Mechanisms for the enhancement of influenza B virus vaccine antigen yield

Satisfactory influenza haemagglutinin (HA) antigen yields are critical for the supply of seasonal trivalent vaccines. Gene reassortment by co-infection with a high-yielding donor strain and an epidemic strain has proved useful for improving HA yields of influenza A viruses but is not generally applicable to influenza B viruses. Selection of high growth influenza B vaccine seed viruses by serial egg passage is generally used but the yields of influenza B viruses can be a rate-limiting step in the production of trivalent vaccines. To obtain enhanced growth of influenza B viruses in embryonated hens’ eggs for vaccine use, the growth of recent isolates of influenza B viruses was studied in eggs and cell cultures under different conditions. Results demonstrate that the need to determine the optimal infectious titre of the inoculum dose used for each influenza B seed virus in order to obtain maximum yields of HA for inclusion in vaccines. Other important determinants of yield are the incubation temperature and the time of harvest. The role of interferon-α, caspase-induced apoptosis and DI RNA as possible causes of variability in the growth of influenza B viruses are also described. Potential donor strains were prepared by approach involving the selection of stable cold-temperature mutants. The three cold-adapted (ca) influenza B viruses (ca B/Malaysia/2506/2004, ca B/Florida/4/2006, ca B/Brisbane/60/2008) which express temperature-sensitive (ts) and ca phenotype, and high growth properties were generated. Reassortants of the novel ca influenza B viruses developed in this thesis prepared by reverse genetics and co-infection with wild-type (wt) epidemic strains also showed enhanced growth, compared with wt parental viruses. This data demonstrate that the genetic signature of the ca influenza B viruses can be transferred to divergent epidemic strains. Further, molecular studies of the three ca influenza B viruses identified that the PB2, PA, NP and NA gene segments are required for the expression of ca and ts phenotype and NP, HA and NA gene segments determine the growth of ca influenza B viruses at 33℃ and the PB2 is required for growth at 25℃. In addition, analysis of deduced amino acid substitutions revealed the association of the ca phenotype with growth enhancement for influenza B viruses. Five loci in four RNA segments, K73 in PB2, E253 in NP, G156, N160 in HA and T146 in NA were found to be sufficient to allow efficient growth of influenza B viruses