Method for producing temperature-sensitive morbillivirus
View Patent ↗It was found that a mutation of an amino acid at a specific position in the P protein of a morbillivirus gives a temperature-sensitivity character to a virus. By introducing this mutation, a virus to which a temperature-sensitivity character has been introduced can be produced. According to this invention, attenuated viruses useful in the preparation of vaccines can be easily produced.
1. An isolated polynucleotide that encodes a Morbillivirus P protein, wherein said Morbillivirus is other than the AIK-C measles virus strain, wherein said P protein has a mutation of an amino acid at a position corresponding to the 439 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, and wherein said mutation confers temperature-sensitivity on the virus when introduced into the virus.
2. The polynucleotide according to claim 1 , wherein said polynucleotide encodes a protein having at least 40% identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein the protein has a mutation of an amino acid at a position corresponding to the 439 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein said mutation confers temperature-sensitivity on the virus when introduced into the virus.
3. The polynucleotide according to claim 1 , wherein the mutation of an amino acid is a substitution to proline.
4. The polynucleotide according to claim 1 , wherein the polynucleotide encodes a measles virus P protein.
5. The polynucleotide according to claim 1 , wherein the P protein has an additional mutation of an amino acid at a position corresponding to the 110 th and/or 275 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein said additional mutation confers temperature-sensitivity on the virus when introduced into the virus.
6. The polynucleotide according to claim 5 , wherein either or both additional mutation(s) is a substitution to tyrosine.
7. An isolated or recombinantly expressed P protein of a Morbillivirus other than the AIK-C measles virus strain
wherein said P protein comprises a mutation of an amino acid at a position corresponding to the 439 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, and wherein said mutation confers temperature-sensitivity on the virus when introduced into the virus.
8. An expression vector into which the isolated polynucleotide according to claim 1 is inserted.
9. The vector according to claim 8 , wherein the vector is used for reconstituting a measles virus on which temperature-sensitivity is conferred.
10. A method for conferring temperature-sensitivity on a Morbillivirus, said method comprising introducing, into a Morbillivirus P protein, a mutation of an amino acid at a position corresponding to 439 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
11. The method of claim 10 , wherein the Morbillivirus P protein has at least 40% identity to the amino acid sequence set forth in SEQ ID NO: 2.
12. The method according to claim 10 , wherein the mutation of an amino acid is a substitution to proline.
13. The method according to claim 10 , wherein the Morbillivirus is a measles virus.
14. The method according to claim 10 , wherein said method further comprises introducing, into the Morbillivirus P protein, an additional mutation of an amino acid at a position corresponding to the 110 th and/or 275 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
15. The method according to claim 14 , wherein each of the mutations of an amino acid at a position corresponding to the 110 th and/or 275 th position is a substitution to tyrosine.
16. An isolated Morbillivirus on which temperature-sensitivity is conferred, wherein said virus comprises a polynucleotide according to claim 1 .
17. The virus according to claim 16 , wherein the virus is an attenuated virus.
18. A pharmaceutical composition comprising the virus according to claim 16 and a pharmacologically acceptable carrier or medium.
19. The pharmaceutical composition according to claim 18 , wherein the pharmaceutical composition is used as a vaccine.
20. The virus according to claim 16 , wherein said virus is obtainable by a method comprising introducing a mutation into the P protein of the virus at an amino acid at a position corresponding to the 439 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
21. The Morbillivirus P protein according to claim 7 , wherein said protein has at least 40% identity to the amino acid sequence set forth in SEQ ID NO: 2.
22. The Morbillivirus P protein according to claim 7 , wherein the mutation of an amino acid is a substitution to proline.
23. The Morbillivirus P protein according to claim 7 , wherein the protein is a measles virus P protein.
24. The Morbillivirus P protein according to claim 7 , wherein the P protein further has an additional mutation of an amino acid at a position corresponding to the 110 th and/or 275 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein said additional mutation further confers temperature-sensitivity on the virus when introduced into the virus.
25. The Morbillivirus P protein according to claim 23 , wherein either or both additional mutation(s) is a substitution to tyrosine.
26. A method for producing a Morbillivirus on which temperature-sensitivity is conferred, said method comprising introducing, into a Morbillivirus P protein, a mutation of an amino acid at a position corresponding to the 439 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
27. The method according to claim 26 , wherein the Morbillivirus P protein has at least 40% identity to the amino acid sequence set forth in SEQ ID NO: 2.
28. The method according to claim 26 , wherein the mutation of an amino acid is a substitution to proline.
29. The method according to claim 26 , wherein the virus is a measles virus.
30. The method according to claim 26 , wherein said method further comprising introducing, into the Morbillivirus P protein, an additional mutation of an amino acid at a position corresponding to the 110 th and/or 275 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
31. The method according to claim 30 , wherein either or both additional mutation(s) is a substitution to tyrosine.
32. The virus according to claim 16 , wherein said P protein has at least 40% identity to the amino acid sequence set forth in SEQ ID NO: 2.
33. The virus according to claim 16 , wherein the mutation of an amino acid is a substitution to proline.
34. The virus according to claim 16 , wherein the virus is a measles virus.
35. The virus according to claim 16 , wherein the P protein further has an additional mutation of an amino acid at a position corresponding to the 110 th and/or 275 th position of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein said additional mutation confers temperature-sensitivity on the virus.
36. The virus according to claim 35 , wherein either or both additional mutation(s) is a substitution to tyrosine.
37. A pharmaceutical composition comprising the virus according to claim 32 and a pharmacologically acceptable carrier or medium.
38. A pharmaceutical composition comprising the virus according to claim 33 and a pharmacologically acceptable carrier or medium.
39. A pharmaceutical composition comprising the virus according to claim 34 and a pharmacologically acceptable carrier or medium.
40. A pharmaceutical composition comprising the virus according to claim 35 and a pharmacologically acceptable carrier or medium.
41. A pharmaceutical composition comprising the virus according to claim 36 and a pharmaceutically acceptable carrier or medium.
42. The polynucleotide according to claim 1 , wherein said protein has at least 60% identity to the amino acid sequence set forth in SEQ ID NO: 2.
43. The polynucleotide according to claim 1 , wherein said protein has at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 2.
44. The Morbillivirus P protein according to claim 7 , wherein said protein has at least 60% identity to the amino acid sequence set forth in SEQ ID NO: 2.
45. The Morbillivirus P protein according to claim 7 , wherein said protein has at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 2.
46. The method according to claim 10 , wherein the Morbillivirus P protein has at least 60% identity to the amino acid sequence set forth in SEQ ID NO: 2.
47. The method according to claim 10 , wherein the Morbillivirus P protein has at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 2.
48. The method according to claim 26 , wherein the Morbillivirus P protein has at least 60% identity to the amino acid sequence set forth in SEQ ID NO: 2.
49. The method according to claim 26 , wherein the Morbillivirus P protein has at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 2.
50. The virus according to claim 16 , wherein said P protein has at least 60% identity to the amino acid sequence set forth in SEQ ID NO: 2.
51. The virus according to claim 16 , wherein said P protein has at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 2.
52. A pharmaceutical composition comprising the virus according to claim 50 and a pharmacologically acceptable carrier or medium.
53. A pharmaceutical composition comprising the virus according to claim 51 and a pharmacologically acceptable carrier or medium.