# US 2014/0154741 A1 · Duck embryonic derived stem cell lines for the production of viral vaccines

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- Number: US 2014/0154741 A1
- Document ID: US20140154741A1
- Status: Published application
- Kind: A1
- Application number: 14/069,423
- Filed: 2013-11-01
- Published: 2014-06-05
- Earliest claimed: 2007-04-24
- Assignees: Valneva SE
- Applicants: Valneva
- Inventors: Fabienne Guehenneux; Karine Moreau; Magali Esnault; Majid Mehtali
- Web page: https://patentgrep.com/patent/US20140154741A1

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 39/17: Newcastle disease virus
- A61K 39/285: Vaccinia virus or variola virus
- A61P 31/16: for influenza or rhinoviruses
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- A61K 2039/525: {Virus}
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/23: Interleukins [IL]
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2710/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/24151: Methods of production or purification of viral material
- C12N 2760/16034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16051: Methods of production or purification of viral material
- C12N 2760/18134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18151: Methods of production or purification of viral material
- C12N 2760/18434: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18451: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

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

## Claims

### Claim at position 1

-23. (canceled)

### Claim 24

A process for obtaining a continuous avian cell line derived from avian embryonic stem cells (ES), wherein said avian cell line is capable of proliferating in a basal medium in the absence of growth factors, and a feeder layer, said process comprising the steps of:
a) isolating bird embryo(s) at a developmental stage around oviposition;
b) suspending avian embryonic stem (ES) cells obtained by dissociating embryo(s) of step a) in a basal culture medium supplemented with:
Insulin Like Growth Factor 1 (IGF-1) and Ciliary Neurotrophic Factor (CNTF) as the only recombinant growth factors; and
animal serum;
c) seeding the suspension of ES cells obtained in step b) on a layer of feeder cells and further culturing the ES cells for at least one passage;
d) withdrawing IGF-1 and CNTF from the culture medium, and further culturing the cells for at least one passage;
e) progressively decreasing the concentration of feeder cells in the culture medium so as to obtain a total withdrawal of feeder layer after several passages, and further culturing the cells; thereby obtaining an adherent continuous avian cell line derived from ES cells capable of proliferating in a basal medium in the absence of growth factors, and a feeder layer.

### Claim 25 (depends on 24)

The process according to claim 24, wherein after step e) the concentration of animal serum in the culture medium is progressively decreased so as to obtain a total withdrawal of animal serum after several passages.

### Claim 26 (depends on 24)

The process according to claim 24, wherein said adherent avian cell line is adapted to suspension culture conditions.

### Claim 27 (depends on 24)

The process according to claim 24, wherein the withdrawal of the growth factors IGF-1 and CNTF from the culture medium in step d) is performed simultaneously.

### Claim 28 (depends on 24)

The process according to claim 24, wherein said bird wherein said bird is selected in the order of Anseriformes.

### Claim 29 (depends on 24)

The process according to claim 24, wherein said bird is a duck.

### Claim 30 (depends on 29)

The process according to claim 29, wherein said duck is a Pekin duck.

### Claim 31 (depends on 29)

The process according to claim 29, wherein said duck is a Muscovy duck.

### Claim 32 (depends on 31)

The process according to claim 31 wherein the egg of said Muscovy duck is incubated to mature before isolating the duck embryo.

### Claim 33 (depends on 24)

The process according to claim 24, wherein said bird is selected in the Order of Galliformes.

### Claim 34 (depends on 33)

The process according to claim 33 wherein said bird is an ev-0 domestic chicken.

### Claim 35 (depends on 24)

The process according to claim 24 wherein said continuous avian cell line has at least one of the following characteristics:
a high nucleo-cytoplasmic ratio,
an endogenous telomerase activity,
a diameter of around 10 μm;
a population doubling time of around 30 hours or less at 37° C.;
said cells express one or more additional markers selected in the group comprising alkaline phosphatase, SSEA-1, EMA-1, ENS-1.

### Claim 36 (depends on 24)

The process according to claim 24 wherein said continuous avian cell line is diploid.

### Claim 37 (depends on 24)

The process according to claim 24 wherein said continuous avian cell line does not produce replication-competent endogenous retrovirus particles.

### Claim 38 (depends on 24)

The process according to claim 24 wherein the genome of said bird does not contain endogenous proviral sequences susceptible to produce replication competent endogenous retroviral particles.

### Claim 39 (depends on 24)

A process of replicating a virus in a continuous avian cell line obtainable by the process according to claim 24, comprising the steps of:
a) infecting cells of said continuous avian cell line with a virus of interest;
b) culturing said infected avian cells in order to replicate said virus; and
c) harvesting the virus in cell culture supernatant and/or inside said cells.

### Claim 40 (depends on 39)

The process according to claim 39, wherein said virus is selected from the group consisting of poxviruses, orthomyxoviruses, paramyxoviruses, herpes viruses, hepadnaviruses, adenoviruses, parvoviruses, reoviruses, circoviruses, coronaviruses, flaviviruses, togaviruses, birnaviruses and retroviruses.

### Claim 41 (depends on 39)

The process according to claim 39 wherein the virus is selected from the group consisting of:
a poxvirus or a recombinant poxvirus selected from the group consisting of Modified Vaccinia Ankara (MVA) virus, Lister-Elstree vaccinia virus, LC16 m8 vaccinia virus, CVI78 vaccinia virus, Fowl pox virus, canary pox virus, ALVAC, NYVAC, juncopox virus, mynah pox virus, pigeonpox virus, psittacine pox virus, quail pox virus, sparrow poxvirus, starling pox virus, and Turkey pox virus;
a paramyxovirus or a recombinant paramyxovirus selected from the group consisting of measles virus, mumps virus, rubella virus, Sendai virus, Respiratory Syncytial virus (RSV), human para-influenza types I and III, Rinderpest virus, canine distemper virus, Newcastle disease virus, and duck para-influenza virus;
an orthomyxovirus, a recombinant orthomyxovirus or a reassorted orthomyxovirus selected from the group consisting of human influenza virus, avian influenza virus, swine influenza virus, equine influenza virus, and feline influenza virus;
a togavirus selected from the group consisting of Sindbis virus, Semliki forest virus, O'nyong'nyong virus, Chikungunya virus, Mayaro virus, Ross river virus, Eastern equine encephalitis virus, Western Equine encephalitis virus, Venezuelan Equine encephalitis virus, and recombinant togaviruses;
a retrovirus selected from the group consisting of reticulo-endotheliosis virus, duck infectious anemia virus, duck spleen necrosis virus, and recombinant retroviruses;
a duck parvovirus or a recombinant parvovirus thereof;
an adenovirus selected from the group consisting of fowl adenovirus, goose adenovirus, duck adenovirus, pigeon adenovirus and recombinant adenoviruses;
an Infectious Bursal Disease virus; and
a flavivirus selected from the group consisting of Dengue virus, Japanese encephalitis virus and West Nile virus.

### Claim 42 (depends on 24)

A process of production of recombinant proteins and peptides, comprising the steps of:
a) genetically modifying the continuous avian cell line obtainable by the process according to claim 24, by transient or stable transfection of cells of the continuous avian cell line with an expression vector that encodes one or more recombinant proteins or peptides;
b) selecting said genetically modified avian cell lines expressing said recombinant proteins or peptides;
c) culturing cells under conditions suitable for production of recombinant proteins and peptides; and
d) purifying the recombinant peptides or proteins.