Neuraminidase-deficient live influenza vaccines
Attenuated, neuraminidase deficient influenza virus, and compositions and methods to prepare that virus, are provided.
1. A composition to prepare a 7 segment influenza A or B virus, comprising:
one or more vectors which include transcription cassettes for vRNA production and transcription cassettes for mRNA production,
wherein the transcription cassettes for vRNA production are a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence; and
wherein the transcription cassettes for mRNA production are a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PA linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB1 linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB2 linked to a PolII transcription termination sequence, and a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus NP linked to a PolII transcription termination sequence,
and wherein the composition does not include sequences corresponding to NA coding or noncoding sequences for vRNA production and sequences for mRNA production of NA.
2. The composition of claim 1 , wherein each transcription cassette is on a plasmid vector.
3. The composition of claim 1 , wherein one or more transcription cassettes are on one or more plasmid vectors.
4. The composition of claim 3 , wherein one plasmid vector has transcription cassettes for vRNA production of PA, PB1, PB2, HA, NP, M and NS.
5. The composition of claim 3 or 4 , wherein one plasmid vector has one of the transcription cassette for mRNA production and another plasmid vector has the other three transcription cassettes for mRNA production.
6. The composition of claim 3 or 4 , which has four plasmid vectors for mRNA production each with one of the transcription cassettes for mRNA production.
7. The composition of claim 3 , wherein one plasmid vector has six of the transcription cassettes for vRNA production and another plasmid vector has the other transcription cassette for vRNA production.
8. The composition of claim 7 , wherein one plasmid vector has one of the transcription cassettes for mRNA production and another plasmid vector has the other three transcription cassettes for mRNA production.
9. The composition of claim 7 , which has four plasmid vectors for mRNA production.
10. The composition of claim 3 , 4 or 7 , wherein one plasmid has the four transcription cassettes for mRNA production.
11. The composition of claim 1 , further comprising a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.
12. The composition of claim 11 , where the transcription cassette comprising the DNA of interest is on a plasmid vector.
13. The composition of claim 12 , wherein the transcription cassette comprising the DNA of interest is on a different plasmid vector than the transcription cassettes for vRNA production.
14. The composition of claim 12 , wherein the transcription cassette comprising the DNA of interest is on a different plasmid vector than one or more of the transcription cassettes for mRNA production.
15. The composition of claim 12 , wherein the transcription cassette comprising the DNA of interest is on a plasmid vector having one of the transcription cassettes for vRNA production.
16. The composition of claim 12 , wherein the transcription cassette comprising the DNA of interest is on a plasmid vector having one of the transcription cassettes for mRNA production.
17. A composition to prepare a 7 segment influenza A or B virus, comprising:
one or more vectors which include transcription cassettes for vRNA production, wherein the transcription cassettes for vRNA production are a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence,
and wherein the composition does not include sequences corresponding to NA coding or noncoding sequences for vRNA production and sequences for mRNA production of NA.
18. The composition of claim 17 , wherein each transcription cassette is on a plasmid vector.
19. The composition of claim 18 , wherein one or more transcription cassettes are on one or more plasmid vectors.
20. The composition of claim 19 , wherein one plasmid vector has transcription cassettes for vRNA production of PA, PB1, PB2, HA, NP, M and NS.
21. The composition of claim 19 or 20 , wherein one plasmid vector has six of the transcription cassettes for vRNA production and another plasmid vector has the other transcription cassette for vRNA production.
22. The composition of claim 20 , further comprising one plasmid vector having one transcription cassette for mRNA production and another plasmid vector having three transcription cassettes for mRNA production, wherein the transcription cassettes encode influenza virus PA, PB1, PB2 and NP.
23. The composition of claim 20 , further comprising four plasmids for mRNA production, each with a different transcription cassette for mRNA production, wherein the transcription cassettes encode influenza virus PA, PB1, PB2 and NP.
24. The composition of claim 17 , further comprising a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.
25. The composition of claim 24 , wherein the transcription cassette comprising the DNA of interest is on a plasmid vector.
26. The composition of claim 25 , wherein the transcription cassette comprising the DNA of interest is on a different plasmid vector than the transcription cassettes for vRNA production.
27. The composition of claim 26 , wherein the transcription cassette comprising the DNA of interest is on a plasmid vector having one of the transcription cassettes for vRNA production.
28. The composition of claim 1 or 17 , wherein the HA is a type A HA.
29. The composition of claim 28 , wherein the HA is a H5 or H3 HA.
30. The composition of claim 1 or 17 , wherein the HA is a type B HA.
31. The composition of claim 1 or 17 , wherein the HA cDNA encodes an avirulent cleavage site.
32. The composition of claim 1 or 17 , wherein the PB2 protein confers a cold sensitive phenotype to the prepared influenza A or B virus.
33. A method to prepare attenuated influenza A or B virus, comprising: contacting a cell with the composition of claim 1 or 17 , in an amount effective to yield attenuated influenza A or B virus.
34. A method to prepare attenuated 7 segment influenza A or B virus, comprising contacting a cell with one or more vectors which include transcription cassettes for vRNA production and transcription cassettes for mRNA production, wherein the transcription cassettes for vRNA production are a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence; and wherein the transcription cassettes for mRNA production are a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PA linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB1 linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB2 linked to a PolII transcription termination sequence, and/or a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus NP linked to a PolII transcription termination sequence,
and wherein the cell is not contacted with sequences corresponding to NA coding or noncoding sequences for vRNA production and sequences for mRNA production of NA so that the attenuated 7 segment influenza A or B virus produced by the cell does not have a NA gene segment and does not have NA protein.
35. A method to prepare attenuated 7 segment influenza A or B virus, comprising contacting a cell with one or more vectors which include transcription cassettes for vRNA production, wherein the transcription cassette for vRNA production are a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence, wherein the cell is contacted with eight transcription cassettes for vRNA,
and wherein the cell is not contacted with sequences corresponding to NA coding or noncoding sequences for vRNA production and sequences for mRNA production of NA so that the attenuated 7 segment influenza virus produced by the cell does not have a NA gene segment and does not have NA protein.
36. The method of claim 34 or 35 , further comprising a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.
37. The method of claim 34 or 35 , further comprising passaging the virus in a different cell type or eggs.
38. The method of claim 34 or 35 , wherein the cells that are transfected are 293T cells or Vero cells.
39. The method of claim 37 , wherein the cells that are transfected are 293T cells and the virus is passaged in Vero cells.
40. The method of claim 34 or 35 , further comprising isolating the virus.
41. An isolated virus prepared by the method of claim 40 .
42. A vaccine comprising the isolated virus of claim 41 .
43. The vaccine of claim 42 , wherein the isolated virus is an influenza A virus.
44. The vaccine of claim 43 , further comprising an influenza B virus.
45. The vaccine of claim 42 , further comprising granulocyte macrophage-colony stimulating factor (GM-CSF).
46. A plasmid for attenuated 7 segment influenza A or B virus production comprising: a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence, and wherein the plasmid does not contain sequences corresponding to NA coding or noncoding sequences for vRNA production and sequences for mRNA production of NA.
47. One or more plasmids for attenuated 7 segment influenza A or B virus production comprising a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter and a PolI transcription termination sequence and a PolII promoter and a PolII transcription termination sequence each operably linked to a DNA segment for influenza virus PA, a transcription cassette comprising a PolI promoter and a PolI transcription termination sequence and a PolII promoter and PolII transcription termination sequence each operably linked to a DNA segment for influenza virus PB1, a transcription cassette comprising a PolI promoter and a PolI transcription termination sequence and a PolII promoter and a PolII transcription termination sequence each operably linked to a DNA segment encoding influenza virus PB2, and a transcription cassette comprising a PolI promoter and a PolI transcription termination sequence and a PolII promoter and a PolII transcription termination sequence each operably linked to a DNA segment for influenza virus NP, and wherein the one or more plasmids do not contain sequences corresponding to NA coding or noncoding sequences for vRNA production and sequences for mRNA production of NA.
48. A method to prepare attenuated 7 segment influenza virus, comprising: contacting a cell with the plasmid of claim 46 or 47 in an effective amount.
49. An isolated population of cells comprising individual cells having transcription cassettes for vRNA production and transcription cassettes for mRNA production for attenuated 7 segment influenza A or B virus production,
wherein the transcription cassettes for vRNA production are a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence; and
wherein the transcription cassettes for mRNA production are a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PA linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB1 linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB2 linked to a PolII transcription termination sequence, and a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus NP linked to a PolII transcription termination sequence,
and wherein the population of cells does not include sequences corresponding to NA coding or noncoding sequences for vRNA production and does not include sequences for mRNA production of NA.
50. The composition of claim 21 , further comprising one plasmid vector having one transcription cassette for mRNA production and another plasmid vector having three transcription cassettes for mRNA production, wherein the transcription cassettes encode influenza virus PA, PB1, PB2 and NP.
51. The composition of claim 21 , further comprising one plasmid vector having one transcription cassette for mRNA production and another plasmid vector having three transcription cassettes for mRNA production, wherein the transcription cassettes encode influenza virus PA, PB1, PB2 and NP.