Production of viral vaccines in suspension on avian embryonic derived stem cell lines

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 chicken 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.

Claims

1. A process of production of a virus which comprises replicating a virus selected from the group consisting of adenoviruses, hepadnaviruses, herpes viruses, orthomyxoviruses, papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses and retroviruses, in cells of an avian cell line wherein said cell line has been obtained by a method comprising the following steps: a. culturing avian embryonic stem cells in a cell culture medium containing the factors allowing their growth, wherein said factors comprise: i. a trophic factor selected from the group consisting of stem cell factor (SCF), Insulin Growth Factor (IGF-1), and basic Fibroblast Growth Factor (bFGF), and ii. a cytokine whose action is through a receptor which is associated with the gp130 protein, selected from the group consisting of leukemia inhibitory factor (LIF), interleukin 11 (IL-11), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), ciliary neurotrophic factor (CNTF), oncostatin, and cardiotrophin and supplemented with animal serum in the presence of a feeder layer; b. passaging by modifying the culture medium so as to obtain the withdrawal of said exogenous growth factors and cytokines, of the serum, and of the feeder layer; c. establishing adherent or non-adherent cell lines capable of proliferating in the absence of said exogenous growth factors, serum, and inactivated feeder layer from said cell culture medium; said process comprising the steps of: a) proliferating said cells in a cultivation vessel, in suspension, in a serum-free medium No. 1; b) infecting said cells with the selected virus when the cell density is of at least 1.5 million cells/ml; c) shortly before infection, simultaneously to infection, or shortly after infection adding serum-free medium No. 2 to the cell culture; and d) further culturing said infected cells in order to allow virus replication; and e) optionally, harvesting said virus, wherein said process comprises an additional step of feeding the cells wherein said feeding step consists in a periodic addition of concentrated solution of glutamine and D-glucose to the culture medium; and wherein glutamine concentration in the culture medium is maintained between 1 mM to 3 mM and D-glucose concentration in the medium is maintained around 1 g/l to 10 g/l of D-glucose.

2. The process of claim 1, wherein the cultivation vessel is a continuous stirred tank bioreactor.

3. The process of claim 1, wherein the serum-free medium No. 1 and the serum-free medium No. 2 have a different composition.

4. The process of claim 1, wherein between 0.25 to 10 volumes of serum-free medium No. 2 is added to serum-free medium No. 1.

5. The process of claim 1, wherein the serum-free medium No. 1 and/or the serum-free medium No. 2 is/are supplemented with at least one ingredient selected from the group consisting of amino-acids, lipids, fatty acids, cholesterol, carbohydrates, protein hydrolyzates of non-animal origin, and a mixture thereof.

6. The process of claim 1, comprising the additional step of feeding the cells with at least one ingredient selected from the group consisting of amino-acids, fatty acids, carbohydrates, protein hydrolyzates of non-animal origin, and a mixture thereof.

7. The process of claim 6, wherein the feeding occurs during steps a) to d), preferably during the steps b) to d).

8. The process of claim 7, wherein the feeding occurs on a daily basis.

9. The process of claim 8, wherein the feeding occurs on a continuous basis.

10. The process of claim 8, wherein the feeding occurs more than one time per day.

11. The process of claim 8, wherein the feeding occurs less than one time per day.

12. The process of claim 6, wherein fatty acids are selected from the group consisting of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid or a mixture thereof.

13. The process of claim 6, wherein protein hydrolyzates of non-animal origin are selected from the group consisting bacteria tryptone, yeast tryptone, plant hydrolyzates, or a mixture thereof.

14. The process according to claim 1, wherein said cells are cultured in suspension as clumped cells.

15. The process according to claim 1, wherein step c) is performed after infection step b), preferably around 1 hour after step b).

16. The process according to claim 1, wherein step b) is carried out at an m.o.i (multiplicity of infection) of about 10 to 10.sup.6, preferably about 10.sup.3 to 10.sup.5, and more preferably about 10.sup.4.

17. The process according to claim 1, wherein the virus is paramyxovirus, preferably measles virus or Newcastle Disease virus (NDV).

18. The process according to claim 1, wherein step b) is performed when the cell density is of at least at least 4 million cells/ml in batch or fed-batch process, preferably 6 million cells/ml.

19. The process according to claim 1, wherein step b) is performed when the cell density is of at least at least 8 million cells/ml in perfusion process, preferably around 9 to 10 million cells/ml.

20. The process according to claim 1, wherein the serum-free culture medium in steps a), b), c) and d) has a pH in a range from 6.5 to 7.8, and wherein cell culture is performed at a temperature comprising between 30 C. to 39 C., preferably at around 37 C.

21. The process according to claim 1, wherein step d) lasts for 2 to 10 days before the harvest.

22. A virus obtained by a process as claimed in claim 1.

Description

FIGURES

(1) FIG. 1: Transmission Electronic Microscopy analysis of EB14 cells

(2) EB14 cells display a typical embryonic stem cells morphology (i.e high nucleo-cytoplasmic ratio) that resemble the phenotype of murine embryonic stem cells (mES). EB14 cells are small round cells with a large nucleus and nucleolus with short pseudopodia extending from the plasma membrane. They are highly metabolically active with a ribosome and mitochondria rich cytoplasm. The cell morphology of EB14 cells is different to the one of chicken embryonic fibroblasts (CEF).

(3) FIG. 2: Karyotyping analysis of EB14 cells at passages 105 and 118

(4) Analysis of EB14 cells cultured in serum-free medium until passages 105 and 118 confirmed the diploid nature of the cells, with the presence of 18 macro-chromosomes and 30 micro-chromosomes (upper panel). This result is in agreement with the chromosome numbers expected for chicken cells (lower panel).

(5) FIG. 3: Telomerase expression in EB14 cells

(6) Telomerase expression in EB14 cells cultured in serum-free medium at different passages was investigated by using Roche telomerase detection kit (Telomerase OCR ELISA). Telomerase is found to be highly expressed in EB14 cells. The high level of telomerase expression is maintained over passages as shown at passage 89, at passage 160 (which correspond to EB14 cell Master Cell Bank), and at passage 179 (which correspond to end of production passages). Canine MDCK cell line are used a negative control and do not express telomerase. Similar absence of telomerase expression was found for CEFs cells (Data not shown).

(7) FIG. 4: ENS1 gene is expressed in EB14 cells

(8) The ENS1 gene was described as being specifically expressed in chicken ES cells (Acloque & al., Mech Dev. 103, p 79-91, 2001). Its expression in EB14 cells was evaluated by RT-PCR. Avian embryonic stem cells (ES cells), avian embryonic cells collected at oviposition and EB14 cells at various passage (P21, P159, P160) are found to strongly express ENS1 gene while avian cell line DF1 (U.S. Pat. No. 5,672,485) and chicken embryonic fibroblasts (CEFs) do not express this gene. Analysis of the housekeeping GAPDH gene is performed in parallel on the same samples to control the presence of the RNAs (lower panel).

(9) FIG. 5: Expression of ES cell-specific markers in EB14 cell line (left panel) and DF1 cell line (right panel)

(10) EB14 cells express EMEA1 and SSEA1 genes while DF1 cell does not. Paxilline gene is an ubiquitous gene used as a control. EB14 cells do not express cell markers TROMA-1, MelM, TRA-1-60 and SSEA3.

(11) FIG. 6: Cell surface expression of receptors SA2-3 and SAa2-6 in EB14 and MDCK cell lines

(12) Cells are incubated with digoxygenin labelled lectins: Sambuca nigra agglutinin lectin specifically binds to Sia2-6Gal, while Maackia amurensis agglutinin lectin specifically binds to Sia2-3Gal. Lectins that bind to cells are revealed with anti-digoxygenin antibody FITC-labelled according to well-known techniques by the man skilled in the art. FITC-labelled cells are numbered with a fluorescent cell sorter (FACS). SAa2-3 and SAa2-6 molecules are been described to be the receptors for the avian and human influenza viruses, respectively.

(13) FIGS. 7A and 7B: Growth kinetics of EB14 cells in a 3 L-fedbatch bioreactor

(14) FIG. 7AEB14-derived biomass was allowed to accumulate at 37 C. in a cell growth medium supplemented with 0.5 yeastolate until a cell density of 5-6.10.sup.6 cells/mL was reached. Then the mixture was 2.9 fold diluted and cell growth kinetic was followed-up over a 10 days period. Cell density of 13 million cells/ml was reached at day 5.

(15) FIG. 7BSplit ratio flexibility for cell growth kinetic of EB-14 cells in a 3 L fedbatch bioreactor: Following a cell seeding with a split ration of 1/10 (0.23 L at a density of 0.4.10.sup.6 cells/mL in 2.1 L final volume), EB14-derived biomass was allowed to accumulate in Excell 65319 (SAFC) growth medium at 37 C. over a 11 days period. L-glutamine (2 mM) and D-(+)-glucose (2 g/L) concentrations were daily adjusted as a fed-batch process, and bioreactor parameters were fixed as follows: rotation speed: 80 rpm, pO.sub.2: 50%, pH: 7.20.

(16) FIG. 8: Influence of production medium and clumps' size for MVA-GFP virus propagation in infected EB14 cells: GFP expression

(17) EB14 were allowed to form small (left panel) or large (right panel) clumps in T175 stirred tank flasks during cell proliferation in a cell growth SFM medium (SAFC: Excell 65319). Clumps were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in several production SFM media (Optipro, Excell 65319, Excell 65629). During a 7 days virus propagation period at 37 C., pictures of UV-exposed infected cells were taken daily. Control: optipro (INVITROGEN) was used as cell growth and production medium.

(18) FIG. 9: Influence of production medium and clumps' size for MVA-GFP virus propagation in infected EB14 cells: infectious virus titration

(19) EB14 were allowed to form small (left panel) or large (right panel) clumps in T175 stirred tank flasks during cell proliferation in a cell growth medium (SAFC Excell 65319). Clumps were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in several production media (Optipro, Excell 65319, Excell 65421, Excell 65625, Excell 65626, Excell 65627, Excell 65628, Excell 65629, G9916). During a 7 days virus propagation period at 37 C., samples were collected daily and TCID.sub.50 titration was performed at the end of the kinetic.

(20) FIG. 10: Influence of production medium and supplements for MVA-GFP virus propagation in infected EB14 cells

(21) EB14 were allowed to form small clumps in T175 stirred tank flasks during cell proliferation in a cell growth medium (SAFC Excell 65319). Cells were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in several production media (from left to the right panel: medium Excell 65319, Excell 65629 or G9916) supplemented or not with 1 yeastolate (supplement 1) and/or 1 fatty acid (supplement 2). During a 7 days virus propagation period at 37 C., samples were collected daily and TCID.sub.50 titration was performed at the end of the kinetic.

(22) FIG. 11: GFP expression in EB-14 cells infected with MVA-GFP virus in a 3 L fed-batch bioreactor

(23) EB14-derived biomass was allowed to accumulate during cell proliferation phase in Excell 65319 growth medium. Cells were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in G9916 production medium. Pictures (magnification 5 or 10) of UV-exposed infected cells at 37 C. were then taken daily (PI: Post-Infection).

(24) FIG. 12: Infectious virus titration of EB14-derived MVA-GFP influenza virus propaged in a 3 L fed-batch bioreactor

(25) EB14-derived biomass was allowed to accumulate during cell proliferation phase in Excell 65319 growth medium. Cells were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in Excell 65319 supplemented with 1 Yeastolate. During a 9 days virus propagation period at 37 C., samples were collected daily and TCID.sub.50 titration was performed at the end of the kinetic and compared with titers obtained on CEF cells.

(26) FIG. 13: Electron micrographs analysis of MVA-GFP virus produced on EB14 cells

(27) EB14 were infected with 10.sup.2TCID/cell of MVA-GFP virus and harvested 18 h, 48 h and 72 h post-infection. Thin sections of fixed and embedded samples were examined by electron microscopy (Dr. D. Spehner, IGBMC, Strasbourg).

(28) FIG. 14: Infectious virus titration of multiple human influenza strains produced on EB14 cells

(29) EB14 cells were infected in T175 stirred tank flasks with 10.sup.4 TCID.sub.50/cell of various A/H3N2, A/H1N1 and B human influenza strains, in presence of 0.75 USP/mL of recombinant trypsin. Samples were collected every 24 h and TCID.sub.50 titer was analyzed at the end of the kinetic by titration of MDCK cells in absence of bovine serum (left panel). Some infected EB14 cells were in parallel analyzed by electron microscopy, revealing the production of influenza virus particles (right panel; Dr. D. Spehner, IGBMC, Strasbourg).

(30) FIGS. 15A and 15B: Productive replication of multiple influenza virus strains in EB14 cells

(31) FIG. 15AWestern blot analysis of haemagglutinin (HA) in EB14 cells infected with various influenza virus strains

(32) EB14 cells are cultured in serum-free medium in T175 shaken flasks and are infected with the indicated viral strains at a multiplicity of infection of 10.sup.4, in presence of 0.75 USP/mL of trypsin. 44 of cell culture supernatants are collected daily and analyzed by electrophoresis through a 10% SDS-PAGE and western-blotting. Proteins were electroblotted to polyvinylidene difluoride membrane and uncleaved (HA0) or post cleavage subunits (HA1 and HA2) of HA were detected by incubation with specific polyclonal anti-HA sheep serum. An anti-sheep-IgG conjugated to peroxydase was used for immunostaining. For each virus strain, HA accumulation from 72 h to 168 h post-infection is compared with increasing amounts of egg-derived standard HA reagents.

(33) FIG. 15BSRID analysis of EB14-derived HA production levels for various influenza viruses

(34) EB14 cells were infected in T175 shaken flasks with 10.sup.4 TCID.sub.50/cell of various A/H3N2, A/H1N1 and B human influenza strains, in presence of 0.75 USP/mL of trypsin. Samples were collected every 24 h and Serial Radial Immunodiffusion (SRID) analysis was performed at the end of the kinetic. For each virus strain, calculation of HA accumulation is related to a dose-response curve of well-defined corresponding standard antigens.

(35) FIGS. 16A and 16B: Production of A/H3N2 influenza virus strains in EB14 cells in 3 L-bioreactors

(36) FIG. 16AGrowth kinetic of EB14 cells infected with A/H3N2/NewYork/55/2005 influenza virus strain

(37) EB14 biomass was allowed to accumulate at 37 C. during cell proliferation phase in a cell growth medium. Cells were then infected with 10.sup.4 TCID.sub.50/cell of A/H3N2/New York/55/2005 influenza virus, the mixture was diluted in Excell 65629 production medium (medium E) supplemented with 0.3 USP/mL of trypsin and temperature was lowered to 33 C. During a 10 days virus propagation period, samples were collected daily and stored at 80 C. Left panel: cell density (10.sup.6 cells/mL), right panel: total cell number (yellow rhombus, 10.sup.7 cells) and viability (red circles, %).

(38) FIG. 16BAnalysis of HA by western-blot and SRID assays

(39) Samples collected from the 3 L bioreactor over a 7 days post-infection period were analyzed for detection and quantification of produced HA with a specific polyclonal anti-HA sheep serum. Left panel: western blot analysis of 4 L of viral supernatant were immunostained with an anti-HA sheep antibody together with an anti-sheep-IgG conjugated to peroxydase. HA accumulation is compared with increasing amounts of egg-derived standards reagents. HA0: uncleaved HA subunit, HA1*&HA2: cleaved HA subunits. Right panel: SRID quantification of 10 L of viral supernatant. Calculation of HA content is related to a dose-response curve of the same standard reagents.

(40) FIGS. 17A and 17B: Production of B influenza virus strain in EB14 cells in 3 L-bioreactor

(41) FIG. 17AGrowth kinetic of EB14 cells infected with B/Johannesburg/5/99 influenza virus strain

(42) EB14 cells were allowed to accumulate at 37 C. during cell proliferation phase in a cell growth medium. Cells were then infected with 10.sup.4 TCID.sub.50/cell of B/Johannesburg/5/99 influenza virus, the mixture was diluted in SAFC Excell 65629 production medium (medium E) supplemented with 0.3 USP/mL of trypsin and temperature was lowered to 33 C. During a 10 days virus propagation period, samples were collected daily and stored at 80 C. Left panel: cell density (10.sup.6 cells/mL), right panel: total cell number (yellow rhombus, 10.sup.7 cells) and viability (red circles, %).

(43) FIG. 17BWestern blot analysis of EB14-derived B/Johannesburg/5/99 influenza virus HA

(44) Samples collected over a 7 days post-infection period were analyzed for detection of produced HA with a specific polyclonal anti-HA sheep serum. 4 L of viral supernatant were used to perform the western blot analysis, where antigens-captured antibodies were immunostained with an anti-sheep-IgG conjugated to peroxydase. HA accumulation is compared with increasing amounts of egg-derived standards antigens. HA0: uncleaved HA subunit, HA1*&HA2: cleaved HA subunits.

(45) FIGS. 18A and 18B: Production of A/H1N1 influenza virus strains in EB14 cells in 30 L-bioreactor

(46) FIG. 18AGrowth kinetic of EB14 cells infected with A/H1N1/NewCaledonia/20/99 influenza virus strain

(47) EB14 cells were allowed to accumulate at 37 C. during cell proliferation phase in a cell growth medium. Cells were then infected with 10.sup.4 TCID.sub.50/cell of A/H1N1/NewCaledonia/20/99 influenza virus, the mixture was diluted in SAFC medium Excell 65629 production medium (medium E) supplemented with 0.3 USP/mL of trypsin and temperature was lowered to 33 C. During a 8 days virus propagation period, samples were collected daily and stored at 80 C. Left panel: cell density (10.sup.6 cells/m L), right panel: total cell number (yellow rhombus, 10.sup.7 cells) and viability (red circles, %).

(48) FIG. 18BHemagglutinin analysis of EB14-derived A/H1N1/NewCaledonia/20/99 influenza virus

(49) Samples collected over a 7 days post-infection period were analyzed for detection and quantification of produced HA with a specific polyclonal anti-HA sheep serum. Left panel: western blot analysis of 4 L of viral supernatant where antigens-captured antibodies were immunostained with an anti-HA sheep antibody together with an anti-sheep-IgG conjugated to peroxydase. HA accumulation is compared with increasing amounts of egg-derived standards reagents. HA0: uncleaved HA subunit, HA1*&HA2: cleaved HA subunits. Right panel: SRID quantification of 10 L of viral supernatant. Calculation of HA content is related to a dose-response curve of the same standard reagents.

EXAMPLES

Example 1: Process of Derivation of EBx Cell Lines

(50) The process of establishment of avian embryonic derived stem cell lines EBx has been previously described in WO03/076601 and WO05/007840. Briefly, this process of establishment of EBx cell lines comprises the following steps: a) isolation, culture and expansion of avian cells, preferably avian embryonic stem cells, in a complete culture medium containing all the factors allowing their growth and in presence of a feeder layer of mouse fibroblasts, preferably inactivated, and supplemented with animal serum; b) passage by modifying the culture medium so as to obtain progressive or total withdrawal of said factors, of said serum and of said feeder layer; c) establishing adherent or non adherent avian cell lines capable of proliferating in a basal medium in the absence of exogenous growth factors, inactivated feeder layer and a low level of serum or no serum;
In the event, the basal medium of step c) still comprises a low level of serum (i.e. around 2% or less), said process may optionally comprises an additional step d) of changing the basal medium containing no more exogenous growth factor, no more inactivated feeder layer and a low level of serum with a medium of culture selected among: a basal medium complemented with serum (i) and diluted with a serum-free medium, then culturing during successive passages said avian cells in the basal medium (i) in which the ratio of serum-free medium is progressively increased up to the complete disappearance of said basal medium containing no exogenous growth factor, no inactivated feeder layer and no serum; a serum-free medium complemented with serum (ii), then culturing during successive passages said avian cells in said medium (ii) in which the ratio of serum is progressively decreased up to the obtaining of a serum-free medium; a serum-free medium (iii), then culturing said avian cells in medium (iii); then maintaining in serum-free medium said avian cells adapted to the medium change.

(51) The term factor allowing their growth as used herein meant growth factor necessary for the survival and the growth of the avian cells in culture. According to the invention, the growth factors comprises trophic factors and cytokines. Trophic factors are mainly SCF, IGF-1 and bFGF. Cytokines are mainly cytokines whose action is through a receptor which is associated with the gp130 protein such as LIF, interleukin 11, interleukin 6, interleukin 6 receptor, CNTF, oncostatin and cardiotrophin.

(52) The avian cells of step a) are cells selected among avian embryonic cells, more preferably among avian embryonic stem cells and avian primary cells. In a preferred embodiment, the cells are totipotent or pluripotent avian embryonic stem cells isolated from a population suspension of dissociated stage X blastodermal cells obtained from an avian embryo, more preferably a chicken embryo (see EYAL-GILADI's classification: EYAL-GILADI and KOCHAN, 1976, <<From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development in the chick>>. General Morphology Dev. Biol. 49: 321-337). These avian embryonic stem cells are characterized by a slow doubling time comprises between 48 to 72 hours in culture at 39 C.

(53) The modification of the culture medium of step b) of the process of establishment EBx cell lines, so as to obtain progressive or total withdrawal of growth factors, serum and/or feeder layer, can be made simultaneously, successively or separately. The sequence of the weaning of the culture medium may be chosen among: feeder layer/serum/growth factors; feeder layer/growth factors/serum; serum/growth factors/feeder layer; serum/feeder layer/growth factors; growth factors/serum/feeder layer; growth factors/feeder layer/serum.
In a preferred embodiment, the sequence of the weaning is growth factors/feeder layer/serum.

(54) This process allows a selection of cell clones which are adapted to these new, increasingly drastic culture conditions until stable lines are obtained which are capable of growing in a serum-depleted medium or in a medium completely free of serum. The established lines EBx are preferably non adherent stem cells which proliferate in suspension in a medium free of exogenous growth factors and serum without feeder cells.

(55) By complete culture medium, it is meant a basal medium complemented with growth factors and animal serum. Example of complete culture medium is described in Pain et al. (1996, Development 122:2339-2348), EP 787,180 and U.S. Pat. No. 6,114,168, U.S. Pat. No. 5,340,740, U.S. Pat. No. 6,656,479 and U.S. Pat. No. 5,830,510. According to the invention, basal medium meant a medium with a classical media formulation that allows, by itself, at least cells survival, and even better, cell growth. Examples of basal media are SFM media as previously described or media such as BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-Ham F12, Ham-F12 and Ham-F10, Iscove's Modified Dulbecco's medium, MacCoy's 5A medium, RPMI 1640. Basal medium comprises inorganic salts (for examples: CaCl.sub.2, KCl, NaCl, NaHCO.sub.3, NaH.sub.2PO.sub.4, MgSO.sub.4, . . . ), aminoacids, vitamins (thiamine, riboflavin, folic acid, D-Ca panthothenate, . . . ) and others components such as glucose, beta-mercaptoethanol, sodium pyruvate.

(56) It is possible to schematically distinguish two families of growth factors: the cytokines and the trophic factors. The cytokines are mainly cytokines whose action is through a receptor which is associated with the gp130 protein. Thus, LIF, interleukin 11, interleukin 6, interleukin 6 receptor, CNTF, oncostatin and cardiotrophin have a similar mode of action with the recruitment at the level of the receptor of a specific chain and the combination of the latter with the gp130 protein in monomeric or sometimes heterodimeric form. The trophic factors are mainly SCF, IGF-1 and bFGF. More preferably, the complete medium comprises basal medium, Insulin Growth factor 1 (IGF-1), Ciliary Neurotrophic factor (CNTF), Interleukine 6 (IL-6), interleukine 6 receptor (IL-6R), Stem cell Factor (SCF), basic Fibroblast Growth Factor (bFGF), optionally interleukine 11 (IL-11) and animal serum. The avian cells, preferably the avian embryonic cells of step a) are cultured during several passages in the complete medium. The medium is complemented by at least one of the growth factors selected in the group of: LIF, IGF-1, CNTF, IL-6, IL-6R, SCF, bFGF, IL-11, oncostatin, cardiotrophin. According to a preferred embodiment, the complete culture medium is basal medium complemented with IGF-1 and CNTF. According to another preferred embodiment, the complete culture medium is basal medium complemented with IGF-1, CNTF, IL-6, IL-6R, SCF, bFGF, optionally IL-11. The concentration of growth factors IGF-1, CNTF, IL-6, IL-6R, SCF, bFGF, optionally IL-11 in the basal medium is comprised between about 0.01 to 10 ng/ml, preferably, 0.1 to 5 ng/ml, and more preferably about 1 ng/ml.

(57) After around passages 3 to 10, the complete medium is depleted in growth factors (step b). Preferably, for each growth factor, the depletion is made directly in one step, from one passage to another. Alternatively, the growth factor depletion is performed gradually, by a progressive decrease of the growth factor concentration in the complete medium. In a more preferred embodiment, the growth factors depletion is performed simultaneously for at least two growth factors. In a preferred embodiment, when the complete culture medium is basal medium complemented with IGF-1 and CNTF. the depletion in growth factors is made in one round of depletion. In another preferred embodiment, when the complete culture medium is basal medium complemented with IGF-1, CNTF, IL-6, IL-6R, SCF, bFGF, optionally IL-11. the depletion in growth factors is made in two rounds of depletion: firstly, SCF, IL6, IL6R, bFGF optionally IL11 are directly removed from the complete medium; the avian cells are then maintained in culture for at least one passage in a complete medium containing IGF1 and CNTF, optionally IL-11, and supplemented with animal serum. Secondly, IGF1 and CNTF, optionally IL-11 are directly removed from the culture medium, which ultimately comprises the basal medium only supplemented with serum. Usually, the medium is totally depleted in growth factors at around passages 20 to 30.

(58) In a preferred embodiment, the deprivation of feeder cells is performed after the deprivation of growth factors. The deprivation of feeder cells is progressive and performed over several passages. The avian cells are now seeded in flask at a lower concentration than in step a), about around 410.sup.4 cell/cm.sup.2 to 510.sup.4 cell/cm.sup.2. The feeder cells are seeded in flask at around 4.210.sup.4 cell/cm.sup.2. Progressively, the concentration of the feeder cells in the flask is decreased. Practically, the same concentration of the feeder cells is used for 2 to 4 passages, then a lower concentration of the feeder cells is used for an additional 2 to 4 passages, and so. The flask is then seeded with around 4.210.sup.4 feeder cells/cm.sup.2, then around 2.210.sup.4 feeder cells/cm.sup.2, then around 1.810.sup.4 feeder cells/cm.sup.2, then around 1.410.sup.4 feeder cells/cm.sup.2, then around 1.110.sup.4 feeder cells/cm.sup.2, then around 0.910.sup.4 feeder cells/cm.sup.2, then around 0.510.sup.4 feeder cells/cm.sup.2. Then the flask is seeded with 6.510.sup.4 avian cells/cm.sup.2 to 7.510.sup.4 avian cells/cm.sup.2 and without feeder cells. In the hypothesis that avian cells are not in good shape following a decrease of feeder cells concentration in the flask, then the avian cells are cultured for additional passages with the same feeder cells concentration before to pursue the feeder cells deprivation.

(59) In another preferred embodiment, the serum deprivation is performed after the growth factor and the feeder cells deprivation. The basal medium is changed by a medium selected among: The basal medium (i) complemented with serum and diluted with a novel serum free medium (ii). Then the avian cells are cultured through successive passages in the medium (i) in which the serum free medium proportion is progressively increased up to the complete disappearing of the basal medium complemented in serum (progressive dilution). A novel serum free medium (ii) complemented with serum. Then the avian cells are cultured through successive passages in the medium (ii) in which the serum proportion is progressively decreased up to the obtaining of a serum-free medium (progressive weaning). A novel serum free medium (ii) non complemented with serum. Then the avian cells are directly in the serum-free medium (ii) (direct weaning).
In a preferred embodiment, the serum deprivation is performed by progressive weaning.

(60) The feeder cells are animal cells that have been preferably inactivated by irradiation or chemically treated with mitomycin. The feeder may be genetically modified to express growth factors such as SCF. Preferably, the feeder cells are mouse fibroblasts cell lines such as STO (American Type Culture Collection ATCC N CRL-1503).

(61) This process leads to the establishment of avian embryonic derived cell lines named EBx which are maintained in culture in vitro over a considerable period of time. Advantageously, the EBx cells obtained in step c) are capable of proliferating for at least 50 days, 100 days, 150 days, 300 days or preferably at least 600 days. The 600 days do not constitute a time limit because the EBx cells obtained are still alive after much longer time periods. For example a Master Cell Bank of EB14 cells has been produced at passage P160 and an EB14 End of Production cell bank has been produced at P184 and EB14 cells are still able to proliferate. Hence, these lines are considered as being able to grow indefinitely in a basic culture medium free of exogenous growth factors, serum and/or inactivated feeder layer. The expression line is understood to mean any population of cells capable of proliferating indefinitely in culture in vitro while retaining to a greater or lesser degree the same morphological and phenotypic characteristics. Of course, the method mentioned above makes it possible to obtain cellular clones derived from cells obtained from established lines. These clones are cells which are genetically identical to the cell from which they are derived by division.

(62) The established cell lines and the cells derived thereof (step c or d) are preferably embryonic derived avian stem cells lines, more precisely those cells are pluripotent avian embryonic derived stem cells. The avian embryonic derived stem cells EBx obtainable by the process of the invention are small, round, individualized cells with a doubling time of around 24 hours or less at 39 C. The cells obtainable by the process of the invention are at least at passage p60, at least p70, at least p80, at least p90, at least p100, at least p110 at least p120, at least p130, at least P150, at least P160, at least P170, at least P180 or later. The avian embryonic derived stem cells according to the invention have at least one of the following characteristics: a high nucleo-cytoplasmic ratio, an endogenous alkaline phosphatase activity, an endogenous telomerase activity, a reactivity with specific antibodies against SSEA-1 (TEC01), SSEA-3, and EMA-1. They express the ENS1 gene;
A doubling time shorter than the doubling time of the avian cells of step a) of the process of the invention (48 to 72 h at 39 C.), of about 24 hours or less in the same culture conditions. These EBx cell lines are capable of proliferating indefinitely in a basal medium, in particular in a medium such as SAFC Excell media, DMEM, GMEM, HamF12 or McCoy supplemented with various additives commonly used by persons skilled in the art. Among the additives, there may be mentioned non-essential amino acids, vitamins and sodium pyruvate, fatty acids, yeast and soy hydrolyzates. However, the cells are able to proliferate in basal medium without glutamine. These cells lines and the cells derived there from have the characteristic to grow either as adherent cells or as suspension cells.

(63) Preferably, the EBx cells of the invention, preferably EB14 cells, have all the above mentioned characteristics and are useful for the production of biologics such as viral vaccines and recombinant peptides and proteins (i.e antibodies, . . . ).

Example 2: Characterization of EB14 Cells

(64) 2.1EB14 Cells Karyotype

(65) Karyotyping analysis of EB14 cells has been performed in Pr. Michel Franck Laboratory, Unit de zootechnie, ethnologie et conomie rurale, Ecole Nationale Vtrinaire, 1 avenue Bourgelat, 69280 Marcy l'Etoile, France.

(66) EB14 cells were karyotyped at two different passages (Passage 105 and 118) by using standard techniques well-known to the man skilled in the art. As expected, EB14 cells at passage 105 and 118 display a diploid karyotype (FIG. 2):

(67) Passage 105: modal number of chromosomes=78 (average mean: 78.41standard deviation: 4.951 over 53 studied metaphases)

(68) Passage 118: modal number of chromosomes=79 (average mean: 79.68standard deviation: 3.733 over 50 studied metaphases).

(69) Chicken genome comprises two types of pairs of chromosomes: macro- and micro-chromosomes. Passage 115 analysis shows that the modal number of macro-chromosomes is 18 with an average mean of 17.82 and a standard-deviation of 0.833 and a modal number of micro-chromosomes of 60 with an average mean of 60.6 and a standard-deviation of 4.7. Passage 118 analysis shows that the modal number of macro-chromosomes is 18 with an average mean of 18.24 and a standard-deviation of 0.797 and a modal number of micro-chromosomes of 60 with an average mean of 61.44 and a standard-deviation of 3.688. There is no significant deviation in chromosomes distribution between the two studied passages. EB14 cell line displays a normal male (ZZ) diploid karyotype at passages 105 and 118 that demonstrates the chromosomal stability of EB14 cells.

(70) 2.2Tumorigenicity Analysis of EB14 Cells in the Immuno-Suppressed New-Born Rat Model

(71) Tumorogenicity if EB14 cells at passage 127 has been assessed in the immuno-suppressed new-born rat model (Sanofi-Aventis, France) (WHO technical report N 878 (1998). Hela cells were used as positive controls. Ten immuno-suppressed new-born rats were injected sub-cutaneously with 10 million EB14 cells and ten additional immuno-suppressed new-born rats were injected sub-cutaneously with 10 million Hela cells. All animals received 0.1 ml of anti-thymocyte rat serum at days 0, +2, +7 and +14. Animals were regularly observed during three weeks to detect nodules at the injection site. After 3 weeks, animals were killed and examined to detect cell proliferation at the injection site and in other organs. No nodules or tumors were observed at the EB14 cells injection site or in distant organs. EB14 are non-tumorigenic in the immuno-suppressed new-born rat model.

(72) 2.3EB14 Cells Express Avian and Human Influenza Virus Receptors

(73) The detection of receptors to avian (Sia2-3Gal) and human (Sia2-6Gal) influenza viruses on EB14 cells is performed by fluorescent cell sorter analysis by using digoxygenin labelled lectins (Boehringer): Sambuca nigra (SNA) agglutinin lectin specifically binds to Sia2-6Gal; Maackia amurensis (MAA) agglutinin lectin specifically binds to Sia2-3Gal.

(74) EB14 and MDCK cell lines were washed in 10 mM HEPES, 150 mM NaCl pH7.5 and resuspended in the same buffer at a 5.10.sup.6 final concentration. Cells were incubated 30 min on ice, then for an additional 15 to 30 minutes in presence of SNA or MAA. Lectin treated cells were washed in 10 mM HEPES, 150 mM NaCl pH7.5, prior to incubation on ice during 15 to 30 minutes with FITC-labelled anti-digoxygenin antibody. Then cells are washed in NaCl 0.9% and FACS analyzed.

(75) EB14 cells express cell surface receptors comprising oligosaccharides with Sia2-6Gal and Sia2-3Gal residues (FIG. 6).

Example 3: MVA Production in EB14 Cells

(76) 3.1Materials and Methods

(77) Recombinant MVA virus encoding green fluorescent protein gene was used. Titration of infectious MVA-GFP viruses was performed on DF-1 cells. In brief, cells were seeded in 96 flat-bottom well plates at a density of 15.10.sup.3 cells/well in DMEM medium (Biowhittaker) supplemented with 5% foetal calf serum (FCS) (SAFC) and 2 mM L-glutamin (Biowhittaker). Twenty-four hours later, cells were infected with ten fold serially diluted samples in DMEM and incubated for one week at 37 C., 5% CO.sub.2 in a humidified atmosphere. Virus infectivity was measured through microscopic observation of global cytopathic effect (CPE) and UV-exposed infected cells. Then TCID50 titers were calculated according the Reed and Muench method (1938, A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-97).

(78) 3.2Infection in Tissue Culture Flasks

(79) 3.2.1Materials

(80) Vessel: F175 flask (Starstedt, Ref. 83 1812502) Orbital Agitator: IKA KS260 or equivalent Sonicator: IKA U50 (monitored with US50-3 probe) Medium: Excell 65319 (SAFC-JRH) with 2.5 mM Glutamine (Cambrex Ref. BE17605E);
3.2.2Methods
Step 1: EB14 Cells Preparation

(81) Cells should be prepared 2 weeks before starting the infection experiment.

(82) Day 0: EB14 cells are seeded in F175 flask at 0.410.sup.6 cells/mL in 25 ml of Excell 65319 medium with 2.5 mM Glutamine [1.sup.st seeding after clumps breaking]. Cells are incubated at 37 C., 7.5% CO.sub.2, Humidified atmosphere under agitation (60 rpm)

(83) Day 1: 25 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(84) Day 2: 50 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(85) Day 3: Cells are numbered. An aliquot of EB14 cells is seeded in a new F175 flask at 0.410.sup.6 cells/mL in 25 ml of Excell 65319 medium with 2.5 mM Glutamine. This represents the dilution +1.

(86) Day 4: 25 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(87) Day 5: 50 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(88) Day 6: Cells are numbered. An aliquot of EB14 cells are seeded in a new F175 flask at 0.410.sup.6 cells/mL in 25 ml of Excell 65319 medium with 2.5 mM Glutamine. This represents the dilution +2.

(89) Then, the amplification of the cells continues this way, up to a dilution comprises preferably between +3 to +7, more preferably +3 to +5. Then one should proceed with step 2 of cells infection.

(90) To obtain a cell culture containing large clumped EBx cells (hereinafter named: large clumps conditions), when the cells are passaged by dilution to larger vessel(s), the EBx cells are not centrifuged, and the clumps are not broken by pipetting and stirring. In the opposite, to obtain a cell culture without a substantial proportion of clumped EBx cells, the clumps are disrupted by pipetting or stirring when passaging the cells (hereinafter named: no clumps conditions).

(91) Step 2: Infection of EB14 Cells with MVA-GFP (Green Fluorescent Protein)

(92) Day 1: EB14 cells are seeded in F175 flask at 0.410.sup.6 cells/mL in 40 mL JRH Excell 65319 medium with 2.5 mM Glutamine. Cells are incubated at 37 C., 7.5% CO.sub.2, Humidified atmosphere under agitation (60 rpm).

(93) Days 2 and 3: Cells are numbered.

(94) Day 4: Cells are numbered. When cell density in the flask is about 410.sup.6 cells/ml, EB14 cells are infected with a MOI of 0.01 TCID.sub.50/cell with 1 ml viral infection mix per flask. Viral infection mix is prepared just before use by virus dilution in Excell 65319 medium with 2.5 mM Glutamine. Each inoculum is sonicated 30 sec (amplitude 100% and continuous cycle) on ice in 15 ml Falcon tube. Inoculum is warmed at room temperature before mixing with the cell culture. Following inoculation, the infected culture medium is incubated 1 h at 37 C. Then, 60 ml of fresh medium Excell 65319 supplemented with 2.5 mM Glutamine, 0.5 Yeastolate and 0.35 ml/L fatty acids is added in the flask. The infected cell culture is further incubated at 37 C. during at least 144 h (nb: the viral production peak is between pi+72 h and pi+120 h).

(95) Cell culture samples (1 mL) are collected every 24 h and keep frozen at 80 C. Prior the sample collection, cell culture is homogenized by gentle pipetting. Virus titration on every collected samples is performed at the end of the experiment using TCID50/mL Reed and Muench's method (1938).

(96) 3.3Infection in Spinner

(97) 3.3.1Materials

(98) Vessel: 500 ml & 1 liter spinner bottle (Corning) Orbital Agitator: IKA KS260 or equivalent Sonicator: IKA U50 (monitored with US50-3 probe) Medium: Excell 65319 (SAFC-JRH) with 2.5 mM Glutamine (Cambrex Ref. BE17605E);
3.3.2Method
Step 1: EB14 Cells Preparation

(99) Cells should be prepared 2 weeks before starting the infection experiment.

(100) Day 0: EB14 cells are seeded in F175 flask at 0.410.sup.6 cells/mL in 25 ml of Excell 65319 medium with 2.5 mM Glutamine [1.sup.st seeding after clumps breaking]. Cells are incubated at 37 C., 7.5% CO.sub.2, Humidified atmosphere under agitation (60 rpm)

(101) Day 1: 25 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(102) Day 2: 50 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(103) Day 3: Cells are numbered. An aliquot of EB14 cells is seeded in a new F175 flask at 0.410.sup.6 cells/mL in 25 ml of Excell 65319 medium with 2.5 mM Glutamine. This represents the dilution +1.

(104) Day 4: 25 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(105) Day 5: 50 ml of Excell 65319 medium with 2.5 mM Glutamine are added.

(106) Day 6: Cells are numbered. An aliquot of EB14 cells are seeded in a new F175 flask at 0.410.sup.6 cells/mL in 25 ml of Excell 65319 medium with 2.5 mM Glutamine. This represents the dilution +2.

(107) Then, the amplification of the cells continues this way, up to a dilution comprises preferably between +3 to +7, more preferably +3 to +5. Then one should proceed with step 2 of cells infection.

(108) To obtain a cell culture containing large clumped EBx cells (hereinafter named: large clumps), when the cells are passaged by dilution to larger vessel(s), the EBx cells are not centrifuged, and the clumps are not broken by pipetting and stirring. In the opposite, to obtain a cell culture without a substantial proportion of clumped EBx cells, the clumps are disrupted by pipetting or stirring when passaging the cells (hereinafter named: no clumps).

(109) Step 2: EB14 Cells Infection with MVA-GFP

(110) Day 1: EB14 cells are seeded in 500 ml (or 1,000 ml) spinner bottle at 0.410.sup.6 cells/mL in 150 mL (or 300 ml) JRH Excell 65319 medium with 2.5 mM Glutamine. Cells are incubated at 37 C., 7.5% CO.sub.2, Humidified atmosphere under agitation (100 rpm).

(111) Days 2 and 3: Cells are numbered.

(112) Day 4: Cells are numbered. When cell density in the spinner is about 410.sup.6 cells/ml, EB14 cells are infected with a MOI of 0.01 TCID.sub.50/cell with 1 ml viral infection mix per spinner bottle. Viral infection mix is prepared just before use by virus dilution in Excell 65319 medium with 2.5 mM Glutamine. Each inoculum is sonicated 30 sec (amplitude 100% and continuous cycle) on ice in 15 ml Falcon tube. Inoculum is warmed at room temperature before mixing with the cell culture. Following inoculation, the infected culture medium is incubated 1 h at 37 C. Then, 60 ml of fresh medium Excell 65319 supplemented with 2.5 mM Glutamine, 0.5 Yeastolate and 0.35 ml/L fatty acids is added in the 500 ml (or 1,000 ml) spinner bottle. The infected cell culture is further incubated at 37 C. during at least 144 h (nb: the viral production peak is between pi+72 h and pi+120 h).

(113) Cell culture samples (1 mL) are collected every 24 h and keep frozen at 80 C. Prior the sample collection, cell culture is homogenized by gentle pipetting. Virus titration on every collected samples is performed at the end of the experiment using TCID50/mL Reed and Muench's method (1938)*. *Reed L & Muench H (1938) A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-97.

(114) 3.4Infection in 3 L-Stirred Tank Bioreactor

(115) 3.4.1Method

(116) Cell Thawing

(117) Cell cryovials are stocked in liquid nitrogen at 196 C.; each cryovial contains 20.10.sup.6 cells. The cryovial are directly thawed in a 37 C. pre-warmed water bath to rapidly thaw the frozen vial. The cell suspension is pipette into a 50 mL PP sterile tube with 30 mL pre-warmed culture medium. The cell suspension is centrifuged 5 min at 30020 g, at room temperature, the supernatant is discarded and the pellet is resuspended in 15 ml of fresh culture medium and gently homogenise. The cell suspension are plated into a T75 cm.sup.2 flask and is incubated at 37 C. under a 7.5% CO.sub.2 atmosphere on an orbital shaker at 50 rpm. After 24 hours and 48 hours of culture, 15 ml of pre-warmed culture medium is added to the cell culture. After 72 hours of culture, a sample is collected (after bulk homogenisation) and a numeration is performed: 40.10.sup.6 cells are expected. Then the first amplification is performed.

(118) First Cell Amplification: Centrifugation, Dissociation and Dilution

(119) The suspension cells are collected from the flask(s) in 50 mL PP sterile tube(s). After 5 min of centrifugation at 30020 g, at room temperature, the supernatant is discarded and 10 mL of prewarmed fresh culture medium is added on the pellet(s). The cell clumps are gently dissociated with a 10 mL pipette and the cell suspensions are pooled in one 50 mL PP sterile tube if necessary. The culture volume is completed up to 20 mL with fresh pre-warmed culture medium if necessary. A numeration is performed using trypan blue to determine cell density and cell viability (cell viability is typically around 80%). In 1 T175 cm.sup.2 flask, 0.4.10.sup.6 cells.mL.sup.1 are seeded in 40 ml of pre-warmed culture medium. The cell culture is incubated at 37 C. under an 7.5% CO.sub.2 atmosphere on an orbital shaker at 50 rpm. At day 2, 60 ml of pre-warmed culture medium is added to the cell culture. At day 3, cell dilution are performed.

(120) Dilution +1 to +5 (No Centrifugation, No Dissociation, Only Dilution).

(121) A sample is taken from the T175 flask (after gently mixing) to perform a numeration using tryptan blue to determine cell density. A sample is taken from the T175 flask (after gently mixing) in order to seed 0.4.10.sup.6 cells.mL.sup.1 in 1 T175 cm.sup.2 flask in a total volume of 25 ml of pre-warmed culture medium. This represents dilution +1.

(122) At day 1, 50 ml of pre-warmed culture medium is added. At day 2, Dilution +2 is performed using the same way that for dilution +1 (see above). The cells amplification is performed this way, up to Dilution +3 to +5. The Inoculum for the 3 L bioreactor can be prepared from Dilution +3 until Dilution +5. Two T175 flasks are prepared as an inoculum.

(123) Cells Seeding in 3 L Stirred-Thank Bioreactor

(124) SeedingDay 0

(125) The inoculum is prepared (320.10.sup.6 cells are needed to inoculate the 3 L-bioreactor). The 2 T175 flasks are pooled. A sample is taken after gently mixing (cells clumps should not be broken) to perform a numeration using trypan blue to determine cell density. A 150 mL cell mix is prepared in order to obtain a cell concentration of 0.40.10.sup.6 cells.mL.sup.1 into the 800 ml final culture volume in the bioreactor.

(126) Prior to seed cells, the pH is set in the vessel to 7.2 (because pH will be decrease by CO.sub.2 surface injection). The pO.sub.2 is set to 50% O.sub.2 saturation (the mass flow controller is adjusted to 100% which correspond to a maximum sparger flow rate to 50 mL.min.sup.1). At the beginning of the process, the pH is maintained by CO.sub.2 surface injection, later, it is controlled by addition of 7.5% NaHCO.sub.3. The surface aeration is started with air at a flow rate of 0.3 mL.min.sup.1.

(127) Culture Follow-Up/Feed Addition (Day 1, Day 2)

(128) Cell numeration is performed on a routine basis. The metabolites such as glutamate, glutamin, lactate and glucose are analyzed all along the culture with the BioProfile Basic software. Concentration of the metabolites is adjusted if necessary. For example, glutamine concentration is adjusted to 2 mM.

(129) Infection with MVA-GFP (Day 3)

(130) After 3 days of culture, the cell density should be higher than 3.10.sup.6 cells.mL.sup.1. If the target cell density is reached (3 to 5.10.sup.6 cells.mL.sup.1), the virus infection is performed at a MOI of 10.sup.2 TCID.sub.50/cell. The virus strain is thawed on ice. The infection mix is prepared in a 50 mL PP sterile tube with 10 mL of production medium. The mix is sonicated during 30 sec (amplitude 100% and continuous cycle) on ice. The infection mix is inoculated into the bioreactor. After 1 hour of viral adsorption, the final production medium is added to the vessel. The 1.5 L Excell 65319 production medium in 1.5 L is supplemented with Yeastolate and Fatty Acids to a final concentration of respectively 0.5 and 0.35 ml/L (final volume: 2.3 L).

(131) Production Following/Feed Addition (Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10)

(132) The MVA viral production peak is reached between 72 h and 120 h post infection. Every day a sample of approximatively 15 ml is collected from the bioreactor to perform cell numeration, cell morphology analysis and to observe CPE. The metabolites such as glutamate, glutamine, lactate and glucose are analyzed all along the culture with the BioProfile Basic software. Concentration of the metabolites is adjusted if necessary. For example, glutamine concentration is adjusted to 2 mM if necessary. The glucose concentration is adjusted to 2 g.L.sup.1 if necessary.

(133) Analyses

(134) Virus titration is carried-out at the end of the experiment using all collected samples.

(135) 3.5Results

(136) 3.5.1Cell Growth Kinetics of EB14 Cells in a 3 L Fedbatch Bioreactor

(137) EB14 cells are routinely cultured in stirred-tank bioreactor. EB14-derived biomass is allowed to accumulate at 37 C. in a cell growth medium until a cell density of 5-6.10.sup.6 cells/mL was reached. Then the mixture is around 3 fold diluted and cell growth kinetic is followed-up over a 10 days period. In such conditions, cell density of 12 to 16 million cells/ml is routinely reached around day 5 to 8 (FIG. 7A). EB14 cells splitting ratio may be increased. FIG. 7B shows a growth kinetic of EB14 cells diluted 10 fold.

(138) 3.5.2EB-14 Cells Flexibility: Plaque Purification of MVA-GFP Virus on Adherent Cells

(139) EB14 cells grow in suspension culture. However, EB14 cells have also the ability to grow in adherence in flask and plates. This feature allow the inventors to perform plaque purification of MVA-GFP on adherent EB14 cells. To do so, tenfold serial dilutions of MVA-GFP virus were inoculated on adherent EB-14 cells seeded in 6 well plates 24 h before at a density of 7.10.sup.4 cells/cm.sup.2. Following virus adsorption, cells were over layered with a mix of 1.2% LMP agarose/2.5% FCS DMEM and incubated at 37 C. for several days. Wells were finally stained with neutral red. Plaque forming unit titration can then be calculated with dilutions providing isolated plaques.

(140) 3.5.3Influence of Production Medium and Clumps Size for MVA-GFP Virus Propagation in Infected EB14 Cells

(141) EB14 were allowed to form small or large clumps in T175 stirred tank flasks during cell proliferation in a cell growth medium (Excell 65319). Clumps were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in several production media (Optipro, Excell 319, Excell 421, Excell 625, Excell 626, Excell 627, Excell 628, Excell 629, G9916). The presence of large clumps of EB14 cells improves virus infection and propagation (FIG. 8), leading to higher MVA virus titers (FIG. 9). The addition of supplements such as yeastolate (supplement 1) and Fatty acids (supplement 2), in the virus production medium further improves MVA virus titers. As shown in FIG. 10, when yeastolate 1 (supplement 1) is added alone into the medium, MVA-GFP viral yield is increased and a synergistic effect is even obtained when adding yeastolate (supplement 1) and Fatty acids (supplement 2) in the cell growth medium. Indeed, fatty acid 1 alone does not increase viral titers.

(142) 3.5.4MVA Virus Production in 3 L-Bioreactor

(143) EB14-derived biomass was allowed to accumulate during cell proliferation phase in Excell 65319 growth medium. Cells were then infected with 10.sup.2 TCID.sub.50/cell of MVA-GFP virus and the mixture was diluted in Excell 65319 production medium. Following addition of Excell 65319, cell density dropped down (day 3), and at day 5, the cell density of infected cells increased and reached 4 million cell/ml. The fact that no centrifugation is performed during cell amplification allows to get large clumps size in culture 2 to 4 days post-infection (FIG. 11). In such conditions, the MVA-GFP productivity is high. Since at day 6 post-infection, the MVA-GFP titer is around 10.sup.8.32 TCID50/ml (Cell concentration of 1.4910.sup.6 cells/ml) which correspond to TCID50/cell=414 (Amplification factor 41,000) (FIG. 12). It appears that when the EB14 cells are amplified without disrupting the clumps (by dilution for example), a higher viral titer is obtained in 3 L-bioreactor compared to the one obtained in absence of large clumps (Amplification factor around 1900 and TCID50/cell=19) into the cell culture.

(144) 3.5.5Normal Ultrastructure of MVA Infected EB14 Cells

(145) EB14 cells infected with MVA virus were analyzed by electron microscopy (Drs. Daniele Spehner & Robert Drillien, IGBMC, Strasbourg France). The maturation of MVA virus produced in EB14 cells is normal and similar to the one observed in primary chicken embryo fibroblasts.

Example 4: Production of Influenza Virus in EB14 Cells

(146) 4.1Materials & Methods

(147) 4.1.1Influenza Virus Infectivity Assay (TCID50)

(148) Titration of infectious influenza viruses was performed on MDCK cells. In brief, cells were seeded in 96 flat-bottom well plates at a density of 3.10.sup.3 cells/well in UltraMDCK medium supplemented with 2.5 mM L-glutamin. Twenty-four hours later, cells were infected with ten fold serially diluted samples in UltraMDCK containing 6 g.mL.sup.1 trypsin-EDTA and incubated for one week at 33 C., 5% CO.sub.2 in a humidified atmosphere. Virus replication was then tested in an HA assay using chicken red blood cells and TCID50 titers were calculated according the Reed and Muench method (1938)*. *Reed L, Muench H, 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-97.

(149) 4.1.2Single Radial Immunodiffusion Assay (SRID)

(150) The concentration of haemagglutinin in samples derived from influenza virus infected-EB14 cells, was determined as described by Wood and colleagues*. Briefly, glass plates were coated with an agarose gel containing anti-Influenza serum (recommended concentration provided by NIBSC). After the gel has set, 10 L of appropriate dilutions of the reference and the samples were loaded in 3 mm punched wells. Following a 18-24 h incubation in a moist chamber at room temperature, plates were soaked in 0.9% NaCl and washed in distilled water. The gel was then pressed and dried. The plates were stained on Coomassie Brillant Blue solution for 15 min and destained twice in a mixture of methanol and acetic acid until clearly defined stained zones became visible. After drying the plates, the diameter of the stained zones surrounding antigen wells were measured in two directions at right angles. Dose-response curves of antigen dilutions against the surface were constructed and the results were calculated according to standard slope-ratio assay methods. *Wood J M. Et al. An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. J Biol Stand. 1977; 5(3):237-47).

(151) 4.1.3Western Blot Analysis of Influenza Hemagglutinin Protein

(152) SDS-PAGE was performed as described by Laemmli U K (1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 259:680-685) in 10% polyacrylamide gel. Denaturated proteins (1% SDS, 70 mM -mercaptoethanol) were transferred to polyvinylidene difluoride membrane (hybond P, Amersham) by a semidry blotting procedure (Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203-209). Blots were blocked for 1 h at room temperature with a mixture composed of 5% fat dry milkpowder in TBST supplemented with 1% FCS (SAFC). Then, the blots were incubated overnight in blocking solution supplemented with specific polyclonal anti-HA sheep serum (1:500 (NIBSC). The blots were washed 6 times with TBST and incubated for 1 h at room temperature with a hrp-conjugated rabbit anti-sheep IgG polyclonal antibody (1:5000 (Rockland) in blocking solution. After 6 washes with TBST, the protein-conjugate complex was finally revealed using chemiluminescence (ECL kit, Amersham) and films (Hyperfilm, Amersham).

(153) 4.2Influenza Virus Infection of EB14 Cells in 3 L-Bioreactor

(154) 4.2.1Materials and Equipment

(155) Cell Thawing Material

(156) T75 cm.sup.2 flasks (Sarstedt, Cat #831813502) Culture medium (serum free medium) L-Glutamin 200 mM (Biowhittaker, Cat #BE17-605E) Orbital agitator IKA KS260 (Fisher Bioblock, Cat #F35044)
Cell Amplification Material T175 cm.sup.2 flasks (Sarstedt, Cat #831812502) Culture medium (serum free medium): Excell 65319 (JRH, Cat #65319-1000M1687) added with 2.5 mM glutamine L-Glutamin 200 mM (Biowhittaker, Cat #BE17-605E) Yeastolate UF Solution 50 (JRH, Cat #58902-100M) D (+) Glucose (45%) (Sigma, Cat #G8769)
Production Material Production medium (serum free medium): Excell 65629 (JRH, Cat #65629) supplemented with 2.5 mM gln Yeastolate UF Solution 50 (JRH, Cat #58902-100M) L-Glutamin 200 mM (Biowhittaker, Cat #BE17-605E) D (+) Glucose (45%) (Sigma, Cat #G8769) Trypsin (Trypzean 1, Sigma, Cat #T3449) 7.5% bicarbonate sodium solution (Sigma, Cat #205-633-8) Influenza virus strain (frozen at 80 C.)
4.2.2Method
Cell Thawing

(157) Cell cryovials are stocked in liquid nitrogen at 196 C.; each cryovial contains 20.10.sup.6 cells). The cryovial is directly thawed into a +37 C. prewarmed water bath. The cell suspension is put into a 50 mL PP sterile tube with 30 mL prewarmed culture medium. After centrifugation (5 min at 30020 g, at room temperature), 15 mL of fresh culture medium is added on the pellet and gently homogenise. The sample is numbered using trypan blue. Numeration has to be 20.10.sup.6 cells and viability has to be >70% to guarantee a good culture. The cell suspension is plated into a T75 cm.sup.2 flask and incubate at +37 C. under an 7.5% CO.sub.2 atmosphere on an orbital shaker at 50 rpm. After 24 hours and 48 Hours of culture, 15 mL of prewarmed culture medium is added to the culture. After 72 hours of culture, a sample is collected (after bulk homogenisation) and numbered: 20 to 30.10.sup.6 cells are expected. Then perform the first amplification is performed.

(158) First Cell Amplification: Centrifugation, Dissociation and Dilution

(159) The suspension cell is collected from the flask(s) in 50 mL PP sterile tube(s) and centrifuge 5 min at 30020 g, at room temperature. 10 mL of prewarmed fresh culture medium is added on the pellet(s). The cell clumps are gently dissociate and cell suspensions is pooled; the volume is completed to 40 mL with fresh pre-warmed culture medium. In 1 T175 cm.sup.2 flask, 0.25.10.sup.6 cells.mL.sup.1 is plated in 40 ml of pre-warmed medium and incubated at +37 C. under an 7.5% CO.sub.2 atmosphere on an orbital shaker at 50 rpm. At day 2, 60 ml of prewarmed culture medium are added. At day 3, a second round of amplification is performed.

(160) Cells Seeding in 3 L Stirred-Thank Bioreactor

(161) SeedingDay 0

(162) The inoculum is prepared (320.10.sup.6 cells are needed to inoculate the 3 L-bioreactor). The 2 T175 flasks are pooled. A sample is taken after gently mixing (cells clumps should not be broken) to perform a numeration using trypan blue to determine cell density. A 150 mL cell mix is prepared in order to obtain a cell concentration of 0.40.10.sup.6 cells.mL.sup.1 into the 800 ml final culture volume in the bioreactor.

(163) Prior to seed cells, the pH is set in the vessel to 7.2 (because pH will be decrease by CO.sub.2 surface injection). The pO.sub.2 is set to 50% O.sub.2 saturation (the mass flow controller is adjusted to 100% which correspond to a maximum sparger flow rate to 50 mL.min.sup.1). At the beginning of the process, the pH is maintained by CO.sub.2 surface injection, later, it is controlled by addition of 7.5% NaHCO.sub.3. The surface aeration is started with air at a flow rate of 0.3 mL.min.sup.1.

(164) Culture Follow-Up/Feed Addition (Day 1, Day 2)

(165) Cell numeration is performed on a routine basis. The metabolites such as glutamate, glutamin, lactate and glucose are analyzed all along the culture with the BioProfile Basic software. Concentration of the metabolites is adjusted if necessary. For example, glutamin concentration is adjusted to 2 mM.

(166) InfectionDay 3

(167) After 3 days of culture, cell density have to be higher than 4-5.10.sup.6 cells.mL.sup.1. If the target cell density is reached, the virus infection is performed at a MOI of 10.sup.4. The vessel temperature is set to 33 C. The virus strain is thawed on ice. The infection mix is prepared in 10 mL of production medium. After inoculation of the infection mix into the bioreactor, viral adsorption is performed during 1 hour. The final production medium is prepared: in 1.5 L of production medium, trypsin is added in order to obtain a final concentration in the vessel of 0.3 U.mL.sup.1 (2.3 L on the whole) and 0.5 Yeastolate is added. The pre-warmed final production medium is then added.

(168) Production Following/Feed Addition (Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10)

(169) Every day a sample of approximatively 15 ml is collected from the bioreactor to perform cell numeration, cell morphology analysis and to observe CPE. The metabolites such as glutamate, glutamine, lactate and glucose are analyzed all along the culture with the BioProfile Basic software. Concentration of the metabolites is adjusted if necessary. For example, glutamine concentration is adjusted to 2 mM if necessary. The glucose concentration is adjusted to 2 g.L.sup.1 if necessary.

(170) Analyses

(171) Virus titration, haemmaglutinin assays (HAU) and HA antigen quantifications (western blot, SRID) are carry out at the end of the experiment using all collected samples.

(172) 4.3Results

(173) The inventors demonstrate that EB14 cells are a reliable and efficient cell substrate for the replication of various strains A and B of influenza virus. Influenza virus production can be performed in various vessels, such as flasks and spinner (data not shown) and bioreactors. Reproducible and efficient fedbatch process of production of influenza virus in 3 L and 30 L stirred tank bioreactors were obtained by the inventors. Productivities above 25 mg/l of haemagglutinin are routinely obtained in flasks and Productivities above 35 mg/l of haemagglutinin are routinely obtained in flasks with strains A and B of influenza virus.