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Definitions

• the present invention pertains to the field of cell lines for the production of biologies, including viruses and proteins.
• the invention relates to specific immortalized avian cell lines expressing telomerase reverse transcriptase (TERT), and exhibiting distinct biologies production patterns. More particularly, the present invention
• IO relates to immortalized avian cell line capable of either amplifying Flaviviridae but not capable of amplifying Vaccinia virus strain Copenhagen (W-COP) nor Modified Vaccinia virus Ankara (MVA), or capable of amplifying both Flaviviridae and Poxviridae.
• W-COP Vaccinia virus strain Copenhagen
• MVA Modified Vaccinia virus Ankara
• the invention further relates to the use of said immortalized avian cell lines and related methods for producing biologies, including viruses and proteins.
• Avian cells have been used for years for the production of viral vaccines. For instance, embryonated chick eggs are able to support the replication of a wide range of human and animal viruses including notably attenuated viruses which have impaired potential to replicate in human or mammalian cells. Embryonated chick eggs used for human
• >0 viral vaccine production must be certified to be free of a defined set of viral and bacterial contamination (specific pathogen-free or SPF). Embryonated chick eggs are expensive and can constitute up to 40% of the cost of viral vaccines. A vaccine lot cannot be released until the SPF supplier verifies that the parental chickens for the embryonated chick eggs used to manufacture the vaccine lot were completely free of any disease.
• Flaviviridae vaccines are for instance manufactured from CEFs.
• the Flaviviridae Yellow fever viral vaccines recommended by the World Health Organization (WHO) are from 17D strain and substrains, which have minor differences in nucleotide sequences and equivalent immunogenicity (CAMACHO L.A.B. et al., Rev Saude Publica 38(5):671-8 (2004); DOS SANTOS CN. , Virus Res. 35:35-41 (1995)).
• WHO World Health Organization
• Flaviviridae viral production process have resulted in the establishment of an efficient methodology for the production of a Yellow fever viral vaccine in CEFs using the 17DD virus strain (FREIRE M.S., Vaccine 23, 2501-2512 (2005)). While the use of CEFs
• CEFs improves the safety profile, efficiency and reliability of the manufacturing process, it also highly time consuming and further increases costs.
• the production of CEFs depends indeed on the availability of SPF eggs.
• the CEFs are prepared from SPF eggs by mincing embryos to establish and amplify viable cells. Typical for primary animal cells the fibroblasts suffer senescence: the doubling time increases with passaging and
• Immortalized cell lines can be maintained or frozen from batch to batch on the production site and are always available for a new production process. Moreover as they are confined at the production plant, they are less subject to contamination by exogenous contaminant. Their use allows a drastic reduction of the manual manipulation needed for the production process. All these properties lead to a reduction
• Immortalized cell lines can moreover be fully characterized and are thus totally compliant with the good laboratory practice and the requirements of the different medical agencies.
• VERO cell line has been described as alternative candidate for Flaviviridae vaccine production in replacement of the approved
• the live attenuated Japanese encephalitis virus vaccine ChimeriVaxTM-JEV was propagated in VERO cells cultured in media supplemented with fetal bovine serum (MONATH et al., Biologicals, 33:131-144 (2005)).
• MONATH et al. Biologicals, 33:131-144 (2005)
• MATEU et al. Trans. R. Soc. Trop. Med. Hyg., 101 (3):289-98 (2007)
• TORINIWA et al. Vaccine, 4;26(29-30):3680-9 (2008) have respectively produced Yellow fever virus
• immortalized cell lines are moreover not limited to the viral vaccines they are designed for but may be extended to recombinant proteins.
• both antibodies and influenza viral vaccines can be propagated on PER.C6® cell line (ECACC number 96022940), a human fetal retinoblast cell line immortalized by transfection with an E1 minigene of adeno virus type 5.
• WO2007/077256 provides methods for immortalizing primary avian cells by transfection with the Cairina moschata telomerase reverse transcriptase (TERT) nucleic acid molecule either by targeted or random insertion of the TERT nucleic acid molecule into the Cairina moschata primary avian cells genome. Based on said methods, especially by random insertion, we have
• compositions and methods include the referenced components or steps, but not excluding others.
• Consisting essentially of when used to define products, compositions and methods shall mean excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable
• an "immortalized avian cell line” refers to an avian cell line that proliferates in culture beyong the Hayflick limit (HAYFLICK L., Clin. Geriatr. Med. 1 (1):15-27 (1985)). More particularly, an “immortalized avian cell line” refers to an avian cell line that is capable of growing in culture for greater than 30 passages that maintain a doubling time in culture of about 1 to about 2 days and have been in continuous culture for greater than about 6 months. An avian cell line is considered immortalized after about 20 to about 25 passages in culture. Immortalized avian cells are differentiated from transformed cells in that unlike transformed cells,
• immortalized avian cells are growth arrested (i.e. avian cells are confluent and subject to contact inhibition) and have a homogenous fibroblast like morphology.
• an "immortalized avian cell line able of amplifying a virus” mean that an immortalized avian cell line of the invention is able after infection by a virus to increase the amount of said virus due to a productive
• reproductive replication refers to the fact that the said virus replicates in the immortalized avian cell line to such an extent that infectious progeny virus is produced, wherein the ratio of output virus to input virus is above 1.
• able of amplifying a virus means that the ratio of output virus to input virus should be above 1.
• Flaviviruses include Flaviviruses, Pestiviruses, Hepaciviruses, GB virus A, GB virus A-like agents, GB virus-B and GB virus-C (also called hepatitis G virus). Classification, genome organisation and replication cycle of Flaviviridae has been well described (LINDENBACH B. D. et al., in D. M. Knipe and P.M. Howley, Fields Virology 5 th Edition, Eds. Lippincott-Raven
• “Flaviviruses” include the “mosquito-borne virus cluster”, the “tick-borne virus cluster” and the “no-vector cluster” (KUNO G. et al., J. Virol. 72, 73-83 (1998)).
• the "mosquito-borne virus cluster” includes Dengue virus, Japanese encephalitis virus,
• Dengue virus (DENV) strains are divided into the four serotypes DEN-1 , DEN-2, DEN-3 and DEN-4.
• Information on the nucleotide sequences of the genomes of DENVs can also be obtained from publicly accessible gene databases such as GenBank: DEN-1
• JEV Japanese encephalitis virus
• JEV includes the strains: P3, SA14, S892, GP78, ThCMAr4492, ThCMAr6793, JaGArOI , Jaoars982, Subin, KE-093/83, Nakayama wild- strain, Nakayama-RFVL, Nakayama-Yoken, LNDG07-02, LNDG07-16 and K94P05. Information on the nucleotide sequences of the genomes of JEVs can also be obtained
• West Nile virus (WNV) strains are including genotypes NY99 and WN02.
• Information on the nucleotide sequences of the genomes of WNVs can also be obtained from publicly accessible gene databases such as GenBank: e.g. GenBank accession number
• the Yellow fever virus (YFV) strains include the strains: Asibi, French viscerotropic virus (FVV), B4.1 , Rendu, Dak1279, 17D, 17DD, 17D-204, Colombia 88, F-204 and C-204 (HAHN et al., Proceedings of the National Academy of Sciences USA 84, 2019-2023 (1987); WANG et al., J. of Gen. Virol. 76, 2749-2755 (1995); BALLINGER-CRABTREE
• Tick-borne virus cluster refers to Tick-borne encephalitis virus (TBEV) (also called Tick-borne encephalitis virus
• Tick-borne meningoencephalitis virus which includes three subtypes: the Western subtype (also called Central European encephalitis virus), the Far Eastern subtype (also called Russian spring/summer encephalitis virus) and the Siberian subtype (KAISER and REINHARD, Infectious Disease Clinics of North America. 22(3):561-575 (2008)).
• Western subtype also called Central European encephalitis virus
• Far Eastern subtype also called Russian spring/summer encephalitis virus
• Siberian subtype Kaberian subtype
• TBEV also includes the viable chimeric vaccine was constructed which contained the C-preM-E or preM-E structural protein genes of a virulent Far Eastern Russian TBEV with the remaining nonstructural protein genes and 5'- and 3'-noncoding sequences derived from DEN4 [TBEV(CME)/DEN4 and
• the "no-vector cluster” includes aba virus, cell fusing agent virus, San Perlita virus,
• IO instance, for aba virus e.g. GenBank accession number AF160193, NC-003676.
• BDV Border disease virus
• BVDV Bovine viral diarrhea virus
• CSFV classical swine fever virus
• BDV includes the 3 genotypes BDV- 1 to -3 (BECKER et al. Virology 311 96-104 (2003)).
• BVDV includes BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2) (HEINZ et al. in
• Hepaciviruses include hepatitis C virus (HCV). Extensive phylogenetic analyses have led to the classification of HCV isolates
• HCV isolates of genotype 1a include without limitation, HCV-1 (CHOO et al., Proc. Natl. Acad. Sci. USA 88, 2451-2455 (1991)), -J1 (OKAMOTO et al., Nucleic Acids Res. 20, 6410-6410 (1992)) and -H (INCHAUSPE et al., Proc. Natl. Acad. Sci. 88, 10292-10296 (1991)).
• Exemplary HCV isolates of genotype 1b include without limitation, HCV-JA (KATO et al., Proc. Natl. Acad., Sci. 87, 9524-9528 (1990)) and BK (TAKAMIZAWA et al., J. Virol. 65, 1105-1113 (1991)).
• Exemplary HCV isolates of genotype 1c include without limitation, HCV-G9 (OKAMOTO et al., J. Gen. Virol. 45, 629-635 (1994)).
• Exemplary HCV isolates of genotype 2a include without limitation, HCV-J6 (OKAMOTO et al., J.
• Exemplary HCV isolates of genotype 2b include without limitation, HCV-J8 (OKAMOTO et al..Virology 188, 331-341 (1992)).
• Exemplary HCV isolates of genotype 2c include without limitation, HCV-BEBE1 (NAKO et al., J. Gen. Virol. 141 , 701-704 (1996)).
• Exemplary HCV isolates of genotype 3a include without limitation, HCV-NZL1 (SAKAMOTO et al., J. Gen. Virol. 75, 1761- 1768 (1994)).
• Exemplary HCV isolates of genotype 3b include without limitation, HCV-Tr (CHAYAMA et al., J. Gen. Virol. 75, 3623-3628 (1994)).
• Exemplary HCV isolates of genotype 4a include without limitation, HCV-ED43 (CHAMBERLAIN et al., J. Gen. Virol. 78, 1341-
• Exemplary HCV isolates of genotype 5a include without limitation, HCV- EUH1480 (CHAMBERLAIN et al., Biochem. Biophys. Res. Commun. 236, 44-49 (1997)).
• Exemplary HCV isolates of genotype 6a include without limitation, HCV-EUHK2 (ADAMS et al., Biochem. Biophys. Res. Commun. 234, 393-396 (1997)).
• “Poxviridae” include Orthopoxviruses
• Orthopoxviruses include Buffalopoxvirus, Camelpoxvirus, Cowpoxvirus, Ectromelia virus, Monkeypoxvirus, Rabbitpoxvirus, Variola virus, Vaccinia virus (W) and its derivatives such as for instance Modified Vaccinia virus Ankara (MVA).
• Capripoxviruses include sheeppox virus, goatpox virus and lumpy skin disease virus.
• Avipoxviruses include Canarypoxvirus and Fowlpoxvirus.
• Parapoxviruses include pseudocowpox, arapoxvirus ovis and orf virus.
• Leporipoxviruses include Myxoma virus. Sequences of the genome of various Poxviridae are available in the art, for example,
• Vaccinia virus includes the W
• W comprising defective I4L and/or F4L gene(s) (see WO2009/065546), and derivatives thereof.
• Modified Vaccinia virus Ankara refers to the highly attenuated W generated by serial passages on CEFs of the W strain Ankara (MAYR A. et al., Infection 3, 6-14 (1975)) and derivatives thereof.
• the MVA virus was deposited before Collection Nationale de Cultures de Microorganismes (CNCM) under depositary N ° 1-721.
• the MVA is fully described in SUTTER et MOSS (Proc. Natl. Acad. Sci. USA 89, 10847-10851 (1992)).
• the genome of the MVA has 5 been mapped and sequenced (ANTOINE et al., Virol. 244, 365-396 (1998) and is available in Genbank under accession number U94848).
• the present invention concerns a method for producing virus, comprising the steps of:
• the present invention relates to an immortalized >0 avian cell line expressing avian telomerase reverse transcriptase (TERT) selected in the group consisting of an immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502, an immortalized avian cell line deposited at the ECACC under accession number 08060501 and derivatives thereof.
• ECACC European Collection of Cell Cultures
• 08060501 an immortalized avian cell line deposited at the ECACC under accession number 08060501 and derivatives thereof.
• These cell lines have been generated from primary Cairina moschata cells as >5 disclosed in WO2007/077256 by random insertion of the avian TERT nucleic acid molecule (SEQ ID NO: 1) into the Cairina moschata primary avian cells genome .
• SEQ ID NO: 1 avian TERT nucleic acid molecule
• the immortalized avian cell line of the invention is able of amplifying at least one Flaviviridae strain while being unable of amplifying Vaccinia virus strain Copenhagen (W-COP) (GOEBEL et al. 1990; Genbank accession number M35027.1) nor Modified Vaccinia virus Ankara (MVA) (Collection Nationale de Cultures de Microorganismes (CNCM) under depositary N ° 1-721).
• W-COP Vaccinia virus strain Copenhagen
• MVA Modified Vaccinia virus Ankara
• CNCM Collection Nationale de Cultures de Microorganismes
• the immortalized avian cell line of the invention able of amplifying at least one Flaviviridae strain while being unable of amplifying Vaccinia virus strain Copenhagen (W-COP) nor Modified Vaccinia virus Ankara (MVA) is an immortalized avian cell line expressing avian telomerase reverse transcriptase
• the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502 or derivatives thereof is able of amplifying at least one Flaviviridae strain selected in the
• ECACC European Collection of Cell Cultures
• accession number 08060502 or derivatives thereof is able of amplifying Yellow fever virus strain (YFV) and Japanese encephalitis virus strain (JEV).
• YFV Yellow fever virus strain
• JEV Japanese encephalitis virus strain
• the immortalized avian cell line of the invention is able of amplifying (i) at least one Flaviviridae strain and (ii) at least one
• the immortalized avian cell line of the invention able of amplifying at least one Flaviviridae strain and at least one Poxviridae strain is an immortalized avian cell line expressing avian telomerase reverse transcriptase (TERT) deposited at the European Collection of Cell Cultures (ECACC) under accession
• TERT avian telomerase reverse transcriptase
• the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 or derivatives thereof is able of amplifying (i) at least one Flaviviridae strain selected in the group consisting of Yellow fever virus strain (YFV) and Japanese encephalitis virus strain (JEV), and more particularly in the group consisting of YFV 17D strain (e.g. ref.
• Poxviridae strain selected in the group 5 consisting of Vaccinia virus strain Copenhagen (W-COP) (GOEBEL et al. 1990;
• the immortalized avian cell line deposited at the IO European Collection of Cell Cultures (ECACC) under accession number 08060501 or derivatives thereof is able of amplifying Yellow fever virus strain (YFV), Japanese encephalitis virus strain (JEV), Vaccinia virus strain Copenhagen (W-COP) and
• MVA Modified Vaccinia virus Ankara
• Derivatives of the deposited Cairina moschata immortalized avian cell lines of the 15 invention refer to Cairina moschata immortalized avian cell lines derived from the deposited ones by, for example :
• said derivatives of the deposited Cairina moschata immortalized avian cell line are obtained by deletion or mutation of ⁇ 1 ,6-fucosyltransferase (FUT8) and/or GDP-mannose 4,6-dehydratase (GMD) genes;
• said derivatives of the deposited Cairina moschata immortalized avian cell line are obtained by deletion or mutation of gene(s) selected in the group consisting of STAT1 gene, STAT2 gene, STAT3 gene and STAT5 gene) ;
• said anti- apoptotic gene(s) is selected in the group consisting of p19E1 B human adenovirus gene, bcl-2 gene, mcl-1 gene, Bcl-xL gene, Bcl-w gene, a1 gene, ICP34.5 herpes simplex virus gene and p35 baculovirus gene) ;
• IO - overexpression of one or more Cairina moschata's genes involved in controlling the cell cycle using vectors which are suitable for increasing the rate of proliferation is selected in the group consisting of p53 gene, p21 gene, p27 gene and p57 gene);
• said gene(s) which encode receptors for the viruses of interest is gene(s) which encode measles virus CD46 receptor).
• said ECACC 08060502 derivatives are able of amplifying at least one Flaviviridae strain while being unable of amplifying Vaccinia virus strain Copenhagen (W-COP) nor Modified Vaccinia virus Ankara (MVA).
• said ECACC 08060501 derivatives are capable of amplifying (i) at least one Flaviviridae strain and (ii) at least one Poxviridae strain. >5
• the immortalized avian cell lines of the invention are adherent cell lines.
• the immortalized avian cell lines of the invention are non-adherent cell lines which proliferate in suspension, in presence or not of (micro)carriers.
• the (micro)carriers used according to the invention can be made of 50 dextran, collagen, polystyrene, polyacrylamide, gelatine, glass, cellulose, polyethylene and/or plastic.
• (Micro)carriers are commercially available such as e.g.
• CytodexTM microcarriers (Pharmacia), CytoporeTM microcarriers (GE Healthcare Life Sciences), HillexTM microcarriers (SoIoHiII Enginnering), Nunc 2D MicroHexTM microcarriers Nunclon TM (Thermo Fisher Scientific), ProNectinTM microcarriers (SoIoHiII Enginnering), Fibra-CellTM discs (New Brunswick Scientific), BioNocllTM microcarriers (Cesco Bioengineering) and CultiSpher-STM microcarriers (Percell Biolytica).
• the present invention also relates to the use of an immortalized avian cell line of the
• viruses includes viruses selected in the group consisting of Flaviviridae, Poxviridae, flu viruses, Paramyxoviridae, adenovirus, adeno-associated virus (AAV), retrovirus (such as e.g. Rous sarcoma virus (RSV); human immunodeficiency virus (HIV)), hepadnaviruses (such as e.g. hepatitis B virus),
• RSV Rous sarcoma virus
• HAV human immunodeficiency virus
• hepadnaviruses such as e.g. hepatitis B virus
• IO herpes viruses such as e.g. HSV-1 ; HSV-2), reoviruses (such as e.g. rotaviruses), coronaviruses (such as e.g. human SARS-CoV; HCoV-NL63), and alphaviruses (such as e.g. chikungunya; Ross river virus (RRV).
• the viruses can be wild type, attenuated, recombinant and/or temperature sensitive viruses.
• the Flaviviridae and Poxviridae have been defined above.
• the Flaviviridae is a wild type, attenuated, recombinant and/or temperature sensitive Flaviviridae.
• the Poxviridae is a wild type, attenuated, recombinant and/or temperature sensitive Poxviridae.
• attenuated virus refers to any virus that has
• the virus is attenuated to the point it is nonpathogenic from a clinical standpoint, i.e. that subjects exposed to the virus do not exhibit a statistically significant increased level of pathology relative to control subjects.
• recombinant virus refers to a virus comprising an exogenous sequence inserted in its genome.
• exogenous sequence refers to a nucleic acid molecule which is not naturally present in the parent virus.
• Recombinant virus can refer to a virus consisting of a virus in which one or more structural proteins has been replaced with foreign nucleic acid sequence of eukaryotic, prokaryotic, viral origin (e.g. structural protein(s) of a second virus). Therefore, according to the invention, “recombinant virus” can be also indifferently called “chimeric virus” or “hybrid virus”. Notably, in the case of Flaviviridae, chimeric
• Flaviviridae can consist of a first Flaviviridae (e.g. a YFV such as for instance YFV 17D strain as previously described) in which the prM and E proteins have been replaced with the prM and E proteins of a second virus (e.g. a JEV, WNV, DENV, St. Louis encephalitis virus or TBEV as previously described).
• a first Flaviviridae e.g. a YFV such as for instance YFV 17D strain as previously described
• a second virus e.g. a JEV, WNV, DENV, St. Louis encephalitis virus or TBEV as previously described.
• ChimeriVaxTM technology has been used to create chimeric vaccine candidates against medically important
• Flaviviridae employs the YFV 17D vaccine virus as a vector in which the prM-E genes are replaced with the prM-E genes from a heterologous Flaviviridae such as JEV, DENV, WNV or St. Louis encephalitis viruses (MONATH et al., Vaccine 20:1004-1018 (2002); PUGACHEV et al., Int. J. Parasitol. 33:567-582 (2003); GUIRAKHOO et al., J. Virol. 78:4761-4775 (2004)).
• the ChimeriVaxTM-JEV vaccine comprising the prM-E
• chimeric Flaviviridae can also be chimeric Flaviviridae as described in WO2006/068307, WO2002/102828, EP0977587, WO98/37911 , WO93/06214, US7569383 and WO01/39802.
• the recombinant virus can further comprise the elements necessary for
• the elements necessary for the expression comprise of the set of elements allowing the transcription of a nucleotide sequence to RNA and the translation of a mRNA to a polypeptide, in particular the promoter sequences and/or regulatory sequences which are effective in the cell to be infected by the recombinant virus, and optionally the sequences required to allow the
• JO excretion or the expression at the surface of the cells for said polypeptide may be inducible or constitutive.
• the promoter is adapted to the recombinant virus selected and to the host cell.
• the literature provides a large amount of information relating to such promoter sequences.
• the elements necessary can, in addition, include additional elements which improve the expression of the exogenous sequence or its maintenance in the host cell. There may be mentioned in particular the intron sequences, secretion signal sequences, nuclear localization sequences, internal sites for reinitiation of translation of the IRES type, poly A sequences for termination of
• temperature sensitive virus refers to a virus derivative which has an impaired growth at or above a certain temperature at which the wild type has a normal growth.
• temperature sensitive viruses as described in BOYD O. et al. (Virology Apr 10;399(2):221-30 (2010)), EP 0 157 528
• influenza viruses also called influenza viruses
• influenza type A virus includes influenza type A virus and its subtypes, influenza type B virus and influenza type C virus.
• Subtypes of influenza type A virus include the different combinations of HA and
• influenza type A viruses 19 classes of NA proteins possible. 19 classes of NA proteins (classified H1-H15) and 9 classes of NA proteins (classified N1-N9) have been identified in influenza type A viruses.
• subtypes of influenza type A virus can be H1 N1 virus, H1 N2 virus, H3N2 virus, H3N8 virus, H5N1 virus, H7N2 virus, H7N3 virus, H7N7 virus and H9N2 virus. Further information on the nucleotide sequences of the genomes of flu viruses can be H1 N1 virus, H1 N2 virus, H3N2 virus, H3N8 virus, H5N1 virus, H7N2 virus, H7N3 virus, H7N7 virus and H9N2 virus. Further information on the nucleotide sequences of the genomes of flu viruses can be
• nucleotide sequences of the genomes of influenza type A viruses can also be obtained from publicly accessible gene databases such as GenBank, EMBL or LANL: e.g. J02144; J02146; J02148; J02151 ; V00603; V01099; V01104; V01106. Further information on the nucleotide sequences of the genomes of influenza type B
• >5 viruses can also be obtained from publicly accessible gene databases such as GenBank, EMBL or LANL: e.g. J02094; J02095; J02096; K00423; K01395; M20168; M20170; M20172. Further information on the nucleotide sequences of the genomes of influenza type C viruses can also be obtained from publicly accessible gene databases such as GenBank, EMBL or LANL: e.g. K01689; M10087; M17700. Further information
• Parenteraviruses include Avulaviruses, Henipaviruses, Morbilliviruses, Respiroviruses, Rubulaviruses, Pneumoviruses and Metapneumoviruses.
• Avulaviruses include Newcastle disease virus (NDV) also called avian paramyxovirus virus 1 (APMV-1).
• APMV-1 avian paramyxovirus virus 1
• Henipaviruses include Hendra virus (HeV) and Nipah virus (NiV).
• Morbilliviruses include measles virus (MV) of strains: Edmonston B
• Respiroviruses include Sendai virus (SeV, also called murine parainfluenza virus 1), bovine parainfluenza virus 3 (BPIV-3), human parainfluenza virus 1 (HPIV-1) and human parainfluenza virus 3 (HPIV-3).
• Sendai virus also called murine parainfluenza virus 1
• bovine parainfluenza virus 3 BPIV-3
• HPIV-1 human parainfluenza virus 1
• HPIV-3 human parainfluenza virus 3
• Rubulaviruses include human
• Pneumoviruses include human respiratory syncytial virus A2 (HRSV-A2), human respiratory syncytial virus B1 (HRSV- B1) and human respiratory syncytial virus S2 (HRSV-S2).
• Metapneumoviruses include human metapneumovirus (HMPV).
• proteins include:
• antibodies e.g. rituximab; trastuzumab; cetuximab; milatuzumab; eculizumab; tocilizumab; nimotuzumab; golimumab; ramcirumab; bapineuzumab; infliximab; bevacizumab; adalimumab; ranibizumab; palivizumab; omalizumab; natalizumab; panitumumab; abciximab;
• trastuzumab cetuximab
• milatuzumab eculizumab
• tocilizumab nimotuzumab
• golimumab golimumab
• ramcirumab bapineuzumab
• infliximab bevacizumab
• adalimumab ranibizumab
• palivizumab omalizuma
• - receptor ligands e.g. hormones; cytokines; growth factors
• - hormones e.g. adrenocorticotropic hormone (ACTH); antidiuretic hormone (ADH, vasopressin); atrial-natriuretic peptide (ANP); calcitonin; cholecystokinin (CCK); corticotropin-releasing hormone (CRH); glucagon; gonadotropin-releasing hormone (GnRH); growth hormone releasing hormone (GHRH); oxytocin; secretin; somatostatin; thyrotropin-releasing hormone (TRH); erythropoietin (EPO); follicle-stimulating hormone (FSH);
• ACTH adrenocorticotropic hormone
• ADH antidiuretic hormone
• ADH antidiuretic hormone
• ADH antidiuretic hormone
• ADH atrial-natriuretic peptide
• ADH atrial-natriuretic peptide
• calcitonin cholecysto
• GH growth hormone
• HCG human chorionic gonadotropin
• IGF-1 insulin
• LH luteinizing hormone
• PTH parathyroid hormone
• PRL prolactin
• TSH thyroid-stimulating hormone
• DHT Testosterone
• DHT dihydrotestosterone
• DHEA dehydroepiandrosterone
• testosterone calciferol
• calcitriol estradiol
• cytokines e.g. interleukins (IL-1 ; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL10; IL-11 ; IL-12; IL13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21 ;
• chemokines CC chemokine ligand 1 (CCL-1); CCL-2; CCL-3 (also called macrophage inflammatory protein 1 alpha (MIP-Ia)); CCL-4 (also called macrophage inflammatory protein 1 beta (MIP-1 ⁇ )); CCL-5 (also called RANTES); CCL-6; CCL-7; CCL-8; CCL-9; CCL-10; CCL-11 ; CCL-12; CCL-13;
• CXCL-1 CXCL-1
• CXCL-2 CXCL-3; CXCL-4 (also called platelet factor 4 (PF4)); CXCL-5; CXCL-6; CXCL-7; CXCL-8; CXCL-9; CXCL-10; CXCL-11 ; CXCL-12; CXCL-13; CXCL-14; CXCL-15; CXCL-16; CXCL-17; C chemokin 1 (XCL-1);
• CX 3 C chemokin 1 CX 3 CL-I
• interferons IFN-alpha; IFN-beta; IFN- gamma
• tumor necrosis factors TNF-alpha; TNF-beta
• EGF epidermal growth factor
• VEGF vascular endothelial growth factor
• BMPs Bone morphogenetic proteins
• EPO Erythropoietin
• FGF Fibroblast growth factor
• G-CSF granulocyte-macrophage colony-stimulating factor
• hPG-CSF human pluripotent granulocyte colony- stimulating factor
• M-CSF macrophage colony-stimulating factor
• GDF9 growth differentiation factor-9
• MIF macrophage migration inhibitory factor
• IGF insulin-like growth factor
• GDF-8 myostatin
• NGF nerve growth factor
• PDGF platelet-derived growth factor
• TPO thrombopoietin
• TPO transforming growth factor alpha(TGF- ⁇ ); transforming growth factor beta (TGF- ⁇ ); placental growth factor (PIGF)), antigens (e.g. antigens of the MHC; leukocyte function 5 associated antigen-1 (LFA-1)),
• ICM-1 intercellular cell adhesion molecule
• NCAMs neural cell adhesion molecules
• VCAM-1 vascular cell adhesion molecule
• PECAM-1 platelet-endothelial cell adhesion molecule
• nectins synaptic cell adhesion molecules
• IO - blood clotting factors e.g. Factor VIII; Factor IX; tPA
• enzymes e.g. alpha-amylase; beta-amylase; cellulose; beta-Glucanase; beta-glucosidase; dextranase; dextrinase; dlucoamylase; hemmicellulase; pentosanase; xylanase; invertase; lactase; naringinase; pectinase; pullulanase; acid proteinase; alkaline protease; bromelain; pepsin;
• alpha-amylase e.g. alpha-amylase; beta-amylase; cellulose; beta-Glucanase; beta-glucosidase; dextranase; dextrinase; dlucoamylase; hemmicellulase; pentosanase; xylanase; invertase; lactase;
• aminopeptidase 15 aminopeptidase; endopeptidase; subtilisin; aminoacylase; glutaminase; lysozyme; penicillin acylase; isomerase; alcohol dehydrogenase; catalase; chloroperoxidase; peroxidase; acetolactate decarboxylase; histidase; cyclodextrin glycosy transferase), fragments thereof and combinations thereof.
• the invention relates to the use of the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502 and derivatives thereof, for the production of viruses and proteins.
• ECACC European Collection of Cell Cultures
• the invention relates to the use of the immortalized
• Flaviviridae preferably selected in the group consisting of Yellow fever virus strain (YFV) and Japanese encephalitis virus strain (JEV), and more particularly in the group consisting of YFV 17D strain and JEV Nakayama wild strain.
• the invention relates to the use of the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502 and derivatives thereof, for the production of proteins, such as for example those selected from the group consisting of cytokines, antibodies and hormones, and even more particularly in the group consisting of IL-2, rituximab and erythropoietin (EPO).
• ECACC European Collection of Cell Cultures
• EPO erythropoietin
• Example 5 A preferred method for producing rituximab is described in Example 6.
• Example 7 A preferred method for producing EPO is described in Example 7.
• the invention relates to the use of the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 and derivatives thereof, for the production
• the invention relates to the use of the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 and derivatives thereof, for the production of viruses and the said virus is a Flaviviridae, preferably selected in the group consisting of Yellow
• example 3 describes a preferred method for producing YFV 17D strain and JEV Nakayama wild strain. According to another preferred embodiment, the invention relates to the use of the
• the invention relates to the use of the immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 and derivatives thereof, for the production of proteins, such as for example those selected from the group consisting of cytokines, antibodies and hormones, and even more particularly in the group consisting of IL-2,
• proteins such as for example those selected from the group consisting of cytokines, antibodies and hormones, and even more particularly in the group consisting of IL-2,
• the present invention also relates to a method for producing a virus comprising the steps of: a) infecting an immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502, with
• the present invention further relates to a method for producing and purifying a wild type, an attenuated and/or a recombinant Orthopoxvirus, comprising the following steps: IO a) preparing a culture of packaging cells; b) infecting the packaging cell culture with an Orthopoxvirus; c) culturing the infected packaging cells until progeny Orthopoxvirus is produced; d) incubation in presence of one or more nucleases;
• Orthopoxviruses in the flow through j) concentrating the flow through obtained in step h) and the flow through obtained in step i); 50 k) diafiltrating the fraction comprising the Orthopoxviruses obtained in step j), wherein said packaging cells are Cairina moschata immortalized avian cell lines comprising a nucleic acid sequence coding a telomerase reverse transcriptase (TERT) covered by patent application WO 2007/077256.
• T3-17490 as deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502 (see Figures 2, 3 and 4) or a derivative 5 thereof;
• T6-17490 as deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 (see Figures 5, 6 and 7) or a derivative thereof.
• the virus is a Flaviviridae, preferably selected in the IO group consisting of Yellow fever virus strain (YFV) and Japanese encephalitis virus strain (JEV), and more particularly in the group consisting of YFV 17D strain and JEV Nakayama wild strain.
• YFV Yellow fever virus strain
• JEV Japanese encephalitis virus strain
• Example 3 describes a preferred method for producing YFV 17D strain and JEV Nakayama wild strain.
• the invention relates to a method for 15 producing a virus comprising the steps of: a) infecting an immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 , with a virus; and b) cultivating the infected avian cell line under conditions which are enabling >0 virus amplification.
• ECACC European Collection of Cell Cultures
• the produced virus is a Flaviviridae, preferably selected in the group consisting of Yellow fever virus strain (YFV) and Japanese encephalitis virus strain (JEV), and more particularly in the group consisting of YFV 17D strain and JEV Nakayama wild strain.
• YFV Yellow fever virus strain
• JEV Japanese encephalitis virus strain
• Example 3 describes a preferred method for
• the produced virus is a Poxviridae, preferably selected in the group consisting of Vaccinia virus strain Copenhagen (W- COP) and Modified Vaccinia virus Ankara (MVA).
• W- COP Vaccinia virus strain Copenhagen
• MVA Modified Vaccinia virus Ankara
• the avian cell lines of the invention are preferably infected at a temperature comprised between 30°C and 37°C, and more preferably at 37 °C as described in Example 3 and 4.
• Step a) of infection of the cell lines of the invention with a virus is performed in an appropriate cell culture medium.
• the cell culture medium can be for instance Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) or Basal Medium Eagle (BME, Invitrogen) which can be optionally supplemented with e.g. serum (e.g. Fetal Calf Serum (FCS)) and/or amino acid(s) (e.g. L-Glutamine).
• DMEM Dulbecco's Modified Eagle's Medium
• BME Basal Medium Eagle
• the cell culture medium can also be a medium free from animal product. Many media free from animal product have
• IO PER.C6® Cells CD 293 AGTTM; CD 293 Medium ; COS-7L Cells, SFM Adapted; EPISERF® Medium; OptiProTM SFM (all available from Invitrogen).
• preferred cell culture medium used for the infection of the avian cell lines of the invention is BME (Invitrogen) supplemented with FCS and L- Glutamine.
• the cell lines of the present invention are infected with a virus at a Multiplicity of Infection (MOI) which depends on the produced virus.
• MOI Multiplicity of Infection
• a Flaviviridae is seeded into the avian cell lines of the invention at a MOI which is preferably comprised between about 0.001 and 0.1. More particularly, when the produced Flaviviridae is YFV or JEV, the MOI is more preferably about 0.001 as described in Example 3, Figure 7A,
• a Poxviridae is seeded into the avian cell lines of the invention at a MOI which is preferably comprised between about 0.0001 and 0.1. More particularly, when the produced Poxviridae is W-COP, the MOI is more preferably about 0.0001 as described in Example 4 and Figure 11 B. When the produced Poxviridae is a MVA, the MOI is more preferably about 0.05 as described
• the infected cell lines are then cultivated under conditions which are enabling virus amplification (i.e. step b)) meaning that the viral genome is transcribed, translated into viral proteins and packaged into infectious viral particles.
• the infected cell lines can be cultivated as adherent cells to surfaces or in suspension,
• the infected avian cell lines of the invention are preferably cultivated at a temperature comprised between 30°C and 37°C, and more preferably at 37 °C as described in Example 3 and 4.
• Step b) of culture of the infected avian cell lines is performed in an appropriate cell culture medium which can be the same or different from the cell culture medium used for the infection of the avian cell lines (in step a)).
• the infected avian cell lines are preferably cultivated for between 1
• the infected avian cell lines are more preferably cultivated for between 1 and 3 days as shown in Figure 7A and Figure 9A.
• the infected avian cell lines are more preferably cultivated for between 1 and 4 days as shown in Figure 8A and Figure 1OA.
• the produced virus is YFV
• the infected avian cell lines are more preferably cultivated for between 1 and 4 days as shown in Figure 8A and Figure 1OA.
• the produced virus is YFV
• the infected avian cell lines are more preferably cultivated for between 1 and 4 days as shown in Figure 8A and Figure 1OA.
• IO virus is W-COP or a MVA
• the infected avian cell lines are more preferably cultivated between 1 and 4 days as shown in Figure 11 (A-B).
• step a) of infection can be preceded by a step of culturing a cell line of the invention in an appropriate cell culture medium which can be the same or different from the cell culture medium used for the infection of the avian cell lines (in
• step a) 15 step a)) and from the cell culture medium used for the culture of the infected avian cell lines (in step b)).
• the avian cell lines are preferably cultivated for between 1 and 3 days, more preferably for 1 day before infection, at a temperature comprised between 30 and 37°C, more preferably at 37°C as described in Example 3 and 4.
• step b) of culture the infected cell line can be followed by a
• nucleases i.e. endonuclease or exonucleases
• Nucleases preferably used according to the present invention are endonucleases. Endonucleases that can be used according to the invention can be classified based on their substrates as follows: deoxyribonucleases (DNases) which
• Endonucleases include but are not limited to DNase I, DNase Il and endodeoxyribonuclease IV.
• Endonucleases RNases include but are not limited to RNase I, RNase III, RNAse E, RNAse F and RNAse P.
• Endonucleases that degrade DNA and RNA include but are not limited to Benzonase®. Endonuclease
• Benzonase® degrades nucleic acid (e.g. DNA; RNA) by hydrolyzing internal phosphodiester bonds between specific nucleotides. Upon complete digestion, all free nucleic acids (e.g. DNA; RNA) present in solution are reduced to 5'-monophosphate terminated oligonucleotides which are 3 to 8 bases in length.
• Benzonaze® has no proteolytic activity.
• Benzonaze® used according to the present invention is preferably pharmaceutically acceptable. Pharmaceutically acceptable Benzonaze® are commercially available (e.g. Eurogentec under the reference ME-0280-10; Merck under the reference e.g. 1.01653.0001).
• the concentration of nuclease(s) used is in a range of 5 to 100 U/ml, preferably in a range of 5 to 50 U/ml, and more preferably 10 U/ml.
• the viruses produced are then recovered from the supernatant and/or from the cells.
• the step of recovering of the viruses produced can IO be preceded by a step allowing the disruption of the cell membrane. This step leads to the liberation of the viruses from the cells.
• the disruption of the cell membrane can be induced by various techniques well known by the one skilled in the art.
• Sonicators are 15 commercially available from e.g. Heraeus PSP, Biologies, Misonix or GlenMills.
• Preferred sonicators used according to the present invention are SONITUBE 20 kHz type SM 20-120-3 and SONITUBE 35 kHz type SM 35-400-3 (Heraeus PSP).
• Microfluidizers are commercially available from e.g. Microfluidics Corporation.
• the avian cell membrane can also be disrupted by using a using a SLM Aminco French press. >0
• the cell membrane can also be disrupted by using a high speed homogenizer.
• High speed homogenizers are commercially available from e.g. Silverson Machines or Ika- Labotechnik.
• the viruses recovered are then purified.
• Purification of the viruses produced can comprise for instance one or more of >5 the following steps:
• Depth filtration includes but is not limited to the use of one or more commercially available products such as Sartopure® filters from Sartorius JO (e.g. Sartopure® PP2), CUNO Incorporated AP series depth filters (e.g.
• CUNO Incorporated CP series depth filters e.g. CP10, CP30, CP50, CP60, CP70, CP90
• CUNO Incorporated HP series depth filters e.g. HP10, HP30, HP50, HP60, HP70, HP90
• CUNO Incorporated Calif series depth filters
• SP series depth filters e.g. SP10, SP30, SP50, SP60, SP70, SP90
• CUNO Delipid and Delipid Plus filters Millipore Corporation CE series depth filters (e.g. CE15, CE20, CE25, CE30, CE35, CE40, CE45, CE50, CE70, CE75),
• HC filters e.g. A1 HC, B1 HC, COHC
• CUNO PolyNetTM Filters e.g. PolyNetTM PB P050, P100, P200, P300, P400, P500, P700
• Millipore Clarigard and Polygard filters CUNO Life Assure filters, ManCel Associates depth filters
• IO e.g. PR 12 UP, PR12, PR 5 UP
• PALL or SeitzSchenk Incorporated filters In order to improve the clarification capacity of the available depth filtration units, it can be useful to couple two or more units with decreasing pore sizes. In this embodiment, the mixture to be clarified passes through the first depth filtration unit where the biggest contaminants are retained and
• the clarification is preferably performed by depth filtration, and more preferably over filters having a pore size of 8 ⁇ m coupled to filters having a pore size of 5 ⁇ m.
• Preferred filters having a pore size of 8 ⁇ m and 5 ⁇ m used according to the present invention are Sartopure® filters commercially
• the depth filtration is preferably performed at a flow rate of 1 L/minute.
• Microfiltration is a pressure driven membrane process that concentrates and purifies large molecules. More specifically, a solution is
• Filters used according to the invention are preferably autoclavable commercially available filters such as for instance Prostak Microfiltration
• a diafiltration which is an improvement of microfiltration (as previously described) and involves diluting said fraction comprising the virus with a solution to effect a reduction in the concentration of the impurities in said fraction.
• the dilution of the fraction comprising the viruses allows washing out more of the impurities from said fraction. It is understood that the diafiltration may be carried out in a batch mode, semi-continuous mode, or a continuous mode.
• the diafiltration can be advantageously used to change
• the buffer in which the virus is comprised For example, it can be useful to exchange the buffer used in the purification process against a pharmaceutically acceptable buffer.
• Filters used for the diafiltration according to the invention allow the rejection of the virus in the retentate and the passage of the small molecules (e.g. proteins) through the filters into the
• Such filters are preferably autoclavable commercially available filters such as for instance Prostak Microfiltration Modules (Millipore).
• the functional groups of the anion exchange adsorbent can be primary, secondary, tertiary or quaternary amino
• DMAE dimethylaminoethyl
• diethylaminoethyl diethylaminoethyl
• the anion exchange adsorbent can consist in, but is not limited to, e.g. a beads-formed matrix or a membrane.
• the matrix can be e.g. agarose, hydrophilic polymer, cellulose, dextran or silica. Chains (e.g. dextran chains) are coupled to the matrix. Functional groups as previously described are
• Anion exchange adsorbents consisting ofbeads-formed matrix used according to the invention are preferably autoclavable such as for instance UNOsphere® Q (BioRad), UNOsphere® S (BioRad), STREAMLINETM Q Sepharose® XL (Amersham Biosciences), STREAMLINETM SP Sepharose®
• the membrane used has a pore size lower than the size of the virus.
• the wild type, attenuated, recombinant and/or temperature sensitive virus produced can be further inactivated so that the outer virion coat has been left intact but the replicative function has been destroyed.
• Preparation of said "whole-killed virus” can take the route of heat or chemicals.
• the chemicals used include for formaldehyde or beta- 5 propiolactone and formalin (CHERTOVA E. et al., AIDS Vaccine (2001)).
• the invention relates to a method for producing proteins comprising the steps of: a) contacting an immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060502, with
• IO at least one recombinant vector comprising a nucleotide sequence coding at least one protein; and b) cultivating the avian cell line under conditions which are enabling the protein to be produced.
• the invention relates to a method for 15 producing a protein comprising the steps of: a) contacting an immortalized avian cell line deposited at the European Collection of Cell Cultures (ECACC) under accession number 08060501 , with a recombinant vector comprising a nucleotide sequence coding the protein; and >0 b) cultivating the avian cell line under conditions which are enabling the protein to be produced.
• ECACC European Collection of Cell Cultures
• the recombinant vector can be of plasmid or viral origin and can, where appropriate, be combined with one or more substances which improve the transfectional efficiency and/or stability of the vector.
• these substances are >5 widely documented in the literature which is available to the skilled person (see for example FEIGNER et al., Proc. West. Pharmacol. Soc. 32,115-121 (1987); HODGSON and SOLAIMAN, Nature Biotechnology 14, 339-342 (1996); REMY et al., Bioconjugate Chemistry, 5, 647-654 (1994)).
• the recombinant vectors are expression vectors.
• the recombinant vector is a plasmid.
• the plasmid which is used in the context of the present invention contains an origin of replication.
• the plasmid can additionally comprise a selection gene which enables the transfected cells to be selected or identified (complementation of an auxotrophic mutation, gene encoding resistance to an antibiotic, etc.).
• the plasmid can contain additional elements which improve its maintenance and/or its stability in the
• the produced protein is a cytokine, more particularly an interleukin, and even more particularly IL-2.
• cytokine more particularly an interleukin
• IL-2 even more particularly IL-2.
• a preferred method for producing IL-2 is described in Example 5.
• the produced protein is an antibody, and more particularly rituximab.
• Rituximab (Rituxan® or MabThera®) is a chimeric murine/human anti-CD20 monoclonal antibody.
• a preferred method for producing rituximab is described in Example 6.
• the produced protein is a
• EPO erythropoietin
• the methods for producing proteins and viruses are free from animal products (except the avian cell lines of the invention) and suitable for an aseptic industrial-scale manufacturing process to ensure a
• animal products refer to any compound or collection of compounds that was produced in or by an animal cell in a living organism.
• the present invention also relates to a purified wild type, attenuated, recombinant and/or temperature sensitive virus obtained by the method as previously described.
• the present invention also relates to a purified whole-killed virus obtained by the method as previously described.
• the present invention also relates to a purified protein obtained by the method as previously described.
• the present invention also relates to a pharmaceutical composition, and more
• a vaccine comprising a purified wild type, attenuated, recombinant, temperature sensitive and/or whole-killed virus obtained by the method as previously described.
• the present invention also relates to a pharmaceutical composition comprising a purified protein obtained by the method as previously described.
• a pharmaceutical composition refers to a composition comprising a pharmaceutically acceptable carrier. Said pharmaceutically acceptable carrier is
• a carrier may contain any solvent, or aqueous or partially aqueous liquid such as nonpyrogenic sterile water.
• the pH of the pharmaceutical composition is, in addition, adjusted and buffered so as to meet the requirements of use in vivo.
• the pharmaceutical compositions may also
• IO include a pharmaceutically acceptable diluent, adjuvant or excipient, as well as solubilizing, stabilizing and preserving agents.
• a formulation in aqueous, nonaqueous or isotonic solution is preferred. It may be provided in a single dose or in a multidose in liquid or dry (powder, lyophilisate and the like) form which can be reconstituted at the time of use with an appropriate diluent.
• Figure 1 depicts light microscopy imaging of the Cairina moschata immortalized avian cell line ECACC 08060502 (passage 39). >0 Figure 2 depicts the Cairina moschata immortalized avian cell line ECACC 08060502 growth curve (from passage 7 to passage 75).
• Figure 3 depicts the Cairina moschata immortalized avian cell line ECACC 08060502 population doubling time evolution (from passage 7 to passage 75).
• Figure 4 depicts light microscopy imaging of the Cairina moschata immortalized avian >5 cell line ECACC 08060501 (passage 45).
• Figure 5 depicts the Cairina moschata immortalized avian cell line ECACC 08060501 growth curve (from passage 15 to passage 51).
• Figure 6 depicts the Cairina moschata immortalized avian cell line ECACC 08060501 population doubling time evolution (from passage 16 to passage 51).
• Figure 7 depicts the Flaviviridae production profile (Yellow Fever Virus 17D(MoI
• FIG. 8 depicts the Flaviviridae production profile (Japanese Encephalitis Virus Nakayama wild strain (MoI 0.001)) of Cairina moschata immortalized avian cell line ECACC 08060502 (Figure 8A) and of VERO cell line ( Figure 8B).
• Figure 9 depicts the Flaviviridae production profile (Yellow Fever Virus 17D (MoI
• Figure 10 depicts the Flaviviridae production profile (Japanese Encephalitis Virus Nakayama wild strain(Mol 0.001)) of the Cairina moschata immortalized avian cell line ECACC 08060501 ( Figure 10A) and of the VERO cell line ( Figure 10B).
• FIG 11 depicts the Poxviridae production profile (Vaccinia Virus strain Copenhagen (W-COP) ( Figure 11 B) and Modified Vaccinia virus Ankara (MVA) ( Figure 11A)) of the Cairina moschata immortalized avian cell line ECACC 08060501.
• Figure 12 depicts the production of rituximab from Cairina moschata immortalized avian cell line ECACC 08060501 ( Figure 12C) and from CHO K1 cell line ( Figure 12A)
• FIG 13 depicts the production of erythropoietin (EPO) from Cairina moschata immortalized avian cell line ECACC 08060502 (T3) and ECACC 08060501 (T6).
• EPO erythropoietin
• T3 Cairina moschata immortalized avian cell line
• T6 ECACC 08060501
• Example 1 Immortalized Cairina moschata cell line ECACC 08060502.
• the Cairina moschata cells ECACC 08060502 (passage 39) have a homogenous fibroblast like morphology ( Figure 1).
• the static monolayer is stable up to 100% confluence and subject to contact inhibition.
• the cells were tested negative for
• the cell line growth curve (from passage 7 to passage 75) ( Figure 2) shows a continuous exponential growth phase from passage 19 to passage 75. Focusing on the evolution of the population doubling time (PDT), a progressive stabilisation and decrease is observed, in particular it can be noted that the PDT is stabilised under the 48h mark during the 10
• PDL population doubling level
• the population doubling time (PDT), also called generation time, is the time 10 needed for one population doubling.
• PDT calculation: PDT ⁇ t * Ln(2)/
• Example 2 Immortalized Cairina moschata cell line ECACC 08060501.
• the Cairina moschata cells ECACC 08060501 (passage 45) have a homogenous fibroblast like morphology ( Figure 4).
• the static monolayer is stable up to 100%
• the number of calculated population doublings (PDL) corresponding to the 51 passages is at least 71 population doublings.
• the Cairina moschata cell line ECACC 08060501 is therefore far beyond the Hayflick limit and is consequently referred as immortalized cell line.
• the population doubling level refers to the number of cell generations
• the population doubling time (PDT), also called generation time, is the time needed for one population doubling.
• PDT calculation: PDT ⁇ t * Ln(2)/
• Example 3 Production of Flaviviridae .
• YFV Yellow Fever virus
• Cairina moschata immortalized avian cell lines ECACC 08060502 and 08060501 were evaluated and compared to VERO cell line which is the reference for Flaviridae propagation.
• Cairina moschata cell lines are grown in Basal Medium Eagle (Invitrogen) supplemented with 10% Foetal Calf Serum (FCS) and 4 mM
• JEV Japanese Encephalitis virus
• Cairina moschata cell lines are grown in Basal Medium Eagle (Invitrogen) supplemented with 10% Foetal Calf Serum (FCS) and 4 mM L-Glutamine and the VERO cell line in Dulbecco's Modified Eagle's Medium (Invitrogen) supplemented with 5% FCS. Infections were performed in the same culture media.
• Basal Medium Eagle Invitrogen
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Foetal Calf Serum
• FCS Dulbecco's Modified Eagle's
• Cairina moschata immortalized avian cell lines ECACC 08060502 and 08060501 and VERO were seeded at respectively 4.10 5 , 2.10 5 and 6.1O 5 CeIIs in 6 well plates and cultivated for 24 hours in humid atmosphere at 37°C, 5% CO2. The medium was then removed and cells infected
• CEFs primary chicken embryo fibroblasts
• An MVA Collection Nationale de Cultures de Microorganismes (CNCM) under depositary N 602 1-721) expressing eGFP was chosen in order to facilitate virus propagation follow up and titration.
• IO removed cells washed once with PBS and 5ml_ BME 10%FCS were then added to each flask.
• Virus was recovered by a freezing- thawing step from cells and supernatant after 24, 48, 72 and 96 hours infection at 37°C,
• the Cairina moschata immortalized avian cell line ECACC 08060502 did not amplify VV-COP.
• Example 5 Production of IL-2.
• IL-2 was quantified by ELISA (Quantikine, RD Systems) and functionality determined in a CTLL assay. Results: Both cell lines ECACC 08060502 and 08060501 produced functional IL-2. Amount was equivalent between both cell lines. Furthermore level of production was independent from the transfection method used, ranging from 4865 IU/mL (CTLL) and
• ELISA 211 ng/mL
• CTL 5672 IU/mL
• 308ng/mL corresponding to a specific activity of 18.4 IU/ng. So the measured specific activities of IL-2 produced in the cells lines are at least equivalent to the 13.16 IU/ng WHO international standard for IL-2 (Human, Jurkat-derived).
• Example 6 Production of rituxan.
• IO showed no fucosylation difference between CHO expressed Rituxan and cell line ECACC 08060501 expressed Rituxan.
• Example 7 Production of erythropoietin (EPO).
• Expression transient assays were performed using a commercially available plasmid obtained from Invitrogen (GeneStorm® Human Clones, ref HK1000 RG001720 ,

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