Methods for enhancing the production of viral vaccines in cell culture

#1. Technical Field

#The present invention relates to methods for the production of virus for vaccine production in cell culture.

#2. Background

#FIGS. 1A and 1B. PKR activity and protein levels in U937-derived stable transfectant cell lines. FIG. 1A Functional PKR activity was determined using a poly(I):poly(C)-cellulose assay for PKR autophosphorylation. Cell extracts were prepared from the different U937 transfectant cell lines following incubation with (+) or without (−) recombinant human IFN-α2 (200 U/mL) as indicated, while L929 cells were similarly treated with mouse IFN-α/β. Lane 1, HeLa; lanes 2 and 3, U937-neo; lane 4, U937-AS1; lane 5, U937-AS3; lane 6, U937-M13; lane 7, U937-M22; lane 8, L929. Positions of the human (68 kDa) and mouse (65 kDa) PKR proteins, and the molecular size standards (80 and 50 kDa) are indicated. FIG. 1B Cell extracts were prepared as above after induction with IFN-α or -γ and PKR protein levels were determined by Western blot analysis.

#FIGS. 2A and 2B. Kinetics of EMCV replication are enhanced in PKR-deficient cells. The different U937 cell lines were challenged with EMCV at 0.1 (FIG. 2A) or 0.001 (FIG. 2B) TCID50/cell. Samples were harvested at the indicated times and viral yields were measured in terms of TCID50.

#The present invention relies upon the discovery by the inventor that the level of interferon production in cells can be regulated by manipulating the expression or activity of certain factors that normally regulate interferon expression and activity in vivo. These factors include certain interferon-specific transcriptional regulators, particularly IRF1, certain interferon receptors, as well as the gene products of certain interferon simulated genes (also called interferon-mediated antiviral responses), particularly PKR and 2-5A synthetase. Suppression or elimination of the expression or activity of any of these factors will result in a lower than normal level of expression of interferon genes. One consequence of this lower than normal interferon expression level is an increased permissiveness of the cell to viral replication. An increased permissiveness of the cell to viral reproduction means that greater viral production can be achieved in that cell relative to a cell having normal interferon expression. Cells having an increased permissiveness to viral replication are useful for a number of applications including vaccine production, sensitive detection of low levels of virus and for the evaluation of antiviral compounds.

#The present inventor has surprisingly found that animal cells that are deficient in interferon-mediated antiviral responses, particularly cells deficient in dsRNA dependent kinase, 2N-5N Oligoadenylate synthetase or both, produce a higher viral yield when infected with an animal virus than cells with normal levels of these proteins. Increases of viral yield by as much as 103 to 104 or more can be obtained using the method of the present invention. The ability to obtain high yields of virus in PKR- or 2-5A synthetase-deficient cell culture makes it possible to produce large amounts of virus within a short time. This is particularly important for production of viral vaccines, most particularly for RNA virus, including influenza virus. The increased permissiveness of the deficient cells to viral replication makes them useful in a method for evaluating antiviral drugs in cell culture and in a method for detecting viral pathogens.

#One aspect of the present invention provides a method for production of a viral vaccine in cell culture which comprises (a) infecting a cell culture with a donor strain animal virus, wherein said cell culture is deficient in the activity of the gene product of an interferon-stimulated gene, (b) culturing the infected cell culture under conditions sufficient to provide efficient virus growth, and (c) harvesting the virus produced. The harvested virus may be additionally prepared for vaccine use by purification, for instance by sterile filtration, ultrafiltration and/or concentration by column chromatography or other methods. The harvested virus may optionally be treated to inactivate the virus for the production of killed viral vaccines.