Method for production of pH stable enveloped viruses
View Patent ↗The present invention provides a method for producing pH-stable enveloped viruses wherein said viruses are used for infection of host cells under low pH conditions and for incubation with cell culture cells under conditions of low pH, as well as influenza viruses obtainable by this method which exhibit a high growth rate in cell culture, increased pH and temperature stability and which have human receptor specificity.
1. A method for producing influenza viruses characterized in that it comprises the following steps:
a) diluting influenza viruses in a solution having a pH of between 5.2 and 5.9;
b) infecting host cells with at least one infectious virus particle of the influenza viruses, wherein:
i) the virus particle is added to said cells; and
ii) said cells and said virus particle are incubated at a pH of between 5.2 and 5.9 to provide a virus/cell complex;
c) cultivating the infected host cells to propagate viruses;
d) harvesting the viruses; and
e) optionally, purifying and/or characterising the viruses.
2. A method according to claim 1 characterized in that the viruses are diluted in step a) in a solution having a pH of between 5.5 and 5.8.
3. A method according to claim 2 characterized in that the viruses are diluted in step a) in a solution having a pH of about 5.6.
4. A method according to claim 1 characterized in that said cells and said virus particle are incubated in step b) at a pH of between 5.5 and 5.8.
5. A method according to claim 4 characterized in that said cells and said virus particle are incubated in step b) at a pH of about 5.6.
6. A method according to claim 1 further comprising the step of adding a macrolide polyene antibiotic to the solution of step a), step b), or c).
7. A method according to claim 1 characterized in that the cells are tissue culture cells.
8. A method according to claim 7 , wherein the tissue culture cells are selected from the group consisting of BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, W1-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0 cells, and PerC6 cells.
9. A method according to claim 1 characterized in that the influenza viruses are selected from the group consisting of influenza A, influenza B, and influenza C.
10. A method according to claim 9 characterized in that the influenza viruses are attenuated influenza viruses.
11. A method according to claim 9 characterized in that the influenza viruses comprise a deletion or modification within the NS1 gene.
12. A method according to claim 9 characterized in that the influenza viruses are cold adapted viruses.
13. A method according to claim 1 characterized in that the viruses are diluted in a buffer which can provide a range of pH values and which is physiological for the cells.
14. A method according to claim 13 characterized in that the buffer is selected from the group consisting of MES buffer, citric buffer, and acetate buffer.
15. A method according to claim 1 characterized in that a cultivation medium is used to cultivate the cells, wherein the cultivation medium is a serum free, low protein medium.
16. A method according to claim 1 characterized in that the viruses are passaged in the host cells for at least one passage.
17. A method according to claim 1 , further comprising the step of inoculating a cell culture with one or more virus particles harvested in step (d).
18. A method according to claim 17 , wherein the cell culture has a volume of at least 200 liters.
19. A method according to claim 6 , wherein the polyene antibiotic is amphotericin B.
20. A method according to claim 1 further comprising the step of adding a derivative of a macrolide polyene antibiotic to the solution of step a), step b), or step c).