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Process of manufacturing viral vaccines in suspension avian embryonic derived stem cell lines

A technology of stem cells and embryos, applied in the field of virus vaccine development and preparation, can solve the problems of heavy workload, long cycle, and large resource consumption

Inactive Publication Date: 2008-06-04
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] - Large quantities of eggs or CEFs to be obtained per production and quality control to be carried out, resulting in a long, labor-intensive and resource-intensive method

Method used

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  • Process of manufacturing viral vaccines in suspension avian embryonic derived stem cell lines
  • Process of manufacturing viral vaccines in suspension avian embryonic derived stem cell lines
  • Process of manufacturing viral vaccines in suspension avian embryonic derived stem cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0159] Example 1: Method for Constructing EBx(R) Cell Line Derivatives

[0160] Methods for the construction of avian embryo-derived stem cells EBx(R) were previously described in WO03 / 076601 and WO05 / 007840. Briefly, the method for EBx(R) cell line construction comprises the following steps:

[0161] a) Isolation, cultivation and expansion of avian cells in complete medium containing all growth factors necessary for growth in the presence of a mouse fibroblast feeder layer (preferably inactivated) supplemented with animal serum, preferably Avian embryonic stem cells;

[0162] b) passaging by improving the medium or completely removing the factors, the serum and the feeder layer to accumulate cells;

[0163] c) establishment of adherent and non-adherent avian cell lines capable of proliferating in basal medium without exogenous growth factors, inactivated feeder layers, and low or no serum;

[0164] If the minimal medium of step c) still contains low levels of serum (e.g. a...

Embodiment 2

[0199] Example 2: Characteristics of EB14 cells

[0200] 2.1 Karyotype of EB14 cells

[0201] The laboratory of Pr. Michel Franck has analyzed the karyotype of EB14 cells (Unite de zootechnie, ethnologie et economie rurale, Ecole Nationale Veterinaire, 1 avenue Bourgelat, 69280 Marcy I'Etoile, France).

[0202] Karyotyping of EB14 cells at two different passages (105 and 118) was performed using standard techniques well known to those skilled in the art. As expected, EB14 cells at passages 105 and 118 were diploid karyotypes (Fig. 2):

[0203] Generation 105: number of chromosomes = 78 (mean: 78.41 - standard deviation: 4.951 for the 53 studied metaphase chromosomes).

[0204] Generation 118: number of chromosomes = 79 (mean: 79.68 - standard deviation: 3.733 for the 50 studied metaphase chromosomes).

[0205] The chicken genome contains two types of chromosome pairs: giant and tiny. The analysis of the 115th generation shows that the number of giant chromosomes is 18, wit...

Embodiment 3

[0215] Example 3: MVA production in EB14 cells

[0216] 3.1 Materials and methods

[0217] A recombinant MVA virus encoding green fluorescent protein was used in the experiment. The titer of infectious MVA-GFP virus was determined using DF-1 cells. Simply put, with 15×10 3 The density of cells / well Seed cells in 96-well flat-bottom culture plate, the medium used is the DMEM medium (Biowhittaker) that has added 5% fetal calf serum (FCS) (SAFC) and 2mM L-glutamine (Biowhittaker) . After 24 hours, infect the cells with 10-fold serially diluted samples of DMEM medium at 37 °C, 5% CO 2 Incubate for 1 week under humidified conditions. Viral infection was determined by microscopic observation of spherocytopathy (CPE) and exposure of infected cells to UV light. Then, the TCID50 titer was calculated using the method of Reed and Muench (1938, Asimple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-97).

[0218] 3.2 - Infection in tissue culture flasks

[0219] ...

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Abstract

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

Description

technical field [0001] The present invention relates to the development and preparation of virus vaccines. The present invention particularly relates to the field of industrial production of viral vectors and vaccines, and more particularly to the application of poultry cells, preferably stem cells derived from chicken embryos, to produce viral vectors and viruses. The invention is particularly suitable for the industrial production of virus vaccines that can prevent human and animal virus infection. Background technique [0002] Mass vaccination is the easiest and most effective way to control virus epidemics, such as national and worldwide flu outbreaks, and it can also prevent a large number of bioterrorist attacks, such as the recent terrorist anthrax attack in the United States. However, many vaccines such as those for influenza and smallpox are produced using egg-based systems, and current vaccine manufacturers cannot cope with the demands of a pandemic or a bioterror...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06A61K39/12C12N7/00A61P31/16A61K39/00C12N5/0735
CPCC12N2501/13C12N2501/115C12N2501/23A61K2039/525C12N5/0606A61K39/145C12N2760/16151C12N2710/24151C12N2501/125C12N2500/99C12N7/00A61K2039/5252C12N2760/16134A61K39/12A61P31/12A61P31/16C12N2500/90C12N5/0609C12N15/86C12N2710/24121C12N2710/24134C12N2760/16021C12N2760/16034C12N2760/16051
Inventor M·梅赫塔利P·尚皮翁-阿诺A·莱昂
Owner ВАЛЬНЕВА
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