Attenuated influenza vaccines and uses thereof

1. A modified influenza A virus comprising a PB1 polymerase comprising one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at an amino acid corresponding to position 319 (L319E/D/N) of SEQ ID NO: 1, a threonine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 323 (T323E/D/Q/N) of SEQ ID NO: 1; and an isoleucine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 342 (I342E/D/Q/N) of SEQ ID NO: 1.

2. The virus of claim 1 , wherein the PB1 polymerase comprises a leucine to glutamic acid substitution (L319E).

3. The virus of claim 1 , wherein the PB1 polymerase comprises a leucine to asparagine substitution (L319N).

4. The virus of claim 1 , wherein the PB1 polymerase further comprises one or more mutations selected from the group consisting of a lysine to glutamic acid substitution at an amino acid corresponding to position 391 (K391E) of SEQ ID NO: 1, a glutamic acid to glycine substitution at an amino acid corresponding to position 581 (E581G) of SEQ ID NO: 1 and an alanine to threonine substitution at an amino acid corresponding to position 661 (A661T) of SEQ ID NO: 1.

5. The virus of claim 1 , wherein the virus further comprises a PB2 polymerase comprising an asparagine to serine substitution at position 265 (N265S).

6. The virus of claim 1 , wherein the virus further comprises an influenza virus nucleoprotein (NP) comprising an aspartic acid to glycine substitution at position 34 (D34G).

7. The virus of claim 1 , wherein the influenza A virus is selected from the group consisting of an H2N2 virus, an H3N2 virus, an H1N1 virus, an H9N2 virus and an H5N1 virus.

8. The virus of claim 1 , wherein the influenza A virus is (A/Puerto Rico/8/34/H1 N1)(PR8), (A/California/04/2007 H1N1) or (A/Ann Arbor/6/60 H2N2).

9. The virus of claim 1 , wherein the virus is a live attenuated influenza A virus with reduced growth from about 37° C. to about 39° C., as compared to an influenza A virus comprising a PB1 polymerase lacking one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at an amino acid corresponding to position 319 (L319E/D/N) of SEQ ID NO: 1, a threonine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 323 (T323E/D/Q/N) of SEQ ID NO: 1; and an isoleucine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 342 (I342 E/D/Q/N) of SEQ ID NO: 1.

10. An immunogenic composition comprising the virus of claim 1 and a pharmaceutically acceptable carrier.

11. A method for eliciting an immune response against an influenza virus in a subject, comprising administering an effective dose of the immunogenic composition of claim 10 to the subject.

12. A method of treating or reducing the risk of influenza infection in a subject, comprising administering to a subject with an influenza infection or at risk of exposure to an influenza infection an effective dose of the immunogenic composition of claim 10 .

13. A recombinant nucleic acid encoding a PB1 polymerase of an influenza A virus, wherein the nucleic acid encodes a PB1 polymerase having one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at an amino acid corresponding to position 319 (L319E/D/N) of SEQ ID NO: 1, a threonine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 323 (T323E/D/Q/N) of SEQ ID NO: 1; and an isoleucine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 342 (I342E/D/Q/N) of SEQ ID NO: 1.

14. A recombinant nucleic acid encoding a PB1 polymerase of an influenza A virus wherein the nucleic acid encodes a PB1 polymerase comprising one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at an amino acid corresponding to position 319 (L319E/D/N) of SEQ ID NO: 1, a threonine to glutamic acid, aspartic acid, glutamine or asparagine substitution at amino acid corresponding to position 323 (T323E/D/Q/N) of SEQ ID NO: 1; a serine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 338 (S338E/D/Q/N) and an isoleucine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 342 (I342E/D/Q/N) of SEQ ID NO: 1; and one or more mutations selected from the group consisting of a lysine to glutamic acid substitution at an amino acid corresponding to position 391 (K391E) of SEQ ID NO: 1, a glutamic acid to glycine substitution at an amino acid corresponding to position 581 (E581G) of SEQ ID NO: 1 and an alanine to threonine substitution at an amino acid corresponding to position 661 (A661T) of SEQ ID NO: 1.

15. A vector comprising the nucleic acid of claim 13 .

16. A method of producing the influenza virus of claim 1 , comprising:

a) transfecting a population of host cells with one or more vectors comprising

(i) nucleic acid sequences encoding the internal genome segments of an influenza A virus and

(ii) a nucleic acid encoding a PB1 polymerase comprising one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at an amino acid corresponding to position 319 (L319E/D/N) of SEQ ID NO: 1, a threonine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 323 (T323E/D/Q/N) of SEQ ID NO: 1; and an isoleucine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 342 (I342 E/D/Q/N) of SEQ ID NO: 1;

b) culturing the host cells; and

c) recovering the influenza A virus.

17. The method of claim 16 , wherein the nucleic acid encoding the PB1 polymerase encodes a PB1 polymerase comprising one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at an amino acid corresponding to position 319 (L319E/D/N) of SEQ ID NO: 1, a threonine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 323 (T323E/D/Q/N) of SEQ ID NO: 1; a serine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 338 (S338 E/D/Q/N) of SEQ ID NO: 1 and an isoleucine to glutamic acid, aspartic acid, glutamine or asparagine substitution at an amino acid corresponding to position 342 (I342 E/D/Q/N) of SEQ ID NO: 1; and one or mutations selected from the group consisting of a glutamic acid to glycine substitution at an amino acid corresponding to position 581 (E581G) of SEQ ID NO: 1 and an alanine to threonine substitution at an amino acid corresponding to position 661 (A661T) of SEQ ID NO: 1.

18. The method of claim 16 , wherein the one or more vectors further comprise a nucleic acid encoding a PB2 polymerase comprising a N265S mutation.

19. The method of claim 16 , further comprising transforming the cells with a nucleic acid encoding an influenza virus nucleoprotein (NP) comprising an aspartic acid to glycine substitution at position 34 (D34G).

20. The method of claim 16 , wherein the influenza A virus is selected from the group consisting of an H2N2 virus, an H3N2 virus, an H1N1 virus, an H9N2 virus and an H5N1 virus.

21. The method of claim 16 , wherein the cells are Vero cells, MDCK cells or CEK cells.

22. A method for producing an influenza immunogen comprising:

(a) infecting a population of cells with the virus of claim 1 ;

(b) culturing the cells;

(c) harvesting the virus from the culture of step b); and

(d) preparing an immunogen with the harvested virus.

23. The method of claim 22 , wherein the cells are mammalian cells or avian cells.

24. A population of isolated cells comprising the virus of claim 1 .