Immortalized porcine alveolar macrophage
The present invention relates to immortalized porcine alveolar macrophages (PAMs), to cell cultures comprising such PAMs, to methods for the immortalization of PAMs, to methods of replicating PRRS virus on immortalized PAMs and to methods for the preparation of vaccines comprising PRRSV.
1. A method of isolating progeny Porcine Respiratory and Reproductive Virus (PRRSV) from an immortalized porcine alveolar macrophage (PAM) cell, wherein said PAM cell is susceptible to PRRSV, expresses an SV40 T antigen, and does not comprise retroviral Long Terminal Repeat DNA capable of retroviral gene expression; wherein said method comprises the steps of:
a) obtaining an aliquot of PAM cells;
b) transfecting said aliquot of PAM cells with a DNA molecule comprising transposons and comprising a gene encoding the SV40 T antigen under the control of a suitable promoter;
c) culturing the transfected PAM cells for at least 5 cell cycles;
wherein immortalized PAM cells are obtained;
d) selecting immortalized PAM cells;
e) further culturing the immortalized PAM cells;
f) contacting the immortalized PAM cells with a PRRSV;
g) allowing the PRRSV to replicate and form progeny PRRSV; and
h) isolating the progeny PRRSV.
2. The method of claim 1 , wherein said culturing of the transfected PAM cells is for at least 10 cell cycles.
3. The method of claim 1 , wherein said method additionally comprises the step of adding an amount of at least 1 ng/ml of macrophage colony stimulating factor (M-CSF) during said culturing of step c).
4. The method of claim 3 , wherein said method additionally comprises the step of adding an amount of at least 1 ng/ml of macrophage colony stimulating factor (M-CSF) during said culturing of step e).
5. The method of claim 1 , wherein said method additionally comprises the step of adding an amount of at least 1 ng/ml of macrophage colony stimulating factor (M-CSF) during said culturing of step e).
6. The method of claim 3 further comprising mixing the isolated progeny PRRSV of step h) with a pharmaceutically acceptable carrier; wherein the PRRSV and the progeny PRRSV are in a live attenuated form.
7. The method of claim 3 , further comprising the step of inactivating the progeny PRRSV after step g) and mixing the inactivated progeny PRRSV with a pharmaceutically acceptable carrier.
8. The method of claim 3 , wherein inactivating the progeny PRRSV is performed after step h).
9. The method of claim 1 , further comprising mixing the isolated progeny PRRSV of step h) with a pharmaceutically acceptable carrier; wherein the PRRSV and the progeny PRRSV are in a live attenuated form.
10. The method of claim 1 , further comprising the step of inactivating the progeny PRRSV after step g) and mixing the inactivated progeny PRRSV with a pharmaceutically acceptable carrier.
11. The method of claim 1 , wherein inactivating the progeny PRRSV is performed after step h).