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 PRRSV from an immortalized porcine alveolar macrophage (PAM), wherein said PAM is susceptible to Porcine Respiratory and Reproductive Virus (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 a cell-containing bronchoalveolar lavage sample from a porcine subject,
b) separating a cellular component from said sample,
c) transfecting said cellular component with a DNA molecule comprising transposons and comprising a gene encoding the SV40 T antigen under the control of a suitable promoter,
d) selecting immortalized PAM cells that have been cultured for at least 5 cell cycles,
e) further culturing the selected immortalized PAM cells,
f) contacting the immortalized PAM cells with the PRRSV,
g) allowing the PRRSV to replicate and form progeny PRRSV, and
h) isolating the progeny PRRSV.
2. The method of claim 1 , wherein PAM cells are selected that have been cultured for at least 10 cell cycles.
3. The method of claim 2 , 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 step d) and while culturing the immortalized PAM.
4. 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.
5. 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.
6. The method of claim 1 , further comprising the step of inactivating the progeny PRRSV after step h) and mixing the inactivated progeny PRRSV with a pharmaceutically acceptable carrier.
7. The method of claim 1 , wherein said method further comprises the step of adding an amount of at least 5 ng/ml of macrophage colony stimulating factor (M-CSF) to the cell-containing bronchoalveolar lavage sample, the cellular component, or both the cell-containing bronchoalveolar lavage sample and the cellular component before the transfection step.
8. 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 step d) or while culturing the PAM.
9. The method of claim 8 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 8 , further comprising the step of inactivating the progeny PRRSV after step a) and mixing the inactivated progeny PRRSV with a pharmaceutically acceptable carrier.
11. The method of claim 8 , further comprising the step of inactivating the progeny PRRSV after step h).