which is the anchor residue of a HLA-B*2705-restricted epitope NP383-391 (SRYWAIRTR) and the HLA-B*08-restricted epitope NP380-388 (ELRSRYWAI). It was shown that these mutations have arisen in the 1993/1994 season and that these mutant variants completely replaced the virus strains containing the wild-type epitopes. Furthermore, T cell recognition was completely abrogated by the R384G mutation. A third example of variation in an influenza virus CTL epitope was found in a newly identified HLA-B*3501-restricted CTL epitope. This immunodominant epitope exhibited extensive amino acid sequence variation and the variants emerged in a chronological order. Again CTL specific for older variants failed to recognize more recent strains of influenza A virus, indicating an escape from CTL immunity. Thus, in addition to the introduction of mutations in the surface glycoproteins like the hemagglutinin, allowing escape from antibody-mediated immunity, there is now evidence that influenza viruses can escape in a similar way from CTL-mediated immunity.