Claims
- 1. An isolated recombinant influenza virus comprising a mutant membrane protein gene which does not encode a functional membrane protein or a functional portion thereof, wherein the mutant membrane protein gene comprises at least two mutations relative to a corresponding membrane protein gene which encodes a functional membrane protein, one of which mutations is not in a region of the membrane protein gene corresponding to the transmembrane domain.
- 2. The isolated recombinant virus of claim 1 wherein the mutant membrane protein gene encodes at least one amino acid substitution.
- 3. The isolated recombinant virus of claim 2 wherein at least one mutation encodes a substitution at the codon for the initiator methionine.
- 4. The isolated recombinant virus of claim 1 wherein one mutation in the mutant membrane protein gene is a stop codon for the codon for the initiator methionine.
- 5. The isolated recombinant virus of claim 1 wherein one mutation in the mutant membrane protein gene is a stop codon in the coding region for the membrane protein.
- 6. The isolated recombinant virus of claim 1 wherein the mutant membrane protein gene comprises a deletion of one or more nucleotides.
- 7. The isolated recombinant virus of claim 6 wherein the deletion alters the reading frame for the membrane protein.
- 8. The isolated recombinant virus of claim 1 wherein the mutant membrane protein gene comprises an insertion of one or more nucleotides.
- 9. The isolated recombinant virus of claim 8 wherein the insertion alters the reading frame for the membrane protein.
- 10. The isolated recombinant virus of claim 1 wherein the mutant membrane protein gene comprises a deletion of one or more nucleotides and encodes an amino acid substitution.
- 11. The isolated recombinant virus of claim 1 wherein the mutant membrane protein gene comprises an insertion of one or more nucleotides and encodes an amino acid substitution.
- 12. The isolated recombinant virus of claim 1 wherein the membrane protein is the M2 protein of influenza A virus.
- 13. The isolated recombinant virus of claim 1 wherein the membrane protein is the NB protein of influenza B virus.
- 14. The isolated recombinant virus of claim 1 wherein the membrane protein is the CM1 protein of influenza C virus.
- 15. The isolated recombinant virus of claim 1 which further comprises a heterologous immunogenic protein of a pathogen or a therapeutic protein.
- 16. The isolated recombinant virus of claim 1 which further comprises a heterologous immunogenic protein gene of a pathogen or a therapeutic protein gene.
- 17. The isolated recombinant virus of claim 1 wherein at least one mutation does not alter the in vitro replication of the virus but is associated with attenuation of the virus in vivo.
- 18. A vaccine comprising the isolated recombinant virus of claim 1.
- 19. A method of preparing a recombinant influenza virus comprising a mutant membrane protein gene which does not encode a functional membrane protein or a functional portion thereof, comprising:
(i) contacting a host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA for NB and NA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the sequence of the cDNA for NB and NA comprises at least two mutations in the NB sequence relative to a corresponding NB gene which encodes a functional membrane protein, one of which mutations is not in the transmembrane domain, the presence of which in the mutant gene, when the mutant gene is transcribed and translated in the host cell, does not yield a functional membrane protein or a functional portion thereof, and optionally yields a functional NA protein, and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and (ii) isolating the virus.
- 20. A method of preparing a recombinant influenza virus comprising a mutant membrane protein gene which does not encode a functional membrane protein or a functional portion thereof, comprising:
(i) contacting a host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a mutant M2 cDNA linked to a transcription termination sequence, wherein the mutant M2 cDNA comprises at least two mutations relative to a corresponding M2 gene which encodes a functional membrane protein, one of which mutations is not in the transmembrane domain, the presence of which in the mutant gene, when the mutant gene is transcribed and translated in the host cell, does not yield a functional membrane protein or a functional portion thereof, and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and (ii) isolating the virus.
- 21. The method of claim 19 or 20 wherein the mutant membrane protein gene encodes at least one amino acid substitution.
- 22. The method of claim 19 or 20 wherein one mutation in the mutant membrane protein gene is a stop codon for the codon for the initiator methionine.
- 23. The method of claim 19 or 20 wherein one mutation in the mutant membrane protein gene is a stop codon in the coding region for the membrane protein.
- 24. The method of claim 19 or 20 wherein the mutant membrane protein gene comprises a deletion of one or more nucleotides.
- 25. The method of claim 24 wherein the deletion alters the reading frame for the membrane protein.
- 26. The method of claim 19 or 20 wherein the mutant membrane protein gene comprises an insertion of one or more nucleotides.
- 27. The method of claim 26 wherein the infection alters the reading frame for the membrane protein.
- 28. The method of claim 19 or 20 wherein at least one mutation encodes a substitution at the initiator methionine.
- 29. The method of claim 19 or 20 wherein the mutant membrane protein gene comprises a deletion of one or more nucleotides and encodes at least one amino acid substitution.
- 30. The method of claim 19 or 20 wherein the mutant membrane protein gene comprises an insertion of one or more nucleotides and encodes an amino acid substitution.
- 31. A method to immunize a vertebrate, comprising: contacting the vertebrate with an effective amount of the recombinant virus of any one of claims 1 to 17.
- 32. The method of claim 31 wherein the vertebrate is an avian.
- 33. The method of claim 31 wherein the vertebrate is a mammal.
- 34. The method of claim 31 wherein the vertebrate is a human.
- 35. A composition comprising a plurality of influenza vectors, comprising:
a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA for NB and NA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the sequence of the cDNA for NB and NA comprises at least two mutations in the sequence for NB relative to a corresponding NB gene which encodes a functional membrane protein, one of which mutations is not in the transmembrane domain, the presence of which in the mutant gene, when the mutant gene is transcribed and translated in the host cell, does not yield a functional membrane protein or a functional portion thereof, and optionally yields a functional NA protein; and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus mutant NB, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2.
- 36. A composition comprising a plurality of influenza vectors, comprising:
a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M1 cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a mutant M2 cDNA segment linked to a transcription sequence, wherein the mutant M2 cDNA comprises at least two mutations relative to a corresponding M2 gene which encodes a functional membrane protein, one of which mutations is not in the transmembrane domain, the presence of which in the mutant gene, when the mutant gent is transcribed and translated in the host cell, does not yield a functional membrane protein or a functional portion thereof; and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2.
- 37. The composition of claim 35 or 36 further comprising a vector comprising a promoter operably linked to a DNA fragment of interest in antisense orientation.
- 38. The composition of claim 37 wherein the vector comprises a DNA fragment which encodes an immunogenic polypeptide or peptide of a pathogen or a therapeutic protein.
- 39. Isolated virus prepared by the method of claim 19 or 20.
- 40. A host cell contacted with the virus of claim 1 or 39.
- 41. An isolated polynucleotide comprising a nucleic acid segment comprising sequences encoding at least one of the proteins of influenza virus B/Lee, or a portion thereof, or the complement of the polynucleotide, wherein the isolated polynucleotide encodes a HA, NA, PB1, PB2, PA, NP, M1, NB, or NS, or a portion thereof, having substantially the same activity as a corresponding polypeptide of one of SEQ ID NOs:1-8.
- 42. An isolated polynucleotide comprising a nucleic acid segment comprising sequences encoding at least one of the proteins of influenza virus B/Lee, or a portion thereof, or the complement of the polynucleotide, wherein the isolated polynucleotide encodes a HA, NA, PB1, PB2, PA, NP, M1, NB, or NS, or a portion thereof, which hybridizes to one of SEQ ID NOs:1-8 or the complement thereof.
- 43. The isolated polynucleotide of claim 42 wherein the isolated polynucleotide has substantially the same nucleotide sequence as one of SEQ ID NOs:1-8 or the complement thereof.
- 44. The isolated polynucleotide of claim 42 wherein the isolated polynucleotide encodes a polypeptide that has substantially the same activity as the polypeptide encoded by one of SEQ ID NOs:1-8.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of the filing date of U.S. application Serial No. 60/464,776, filed Apr. 23, 2003, and U.S. application Serial No. 60/465,328, filed Apr. 24, 2003, the disclosures of which are incorporated by reference herein.
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made with a grant from the Government of the United States of America (grant AI-47446 from the National Institutes of Health). The Government may have certain rights in the invention.
Provisional Applications (2)
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Number |
Date |
Country |
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60464776 |
Apr 2003 |
US |
|
60465328 |
Apr 2003 |
US |