Enhancement of influenza viral yields by transgenic embryonated eggs

Egg-based propagation of virus for influenza vaccine has been used for more than 60 years. At the moment for writing this grant proposal, almost all marketed influenza vaccines are grown in embryonated eggs. Egg-based vaccines have been considered to be relative safe. However, the yields for human strains sometimes could not reach a satisfactory amount. Thus, there is a need to increase viral yields in eggs. We found that hSLU7 significantly enhanced viral production in avian cells. It is because that the human splicing factor, hSLU7, can enhance viral production through increasing the level of M2 ion channel protein that is translated from an alternative spliced M mRNA (M2 mRNA). In contrast, avian SLU7 cannot regulate alternative splicing of M mRNA. M2 protein would increase viral yields in avian cells. Therefore, this proposal would like to deliver human SLU7 gene (hSLU7) into chicken embryonated eggs to increase viral yields for vaccine production. This would be also a pioneer approach of a “proof-of-concept” study that transferring a host specific factor into production system would promote vaccine yields. The concept would not only be applied in chicken egg-based system, but also could be used in cell-based production systems, i.g. Vero (a monkey cell line) or MDCK (a canine cell line) cells that are currently in late-stage development as substrates for the growth of influenza virus. Three specific aims of the proposal were summarized: 1. To transfer hSLU7 into embryonated eggs by the RCAS (Replication Competent ALV Long Terminal Repeat with a Splice acceptor) system. An avian leucosis virus (ALV) which contains env (envelope proteins), pol (polymerase) and gag (internal proteins) will be applied as the vector. We would like to take the advantage that the host restriction of ALV is quite stringent so that ALV is unlikely to infect humans. Therefore, use of ALV would be safe in the procedure for vaccine production. 2. To evaluate the viral yields in hSLU7-transferred embryonated eggs (hSLU7-eggs). Viral titer will be determined by plaque assay. Moreover, haemagglutinin (HA) antigen will be quantified by a single radial immune-diffusion (SRID) assay. 3. To analyze the composition of the vaccines that produced by hSLU7-eggs. Seasonal influenza vaccines are usually trivalent. Each dose would contain 15 ❍g of each two influenza A subtypes (e.g. H1N1 and H3N2) and 15 ❍g of one influenza B strain. Therefore, H1N1 , H3N2 and influenza B viruses will be inoculated into hSLU7-eggs and the HA antigens for each virus will be analyzed.

Project ID:PC10202-0502
External Project ID:NSC101-2321-B182-016-MY2