Immortalized avian cell lines for virus production

Abstract

The present invention relates to immortalized avian cell lines suitable for production of biologicals or viruses for vaccination. In particular, the cell lines are derived from primary cells which are transformed with at least two viral or cellular genes, one of which causes cell cycle progression whereas the other interferes with innate protective mechanisms of the cell induced by dysregulated replication. The invention moreover relates to the production of said immortalized cell lines and their use for producing biologicals or viruses for vaccination.

1 - 14 . (canceled) 
     
     
         15 . An avian cell line free of reverse transcriptase activity and permissive for modified vaccinia virus Ankara (MVA). 
     
     
         16 . The cell line of  claim 15 , wherein the cell line is infected with and replicates MVA. 
     
     
         17 . The cell line of  claim 15 , wherein the cell line is not a neoplastic cell line derived from naturally occurring or spontaneous tumors. 
     
     
         18 . The cell line of  claim 15 , wherein the cell line does not release infectious virus particles from endogenous retroviruses. 
     
     
         19 . The cell line of  claim 15 , wherein the cell line does not release infectious virus particles from endogenous retroviruses, is infected with MVA and replicates MVA. 
     
     
         20 . The cell line of  claim 15 , wherein the cells are immortalized. 
     
     
         21 . The cell line of  claim 20 , wherein the immortalized cells are derived from primary cells, cells from isolated body segments or separated individual organs. 
     
     
         22 . The cell line of  claim 15 , further comprising a first gene encoding an expression product capable of overcoming G1 checkpoint control and a second gene encoding an expression product capable of preventing apoptosis induced by expression product of the first gene. 
     
     
         23 . The cell line of  claim 15 , further comprising an antitumor gene. 
     
     
         24 . The cell line of  claim 22 , wherein the expression product of the first gene is capable of mediating disruption of complexes between retinoblastoma proteins and E2F transcription factors; and/or wherein the expression product of the second gene is capable of preventing transcriptional activation by p53. 
     
     
         25 . The cell line of  claim 15 , which is immortalized with a combination of viral and/or cellular genes (gene(s)), wherein the cell line comprises a combination of viral and/or cellular genes including:
 at least one first gene encoding an expression product capable of affecting the function of the retinoblastoma protein by mediating disruption of complexes between retinoblastoma proteins and E2F transcription factors; and   at least one second gene encoding an expression product capable of affecting the p53 protein or a family member thereof;   wherein the first gene is a viral gene selected from the group consisting of an adenovirus E1A gene from mastadenoviruses, an E7 gene of papillomaviruses, an orf 22 gene of avian adenoviruses, and E43 open reading frames from ovine attadenovirus; or is a cellular gene mediating disruption of complexes between retinoblastoma proteins and E2F transcription factors and being selected from Cyclins D1, D2 and D3, and a mutated CDK4 not susceptible to inactivation by p16INK4a; and   wherein the second gene is a viral gene coding for a protein preventing induction of growth arrest and apoptosis by p53 selected from the group consisting of adenovirus E1B55K protein of all groups, GAM-1 of chicken embryo lethal orphan virus CELO and E6 protein of papillomaviruses; or is mdm2 being a cellular gene preventing growth arrest and apoptosis by p53.   
     
     
         26 . The cell line of  claim 25 , wherein the first gene is selected from the group consisting of an E1A gene from mastadenovirus of group C, and an E7 gene from a low-risk human papilloma virus (HPV) and/or wherein the second gene codes for an E6 protein from a low-risk HPV. 
     
     
         27 . The cell line of  claim 15 , which is immortalized with
 (i) E1A and E1B encoding genes of an adenovirus from the genus  Mastadenovirus ; and/or   (ii) orf22 and GAM-1 encoding genes from the genus aviadenovirus CELO.   
     
     
         28 . The cell line of  claim 15 , further comprising non-natural functional sequences selected from the group consisting of transgenes, promoters, enhancers and selection markers. 
     
     
         29 . The cell line of  claim 15 , wherein the cell line is free of animal serum. 
     
     
         30 . The cell line of  claim 15 , wherein the cell line is a duck cell line. 
     
     
         31 . The cell line of  claim 30 , wherein the duck cell line is a cell line originating from duck somites, duck neuronal tissue or duck retina. 
     
     
         32 . The cell line of  claim 15 , wherein the cell line is cell line 12A07-A10 (DSM ACC 2695). 
     
     
         33 . A method for producing MVA, which comprises
 (i) contacting MVA with an avian cell line as defined in claim  1 ; and   (ii) cultivating said MVA on said avian cell line.   
     
     
         34 . The method of  claim 33 , wherein the avian cell line does not release infectious virus particles from endogenous retroviruses. 
     
     
         35 . The method of  claim 33 , wherein the avian cell line is a duck cell line.