Immortalised chicken embryo fibroblasts
The present invention relates to immortalized chicken embryo fibroblasts, to cell cultures comprising such immortalized cells, to vaccines comprising such cells, to methods for the propagation of avian viruses on such cells, and to methods for the preparation of such cells and such vaccines.
1. A stably transfected immortalized chicken embryo fibroblast (CEF), wherein said immortalized CEF (i) expresses an SV40 T antigen, (ii) expresses a chicken telomerase (cTERT) under the control of a heterologous promoter, and (iii) does not comprise an exogenous retroviral Long Terminal Repeat (LTR) DNA.
2. A cell culture comprising the immortalized CEF of claim 1 .
3. The cell culture of claim 2 , which is infected with an avian virus or avian viral vector.
4. The cell culture of claim 3 , wherein the avian virus or avian viral vector is selected from the group consisting of Marek's Disease virus (MDV), the MDV-related Herpes virus of turkey (HVT), Newcastle Disease virus (NDV), Infectious Bronchitis virus (IBV), Infectious Bursal Disease virus (IBDV), Egg Drop Syndrome virus (EDSV), Turkey Rhinotracheitis virus (TRT), Reovirus (RV) and a HVT vector; wherein said HVT vector comprises an IBDV VP2-gene, an IBV-spike protein gene, an avian influenza HA gene, an ILT gD/gI protein gene, or an NDV F-gene.
5. A method of preparing the immortalized CEF of claim 1 , comprising the steps of:
a) obtaining primary CEF cells;
b) transfecting said primary CEFs with:
i) a first DNA molecule free of LTR sequences, comprising transposon inverted repeats and further comprising a gene encoding the SV40 T antigen under the control of a suitable promoter;
ii) a second DNA molecule free of LTR sequences, comprising transposon inverted repeats and further comprising a gene encoding chicken telomerase (cTERT) under the control of a suitable promoter; and
iii) a third DNA molecule comprising a gene encoding transposase under the control of a suitable promoter; and
c) selecting CEF cells that have been cultured for at least 45 cell cycles.
6. A method of preparing the immortalized CEF of claim 1 , comprising the steps of:
a) obtaining primary CEF cells;
b) transfecting said primary CEF cells with a single DNA molecule free of LTR sequences, comprising (i) transposon inverted repeats and a gene encoding the SV40 T antigen under the control of a suitable promoter, (ii) a gene encoding chicken telomerase under the control of a suitable promoter, and (iii) a gene encoding transposase under the control of a suitable promoter; and
c) selecting CEF cells that have been cultured for at least 45 cell cycles.
7. A method of preparing the immortalized CEF of claim 1 , comprising the steps of:
a) obtaining primary CEF cells;
b) transfecting said primary CEF cells with (i) a first DNA molecule free of LTR sequences, comprising transposon inverted repeats, a gene encoding the SV40 T antigen under the control of a suitable promoter, and a gene encoding chicken telomerase under the control of a suitable promoter, and (ii) a second DNA molecule comprising a gene encoding transposase under the control of a suitable promoter; and
c) selecting CEF cells that have been cultured for at least 45 cell cycles.
8. The method of claim 5 , wherein said CEF cells have been cultured for at least 100 cell cycles.
9. The method of claim 6 , wherein said CEF cells have been cultured for at least 100 cell cycles.
10. The method of claim 7 , wherein said CEF cells have been cultured for at least 100 cell cycles.
11. A method for replicating an avian virus or avian viral vector, wherein said method comprises the steps of:
a) culturing the immortalized CEF of claim 1 ;
b) contacting the immortalized CEF with the avian virus or avian viral vector;
c) allowing the avian virus or avian viral vector to replicate; and
d) isolating a progeny virus or vector from said immortalized CEF.
12. The method of claim 11 , wherein the avian virus or avian viral vector is selected from the group of avian viruses consisting of Marek's Disease virus (MDV), the MDV-related Herpes virus of turkey (HVT), Newcastle Disease virus (NDV), Infectious Bronchitis virus (IBV), Infectious Bursal Disease virus (IBDV), Egg Drop Syndrome virus (EDSV), Turkey Rhinotracheitis virus (TRT), Reovirus (RV), a HVT vector; wherein said HVT vector comprises an IBDV VP2-gene, an IBV-spike protein gene, an avian influenza HA gene, an ILT gD/gI protein gene, or an NDV F-gene.
13. The method of claim 11 , wherein the avian virus or avian viral vector is selected from the group of avian viruses consisting of a MDV and a MDV vector virus.
14. A method of preparing a vaccine comprising an avian virus or an avian viral vector, comprising the step of mixing the cell culture of claim 3 with a pharmaceutically acceptable carrier.
15. The method of claim 14 , wherein the avian virus or an avian viral vector is an MDV.
16. The method of claim 14 , wherein the MDV or MDV viral vector is in a live attenuated form.
17. A vaccine comprising the cell culture of claim 3 , and a pharmaceutically acceptable carrier.
18. A method of preparing a vaccine comprising an avian virus or an avian viral vector, wherein the method comprises the steps of:
a) infecting the cell culture of claim 2 with an avian virus or an avian viral vector;
b) replicating said avian virus or an avian viral vector;
c) isolating a progeny avian virus or avian viral vector; and
d) mixing the progeny avian virus or avian viral vector with a pharmaceutically acceptable carrier.