Influenza virus mutants and uses therefor
Disclosed herein are compositions and methods related to mutant viruses, and in particular, mutant influenza viruses. The mutant viruses disclosed herein include a mutant M2 sequence, and are useful in immunogenic compositions, e.g., as vaccines. Also disclosed herein are methods, compositions and cells for propagating the viral mutants, and methods, devices and compositions related to vaccination.
1. A recombinant influenza A virus comprising a mutant M gene, wherein the mutant M gene comprises two stop codons at nucleotides 786-791 of SEQ ID NO: 1 or at a position corresponding to nucleotides 786-791 of SEQ ID NO: 1 with respect to the M gene coding sequence, and a deletion of nucleotides 792-842 of SEQ ID NO: 1 or at a position corresponding to nucleotides 792-842 of SEQ ID NO: 1 with respect to the M gene coding sequence.
2. A recombinant influenza A virus comprising a mutant M gene, wherein the mutant M gene comprises two stop codons at nucleotides 786-791 of SEQ ID NO: 1 or at a position corresponding to nucleotides 786-791 of SEQ ID NO: 1 with respect to the M gene coding sequence, and a G to C substitution at nucleotide 52 of SEQ ID NO: 1, or at a position corresponding to nucleotide 52 of SEQ ID NO: 1 with respect to the M gene coding sequence.
3. A recombinant influenza A virus comprising a mutant M gene, wherein the mutant M gene comprises two stop codons at nucleotides 786-791 of SEQ ID NO: 1 or at a position corresponding to nucleotides 786-791 of SEQ ID NO: 1 with respect to the M gene coding sequence, and a deletion of nucleotides 792-842 of SEQ ID NO: 1 or at a position corresponding to nucleotides 792-842 of SEQ ID NO: 1, and a G to C substitution at nucleotide 52 of SEQ ID NO: 1 or at a position corresponding to nucleotide 52 of SEQ ID NO: 1 with respect to the M gene coding sequence.
4. The recombinant influenza virus of claim 1 , wherein mutation of the M gene results in failure of the virus to express the M2 protein, or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.
5. The recombinant influenza virus of claim 1 , wherein the mutant M gene does not revert to wild-type or to a non-wild-type sequence encoding a functional M2 protein for at least 10 passages in an in vitro host cell system, wherein the host cell is modified to produce a wild-type version of the mutant gene, thereby providing the gene product to the virus in trans.
6. The recombinant virus of claim 1 , wherein the virus is non-pathogenic in a mammal infected with the virus.
7. The recombinant virus of claim 5 , wherein the in vitro cell system comprises Chinese Hamster Ovary cells or Vero cells.
8. A composition comprising the recombinant influenza A virus of claim 1 .
9. A composition comprising the recombinant influenza A virus of claim 2 .
10. A composition comprising the recombinant influenza A virus of claim 3 .
11. The composition of claim 8 , wherein mutation of the M gene results in failure of the virus to express the M2 protein, or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.
12. The composition of claim 8 , wherein the composition is non-pathogenic to a mammal administered the composition.
13. The composition of claim 8 , further comprising an adjuvant.
14. A method for propagating a recombinant influenza virus, comprising: contacting a host cell with the recombinant influenza virus of claim 1 and incubating the host cell for a sufficient time and under conditions suitable for viral replication, wherein the host cell is modified to produce a wild-type version of the influenza M gene, thereby providing the gene product to the virus in trans.
15. The method of claim 14 , further comprising isolating progeny virus particles.
16. The method of claim 14 , further comprising formulating the virus particles into a vaccine.
17. The method of claim 14 , wherein the virus fails to express the M2 protein, or expresses a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.
18. The method of claim 14 , wherein the virus is non-pathogenic to a mammal administered the virus.
19. The method of claim 14 , wherein the mutant M gene does not revert to wild-type or to a non-wild-type sequence encoding a functional M2 protein for at least 10 passages of the host cell.
20. The method of claim 14 , wherein the host cell is a CHO cell or a Vero cell.