WO2007135133A1

WO2007135133A1 - Avian cell lines derived from primordial germ cells useful for the production of substances of interest

Info

Publication number: WO2007135133A1
Application number: PCT/EP2007/054898
Authority: WO
Inventors: Majid Mehtali
Original assignee: Vivalis
Priority date: 2006-05-19
Filing date: 2007-05-21
Publication date: 2007-11-29

Abstract

The present invention relates to a method for producing avian cell lines derived from PGC or PGC derived EG, comprising gradual or complete withdrawal of growth factors, serum and/or feeder layer so that the established lines are adherent or non-adherent cells capable of proliferating indefinitely in a basic culture medium. The invention also relates to the use of such cell lines for the production of substances of interest, mainly viral vaccines and recombinant proteins.

Description

Avian cell lines derived from Primordial Germ Cells useful for the production of substances of interest

The present invention relates to a method of establishment avian cell lines from primordial germinal cells, said method comprising progressive withdrawal of growth factors, serum and/or feeder layer from the cell culture medium. The invention also relates to the use of such avian cell lines for the production of viral vaccines and recombinant proteins of interest.

Mass vaccination would be the simplest and most effective approach to control viral pandemics, such as for example pandemic and inter-pandemic flu outbreaks, but also to prevent bioterrorist threat, such as the recent terrorist acts involving anthrax in the USA. For many years, viral vaccines such as for example, measles, mumps, rubella, influenza and smallpox vaccines are produced on egg-based systems, and mainly on embryo nated eggs or chicken embryonic fibroblasts (CEFs). Despite being reliable for more than 50 years, egg-based systems shows its limitations that include: a lengthy, cumbersome and resource-consuming manufacturing process that requires the procurement and quality control of large quantities of eggs or CEFs for each individual production campaign; the need in many cases to use costly specific pathogen free (SPF) chicken embryos; the risks of insufficient supply of eggs in cases of epidemic infections in donor chicken flocks; - the inflationist costs associated with the use of bovine sera originating from

BSE-exempt countries; the allergenicity of eggs in some individuals; the inability to use eggs for the propagation of viruses that are highly virulent and lethal to chickens. Moreover, it is most likely that the current production capacity of vaccines manufacturers would not suffice to cover the needs in a case of a pandemics of avian influenza or a bioterrorist attack with smallpox virus for example. While the egg-based and CEFs production process remain relatively reliable process, an efficient cell-based production system would represent a significant improvement in providing a faster, cheaper and less cumbersome method for growing viruses. Moreover, in the event of a flu pandemic, a cell culture based manufacturing process offers additional advantages: the production of the influenza vaccine can start immediately after the pandemic strain has been identified, isolated and distributed; there is no need to wait for the development of so-called High Growth Reassortants (viruses adapted to high yield growth in embryonated hens eggs) necessary for production in eggs; the availability of the first vaccine batch would be approximately 9 weeks after the receipt of the strain, instead of 6-9 months with the egg-derived process; a cell- derived process allows the production of strains that cannot be adequately grown in eggs (e.g. Avian Hong Kong Flu in 1997); there is no problem of egg shortage during pandemics. Moreover, the use of cell lines for manufacture of viral vaccines, instead of egg or CEF platforms, would have the additional following advantages in connection with the safety of the vaccine: no antibiotic additives present in the vaccine formulation; no toxic preservatives (such as thiomersal) needed; reduced endotoxin levels; growth in protein and serum free media (no adventitious agent/BSE); high purity of virus vaccine preparation.

There is therefore an urgent need to improve the current viral vaccine production technologies based on eggs or chicken-embryonic fibroblasts. The development of cell- culture platforms as an alternative to the eggs and CEF production systems for the manufacture of viral vaccines is likely the most rapid and promising solution to overcome current vaccine production bottlenecks and time constrains. Moreover, cell- culture production technologies would improve possibilities of up-scaling of viral vaccines production capacities in face of a pandemic or a terrorist attack. In addition such cell culture production technology would be of major interest for the production of recombinant proteins, such as monoclonal antibodies for example.

Based on these specific requirements, the inventor has taken advantage of its expertise in avian biology and in avian embryonic stem (ES) cells to undertake the development of stable avian cell lines that enables the efficient replication of human and veterinarian viral vaccines and vaccine candidates, but also the production of proteins of interest, and that fulfil the industrial, regulatory and medical specifications. Using a proprietary process (see WO 03/076601 and WO 05/007840), the inventor has thus generated a series of well characterized and documented cell lines (the EBx® cells) that are derived from chicken ES or duck cells with no steps of genetic, chemical or viral immortalization. EBx® cells have been generated using a fully documented process, and taking in consideration all regulatory requirements (eg. regular monitoring of the sanitary status of the avian flocks, use of serum from BSE-free countries, use of pronase instead of trypsin, availability of certificates of origin for all components included in the process, ...).

Today, the inventor is proposing to use a selected population of avian embryonic stem cells, either the primordial germ cells (PGC) or Embryonic Germ cells (EG) that derived from PGC, to undertake the development of stable avian cell lines that enable the efficient replication of human and veterinarian viral vaccines and vaccine candidates, but also the production of proteins of interest, and that fulfil the industrial, regulatory and medical specifications.

Stem cells are cells identified by their culture in vitro from an embryo, from part of an embryo or even from an adult tissue. The expression "stem cell" is understood to mean any multi-, pluri- or toti-potent cell of embryonic or adult origin which has a capacity for self-renewal and is capable of giving specialized differentiated cells. In other words, any non-cancerous cell capable of dividing indefinitely in culture and of giving a daughter cell having the same capacity for proliferation and differentiation as the mother cell from which it is derived. These isolated cells exhibit particular morphological and immuno-cytochemical characteristics. It is also possible to distinguish the notion of:

- embryonic stem cells (ES cells), stem cells which have the characteristic feature of being obtained from culturing parts or all of a very early embryo (e.g blastula stage). These ES cells exhibit in vitro all the characteristics of a stem cell, and in vivo the unique capacity of contributing to the morphogenesis of an embryo and of participating in germline colonization when they are re-implanted in any manner whatsoever in a recipient embryo. Primordial Germ Cells (PGC) which are the progenitors of the sperm or ovocyte cells that develop after sexual maturity are pluripotent ES cells and constitutes a subtype of ES cells. Embryonic Germ cells (EG) are also pluripotent ES cells that derived from PGCs. The morphology of EG cells is similar to that of ES cells; EG cells may contribute to somatic tissue when injected into a stage X of EYAL-GILADI classification (EYAL-GILADI and

KOCHAV, 1976 « From cleavage to primitive streack 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) chicken recipient. - somatic stem cells (SSC), cells which have all the characteristics of stem cells when they are cultured in vitro, but which, unlike ES cells, do not have the potential to colonize in vivo the gonads after infection into an embryo. They contribute solely to the morphogenesis of the somatic tissues in the embryo.

Unlike already differentiated primary cells, stem cells do not exhibit an easily identifiable characteristic state of morphological differentiation (fibroblasts, adipocytes, macrophage, and the like), but are rather characterized by a state of proliferation and of non-differentiation. This state results in different behaviors such as a rapid proliferation in vitro, a characteristic morphology, the presence of different markers, variable requirements for growth factors and an ability to respond to particular stimuli for induction of differentiation. As used herein, the terms primordial germ cells (PGCs) mean cells exhibiting a

PGC morphology and which contribute exclusively to the germline in recipient embryos. A PGC phenotype may be established by (1) the germline specific genes CVH and Dazl are strongly transcribed in this cell line, (2) the cells strongly express the CVH protein, (3) the cells do not contribute to somatic tissues when injected into a Stage X nor a Stage 12-17 (Hamburger & Hamilton) recipient embryo, (4) the cells give rise to

EG cells (see below), or (5) the cells transmit the PGC genotype through the germline when injected into Stage 12-17 embryos (Hamburger & Hamilton, 1951, A series of normal stages in the development of chick embryo, J. Morphol. 88:49-92; Tajima et al., 1993, Theriogenology 40, 509-519; Naito et al., 1994, MoI. Reprod. Dev., 39, 153-161; Naito et al., 1999, J. Reprod. Fert. 117, 291-298). In vitro method to culture PGCs over a long period of time has been previously described (see WO 2001/11019). As used herein, the term embryonic germ (EG) cells means cells derived from PGCs and are analogous in function to murine EG cells. The morphology of chicken EG cells is similar to that of chicken ES cells and chicken EG cells contribute to somatic tissues when injected into a Stage X (Eyal-Giladi's classification) chicken recipient. The instant invention provides a process for obtaining avian cell lines derived from stem cells, preferably avian cell lines derived from embryonic stem cells, and more preferably avian cell lines derived from primordial germ cells (PGC) or embryonic germ cells (EG) derived from PGCs, said process comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification (EYAL-GILADI and KOCHAV, 1976,

« From cleavage to primitive streack 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) and hatching, preferably around oviposition; b) optionally, isolating PGC cells from said avian embryo(s); c) optionally, inducing differentiation of PGC cells comprised in avian embryonic stem cells obtained by dissociating avian embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing avian embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), or culturing EG cells of step c) in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent avian cell lines derived from PGC cells or from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum. The instant invention provides a process for obtaining avian cell lines derived from primordial germ cells (PGC), said process comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage VI of Eyal-Giladi's classification and hatching, preferably around oviposition; b) optionally, isolating PGC from said avian embryo(s); c) culturing avian embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth and a feeder layer, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, e) establishing adherent or non adherent avian cell lines derived from PGC cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum. The instant invention also provides a process for obtaining avian cell lines derived from PGC derived EG, said process comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage VI of Eyal-Giladi's classification and hatch, preferably around oviposition; b) isolating PGC from said avian embryo(s); c) inducing differentiation of PGC cells comprised in avian embryonic stem cells obtained by dissociating avian embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing avian EG cells of step c) in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent avian cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum. The avian cells of step a) are cells selected among avian stem cells that comprises embryonic and somatic stem cells. More preferably, the avian cells of step a) are cells selected among avian embryonic stem cells and are preferably PGCs. In avian species, PGCs arise from the epiblast and migrate to the hypoblast of the area pellucida (the germinal crescent) at stage IX (Eyal-Giladi's classification), approximatively 18 to 19 hour after incubation (EY AL-GILADI and KOCHAV, 1976, « From cleavage to primitive streack 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; Hamburger & Hamilton, 1951, A series of normal stages in the development of chick embryo, J. Morphol. 88:49-92). PGCs move from the germinal crescent into the blood stream at stage 10 to 25 (Hamburger & Hamilton's classification), (Ukeshima et al., 1991, J. Electron Microscop., 40:124-128) and circulate in the vascular system until stage 17 (2.5 days of incubation) when they reach the region of the germinal ridges, in which they finally concentrate and colonize (Nieuwkoop and Sutasurya, 1979, The Migration of the primordial germ cells. In: Primordial germ cell in Chordates. London: Cambridge University Press pi 13-127). Methods for isolation of PGCs from donor avian embryos have been reported in the literature and can be effected by one skilled in the art (See, e.g. JP 924997 published Sept. 7, 1993, Pub. N° 05-227947; Chang et al., 1992, Cell Biol. Int. 19(2): 143-149; Naito et al., 1994, MoI. Reprod. Dev. 39:153-161; Yasuda et al., 1992, J. Reprod. Fert, 96:521-528; Chang et al., 1992, Cell Biol. Int. Reporter, 16(9):853-857). According to a preferred embodiment, PGCs are collected from embryonic blood collected from the dorsal aorta of a chicken embryo at stage 12- 14 of Hamburger & Hamilton's classification. In another preferred embodiment, PGCs were collected from the germinal crescent by mechanical dissection of chicken embryo or from the gonads. However, as discussed above, others methods for isolating PGCs are known and alternatively be used.

EG cells may be established by sub-culturing PGCs retrieved from the germinal crescent or embryonic blood or from gonads (Park et al., 2003, MoI. Reprod. Dev. 65:389-395). PGCs maintained in culture with conventional techniques often do not proliferate and multiply. In the absence of robust growth, the PGC culture are "terminal" and cannot be maintained indefinitely. Over the time, these terminal cell cultures are degraded and the cells lose their unique PGC morphology and revert to EG cells. EG cells acquire a different morphology from PGCs, lose their restriction to the germ line and gain the ability to contribute to somatic tissues when injected into early stages of embryonic development. In another preferred embodiment, the avian cells of step a) are totipotent or pluripotent avian embryonic stem cells isolated from a population suspension of dissociated stage X blastodermal cells (Eyal-Giladi's classification) obtained from an avian embryo, more preferably a chicken embryo. According to a preferred embodiment, the avian embryonic stem cells according to step a) of the invention are collected from avian embryo at oviposition, that is to say when the egg is laid. According to Sellier et al (2006, J. Appl. Poult. Res., 15:219-228), oviposition corresponds to the following development stages according to Eyal-Giladi's classification (EYAL-GILADI's classification: EYAL-GILADI and KOCHAN, 1976, « From cleavage to primitive streack 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):

Muscovy duck: stage VII Guinea fowl: stage VII - VII I - Turkey: stage VII-VIII

Pekin duck: stage VIII Chicken: Stage X Japanese Quail: stage XI Goose: stage XI. Preferably, the chicken embryonic stem (ES) cells, preferably from ev-0 chicken strain, of step a) is obtained by dissociating embryo(s) at around stage X (oviposition) of Eyal-Giladi's classification.

Preferably, the duck embryonic stem (ES) cells of step a) is obtained by dissociating embryo(s) at around stage VIII (oviposition) of Eyal-Giladi's classification. Alternatively, the avian embryonic stem cells according to step a) of the invention are collected from embryo before oviposition. The main limitations encountered before oviposition is the fact that the egg has to be surgically removed from hens and that the amount of ES cells per embryo is less important. Moreover at very early stages of avian embryo development, ES cells are not well individualized rendering difficult in vitro culture of ES cells. A man skilled in the Art will be able to define the timeframe prior egg laying that allows to collect avian ES cells.

Alternatively, the avian embryonic stem cells according to step a) of the invention may be collected from avian embryo after oviposition up to hatching. However, avian embryonic stem cells will progressively enter into differentiation to generate differentiated tissues; therefore, it is preferred to collect avian ES not to long after the lay. A man skilled in the Art will be able to define the timeframe after egg laying that allows to collect avian embryonic stem cells.

The term « avian » as used herein is intended to refer to any species, subspecies or race of organism of the taxonomic class « ova », such as, but not limited to, such organisms as chicken, turkey, duck, goose, quails, pheasants, parrots, finches, hawks, crows, ostrich, emu and cassowary. The term "avian", "ava", "bird" as used herein is intended to have the same meaning and will be used indistinctly.

In a preferred embodiment, "birds" refer to any animal of the taxonomix order:

- "Anseriformes" (i.e duck, goose, swan and allies). The order Anseriformes contains about 150 species of birds in three families: the Anhimidae (the screamers), Anseranatidae (the Magpie-goose), and the Anatidae, which includes over 140 species of waterfowl, among them the ducks, geese, and swans. All species in the order are highly adapted for an aquatic existence at the water surface. All are web-footed for efficient swimming (although some have subsequently become mainly terrestrial).

- "Galliformes" (i.e chicken, quails, turkey, pheasant and allies). The Galliformes is an order of birds containing the chicken, turkeys, quails and pheasants. About 256 species are found worldwide. Chicken include various strains of Gallus gallus (i.e chicken), such as White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partidge-colored and other poultry commonly bred.

- "Columbiformes" (i.e Pigeon and allies). The bird order Columbiformes includes the very widespread doves and pigeons.

According to a preferred embodiment, the bird of the invention are selected among the birds that does not comprises avian leucosis virus E (ALV-E) and endogenous avian virus (EAV) proviral sequences in its genome. A man skilled in the art is able to determine whether ALV-E and EAV sequences are present in a bird genome (Johnson et Heneine, 2001, J. Virol, 75:3605-3612; Weissmahr et al, 1997, J.

Virol, 71:3005-3012). Preferably the bird is selected in the group comprising Anseriformes (i.e duck, goose, swan), turkeys, quails, Japanese quail, Guinea fowl, Pea Fowl. Therefore, cells derived from such bird do not produce replication-competent endogenous ALV-E and/or EAV particles. In a preferred embodiment, the bird of the present invention is selected among the group comprising ducks, geese, swans, turkeys, quails and Japanese quails, Guinea Fowls and Pea Fowls. According to a more preferred embodiment, the bird is a duck, more preferably a Pekin or Moscovy ducks. According to a more preferred embodiment, the bird is a Pekin duck. Therefore, the instant invention provides a process for obtaining continuous diploid duck cell lines derived from embryonic stem cells (ES), preferably from PGCs or EGs cells, wherein said duck cell lines do not produce replication-competent endogenous retrovirus particles. According to a second preferred embodiment, the bird of the invention are selected among the birds that does not comprises complete ALV-E proviral sequences in its genome but eventually EAV proviral sequences. A man skilled in the art is able to determine whether partial or complete ALV-E and EAV sequences are present in a bird genome (Johnson and Heneine, 2001). Several chicken strains have been selected by breeding that do not contain complete ALV-E proviral sequences (i.e: ev-0 strain) and therefore do not produce infectious ALV-E retroparticles, such as:

- Line 0 domestic chicken of East Lansing USDA poultry stock (ELL-O strain). The East Lansing Line-0 chickens do not contain any endogenous viral (ev) loci related to ALV (Dunwiddie and Faras, 1985, Proc. Natl. Acad. Sci USA, 82:5097-5101).

Line 22 white leghorn chicken of Charles River (SPAFAS). Lines DE and PEI l from Institut National de Ia Recherche Agronomique (Domaine de Magneraud, Surgeres, France). Therefore, cells derived from ev-0 birds do not produce replication-competent endogenous ALV-E particles. According to a preferred embodiment, the bird of the invention is an ev-0 domestic chicken {Gallus Gallus subspecies domesticus), preferably selected among ELL-O, Line 22, DE and PEI l. Usually, ev-0 chickens still contain EAV proviral sequence but so far no infectious EAV isolates have been identified. Therefore, the instant invention provides a process for obtaining continuous diploid chicken cell lines derived from embryonic stem cells (ES), preferably from

PGCs or EGs of ev-0 chicken strains, wherein said ev-0 chicken cell lines do not produce replication-competent endogenous retrovirus particles. In the instant invention, by the term "endogenous retroviral particle" or "endogenous retrovirus particle", terms that could be used indistinctively, it is meant a retroviral particle or retrovirus encoded by and/or expressed from ALV-E or EAV proviral sequences present in some avian cell genomes. In the birds, ALV-E proviral sequences are known to be present in the genome of domestic chicken (except Line-0 chicken), red jungle fowl and Ringneck Pheasant. In the birds, EAV proviral sequences are known to be present in all genus gallus that includes domestic chicken, Line-0 chicken, red jungle fowl, green jungle fowl, grey jungle fowl, Ceylonese jungle fowl and allies) (see Resnick et al, 1990, J. Virol, 64:4640-4653). In a preferred embodiment, the avian cell of the present invention is a chicken cell. More preferably, the avian cell line derives from chicken PGCs or chicken EGs. Alternatively, the avian cell line derives from duck PGCs or duck EGs.

Without to be bound by a theory, the defined cell culture conditions of avian ES cells followed by the progressive weaning in grow factors, feeder layer and serum, allow to adapt and select cells that maintain most of the desirable feature of ES cells

(stability of karyotype, indefinite proliferation, expression of ES markers) but in addition display industrial- friendly characteristics like growth in suspension up to high cell densities in serum- free medium. Telomerase constitutes one of the most important ES markers. Due to the sustained and maintained telomerase expression over the cell passages, avian cell lines, named EBx®, obtained by the process of the invention, are continuous (i.e immortal) but in addition are genetically stable (i.e diploid).

Thus, according to a first preferred embodiment, the invention provides a process of establishing adherent or non adherent avian cell lines derived from PGC cells comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage IX of EYAL-GILADI classification and around stage 15 of Hamburger & Hamilton's classification; b) isolating PGC from said avian embryo(s); c) culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth and a feeder layer, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, e) establishing adherent or non adherent avian cell lines derived from PGC cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum. Thus, according to a second preferred embodiment, the invention provides a process of establishing adherent or non adherent avian cell lines derived from PGC derived EG cells comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage

IX of EYAL-GILADI classification and around stage 15 of Hamburger & Hamilton's classification; b) isolating PGC from said avian embryo(s); c) inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing avian EG cells of step c) in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent avian cell lines derived from PGC derived cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum. In step c) of inducing differentiation of PGC cells into EG cells, the differentiation of PGC cells into EG cells can be obtain spontaneously after maintening the PGC in culture by the conventional technique. Indeed, the PGC cannot be cultured indefinitely by the conventional technique and spontaneously revert into EG cells at the end of the culture.

Thus, according to a third preferred embodiment, the invention provides a process of establishing adherent or non adherent avian cell lines derived from PGC cells comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage IX of EYAL-GILADI classification and around stage 15 of Hamburger & Hamilton's classification; b) isolating PGC from said avian embryo(s); c) culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and a feeder layer, wherein said complete medium is a conditioned culture medium comprising growth factors and optionally supplemented with exogenous growth factors, allowing PGC cell growth, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of growth factors, of the animal serum and of the feeder layer, wherein said conditioned culture medium, optionally supplemented with exogenous growth factors, is progressively replace by a synthetic basal culture medium, so as to obtain a basal culture medium comprising no exogenous growth factors, no feeder cells and containing a low level of animal serum or no animal serum; e) establishing adherent or non adherent avian cell lines derived from PGC cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum. Thus, according to a fourth preferred embodiment, the invention provides a process of establishing adherent or non adherent avian cell lines derived from PGC derived EG cells comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage IX of EYAL-GILADI classification and around stage 15 of Hamburger & Hamilton's classification; b) isolating PGC from said avian embryo(s); c) inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing avian EG cells of step c) in a complete culture medium supplemented with animal serum and a feeder layer, wherein said complete medium is a conditioned culture medium comprising growth factors and optionally supplemented with exogenous growth factors, allowing EG cells growth, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of growth factors, of the animal serum and of the feeder layer, wherein said conditioned culture medium, optionally supplemented with exogenous growth factors, is progressively replace by a synthetic basal culture medium, so as to obtain a basal culture medium comprising no exogenous growth factors, no feeder cells and containing a low level of animal serum or no animal serum; e) establishing adherent or non adherent avian cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

Thus, according to a fifth preferred embodiment, the invention provides a process of establishing adherent or non adherent avian cell lines derived from ES cells comprising the steps of: a) isolating avian embryo(s) at developmental stages comprise between stages

VI and X of Eyal-Giladi's classification; b) culturing avian embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth and a feeder layer, and; c) passage by modifying the culture medium so as to obtain a progressive withdrawal of said exogenous growth factors, of the animal serum and of the feeder layer, d) establishing adherent or non adherent avian cell lines derived from PGC cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

More specifically, the instant invention provides a process for obtaining chicken cell lines derived from stem cells, preferably chicken cell lines derived from embryonic stem cells, and more preferably chicken cell lines derived from primordial germ cells

(PGC), said process comprising the steps of: a) isolating chicken embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification and hatching, preferably around oviposition (stage X of EYAL-GILADI classification); b) optionally, isolating PGC cells from said chicken embryo(s); c) culturing chicken embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, e) establishing adherent or non adherent chicken cell lines derived from PGC cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

According to a second invention, the instant invention also provides a process for obtaining chicken cell lines derived from stem cells, preferably chicken cell lines derived from embryonic stem cells, and more preferably chicken cell lines derived from embryonic germ cells (EG) derived from PGCs, said process comprising the steps of: a) isolating chicken embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification and hatching, preferably around oviposition (stage X of EYAL-GILADI classification); b) optionally, isolating PGC cells from said avian embryo(s); c) inducing differentiation of PGC cells comprised in avian embryonic stem cells obtained by dissociating avian embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing EG cells derived from PGC cells in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent avian cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

The instant invention provides a process for obtaining duck cell lines derived from stem cells, preferably duck cell lines derived from embryonic stem cells, and more preferably duck cell lines derived from primordial germ cells (PGC), said process comprising the steps of: a) isolating duck embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification (see Sellier et al, 2006, J. Appl. Poult. Res., 15:219-228) and hatching, preferably around oviposition (stage VII of EYAL- GILADI classification); b) optionally, isolating PGC cells from said duck embryo(s); c) culturing duck embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, e) establishing adherent or non adherent duck cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

The instant invention also provides a process for obtaining duck cell lines derived from stem cells, preferably duck cell lines derived from embryonic stem cells, and more preferably duck cell lines derived from embryonic germ cells (EG) derived from PGCs, said process comprising the steps of: a) isolating duck embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification (see Sellier et al., 2006, J. Appl. Poult. Res., 15:219-228) and hatching, preferably around oviposition (stage VII of EYAL- GILADI classification); b) optionally, isolating PGC cells from said duck embryo(s); c) inducing differentiation of PGC cells comprised in duck embryonic stem cells obtained by dissociating duck embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing EG cells derived from PGC cells in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent duck cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

In the event, the basal medium still comprises a low level of serum (i.e. around 2% or less) at the end of the process of cell lines establishment, said process may optionally comprises an additional step of changing the basal medium to a serum free medium (SFM), by performing one of the following additional step: i)- the basal medium comprising low level of serum is diluted with a SFM medium, then during successive passages the ratio of SFM medium is progressively increased up to the complete disappearance of said basal medium; ii)- the basal medium comprising low level of serum is replaced in one passage by SFM medium complemented with serum, then the cell lines are cultured during successive passages in said SFM medium in which the ratio of serum is progressively decreased up to the obtaining of a SFM medium without serum; iii)- the basal medium comprising low level of serum is replaced in one passage by a serum-free medium (SFM); then maintaining in SFM medium said avian cell lines derived from PGC or PGC derived EG.

According to a preferred embodiment, the serum of the invention is animal serum, preferably foetal calf serum or chick serum.

The modification of the culture medium in the process of the invention, so as to obtain progressive withdrawal of growth factors, serum and/or feeder layer, can be performed simultaneously, successively or separately. The sequence of the modification of the culture medium is chosen among the following sequences of weaning: - progressive withdrawal of feeder layer / progressive withdrawal of serum / progressive withdrawal of growth factors;

- progressive withdrawal of feeder layer / progressive withdrawal of growth factors / progressive withdrawal of serum; - progressive withdrawal of serum / progressive withdrawal of growth factors / progressive withdrawal of feeder layer;

- progressive withdrawal of serum / progressive withdrawal of feeder layer / progressive withdrawal of growth factors;

- progressive withdrawal of growth factors / progressive withdrawal of serum / progressive withdrawal of feeder layer;

- progressive withdrawal of growth factors / progressive withdrawal of feeder layer / progressive withdrawal of serum.

In a preferred embodiment, the sequence of the modification of the culture medium is progressive withdrawal of growth factors / progressive withdrawal of feeder layer / progressive withdrawal of serum.

More precisely, the process comprises the seeding of culture flasks with around 10³/cm² to around 10⁵/cm² avian cells in a complete culture medium supplemented with animal serum and a feeder layer. By "complete culture medium", it is meant a basal medium, preferably a basal synthetic medium, complemented with at least one growth factor and animal serum. Example of complete culture medium is described in WO 03/076601, WO 05/007840, EP 0 787 180, US 6,114,168, US 5,340,740, US 6,656,479, US 5,830,510 and in a Pain et al. (1996, Development 122:2339-2348). 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 BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199,

DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 and Ham-F10, Iscove's Modified Dulbecco's medium, MacCoy's 5A medium, RPMI 1640. Basal medium comprises inorganic salts (for examples: CaCl₂, KCl, NaCl, NaHCO₃, NaH₂PO₄, MgSO₄, ...), amino-acids, vitamins (thiamine, riboflavin, folic acid, D-Ca panthothenate, ...) and others components such as glucose, beta-mercapto-ethanol, sodium pyruvate. Preferably basal medium is a synthetic medium. Alternatively, the complete culture medium is a conditioned medium, preferably BRL conditioned medium. By way of example, BRL conditioned media is prepared according to art-recognized techniques, such as described by Smith and Hooper (1987, Dev. Biol, 121:1-9). BRL cells are available from ATCC accession number CRL-1442. Conditioned medium may be supplemented with exogenous growth factors as described below.

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. 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 gpl30 protein. Thus, leukemia inhibitory factor (LIF), interleukin 11, interleukin 6, interleukin 6 receptor, Ciliary Neurotrophic factor (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 gpl30 protein in monomeric or sometimes hetero-dimeric form. The trophic factors are mainly Stem cell Factor (SCF), Insulin Growth factor 1 (IGF-I) and Fibroblast Growth Factor (FGF), preferably basic FGF (bFGF) or human FGF (hFGF). The complete culture medium according to the invention comprises basal medium, preferably basal synthetic medium, and at least one cytokine whose action is through a receptor which is associated with the gpl30 protein and/or at least one trophic factors. Preferably, the complete culture medium according to the invention comprises basal medium and at least one growth factor selected in the group consisting of Leukemia Inhibitory factor (LIF), Insulin Growth factor 1 (IGF-I), Ciliary Neurotrophic factor (CNTF), Interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), Stem cell Factor (SCF), Fibroblast Growth Factor, preferably bFGF, optionally interleukin 11 (IL-11).

According to a first preferred embodiment, the complete culture medium is basal medium complemented with at least IGF-I and CNTF. According to a second preferred embodiment, the complete culture medium is basal medium complemented with at least IGF-I, CNTF, SCF and FGF, preferably bFGF, and optionally IL-I l. According to a third preferred embodiment, the complete culture medium is basal medium complemented with at least IGF-I, CNTF, IL-6, IL-6R, SCF, bFGF, optionally IL-11. According to another embodiment, the complete culture medium of step a) is a conditioned culture medium comprising growth factors (i.e expressed by BRL cells for example) and optionally supplemented with at least one exogenous growth factors selected in the group consisting of: Leukemia Inhibitory factor (LIF), Insulin Growth factor 1 (IGF-I), Ciliary Neurotrophic factor (CNTF), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), Stem cell Factor (SCF), Fibroblast Growth Factor (FGF), interleukin 11 (IL-I l).

The concentration of growth factors IGF-I, CNTF, IL-6, IL-6R, SCF, bFGF, 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. The avian stem cells, preferably the avian PGCs of step a) are cultured during several passages in the complete medium.

After around passages 3 to 15, the complete medium is depleted in growth factors. 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-I 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-I, CNTF, SCF, FGF, optionally IL-I l, the depletion in growth factors is made in two rounds of depletion: in the first round, SCF and FGF and optionally ILI l are directly removed from the complete medium, then the avian cells are maintained in culture for at least one passage in a complete medium containing IGFl and CNTF. Secondly, IGFl and CNTF are directly removed from the culture medium, which ultimately comprises the basal medium optionally only supplemented with animal serum. In another preferred embodiment, when the complete culture medium is basal medium complemented with IGF-I, CNTF, IL-6, IL-6R, SCF, FGF, optionally IL-I l, the depletion in growth factors is made in two rounds of depletion: in the first round, SCF, IL6, IL6R, FGF, optionally ILl 1 are directly removed from the complete medium, then the avian cells are maintained in culture for at least one passage in a complete medium containing IGFl and CNTF, and supplemented with animal serum. Secondly, IGFl and CNTF are directly removed from the culture medium, which ultimately comprises the basal medium optionally supplemented with animal serum and/or feeder cells. Usually, the medium is totally depleted in growth factors at around passages 20 to 40. In a preferred embodiment, the deprivation of feeder cells is performed after the deprivation of growth factors. 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 N⁰CRL- 1503).

The deprivation of feeder cells is progressive and performed over several passages. The avian cells are now preferably seeded in flask at a concentration about around 10³ cell/cm² to 10⁵ cell/cm². The feeder cells are seeded in flask at around 10⁴ to 10⁵ cells/cm². 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 5 passages, then a lower concentration of the feeder cells is used for an additional 2 to 5 passages, and so. For example, the flask is seeded with around 4 xlO⁴ feeder cells/cm², then around 2 x 10⁴ feeder cells/cm², then around 10⁴ feeder cells/cm², then around 0.5x 10⁴ feeder cells/cm², then around 10³ feeder cells/cm², then around 0.5 x 10³ feeder cells/cm², then around 10² feeder cells/cm². Then the flask is seeded with avian cells at a concentration about around 10³ cell/cm² to 10⁵ cell/cm² but 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. In the 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) supplemented with animal 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.

The expression serum-depleted is understood to mean a gradual reduction of the concentration of serum spread out over time. This method allows a selection of clones which adapt to these new, increasingly drastic 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 invention also relates to the established avian stem cell lines of the invention. This process of the invention will lead to the establishment of avian cell lines derived from embryonic stem cells, and more preferably derived from avian PGCs or PGC derived EG; said avian cell lines are maintained in culture in vitro over a long period of time. The invention also relates to the avian cell lines derived from PGC or PGC derived EG, more preferably to the chicken or duck cell lines derived from PGC or PGC derived EG. The avian stem cell lines of the invention will be 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 and cells can be cultured for longer time periods. Avian, preferably chicken or duck, cells of the invention derived from PGC or PGC derived EG, will be able to grow indefinitely in a basic culture medium, free of exogenous growth factors, with no (or reduced level of) animal serum and with no feeder layer. For example, avian cell lines of the invention are capable of proliferating for at least 10 generations, at least 20 generations, at least 30 generations, at least 40 generation, for at least 100 generations.

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. In the present invention, avian cells and avian cell lines will have the same meaning and use interchangeably; avian cell lines being composed of avian cells. The established avian cells obtainable by the process of invention are round, individualized cells with a doubling time comprise between 16 hours to 72 hours, preferable around 18 to 30 hours at 39°C.

The avian cells derived from PGC, EG or ES 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, an expression of cellular markers selected in the group comprising SSEA-I (TECOl), EMA-I, DAZL, VASA. Preferably, the avian cells derived from PGC, EG or ES cells have all the above mentioned characteristics and are useful for the production of biologies such as viral vaccines and recombinant peptides and proteins (i.e. antibodies, ...).

These avian cells obtainable by the process of the invention are capable of proliferating indefinitely in a basal medium, in particular in a medium such as SAFC Biosciences (Lenaxa, Ka USA) 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 hydro lyzates. Duck 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.

According to a preferred embodiment, basal medium is a serum-free medium. According to the present invention, "serum-free medium" (SFM) meant a cell culture medium ready to use, that is to say that it does not required serum addition allowing cells survival and cell growth. This medium is not necessary chemically defined, and may contained hydrolyzates of various origin, from plant for instance. Preferably, said

SFM are "non animal origin" qualified, that is to say that it does not contain components of animal or human origin (FAO status: "free of animal origin"). In SFM, the native serum proteins are replaced by recombinant proteins. Alternatively SFM medium according to the invention does not contain protein (PF medium: "protein free medium") and/or are chemically defined (CDM medium: "chemically defined medium"). SFM media present several advantages: (i) the first of all being the regulatory compliance of such media (indeed there is no risk of contamination by adventitious agents such as BSE, viruses); (ii) the optimization of the purification process; (iii) the better reproducibility in the process because of the better defined medium. Example of commercially available SFM media are: VP SFM (InVitrogen Ref. 11681-020, catalogue 2003), Opti Pro (InVitrogen Ref. 12309-019, catalogue 2003), Episerf (InVitrogen Ref. 10732-022, catalogue 2003), Pro 293 S-CDM (Cambrex Ref. 12765Q, catalogue 2003), LC17 (Cambrex Ref. BESP302Q), Pro CHO 5-CDM (Cambrex Ref. 12-766Q, catalogue 2003), HyQ SFM4CHO (Hyclone Ref. SH30515- 02), HyQ SFM4CHO-Utility (Hyclone Ref. SH30516.02), HyQ PF293 (Hyclone Ref. SH30356.02), HyQ PF Vero (Hyclone Ref. SH30352.02), Ex cell 293 medium (JRH Biosciences Ref. 14570-1000M), Ex cell 325 PF CHO Protein free medium (JRH Biosciences Ref. 14335-1000M), Ex cell VPRO medium (JRH Biosciences Ref. 14560- 1000M), Ex cell 302 serum free medium (JRH Biosciences Ref. 14312-1000M).

The instant invention also provides a process of replicating a virus in avian cells, preferably the chicken or duck cells derived from PGC, EG or ES of the invention. The process for replicating viruses comprises the steps of inoculating avian cells, preferably the chicken or duck cells derived from PGC, EG or ES of the invention with virus and culturing said cells in a culture medium, preferably in a serum-free medium, until viral replication occurs and newly virus particules are produced. Said process may comprise the additional step of harvesting the virus in cell culture supernatant and/or inside said cells.

The term "virus" as used herein includes not only naturally occurring viruses but also attenuated viruses, temperature sensitive virus, low-temperature adapted virus, reassortant viruses, vaccine strains, as well as recombinant viruses and viral vectors. The virus of the invention are preferably selected from the group consisting of adenoviruses, hepadnaviruses, herpes viruses, orthomyxoviruses, papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses and retroviruses. In a preferred embodiment, the viruses, the related viral vectors, viral particles and viral vaccines belong to the family of poxviruses, and more preferably to the chordopoxviridae. In one embodiment, the virus or the related viral vectors, viral particles and viral vaccines is an avipoxvirus selected among fowlpox virus, canary pox virus (i.e ALVAC), juncopox virus, mynah pox virus, pigeon pox virus, psittacine pox virus, quail poxvirus, sparrow poxvirus, starling poxvirus, turkey poxvirus. According to another preferred embodiment, the virus is a vaccinia virus selected among Lister- Elstree vaccinia virus strain, modified vaccinia virus such as Modified Vaccinia virus Ankara (MVA) which can be obtained from ATCC (ATCC Number VR- 1508), NYVAC (Tartaglia et al, 1992, Virology 188:217-232), LC16m8 (Sugimoto et Yamanouchi, 1994, Vaccine 12:675-681), CVI78 (Kempe et al., 1968, Pediatrics 42:980-985) and other recombinant or non-recombinant vaccinia virus.

In another preferred embodiment, the viruses, the related viral vectors, the viral particles and vaccines belong to the family of ortho-myxo viruses, in particular influenza virus. The influenza virus is selected from the group consisting of human influenza virus, avian influenza virus, equine influenza virus, swine influenza virus, feline influenza virus. Influenza virus is preferably selected in strains A, B and C. Among strains A, one can recite viruses with different subtypes of haemagglutinin and neuraminidase, such as without limitation HlNl, H2N2, H3N2, H4N2, H4N6, H5N1, H5N2, H7N7 et H9N2. Among HlNl strains, one can recite A/Porto Rico/8/34, A/New Caledonia/20/99, A/Beijing/262/95, A/Johannesburg/282/96, A/Texas/36/91, A/Singapore. Among strains H3N2, one can recite A/Panama/2007/99, A/Moscow/10/99, A/Johannesburg/33/94. Among B strains, one can recite without limitation B/Porto Rico/8/34, B/Johannesburg/5/99, B/Vienna/1/99, B/Ann Arbor/1/86, B/Memphis/1/93, B/Harbin/7/94, N/Shandong/7/97, B/Hong Kong/330/01,

B/Yamanashi/166/98. The influenza Virus of the invention is selected among wild type virus, primary viral isolate obtained from infected individual, recombinant virus, attenuated virus, temperature sensitive virus, low-temperature adapted virus, reassortant virus, reverse genetic engineered virus. When the virus of the invention is influenza virus, the process of the invention comprises the additional step of adding proteolytic enzyme in the culture medium in conditions that allow virus propagation. The addition of proteolytic enzyme is performed before virus infection of cell culture. The addition of proteolytic enzyme is performed simultaneously to virus infection of cell culture. In a preferred embodiment, the addition of proteolytic enzyme is performed after virus infection of cell culture, that is to say between few minutes to several hours. The proteolytic enzyme is selected from the group consisting of trypsine, chymotrypsine, thermo lysine, pepsine, pancreatine, Ia papaϊne, Ia pronase, subtilisine A, elastase, furine and carboxypeptidase. According to a preferred embodiment, the enzyme is trypsine. Preferably, the proteolytic enzyme is a recombinant protein of procaryotic origin.

In another preferred embodiment, the viruses, the related viral vectors, the viral particles and vaccines belong to the family of paramyxoviruses, in particular measles virus, Newcastle Disease virus, mumps virus and rubella viruses.

In another preferred embodiment, the viruses, the related viral vectors, the viral particles and vaccines belong to the family of birnavirus, in particular Infectious Bursal Disease virus. Recombinant viruses include but are not limited to viral vectors comprising a heterologous gene. In some embodiments, a helper function(s) for replication of the viruses is provided by the host cell, a helper virus, or a helper plasmid. Representative vectors include but are not limited to those that will infect avian or mammalian cells.

The invention also relate to the virus obtained or obtainable by a process of the invention. The instant invention also relates to the vaccine containing the virus of the invention. The process of manufacturing a viral vaccine comprises the process of replicating a virus according to the invention wherein the step of virus harvest is comprising at least one step selected among filtering, concentrating, freezing and stabilizing by addition of stabilizing agent. The virus harvest is performed according to technologies well-known to the man skilled in the art. According to a preferred embodiment, the step of harvesting said virus comprises collecting cell culture supernatant obtained from centrifugation of cell culture, then filtering, concentrating, freezing and stabilizing virus preparation by addition of stabilizing agent. For example, for influenza virus see Furminger, In Nicholson, Webster and Hay (Eds) Textbook of influenza, chapter 24 pp324-332.

The process of manufacturing a viral vaccine according to the invention may also comprise the additional step of inactivation of harvested virus. Inactivation is preferably performed by treatment with formaldehyde, beta-propio lactone, ether, ether and detergent (i.e such as Tween 80™), cetyl-trimethyl ammonium bromide (CTAB) and Triton N102, sodium deoxycholate and tri(N-butyl)phosphate.

According to another embodiment, the invention also relates to a process of preparation of viral antigenic proteins from the virus obtainable by a process of the invention, said process comprises the additional steps of: a) optionally, incubating cell culture supernatant comprising whole virus with a desoxyribonucleic acid restriction enzyme, preferably DNAses (see EC3.1.21 and EC3.1.22 classification) and nucleases (see EC3.1.30 and EC3.1.31 classification). Preferably, DNA digestion enzyme is benzonase

(Benzon nuclease) or DNase I; b) adjunction of cationic detergent. Examples of cationic detergent are; without limitation: cetyl-trimethyl ammonium salt such as CTAB, myristyl-trimethyl ammonium salt, lipofectine, DOTMA and Tween™; c) isolation of antigenic proteins. This latter step may be realized by centrifugation or ultrafiltration.

The virus in the vaccine may be present either as intact virus particles, or as disintegrated virus particles. According to an embodiment, the vaccine is a killed or inactivated vaccine. According to another embodiment, the vaccine is a live attenuated vaccine wherein said vaccines mainly comprises avian cells culture supernatant obtainable by the process of the invention, preferably without serum, optionally filtered and/or concentrated and comprising said virus. According to a third embodiment, the vaccine is comprising viral antigenic proteins obtainable from a virus prepared according to the process of the invention. The invention also pertain to provide a vaccine containing isolated proteins of the virus. The invention also pertains to provide a vaccine comprising infected avian cells, preferably chicken or duck cells derived from PGC, EG or ES obtainable by the process of the invention.

The vaccine of the invention may comprised the virus of the invention in combination with pharmaceutically acceptable substances which increase the immune response. Non limitating examples of substances which increase the immune response comprises complete Freund adjuvant, saponine, aluminium hydroxide salts, lyso lecithin, plutonic polyols, polyanions, peptides, bacilli Calmette-Guerin (BCG) and corynebacterium parvum. Example of synthetic adjuvant is QS-21. In addition, immuno-stimulating proteins (interleukins 111, 112, IL3, IL4, IL 12, ILl 3, granulocyte- macrophage-colony-stimulating factor, ...) may be used to enhance the vaccine immune response.

The vaccine of the invention is preferably a liquid formulation, a frozen preparation, a dehydrated and frozen preparation, optionally adapted to intra-nasal route of administration.

The vaccine of the invention is preferably use for the prophylactic and/or therapeutic treatment of a human or animal infected by a virus preferably chosen among Table 1. More preferably, the vaccine of the invention is preferably use for the prophylactic and/or therapeutic treatment of a human infected by a virus selected among smallpox and influenza, measles, mumps and rubella viruses. The recombinant viral vaccine of the invention may also be used for the prophylactic and/or therapeutic treatment of chronic diseases such as cancer or infectious diseases, such as AIDS.

Table 1

The avian cell lines of the invention, preferably the chicken or duck cell lines derived from PGC, EG or ES cells of the invention are useful to generate and produce re-assorted virus. The virus with a segmented genome, such as influenza virus may be re-assorted. When infecting simultaneously avian cells of the invention with at least two different strains of influenza virus, a mix of segmented genome from two different strains is present in the same host cell. During virus assembly, all combination of genomic segments can theoretically be generated. Specific re-assorted virus may thus be isolated by selecting or eliminating, with an antibody for example, virus with a desired traits (See Kilnourne E. D in Plotkin SA and Mortimer E. A. Eds, Vaccines 1994). The avian cell lines of the invention, preferably the chicken or duck cell lines derived from PGC, EG or ES of the invention are also usefull to generate and produce influenza virus by reverse genetics (See Enami, Proc. Natl. Acad.Sci. USA 87:3802-3805 (1990); Enami et Palese, J. Virol. 65:2511-2513 (1991); Luytjes, Cell 59: 1107-1113 (1989)).

The invention also relates to the diagnostic composition containing viruses of the invention or constituents thereof.

The avian cell lines of the invention, preferably the chicken or duck cell lines derived from PGC, EG or ES cells of the invention are also useful to produce recombinant protein of interest by using techniques known by the man skilled the art. The invention provides a process for production of recombinant protein of interest comprising the steps of: a) transiently or stably genetically modifying the avian cells of the invention, preferably the chicken or duck cells derived from PGC, EG or ES cells of the invention, with at least one expression vector in order to produce a recombinant protein of interest; b) culturing said modified avian cells under suitable conditions and in suitable cell culture medium; and c) harvesting the recombinant product of interest from the avian cells culture, from the suitable medium, or from both.

Examples of proteins of interest that can be advantageously produced by avian cell lines of this invention include, without limitation, cytokines, cytokine receptors, growth factors (e.g. EGF, HER-2, FGF-alpha, FGF-beta, TGF-alpha, TGF-beta, PDGF, IGF-I, IGF-2, NGF), growth factor receptors, including fragment of the protein thereof. Other non-limiting examples include growth hormones (e.g. human growth hormone, bovine growth hormone); insulin (e.g., insulin A chain and insulin B chain), pro-insulin, erythropoietin (EPO), colony stimulating factors (e.g. G-CSF, GM-CSF, M-CSF); interleukins (e.g. IL-I through IL-12); vascular endothelial growth factor (VEGF) and its receptor (VEGF-R), interferons (e.g. IFN-alpha, beta and gamma), tumor necrosis factor (TNF) and their receptors (TNFR-I and TNFR-2), thrombopoietin (TPO), thrombin, brain natriuretic peptide (BNP); clotting factors (e.g. FactorVIII, Factor IX, von Willebrands factor and the like), anti-clotting factors; tissue plasminogen activator (TPA), urokinase, follicle stimulating hormone (FSH), luteinizing hormone (LH), calcitonin, CD proteins (e. g., CD2, CD3, CD4, CD5, CD7, CD8, CDl Ia, CDl Ib, CD18, CD19, CD25, CD33, CD44, CD45, CD71, etc.), CTLA proteins (e.g.CTLA4); T-cell and B-cell receptor proteins, bone morphogenic proteins (BNPs, e.g. BMP-I, BMP-2, BMP-3, etc. ), neurotrophic factors, e.g. bone derived neurotrophic factor (BDNF), neurotrophins, e.g. rennin, rheumatoid factor, RANTES, albumin, relaxin, macrophage inhibitory protein (e.g. MIP-I, MIP-2), viral proteins or antigens, surface membrane proteins, ion channel proteins, enzymes, regulatory proteins, antibodies, immunomodulatory proteins, (e.g. HLA, MHC, the B7 family), homing receptors, transport proteins, superoxide dismutase (SOD), G-protein coupled receptor proteins (GPCRs), neuromodulatory proteins, Alzheimer's Disease associated proteins and peptides, (e.g. A-beta) and others as known in the art. Fusion proteins and polypeptides, chimeric proteins and polypeptides, as well as fragments or portions, or mutants, variants, or analogs of any of the aforementioned proteins and polypeptides are also included among the suitable proteins, polypeptides and peptides that can be produced by the methods of the present invention. In a preferred embodiment, the protein of interest is an antibody. The term "antibody" as used herein refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof. The term "antibody" refers to a homogeneous molecular entity, or a mixture such as a polyclonal serum product made up of a plurality of different molecular entities, and broadly encompasses naturally-occurring forms of antibodies (for example, IgD, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies. The term "antibody" also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatised variants thereof that retains the ability to specifically bind an epitope. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. A monoclonal antibody is capable of selectively binding to a target antigen or epitope. Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, single chain antibodies (scFvs), Fab fragments, F(ab')2 fragments, disulfide- linked Fvs (sdFv) fragments, anti-idiotypic (anti-Id) antibodies, intra-bodies, synthetic antibodies, and epitope-binding fragments of any of the above. The term "antibody" also refers to fusion protein that includes a region equivalent to the Fc region of an immunoglobulin.

The invention also relates to the use of the biological product of interest of the invention produced by the process of the invention as a medicament.

The examples below explain the invention in more detail. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

EXAMPLES Primordial germ Cells arise from the central region of the blastoderm (Ginsburg and

Eyal-Giladi (1987) Development 101 (2):209-219; Karagenc et al. (1996) Dev. Genet. 19(4):290-301; Petitte et al.(1997) Poult.Sci.76(8): 1084-1092. Then they move to an anterior, extra-embryonic site, the germinal crescent until collected by the vasculature between 2.5 and 5 days of embryonic development to reach the germinal ridge. They colonize the germinal ridge where they eventually differentiate into oocytes or spermatocytes. The three main sources of PGC are the germinal crescent, the blood stream at 2.5 days of embryonic development and the gonads at 5.5 days of embryonic development. The germ cells from the germinal crescent and from the blood have the intrinsic potentiality to colonize the gonads. If the culture conditions were appropriate to retain the germ status, the cells from the germinal crescent or from the blood stream would surely be the most promising cells to achieve the germ-line transmission upon engraftment into recipient embryo after in vitro amplification. The number of cells accessible from these two sources is very limited, about 50 cells for the germinal crescent and 200 cells from the blood. The number of cells accessible from the gonads is much more important, about 1500 cells, and, although they have already homed, they still seem to get to potentiality to colonize the gonads when injected in the blood stream of embryos incubated for 2.5 days (Park et al. (2003) Biol. Reprod. 68(5): 1657-1662; Naito et al. (1994) MoI. Reprod.Dev. 68(5): 1657-1662. The respective number of cells in these 3 locations is approximately 50, 200 and 1500 cells. The establishment of a PGC derived stem cell lines requires an access to a large number of cells. For this reason the inventor isolated germ cells from the gonads of chicken embryos incubated for 5.5 days to improve in vitro culture conditions. However, other isolation process of PGC can be envisioned. Materials and methods

1- Isolation ofGonadic Primordial Germ Cells Samples Preparation: - Each egg is broken on 150 mm Petri dish; the embryo is collected in another 150 mm dish in IX Phosphate Buffered Saline buffer (IX PBS);

The head of embryo is discarded with a tweezers and the remaining body is transferred in a 100 mm Petri dish containing IX PBS; Approximatively 10-20 embryos are prepared at the same time. Mesonephros preparation

A sample placed in IX PBS is observed with binoculars.

With fine tweezers, tissues and organs located in the abdomen are cautiously removed.

By squeezing the sample under the mesonephros, the mesonephros is carefully removed.

Gonad dissection:

Gonads are dissected from mesonephros and placed in 1.5 ml of IX PBS. Gonad dissociation

• Perforin 15 secondes of bench top centrifugation

• Add 300 μl of trypsine 0,25 X (pre-warmed at 37°C)

• Incubate at 37°C for 6 minutes • Add 1.3 ml of culture medium supplemented with 10% fetal calf serum (FCS) and antibiotics

• Filtrate through 70 μm

• Centrifuge 400 g for 10 minutes at 4°C

• Discard supernatant and resuspent pellet in 0.5 to ImI of IX PBS. • Centrifuge 400 g for 5 minutes at 4°C

• 400 g 5' 4°C

• Discard supernatant and resuspent cell pellet in 400 ull of IX PBS/10% FCS. Germ cells labeling

• Incubate cell pellet 30 minutes on ice. • Primary antibody:

• Centrifuge cell at 400 g 5 minutes at 4°C; Discard supernatant and resuspend pellet in 400 μl of primary antibody directed against SSEA-I (dilution 1/10); Incubate 30 minutes on ice;

• Secondary antibody: • Centrifuge cell at 400 g 5 minutes at 4°C; Discard supernatant and resuspend pellet in 500 ul of PBS/10% SVF;

• Centrifuge cell at 400 g 5 minutes at 4°C; Discard supernatant;

• Add 400 μl of FITC labelled- secondary antibody / tube (dilution 1/50)

• Incubate 30 minutes on ice in darkness; • Centrifuge cell at 400 g 5 minutes at 4°C; discard supernatant

• Resuspend pellet in 800 μl of PBS/10% SVF;

• Centrifuge cell at 400 g 5 minutes at 4°C; discard supernatant Periodic Acid Schiff labeling

Apply cells on the slide and let them dry one hour under laminar flow hood - Incubate 7 minutes in Periodic acid then rinse the slide with water; Incubate with Schiff reagents during 15 minutes, then rinse 3 minutes with water;

Dry slide and cover with a cover-slip. Results Culture ofgPGC

A process of isolation of PGC by cell sorting, in particular the isolation of gonads from embryos incubated for 5.5 days, as well as the dissociation of the gonads and the labelling of the cells, was established which proved robust since gonad PGC could be reproducibly isolated with an average of 600 cells isolated per gonad (table 2). The isolated PGC are cultured in a presence of a feeder layer in a complete culture medium consisting of a basal synthetic medium complemented with calf foetal serum at a final concentration of about 10 % and with the following growth factors Insulin Growth factor 1 (IGF-I), Ciliary Neurotrophic factor (CNTF), interleukin 6 (IL- 6), interleukin 6 receptor (IL-6R), Stem cell Factor (SCF), Fibroblast Growth Factor type b (bFGF) and interleukin 11 (IL-I l). The concentration of growth factors IGF-I, CNTF, IL-6, IL-6R, SCF, bFGF and IL-I l in the basal medium is about 1 ng/ml. The feeder cells of the feeder layer is constituted of cells from mouse fibroblasts cell line STO (American Type Culture Collection ATCC N⁰CRL- 1503), feeder cells that have been inactivated by irradiation or chemically treated with mitomycin. Deprivation of the exogenous growth factors, feeder cells and animal serum

After around 10-15 passages, the complete supplemented medium is depleted in growth factors.

The growth factor depletion is made in two rounds of depletion: in the first round, SCF, IL6, IL6R, FGF and ILl 1 are directly removed from the complete medium, then the avian cells are maintained in culture for at least one passage in a feeder layer and in a complete medium containing IGFl and CNTF, and supplemented with animal serum. Secondly, IGFl and CNTF are directly removed from the culture medium, which ultimately comprises the basal medium optionally supplemented with animal serum and/or feeder cells. Usually, the medium is totally depleted in growth factors at around passages 20 to 40.

The progressive deprivation of feeder cells is performed after the deprivation of growth factors. The same concentration of the feeder cells is used for 2 to 5 passages, with around 4 xlO⁴ feeder cells/cm², then around 2 x 10⁴ feeder cells/cm², then around 10⁴ feeder cells/cm², then around 0.5x 10⁴ feeder cells/cm², then around 10³ feeder cells/cm², then around 0.5 x 10³ feeder cells/cm², then around 10² feeder cells/cm². Then the flask is seeded with the PGC cells at a concentration about around 10³ cell/cm² to 10⁵ cell/cm² but without feeder cells.

Table 2: Quantitative aspects of PGC isolation from embryonic gonads

Claims

1. Process for obtaining avian cell lines derived from primordial germ cells (PGC) or embryonic germ cells (EG) derived from PGC, said process comprising the steps of: a) isolating avian embryo(s) at a developmental stage comprises between stage

VI of EYAL-GILADI classification and hatching; b) optionally, isolating PGC cells from said avian embryo(s); c) optionally, inducing differentiation of PGC cells comprised in avian embryonic stem cells obtained by dissociating avian embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing avian embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), or culturing EG cells of step c) in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent avian cell lines derived from PGC cells or from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

2. Process for obtaining chicken cell lines derived from PGC, said process comprising the steps of: a) isolating chicken embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification and hatching; b) optionally, isolating PGC cells from said chicken embryo(s); c) culturing chicken embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, e) establishing adherent or non adherent chicken cell lines derived from PGC cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

3. Process for obtaining chicken cell lines derived from embryonic germ cells (EG) derived from PGC, said process comprising the steps of: a) isolating chicken embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification and hatching; b) optionally, isolating PGC cells from said avian embryo(s); c) inducing differentiation of PGC cells comprised in avian embryonic stem cells obtained by dissociating avian embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing EG cells derived from PGC cells in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent avian cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

4. Process for obtaining duck cell lines derived from PGC said process comprising the steps of: a) isolating duck embryo(s) at a developmental stage comprises between stage VI of EYAL-GILADI classification and hatching; b) optionally, isolating PGC cells from said duck embryo(s); c) culturing duck embryonic stem cells comprising PGC cells obtained by dissociating embryo(s) of step a) or culturing PGC cells isolated in step b), in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; d) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, e) establishing adherent or non adherent duck cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

5. Process for obtaining duck cell lines derived from embryonic germ cells (EG) derived from PGC, said process comprising the steps of: a) isolating duck embryo(s) at a developmental stage comprises between stage

VI of EYAL-GILADI classification and hatching; b) optionally, isolating PGC cells from said duck embryo(s); c) inducing differentiation of PGC cells comprised in duck embryonic stem cells obtained by dissociating duck embryo(s) of step a) or inducing differentiation of PGC cells isolated in step b) into EG cells; d) culturing EG cells derived from PGC cells in a complete culture medium supplemented with animal serum and containing exogenous growth factors allowing their growth on a feeder layer, and; e) passage by modifying the culture medium so as to obtain a progressive withdrawal of said growth factors, of the animal serum and of the feeder layer, f) establishing adherent or non adherent duck cell lines derived from PGC derived EG cells capable of proliferating in a basal medium in the absence of growth factors and feeder layer and containing a low level of animal serum or no animal serum.

6. The process according to claims 1 to 5 wherein the complete culture medium of step a) is a basal cell culture medium supplemented with at least one growth factor selected in the group consisting of: Leukemia Inhibitory factor (LIF), Insulin Growth factor 1 (IGF-I), Ciliary Neurotrophic factor (CNTF), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), Stem cell Factor (SCF), Fibroblast Growth Factor (FGF), interleukin 11 (IL-

11).

7. The process according to claim 6 wherein the complete culture medium of step a) is a basal cell culture medium supplemented with at least IGF-I and CNTF.

8. The process according to claim 6 wherein the complete culture medium of step a) is a basal cell culture medium supplemented with at least IGF-I, CNTF, SCF and FGF.

9. The process according to claim 6 wherein the complete culture medium of step a) is a basal cell culture medium supplemented with at least IGF-I, CNTF, SCF, FGF, IL-6 and IL-6R.

10. The process according to claims 1 to 9 wherein the sequence of the modification of the culture medium is "progressive withdrawal of growth factors / progressive withdrawal of feeder layer / progressive withdrawal of serum".

11. Avian cell lines derived from stem cells obtainable by the process according to claim 1 to 10 wherein cells of said avian cell lines have at least one of the following characteristics: a high nucleo-cytoplasmic ratio, an endogenous alkaline phosphatase activity, - an endogenous telomerase activity, an expression of cellular markers selected in the group comprising SSEA-I (TECOl), EMA-I, DAZL, VASA.

12. A process for replicating viruses comprising the steps of inoculating avian cells according to claims 11 with virus and culturing said cells in a culture medium until viral replication cells occurs and newly virus particules are produced.

13. The process according to claim 12, wherein said virus is selected from the group consisting of adenoviruses, hepadnaviruses, herpes viruses, orthomyxoviruses, papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses and retroviruses.

14. The process according to claim 13 wherein the virus is a naturally occuring poxvirus or a recombinant poxvirus selected among the group consisting of Modified Vaccinia Ankara (MVA) virus, Fowl pox virus, canary pox virus (i.e ALVAC), juncopox virus, mynah pox virus, pigeonpox virus, psittacine pox virus, quail pox virus, sparrow poxvirus, starling pox virus, turkey pox virus.

15. The process according to claim 13 wherein the virus is a naturally occuring paramyxovirus or a recombinant paramyxovirus selected among the group consisting of measles virus, mumps virus, rubella virus and Newcastle disease virus.

16. The process according to claim 13 wherein the virus is selected among human influenza virus, avian influenza virus, swine influenza virus, equine influenza virus, feline influenza virus.

17. A virus obtained by a process as claimed in one of claims 12 to 16.

18. A vaccine containing virus as claimed in claim 17, if appropriate in combination with substances which increase the immune response.

19. A vaccine containing viral antigenic proteins obtainable from a virus as claimed in claim 17, if appropriate in combination with substances which increase the immune response.

20. A vaccine comprising avian cells derived from PGC or PGC derived EG as claimed in claim 11 and infected with a virus.

21. A process for production of recombinant protein of interest comprising the steps of: a) transiently or stably genetically modifying an avian cell line as claimed in claim 11 with at least one expression vector in order to produce a recombinant protein of interest; b) culturing said modified avian cells under suitable conditions and in suitable cell culture medium; and c) harvesting the recombinant product of interest from the avian cells, from the suitable medium, or from both.

22. The process of claim 21 wherein the recombinant protein of interest is an antibody molecule or a fragment thereof.

23. The protein of interest produced by a process according to claims 21 and 22 as a medicament.