Screening methods for identifying viral proteins with interferon antagonizing functions and potential antiviral agents
View Patent ↗The present invention relates, in general, to a screening method for identifying novel viral proteins with interferon antagonizing function using a transfection-based assay, and the use of such proteins in isolating various types of attenuated viruses for the development of vaccine and pharmaceutical formulations. The invention also relates to the use of viral interferon antagonists in screening assays to identify potential anti-viral agents. The invention further relates to protocols utilizing interferon antagonists, e.g., NS1, to enhance gene therapy or DNA vaccination based on their ability to increase gene expression.
1. A method for propagating an attenuated influenza virus with a mutation in the genomic segment that encodes NS1, which virus has an impaired ability to antagonize a cellular interferon response, the method comprising:
a. infecting cells engineered to express a viral interferon antagonist from a different influenza type with said virus; and
b. culturing the infected cells under conditions that are suitable for propagation of said virus.
2. The method of claim 1 , wherein the attenuated influenza virus is an influenza A virus.
3. The method of claim 1 , wherein the attenuated influenza virus has a mutation in the coding region of NS1.
4. The method of claim 1 , wherein the attenuated influenza virus is a delNS1 influenza virus.
5. The method of claim 2 , wherein the viral interferon antagonist is the NS1 protein of an influenza B virus.
6. The method of claim 3 , wherein the viral interferon antagonist is the NS1 protein of an influenza B virus.
7. The method of claim 4 , wherein the viral interferon antagonist is the NS1 protein of an influenza B virus.
8. The method of claim 2 , wherein the viral interferon antagonist is the NS1 protein of an influenza C virus.
9. The method of claim 3 , wherein the viral interferon antagonist is the NS1 protein of an influenza C virus.
10. The method of claim 4 , wherein the viral interferon antagonist is the NS1 protein of an influenza C virus.
11. The method of claim 1 , wherein the attenuated influenza virus is an influenza B virus or an influenza C virus.
12. A method for vaccine production comprising:
a. propagating an attenuated influenza virus with a mutation in the genomic segment that encodes NS1, which virus has an impaired ability to antagonize a cellular interferon response, in cells that express a heterologous viral interferon antagonist; and
b. collecting progeny virus, wherein the virus is grown under conditions and to sufficient quantities so that the progeny virus is suitable for formulation into a vaccine.
13. The method of claim 12 , wherein the influenza virus is an influenza A virus.
14. The method of claim 12 , wherein the influenza virus has a mutation in the coding region of NS1.
15. The method of claim 12 , wherein the influenza virus is a delNS1 influenza virus.
16. The method of claim 13 , wherein the heterologous viral interferon antagonist is the NS1 protein of an influenza B virus.
17. The method of claim 14 , wherein the heterologous viral interferon antagonist is the NS1 protein of an influenza B virus.
18. The method of claim 15 , wherein the heterologous viral interferon antagonist is the NS1 protein of an influenza B virus.
19. The method of claim 13 , wherein the heterologous viral interferon antagonist is the NS1 protein of an influenza C virus.
20. The method of claim 14 , wherein the heterologous viral interferon antagonist is the NS1 protein of an influenza C virus.
21. The method of claim 15 , wherein the heterologous viral interferon antagonist is the NS1 protein of an influenza C virus.
22. The method of claim 12 , wherein the heterologous viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
23. The method of claim 13 , wherein the heterologous viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
24. The method of claim 14 , wherein the heterologous viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
25. The method of claim 15 , wherein the heterologous viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
26. A cell infected with an attenuated influenza virus with a mutation in the genomic segment that encodes NS1, which virus has an impaired ability to antagonize a cellular interferon response, wherein the cell recombinantly expresses a viral interferon antagonist from a different influenza type.
27. The cell of claim 26 , wherein the attenuated influenza virus is an influenza A virus.
28. The cell of claim 26 , wherein the attenuated influenza virus has a mutation in the coding region of NS1.
29. The cell of claim 26 , wherein the attenuated influenza virus is a delNS1 influenza virus.
30. The cell of claim 27 , wherein the viral interferon antagonist is the NS1 protein of an influenza B virus.
31. The cell of claim 28 , wherein the viral interferon antagonist is the NS1 protein of an influenza B virus.
32. The cell of claim 29 , wherein the viral interferon antagonist is the NS1 protein of an influenza B virus.
33. The cell of claim 27 , wherein the viral interferon antagonist is the NS1 protein of an influenza C virus.
34. The cell of claim 28 , wherein the viral interferon antagonist is the NS1 protein of an influenza C virus.
35. The cell of claim 29 , wherein the viral interferon antagonist is the NS1 protein of an influenza C virus.
36. The cell of claim 26 , wherein the attenuated influenza virus is an influenza B virus or an influenza C virus.
37. The cell of claim 26 which is a MDCK cell.
38. The cell of claim 27 which is a MDCK cell.
39. The cell of claim 28 which is a MDCK cell.
40. The cell of claim 29 which is a MDCK cell.
41. The cell of claim 26 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
42. The cell of claim 27 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
43. The cell of claim 28 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
44. The cell of claim 29 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
45. A method for propagating an attenuated influenza virus with a mutation in the genomic segment that encodes NS1, which virus has an impaired ability to antagonize a cellular interferon response, the method comprising:
a. infecting cells engineered to express a non-influenza viral interferon antagonist with said attenuated influenza virus; and
b. culturing the infected cells under conditions that are suitable for propagation of said attenuated influenza virus.
46. The method of claim 45 , wherein the attenuated influenza virus is an influenza A virus.
47. The method of claim 45 , wherein the attenuated influenza virus has a mutation in the coding region of NS1.
48. The method of claim 45 , wherein the attenuated influenza virus is a delNS1 influenza virus.
49. The method of claim 45 , wherein the attenuated influenza virus is an influenza B virus or an influenza C virus.
50. The method of claim 45 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
51. The method of claim 46 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
52. The method of claim 47 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
53. The method of claim 48 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
54. The method of claim 49 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
55. A cell infected with an attenuated influenza virus with a mutation in the genomic segment that encodes NS1, which virus has an impaired ability to antagonize a cellular interferon response, wherein the cell recombinantly expresses a non-influenza viral interferon antagonist.
56. The cell of claim 55 , wherein the attenuated influenza virus is an influenza A virus.
57. The cell of claim 55 , wherein the attenuated influenza virus has a mutation in the coding region of NS1.
58. The cell of claim 55 , wherein the attenuated influenza virus is a delNS1 influenza virus.
59. The cell of claim 55 , wherein the attenuated influenza virus is an influenza B virus or an influenza C virus.
60. The cell of claim 55 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
61. The cell of claim 56 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
62. The cell of claim 57 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
63. The cell of claim 58 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
64. The cell of claim 59 , wherein the non-influenza viral interferon antagonist is the VP35 protein of Ebola virus, the NS2 protein of respiratory syncytial virus (RSV), the E3L protein of vaccinia virus, or the ICP34.5 protein of herpes simplex virus type 1 (HSV-1).
65. The cell of claim 55 which is a MDCK cell.
66. The cell of claim 56 which is a MDCK cell.
67. The cell of claim 57 which is a MDCK cell.
68. The cell of claim 58 which is a MDCK cell.
69. The cell of claim 55 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
70. The cell of claim 56 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
71. The cell of claim 57 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.
72. The cell of claim 58 which is a human embryonic kidney cell, a human osteogenic sarcoma cell, a HeLa cell, a Cos cell, or an NIH3T3 cell.