Hepatitis-A viruses adapted to human fibroblast cells

Abstract

Hepatitis-A virus, suitable for use in human or animal vaccines, is prepared by adapting the virus first to human kidney cells and subsequently adapting the thus-altered virus to human fibroblast strains. In the process the growth rate of Hepatitis-A virus on the noted tissues is materially increased. This HAV is also a useful antigen in diagnostic test procedures.

What is claimed is: 
     
       1. A process which comprises culturing unmodified HAV on human kidney cells (HKC) and harvesting .[.the thus-.]. produced .Iadd.cell-free .Iaddend.viruses. 
     
     
       2. A process which comprises culturing on HKC isolated .Iadd.cell-free .Iaddend.viruses produced by the process of claim 1 and isolating thus-produced .Iadd.cell-free .Iaddend.viruses. 
     
     
       3. A process for increasing the growth rate of HAV cultured on HKC which comprises (a) the process of claim 2, (b) culturing the resulting isolated .Iadd.cell-free .Iaddend.viruses on HKC and isolating thus-produced .Iadd.cell-free .Iaddend.viruses and (c) repeating step (b) a number of times, each with isolated .Iadd.cell-free .Iaddend.viruses from the immediately preceding culturing on HKC. 
     
     
       4. A process of claim 3 which comprises from about five to ten successive culturing steps. 
     
     
       5. A process according to claim 2 or claim 3 which comprises isolating from the final culturing step those viruses which grow most rapidly and/or are produced in largest quantities. 
     
     
       6. A process according to claim 2 or claim 3 which comprises (a) selecting from each culturing step those viruses which grow most rapidly and/or are produced in largest quantities and (b) culturing only thus-selected viruses in any immediately succeeding culturing step. 
     
     
       7. A process of claim 1 wherein the HAV is Hepatitis-A virus isolate of human origin. 
     
     
       8. A process of claim 7 wherein the HAV consists essentially of that isolated from stool. 
     
     
       9. A process of claim 1 which consists essentially of culturing HAV on HKC. 
     
     
       10. Modified HAV adapted to HKC (HAV/HKC) and having a rate of growth and/or a rate of proliferation significantly greater than that of unmodified HAV cultured on HKC. 
     
     
       11. A process which comprises culturing the modified virus of claim 10 on human fibroblast strains (HFS). 
     
     
       12. A process which comprises culturing on HFS isolated viruses produced by the process of claim 11 and isolating thus-produced viruses. 
     
     
       13. A process for increasing the growth rate of HAV/HKC cultured on HFS which comprises (a) the process of claim 12, (b) culturing the resulting isolated viruses on HFS and isolating thus-produced viruses and (c) repeating step (b) a number of times, each with isolated viruses from the immediately preceding culturing on HFS. 
     
     
       14. A process of claim 13 which comprises from about 50 to 75 successive culturing steps. 
     
     
       15. A process according to claim 13 or claim 14 which comprises (a) selecting from each culturing step those viruses which grow most rapidly and/or proliferate in greatest quantities and (b) culturing only thus-selected viruses in any immediately succeeding culturing step. 
     
     
       16. Modified HAV according to claim 10 and further adapted to HFS (HAV/HFS) and having a rate of growth and/or a rate of proliferation significantly greater than that of modified HAV according to claim .[.8.]. .Iadd.10 .Iaddend.and cultured on HFS. 
     
     
       17. A process which comprises: (a) culturing unmodified HAV on human kidney cells (HKC) and harvesting thus-produced viruses,   (b) culturing isolated viruses from step (a) on EIKC and isolating thus-produced viruses,   (c) culturing isolated viruses from step (b) on HKC and isolating thus-produced viruses,   (d) repeating step (c) a number of times, each with isolated viruses from the immediately preceding culturing on HKC to obtain HAV adapted to HKC (HAV/HKC) and having a rate of growth and/or a rate of proliferation significantly greater than that of HAV cultured on HKC, the isolated viruses cultured in and isolated from the products of steps (b) through (d) being those which grow most rapidly and/or are produced in largest quantities;   (e) culturing HAV/HKC isolated from step (d) on human fibroblast strains (HFS) and isolating thus-produced viruses,   (f) culturing isolated viruses from step (e) on HFS and isolating thus-produced viruses,   (g) culturing isolated viruses from step (f) on HFS and isolating thus-produced viruses,   (h) repeating step (g) a number of times, each with isolated viruses from the immediately preceding culturing on HFS to obtain HAV adapted to HFS (HAV/HFS) and having a rate of growth and/or a rate of proliferation significantly greater than that of HAV/HKC cultured on HFS, the isolated viruses cultured in and isolated from the products of steps (e) through (h) being those which grow most rapidly and/or are produced in largest quantities. .Iadd.   
     
     
       18.  A process which comprises culturing unmodified HAV on human kidney cells (HKC) in a culture medium until supernatant in the culture medium is positive for HAV, inocculating fresh HKC with the supernatant, culturing again until supernatant is positive for HAV, and harvesting thus-produced viruses from the latter supernatant. .Iaddend. .Iadd. 
     
     
       19.  A process of claim 1 which comprises culturing unmodified HAV on human kidney cells (HKC) and harvesting, directly from culture supernatant, produced cell-free viruses which are free from cell particles without further separation. .Iaddend. .Iadd.20. A process of claim 1 which comprises culturing unmodified HAV on human kidney cells (HKC) until cell-free viruses pass through intact cell walls into culture supernatant, and harvesting produced cell-free viruses from the supernatant. .Iaddend.