Compositions and methods for the production of alpha-herpesviruses
The present invention relates to virus growth media that improve the yield of alpha-herpesviruses (e.g., HSV-2) grown in cell cultures. The growth media of the invention include two additives, a disaccharide and a lipid mixture, that can be added to serum-free or serum-enriched growth media to improve the efficiency of virus production. The invention further provides methods of producing alpha-herpesviruses (e.g., HSV-2) in such growth media.
1. A composition comprising (i) a growth medium supplemented with between 5 mM and 500 mM of a disaccharide and a lipid mixture, (ii) Vero cells that express herpes simplex virus 2 (HSV-2) polypeptides U L 5 and U L 29, and (iii) an HSV-2 having inactivating mutations in U L 5 and U L 29.
2. The composition of claim 1 , wherein said disaccharide is sucrose, lactose, maltose, trehalose, or cellobiose, or a mixture thereof.
3. The composition of claim 1 , wherein said disaccharide is present in said medium at 50 mM or 80 mM, or between 50 mM and 80 mM.
4. The composition of claim 1 , wherein said medium further comprises methyl-β-cyclodextrin and/or serum.
5. The composition of claim 4 , wherein said serum is fetal bovine serum (FBS).
6. The composition of claim 1 , wherein said lipid mixture is from a plant, is synthetic, comprises cholesterol, and/or comprises stigmastanol.
7. The composition of claim 1 , wherein said medium increases production of said HSV-2 in the cells by at least 10% relative to HSV-2 production in cells grown in a medium lacking said disaccharide and said lipid mixture.
8. A method of increasing herpesvirus production in cells, said method comprising culturing the composition of claim 1 , wherein said composition increases production of the herpesvirus by at least 10% relative to a herpesvirus cultured in cells grown in a medium lacking said disaccharide or said lipid mixture, wherein said herpesvirus is a replication-defective herpesvirus having inactivating mutations in U L 5 and U L 29, optionally in combination with an inactivating mutation in U L 41.
9. The method of claim 8 , wherein said cells are AV529-19 cells.
10. The method of claim 8 , wherein said cells express the herpesvirus polypeptides U L 5 and/or U L 29.
11. The method of claim 8 , wherein said disaccharide is sucrose, lactose, maltose, trehalose, or cellobiose, or a mixture thereof.
12. The method of claim 8 , wherein said disaccharide is present at 50 mM or 80 mM, or between 50 mM and 80 mM.
13. The method of claim 8 , wherein said medium further comprises methyl-β-cyclodextrin and/or serum.
14. The method of claim 13 , wherein said serum is fetal bovine serum (FBS).
15. The method of claim 8 , wherein said lipid mixture is from a plant, is synthetic, comprises cholesterol, and/or comprises stigmastanol.
16. The method of claim 8 , wherein said method increases production of a herpesvirus in the cells by at least 10% relative to herpesvirus production in cells grown in a composition lacking said disaccharide and said lipid mixture.
17. The composition of claim 1 , wherein said HSV-2 has an inactivating mutation in U L 41.
18. The composition of claim 1 , wherein said HSV-2 is strain dl5-29.
19. The composition of claim 18 , wherein said Vero cells are AV529-19 cells.
20. A composition comprising (i) a growth medium supplemented with between 5 mM and 500 mM of a disaccharide and a lipid mixture, (ii) Vero cells engineered to express herpes simplex virus polypeptides U L 5 and U L 29, and (iii) a herpes simplex virus 2 (HSV-2) having inactivating mutations in U L 5 and U L 29.
21. The composition of claim 20, wherein said disaccharide is sucrose, lactose, maltose, trehalose, or cellobiose, or a mixture thereof.
22. The composition of claim 20, wherein said disaccharide is present in said medium at 50 mM or 80 mM, or between 50 mM and 80 mM.
23. The composition of claim 20, wherein said medium further comprises methyl-β-cyclodextrin and/or serum.
24. The composition of claim 20, wherein said lipid mixture is from a plant, is synthetic, comprises cholesterol, and/or comprises stigmastanol.
25. The composition of claim 20, wherein said medium increases production of said HSV-2 in the cells by at least 10% relative to HSV-2 production in cells grown in a medium lacking said disaccharide and said lipid mixture.
26. The composition of claim 20, wherein said HSV-2 is strain dl5-29.
27. The composition of claim 20, wherein said Vero cells are AV529-19 cells.