NIAID CEIRS | Research Publication Commentary
The majority of influenza vaccines, including most annual seasonal vaccines and stockpiled vaccines against strains with pandemic potential, are produced in chicken eggs. These egg-based vaccines depend on a large supply of eggs, are time-consuming to produce, and cannot be administered to people with egg allergies. Cell-based vaccines, made by growing viruses in mammalian cells, offer several advantages over the current egg-based methods. These mammalian cells are readily available and can be rapidly cultured and used to produce influenza vaccine in the event of an outbreak. Moreover, the resulting vaccine can be safely administered to individuals with egg allergies. However, some vaccine virus strains grow poorly in cell culture, limiting the quantity and increasing the time necessary to produce the vaccine. To address these limitations, Dr. Jihui Ping and his colleagues from the Kawaoka group at the University of Wisconsin-Madison, part of the Center for Research on Influenza Pathogenesis (CRIP), undertook a comprehensive study to develop a vaccine “backbone” that can grow efficiently in cell culture systems.
An influenza vaccine strain is made up of the vaccine backbone (containing the six viral gene segments encoding the internal proteins) and the hemagglutinin (HA) and neuraminidase (NA) surface protein genes selected from the circulating virus. The ideal backbone for a cell culture-based influenza vaccine would confer increased growth in two common mammalian cell types – Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cells. The backbone should also support the production of high quantities of the HA protein, the main antigenic component in the vaccine that confers protection from influenza.
In this study, Ping and colleagues introduced random mutations into various gene segments of the influenza vaccine backbone in an attempt to boost the growth rate of the vaccine strain. The authors generated tens of thousands of individual variants, each possessing random amino acid changes in one or more viral proteins. Viruses containing the mutations were then passaged in MDCK and Vero cells, and those that produced more virus were selected for further analysis. In addition, the authors tested a number of mutations previously shown to increase the growth of different influenza virus strains. The lead candidate, PR8-HY#1, possesses seven amino acid mutations across five viral gene segments that contribute to increased virus growth. PR8-HY#1 was subsequently used as the backbone to study how well candidate vaccine strains grew in MDCK cells, Vero cells, and chicken eggs. The candidates included seasonal H1N1 and H3N2 strains as well as influenza viruses with pandemic potential (H5N1 and H7N9).
Using the PR8-HY backbone in cell culture, the seasonal vaccine strains and those with pandemic potential replicated to significantly higher levels than the control virus (up to 220-fold), depending on the cell type and virus strain. The increased virus titers were accompanied by an increase in production of the HA protein, the main antigenic component of an influenza vaccine. Moreover, the PR8-HY vaccine backbone increased vaccine virus titers and HA protein levels in chicken eggs, suggesting that this backbone could be useful to increase vaccine virus yields in the traditional egg-based method as well.
To test whether the mutations that confer increased virus yield affected the genetic stability of the virus, Ping and colleagues passaged PR8-HY virus in eggs and Vero cells. None of the high-yield mutations reverted to wild-type, nor were any additional mutations identified in the viral genome, suggesting that the yield-enhancing mutations are genetically stable thereby supporting the use of this virus backbone to produce influenza vaccine.
In this study, Ping and colleagues employed “gain-of-function experiments” – techniques that have the potential to confer increased replication, transmission, or virulence – to increase the growth rate of the vaccine strain. All experiments were completed before the U.S. Government announced a research pause on certain gain-of-function studies on 17 October 2014. Commenting on the significance of these findings, corresponding author Dr. Kawaoka stated that, “Our findings have the potential to increase vaccine virus yields significantly. Vaccine viruses that replicate efficiently are highly desired and could save millions of lives during a pandemic, when large amounts of novel vaccines have to be produced in the shortest time possible.” In summary, the high-yield PR8-HY vaccine virus backbone created by Ping and colleagues greatly improves the production of virus in cell culture-based systems and chicken eggs and could lead to faster and more efficient production of influenza vaccines.