Production of poxviruses with adherent or non adherent avian cell lines
The present invention relates to a method for replicating pox viruses such as vaccinia virus comprising the steps of inoculating avian embryonic stem cells with viral particles and culturing said cells in a basal medium until cells lysis occurs and newly produced viral particles are released in said medium.
1. A method for replicating a native or recombinant vaccinia virus comprising the steps of:
1) producing avian embryonic derived stem cells by:
a) culturing avian embryonic cells in a complete culture medium complemented in serum containing:
i) exogenous growth factors comprising the trophic factors SCF, IGF-1 and bFGF, and cytokines whose action is through a receptor which is associated with the gp130 protein, said cytokines being selected from the group consisting of LIF, interleukin 11, interleukin 6, interleukin 6 receptor, CNFT, oncostatin and cardiotrophin; and
ii) a feeder layer;
b) passage by modifying the culture medium so as to obtain the withdrawal of said growth factors, of the serum and/or of the feeder layer;
c) establishing adherent or non-adherent cell lines capable of proliferating in a basal medium in the absence of exogenous growth factors, serum and/or inactivated feeder layer;
2) inoculating the resultant avian embryonic derived stem cells with viral particles of said vaccinia virus; and
3) culturing said cells in a basal medium until cell lysis occurs and newly produced viral particles of said vaccinia virus are released in said medium.
2. The method according to claim 1 , wherein said avian embryonic cells are chicken or duck embryonic cells.
3. The method according to claim 1 , wherein the feeder layer is inactivated.
4. The method according to claim 1 , wherein the avian embryonic derived stem cells produced in step 1) are non-adherent stem cells which proliferate in suspension in a medium free of exogenous growth factors, serum and feeder cells.
5. The method according to claim 1 , wherein said vaccinia virus is the native Modified Vaccinia virus Ankara (MVA) or a recombinant thereof.
6. The method according to claim 1 , wherein the avian embryonic derived stem cells produced in step 1) are capable of proliferating for at least 600 days.
7. The method according to claim 1 , wherein the avian embryonic derived cell lines produced in step 1) are non-adherent stem cells which proliferate in suspension in a medium free of exogenous growth factors.
8. The method according to claim 1 , wherein the avian embryonic derived cell lines produced in step 1) are non-adherent stem cells which proliferate in suspension in a medium free of serum (serum-free medium).
9. The method according to claim 1 , wherein the avian embryonic derived cell lines produced in step 1) are non-adherent stem cells which proliferate in suspension in a medium free of exogenous growth factors and serum.
10. The method according claim 1 , wherein the avian embryonic derived cell lines produced in step 1) have at least one of the following characteristics:
a high nucleo-cytoplasmic ratio,
an endogenous alkaline phosphatase activity,
an endogenous telomerase activity,
a reactivity with specific antibodies selected from the group of antibodies consisting of SSEA-1 (TEC01), SSEA-3 and EMA-1.
11. The method according to claim 1 , wherein said basal medium is selected from the group consisting of DMEM, GMEM, HamF12 and McCoy basal medium supplemented with additives.