Animal cells and processes for the replication of influenza viruses

Abstract

Animal cells are described which can be infected by influenza viruses and which are adapted to growth in suspension in serum-free medium. Processes for the replication of influenza viruses in cell culture using these cells are furthermore described, as well as vaccines which contain the influenza viruses obtainable by the process or constituents thereof.

1 . A process for making a vaccine for administration to humans or animals, the process comprising:
 (a) incubating MDCK cells in a serum-free medium;   (b) adding influenza viruses to the serum-free medium to infect the MDCK cells incubated in step (a), wherein protease is present in the serum-free medium or added to the serum-free medium before, during, or after infection with the influenza viruses;   (c) culturing the MDCK cells infected in step (b) to replicate the influenza viruses;   (d) isolating the influenza viruses replicated in step (c); and   (e) formulating the influenza viruses isolated in step (d) to provide the vaccine, wherein the vaccine contains disintegrated viruses.   
     
     
         2 . The process of  claim 1 , wherein the MDCK cells incubated in step (a) are incubated in a perfusion system. 
     
     
         3 . The process of  claim 1 , wherein the MDCK cells incubated in step (a) are incubated in a batch process. 
     
     
         4 . The process of  claim 1 , wherein the MDCK cells infected in step (b) are cultured in step (c) at a regulated pH and pO 2 . 
     
     
         5 . The process of  claim 1 , wherein the serum-free medium is a protein-free medium. 
     
     
         6 . The process of  claim 1 , wherein the influenza viruses replicated in step (c) are isolated in step (d) by a process comprising centrifugation. 
     
     
         7 . The process of  claim 6 , wherein the influenza viruses replicated in step (c) are isolated in step (d) by a process comprising sucrose gradient centrifugation. 
     
     
         8 . The process of  claim 1 , wherein the MDCK cells are of the cell line MDCK 33016 (DSM ACC 2219). 
     
     
         9 . The process of  claim 1 , wherein the influenza viruses isolated in step (d) are inactivated by formaldehyde. 
     
     
         10 . The process of  claim 1 , wherein the vaccine comprises an adjuvant. 
     
     
         11 . The process of  claim 1 , wherein the influenza viruses isolated in step (d) are disintegrated using a detergent, prior to step (e). 
     
     
         12 . The process of  claim 1 , wherein the influenza viruses isolated in step (d) are disintegrated using a solvent, prior to step (e). 
     
     
         13 . The process of  claim 1 , wherein the influenza viruses isolated in step (d) are disintegrated using a detergent and a solvent, prior to step (e). 
     
     
         14 . The process of  claim 1 , wherein the vaccine is a subunit vaccine. 
     
     
         15 . The process of  claim 1 , wherein step (e) comprises adjusting the vaccine containing disintegrated viruses to a desired amount of antigen. 
     
     
         16 . The process of  claim 1 , wherein the protease is trypsin and the trypsin is present in the serum-free medium, or added to the serum-free medium before, during, or after infection with influenza viruses, at a concentration from 1 to 200 μg/ml. 
     
     
         17 . The process of  claim 1 , wherein the influenza viruses have a genome segment from strain A/PR/8134. 
     
     
         18 . The process of  claim 1 , wherein the MDCK cells are incubated adherently. 
     
     
         19 . The process of  claim 18 , wherein the MDCK cells are incubated adherently on microcarriers. 
     
     
         20 . The process of  claim 1 , wherein the MDCK cells are incubated in suspension. 
     
     
         21 . The process of  claim 1 , wherein the MDCK cells infected in step (b) are cultured in step (c) at a temperature below 37° C. 
     
     
         22 . The process of  claim 21 , wherein the MDCK cells infected in step (b) are cultured in step (c) at a temperature of 30° C. to 36° C. 
     
     
         23 . The process of  claim 21 , wherein the MDCK cells infected in step (b) are cultured in step (c) for 2 to 10 days. 
     
     
         24 . The process of  claim 1 , wherein the process is performed in a culture vessel that is not opened after adding the influenza viruses in step (b) or before isolating the influenza viruses in step (d). 
     
     
         25 . The process of  claim 1 , wherein (i) the influenza viruses replicated in step (c) are isolated in step (d) by a process comprising centrifugation, (ii) the influenza viruses isolated in step (d) are inactivated by formaldehyde, and (iii) the MDCK cells are incubated adherently. 
     
     
         26 . A process for making a vaccine for administration to humans or animals, the process comprising:
 (a) incubating MDCK cells which can be infected by influenza viruses in a serum-free medium;   (b) adding the influenza viruses to the serum-free medium to infect the MDCK cells incubated in step (a), wherein protease is present in the serum-free medium or added to the serum-free medium before, during, or after infection with influenza viruses;   (c) culturing the MDCK cells infected in step (b) to replicate the influenza viruses;   (d) isolating the influenza viruses replicated in step (c) by a process comprising centrifugation; and   (e) formulating the influenza viruses replicated in step (c) to provide the vaccine, wherein the vaccine contains live attenuated viruses.   
     
     
         27 . The process of  claim 26 , wherein step (e) comprises adjusting the influenza viruses isolated in step (d) to a desired titer. 
     
     
         28 . The process of  claim 26 , wherein step (e) further comprises lyophilizing the viruses. 
     
     
         29 . The process of  claim 26 , wherein step (e) further comprises stabilizing the viruses in liquid form. 
     
     
         30 . A process for making a vaccine for administration to humans or animals, the process comprising:
 (a) incubating MDCK cells adherently in a serum-free medium;   (b) adding influenza viruses, having a genome segment from strain A/PR/8/34, to the serum-free medium to infect the MDCK cells incubated in step (a), wherein protease is present in the serum-free medium or added to the serum-free medium before, during, or after infection with the influenza viruses;   (c) culturing the MDCK cells infected in step (b) to replicate the influenza viruses;   (d) isolating the influenza viruses replicated in step (c);   (e) inactivating the influenza viruses isolated in step (d) by formaldehyde; and   (f) formulating the influenza viruses inactivated in step (e) to provide the vaccine, wherein the vaccine contains disintegrated viruses.