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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5254—Virus avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/542—Mucosal route oral/gastrointestinal
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16151—Methods of production or purification of viral material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16171—Demonstrated in vivo effect

Definitions

• HlNl swine-origin influenza A
• HlNl swine- origin influenza A
• backbone segments of attenuated influenza viruses
• the present invention provides new and/or newly isolated swine influenza Hl hemagglutinin variants that are capable of use for the production of numerous types of vaccines as well as in research, diagnostics, etc.
• the present invention further provides methods of improving the replication efficiency of Hl influenza viruses. Numerous other benefits will become apparent upon review of the following.
• reassortant influenza strains suitable for use in the development of HlNl vaccines were generated by reverse genetics.
• the initially isolated reassortant viruses comprising the wild type HA and NA genome segments, which grew poorly in eggs, were modified such that their growth in eggs is significantly improved.
• Sequence analysis of the modified reassortant viruses indicated that amino acid changes at positions 119, 153, 154 and 186 of the Hl swine hemagglutinin were responsible for the growth advantage in eggs and MDCK cells. Amino acid substitutions at HA residue 155, previously shown to increase swine 1976 virus replication, were also found to increase reassortant A/CA/7/09 virus replication in eggs.
• Viruses having amino acid changes at HA residues 119 and 186 increased egg growth without significantly altering virus antigenicity.
• the Hl HA variants described herein conferred increased growth phenotype on 6:2 reassortant viruses comprising the 6 internal genome segment of the PR8 or A/ Ann Arbor/6/60 virus strains.
• the variant viruses comprising the ca A/Ann Arbor/6/60 backbone and an Hl HA variant having an amino acid substitution at HA residue 119 or 186 were attenuated and very immunogenic in ferrets.
• the present invention includes polypeptide and polynucleotide sequences of influenza hemagglutinin as well as vectors, viruses, vaccines, compositions and the like comprising such sequences and methods of their use. Additional features of the invention are described in more detail herein.
• an isolated or recombinant HA polypeptide of the invention is an Hl HA polypeptide.
• the isolated or recombinant HA polypeptide of the invention may be a swine influenza HA polypeptide.
• an isolated or recombinant HA polypeptide of the invention may comprise a non naturally occurring amino acid at the position corresponding to amino acid residue position 119 of SEQ ID NO: 1 or the position corresponding to amino acid residue position 186 of SEQ ID NO: 1.
• an isolated or recombinant HA polypeptide of the invention may comprise a non naturally occurring amino acid at the position corresponding to amino acid residue position 119 of SEQ ID NO: 1 and the position corresponding to amino acid residue position 186 of SEQ ID NO: 1.
• an isolated or recombinant HA polypeptide of the invention comprises at least one amino acid substitution selected from the group consisting of Kl 19E, Kl 19N and A186D.
• an isolated or recombinant HA polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution selected from the group consisting of Kl 19E, Kl 19N and A186D.
• an isolated or recombinant HA polypeptide of the invention may comprise a glutamic acid (E) at the amino acid residue corresponding to the amino acid residue of position 119 of SEQ ID NO: 1, and an aspartic acid (D) at the amino acid residue corresponding to the amino acid residue of position 186 of SEQ ID NO: 1.
• an isolated or recombinant HA polypeptide of the invention may comprise an asparagine (N) at the amino acid residue corresponding to the amino acid residue of position 119 of SEQ ID NO: 1, and an aspartic acid (D) at the amino acid residue corresponding to the amino acid residue of position 186 of SEQ ID NO: 1.
• an isolated or recombinant HA polypeptide of the invention may further comprise the H273Y substitution.
• an isolated or recombinant HA polypeptide of the invention may further comprise the D222G and/or Q223R substitution.
• an HA polypeptide of the invention comprises the amino acid sequence of SEQ ID NO: 6 or 8.
• an HA polypeptide of the invention comprises an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• an HA polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• an HA polypeptide of the invention comprises the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• an HA polypeptide of the invention comprises an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1- 327 of SEQ ID NO:6 or 8.
• an HA polypeptide of the invention comprises the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• a nucleic acid of the invention comprises the nucleotide sequence of SEQ ID NO:7.
• a nucleic acid of the invention comprises a nucleotide sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the nucleotide sequence of SEQ ID NO:7.
• a nucleic acid of the invention comprises the nucleotide sequence of SEQ ID NO: 7 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 nucleotide substitutions, deletions or insertions.
• a nucleic acid of the invention comprises the nucleotide sequence of residues 84 to 1064 of SEQ ID NO:7.
• a nucleic acid of the invention comprises a nucleotide sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the nucleotide sequence of 84 to 1064 of SEQ ID NO:7.
• a nucleic acid of the invention comprises the nucleotide sequence of 84 to 1064 of SEQ ID NO:7 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 nucleotide substitutions, deletions or insertions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/ Ann Arbor/6/60, a first genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8, and a second genome segment encoding a neuramidinase polypeptide.
• the first genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• the first genome segment may encode an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the second genome segment comprises the nucleotide sequence of SEQ ID NO:5.
• the neuramidinase polypeptide comprises the amino acid sequence of SEQ ID NO:4.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 logio PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 logio PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in tissue cultures.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/ Ann Arbor/6/60, a first genome segment encoding an HA polypeptide comprising the amino acid sequence of residues 1- 327 SEQ ID NO:6 or 8, and a second genome segment encoding a neuramidinase polypeptide.
• the first genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 SEQ ID NO:6.
• the first genome segment may encode an HA polypeptide comprising the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the second genome segment comprises the nucleotide sequence of SEQ ID NO:5.
• the neuramidinase polypeptide comprises the amino acid sequence of SEQ ID NO:4.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 log 10 PFU/ml , at least about 8 log 10 PFU/ml, at least about 8.5 log 10 PFU/ml, at least about 9 log 10 PFU/ml, at least about 9.5 log 10 PFU/ml, at least about 10 log 10 PFU/ml, at least about 10.5 log 10 PFU/ml or at least about 11 log 10 PFU/ml in embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 log 10 PFU/ml , at least about 8 log 10 PFU/ml, at least about 8.5 log 10 PFU/ml, at least about 9 log 10 PFU/ml, at least about 9.5 log 10 PFU/ml, at least about 10 log 10 PFU/ml, at least about 10.5 log 10 PFU/ml or at least about 11 log 10 PFU/ml in tissue cultures.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/Puerto Rico/8/34, a first genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8, and a second genome segment encoding a neuramidinase polypeptide.
• the first genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• the first genome segment may encode an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the second genome segment comprises the nucleotide sequence of SEQ ID NO:5.
• the neuramidinase polypeptide comprises the amino acid sequence of SEQ ID NO:4.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 log 10 PFU/ml , at least about 8 log 10 PFU/ml, at least about 8.5 log 10 PFU/ml, at least about 9 log 10 PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 log 10 PFU/ml, at least about 10.5 log 10 PFU/ml or at least about 11 log 10 PFU/ml in embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 log 10 PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 log 10 PFU/ml, at least about 9 log 10 PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in tissue cultures.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/Puerto Rico/8/34, a first genome segment encoding an HA polypeptide comprising the amino acid sequence of residues 1- 327 SEQ ID NO:6 or 8, and a second genome segment encoding a neuramidinase polypeptide.
• the first genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8.
• the first genome segment may encode an HA polypeptide comprising the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the second genome segment comprises the nucleotide sequence of SEQ ID NO:5.
• the neuramidinase polypeptide comprises the amino acid sequence of SEQ ID NO:4.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 logio PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 logio PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in tissue cultures.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/ Ann Arbor/6/60, a first genome segment comprising the nucleotide sequence of SEQ ID NO:7, and a second genome segment encoding a neuramidinase polypeptide.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/ Ann Arbor/6/60, a first genome segment comprising the nucleotide sequence of residues 84-1064 of SEQ ID NO:7, and a second genome segment encoding a neuramidinase polypeptide.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/Puerto Rico/8/34, a first genome segment comprising the nucleotide sequence of SEQ ID NO:7, and a second genome segment encoding a neuramidinase polypeptide.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments from A/Puerto Rico/8/34, a first genome segment comprising the nucleotide sequence of residues 84-1064 of SEQ ID NO:7, and a second genome segment encoding a neuramidinase polypeptide.
• the second genome segment comprises the nucleotide sequence of SEQ ID NO:5.
• the neuramidinase polypeptide comprises the amino acid sequence of SEQ ID NO:4.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 log 10 PFU/ml, at least about 8.5 log 10 PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 log 10 PFU/ml, at least about 10 log 10 PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 logio PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in tissue cultures.
• the invention comprises a sterile composition with one or more polypeptide listed above, or fragments thereof.
• the invention also encompasses immunogenic compositions comprising an immunologically effective amount of one or more of the polypeptides described above, as well as methods for stimulating the immune system of an individual to produce a protective immune response against an influenza virus comprising administering to the individual an immunologically effective amount of one or more of the polypeptides described above.
• an immunogenic composition of the invention comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8.
• an immunogenic composition of the invention comprises a polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO:6 or 8.
• an immunogenic composition of the invention comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• an immunogenic composition of the invention comprises a polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• an immunogenic composition of the invention comprises a polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 of SEQ ID NO: 6 or 8.
• an immunogenic composition of the invention comprises a polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• an immunogenic composition of the invention is a monovalent immunogenic composition comprising a single reassortant influenza virus. In one embodiment, an immunogenic composition of the invention is a trivalent immunogenic composition comprising three reassortant influenza viruses. In one embodiment, an immunogenic composition of the invention is a trivalent immunogenic composition comprising two reassortant influenza A viruses and a reassortant influenza B virus. In one embodiment, an immunogenic composition of the invention is a trivalent immunogenic composition comprising a reassortant influenza A virus of the Hl type, a reassortant influenza A virus of the H3 type and a reassortant influenza B virus.
• an immunogenic composition of the invention is a tetravalent immunogenic composition comprising four reassortant influenza viruses.
• an immunogenic composition of the invention is a tetravalent immunogenic composition comprising two reassortant influenza A viruses and two reassortant influenza B viruses.
• an immunogenic composition of the invention is a tetravalent immunogenic composition comprising a reassortant influenza A virus of the Hl type, a reassortant influenza A virus of the H3 type, a reassortant influenza B virus of the Victoria lineage and a reassortant influenza B virus of the Yamagata lineage.
• an immunogenic composition of the invention comprises a reassortant or recombinant influenza virus comprising a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8.
• an immunogenic composition of the invention comprises a reassortant or recombinant influenza virus comprising a genome segment encoding an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• an immunogenic composition of the invention comprises a reassortant or recombinant influenza virus comprising a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• an immunogenic composition of the invention comprises a reassortant or recombinant influenza virus comprising a genome segment encoding an HA polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• an immunogenic composition of the invention comprises a reassortant or recombinant influenza virus comprising a genome segment encoding an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• an immunogenic composition of the invention comprises a reassortant or recombinant influenza virus comprising a genome segment encoding an HA polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the invention encompasses a reassortant influenza virus that comprises a genome segment encoding one or more of the polypeptides described above. Further, the invention encompasses immunogenic compositions comprising an immunologically effective amount of such reassortant influenza virus.
• a reassortant influenza virus of the invention is a 6:2 reassortant virus comprising 6 internal genome segments from one or more donor virus (e.g. A/AA/6/60 or A/Puerto Rico/8/34, which is more commonly known as PR8) and further comprising 2 genome segments (typically and preferably encoding HA and NA or fragments thereof).
• donor virus e.g. A/AA/6/60 or A/Puerto Rico/8/34, which is more commonly known as PR8
• Immunogenic compositions comprising such reassortant (recombinant) virus are also features of the invention.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/ Ann Arbor/6/60 and a genome segment encoding an HA polypeptide.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 having at least one substitution, deletion or insertion.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 having the H273Y substitution.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 having the D222G and/or Q223R substitution.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO:1 having at least one amino acid substitution selected from the group consisting of Kl 19E, Kl 19N and A186D.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Ann Arbor/6/60 and a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO:1 having the D222G, Kl 19E and A186D substitutions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/ Ann Arbor/6/60 and a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO:1 having Q223R and A186D substitutions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/ Ann Arbor/6/60 and a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO:1 having Q223R, H273Y and A186D substitutions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Ann Arbor/6/60 and an HA genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8.
• the HA genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• the HA genome segment may encode an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the HA genome segment may encode an HA polypeptide comprising the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8.
• the HA genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 SEQ ID N0:6 or 8.
• the HA genome segment may encode an HA polypeptide comprising the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Puerto Rico/8/34 (PR8) and a genome segment encoding an HA polypeptide.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 having at least one substitution, deletion or insertion.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 having the D222G and/or Q223R substitution.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO:1 having at least one amino acid substitution selected from the group consisting of Kl 19E, Kl 19N and A186D.
• the HA polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 having the H273Y substitution.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Puerto Rico/8/34 (PR8) and a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 1 having the D222G, Kl 19E and A186D substitutions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Puerto
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Puerto Rico/8/34 (PR8) and a genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 1 having the Q223R, H273Y and A186D substitutions.
• a reassortant or recombinant influenza virus of the invention comprises 6 internal genome segments of A/Puerto Rico/8/34 (PR8) and an HA genome segment encoding an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8.
• the HA genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• the HA genome segment may encode an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the HA genome segment may encode an HA polypeptide comprising the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8.
• the HA genome segment may encode an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8.
• the HA genome segment may encode an HA polypeptide comprising the amino acid sequence of residues 1-327 SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• reassortant influenza viruses comprising a polynucleotide encoding a polypeptide of the invention.
• such reassortant viruses are 6:2 reassortant viruses comprising 6 internal genome segments from one or more donor virus (e.g., A/AA/6/60 or A/Puerto Rico/8/34), a first genome segment encoding a hemagglutinin polypeptide of the invention, and a second genome segment encoding a neuramidinase polypeptide.
• Immunogenic compositions comprising immunologically effective amounts of such a reassortant/recombinant influenza virus are also within purview of the current invention.
• Reassortant or recombinant viruses of the invention display high rate of replication in tissue cultures or embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 log 10 PFU/ml, at least about 9 log 10 PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in embryonated eggs.
• a reassortant or recombinant influenza virus grows to a titer of at least about 7.5 logio PFU/ml , at least about 8 logio PFU/ml, at least about 8.5 logio PFU/ml, at least about 9 logio PFU/ml, at least about 9.5 logio PFU/ml, at least about 10 logio PFU/ml, at least about 10.5 logio PFU/ml or at least about 11 logio PFU/ml in tissue cultures.
• the invention comprises immunogenic compositions having an immunologically effective amount of one or more of the above described reassortant influenza viruses of the invention.
• an immunogenic composition of the invention comprises a live virus of the invention.
• Other embodiments include methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus comprising administering to the individual an immunologically effective amount of one or more of the reassortant influenza viruses described above (optionally in a physiologically effective carrier).
• the invention also encompasses vaccines comprising a recombinant or reassortant influenza virus of the invention.
• a vaccine of the invention is a split vaccine or killed vaccine.
• a vaccine of the invention is a live attenuated influenza virus vaccine.
• a vaccine of the invention may be a monovalent, bivalent, trivalent or tetravalent vaccine.
• a vaccine of the invention may be a monovalent, bivalent, trivalent or tetravalent vaccine.
• the present invention also relates to methods and compositions for increasing the replication capacity of influenza viruses in, for example, embryonated hens' eggs and/or cell culture.
• the invention is based, in part, on the identification of particular Hl protein amino acids associated with increased replication capacity.
• an Hl HA gene encoding an Hl HA protein that comprises one or more of these particular amino acids, improved influenza viral yields can be achieved.
• a method for increasing the replication capacity of a reassortant or recombinant influenza virus comprising altering an amino acid residue of the hemagglutinin polypeptide at a position corresponding to the amino acid residue of position 119 or 186 of SEQ ID NO: 1, thereby increasing the replication capacity of the reassortant or recombinant influenza virus.
• a recombinant influenza virus produced by the method is also provided.
• the present invention also relates to methods and compositions for increasing the replication capacity of HlNl reassortant influenza viruses in, for example, embryonated hens' eggs and/or cell culture.
• a method is provided for increasing the replication capacity of a HlNl reassortant or recombinant influenza virus comprising altering an amino acid residue of the hemagglutinin polypeptide at a position corresponding to the amino acid residue of position 119 or 186 of SEQ ID NO: 1, thereby increasing the replication capacity of the reassortant or recombinant influenza virus.
• a recombinant influenza virus produced by the method is also provided.
• the replication capacity of a reassortant or recombinant influenza virus of the invention which comprises a hemagglutinin polypeptide of the invention having a non naturally occurring amino acid is increased at least 2-fold, at least 4- fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold, or at least 100-fold relative to the same reassortant or recombinant influenza virus not comprising a non naturally occurring amino acid at the same position.
• the replication capacity of a reassortant or recombinant influenza virus of the invention which comprises a hemagglutinin polypeptide of the invention having a non naturally occurring amino acid is increased at between 2-fold and 10-fold, between 2-fold and 20-fold, between 2-fold and 40-fold, between 2-fold and 100-fold, between 5-fold and 10-fold, between 5-fold and 20-fold, between 5-fold and 40-fold, or between 5-fold and 100-fold relative to the same reassortant or recombinant influenza virus not comprising a non naturally occurring amino acid at the same position.
• the peak titer in embryonated eggs for a reassortant or recombinant influenza virus of the invention which comprises a hemagglutinin polypeptide of the invention having a non naturally occurring amino acid is increased at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold, or at least 100-fold relative to the same reassortant or recombinant influenza virus not comprising a non naturally occurring amino acid at the same position.
• the peak titer in embryonated eggs for a reassortant or recombinant influenza virus of the invention which comprises a hemagglutinin polypeptide of the invention having a non naturally occurring amino acid is increased at between 2-fold and 10-fold, between 2-fold and 20-fold, between 2-fold and 40-fold, between 2-fold and 100-fold, between 5-fold and 10-fold, between 5-fold and 20-fold, between 5-fold and 40-fold, or between 5 -fold and 100-fold relative to the same reassortant or recombinant influenza virus not comprising a non naturally occurring amino acid at the same position.
• a recombinant influenza virus which comprises an HA polypeptide of an HlNl virus which polypeptide comprises an amino acid selected from the group consisting of Kl 19E, Kl 19N and A186D.
• a recombinant influenza virus is provided which comprises an HA polypeptide of an HlNl virus which polypeptide comprises an amino acid selected from the group consisting of Kl 19X and A186X, wherein X is any amino acid not naturally occurring in said HlNl virus.
• the recombinant influenza viruses of the invention are 6:2 reassortant viruses comprising at least 5 or 6 segments of (or derived from) an attenuated virus.
• the recombinant influenza viruses of the invention are 6:2 reassortant viruses comprising at least 5 or 6 segments of (or derived from) an attenuated virus selected from the group consisting of: Ann Arbor/6/60, A/Puerto Rico/8/34, A/Leningrad/I 34/17/57, and A/Leningrad/I 7.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the recombinant influenza viruses of the invention are 6:2, or 7:1 reassortant viruses comprising an HA polypeptide of the invention.
• the recombinant influenza viruses of the invention are 6:2, or 7:1 reassortant viruses comprising an HA polypeptide of the invention and at least 5 or 6 segments of (or derived from) an attenuated virus selected from the group consisting of: A/Ann Arbor/6/60, A/Puerto Rico/8/34, A/Leningrad/I 34/17/57, and A/Leningrad/I 7.
• the recombinant influenza viruses of the invention comprise an HA polypeptide of (or derived from) A/CA/04/09 or A/CA/07/09 having 1, 2, or 3 substitutions (e.g., at positions 119, 186). In one embodiment, the recombinant influenza viruses of the invention comprise an HA polypeptide of (or derived from) A/CA/04/09 or A/CA/07/09 having a glutamic acid or an asparagine at amino acid position 119 and an aspartic acid at position 186.
• the recombinant influenza viruses of the invention comprise an HA polypeptide of (or derived from) A/CA/04/09 or A/CA/07/09 having an amino acid substitution at amino acid position 119 and position 186. In one embodiment, the recombinant influenza viruses of the invention comprise an HA polypeptide of (or derived from) an HlNl swine flu virus having an amino acid substitution at amino acid position 119 and position 186.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of SEQ ID NO:6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8.
• the recombinant influenza viruses of the invention comprise an HA polypeptide comprising the amino acid sequence of residues 1-327 of SEQ ID NO:6 or 8 comprising less than 3, less than 5, less then 10, less then 15, less than 20, less than 25, less than 30 amino acid substitutions, deletions or insertions.
• the exact position of an amino acid can vary depending on the particular influenza strain used in the vectors, methods, and viruses of the invention.
• the HA protein of a particular influenza strain may comprise an insertion or deletion in the HA gene encoding the HA protein such that the position corresponding to position 119 of SEQ ID NO:1 is found at, for example, residue 93 or 97 of the HA protein of that particular influenza strain.
• position 119 and 186 of the A/CA/07/09 HA polypeptide corresponds to position 118 and 185, respectively of the A/South Dakota/6/07 Hl HA polypeptide.
• One skilled in the art can readily recognize whether a particular amino acid position corresponds to a position that, when altered, is associated with increased replication capacity using techniques conventional to the art.
• One such conventional technique is to align the amino acid sequence of SEQ ID NO: 1 and the particular influenza strain HA polypeptide using algorithms available in the art.
• Figure 1 Plaque morphology of variants selected from MDCK cells and the indicated variants containing introduced amino acid changes (A). Plaque assay was performed in MDCK cells, incubated at 33 0 C for 4 days and immunostained with polyclonal antiserum against influenza A virus. The plaque morphology of double mutant V5- 119E/186D is compared with single 119E and 186D mutant in panel B.
• Figure 2 HAl protein sequence comparison of A/California/7/09, A/Swine/Iowa/1/1976 (nucleotide sequence accession code CY022069), A/Swine/1931 (nucleotide sequence accession code CY009628), and A/South Dakota/6/07. The amino acids at positions 119, 153, 154, 155, 186 and 278 are highlighted.
• FIG. 1 Protein composition of virus preparations purified from embryonated eggs.
• Figure 4 Protein composition of virus preparations purified from embryonated eggs.
• Figure 5. Protein composition of purified virus preparations harvested from embryonated eggs at (A) 48 hrs and (B) 60 hrs.
• Figure 6. Quantitative comparison of the HAl protein produced by two reassortant HlNl viruses in embryonated eggs.
• nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 90%, preferably 91%, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
• variant refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
• the variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
• a variant can have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan.
• Analogous minor variation can also include amino acid deletion or insertion, or both.
• Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software. Examples of conservative substitutions are also described herein.
• the "neuraminidase" polypeptides of the invention show immunological cross reactivity with one or more known neuraminidase molecule from an influenza virus.
• hemagglutinin polypeptides of the invention may show immunological cross-reactivity with one or more known hemagglutinin molecule from an influenza virus. Again, the literature is replete with examples of such known hemagglutinin molecules.
• Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins.
• Non-expressed regulatory sequences include “promoters” and “enhancers,” to which regulatory proteins such as transcription factors bind, resulting in transcription of adjacent or nearby sequences.
• a "tissue specific” promoter or enhancer is one that regulates transcription in a specific tissue type or cell type, or types.
• Expression of a gene or "expression of a nucleic acid” typically means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing) or transcription of RNA into mRNA, translation of RNA into a polypeptide (possibly including subsequent modification of the polypeptide, e.g., post-translational modification), or both transcription and translation, as indicated by the context.
• ORF is a possible translational reading frame of DNA or RNA (e.g., of a gene), which is capable of being translated into a polypeptide. That is, the reading frame is not interrupted by stop codons.
• ORF does not necessarily indicate that the polynucleotide is, in fact, translated into a polypeptide.
• vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
• Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell.
• a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome - conjugated DNA, or the like, that is not autonomously replicating.
• the vectors of the present invention are plasmids.
• An "expression vector” is a vector, such as a plasmid that is capable of promoting expression, as well as replication of a nucleic acid incorporated therein.
• the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.
• a "bi-directional expression vector” is characterized by two alternative promoters oriented in the opposite direction relative to a nucleic acid situated between the two promoters, such that expression can be initiated in both orientations resulting in, e.g., transcription of both plus (+) or sense strand, and negative (-) or antisense strand RNAs.
• isolated refers to a biological material, such as a virus, a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it in its naturally occurring environment.
• the isolated biological material optionally comprises additional material not found with the biological material in its natural environment, e.g., a cell or wild-type virus.
• the material can have been placed at a location in the cell (e.g., genome or genetic element) not native to such material found in that environment.
• a naturally occurring nucleic acid e.g., a coding sequence, a promoter, an enhancer, etc.
• a locus of the genome e.g., a vector, such as a plasmid or virus vector, or amplicon
• Such nucleic acids are also referred to as "heterologous" nucleic acids.
• An isolated virus for example, is in an environment (e.g., a cell culture system, or purified from cell culture) other than the native environment of wild-type virus (e.g., the nasopharynx of an infected individual).
• chimeric when referring to a virus, indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral strain or source.
• chimeric when referring to a viral protein, indicates that the protein includes polypeptide components (i.e., amino acid subsequences) derived from more than one parental viral strain or source.
• polypeptide components i.e., amino acid subsequences
• such chimeric viruses are typically reassortant/recombinant viruses.
• a chimera can optionally include, e.g., a sequence (e.g., of HA and/or NA) from an A influenza virus placed into a backbone comprised of, or constructed/derived from a B influenza virus (e.g., B/AA/1/66, etc.) or a B influenza virus sequence placed into an A influenza virus backbone (i.e., donor virus) such as, e.g., A/AA/6/60, etc.
• a sequence e.g., of HA and/or NA
• a influenza virus backbone i.e., donor virus
• the term "recombinant" indicates that the material (e.g., a nucleic acid or protein) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state.
• an influenza virus is recombinant when it is produced by the expression of a recombinant nucleic acid.
• a "recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis;
• a "recombinant polypeptide” or “recombinant protein” is a polypeptide or protein which is produced by expression of a recombinant nucleic acid;
• a "recombinant virus,” e.g., a recombinant influenza virus is produced by the expression of a recombinant nucleic acid.
• reassortant when referring to a virus (typically herein, an influenza virus), indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral strain or source.
• a 7: 1 reassortant includes 7 viral genome segments (or gene segments) derived from a first parental virus, and a single complementary viral genome segment, e.g., encoding a hemagglutinin or neuraminidase described herein.
• a 6:2 reassortant includes 6 genome segments, most commonly the 6 internal genome segments from a first parental virus, and two complementary segments, e.g., hemagglutinin and neuraminidase genome segments, from one or more different parental virus.
• Reassortant viruses can also, depending upon context herein, be termed as “chimeric” and/or "recombinant.”
• nucleic acid when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• the term includes such methods as “infection,” “transfection,” “transformation,” and “transduction.”
• a variety of methods can be employed to introduce nucleic acids into cells, including electroporation, calcium phosphate precipitation, lipid mediated transfection (lipofection), etc.
• host cell means a cell that contains a heterologous nucleic acid, such as a vector or a virus, and supports the replication and/or expression of the nucleic acid.
• Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells.
• Exemplary host cells can include, e.g., Vero (African green monkey kidney) cells, BHK (baby hamster kidney) cells, primary chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g., COSl, C0S7 cells), etc.
• host cells can optionally include eggs (e.g., hen eggs, embryonated hen eggs, etc.).
• influenza virus is an amount sufficient to enhance an individual's (e.g., a human's) own immune response against a subsequent exposure to influenza virus.
• Levels of induced immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay.
• a "protective immune response" against influenza virus refers to an immune response exhibited by an individual (e.g., a human) that is protective against disease when the individual is subsequently exposed to and/or infected with wild-type influenza virus.
• the wild-type influenza virus can still cause infection, but it cannot cause a serious or life -threatening infection.
• the protective immune response results in detectable levels of host engendered serum and secretory antibodies that are capable of neutralizing virus of the same strain and/or subgroup (and possibly also of a different, non- vaccine strain and/or subgroup) in vitro and in vivo.
• an "antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• a typical immunoglobulin (antibody) structural unit comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
• Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
• pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond.
• the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer.
• the Fab' monomer is essentially a Fab with part of the hinge region (see Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1999) for a more detailed description of other antibody fragments).
• antibody includes antibodies or fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
• Antibodies include, e.g., polyclonal antibodies, monoclonal antibodies, multiple or single chain antibodies, including single chain Fv (sFv or scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.
• influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein.
• the genome of influenza viruses is composed of eight segments of linear (-) strand ribonucleic acid (RNA), encoding the immunogenic hemagglutinin (HA) and neuraminidase (NA) proteins, and six internal core polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins (M); non-structural proteins (NS); and 3 RNA polymerase (PA, PBl, PB2) proteins.
• RNA nucleocapsid nucleoprotein
• M matrix proteins
• NS non-structural proteins
• PA 3 RNA polymerase
• Each of the eight genomic segments is packaged into ribonucleoprotein complexes that contain, in addition to the RNA, NP and a polymerase complex (PBl, PB2, and PA).
• the hemagglutinin molecule consists of a surface glycoprotein and acts to bind to N-AcetylNeuraminic acid (NeuNAc), also known as sialic acid, on host cell surface receptors.
• the polypeptides of the invention can act to bind NeuNAc whether in vitro or in vivo. Such action can in some embodiments also be done by fragments of hemagglutinin which retain hemagglutinin activity.
• Hemagglutinin is made up of two subunits, HAl and HA2 and the entire structure is about 550 amino acids in length and about 220 kD.
• Neuraminidase molecules cleave terminal sialic acid residues from cell surface receptors of influenza virus, thereby releasing virions from infected cells. Neuraminidase also removes sialic acid from newly made hemagglutinin and neuraminidase molecules.
• the polypeptides of the invention (and polypeptides encoded by the polynucleotides of the invention) can act to cleave sialic acid residues whether in vitro or in vivo.
• neuraminidase polypeptides of the invention show immunological cross reactivity with one or more known neuraminidase molecule from an influenza virus.
• the literature is replete with examples of such known neuraminidases (e.g., in GenBank, in publications from the CDC, etc.).
• the hemagglutinin polypeptides of the invention show immunological cross- reactivity with one or more known hemagglutinin molecule from an influenza virus. Again, the literature is replete with examples of such known hemagglutinin molecules.
• Influenza is commonly grouped into influenza A and influenza B categories, as well as a typically less important C category. Influenza A and influenza B viruses each contain eight segments of single stranded RNA with negative polarity.
• the influenza A genome encodes eleven polypeptides. Segments 1-3 encode three polypeptides, making up a RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2. The remaining polymerase proteins PBl and PA are encoded by segment 2 and segment 3, respectively.
• segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the PBl coding region.
• Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection.
• HA hemagglutinin
• Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA.
• Segment 6 encodes a neuraminidase (NA) envelope glycoprotein.
• Segment 7 encodes two matrix proteins, designated Ml and M2, which are translated from differentially spliced mRNAs.
• Segment 8 encodes NSl and NS2, two nonstructural proteins, which are translated from alternatively spliced mRNA variants.
• the eight genome segments of influenza B encode 11 proteins. The three largest genes code for components of the RNA polymerase, PBl, PB2 and PA.
• Segment 4 encodes the HA protein.
• Segment 5 encodes NP.
• Segment 6 encodes the NA protein and the NB protein.
• NSl is translated from the full length RNA
• NS2 is translated from a spliced mRNA variant.
• Influenza types A and B are typically associated with influenza outbreaks in human populations. However, type A influenza also infects other species as well, e.g., birds, pigs, and other animals.
• the type A viruses are categorized into subtypes based upon differences within their hemagglutinin and neuraminidase surface glycoprotein antigens. Hemagglutinin in type A viruses has 16 known subtypes and neuraminidase has 9 known subtypes. In humans, currently only about 4 different hemagglutinin and 2 different neuraminidase subtypes are known, e.g., Hl, H2, H3, H5, Nl, and N2.
• influenza A has been active in humans, namely, HlNl and H3N2.
• H1N2 has recently been of concern.
• Influenza B viruses are not divided into subtypes based upon their hemagglutinin and neuraminidase proteins.
• Different strains of influenza can be categorized based upon, e.g., the ability of influenza to agglutinate red blood cells (RBCs or erythrocytes). Antibodies specific for particular influenza strains can bind to the virus and, thus, prevent such agglutination. Assays determining strain types based on such inhibition are typically known as hemagglutinin inhibition assays (HI assays or HAI assays) and are standard and well known methods in the art to characterize influenza strains. Of course, those of skill in the art will be familiar with other assays, e.g., ELISA, indirect fluorescent antibody assays, immunohistochemistry, Western blot assays, etc. with which to characterize influenza strains and the use of and discussion herein of HI assays should not be necessarily construed as limiting.
• HI assays hemagglutinin inhibition assays
• erythrocyte samples in typical HI assays, sera to be used for typing or categorization, which is often produced in ferrets, is added to erythrocyte samples in various dilutions, e.g., 2-fold, etc. Optical determination is then made whether the erythrocytes are clumped together (i.e., agglutinated) or are suspended (i.e., non-agglutinated). If the cells are not clumped, then agglutination did not occur due to the inhibition from antibodies in the sera that are specific for that influenza. Thus, the types of influenza are defined as being within the same strain.
• one strain is described as being “like” the other, e.g., strain x is a "y-like” strain, etc.
• strain x is a "y-like” strain, etc.
• strains are typically categorized based upon their immunologic or antigenic profile.
• An HAI titer is typically defined as the highest dilution of a serum that completely inhibits hemagglutination. See, e.g., Schild, et al., Bull.
• such same strains that are within various vectors are also included.
• typically ones used for plasmid reassortment and rescue such as A/ Ann Arbor/6/60 or B/Ann Arbor/1/66, A/Puerto Rico/8/34, B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, or B/England/2608/76, etc.
• similar strain should be taken to indicate that a first influenza virus is of the same or related strain as a second influenza virus. In typical embodiments such relation is commonly determined through use of an HAI assay.
• Influenza viruses that fall within a four- fold titer of one another in an HAI assay are, thus, of a "similar strain.”
• Those of skill in the art will be familiar with other assays, etc. to determine similar strains, e.g., FRID, neutralization assays, etc.
• the current invention also comprises such similar strains (i.e., strains similar to the ones present in the sequence listing herein) in the various plasmids, vectors, viruses, methods, etc. herein.
• influenza virus vaccines have primarily been produced in embryonated hen eggs using strains of virus selected or based on empirical predictions of relevant strains. More recently, reassortant viruses have been produced that incorporate selected hemagglutinin and/or neuraminidase antigens in the context of an approved attenuated, temperature sensitive master strain. Following culture of the virus through multiple passages in hen eggs, influenza viruses are recovered and, optionally, inactivated, e.g., using formaldehyde and/or ⁇ -propiolactone (or alternatively used in live attenuated vaccines). Thus, it will be appreciated that HA and NA sequences (as in the current invention) are quite useful in constructing influenza vaccines.
• the cultures are maintained in a system, such as a cell culture incubator, under controlled humidity and CO 2 , at constant temperature using a temperature regulator, such as a thermostat to insure that the temperature does not exceed 35 0 C.
• a temperature regulator such as a thermostat to insure that the temperature does not exceed 35 0 C.
• Reassortant influenza viruses can be readily obtained by introducing a subset of vectors corresponding to genomic segments of a master influenza virus, in combination with complementary segments derived from strains of interest (e.g., HA and NA antigenic variants herein).
• the master strains are selected on the basis of desirable properties relevant to vaccine administration.
• the master donor virus strain may be selected for an attenuated phenotype, cold adaptation and/or temperature sensitivity. As explained elsewhere herein and, e.g., in U.S. Patent Application No.
• various embodiments of the invention utilize A/ Ann Arbor (AA)/6/60 or B/Ann Arbor/1/66 or A/Puerto Rico/8/34, or B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, or B/England/2608/76 influenza strain as a "backbone" upon which to add HA and/or NA genes (e.g., such as those sequences listed herein, etc.) to create desired reassortant viruses.
• AA Ann Arbor
• B/Ann Arbor/1/66 or A/Puerto Rico/8/34
• B/Leningrad/14/17/55 B/14/5/1
• B/USSR/60/69 B/Leningrad/179/86
• B/Leningrad/14/55 or B/England/2608/76 influenza strain as a "backbone" upon which to add HA and/or NA genes (
• a 6:2 reassortant 2 genes (i.e., NA and HA) would be from the influenza strain(s) against which an immunogenic reaction is desired, while the other 6 genes would be from the Ann Arbor strain, or other backbone strain, etc.
• the Ann Arbor virus is useful for its cold adapted, attenuated, temperature sensitive attributes.
• the HA and NA sequences herein are capable of reassortment with a number of other virus genes or virus types (e.g., a number of different "backbones" such as A/Puerto Rico/8/34, etc., containing the other influenza genes present in a reassortant, namely, the non-HA and non-NA genes).
• reassortant HlNl and H1N2 viruses in which the internal protein genes of A/ Ann Arbor (AA)/6/60 (H2N2) cold adapted (ca) virus confer the cold adapted, attenuation and temperature sensitive phenotypes of the AA ca virus on the reassortant viruses (i.e., the ones having the hemagglutinin and neuraminidase genes from the non-Ann Arbor strain).
• the reassortants can also comprise 7:1 reassortants. In other words, only the HA or the NA is not from the backbone or MDV strain.
• the sequences herein can optionally have specific regions removed (both or either in the nucleic acid sequence or the amino acid sequence). For example, for those molecules having a polybasic cleavage site, such sites can optionally be removed.
• Such cleavage sites are, e.g., modified or altered in their sequences in comparison to the wild-type sequences from which such sequences are derived (e.g., to disable the cleavage or reduce the cleavage there, etc.). Such modifications/alterations can be different in different strains or sequences due to the various sequences of the cleavage sites in the starting sequences.
• polybasic residues are typically removed in some HA sequences, (as compared to wt).
• polybasic cleavage sites can be modified in a number of ways (all of which are contained within the invention).
• the polybasic cleavage site can be removed one amino acid at a time (e.g., one R removed, two Rs removed, RRK removed, or RRKK removed).
• an amino acid residue directly upstream of the cleavage site can also be removed or altered (e.g., from an R to a T, etc.); also, the nucleotides encoding the amino acid residue directly after the cleavage site can also be modified.
• temperature sensitive indicates, e.g., that the virus exhibits a 100 fold or greater reduction in titer at 39 0 C relative to 33 0 C for influenza A strains, or that the virus exhibits a 100 fold or greater reduction in titer at 37 0 C relative to 33 0 C for influenza B strains.
• the term “cold adapted” indicates that the virus exhibits growth at 25 0 C within 100 fold of its growth at 33 0 C, while the term “attenuated” (att) indicates that the virus replicates in the upper airways of ferrets but is not detectable in their lung tissues, and does not cause influenza-like illness in the animal.
• viruses with intermediate phenotypes i.e., viruses exhibiting titer reductions less than 100 fold at 39 0 C (for A strain viruses) or 37 0 C (for B strain viruses), or exhibiting growth at 25 0 C that is more than 100 fold than its growth at 33 0 C (e.g., within 200 fold, 500 fold, 1000 fold, 10,000 fold less), and/or exhibit reduced growth in the lungs relative to growth in the upper airways of ferrets (i.e., partially attenuated) and/or reduced influenza like illness in the animal, are also useful viruses and can be used in conjunction with the HA and NA sequences herein.
• the present invention can utilize growth, e.g., in appropriate culture conditions, of virus strains (both A strain and B strain influenza viruses) with desirable properties relative to vaccine production (e.g., attenuated pathogenicity or phenotype, cold adaptation, temperature sensitivity, etc.) in vitro in cultured cells.
• Influenza viruses can be produced by introducing a plurality of vectors incorporating cloned viral genome segments into host cells, and culturing the cells at a temperature not exceeding 35 0 C. When vectors including an influenza virus genome are transfected, recombinant viruses suitable as vaccines can be recovered by standard purification procedures.
• reassortant viruses incorporating the six internal gene segments of a strain selected for its desirable properties with respect to vaccine production, and the immunogenic HA and NA segments from a selected, e.g., pathogenic strain such as those in the sequence listing herein, can be rapidly and efficiently produced in tissue culture.
• the system and methods described herein are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B viruses, including viruses suitable for use as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration.
• a single Master Donor Virus (MDV) strain is selected for each of the A and B subtypes.
• the Master Donor Virus strain is typically chosen for its favorable properties, e.g., temperature sensitivity, cold adaptation and/or attenuation, relative to vaccine production.
• exemplary Master Donor Strains include such temperature sensitive, attenuated and cold adapted strains of A/ Ann Arbor/6/60 and B/Ann Arbor/1/66, respectively, as well as others mentioned throughout.
• a selected master donor type A virus (MDV-A), or master donor type B virus (MDV-B) is produced from a plurality of cloned viral cDNAs constituting the viral genome.
• MDV-A master donor type A virus
• MDV-B master donor type B virus
• Embodiments include those wherein recombinant viruses are produced from eight cloned viral cDNAs.
• Infectious recombinant MDV-A or MDV-B virus can be then recovered following transfection of plasmids bearing the eight viral cDNAs into appropriate host cells, e.g., Vera cells, co-cultured MDCK/293T or MDCK/COS7 cells.
• appropriate host cells e.g., Vera cells
• MDCK/293T or MDCK/COS7 cells e.g., Vera cells
• the invention is useful, e.g., for generating 6:2 reassortant influenza vaccines by co-transfection of the 6 internal genes (PBl, PB2, PA, NP, M and NS) of the selected virus (e.g., MDV-A, MDV-B) together with the HA and NA derived from different corresponding type (A or B) influenza viruses e.g., as shown in the sequence listings herein.
• 6:2 reassortant influenza vaccines by co-transfection of the 6 internal genes (PBl, PB2, PA, NP, M and NS) of the selected virus (e.g., MDV-A, MDV-B) together with the HA and NA derived from different corresponding type (A or B) influenza viruses e.g., as shown in the sequence listings herein.
• the HA segment is favorably selected from a pathogenically relevant Hl, H3 or B strain, as is routinely performed for vaccine production.
• the HA segment can be selected from a strain with emerging relevance as a pathogenic strain such as those in the sequence listing herein.
• Reassortants incorporating seven genome segments of the MDV and either the HA or NA gene of a selected strain (7:1 reassortants) can also be produced. It will be appreciated, and as is detailed throughout, the molecules of the invention can optionally be combined in any desired combination.
• the HA and/or NA sequences herein can be placed, e.g., into a reassortant backbone such as A/AA/6/60, B/AA/1/66, A/Puerto Rico/8/34 (i.e., PR8), etc., in 6:2 reassortants or 7:1 reassortants, etc.
• a reassortant backbone such as A/AA/6/60, B/AA/1/66, A/Puerto Rico/8/34 (i.e., PR8), etc.
• Such 2 genome segments are preferably the HA and NA genes.
• any of the sequences of the invention can optionally be present with any other sequence of the invention.
• Typical, and preferred, embodiments comprise HA and NA from the same original wild-type strains however (or modified wild-type strains such as those with modified polybasic cleavage sites).
• typical embodiments can comprise a 6:2 reassortant having 6 internal genome segments from a donor virus such as A/AA/6/60 and the HA and NA genome segments described herein.
• the invention also includes such reassortant viruses wherein the HA and NA genome segments are from similar strains.
• the above references are specifically incorporated herein in their entirety for all purposes, e.g., especially for their teachings regarding plasmids, plasmid rescue of virus (influenza virus), multi-plasmid systems for virus rescue/production, etc.
• the HA and NA sequences of the current invention are optionally utilized in such plasmid reassortment vaccines (and/or in other ts, cs, ca, and/or att viruses and vaccines).
• the HA and NA sequences, etc. of the invention are not limited to specific vaccine compositions or production methods, and can, thus, be utilized in substantially any vaccine type or vaccine production method which utilizes strain specific HA and NA antigens (e.g., the sequences of the invention).
• influenza vaccine is FluMist (Medlmmune Vaccines Inc., Mt. View, CA) which is a live, attenuated vaccine that protects children and adults from influenza illness (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N Engl J Med 338:1405-12; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282: 137-44).
• the methods and compositions of the current invention are preferably adapted to/used with production of FluMist vaccine.
• FluMist vaccine strains contain, e.g., HA and NA gene segments derived from the wild-type strains to which the vaccine is addressed (or, in some instances, to related strains) along with six gene segments, PBl, PB2, PA, NP, M and NS, from a common master donor virus (MDV).
• MDV master donor virus
• MDV-A The MDV for influenza A strains of FluMist (MDV-A), was created by serial passage of the wild-type A/ Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue culture at successively lower temperatures (Maassab (1967) Adaptation and growth characteristics of influenza virus at 25 degrees CNature 213:612-4). MDV-A replicates efficiently at 25 0 C (ca, cold adapted), but its growth is restricted at 38 and 39 0 C (ts, temperature sensitive). Additionally, this virus does not replicate in the lungs of infected ferrets (att, attenuation).
• the ts phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its replication in all but the coolest regions of the respiratory tract. The stability of this property has been demonstrated in animal models and clinical studies.
• the ts property of MDV-A does not revert following passage through infected hamsters or in shed isolates from children (for a recent review, see Murphy & Coelingh (2002) Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines Viral Immunol 15:295-323).
• the system and methods described previously are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B viruses, including viruses suitable for use as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration.
• the sequences, methods, etc. of the current invention are optionally used in conjunction with, or in combination with, such previous work involving, e.g., reassorted influenza viruses for vaccine production to produce viruses for vaccines.
• FluMist TM vaccine the current invention can be used in other vaccine formulations.
• recombinant and reassortant viruses of the invention can be administered prophylactically in an immunologically effective amount and in an appropriate carrier or excipient to stimulate an immune response specific for one or more strains of influenza virus as determined by the HA and/or NA sequence.
• the carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, allantoic fluid from uninfected hen eggs (i.e., normal allantoic fluid or NAF), or combinations thereof.
• a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc.
• a related aspect of the invention provides methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus.
• an immunologically effective amount of a recombinant influenza virus e.g., an HA and/or an NA molecule of the invention
• an immunologically effective amount of a polypeptide of the invention e.g., an immunologically effective amount of a polypeptide of the invention
• an immunologically effective amount of a nucleic acid of the invention is administered to the individual in a physiologically acceptable carrier.
• the influenza viruses of the invention are administered in a quantity sufficient to stimulate an immune response specific for one or more strains of influenza virus (i.e., against the HA and/or NA strains of the invention).
• administration of the influenza viruses elicits a protective immune response to such strains.
• influenza viruses are provided in the range of about 1-1000 HID50 (human infectious dose), i.e., about 10 5 - 10 8 pfu (plaque forming units) per dose administered.
• the dose will be adjusted within this range based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors.
• the prophylactic vaccine formulation is systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needle-less injection device.
• the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery results in a protective systemic immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of entry of the influenza virus.
• Attenuated live virus vaccines are often preferred, e.g., an attenuated, cold adapted and/or temperature sensitive recombinant or reassortant influenza virus. See above. While stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect.
• the attenuated recombinant influenza of this invention as used in a vaccine is sufficiently attenuated such that symptoms of infection, or at least symptoms of serious infection, will not occur in most individuals immunized (or otherwise infected) with the attenuated influenza virus.
• the attenuated influenza virus can still be capable of producing symptoms of mild illness (e.g., mild upper respiratory illness) and/or of dissemination to unvaccinated individuals.
• its virulence is sufficiently abrogated such that severe lower respiratory tract infections do not occur in the vaccinated or incidental host.
• an immune response can be stimulated by ex vivo or in vivo targeting of dendritic cells with influenza viruses comprising the sequences herein.
• proliferating dendritic cells are exposed to viruses in a sufficient amount and for a sufficient period of time to permit capture of the influenza antigens by the dendritic cells.
• the cells are then transferred into a subject to be vaccinated by standard intravenous transplantation methods.
• While stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect.
• multiple administrations may be required to elicit sufficient levels of immunity. Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against wild-type influenza infection.
• adults who are particularly susceptible to repeated or serious influenza infection such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses.
• Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.
• the formulation for prophylactic administration of the influenza viruses also contains one or more adjuvants for enhancing the immune response to the influenza antigens.
• Suitable adjuvants include: complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59.
• prophylactic vaccine administration of influenza viruses can be performed in conjunction with administration of one or more immunostimulatory molecules.
• Immunostimulatory molecules include various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
• the immunostimulatory molecules can be administered in the same formulation as the influenza viruses, or can be administered separately. Either the protein (e.g., an HA and/or NA polypeptide of the invention) or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.
• a vector of the invention comprising a heterologous polynucleotide encoding a therapeutically or prophylactically effective HA and/or NA polypeptide (or peptide) or HA and/or NA RNA (e.g., an antisense RNA or ribozyme) into a population of target cells in vitro, ex vivo or in vivo.
• HA and/or NA polypeptide or peptide
• NA RNA e.g., an antisense RNA or ribozyme
• the polynucleotide encoding the polypeptide (or peptide), or RNA, of interest is operably linked to appropriate regulatory sequences, e.g., as described herein.
• heterologous coding sequence is incorporated into a single vector or virus.
• the vector in addition to a polynucleotide encoding a therapeutically or prophylactically active HA and/or NA polypeptide or RNA, the vector can also include additional therapeutic or prophylactic polypeptides, e.g., antigens, co-stimulatory molecules, cytokines, antibodies, etc., and/or markers, and the like.
• vaccinating an individual with attenuated influenza virus of a particular strain of a particular subgroup can induce cross-protection against influenza virus of different strains and/or subgroups
• cross-protection can be enhanced, if desired, by vaccinating the individual with attenuated influenza virus from at least two, at least three, or at least four influenza virus strains or substrains, e.g., at least two of which may represent a different subgroup.
• vaccinating an individual with at least four strains or substrains of attenuated influenza virus may include vaccinating the individual with at least two strains or substrains of influenza A virus and at least two strains or substrains of influenza B virus.
• Vaccinating the individual with the at least four strains or substrains of attenuated influenza virus may include vaccinating the individual with at least three strains or substrains of influenza A virus and at least one strain or substrain of influenza B virus.
• the vaccination of the individual with at least four influenza virus strains or substrains may require administration of a single tetravalent vaccine which comprises all of the at least four attenuated influenza virus strains or substrains.
• the vaccination may alternatively require administration of multiple vaccines, each of which comprises one, two, or three of the attenuated influenza virus strains or substrains.
• vaccine combinations can optionally include mixes of pandemic vaccines and non-pandemic strains.
• Vaccine mixtures can comprise components from human strains and/or non-human influenza strains (e.g., avian and human, etc.).
• the attenuated influenza virus vaccines of this invention can optionally be combined with vaccines that induce protective immune responses against other infectious agents.
• a vaccine of the invention is a trivalent vaccine comprising three reassortant influenza viruses.
• a vaccine of the invention is a trivalent vaccine comprising two reassortant influenza A viruses and a reassortant influenza B virus.
• a vaccine of the invention is a trivalent vaccine comprising a reassortant influenza A virus of the Hl type, a reassortant influenza A virus of the H3 type and a reassortant influenza B virus.
• a vaccine of the invention is a tetravalent vaccine comprising four reassortant influenza viruses.
• a vaccine of the invention is a tetravalent vaccine comprising two reassortant influenza A viruses and two reassortant influenza B viruses.
• a vaccine of the invention is a tetravalent vaccine comprising a reassortant influenza A virus of the Hl type, a reassortant influenza A virus of the H3 type, a reassortant influenza B virus of the Victoria lineage and a reasortant influenza B virus of the Yamagata lineage.
• Negative strand RNA viruses can be genetically engineered and recovered using a recombinant reverse genetics approach (USPN 5,166,057 to Palese et al.). Such method was originally applied to engineer influenza viral genomes (Luytjes et al. (1989) Cell 59:1107-1113; Enami et al. (1990) Proc. Natl. Acad. Sci. USA 92:11563-11567), and has been successfully applied to a wide variety of segmented and nonsegmented negative strand RNA viruses, e.g., rabies (Schnell et al. (1994) EMBO J. 13: 4195-4203); VSV (Lawson et al. (1995) Proc. Natl.
• influenza viruses in general (and those of the invention as well) are negative stranded RNA viruses.
• influenza viruses as comprising, e.g., the sequences of Figure 1, etc.
• the nucleotide sequences in Figure 1 comprise DNA versions (e.g., coding plus sense, etc.) of the genes (along with some untranslated regions in the nucleotide sequences).
• DNA versions e.g., coding plus sense, etc.
• Those of skill in the art can easily convert between RNA and DNA sequences (e.g., changing U to T, etc.), and between complementary nucleotide sequences (whether RNA or DNA), etc.
• nucleotide sequence can easily convert from a nucleotide sequence to the corresponding amino acid sequence or to a corresponding complementary sequence (whether DNA or RNA), etc.
• HA and/or NA sequences are described within DNA vectors, e.g., plasmids, etc., then the corresponding DNA version of the sequences are typically to be understood.
• nucleic acids of the invention include the explicit sequences in the sequence listings herein, as well as the complements of such sequences (both RNA and DNA), the double stranded form of the sequences in the sequence listings, the corresponding RNA forms of the sequences in the sequence listings (either as the RNA complement to the explicit sequence in the sequence listing or as the RNA version of the sequence in the sequence listing, e.g., of the same sense, but comprised of RNA, with U in place of T, etc.).
• the present invention also relates to host cells that are introduced
• Host cells are genetically engineered (i.e., transduced, transformed or transfected) with a vector, such as an expression vector, of this invention.
• a vector such as an expression vector, of this invention.
• the vector can be in the form of a plasmid, a viral particle, a phage, etc.
• appropriate expression hosts include: bacterial cells, such as E.
• coli coli, Streptomyces, and Salmonella typhimurium
• fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa
• insect cells such as Drosophila and Spodoptera frugiperda.
• Suitable host cells for the replication of influenza virus include, e.g., Vero cells, BHK cells, MDCK cells, 293 cells and COS cells, including 293T cells, COS7 cells or the like.
• co- cultures including two of the above cell lines, e.g., MDCK cells and either 293T or COS cells are employed at a ratio, e.g., of 1 : 1 , to improve replication efficiency.
• cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and CO 2 concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2).
• a standard commercial culture medium such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and CO 2 concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2).
• the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and/or additional nutrients, such as L-glutamine, sodium pyruvate, non-essential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin, ⁇ -mercaptoethanol, and the like.
• the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the inserted polynucleotide sequences, e.g., through production of viruses.
• the culture conditions such as temperature, pH and the like, are typically those previously used with the particular host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3 rd edition, Wiley- Liss, New York and the references cited therein.
• Cells for production of influenza virus can be cultured in serum-containing or serum free medium. In some cases, e.g., for the preparation of purified viruses, it is typically desirable to grow the host cells in serum free conditions.
• Cells can be cultured in small scale, e.g., less than 25 ml medium, culture tubes or flasks or in large flasks with agitation, in rotator bottles, or on microcarrier beads (e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylamine beads, such as Hillex, SoIoHiIl, Ann Arbor) in flasks, bottles or reactor cultures.
• microcarrier beads are small spheres (in the range of 100-200 microns in diameter) that provide a large surface area for adherent cell growth per volume of cell culture.
• a single liter of medium can include more than 20 million microcarrier beads providing greater than 8000 square centimeters of growth surface.
• Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, MN); NBS bioreactors (New Brunswick Scientific, Edison, NJ); laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsoder, Germany).
• a regulator e.g., a thermostat, or other device for sensing and maintaining the temperature of the cell culture system and/or other solution, is employed to insure that the temperature is at the correct level during the appropriate period (e.g., virus replication, etc.).
• a regulator e.g., a thermostat, or other device for sensing and maintaining the temperature of the cell culture system and/or other solution
• the temperature is at the correct level during the appropriate period (e.g., virus replication, etc.).
• vectors comprising influenza genome segments are introduced (e.g., transfected) into host cells according to methods well known in the art for introducing heterologous nucleic acids into eukaryotic cells, including, e.g., calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents.
• vectors e.g., plasmids
• host cells such as COS cells, 293T cells or combinations of COS or 293T cells and MDCK cells
• TransIT-LTl Minis
• approximately 1 ⁇ g of each vector is introduced into a population of host cells with approximately 2 ⁇ l of TransIT-LTl diluted in 160 ⁇ l medium, preferably serum- free medium, in a total volume of 200 ⁇ l.
• the DNA:transfection reagent mixtures are incubated at room temperature for 45 minuets followed by addition of 800 ⁇ l of medium.
• transfection mixture is added to the host cells, and the cells are cultured as described via other methods well known to those skilled in the art. Accordingly, for the production of recombinant or reassortant viruses in cell culture, vectors incorporating each of the 8 genome segments, (PB2, PBl, PA, NP, M, NS, HA and NA, e.g., of the invention) are mixed with approximately 20 ⁇ l TransIT-LTl and transfected into host cells.
• vectors incorporating each of the 8 genome segments, (PB2, PBl, PA, NP, M, NS, HA and NA, e.g., of the invention) are mixed with approximately 20 ⁇ l TransIT-LTl and transfected into host cells.
• serum-containing medium is replaced prior to transfection with serum-free medium, e.g., Opti-MEM I, and incubated for 4-6 hours.
• serum-free medium e.g., Opti-MEM I
• electroporation can be employed to introduce such vectors incorporating influenza genome segments into host cells.
• plasmid vectors incorporating an influenza A or influenza B virus are favorably introduced into Vera cells using electroporation according to the following procedure.
• approximately 5 x 10 6 Vera cells, e.g., grown in Modified Eagle's Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in an electroporation cuvette. Twenty micrograms of DNA in a volume of up to 25 ⁇ l is added to the cells in the cuvette, which is then mixed gently by tapping.
• MEM Modified Eagle's Medium
• FBS Fetal Bovine Serum
• Electroporation is performed according to the manufacturer's instructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plus connected) at 300 volts, 950 microFarads with a time constant of between 28-33 msec.
• the cells are remixed by gently tapping and approximately 1-2 minutes following electroporation 0.7 ml MEM with 10% FBS is added directly to the cuvette.
• the cells are then transferred to two wells of a standard 6 well tissue culture dish containing 2 ml MEM, 10% FBS.
• the cuvette is washed to recover any remaining cells and the wash suspension is divided between the two wells. Final volume is approximately 3.5 mL.
• the cells are then incubated under conditions permissive for viral growth, e.g., at approximately 33 0 C for cold adapted strains.
• a number of expression systems such as viral- based systems, can be utilized.
• a coding sequence is optionally ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing the polypeptides of interest in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81 :3655-3659).
• transcription enhancers such as the rous sarcoma virus (RSV) enhancer
• RSV rous sarcoma virus
• a host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
• modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
• Post-translational processing which cleaves a precursor form into a mature form, of the protein is sometimes important for correct insertion, folding and/or function. Additionally proper location within a host cell (e.g., on the cell surface) is also important.
• Different host cells such as COS, CHO, BHK, MDCK, 293, 293T, C0S7, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure the correct modification and processing of the current introduced, foreign protein.
• stable expression systems are optionally used.
• cell lines, stably expressing a polypeptide of the invention are trans fected using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene.
• a selectable marker gene For example, following the introduction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
• the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
• resistant clumps of stably transformed cells e.g., derived from single cell type, can be proliferated using tissue culture techniques appropriate to the cell type.
• Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
• the cells expressing said protein can be sorted, isolated and/or purified.
• the protein or fragment thereof produced by a recombinant cell can be secreted, membrane-bound, or retained intracellularly, depending on the sequence (e.g., depending upon fusion proteins encoding a membrane retention signal or the like) and/or the vector used.
• Expression products corresponding to the nucleic acids of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like.
• non-animal cells such as plants, yeast, fungi, bacteria and the like.
• details regarding cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
• a number of expression vectors can be selected depending upon the use intended for the expressed product. For example, when large quantities of a polypeptide or fragments thereof are needed for the production of antibodies, vectors that direct high-level expression of fusion proteins that are readily purified are favorably employed. Such vectors include, but are not limited to, multifunctional E.
• coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the coding sequence of interest, e.g., sequences comprising those found herein, etc., can be ligated into the vector in-frame with sequences for the amino -terminal translation initiating methionine and the subsequent 7 residues of beta-galactosidase producing a catalytically active beta galactosidase fusion protein; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison WI); and the like.
• BLUESCRIPT Stratagene
• yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used for production of the desired expression products.
• constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
• mutagenesis are optionally used in the present invention, e.g., to produce and/or isolate, e.g., novel or newly isolated HA and/or NA molecules and/or to further modify/mutate the polypeptides (e.g., HA and NA molecules) of the invention.
• They include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like.
• Suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like.
• Mutagenesis e.g., involving chimeric constructs, is also included in the present invention.
• mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.
• Oligonucleotides for use in mutagenesis of the present invention, e.g., mutating libraries of the HA and/or NA molecules of the invention, or altering such, are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts 22(20): 1859-1862, (1981) e.g., using an automated synthesizer, as described in Needham-VanDevanter et al, Nucleic Acids Res, 12:6159-6168 (1984).
• essentially any nucleic acid can be custom or standard ordered from any of a variety of commercial sources
• peptides and antibodies can be custom ordered from any of a variety of sources.
• the present invention also relates to host cells and organisms comprising a HA and/or NA molecule or other polypeptide and/or nucleic acid of the invention or such HA and/or NA or other sequences within various vectors such as 6:2 reassortant influenza viruses, plasmids in plasmid rescue systems, etc.
• Host cells are genetically engineered (e.g., transformed, transduced or transfected) with the vectors of this invention, which can be, for example, a cloning vector or an expression vector.
• the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
• the vectors are introduced into cells and/or microorganisms by standard methods including electroporation (see, From et al., Proc Natl Acad Sci USA 82, 5824 (1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide a variety of appropriate transformation methods. See, above.
• Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which can be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors, etc.
• Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook).
• kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanTM, from Stratagene; and, QIAprepTM from Qiagen).
• the isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms.
• Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
• the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
• Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, B., et al., Protein Expr Purif 6435:10 (1995); Ausubel, Sambrook, Berger (all supra).
• a catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds.) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. See, above.
• a selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
• a secreted polypeptide product e.g., a HA and/or NA polypeptide as in a secreted fusion protein form, etc.
• a virus particle containing one or more HA and/or NA polypeptide of the invention is produced from the cell.
• cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
• Eukaryotic or microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art. Additionally, cells expressing a HA and/or a NA polypeptide product of the invention can be utilized without separating the polypeptide from the cell. In such situations, the polypeptide of the invention is optionally expressed on the cell surface and is examined thus (e.g., by having HA and/or NA molecules, or fragments thereof, e.g., comprising fusion proteins or the like) on the cell surface bind antibodies, etc. Such cells are also features of the invention.
• Expressed polypeptides can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the tagging systems known to those skilled in the art), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Also, high performance liquid chromatography (HPLC) can be employed in the final purification steps.
• HPLC high performance liquid chromatography
• the viruses are typically recovered from the culture medium, in which infected (transfected) cells have been grown.
• crude medium is clarified prior to concentration of influenza viruses.
• Common methods include ultrafiltration, adsorption on barium sulfate and elution, and centrifugation.
• crude medium from infected cultures can first be clarified by centrifugation at, e.g., 1000-2000 x g for a time sufficient to remove cell debris and other large particulate matter, e.g., between 10 and 30 minutes.
• the clarified medium supernatant is then centrifuged to pellet the influenza viruses, e.g., at 15,000 x g, for approximately 3-5 hours.
• the virus is concentrated by density gradient centrifugation on sucrose (60%- 12%) or potassium tartrate (50%- 10%). Either continuous or step gradients, e.g., a sucrose gradient between 12% and 60% in four 12% steps, are suitable. The gradients are centrifuged at a speed, and for a time, sufficient for the viruses to concentrate into a visible band for recovery.
• an appropriate buffer such as STE (0.01 M Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA
• PBS phosphate buffered saline
• virus is elutriated from density gradients using a zonal-centrifuge rotor operating in continuous mode. Additional details sufficient to guide one of skill through the preparation of influenza viruses from tissue culture are provided, e.g., in Furminger. Vaccine Production, in Nicholson et al. (eds.) Textbook of Influenza pp. 324- 332; Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation, in Cohen & Shafferman (eds.) Novel Strategies in Design and Production of Vaccines pp. 141-151, and United States Patent No. 5,690,937. If desired, the recovered viruses can be stored at -8O 0 C in the presence of sucrose-phosphate-glutamate (SPG) as a stabilizer
• SPG sucrose-phosphate-glutamate
• Expressed polypeptides of the invention can contain one or more modified amino acids.
• the presence of modified amino acids can be advantageous in, for example, (a) increasing polypeptide serum half-life, (b) reducing/increasing polypeptide antigenicity, (c) increasing polypeptide storage stability, etc.
• Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means (e.g., via PEGylation).
• Non- limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like, as well as amino acids modified by conjugation to, e.g., lipid moieties or other organic derivatizing agents.
• References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ. Fusion Proteins
• the present invention also provides fusion proteins comprising fusions of the sequences of the invention (e.g., encoding HA and/or NA polypeptides) or fragments thereof with, e.g., immunoglobulins (or portions thereof), sequences encoding, e.g., GFP (green fluorescent protein), or other similar markers, etc. Nucleotide sequences encoding such fusion proteins are another aspect of the invention. Fusion proteins of the invention are optionally used for, e.g., similar applications (including, e.g., therapeutic, prophylactic, diagnostic, experimental, etc. applications as described herein) as the non-fusion proteins of the invention.
• similar applications including, e.g., therapeutic, prophylactic, diagnostic, experimental, etc. applications as described herein
• the proteins of the invention are also optionally fused with, e.g., sequences which allow sorting of the fusion proteins and/or targeting of the fusion proteins to specific cell types, regions, etc.
• polypeptides of the invention can be used to produce antibodies specific for the polypeptides given herein and/or polypeptides encoded by the polynucleotides of the invention, e.g., those shown herein, and conservative variants thereof.
• Antibodies specific for the above mentioned polypeptides are useful, e.g., for diagnostic and therapeutic purposes, e.g., related to the activity, distribution, and expression of target polypeptides.
• such antibodies can optionally be utilized to define other viruses within the same strain(s) as the HA/NA sequences herein.
• Antibodies specific for the polypeptides of the invention can be generated by methods well known in the art. Such antibodies can include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Polypeptides do not require biological activity for antibody production (e.g., full length functional hemagglutinin or neuraminidase is not required). However, the polypeptide or oligopeptide must be antigenic. Peptides used to induce specific antibodies typically have an amino acid sequence of at least about 4 amino acids, and often at least 5 or 10 amino acids. Short stretches of a polypeptide can be fused with another protein, such as keyhole limpet hemocyanin, and antibody produced against the chimeric molecule.
• Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of, e.g., at least about 0.1 ⁇ M, at least about 0.01 ⁇ M or better, and, typically and at least about 0.001 ⁇ M or better.
• humanized antibodies are desirable. Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Patent 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed.) (1995) Antibody Engineering, 2 nd Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ (Paul).
• NUCLEIC ACID AND POLYPEPTIDE SEQUENCE VARIANTS As described herein, the invention provides for nucleic acid polynucleotide sequences and polypeptide amino acid sequences, e.g., hemagglutinin and neuraminidase sequences, and, e.g., compositions and methods comprising said sequences. Examples of said sequences are disclosed herein. However, one of skill in the art will appreciate that the invention is not necessarily limited to those sequences disclosed herein and that the present invention also provides many related and unrelated sequences with the functions described herein, e.g., encoding a HA and/or a NA molecule. One of skill will also appreciate that many variants of the disclosed sequences are included in the invention.
• sequences disclosed herein are conservative variations of the disclosed sequences that yield a functionally identical sequence.
• Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence are considered to be included in the invention.
• Unique subsequences of the sequences disclosed herein, as determined by, e.g., standard sequence comparison techniques, are also included in the invention.
• any of a variety of nucleic acid sequences encoding polypeptides and/or viruses of the invention are optionally produced, some which can bear lower levels of sequence identity to the HA and NA nucleic acid and polypeptide sequences herein.
• the following provides a typical codon table specifying the genetic code, found in many biology and biochemistry texts.
• the codon table shows that many amino acids are encoded by more than one codon.
• the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
• the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.
• silent variations are one species of “conservatively modified variations,” discussed below.
• each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine, and TTG, which is ordinarily the only codon for tryptophan
• TTG which is ordinarily the only codon for tryptophan
• each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence.
• the invention therefore, explicitly provides each and every possible variation of a nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices.
• substitutions i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
• conservative amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct such as those herein. Such conservative variations of each disclosed sequence are a feature of the present invention.
• Constant variation of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences, see, Table 2 below.
• substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 3%, 2% or 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
• “conservative variations” of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 4%, 3%, 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.
• substitutions of a small percentage, typically less than 5%, more typically less than 4%, 3%, 2% or 1%, of the amino acids of the polypeptide sequence with a conservatively selected amino acid of the same conservative substitution group.
• sequences which do not alter the encoded activity of a nucleic acid molecule is a conservative variation of the basic nucleic acid.
• nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
• nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 90%, preferably 91 %, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
• substantially identical sequences are typically considered to be “homologous,” without reference to actual ancestry.
• “substantial identity” exists over a region of the amino acid sequences that is at least about 200 residues in length, more preferably over a region of at least about 250 residues, and most preferably the sequences are substantially identical over at least about 300 residues, 350 residues, 400 residues, 425 residues, 450 residues, 475 residues, 480 residues, 490 residues, 495 residues, 499 residues, 500 residues, 502 residues, 559 residues, 565 residues, or 566 residues, or over the full length of the two sequences to be compared when the amino acids are hemagglutinin or hemagglutinin fragments or which is substantially identical over at least about 350 amino acids; over at least about 400 amino acids; over at least about over at least about 436 amino acids, over at least about 450 amino acids; over at least about 451 amino acids; over at least about 4
• sequence comparison and homology determination typically one sequence acts as a reference sequence to which test sequences are compared.
• test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J MoI Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc Natl Acad Sci USA 85:2444 (1988), by computerized implementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by visual inspection (see generally, Ausubel et al, supra).
• HSPs high scoring sequence pairs
• initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc Natl Acad Sci USA 90:5873-5787 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment.
• PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J. MoI. Evol. 35:351-360. The method used is similar to the method described by Higgins & Sharp (1989) CABIOS5 :151-153.
• the program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.
• the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
• This cluster can then be aligned to the next most related sequence or cluster of aligned sequences.
• Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
• the program can also be used to plot a dendogram or tree representation of clustering relationships. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison.
• An additional example of an algorithm that is suitable for multiple nucleic acid, or amino acid, sequence alignments is the CLUSTALW program (Thompson, J. D. et al. (1994) Nucl. Acids. Res. 22: 4673-4680).
• CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology.
• Gap open and Gap extension penalties can be, e.g., 10 and 0.05 respectively.
• the BLOSUM algorithm can be used as a protein weight matrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919.
• the present invention provides digital systems, e.g., computers, computer readable media and integrated systems comprising character strings corresponding to the sequence information herein for the nucleic acids and isolated or recombinant polypeptides herein, including, e.g., the sequences shown herein, and the various silent substitutions and conservative substitutions thereof.
• Integrated systems can further include, e.g., gene synthesis equipment for making genes corresponding to the character strings.
• Various methods known in the art can be used to detect homology or similarity between different character strings, or can be used to perform other desirable functions such as to control output files, provide the basis for making presentations of information including the sequences and the like. Examples include BLAST, discussed supra.
• Computer systems of the invention can include such programs, e.g., in conjunction with one or more data file or data base comprising a sequence as noted herein.
• homology determination methods have been designed for comparative analysis of sequences of biopolymers, for spell-checking in word processing, and for data retrieval from various databases.
• models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings corresponding to the sequences herein (e.g., word-processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).
• word processing software e.g.,
• Microsoft WordTM or Corel WordPerfectTM can be adapted to the present invention by inputting a character string corresponding to one or more polynucleotides and polypeptides of the invention (either nucleic acids or proteins, or both).
• a system of the invention can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system) to manipulate strings of characters corresponding to the sequences herein.
• a user interface e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system
• specialized alignment programs such as BLAST can also be incorporated into the systems of the invention for alignment of nucleic acids or proteins (or corresponding character strings).
• Systems in the present invention typically include a digital computer with data sets entered into the software system comprising any of the sequences herein.
• the computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, OS2TM
• Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, PERL, Fortran, Basic, Java, or the like.
• Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display), or others.
• Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others.
• the box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements.
• Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.
• the computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
• the software then converts these instructions to appropriate language for instructing the operation, e.g., of appropriate mechanisms or transport controllers to carry out the desired operation.
• the software can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of sequences herein), comparisons of samples for differential gene expression, or other operations.
• kits of the invention contains one or more nucleic acid, polypeptide, antibody, or cell line described herein (e.g., comprising, or with, a HA and/or NA molecule of the invention).
• the kit can contain a diagnostic nucleic acid or polypeptide, e.g., antibody, probe set, e.g., as a cDNA micro-array packaged in a suitable container, or other nucleic acid such as one or more expression vector.
• the kit typically further comprises, one or more additional reagents, e.g., substrates, labels, primers, for labeling expression products, tubes and/or other accessories, reagents for collecting samples, buffers, hybridization chambers, cover slips, etc.
• additional reagents e.g., substrates, labels, primers, for labeling expression products, tubes and/or other accessories, reagents for collecting samples, buffers, hybridization chambers, cover slips, etc.
• the kit optionally further comprises an instruction set or user manual detailing preferred methods of using the kit components for discovery or application of diagnostic sets, etc.
• the kit can be used, e.g., for evaluating a disease state or condition, for evaluating effects of a pharmaceutical agent or other treatment intervention on progression of a disease state or condition in a cell or organism, or for use as a vaccine, etc.
• the present invention provides system kits embodying the methods, composition, systems and apparatus herein.
• System kits of the invention optionally comprise one or more of the following: (1) an apparatus, system, system component or apparatus component; (2) instructions for practicing methods described herein, and/or for operating the apparatus or apparatus components herein and/or for using the compositions herein.
• the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.
• kits can include one or more translation system as noted above (e.g., a cell) with appropriate packaging material, containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like.
• products of the translation systems e.g., proteins such as HA and/or NA molecules
• kit form e.g., with containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like.
• kits can comprise various vaccines (e.g., produced through plasmid rescue protocols) such as live attenuated vaccine (e.g., FluMist) comprising the HA and/or NA sequences herein.
• vaccines e.g., produced through plasmid rescue protocols
• live attenuated vaccine e.g., FluMist
• any of the vaccine components and/or compositions e.g., reassorted virus in allantoic fluid, etc.
• additional components such as, buffer, cells, culture medium, useful for packaging and infection of influenza viruses for experimental or therapeutic vaccine purposes
• the kit contains, in addition to the above components, additional materials which can include, e.g., instructions for performing the methods of the invention, packaging material, and a container.
• FDA Food and Drug Administration
• Site-directed mutagenesis was performed to introduce specific changes into the HA genes using the QuikChange® Site-Directed Mutagenesis kit (Stratagene) and the HA sequence was confirmed by sequencing analyses.
• the 6:2 reassortant vaccine strains were generated by co-transfecting 8 cDNA plasmids encoding the HA and NA of the HlNl virus and the 6 internal gene segments of cold adapted (ca) A/ Ann Arbor/6/60 (MDV-A, master donor virus for type A influenza virus) into co-cultured 293T and MDCK cells.
• the vaccine strains used for manufacture are produced in serum- free Vero/CEK cells by electroporation. Viruses were propagated in the allantoic cavities of 10- to 11 -day-old embryonated chicken eggs. The HA and NA sequences of the rescued viruses were verified by sequencing of RT-PCR cDNAs amplified from vRNA.
• Virus titration Virus titers were measured by the fluorescence focus assay and expressed as log 1 OFFU (fluorescent focus units Unit)/ml (Forrest et al. (2008) Clin Vaccine Immunol 15: 1042-1053). Virus plaque morphology was examined by plaque assay as previously described (Jin et al., (2003) Virology 306, 18-24).
• Receptor-binding assay Chicken red blood cells (cRBCs) (HEMA Resource and Supply, Inc.) were desialylated and re-sialylated as previously described (). 100 ⁇ l of 10% cRBCs was incubated with 50 mU Vibrio cholerae neuraminidase (Sigma, St.
• resialylated cRBCs were resuspended as 0.5% (v/v) in PBS after washing three times with PBS.
• Haemagglutination assays were carried out in V- bottomed 96 well microtiter plates for the binding activity. 50 ⁇ l of two-fold serially diluted viruses were incubated with 50 ⁇ l of 0.5% cRBC, ⁇ 2,3, or ⁇ 2,6 resialylated cRBC at room temperature for 60 min.
• the HA titer was defined as the highest dilution that hemagglutinate the RBCs.
• Ferret studies 8-10 weeks old male and female ferrets from Simonson
• Virus titers in the lung and NT were determined by the EID50 assay and expressed as 50% egg infectious dose per gram of tissue (loglOEID50/g). Ferrets that were assigned for immunogenicity studies were bled on days 14, 21 and 28 days postinfection and sera were assayed for antibody titers by HAI.
• H INl -specific antibody level in post-infected ferret sera against homologous and heterologous viruses was determined by HAI assay.
• ferret sera Prior to serologic analysis, ferret sera were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) that was reconstituted in 10 mL of 0.9 % NaCl per vial.
• RDE receptor-destroying enzyme
• 0.1 mL serum was mixed with 0.15 mL RDE and incubated at 37 0 C for 18 hr and adjusted to a final 1 :4 dilution by adding 0.15 mL of 0.9 % sodium citrate followed by incubation at 56 0 C for 45 min.
• Strain- specific serum HAI titers were determined using 0.5% tRBC and the HAI titers are presented as the reciprocal value of the highest serum dilution that inhibited hemagglutination.
• the HA and NA gene segments from wt A/CA/4/09 were cloned from infected MDCK cell RNA following RT-PCR amplification. A total of 12 cDNA clones from each HA or NA were found to be identical by nucleotide sequence analysis. Plasmids representing this HA sequence and the NA sequence of wt A/CA/4/09 were transfected into 293/MDCK cells together with plasmids representing the six internal protein gene segments of MDV-A, however, no viable reassortant progeny could be recovered either on MDCK cells or eggs.
• the HA cDNAs cloned from the wt A/CA/4/09 egg isolate were heterogeneous at two amino acid positions (Table 3).
• the HA plasmid cloned from A/CA/7/09 that did not have amino acid change at residues of 222 or 223 could be rescued to produce progeny virus. This indicated that T 197 A change in CA/7/09 was responsible for the efficient rescue of A/CA/7/09. Other A/CA/7/09 clones had changes of either D222G or Q223R, viruses containing D222G or Q223R in HA were also rescued. Due to the high degree of similarity between the HAs of A/CA/4/09 and A/CA/7/09 and since the NA sequences were identical between these two strains, the variants derived from A/CA/7/09 were developed further. Table 3. HA sequences and virus titers of novel HlNl variants
• Virus titers are mean titers from at least three virus stocks; NA: not applicable Selection of vaccine strains with better growth in embryonated chicken eggs.
• the rescued A/CA/4/09 and A/CA/7/09 reassortant vaccine strains replicated in chicken embryonated eggs at titers of 7.4 to 7.8 logioFFU/ml, which was lower than the seasonal HlNl vaccine strains by at least 10-fold.
• the recovered A/CA/7/09 reassortant vaccine candidates formed very small plaques in MDCK cells.
• A/CA/7/09 candidates (V5 and V6) were passaged twice in MDCK cells at moi of 4.0, 0.4 and 0.04, then the viruses present in the supernatants were examined by plaque assay.
• All the MDCK- passaged viruses contained plaques that were much larger (2-4 mm) than the parental viruses (Fig.l).
• the HA gene segment of twelve plaques from MDCK passaged V5 and V6 vaccine candidates were sequenced.
• the HA of each of the large plaque morphology isolates had single amino acid changes at one of the following positions: Kl 19N or Kl 19E, K153E, K154E derived from V5 and A186D from V6.
• the HA sequence of A/CA/7/09 was also compared with two earlier HlNl viruses that were originated from swine, A/swine/Iowa/1/1976 and A/swine/1931 , and a recent human HlNl virus, A/South Dakota/6/07 (A/SD/07).
• Kl 19, K153 and K154 are highly conserved among the swine HlNl viruses.
• A/SD/6/07 also contained Kl 19 and Kl 54 residues.
• the amino acids at 186 are more diverse among the four viruses shown in Fig. 2.
• a previous study (Both et al.
• V5 was also introduced into V6 and V6-186D to evaluate the influence of single and double amino acid change on virus replication in eggs.
• the 119N mutation found in V5 was also introduced into V6 and V6-186D to evaluate the influence of single and double amino acid changes on virus replication in eggs.
• HA sequence analysis also showed that A/CA/09 strains contained a unique glycosylation site at residue 278 (Fig. 2). To evaluate if this additional glycosylation site affected virus growth, a T278K change was introduced into V5 and V6, respectively. The rescued viruses were examined for their growth in eggs. As shown in Table 4, most of the variants containing the introduced HA mutations grew significantly better than V5 and V6.
• V5-278K and V6-278K had titers of 7.6 and 7.7 LogioFFU/ml that were similar to V5 and V6. Thus, removal of the 278 glycosylation site had minimal impact on virus titers in eggs.
• the changes at the 119, 186, 153, 154, 155 and 186 sites increased virus titers by 0.2 to 1.2 logioFFU/ml.
• the combined 119E and 186D change in V5 resulted in a slightly higher titer than either 119E or 186D on their own.
• Table 4 A/CA/07/09 HA variants identified from MDCK cells and introduced by reverse genetics. Amino acid residue numbers of Hl HA are shown in row 2; corresponding amino acid residue numbers in H3 HA are shown in row 3.
• Virus titers are mean titers from at least three virus stocks
• A/CA/7/09 variants were evaluated for their reactivity with different postinfection ferret sera using the HAI assay (Table 5).
• V5-119N 8192 512 2048 1024
• A/CA/7/09 vaccine candidates are attenuated but immunogenic in ferrets.
• ferrets were inoculated with 7.0 logioFFU of virus intranasally in 0.2 ml of dose volume and virus replication in the upper and lower respiratory tracts of ferrets was determined by EID 50 assay.
• EID 50 assay As shown in Table 6, all A/CA/7/09 variants replicated efficiently in the NT tissues, but no virus was detected in the lungs.
• V5-119E 4.2 ⁇ 0.33 ⁇ 1.5 813 645 645 323 406 64 256 V5-119N 4.5 ⁇ 0.33 ⁇ 1.5 256 203 256 256 323 64 161 V5-186D 4.9 ⁇ 0.20 ⁇ 1.5 813 645 645 645 128 256
• V5 (without 222 and 223 change) preferred to bind to ⁇ 2-6 SA than ⁇ 2-3 SA.
• V6 (Q223R) could only bind to ⁇ 2-3 SA resialylated RBC, confirming that these two residues affected virus receptor binding specificity.
• the V5-119E/N and V5-186D viruses had similar binding specificity as V5.
• the double HA mutant V5-119E/186D preferred binding to ⁇ 2-6 SA better than ⁇ 2-3 SA.
• the 119E and 186D residues introduced into V6 could not restore their binding to ⁇ 2-6 SA (data not shown). Thus, the change in the 119 and 186 residues do not significantly affect virus receptor binding specificity.
• V5-119N 512 64 64 ⁇ 2
• V5-119E 512 128 128 ⁇ 2
• V5-186D 512 32 64 ⁇ 2
• Reassortant viruses with "MDVA” in their designation comprise 6 internal genome segment of A/ Ann Arbor/6/60.
• Reassortant viruses with "PR8” in their designation comprise 6 internal genome segment of PR8.
• 6:2 reassortant influenza viruses comprising the 6 internal genome segments from PR8 and the HA and NA genome segments from A/C A/7/09 were generated. Additional 6:2 reassortant viruses comprising the 6 internal genome segments from PR8, the NA genome segment from A/CA/7/09, and an HA genome segment encoding an Hl HA variant polypeptide described herein were generated. The 6:2 reassortant viruses comprising different A/CA/7/09 HA variants, along with A/Texas/5/2009 RG 15 (A/TX/7/09 RG15) and A/California/07/09 NYMC X179A, were expanded in chicken embryonated eggs and their peak titers were determined by FFA.
• Virus strains assayed are shown in Table 8.
• 6:2 PR8-South Dakota is a 6:2 reassortant virus comprising the 6 internal genome segments from PR8 and the HA and NA genome segments from A/South Dakota/6/07.
• 6:2AA-V5-119E/186D is a 6:2 reassortant virus comprising the 6 internal genome segments from A/ Ann Arbor/6/60, the NA genome segment from A/CA/7/09, and the V5-119E/186D variant HA genome segment.
• PR8 reassortant viruses comprising Hl HA variant genome segments described herein had comparable titers to X 179 A, the highest yield strain available for TIV manufacture.
• HA protein yield of some of the reassortant viruses listed in Table 8. Selected viruses were amplified by infecting 50 eggs each with an MOI of 10 FFU/egg. Viruses were harvested after incubation at 33°C for 62 hrs. Equal amount of allantoic fluid (250ml) were then purified by sucrose gradient. Virus proteins were analyzed by SDS PAGE followed by Coomassie blue staining.
• FIG. 4A A representative sample of the results is shown in Figure 4.
• the total protein yield of X179A, 6:2 PR8-V6-186D and 6:2AA-V5-119E/186D was 460, 300, and 350 ug respectively.
• 6:2 AA-V5-119E/186D had the highest HA protein yield, even though 6:2 AA-V5-119E/186D had a lower total protein yield than A/CA/7/09 X179A.
• the lower total protein yield observed for 6:2AA-V5-119E/186D in this experiment was likely due to assay variation.
• the total protein yield of 6:2 PR8-V5-119E/186D and X179A was 780 and 720 ug respectively.
• the HA protein yield of 6:2 PR8-V5-119E/186D was also higher than that of A/CA/7/09 X179A.
• Reassortant viruses comprising the PR8 backbone and a variant A/California/7/09 Hl polypeptide
• Additional 6:2 reassortant influenza viruses comprising 6 internal genome segments from PR8, a variant of the A/CA/7/09 Hl HA genome segment, and the A/CA/7/09 NA genome segment were generated.
• the Hl HA amino acid variations are summarized in Table 9.
• Reassortant viruses comprising variant A/CA/7/09 Hl HA and the PR8 backbone.
• Samples prepared from AA-V5-ED (comprising the V5-119E/186D variant HA and the A/ Ann Arbor/6/60 backbone) and X179A viruses were included as controls.
• the PR8-C virus produced higher yield of the HA polypeptide than X 179 A.
• the HA yield of PR8-C and AA-V5-ED relative to the HA yield of X179A was further measured using SDS-PAGE followed by Coomassie blue staining.
• Various volumes (10, 7.5, 5 and 2.5 microliter) of the PR8-C and AA-V5-ED samples and 10 microliter of the X179A sample were analyzed by SDS-PAGE.
• the relative amount of HAl protein found in each sample was measured using image analysis software. The result of the measurement is shown in Figure 6.
• the AA-V5-ED and PR8-C viruses produce 1-1.5 times more HA protein than X 179 A.

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