Vaccines comprising mutant attenuated influenza viruses

1. A vaccine comprising an effective amount of a live, attenuated isolated recombinant influenza virus comprising a mutant M viral segment that is mutated so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein comprising an extracellular domain or a portion thereof, a deletion of a cytoplasmic tail and either lacking a transmembrane domain or having a mutated transmembrane domain, wherein the attenuated isolated recombinant influenza virus is a reassortant virus comprising at least six viral segments from a master vaccine strain and at least one viral segment from a strain different from the master vaccine strain, wherein the at least one viral segment from a strain different from the master vaccine strain is HA.

2. The vaccine of claim 1 , wherein the live, attenuated isolated recombinant influenza virus is present at a concentration of 1-50 billion virus particles per 0.5 ml.

3. The vaccine of claim 1 , wherein the vaccine comprises 0.1 to 200 micrograms influenza virus HA per influenza virus strain.

4. The vaccine of claim 3 , wherein the vaccine comprises 1 to 50 micrograms influenza virus HA per influenza virus strain.

5. The vaccine of claim 1 , wherein the mutant M2 protein consists of the extracellular domain.

6. The vaccine of claim 1 , wherein the mutant M2 protein lacks the transmembrane domain of M2.

7. The vaccine of claim 1 , wherein the HA comprises influenza A H1.

8. The vaccine of claim 1 , wherein the HA comprises influenza A H2.

9. The vaccine of claim 1 , wherein the HA comprises influenza A H3.

10. The vaccine of claim 1 , wherein the HA comprises influenza A H5.

11. A vaccine comprising an effective amount of a live, attenuated isolated recombinant influenza virus comprising a mutant M viral segment that is mutated so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein comprising an extracellular domain or a portion thereof, a deletion of a cytoplasmic tail and either lacking a transmembrane domain or having a mutated transmembrane domain, wherein the recombinant virus comprises influenza A hemagglutinin (HA) H2 or H3.

12. The vaccine of claim 11 , wherein the live, attenuated isolated recombinant influenza virus is present at a concentration of 1 to 50 billion virus particles per 0.5 ml.

13. The vaccine of claim 11 , wherein the vaccine comprises 0.1 to 200 micrograms influenza virus HA per influenza virus strain.

14. The vaccine of claim 13 , wherein the vaccine comprises 1 to 50 micrograms influenza virus HA per influenza virus strain.

15. The vaccine of claim 11 , wherein the mutant M2 protein consists of the extracellular domain.

16. The vaccine of claim 11 , wherein the mutant M2 protein lacks the transmembrane domain of M2.

17. The vaccine of claim 11 , wherein the recombinant virus comprises influenza A H2.

18. The vaccine of claim 11 , wherein the recombinant virus comprises influenza A H3.

19. The vaccine of claim 11 , wherein the attenuated isolated recombinant influenza virus is a reassortant virus.

20. The vaccine of claim 19 , wherein the reassortant comprises at least six viral segments from a master vaccine strain.

21. A method to immunize a mammal comprising administering to the mammal a vaccine dose comprising an effective amount of a live, attenuated isolated recombinant influenza virus comprising a mutant M viral segment that is mutated so that upon viral infection of a cell the mutant M gene expresses a functional M1 protein and a mutant M2 protein comprising an extracellular domain or portion thereof, a deletion of a cytoplasmic tail and either lacking a transmembrane domain or having a mutated transmembrane domain, wherein the attenuated isolated recombinant influenza virus is a reassortant virus comprising at least six viral segments from a master vaccine strain and at least one viral segment from a strain different from the master vaccine strain, wherein the at least one viral segment from a strain different from the master vaccine strain is HA.

22. The method of claim 21 , wherein up to 1×10 9 to 50×10 9 virus particles per 0.5 mL live, attenuated isolated recombinant influenza virus is present in the vaccine dose.

23. The method of claim 21 , wherein 10 3 to 10 7 PFU/kg live, attenuated isolated recombinant influenza virus is present in the vaccine dose.

24. The method of claim 21 , wherein 0.1 to 200 micrograms of influenza virus HA per influenza virus strain is present in the vaccine dose.

25. The method of claim 24 , wherein 1 to 50 micrograms influenza virus HA per influenza virus strain is present in the vaccine dose.

26. The method of claim 21 , wherein the mutant M2 protein consists of the extracellular domain.

27. The method of claim 21 , wherein the mutant M2 protein lacks the transmembrane domain.

28. The method of claim 21 , wherein the attenuated isolated recombinant virus comprises influenza A HA.

29. The method of claim 28 , wherein the recombinant virus comprises influenza A H1 HA.

30. The method of claim 28 , wherein the recombinant virus comprises influenza A H2 HA.

31. The method of claim 28 , wherein the recombinant virus comprises influenza A H3 HA.

32. The method of claim 28 , wherein the recombinant virus comprises influenza A H5 HA.

33. The method of claim 21 , wherein the vertebrate is a human.

34. A method of preparing an isolated virus for the vaccine of claim 1 comprising: contacting a cell expressing M2 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 NA 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 mutant 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 mutant M cDNA expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and either lacking a transmembrane domain or having a mutated transmembrane domain; and

wherein the transcription cassettes for mRNA production are a transcription cassette comprising a PolI 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 PolI 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 PolI transcription termination sequence; and

isolating virus from the cell.

35. The method of claim 34 further comprising preparing the vaccine of claim 1 comprising the isolated virus.