Avian Cells for Improved Virus Production

Abstract

The present Invention provides as avian cell in which the expression or activity of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3) is reduced. The invention also provides methods for passaging viruses in avian cells, embryos and/or avian cell lines which have reduced expression of one or more IFITM genes and methods which involve investigating the sequence of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3).

1 . An isolated chicken cell or a chicken cell within an embryonated egg,
 wherein the isolated chicken cell or the chicken cell within the embryonated egg comprises a mutation in, or a knockout of, an endogenous interferon-inducible transmembrane protein 2 (IFITM2) gene that encodes an IFITM2 protein in a comparable wildtype chicken cell that lacks said mutation,   wherein the IFITM2 protein encoded by said endogenous IFITM2 gene in the wildtype chicken cell has an amino acid sequence at least 95% identical to SEQ ID NO: 2,   wherein the mutation or knockout reduces expression and/or activity of the IFITM2 protein, and   wherein the isolated chicken cell or the chicken cell within the embryonated egg has reduced expression and/or activity of said IFITM2 protein compared to the wildtype chicken cell that lacks said mutation or knockout.   
     
     
         2 . (canceled) 
     
     
         3 . The isolated chicken cell or the chicken cell within the embryonated egg of  claim 1 , which is a chicken embryonic fibroblast (CEF). 
     
     
         4 . The isolated chicken cell or the chicken cell within the embryonated egg of  claim 1  that comprises the mutation that reduces the expression and/or activity of the, IFITM2 protein. 
     
     
         5 . The isolated chicken cell or the chicken cell within the embryonated egg of  claim 1  that comprises the knockout of the endogenous IFITM2 gene. 
     
     
         6 . (canceled) 
     
     
         7 . A method for propagating a virus comprising:
 (i) transducing the isolated chicken cell or the chicken cell within the embryonated egg of  claim 1  with a virus; and;   (ii) passaging the transduced chicken cell to propagate the virus.   
     
     
         8 . A method for producing a vaccine comprising:
 (i) propagating a virus according to the method of  claim 7 ; and,   (ii) incorporating the propagated virus into a vaccine.   
     
     
         9 . A method for generating an attenuated virus, comprising the following steps:
 (i) transducing the isolated chicken cell or the chicken cell within the embryonated egg of  claim 1  with a virus; and,   (ii) passaging the transduced chicken cell such that the virus completes multiple rounds of infection and becomes attenuated.   
     
     
         10 . A method for producing a vaccine comprising:
 (i) generating an attenuated virus according to  claim 9 ;   (ii) propagating the attenuated virus; and,   (iii) incorporating the virus propagated in (ii) into a vaccine.   
     
     
         11 . The method according to  claim 7 , wherein the virus enters the isolated chicken cell or the chicken cell within the embryonated ego through acidic endosomes. 
     
     
         12 . A transgenic chicken in which the expression and/or activity of one or more of the following polypeptides, or a homologue thereof: Chicken IFITM1 (SEQ ID NO: 1); Chicken IFITM2 (SEQ ID NO: 2) and Chicken IFITM3 (SEQ ID NO: 3) is reduced, knocked-down or knocked-out. 
     
     
         13 . A transgenic chicken whose germ cells and somatic cells comprise a heterogenic version of one or more nucleotide sequences encoding for one of the following polypeptides, or a homologue thereof: Chicken IFITM1 (SEQ ID NO: 1); Chicken IFITM2 (SEQ ID NO: 2) and Chicken IFITM3 (SEQ ID NO: 3) which nucleotide sequence was introduced into said chicken, or an ancestor of said chicken, at an embryonic stage. 
     
     
         14 - 16 . (canceled) 
     
     
         17 . An avian cell according to  claim 1  which is derived from a transgenic avian animal. 
     
     
         18 - 28 . (canceled) 
     
     
         29 . The isolated chicken cell or a chicken cell within an embryonated egg of  claim 1 , wherein the IFITM2 protein encoded by said endogenous IFITM2 gene in the equivalent wildtype chicken cell comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         30 . The isolated chicken cell or an chicken cell within an embryonated egg of  claim 1 , wherein the wild-type chicken cell has a gene encoding an interferon-inducible transmembrane protein (IFITM1) with at least 95% sequence identity to SEQ ID NO: 1; and wherein the isolated chicken cell or the cell within the embryonated egg has a mutation or knockout of the gene that encodes the IFITM1, said mutation or knockout reducing expression and/or activity of the IFITM1. 
     
     
         31 . The isolated chicken cell or an chicken cell within an embryonated egg of  claim 1 , wherein the wild-type chicken cell has a gene encoding an interferon-inducible transmembrane protein (IFITM3) with at least 95% sequence identity to SEQ ID NO: 3; and wherein the isolated chicken cell or the cell within the embryonated egg has a mutation or knockout of the gene that encodes the IFITM3, said mutation or knockout reducing expression and/or activity of the IFITM3.