Influenza vaccine production in liquid cell culture

Abstract

Infectivity and replication of influenza viruses in successive numbers of cells of the same liquid cell culture is assured by including a protein hydrolyzing enzyme in the culture during virus incubation. Technique overcomes "one-step growth cycle" of virus and allows commercial influenza vaccine production from liquid cell cultures instead of from more costly embryonated chicken eggs. Resulting vaccine is thus substantially free of egg proteins.

We claim: 
     
       1. A method of preparing an influenza virus vaccine which comprises the steps of: (a) infecting a portion of the cells of a liquid cell culture substantially free of proteolytic enzyme with an influenza virus;   (b) incubating the infected culture in the presence of a proteolytic enzyme under conditions sufficient to assure further infection and replication of the virus in cells other than the originally infected cells and an EID 50  /ml titer of at least about 10 7  ;   (c) harvesting the virus from the culture, and;   (d) preparing a vaccine from the harvested virus.   
     
     
       2. The method of claim 1 wherein the cell culture of step (a) comprises a confluent monolayer of cells. 
     
     
       3. The method of claim 1 wherein the cell culture of step (a) comprises a liquid suspension of cells. 
     
     
       4. The method of claim 1 wherein the vaccine preparation of step (d) includes the step of inactivating the harvested virus. 
     
     
       5. The method of claim 1 wherein the vaccine preparation of step (d) includes the step of attenuating the harvested virus. 
     
     
       6. The method of claim 1 wherein the proteolytic enzyme of step (b) is selected from trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxypeptidase. 
     
     
       7. The method of claim 1 wherein the virus is an equine influenza virus. 
     
     
       8. The method of claim 7 wherein the virus is a strain selected from equine influenza A1 and equine influenza A2 virus strains. 
     
     
       9. The method of claim 1 wherein the virus is a human influenza virus. 
     
     
       10. The method of claim 9 wherein the virus is selected from B/Hong Kong, A/Texas, and A/USSR virus strains. 
     
     
       11. The method of claim 1 wherein the cells of the cell culture are dog kidney cells. 
     
     
       12. The method of claim 1 wherein the cell culture of step (a) is a dog kidney cell culture, the virus of step (a) is an equine influenza virus, and the enzyme of step (b) is trypsin. 
     
     
       13. The method of claim 1 wherein the cell culture of step (a) is a dog kidney cell culture, the virus of step (a) is a human influenza virus, and the enzyme of step (b) is trypsin. 
     
     
       14. The method of claim 1 wherein the enzyme of step (b) is present in an amount sufficient to assure infectivity and replication of the virus in culture cells other than the cells comprising that portion infected in step (a). 
     
     
       15. The method of claim 14 wherein the amount of trypsin ranges from about 4 to about 25 micrograms per ml of culture media. .Iadd. 
     
     
       16.  A method of preparing an influenza virus vaccine which comprises the steps of (a) infecting a portion of the cells of a liquid cell culture substantially free of proteolytic enzyme with an influenza virus;   (b) incubating the already infected culture in the presence of a proteolytic enzyme present in an amount sufficient to assure further infection and replication of the virus in cells other than the originally infected cells until the EID 50  /ml titer has increased by a factor of at least about 10 2  ;   (c) harvesting the virus from the culture; and   (d) preparing a vaccine from the harvested virus. .Iaddend. .Iadd.   
     
     
       17.  The method of claim 16 wherein the proteolytic enzyme is present at a concentration of between about 4 and 25 micrograms per ml of culture medium. .Iaddend. .Iadd.18. The method of claim 16 wherein the cells are incubated with the infecting inoculum for at least about one hour before the proteolytic enzyme is added. .Iaddend. .Iadd.19. The method of claim 16 wherein the proteolytic enzyme is selected from the group consisting of trypsin, chymotrypsin, pepsin, pancreatin, pronase and carboxypeptidase. .Iaddend. .Iadd.20. The method of claim 19 wherein the proteolytic enzyme is trypsin. .Iaddend. .Iadd.21. The method of claim 20 wherein the trypsin is present at a concentration of between about 4 and 25 micrograms per ml of culture medium. .Iaddend. .Iadd.22. The method of claim 21 wherein the cells are incubated with the infecting inoculum for at least about one hour before the trypsin is added. .Iaddend. .Iadd.23. The method of claim 
     
     
        22 wherein the virus is an equine influenza virus. .Iaddend. .Iadd.24. The method of claim 23 wherein the virus is a strain selected from the group consisting of the A1 and A2 equine influenza strains. .Iaddend. .Iadd.25. The method of claim 22 wherein the virus is a human influenza virus. .Iaddend. .Iadd.26. The method of claim 25 wherein the virus is selected from the group consisting of the B/Hong Kong, A/Texas, and A/USSR human influenza strains. .Iaddend. .Iadd.27. The method of claim 23 or claim 24 or claim 25 or claim 26 wherein the cells of the cell culture are dog kidney cells. .Iaddend. .Iadd.28. The method of claim 16 wherein the cell culture which is infected is a liquid cell suspension culture. .Iaddend. .Iadd.29. The method of claim 28 wherein the cell culture is maintained as a liquid cell suspension culture until the virus is harvested. .Iaddend. .Iadd.30. The method of claim 16 wherein the cell culture is incubated with the proteolytic enzyme as a confluent monolayer of cells. .Iaddend. .Iadd.31. The method of claim 30 wherein the cell culture is incubated with the proteolytic enzyme in a roller bottle, Roux bottle or Povitsky flask. .Iaddend. .Iadd.32. The method of claim 31 wherein the cell culture is incubated with the proteolytic enzyme in a 
     
     
        roller bottle. .Iaddend. .Iadd.33.  A method of preparing an equine influenza vaccine which is essentially free of egg proteins which comprises the steps of (a) incubating a liquid cell culture with an amount of an inoculum of the influenza virus sufficient to infect a portion but not all of the cells in the absence of any substantial amount of proteolytic enzyme for between about 1 to 72 hours;   (b) combining the infected cell culture with between about 4 and 25 micrograms per ml of culture of trypsin and incubating until maximum cytopathic effect is observed and the EID 50  /ml titer has increased by a factor of at least about 10 2  ;   (c) harvesting the virus from the culture; and   
     
     
       (d) preparing a vaccine from the culture. .Iaddend. .Iadd.34.  The method of claim 33 wherein the cells of the culture are dog kidney cells. .Iaddend.