Crucell Holland B.v.

Alphonsus G C M UytdeHaag

Maria Grazia Pau

Govert Johan Schouten

Dirk J E Opstelten

Jaap Goudsmit

Ronald H P Brus

Means and methods for producing mammalian viruses, the method comprising infecting a culture of immortalized human cells with a virus, incubating the culture infected with virus to propagate the virus under conditions that permit growth of the virus, and to form a virus-containing medium, and removing the virus-containing medium. The viruses can be harvested and be used for the production of vaccines. Advantages include that human cells of the present invention can be cultured under defined serum-free conditions and the cells show improved capability for propagating virus. Methods are provided for producing, in cultured human cells, influenza virus and vaccines derived thereof. This method eliminates the necessity of using whole chicken embryos for the production of Influenza vaccines. The method also provides for the continuous or batch-wise removal of culture media. As such, the present invention allows the large-scale continuous production of viruses to a high titer.

Production of vaccines

Disclosed are immortalized human embryonic retina cells, having a nucleic acid sequence encoding an adenoviral E1A protein integrated into the genome of the cells, and further comprising a nucleic acid sequence encoding an enzyme involved in post-translational modification of proteins, such as a sialyltransferase, wherein the nucleic acid sequence encoding the enzyme involved in post-translational modification of proteins is under control of a heterologous promoter. Methods for producing recombinant proteins from such cells and obtaining such recombinant proteins having increased sialylation are provided as are novel compositions of isoforms of erythropoietin.

Methods of producing erythropoietin isoforms comprising Lewis-X structures and high sialic acid content and compositions of the same

Means and methods are provided for the production of mammalian viruses comprising: infecting a culture of immortalized human cells with the virus, incubating the culture infected with virus to propagate the virus under conditions that permit growth of the virus, and to form a virus-containing medium, and removing the virus-containing medium. The viruses can be harvested and be used for the production of vaccines. Advantages are that human cells of the present invention can be cultured under defined serum free conditions, and the cells show improved capability for propagating virus. In particular, methods are provided for producing, in cultured human cells, influenza virus and vaccines derived thereof. This method eliminates the necessity to use whole chicken embryos for the production of influenza vaccines. The method provides also for the continuous or batchwise removal of culture media. As such, the invention allows the large-scale, continuous production of viruses to a high titer.

The present invention relates to novel vaccines containing (whole-inactivated) West Nile Viruses and/or West Nile viral proteins derived therefrom, produced on human cells, wherein the human cells comprise a sequence encoding at least one early region-1 (E1) gene product of an adenovirus. The cells are preferably cultured in suspension to very high densities and under serum-free conditions. Herein, it is disclosed that use of such cells results in high titers of West Nile Virus produced.

Vaccines against West Nile Virus

Disclosed are immortalized human embryonic retina cells, having a nucleic acid sequence encoding an adenoviral E1A protein integrated into the genome of the cells, and further comprising a nucleic acid sequence encoding an enzyme involved in post-translational modification of proteins, such as a sialyltransferase, wherein said nucleic acid sequence encoding the enzyme involved in post-translational modification of proteins is under control of a heterologous promoter. Methods for producing recombinant proteins from such cells and obtaining such recombinant proteins having increased sialylation are provided as are novel compositions of isoforms of erythropoietin.

Compositions of erythropoietin isoforms comprising Lewis-X structures and high sialic acid content

Novel means and methods are provided for the production of mammalian viruses comprising, infecting a culture of immortalized human cells with the virus, incubating the culture infected with virus to propagate the virus under conditions that permit growth of the virus, and to form a virus-containing medium, and removing the virus-containing medium. The viruses can be harvested and be used for the production of vaccines. Advantages are that human cells of the present invention can be cultured under defined serum free conditions, and the cells show improved capability for propagating virus. In particular, methods are provided for producing, in cultured human cells, influenza virus and vaccines derived thereof. This method eliminates the necessity to use whole chicken embryos for the production of influenza vaccines. The method provides also for the continuous or batchwise removal of culture media. As such, the present invention allows the large-scale, continuous production of viruses to a high titer.

The present invention provides novel methods for determining whether a compound influences a phase in the life cycle of a virus comprising providing a cell with the compound and with at least a fragment of the virus sufficient for performing the phase and determining whether the phase is influenced in the cell, the cell comprising a nucleic acid encoding an adenovirus early protein or a functional part, derivative and/or analogue of the adenovirus early protein. In another aspect, the invention provides the use of a cell, the cell comprising nucleic acid encoding an adenovirus early protein, for screening a library of compounds for the presence of a compound capable of influencing a phase in the life cycle of a virus capable of entering the cell. The invention also provides novel methods for identifying a compound with antiviral activity comprising providing a cell with at least a fragment of a virus, the fragment capable of performing a step in the life cycle of the virus, providing the cell with a compound and determining whether the compound is capable of influencing the step in the life cycle of the virus, wherein the cell comprises a nucleic acid encoding an adenovirus early protein or a functional part, derivative and/or analogue of the adenovirus early protein.

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Vleuten, Netherlands

Alphonsus G C M UytdeHaag