Title of Invention

"A METHOD FOR IN VITRO REPLICATING RECOMBINANT VACCINIA VIRUS"

Abstract A method for in vitro replicating recombinant vaccinia virus comprising the steps of: A) inoculating adherent or non-adherent cells from an avian cell line with viral particles of said vaccinia virus wherein said avian cell line is obtained by a process consisting of: a) culturing avian embryonic cells in a complete culture medium, said complete culture medium comprising: a basal medium selected from the group of basal media consisting of BMR (basal Eagle Medium), MEM (minimum Eagle Medium), medium 190, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 and Ham-FlO, Iscove's Modified Dulbecco's medium, MacCoy's 5A medium, RPMI 1640; and complemented in fetal calf serum at a concentration of 12 to 8 % and containing the growth factors comprising the trophic factors SCF, IGF- 1 and bFGF, and the cytokines selected from the group of cytokines consisting of interleukin 6, interleukin 6 receptor and, optionally interleukin 11; and an inactivated feeder layer of STO cells, b) after about twenty passages, the culture medium is modified: by gradual withdrawal of said growth factors and of said cytokine; and by gradual withdrawal of the feeder layer; and by decreasing the fetal calf serum concentration until small proportions of 5% are reached, c) the adherent or non-adherent cell lines obtained being capable of proliferating in a basal medium in the absence of exogenous growth factors and cytokine, serum and the inactivated feeder layer, the non adherent cell lines being obtained by sowing at least 1 x 106 cells/mL in a bacteriological dish followed by several passages with simple dilution. B) culturing said inoculated cells in a basal medium until cells lysis occurs and newly produced viral particles of said vaccinia virus are released in said medium.
Full Text Avian cell culturing in serum-free medium
The present invention relates to a method for producing avian cells, preferably avian stem cells, which are capable of growing in a serum-free medium as well as culturing avian stem or primary cells in a serum-free medium. The invention further relates to the use of these cultures for manufacturing vaccines, in particular a vaccine against smallpox and influenza, and to the production of antibodies and recombinant proteins.
For several decades, various cell types of diverse origin have been used for producing vaccines and recombinant molecules of therapeutic value such as proteins and in particular antibodies. These cells are of human, simian, canine, bovine or avian origin. Upon culturing, these cells have generally undergone alterations in their genome, converting them and providing them with specific properties such as immortality. Viral infections and spontaneous mutations in the key loci for the control of the cell cycle are amongst the best known immortalization events. As an example, the most used lines include CHO (Chinese Hamster Ovary), HEK 293 (Human Embryonic Kidney), Vero (Vervet Origin), MDCK (Madin-Darby canine kidney), BHK21 (Baby Syrian Hamster Kidney), Hela (derived from cervical carcinoma), COS7 (Monkey Kidney), HEP2 (human epithelial like larynx carcinoma cells), MDBK (Bovine Kidney) and MRC5 (Fetal lung fibroblast), and QT (quail cells). Avian stem cells of the fibroblastic type (CEF, Chicken Embryonic Fibroblasts) are most generally used for producing vaccines such as influenza vaccine.
These various cells useful for producing vaccines and therapeutic molecules are likely to expose indirectly the future patient to various biological hazards due to in vitro culturing conditions. In fact, the cells are generally maintained in commercially available traditional culture media which require to be supplemented with animal serum, such as foetal calf serum, whether adult or new-born. Further, they can contain lyophilysates or hydrolysates of animal, plant or other origin.
Serum is an outstanding nutrient source for a cell. It provides minerals, vitamins, protection and growth factors, hormones, and various metabolites such as cholesterol, albumin, transferrin, and binding factors. However, it has many disadvantages or drawbacks in use. More specifically, for pharmaceutical companies:
its price is high

the variability between batches related to biological variability of donating animals is high
it is a mixture which is not chemically determined
the presence of animal proteins in the production of recombinant proteins makes the purification process more intricate,
the risks of transmission of pathogenic agents such as bacteria, fungi, viruses and prion (BSE) are not insignificant.
Following the discovery in the 1980's of the prion's involvement in the aetiology of the Bovine Spongiform Encephalopathy (BSE), the health authorities responded and published very stringent procedures and guidance relating to the use of cell culture media containing animal-derived components, such as serum or certain protein hydrolysates, in the various vaccine and recombinant molecule products. Traceability of the materials used (origin, production route, analysis certificates) has become an absolute necessity. Accordingly, constraints related to cultures and to production of vaccines and molecules for therapeutic purposes have increased for pharmaceutical companies. Health authorities currently put a lot of pressure aiming towards improving culturing conditions during establishment of cell lines and their amplification. The tendency is to remove completely any animal products or products derived in these media.
Accordingly, cell media containing no animal elements are favoured. Furthermore, the sera used preferably come from countries having no BSE occurrence (USA, Australia, New-Zealand, and until very recently Canada). The serum is analysed before use for the presence of any known and recorded pathogen (bacteria, fungi, mollicutes, and viruses...). In order to prevent any viral contamination before use, the sera are further irradiated at very high doses (several tens of kiloGrey).
Consequently, manufacturers of culture media develop culture media which are optimized for one cell type (e.g., HEK 293, CHO, Vero, MDCK.) which do not require serum supplementation. These media are known as serum-free. Now, the vast majority of cell lines are not capable of being cultured in the absence of serum on serum-free media. Transferring a culture from a traditional medium supplemented with serum to a serum-free medium is a delicate operation and requires adaptation and selection work which is not straightforward.
The use of these serum-free media causes various stresses to the cells being cultured. Thus, the cells undergo a stress related to adaptation or habituation to the aew culturing conditions. This adaptation time usually generates a high death rate

and a high selection of the population. Also, the cells being cultured undergo a physical stress caused by agitation of the cultures, as a consequence of shear forces which are a lot greater in serum-free media containing few or no BSA (bovine serum albumin); this protein is indeed known to solidify the membrane and thus protect the cells from the shear forces intended to facilitate the cell agitation and transportation. In general, BSA can be replaced by a substitute such as pluronic acid F68 or polyethylene glycol. Further, for adherent cells, a problem of binding to the support has to be pointed out, with the binding factors being in general provided by the serum in traditional culture media.
Now, as far as we know, no avian cell line has been maintained or amplified in culture on such serum-free media. The object of the present invention is to develop avian stem cell models which are capable of proliferating for an extended period, both in suspension or adherent, without adding feeder cells, growth factors and/or serum, in a serum-free culture medium. The present invention accordingly provides a method for adapting avian stem cells to serum-free media. More specifically, the invention relates to a method for directly adapting avian stem cells to serum-free media, for carrying out adaptation over several passages for numerous serum-free media, or for carrying out such adaptation at an early stage on avian stem cells which a still dependent on the feeder layer. The inventor has further shown that it is possible to maintain the avian stem cells in culture for an extended period in a serum-free medium and to carry out cell freeze-thaw cycles in such serum-free media.
The present invention also relates to avian stem cell lines, avian primary cell lines, avian lines, either differentiated or undifferentiated with an ability to grow during long periods in serum-free media. These cells according to the invention are an original and efficient support for the production of protein molecules, in particular recombinant proteins for therapeutic purposes such as antibodies, or vaccines [vaccinia virus (EP application No. 03291813.8), influenza virus and the like) due to their avian origin (WO03/076601). The cell lines according to the invention are advantageous in that they have not undergone an immortalization process by site directed, viral, or chemical mutagenesis, or other processes. They are derived by selection of a population under specific culture and adaptation conditions described below and optimized so that the cells can be employed in various production methods, both in suspension or adherent. Avian stem cells according to the invention have the ability to achieve a high number of culture passages without undergoing a senescence process, and thus generate a high biomass production. The method according to the invention provides cell densities in serum-free media

which are similar to those obtained in traditional media requiring serum supplementation.
The cells of the invention are therefore intended to replace cell lines currently employed for various vaccines or peptides or recombinant proteins because they have an unquestionable advantage in term of regulation in view of their ability to grow in serum-free media exempt from animal-derived compounds. The inventor has thus tested a variety of serum-free media commercially available from various specialized companies.
Finally, the inventor has demonstrated that avian stem cells according to the invention are likely to differentiate massively and homogeneously by using particular serum-free media. The invention therefore also relates to differentiation methods and to the use of these serum-free media in the presence of specific inducers to induce avian stem cell differentiation.
DESCRIPTION
The present invention relates to a method for producing avian cell lines which are capable of growing in a serum-free medium, characterised in mat it comprises the following steps:
a) culturing avian cells in a traditional medium supplemented with serum and containing all factors supporting cellular growth. Preferably me cell culture is made on a mat of inactivated feeder cells (feeder layer),
b) providing at least one culture passage by modifying the culture medium so as to obtain gradual or total withdrawal of the serum,
c) establishing adherent or non-adherent cell lines capable of proliferating in a basal medium in the absence of serum.
More specifically, the present invention relates to a method for adapting avian cells to a serum-free medium characterised in that it comprises the steps of:
a) culturing the avian cells in a traditional culture medium supplemented with serum;
b) exchanging the traditional medium from step a) with a culture medium selected from the group consisting of
• a traditional medium (i) supplemented with serum and diluted with a serum-free medium, followed by culturing by sequential passages said avian cells in the medium (i) wherein the level of serum-free medium is

gradually increased until the traditional medium supplemented with serum has completely disappeared (gradual dilution);
• a serum-free medium supplemented with serum (ii), followed by culturing by sequential passages said avian cells in the medium (ii) wherein the level of serum is gradually decreased until a serum-free medium is obtained (gradual withdrawal);
• a serum-free medium (iii), followed by culturing said avian cells in the medium (iii) (direct passage);
c) maintaining in culture in a serum-free medium said avian cells which have adapted to the change of medium and which have been selected.
The avian cells according to the invention are cells selected from the group consisting of stem or primary avian cells, adherent or proliferating in suspension. The avian stem cells are totipotent or pluripotent cells.
"Traditional medium" is intended to refers to any basal medium requiring for its use at least one supplementation with foetal calf serum. These media include, but are not limited to, DMEM (Dulbecco's Modified Eagle Medium and derivatives thereof such as DMEM-F12, HAM-F10, HAM-F12, Iscove's Modified Dulbecco Medium, McCoy's 5A, medium 199, MEM, GMEM (Glasgow Minimum Essential Medium), and RPMI1640. Optionally, according to the type of cells cultured with this traditional medium, it may further be necessary to supplement the medium with at least one of the following compounds: L-glutamine, sodium pyruvate, beta mercaptoethanol, amino acids, vitamins, and recombinant growth factors.
Avian cells, including avian stem cells, may be cultured in step a) in a traditional medium such as described in [Pain B et al. (1996), Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities; Development 122:2339-2348], EP 787 180 and US 6,114,168. The medium disclosed in US 5,340,740, US 5,656,479 and US 5,830,510 can also be used.
"Serum-free medium" relates to a ready-for-use medium, i.e. not requiring a serum addition, capable of supporting cell growth and of maintaining them in culture (SFM - Serum-Free Medium). This medium does not have to be chemically defined, can contain hydrolysates of various origins, of plant origin for example. Preferably, these are known as "non-animal origin" media, i.e. do not contain animal or human-derived elements (FAO - free of animal origin - status). In these media, the serum native proteins are replaced by recombinant proteins

synthetically produced or derived from recombinant sources. These proteins include for example insulin and/or transferrin. Some serum-free media do not contain a protein (PF - protein free - media) and/or are chemically defined (CDM - chemically defined media). As indicated by their name, the former contain no protein. They have been primarily developed for producing recombinant proteins or antibodies and facilitate the purification step. The latter have a fully defined composition, with all the component molecules having a perfectly known chemical structure.
The advantages of these media are numerous since these media are exempt of serum, which prevents any biological hazards of zoonosis related to the use of serum (exposition hazards to BSE and viruses), and they provide a better production quality related to a better traceability and an optimised purification. Furthermore, there is less variability from batch to batch of media since they are better defined (SFM and PF media) or fully defined (CDM media).
The method for producing and culturing avian cells capable of growing in a serum-free medium according to the invention further comprises the step of d) isolating (cloning) one or more avian cells, followed by culturing said isolated avian cells in a serum-free medium. It is within the scope of the invention to isolate, for example by limiting dilution, an avian cell obtained by the method of the invention in order to have a clone population of avian cells capable of growing in a serum-free medium. Alternatively, step d) according to the invention may consist in isolating several avian cells capable of growing in a serum-free medium in order to enrich the population of avian cells obtained by the method of the invention with cells having particular or superior capabilities or characteristics.
According to a first embodiment, the method according to the invention is characterised in that the culture medium (i) comprises 10% to 60% of serum-free medium and respectively 90% to 40% of traditional culture medium, preferably from 25% to 50% of serum-free medium, respectively 75% to 50% of traditional culture medium. Also, the method according to the invention is characterised in that the culture medium (ii) comprises between 2% and 7.5% of serum, preferably 3%.
The decision as to whether increase the level of serum-free medium in the culture medium (i) or reduce the level of serum in the culture medium (ii) is dependent on the rate of cell proliferation and the morphology of avian cells in culture. When the cells show adaptation problems to the culture medium (i), (ii) or (iii), or adaptation

problems to medium (i) in which the level of serum-free medium has been increased, or to medium (ii) in which the level of serum has been decreased, upon sequential culture passages, said cells are maintained over several passages in the culture medium in which the cells show adaptation problems, before the dilution or withdrawal process is resumed. In fact, the cells during the process must keep their ability to undergo a division at least once every 48 hours. The presence of mitotic figure is essential. The cell morphology should not be close to those of vacuolated, necrosed, apoptotic or senescent cells. For instance, an abrupt decrease of the number of living cells (mortality rate observed to be more than about 50%) indicates that it is necessary to wait for one or two additional passages without modifying the medium or with a less sensitive decrease of the serum concentration.
According to another preferred embodiment of the invention, adaptation of avian cells to serum-free media, in particular avian stem cells, adherent or proliferating in suspension, is carried out by complete withdrawal in a single step, without a prior dilution step or gradual serum withdrawal step. Preferably, the cells are cultured in a traditional medium comprising from 2 to 3% of serum before direct passage in serum-free medium.
The method according to the invention can further comprise a step of engaging differentiation of avian stem cells, adherent or proliferating in suspension, which comprises adding to the serum-free medium at least one inducer selected from the group consisting of global inducers retinoic acid and dimethyl sulfoxide (DMSO) or specific inducers, including EGF, bFGF, NGF, TNF, IL6, SCF, IL11 and CNTF. The differentiated avian stem cell lines resulting from the avian cells according to the invention are also provided by the invention. This differentiated cells can also be precursor cells, which correspond to cells from an adult or embryonic tissue partially differentiated (by contrast with a totipotent stem cell) and which are again capable of undergoing division and differentiation. These differentiated cells can be characterised by the expression of specific molecular labels. "Differentiated cell" refers to any cell of a specialised adult or embryonic tissue exhibiting specific labels or assuming specific physiological functions. It is possible, in one particular aspect of the invention, especially for particular isolates or clones derived from a particular isolate obtained during the establishing operation, that these stem cells can contribute to the germinal cell line. In this case, these stem cells established as cell lines would be embryonic stem cells.
The process according to the invention can thus lead to the establishment of cell lines. For the purpose of the invention, "establishment of a line" is understood to

refer to maintaining the cells in an in vitro culture over a considerable period of time. Advantageously, the cells derived from the lines 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. Hence, these lines are considered as being able to grow indefinitely in a serum-free medium. The term "line" is understood to mean any population of cells capable of proliferating indefinitely in an in vitro culture while retaining to a greater or lesser degree the same morphological and phenotypic characteristics.
In a second aspect, the invention relates to the use of serum-free culture medium optionally comprising one or more growth factors for culturing and freezing avian stem or primary cells, both adherent or non-adherent. In fact, it has been shown that the above-mentioned lines can be amplified under the above conditions, freezed without serum and recultured in a serum-free medium.
In a third aspect, the invention relates to a method for producing live or attenuated vaccines, viruses or viral particles, peptides or recombinant proteins, preferably for therapeutic purposes, such as antibodies, characterised in mat it comprises culturing avian cells in a serum-free medium according to the invention.
Accordingly, cells derived from established lines can be genetically modified in a stable or transitional way by means of techniques known to the person skilled in the art. Thus for example to produce a peptide or a recombinant protein of interest, such as an antibody, the cells according to the invention can be genetically modified in a stable way by homologous recombination or by insertion transgenesis. The recombination, insertion and/or expression vectors that enable to carry out these genetic modifications are well-known to the person skilled in the art. Examples include retrovirus vectors or minichromosomes.
More specifically, the method according to the invention for producing live or attenuated viruses or viral particles, or any of their recombinant derivatives, further comprises:
inoculating said avian cells with said virus or viral particles with an m.o.i. (multiplicity of infection) ratio preferably in the range of between 0.01 and 0.5, and
culturing said cells in a serum-free medium until the cellular lysis and the release of new viruses or new viral particles occurs in the culture medium.

Avian cells according to the invention are preferably capable to support the replication of live or attenuated viruses, whether they are recombinant or not. Nevertheless, the cells according to the invention can be transitionally or stably modified in time by introducing or expressing the one or more components required to achieve the complete viral cycle of the virus within the cell. Accordingly, for example cells can be modified so as to over-express the virus receptor on the cell surface.
Said virus or viral particle, or a recombinant derivative thereof, is selected from the group consisting of adenoviruses, hepadnaviruses, herpesviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses and retroviruses. More specifically, the poxvirus is an avipoxvirus, preferably selected from the group consisting of fowl poxvirus, junco poxvirus, mynah poxvirus, pigeon poxvirus, canary poxvirus, psittacine poxvirus, quail poxvirus, sparrow poxvirus, starling poxvirus and turkey poxvirus. According to a more preferred embodiment, the poxvirus is selected from the group consisting of native or recombinant vaccinia virus and smallpox virus. In the case of vaccinia, it is preferably the strain Modified Vaccinia virus Ankara (MVA) (ATCC No. VR-1508).
According to another preferred embodiment, the orthomyxovirus is influenza virus. According to another preferred embodiment, the paramyxovirus is selected from the group consisting of measles virus, mumps virus, and rubella virus.
One of the objects of the present invention also relates to the peptide or recombinant protein obtained by carrying out the production method according to the invention. Such a peptide or protein can form an antigen specific to a viral or bacterial disease, or form an antigenic label for a tumor disease. As such, it can be used as a vaccine. Alternatively the recombinant protein is a humanised or human monoclonal antibody.
One of the objects of the present invention further relates to the anti-infection vaccine obtained by carrying out the production method according to the invention or comprising a peptide or a protein according to the invention. Preferably it is a vaccine against influenza or a vaccine against smallpox.
The present invention also relates to a cell culture comprising stem, primary or differentiated, adherent or non-adherent, avian cells, and a serum-free medium.

Preferably, this cell culture has a cell density in serum-free medium equal to or higher than lxl06 cells/ml.
The invention also relates to the use of said cell culture for the production by avian stem cells, adherent or proliferating in suspension in a serum-free medium, of vaccines, live or attenuated viruses, or recombinant viral particles, peptides or recombinant proteins, preferably for therapeutic purposes such as antibodies.
Non limiting examples of embodiments of the invention are given herein below.
FIGURES
Figures 1A to 1K: Photographs illustrating adherent avian stem cells S86N 45 at late passages (between p104 and p1 42) taken after 48 hours of contact with various serum-free media, supplemented with serum.
Figures 2A to 2F: Adherent avian stem cells S86N 45 at late passages (between pl04 and pl42) after 48 hours of incubation in the presence of the control medium (HAM-F12 0% FCS) or various serum-free medium media.
Figures 3A to 3E: Example of differentiation induced by serum-free media on avian stem cells. 4 phenotypes are presented by way of an example of massive and quick induction.
Figures 4A to 4D: Morphology of avian stem cells Valo B at passage 15 under various conditions. Photograph 4A illustrates the morphology of Valo B pi 5 cells, after 48 hours of culture in the control medium (DMEM-F12 min 10% FCS). Photograph 4B illustrates the same cells after 72 hours of incubation in the control medium, without serum. Photographs 4C and 4D show the morphology of Valo B cells and the density obtained after incubation for 72 hours in two serum-free media.
Figures 5A to C: Example of differentiation induced by two serum-free media on Valo B cells after 48 hours of incubation
Photograph 5 A illustrates the morphology of early stem cells Valo B. Photographs 5B and 5C demonstrate a very clear morphology change of the cells upon contact with serum-free media, as used herein after having been supplemented with 10% FCS.

EXAMPLES
MATERIALS AND METHODS:
• The cells used: EB1 and EB14 cells:
Avian stem cells derived from chick embryos of the strain S86N, maintained in long term culture and proliferating in suspension. These cells are cultured in various commercial media (preferably DMEM-F12, McCoy's 5A, HAMF12) supplemented with 10% foetal calf serum (FCS), with glutamine (2mM), with amino acids 1%, with vitamins 1%, with sodium pyruvate ImM, and with beta mercaptoethanol (1mM). The technique for obtaining these cells has been previously disclosed (WO03/076601 and FR02/02945).
S86N 45 and S86N16 cells:
Avian stem cells derived from chick embryos of the strain S86N, maintained in long term culture beyond 140 passages. These adherent cells are independent for their proliferation of specific factors and feeder. The technique for obtaining these cells has been previously disclosed (WO03/076601 and FR02/02945).
ValoB cells:
Adherent avian stem cells derived from chick embryos of the strain White Legom, maintained in long term culture in the presence of factors and a feeder layer. They are used at passage 12 and 15. Their growth is charactarised by their dependence to IGF1 (Insulin Growth Factor 1), CNTF (Ciliary Neurotrophic Factor) and feeder. These cells have been developed according to the technique disclosed in the documents WO03/076601 and FR02/02945.
STO cells:
Line of mouse fibroblasts (CRL-1503, from ATCC). These cells are used after irradiation (45 grey) as feeder cells. The irradiation enables to stop definitely the cell cycle so as to prevent it from being invaded by STO cells from co-cultures of avian stem cells. These irradiated cells secrete amongst others growth factors which are important for maintaining in culture the culturing strains (Robertson, 1987), together with an extracellular matrix assisting cell adhesion. The irradiated STO feeder is seeded 24 hours prior to use of the 6-well plates used in our tests. 0.2 x 106 cells per well are seeded under 2 ml.
• The commercial traditional media used:

Culture medium for supporting S86N 45 cells: HamF12, supplemented with 10% FCS, with glutamine 2mM, with amino acids 1%, with vitamins 1%, with sodium pyruvate1mM, with beta mercaptoethanol ImM.
Culture medium for supporting ValoB cells: DMEM F12, supplemented with 10% FCS, with glutamine 2mM, with amino acids 1%, with vitamins 1%, with sodium pyruvate 1mM, with beta mercaptoethanol 1mM, 1 ng/mL 1GF1 and 1 ng/mL CNTF. This medium is named DMEMF12 min, 10% FCS.
Culture medium for STO cells: DMEM, supplemented with 4% FCS and with glutamine 2mM.
The avian stem cells are maintained at 39°C at 7.5% of CO2 and the STO cells are maintained at 37°C, at 7.5% of C02.
• The serum-free media used:
The serum-free media tested are supplied by various companies specializing in the
field of cell culture. They include:
Company InVitrogen: VP SFM (Ref. 11681-020, catalog 2003), Opti Pro (Ref.
12309-019, catalog 2003), Episerf (Ref. 10732-022, catalog 2003);
Company Cambrex: Pro 293 S-CDM (Ref. 12765Q, catalog 2003), LCI 7 (Ref.
BESP302Q, ex-catalog), Pro CHO 5-CDM (Ref. 12-766Q, catalog 2003):
Company Hyclone: HyQ SFM4CHO (Ref. SH30515-02), HyQ SFM4CHO-Utility
(Ref. SH30516.02), HyQ PF293 (Ref. SH30356.02) HyQ PF Vero (Ref.
SH30352.02);
Company JRH Biosciences: Ex cell 293 medium (Ref. 14570-1000M), Ex cell 325
PF CHO Protein free medium (Ref. 14335-1000M), Ex cell VPRO medium (Ref.
14560-1000M), Ex cell 302 serum free medium (Ref. 14312-1000M).
They were all used without any supplementation except for the supplementations with serum described in the Examples.
EXAMPLE 1: PROCESS FOR PRODUCING AN AVIAN CELL CULTURE IN A SERUM.-FREB MEDIUM
L Results
Some serum-free media developed for specific lines which are neither stem cells nor avian-derived cells are not toxic for avian stem cells (see Figure 1).

S86N 45 cells are seeded in 6-well plates under various conditions. 0.2 x 106 cells per well are seeded under 5 ml of control medium (medium HAM-F12 10% FCS for maintenance) or in various serum-free media supplemented with 10% FCS (see Figures 1A to 1K). No adaptation to these serum-free media, i.e. SFM, PF or CDM, was carried out. The cells were directly seeded into them. The media were changed every day.
The assessment of the serum-free media takes into account the cell viability at 24 h and at 48 h after seeding, their morphology and their density.
Out of the 14 media tested, 11 provide a good proliferation of the avian cells (see Table I). Figures 1A to 1K illustrate the results obtained with some of them. Three serum-free media were found to induce a very high mortality of the avian stem cells, even when they are supplemented with 10% serum. The toxicity of these media is so high that virtually no cell remains in the culture wells after 2 days of culture.
It was also found that globally the proliferation of adherent avian stem cells is at least equal to the one obtained in the control medium (medium HAMF12 supplemented with serum and additives (see Materials and Methods). When these serum-free media are supplemented with 5% FCS in place of 10%, no significant difference in proliferation can be seen. On the contrary, if the control medium is only supplemented with 5% serum, the proliferation is slowed down during the time required for adaptation.
Table I: Report of the results obtained with various serum-free media supplemented with serum, in terms of proliferation and morphology. These results were obtained on S86N 45 cells.
(Table Removed)


+++: proliferation greater than the traditional basal medium specifically
supplemented
++: proliferation equivalent to the one obtained in the traditional basal medium
specifically supplemented
+: proliferation lower than the one obtained in the traditional basal medium
specifically supplemented
EXAMPLE 2: PRODUCTION OF ADHERENT AVIAN STEM CELL CULTURES IN A SERUM-FREE MEDIUM FROM AVIAN STEM CELLS AT LATE PASSAGES
We have shown that adherent avian stem cells at late passages can proliferate in certain serum-free media without added serum and without adaptation (Figures 2B to 2F). They do not proliferate at all in the serum-free control medium in which all cells died after 48 hours (Figure 2A).
The S86N 45 cells are seeded in 6-well plates under various conditions. 0.2 x 106 cells per well are seeded under 5 ml of control medium (medium HAMF12 without added serum) or in various serum-free media with no added culture supplement. No adaptation to these serum-free media, i.e. SFM, PF or CDM, was carried out. The cells were directly seeded into them. The media were changed every day. The assessment of the serum-free media takes into account the cell viability at 24 h and at 48 h after seeding, their morphology and their density.

It was thus shown that out of the 14 serum-free media tested, 5 of them provide, without any adaptation, cell growth over 48 hours and maintenance in a serum-free culture.
EXAMPLE 3: INDUCTION OF THE DIFFERENTIATION OF THE AVIAN CELLS OF EXAMPLE 2 IN A SERUM-FREE MEDIUM
We have shown that adherent avian stem cells at late passages can be induced rapidly, in a massive and homogeneous way, in various differentiation routes by certain serum-free media in the presence or in the absence of serum (Figure 3).
This was evidenced by means of assessment tests carried out in various serum-free media available.
The S86N 45 cells are seeded in 6-well plates under various conditions. 0.2 x 106 cells per well are seeded under 5 ml of control medium (medium HAM-F12 supplemented with serum) or in various serum-free media with no added culture supplement or in the presence of 10% serum (see Figure 3). No adaptation to these serum-free media, i.e. SFM, PF or CDM, was carried out. The cells were directly seeded into them. The media were changed every day. The morphology of the cells is noted each day after seeding. Marked differences were observed by 24 hours of culture. The various phenotypes obtained are illustrated in Figures 3B to 3E. The reference morphology of the non-differentiated avian stem cells at late passage is represented in Figure 3 A.
The main differentiation phenomena reported include, by way of example:
* retraction of the short cytoplasmic extensions characteristic of our avian stem cells (see Figure 3B),
* occurrence of outgrowths of the "neurite" type (see Figure 3C)
* change of the cell shape to more cubical (Figure 3E)
* lengthening and spreading of the cells (Figure 3D).
EXAMPLE 4: PRODUCTION OF ADHERENT AVIAN STEM CELL CULTURES IN A SERUM-FREE MEDIUM FROM AVIAN STEM CELLS AT EARLY PASSAGE
We have shown that adherent avian stem cells at early passage can proliferate in certain serum-free media with or without added serum and without adaptation (Figures 4C and 4D). They do not proliferate at all in the serum-free control medium in which all cells died after 72 hours of culture (Figure 4B).

15-passage Valo B cells are seeded in 6-well plates under various conditions. These 6-well plates were prepared at least 24 hours prior to use, with each well being seeded with 0.2 x 106 STO cells irradiated at 45 grey. At DO, 0.2 x 106 early stem cells per well are seeded under 5 ml of HAMF12 control medium (supplemented with factors and supplemented or non-supplemented with serum), or in various serum-free media supplemented or non-supplemented with 10% FCS. No adaptation to these serum-free media, i.e. SFM, PF or CDM, was carried out. The cells were directly seeded into them. The media are changed every day.
The assessment of the serum-free media takes into account the cell viability at 24, 48 and 72 hours after seeding, their morphology and their density.
As for S86N 45 avian cells, it was shown that the early avian stem cells could proliferate in serum-free media non-supplemented with serum. However some are very toxic. These medium assessments resulted in showing the possibility to culture early avian stem cells directly with no adaptation in certain serum-free media (Figures 4C and 4D), without supplementation with serum.
EXAMPLE 5; INDUCTION OF THE DIFFERENTIATION OF THE AVIAN CELLS OF EXAMPLE 4 IN A SERUM -FREE MEDIUM
Early avian stem cells are induced by certain serum-free media in a plurality of differentiation routes (see Figures 5).
12-passage Valo B cells are seeded in 6-well plates under various conditions. These 6-well plates were prepared at least 24 hours prior to use, with each well being seeded with 0.2 x 106 STO cells irradiated at 45 grey. At DO, 0.2 x 106 early avian stem cells per well are seeded under 5 ml of HAMF12 control medium (supplemented with factors and supplemented or non-supplemented with serum), or in various serum-free media supplemented or non-supplemented with 10% FCS. No adaptation to these serum-free media, i.e. SFM, PF or CDM, was carried out. The cells were directly seeded into them. The media are changed every day.
It was shown that certain serum-free media in the presence of serum can induce massive and homogeneous differentiations of the Valo B p12 avian stem cells (Figures 5B and C). This differentiation induction also occurs in the absence of serum (Figure 4C).

EXAMPLE 6: GRADUAL ADAPTATION OF AVIAN STEM CELLS TO SERUM-FREE MEDIA
Avian stem cells, adherent or proliferating in suspension, whether late-passage or early-passage, can be adapted to various serum-free media over several passages. Such adaptation can be carried out by any one of the following two distinct procedures:
1- Gradual dilution of the control medium (in general supplemented in serum, gltamine, vitamins, sodium pyruvate, non-essential amino acids, beta mercapto-ethanol, and optionally factors) in serum-free medium: this technique is known as gradual dilution.
2- Direct passage into serum-free medium, supplemented with serum, followed with a gradual serum withdrawal: this technique is known as gradual withdrawal.
Both techniques provide, after a gradual withdrawal carried out over 2 to 3 passages or more, avian stem cells, both adherent or in suspension, which proliferate in the serum-free medium.
6.1 Examples of adaptation by gradual dilution:
TO: means the number of passages undergone by the line before the start of the
adaptation.
In this process the serum-free medium used is not supplemented with serum prior
to use.
TO Passage: the avian stem cells , both adherent or in suspension, are cultured in a mixture of 75% control medium/25% serum-free medium. The medium is changed at the same ratio every day for adherent cells. As regards to the suspension cells, medium is added to the cultures at the same ratio.
T+l Passage: after dissociation of the adherent cells (or dilution of the suspension cells) the cells are incubated in a mixture consisting of 50% of control medium and 50% of serum-free medium. The medium is changed at the same ratio every day for adherent cells. As regards to suspension cells, medium is added to the cultures at the same ratio.
T+2 Passage: after dissociation of the adherent cells (or dilution of the suspension cells) the cells are incubated in a mixture consisting of 25% of control medium and 75% of serum-free medium. The medium is changed at the same ratio every day

for adherent cells. As regards to suspension cells, medium is added to the cultures at the same ratio.
T+3 Passage: after dissociation of the adherent cells (or dilution of the suspension cells) the cells are incubated in pure serum-free medium.
The criteria allowing to change from one step to the next one are primarily the proliferation rate and the morphology since avian stem cells are concerned.
It is important to avoid selecting the populations too drastically, and to induce mortality. In fact, the cells during the process should keep their ability to undergo a division at least once every 48 hours. The presence of mitotic figure is essential. The cell morphology should not be close to those of vacuolated, necrosed, apoptotic or senescent cells.
If the cells show adaptation problems at one step it is important to maintain them over several passages in the culture medium in which the cells show adaptation problems, before the withdrawal process is resumed. The medium is then only half-renewed for adherent cells. For suspension cells, the daily added volume is reduced. It is also possible in the most intricate cases to add intermediate steps to the gradual dilution withdrawal.
High variability of the serum-free media efficiencies lead us to conclude that in some cases all those steps constituting the dilution withdrawal process are not absolutely necessary; more particularly the TO-passage step (mixture of 75% control medium/25% serum-free medium) does not seem to be unavoidable. The adaptation thus starts directly with the mixture of 50% control medium/50% serum-free medium and follows according to the scheme given above.
6.2 Example of adaptation by serum withdrawal
TO Passage: the avian stem cells, both adherent or in suspension, are cultured in a serum-free medium supplemented with 5% FCS. The medium is changed every day for adherent cells. As regards to suspension cells, medium is added every day to the cultures.
T+l Passage: after dissociation of the adherent cells (or dilution of the suspension cells) the cells are incubated in a serum-free medium supplemented with 2.5% FCS. The medium is changed every day for adherent cells. As regards to suspension cells, medium is added daily to the cultures.

T+2 Passage: after dissociation of the adherent cells (or dilution of the suspension cells) the cells are incubated in a serum-free medium supplemented with 1.25% FCS. The medium is changed every day for adherent cells. As regards to suspension cells, medium is added daily to the cultures.
T+3 Passage: after dissociation of the adherent cells (or dilution in the case of suspension cells) the cells are incubated in pure serum-free medium.
If the cells show adaptation problems at one step it is important to maintain them over several passages in the culture medium in which the cells show adaptation problems, before the withdrawal process is resumed. The medium is then only half-renewed for adherent cells. For suspension cells, the daily added volume is reduced. It is also possible in the most intricate cases to add intermediate steps to the gradual dilution withdrawal.
High variability of the serum-free media efficiencies lead us to conclude that in some cases all those steps constituting the dilution withdrawal process are not absolutely necessary; more particularly the TO-passage step (serum-free medium supplemented with 5% serum) does not seem to be unavoidable. The adaptation thus starts directly with the serum-free medium supplemented with 3% serum, then follows with a step in serum-free media supplemented with 2% and then with 1% serum.
EXAMPLE 7: DIRECT ADAPTATION OF ADHERENT OR SUSPENSION AVIAN STEM CELLS TO SERUM-FREE MEDIA
Avian stem cells, adherent or proliferating in suspension, whether late-passage or
early-passage, can be cultured directly in various serum-free media. Any of the
following two techniques may be used.
First technique:
Avian stem cells, both adherent or in suspension, can be adapted directly from the
control medium usually supplemented with 10% serum without any intermediate
step.
Passage into serum-free medium then occurs in one single step. For adherent avian
stem cells the serum-free medium is not systematically renewed during the next
first few days of culture. However, it is preferred to renew at least half of it in
order to accustom the cells more rapidly to further total changes. For suspension
avian stem cells, serum-free medium is not systematically added every day.
However it is preferred to add a small amount of serum-free media (at least 25% of

the total volume) until the first dilution. If the cells do not undergo any major event, large volumes can be added over the next dilution steps.
Second technique:
Adherent or suspension avian stem cells can be previously accustomed to a medium less rich in serum prior to passage into serum-free medium.
This step enables to accustom the cells to proliferate in a medium poor in serum, which facilitates single-step direct withdrawal. This serum withdrawal is done gradually, one passage after the next, until cells are obtained which proliferate in 3 to 2% serum followed by the abrupt change to serum-free medium. Passage to serum-free medium is thus done in one single step. For adherent avian stem cells, the serum-free medium is not renewed systematically during the next first few days of culture. However it is preferred to renew at least half of it in order to accustom the cells more rapidly to further total changes. For suspension avian stem cells, serum-free medium is not systematically added every day. However it is preferred to add a small amount of serum-free media (at least 25% of the total volume) until the first dilution. If the cells do not undergo any major event, large volumes can be added over the next dilution steps.
Selection of the one-step adaptation technique is done according to the efficiency of the serum-free media tested. In the case where the serum-free medium is efficient (in terms of proliferation, cellular adhesion and differentiation), the first technique will be preferred. In the case where the efficiency of the serum-free media is poor, the second one will be carried out preferentially.
EXAMPLE 8: FREEZING OF SERUM-FREE ADHERENT OR SUSPENSION AVIAN STEM CELLS
The cells are cultured in a serum-free medium. They are frozen during exponential growth phase.
The adherent cells are dissociated, numerated, and taken up at between 10 to 20 million cells per mL in a volume X of conditioned supernatant harvested before dissociation. A cell suspension results. A volume of serum-free medium supplemented with 20% DMSO (dimethyl sulfoxide), equivalent to volume X, is added dropwise onto the cell suspension. Thus, the final mixture contains 10% DMSO. The cells are then distributed into cryotubes (in an amount of 3 to 20 millions per cryotube), placed at -80C in devices providing a temperature decrease of 1 degree per minute. After 24 hours at -80C, the cells can be put into nitrogen.

The same procedure is applied for avian stem cells proliferating in suspension, except that they are not subjected to a first stage mechanical dissociation before numeration and freezing.
EXAMPLE 9: CONTROL OF SERUM LEVEL FOR THE PROLIFERATION OF CELL LINES
For producing such cell lines, the culture media used are traditional culture media comprising a basal medium (DMEM, GMEM, HAM-F12, McCoy, etc.) supplemented with various additives such as non-essential amino acids, vitamins, and sodium pyruvate. This complex medium comprises foetal calf serum, which remains a main element of the culture, although components from different origins, including plant components, can be gradually used. A process for controlling and accustoming the cells to relatively small quantities of foetal calf serum is provided. This enables to maintain the cells in large proliferation (division time > 1) with low serum quantities (e.g. 2% in the case of S86N 16 cells). The doubling time and the average division times were also calculated and reported in Table II. It should be noted that the average division time increases when the amount of serum decreases. Nevertheless a catching-up phase is seen after a period of time in culture under the above-mentioned conditions. This time remains nevertheless lower than 24 h (d>l), which represents already a very advantageous proliferation from an industrial point of view, even at serum concentrations of 2%, which is already quite low. Improvements to the various metabolites to be used can be envisaged in order to increase this time and to further optimize the culture conditions.
Tableau II:
(Table Removed)
The examples are taken from between passages p204 and pi79 for die 10% condition, between passages p198 and p176 for the 7,5% condition, between passages p224 and p201 for the 3,75% condition, and between passages p216 and p199 for the 2% condition.
EXAMPLE 10: INFECTION OF AVIAN CELL CULTURES IN A SERUM-FREE MEDIUM
Adherent and non-adherent cells are infectable by various viruses and retroviruses, including avian viruses and retroviruses. These cells can also act as a replication substrate for producing viral stocks intended to be used in the production of live, attenuated or inactivated human and veterinary vaccines, as desired. Viruses of

interest include the ones belonging to the classes of adenoviruses (such as Human Adenovirus C, Fowl Adenovirus A, Ovine Adenovirus D, Turkey Adenovirus B), circoviridae (such as Chicken Anemia Virus, CAV), some coronaviruses, such as the virus of avian infectious bronchitis (IBV), flaviviruses (such as Yellow fever virus and Hepatitis C virus), hepadnaviruses (such as Hepatitis B virus and Avihepadnavirus such as Duck Hepatitis B virus); herpesviruses (such as Gallid herpesvirus, HSV (Herpes simplex Virus), and Human Herpesvirus 1, 3 and 5), orthomyxoviruses (such as influenza virus: Influenza virus A, Influenza virus B, and Influenza virus C), papovaviruses (such as polyomavirus and more particularly Simian virus 40), paramyxoviruses (such as measles virus, mumps virus, and rubella virus and respiroviruses and pneumoviruses such as Human respiratory syncytial virus and Metapneumovirus such as Avian pneumovirus), picornaviruses (such as polio virus, hepatitis A virus, and Encephalomyocarditis virus and Foot-and-mouth disease virus), poxviruses (such as fowl poxviruses or avopoxviruses, including canary poxviruses, junco poxviruses, mynah poxviruses, pigeon poxviruses, psittacine poxviruses, quail poxviruses, sparrow poxviruses, starling poxviruses, and turkey poxviruses), reoviruses (such as rotaviruses), retroviruses (such as ALV, avian leukosis virus, Gammaretroviruses such as Murine leukaemia virus, Lentiviruses such as Human immunodeficiency viruses 1 and 2) and Togaviridae such as Rubivirus, in particular Rubella virus.
EXAMPLE 11: PROTOCOL FOR INFECTING NON-ADHERENT AVIAN CELL CULTURES IN A SERUM-FREE MEDIUM WITH A VIRUS
Amplification of the cells:
EB1 or EB14 cells adapted in a serum-free medium can be seeded at a concentration of 0.2 x 106 cells/ml for an initial volume of 50 ml in general. They are maintained in culture at 39°C and at 7.5% CO2, with stirring. Fresh medium is added every day for the 3 to 4 days for which the amplification lasts in order to reach a cell concentration of 1 to 3 x 106 cells/ml for a final culture volume of 100 to 250 ml.
The cells in suspension are collected and centrifuged for l0min at 1 000 rpm approximately. The pellet is resuspended in 20 to 50 ml of 1X PBS (Phosphate buffer Salt). The cells are then numerated, centrifuged and the pelleted cells are taken up in a serum-free medium at a final concentration of 3 to 5 x 106 cells/ml. Several tubes are then prepared under these conditions containing 3 to 5 x 106 cells per tube.

Preparation of the virus and infection:
The viral stock having a known titer is rapidly thawed to 37°C and diluted in serum-free medium at a titer of 10 x to 1 000 x the concentration necessary for the final infection. The cells are infected with the virus of interest at an m.o.i. (multiplicity of infection) of 0.01 to 0.5 according to the types of virus, which involves adding between 0.1 and 10% volume/volume of viral suspension to the cellular pellet. After incubating for 1 hour at an optimum temperature for the virus, in general from 33 to 37°C, the cells are again centrifuged and the medium removed with care. This step is found to be often necessary in order to limit the effect of the initial virus in the subsequent process. One of the possibilities is to directly dilute the cells without centrifuging them again with serum-free medium to a final concentration of 0.2 to 1 x 106 cells/ml and to incubate them again.
Harvesting of the supernatant and of the cells:
After 2 to 4 days of incubation, depending on the viral kinetics and the potential cytopathic effect of certain viruses, the medium containing the cells or the cellular debris is harvested. Depending on the viruses, only the pellet or the supernatant may be of interest and contain the viral particles. The cells are harvested and centrifuged. The collected supernatant is centrifuged again for 5 to 10 minutes at 2 500 rpm, and stored at -80°C before purification of the particles. An aliquot is collected in order to carry out the titration. The cellular pellet is taken up in 5 ml of serum-free medium, sonicated and centrifuged for 5 to 10 minutes at 2 500 rpm. The supernatant obtained is stored at -80°C until purification and titration of an aliquot. The viral infection and production efficiencies are compared between the various conditions performed. For the viruses with cytopathic effects, the titrations are in general carried out by the lysis plaque technique.
EXAMPLE 12: PROTOCOL FOR INFECTING AN ADHEENT AVLAN CELL CULTURE IN A SERUM-FREE MEDIUM WITH A VIRUS
Preparation of the cells:
The cells (S86N 45), previously adapted to a serum-free medium, are seeded 24 hours prior to infection into 175 flasks at a concentration of between 0.03 and 0.06 x 106 cells/cm2 in a serum-free medium. They are maintained at 39°C and 7.5% CO2-
Infection:
The viral stock having a known titer is rapidly thawed at 37°C and diluted in serum-free medium at a titer of 10 x to 1 000 x the concentration necessary for the final infection. The cells are infected with the virus of interest at an m.o.i.

(multiplicity of infection) of 0.01 to 0.5 according to the types of virus, which involves adding between 0.1 and 10% volume/volume of viral suspension to the cellular pellet. The infection is generally carried out in a minimum of medium (from 5 to 10 ml for a 75 cm2 flask). After incubating for 1 hour at the optimum temperature for the virus, in general from 33 to 37°C, 20 ml of serum-free medium are added to the flasks. In a particular case, the cells can be washed with PBS in order to remove the particles which might be attached to the cells. In the case of a cytopathic virus, the cells are observed daily after the infection in order to monitor the appearance of the cell lysis plaque, which indicates good progress of the infection.
Harvesting of the supernatant and of the cells:
After 2 to 4 days of incubation, depending on the viral kinetics and the potential cytopathic effect of certain viruses, the medium containing the supernatant, the cells and the cellular debris are harvested. Depending on the viruses, only the pellet or the supernatant may be of interest and contain the viral particles. The cells are harvested and centrifuged. The collected supernatant is centrifuged again for 5 to 10 minutes at 2 500 rpm, and stored at -80°C before purification of the particles. An aliquot is collected in order to carry out the titration. The cellular pellet is taken up in 5 ml of serum-free medium, sonicated and centrifuged for 5 to 10 minutes at 2 500 rpm. The supernatant obtained is stored at -80°C until purification and titration of an aliquot. The viral infection and production efficiencies are compared between the various conditions performed. For the viruses with cytopathic effect, the titrations are in general carried out by the lysis plaque technique.



We claim:
1. A method for in vitro replicating recombinant vaccinia virus comprising:
a) inoculating viral particles of said vaccinia virus on adherent or nonadherent cells from an avian cell line cultured in a culture medium, the method characterized in that after twenty passages of the said cells in a basal culture medium containing all the factors allowing their growth and an inactivated feeder layer, a gradual withdrawal of growth factors, cytokine and feeder layer is carried out; and the fetal calf serum concentration present in the said culture medium is decreased to a concentration up to zero percent to produce cells capable of indefinite proliferation in a basal medium;
b) culturing said inoculated cells in a basal medium until cell lysis occurs and newly produced viral particles of said vaccinia virus are released in the said medium.

2. A method as claimed in claim 1, wherein said vaccinia virus is the Modified Vaccinia virus Ankara (MVA), the Lister-Elstree vaccinia virus, the, NYVAC virus, the LC16m8 virus or the CV178 virus.
3. A method as claimed in claim 1, wherein said basal medium is selected from the group consisting of DMEM, GMEM, HamF12 or McCoy supplemented with additives selected from the group consisting of nonessential amino acids, vitamins and sodium pyruvate.


Documents:

185-DELNP-2006-Abstract-(15-01-2009).pdf

185-DELNP-2006-Abstract-(27-01-2009).pdf

185-delnp-2006-abstract.pdf

185-DELNP-2006-Claims-(02-03-2009).pdf

185-DELNP-2006-Claims-(15-01-2009).pdf

185-DELNP-2006-Claims-(23-01-2009).pdf

185-DELNP-2006-Claims-(27-01-2009).pdf

185-DELNP-2006-Claims-(27-03-2009).pdf

185-delnp-2006-claims.pdf

185-delnp-2006-complete specification (granted).pdf

185-DELNP-2006-Correspondence-Others-(02-03-2009).pdf

185-DELNP-2006-Correspondence-Others-(15-01-2009).pdf

185-DELNP-2006-Correspondence-Others-(16-03-2011).pdf

185-DELNP-2006-Correspondence-Others-(23-01-2009).pdf

185-DELNP-2006-Correspondence-Others-(27-01-2009).pdf

185-DELNP-2006-Correspondence-Others--(27-03-2009).pdf

185-delnp-2006-correspondence-others-1.pdf

185-delnp-2006-correspondence-others.pdf

185-DELNP-2006-Description (Complete)-(15-01-2009).pdf

185-DELNP-2006-Description (Complete)-(23-01-2009).pdf

185-delnp-2006-description (complete).pdf

185-DELNP-2006-Drawings-(15-01-2009).pdf

185-DELNP-2006-Drawings-(23-01-2009).pdf

185-delnp-2006-drawings.pdf

185-DELNP-2006-Form-1-(15-01-2009).pdf

185-DELNP-2006-Form-1-(27-01-2009).pdf

185-delnp-2006-form-1.pdf

185-delnp-2006-form-18.pdf

185-DELNP-2006-Form-2-(15-01-2009).pdf

185-DELNP-2006-Form-2-(27-01-2009).pdf

185-delnp-2006-form-2.pdf

185-DELNP-2006-Form-27-(16-03-2011).pdf

185-DELNP-2006-Form-3-(15-01-2009).pdf

185-delnp-2006-form-3.pdf

185-DELNP-2006-Form-5-(15-01-2009).pdf

185-delnp-2006-form-5.pdf

185-DELNP-2006-GPA-(15-01-2009).pdf

185-delnp-2006-gpa.pdf

185-DELNP-2006-Others-Document-(15-01-2009).pdf

185-DELNP-2006-Others-Document-(23-01-2009).pdf

185-delnp-2006-pct-101.pdf

185-delnp-2006-pct-210.pdf

185-delnp-2006-pct-301.pdf

185-delnp-2006-pct-304.pdf

185-delnp-2006-pct-308.pdf

185-DELNP-2006-Petition-137-(15-01-2009).pdf

185-DELNP-2006-Petition-138-(15-01-2009).pdf


Patent Number 231096
Indian Patent Application Number 185/DELNP/2006
PG Journal Number 11/2009
Publication Date 13-Mar-2009
Grant Date 02-Mar-2009
Date of Filing 10-Jan-2006
Name of Patentee VIVALIS
Applicant Address LIEUDIT LA CORBIERE, 49450, ROUSSAY, FRANCE
Inventors:
# Inventor's Name Inventor's Address
1 FABIENNE GUEHENNEUX 115, AVENUE DE LA FERRIERE, 44700 ORVAULT, FRANCE
2 BERTRAND PAIN 4, BIS, PLACE BIR-HAKEIM, 69003 LYON, FRANCE
PCT International Classification Number C12N 7/00
PCT International Application Number PCT/IB2004/002621
PCT International Filing date 2004-07-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 03291813.8 2003-07-22 EPO
2 0314389 2003-12-09 EPO