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.