Duck embryonic derived stem cell lines for the production of viral vaccines

Abstract

The present invention relates to the development and manufacturing of viral vaccines. In particular, the invention relates to the field of industrial production of viral vectors and vaccines, more in particular to the use of avian embryonic stem cells, preferably the EBx® cell line derived from duck embryonic stem cells, for the production of viral vectors and viruses. The invention is particularly useful for the industrial production of viral vaccines to prevent viral infection of humans and animals.

1 . A process for obtaining continuous diploid avian cell lines, named EBx®, derived from avian embryonic stem cells (ES), wherein said avian cell lines do not produce replication-competent endogenous retrovirus particles, said process comprising the steps of:
 a) isolating bird embryo(s) at a developmental stage around oviposition, wherein the genome of said bird does not contain endogenous proviral sequences susceptible to produce replication competent endogenous retroviral particles;   b) suspending avian embryonic stem (ES) cells obtained by dissociating embryo(s) of step a) in a basal culture medium supplemented with:
 Insulin Growth Factor 1 (IGF-1) and Ciliary Neurotrophic Factor (CNTF); 
 animal serum; and 
 optionally, growth factors selected from the group consisting of Interleukin 6 (IL 6), Interleukin 6 Receptor (IL 6R), Stem Cell Factor (SCF) and Fibroblast Growth Factor (FGF); 
   c) seeding the suspension of ES cells obtained in step b) on a layer of feeder cells and further culturing the ES cells for at least one passage;   d) optionally withdrawing all the growth factors selected from the group consisting of IL-6, IL-6R, SCF, FGF from the culture medium and further culturing the ES cells for at least one passage;   e) withdrawing IGF-1 and CNTF from the culture medium, and further culturing the cells for at least one passage;   f) progressively decreasing the concentration of feeder cells in the culture medium so as to obtain a total withdrawal of feeder layer after several passages, and further culturing the cells;   g) optionally, progressively decreasing the concentration of animal serum in the culture medium so as to obtain a total withdrawal of animal serum after several passages; and   h) obtaining adherent avian EBx® cell lines derived from ES cells capable of proliferating in a basal medium in the absence of growth factors, feeder layer optionally without animal serum, and wherein said continuous diploid avian cell lines do not produce replication-competent endogenous retrovirus particles;   i) optionally, further adapting adherent avian EBx® cell lines to suspension culture conditions.   
   
   
       2 . The process according to  claim 1  wherein said bird is of the Order  Anseriformes.    
   
   
       3 . The process according to  claim 2  wherein said bird is a duck, preferably a Pekin duck. 
   
   
       4 . The process according to  claim 1  wherein said bird is of the Order  Galliformes.    
   
   
       5 . The process according to  claim 4  wherein said bird is an ev-0 domestic Chicken. 
   
   
       6 . Continuous diploid avian cell lines obtainable by the process according to  claim 1  wherein cells of said avian cell lines have at least one of the following characteristics: a high nucleo-cytoplasmic ratio, an endogenous telomerase activity, a diameter of around 10 [mu]m; a population doubling time of around 30 hours or less at 37<0>C;—and optionally, said cells may express one or more additional markers selected from the group consisting of alkaline phosphatase, SSEA-1, EMA-1, ENS-1, and wherein said cells do not produce replication-competent endogenous retrovirus particles. 
   
   
       7 . A process of replicating a virus in the continuous diploid avian cells of  claim 6 , comprising the steps of:
 a) infecting said avian cells with a virus of interest;   b) culturing said infected avian cells in order to replicate said virus; and   c) harvesting the virus in cell culture supernatant and/or inside said cells.   
   
   
       8 . The process according to  claim 7 , wherein said virus is selected from the group consisting of poxviruses, orthomyxoviruses, paramyxoviruses, herpes viruses, hepadnaviruses, adenoviruses, parvoviruses, reoviruses, circoviruses, coronaviruses, flaviviruses, togaviruses, birnavriruses and retroviruses. 
   
   
       9 . The process according to  claim 8  wherein the virus is a poxvirus or a recombinant poxvirus selected from the group consisting of Modified Vaccinia Ankara (MVA) virus, Lister-Elstree vaccinia virus, LC16m8 vaccinia virus, CVI78 vaccinia virus, Fowl pox virus, canary pox virus (i.e ALVAC), NYVAC, juncopox virus, mynah pox virus, pigeonpox virus, psittacine pox virus, quail pox virus, sparrow poxvirus, starling pox virus, and turkey pox virus. 
   
   
       10 . The process according to  claim 7  wherein the virus is a paramyxovirus or a recombinant paramyxovirus preferably selected from the group consisting of measles virus, mumps virus, rubella virus, Sendai virus, Respiratory Syncythial virus (RSV), human para-influenza types I and III, Rinderpest virus, canine distemper virus, Newcastle disease virus, and duck para-influenza virus. 
   
   
       11 . The process according to  claim 7  wherein the virus is an orthomyxovirus, a recombinant orthomyxovirus or a reassorted orthomyxovirus selected from the group consisting of human influenza virus, avian influenza virus, swine influenza virus, equine influenza virus, and feline influenza virus. 
   
   
       12 . The process according to  claim 7  wherein the virus is a togavirus preferably selected from the group consisting of Sinbis virus, Semliki forest virus, O'nyong'nyong virus, Chikungunya virus, Mayaro virus, Ross river virus, Eastern equine encephalitis virus, Western Equine encephalitis virus, Venezuelan Equine encephalitis virus, and a recombinant togavirus thereof. 
   
   
       13 . The process according to  claim 7  wherein the virus is a retrovirus selected from the group consisting of virus, duck infectious anemia virus, suck spleen necrosis virus, and a recombinant retrovirus thereof. 
   
   
       14 . The process according to  claim 7  wherein the virus is a parvovirus preferably a duck parvovirus or a recombinant parvovirus thereof. 
   
   
       15 . The process according to  claim 7  wherein the virus is an adenovirus preferably selected from the group consisting of fowl adenovirus, goose adenovirus, duck adenovirus and pigeon adenovirus and a recombinant adenovirus thereof. 
   
   
       16 . The process according to  claim 7  wherein the virus is a birnavirus preferably Infectious Bursal Disease virus. 
   
   
       17 . The process according to  claim 7  wherein the virus is a flavivirus preferably selected from the group consisting of Dengue virus, Japanese encephalitis virus and West Nile virus. 
   
   
       18 . A virus obtained by the process according to  claim 7 . 
   
   
       19 . A vaccine containing virus according to  claim 18 , if appropriate in combination with pharmaceutically acceptable substances which increase the immune response. 
   
   
       20 . The vaccine according to  claim 19  wherein the virus is selected from the group consisting of a virus present as an intact virus particle and a virus present as a disintegrated virus particle. 
   
   
       21 . A vaccine containing at least one viral antigenic protein obtained from a virus according to  claim 18 , if appropriate in combination with pharmaceutically acceptable substances which increase the immune response. 
   
   
       22 . A vaccine comprising avian cells derived from avian cell lines according to  claim 6 , and infected with a virus. 
   
   
       23 . A process of production of recombinant proteins and peptides, comprising the steps of:
 a) genetically modifying the continuous diploid avian cell lines obtainable by the process according to  claim 1 , by transient or stable transfection of an expression vector;   b) optionally, selecting said modified continuous diploid avian cell lines expressing said recombinant proteins or peptides; and   c) purifying the recombinant peptides or proteins expressed by said genetically modified continuous diploid avian cell lines.