Process for preparing live smallpox vaccine
View Patent ↗A safer live smallpox vaccine, which contains a lowered content of revertants, is provided. A process for manufacturing a live smallpox vaccine which comprises steps of: inoculating a master seed solution of an attenuated vaccinia virus to an appropriate number of containers (1 to n wherein n is an integer) of rabbit kidney cells and incubating them; inoculating a portion of the cultured solution obtained from each container to RK-13 cells and to Vero E6 cells and incubating them to thereby select containers which contain a cultured solution that forms plaques in RK-13 cells but not in Vero E6 cells; and preparing a drug substance of vaccine using the aforementioned cultured solution (working seed solution), and a live smallpox vaccine prepared in the aforementioned process.
1. A process for preparing a working seed solution of a vaccine containing as an active ingredient a mutant virus that is likely to generate revertants, which comprises the following steps (1) to (3):
(1) inoculating a solution of a mutant virus to 1 to n containers, wherein n is an integer, of an isolated animal cell and incubating-them;
(2) diluting a portion of the cultured solution from each container and inoculating the diluted solution to RK-13 cells and to Vero cells and incubating them; and
(3) selecting containers which contain a cultured solution that forms plaques in RK-13 cells but does not form plaques in Vero cells and pooling each cultured solution obtained from selected containers.
2. The process of claim 1 , wherein a viral content of the diluted solution in (2) above is 1×10 4 PFU or more.
3. The process of claim 1 , wherein the isolated animal cell is a primary cell.
4. The process of claim 3 , wherein the primary cell is derived from rabbit kidney.
5. The process of claim 1 , wherein the mutant virus is an attenuated vaccinia virus.
6. The process of claim 5 , wherein the attenuated vaccinia virus is the LC16m8 strain.
7. A process for manufacturing a smallpox vaccine, which comprises inoculating the working seed solution obtained through the process of claim 5 above to an isolated animal cell, incubating and then collecting a viral solution from the culture.
8. The process of claim 7 , wherein the concentration is a MOI of 0.001 to 5, at which the working virus solution is inoculated, and wherein the number of the isolated animal cells is 1×10 5 to 1×10 12 .
9. The process of claim 7 , wherein the animal cell is a primary cell.
10. The process of claim 9 , wherein the primary cell is derived from rabbit kidney.
11. The process of claim 8 , wherein the MOI is 0.1 to 1.
12. The process of claim 8 , wherein the number of the isolated animal cells is 0.5×10 8 to 3×10 8 .