Method for adapting influenza viruses to Vero cells

1. A method for adapting an influenza virus to Vero cells, comprising:

infecting Vero cells with an influenza virus at a first infectious dose;

cultivating infected Vero cells in a spinner flask to produce viral activities;

harvesting a first influenza virus;

infecting Vero cells with said first influenza virus at a second infectious dose to produce viral activities, wherein said second infectious dose is greater than or equal to said first infectious dose;

harvesting a second influenza virus; and

repeating the process, and harvesting a Vero cell adapted influenza virus,

wherein the Vero cells are attached to microcarriers.

2. A method of claim 1 , wherein at least one of said first infectious dose and said second infectious dose is expressed by MOI, and the MOI is 0,00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, or any value between 0.00001 and 2.0.

3. A method of claim 1 , wherein at least one of said first infectious dose and said second infectious dose is expressed by MOI, and the MOI of influenza virus is 0.00001, 0.00004, 0.00013, 0.0002, 0.0011, 0.0013, 0.002 0.0022, 0.0026, 0.0072, 0.008, 0.018, 0.019, 0.030, 0.033, 0.05, 0.16, 0.22, 0.25, 0.47, 0.5, 0.63, 1.0, 1.58, 1.6, or 2.0.

4. A method of claim 3 , wherein said first infectious dose is 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of said second infectious dose.

5. A method of claim 1 , wherein said viral activities comprise viral replication, viral production, hemagglutinin (HA) titer or neuraminidase (NA) titer.

6. A method of claim 1 , further comprising passing an influenza virus in the Vero cells with a number of passages of 5-10.

7. A method of claim 6 , wherein said number of passages is 5, 6, 7, 8, 9, or 10.

8. A method of claim 7 , wherein said influenza virus is acquired from embryonated chicken eggs, allantoic cavity of chicken embryo, or amniotic membrane of chicken embryo.

9. A method of claim 8 , wherein said influenza virus is selected from a group including A/California/7/2009(H1N1)pdm09, A/Michigan/45/2015(H1N1)pdm09, A/Switzerland/9715293/2013(H3N2), A/Hong Kong/4801/2014(H3N2), B/Brisbane/60/2008, and B/Phuket/3073/2013.

10. A method of claim 8 , comprising using said Vero cell adapted influenza viruses to produce a composition, wherein said composition comprises polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic or polyamide compound, at least one positive ion, influenza viruses and/or influenza antigens.

11. A method of claim 10 , wherein at least one of said influenza antigens is selected from a group including HA and NA; wherein said polyamide compound is selected from a group including spermidine sault, spermidine, N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butandiamine, spermine BR, spermine, OS-dimethylphosphoramidothioate, polylysine, and aminoglycoside; and wherein said positive ion is selected from a group including calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, and zinc.

12. A method of claim 10 , comprising administrating said composition by nasal spray, intramuscular delivery, intravenous delivery, and/or oral administration.

13. A method of claim 1 , comprising cultivating the Vero cells in a single-use bioreactor.

14. A method of claim 2 , wherein at least one of said first infectious dose and said second infectious dose is expressed by MOI, and the MOI of influenza virus is 0.00001, 0.00004, 0.00013, 0.0002, 0.0011, 0.0013, 0.002 0.0022, 0.0026, 0.0072, 0.008, 0.018, 0.019, 0.030, 0.033, 0.05, 0.16, 0.22, 0.25, 0.47, 0.5, 0.63, 1.0, 1.58, 1.6, or 2.0.

15. A method of claim 2 , wherein said viral activities comprise viral replication, viral production, hemagglutinin (HA) titer or neuraminidase (NA) titer.

16. A method of claim 2 , further comprising passing an influenza virus in the Vero cells with a number of passages of 5-10.

17. A method of claim 11 , comprising administering said composition by nasal spray, intramuscular delivery, intravenous delivery, and/or oral administration.

18. A method comprising:

infecting Vero cells with an influenza virus at a first infectious dose;

cultivating infected Vero cells in a spinner flask to produce viral activities;

harvesting a first influenza virus;

infecting Vero cells with said first influenza virus at a second infectious dose to produce viral activities, wherein said second infectious dose is greater than or equal to said first infectious dose;

harvesting a second influenza virus;

repeating the process, and harvesting a Vero cell adapted influenza virus,

wherein the steps of harvesting and infecting are both performed 5-10 times; and

wherein the Vero cells attached to microcarriers; and

using said Vero cell adapted influenza viruses to produce a composition, wherein said composition comprises polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic or polyamide compound, at least one positive ion, influenza viruses and/or influenza antigens.

19. The method of claim 18 , wherein said influenza virus is selected from a group including A/California/7/2009(H1N1)pdm09, A/Michigan/45/2015(H1N1)pdm09, A/Switzerland/9715293/2013(H3N2), A/Hong Kong/4801/2014(H3N2), B/Brisbane/60/2008, and B/Phuket/3073/2013.

20. The method of claim 18 , wherein at least one of said influenza antigens is selected from a group including HA and NA; wherein said polyamide compound is selected from a group including spermidine sault, spermidine, N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butandiamine, spermine BR, spermine, OS-dimethylphosphoramidothioate, polylysine, and aminoglycoside; and wherein said positive ion is selected from a group including calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, and zinc.