WO2010123519A1 - Séquences de réplikine pour la prévention/le traitement de la grippe et la détermination de l'infectiosité/la létalité - Google Patents

Séquences de réplikine pour la prévention/le traitement de la grippe et la détermination de l'infectiosité/la létalité Download PDF

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Publication number
WO2010123519A1
WO2010123519A1 PCT/US2009/061108 US2009061108W WO2010123519A1 WO 2010123519 A1 WO2010123519 A1 WO 2010123519A1 US 2009061108 W US2009061108 W US 2009061108W WO 2010123519 A1 WO2010123519 A1 WO 2010123519A1
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WIPO (PCT)
Prior art keywords
peptide
seq
isolated
polypeptide
vaccine
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PCT/US2009/061108
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English (en)
Inventor
Samuel Bogoch
Elenore S. Bogoch
Samuel Winston Bogoch
Anne Elenore Borsanyi
Original Assignee
Samuel Bogoch
Bogoch Elenore S
Samuel Winston Bogoch
Anne Elenore Borsanyi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Priority claimed from PCT/US2009/041565 external-priority patent/WO2009132209A2/fr
Priority claimed from US12/429,044 external-priority patent/US20090269367A1/en
Priority claimed from US12/538,027 external-priority patent/US20100144589A1/en
Application filed by Samuel Bogoch, Bogoch Elenore S, Samuel Winston Bogoch, Anne Elenore Borsanyi filed Critical Samuel Bogoch
Priority to EP20090743995 priority Critical patent/EP2421546A1/fr
Priority to AU2009344843A priority patent/AU2009344843A1/en
Priority to CA2759325A priority patent/CA2759325A1/fr
Priority to SG2011076536A priority patent/SG175275A1/en
Priority to NZ595812A priority patent/NZ595812A/xx
Publication of WO2010123519A1 publication Critical patent/WO2010123519A1/fr
Priority to IL215770A priority patent/IL215770A0/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N2760/16122—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
    • 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
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the present invention relates to therapies for preventing and treating influenza virus, methods of predicting and differentiating infectivity and lethality of influenza outbreaks, and compounds for diagnostic, therapeutic, and/or preventive purposes in influenza.
  • Influenza is an acute respiratory illness of global importance in humans and animals (both domesticated and wild) including, but not limited to, horses, pigs, chickens, ducks, turkeys, ferrets, and wild birds. Virulent and lethal outbreaks of influenza continue to threaten global health. As demonstrated by the HlNl influenza pandemic of 2009, researchers, government officials, and medical practitioners are acutely aware of the continuing threat of pandemics of virulent and lethal influenza requiring new methods of treatment and novel therapeutic compounds.
  • influenza vaccines have remained the most effective defense against influenza virus.
  • influenza has continued to remain an emergent or re- emergent infectious threat.
  • influenza virus strain that is both relatively more infective and relatively more lethal is an influenza strain that will likely cause increased morbidity and mortality in an outbreak.
  • public health researchers and government officials have advanced knowledge of the infectivity and lethality of an influenza strain, they have crucial additional time for preparations of vaccines and other health measures in advance of a spreading outbreak.
  • Early differentiation of infectivity and lethality of a strain of influenza that is causing an outbreak is of significant importance and utility to those coordinating a response to the outbreak and to those designing vaccines and other health measures in response to an outbreak. For example, early differentiation of infectivity and lethality of a strain of influenza virus causing an outbreak allows for a design of therapies that target the infectivity of a virus, the lethality of a virus, or both,
  • Replikin peptides are a family of small peptides that have been correlated with the phenomenon of rapid replication in influenza, malaria, West Nile virus, foot and mouth disease, and many other pathogens. Replikin peptides have likewise been generally correlated with the phenomenon of rapid replication in viruses, organisms, and malignancies.
  • the present invention provides methods of differentiating the infectivity of an influenza virus isolate or strain of influenza virus from the lethality of the influenza virus isolate or strain of influenza virus and compounds for diagnostic, therapeutic, and/or preventive purposes in influenza including any strain of influenza.
  • a first non-limiting aspect of the present invention provides an isolated or synthesized protein fragment, polypeptide, or peptide comprising at least one peptide A where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the amino acid sequence of the protein fragment, polypeptide, or peptide partially matches the amino acid sequence of an expressed whole protein wherein at least one, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred or more amino acid residues of the amino acid sequence of the expressed whole protein are not present in the protein fragment, polypeptide, or peptide.
  • the amino acid sequence of said protein fragment, polypeptide, or peptide partially matches the amino acid sequence of an expressed whole protein wherein at least one, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of the amino acid sequence of at least one terminus of the expressed whole protein are not present at at least one terminus of said protein fragment, polypeptide, or peptide.
  • the isolated or synthesized protein fragment, polypeptide, or peptide consists of 7 to about 50 amino acids comprising at least one peptide A, wherein said peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the isolated or synthesized protein fragment, polypeptide, or peptide consists of a peptide A that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66, where the length of peptide A is no more than one, five, ten, twenty, thirty, forty, or fifty amino acid residues longer than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32- 66 with which it is homologous.
  • peptide A is no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues longer than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 with which it is homologous.
  • the isolated or synthesized protein fragment, polypeptide, or peptide consists of any one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a further non- limiting embodiment provides a peptide consisting of SEQ ID NO(s):
  • a further non-limiting embodiment provides a peptide consisting of SEQ ID NO(s): 21, 22, 23, 24, 25, 26, 27, or 28.
  • a further non- limiting embodiment of the first aspect of the invention provides an isolated or synthesized protein fragment, polypeptide, or peptide comprising at least one peptide
  • peptide A where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 32-66.
  • Another non-limiting embodiment provides a peptide consisting of at least one of SEQ ID NO(s): 32-66.
  • any peptide of SEQ ID NO(s): 32-66 is provided as comprised in an immunogenic composition and/or comprised in a vaccine.
  • Another non-limiting embodiment of the first aspect of the invention provides a biosynthetic composition consisting essentially of a peptide of SEQ ID NO(s): 1-66.
  • a further non- limiting embodiment provides a biosynthetic composition consisting of a peptide of SEQ ID NO(s): 1-66.
  • Another non-limiting embodiment of the first aspect of the invention provides a protein fragment, polypeptide, or peptide consisting essentially of at least one of SEQ ID NO(s):
  • an isolated protein fragment, polypeptide, or peptide is chemically synthesized by solid phase methods.
  • a second non- limiting aspect of the present invention provides an immunogenic composition comprising at least one protein fragment, polypeptide, or peptide of any one of the above-listed protein fragments, polypeptides, or peptides.
  • the immunogenic compound comprises at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • Another non-limiting embodiment provides an immunogenic composition comprising at least one peptide of SEQ ID NO(s): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and SEQ ID NO(s): 21, 22, 23, 24, 25, 26, 27, and 28.
  • a third non-limiting aspect of the present invention provides a vaccine comprising at least one protein fragment, polypeptide, or peptide of any one of the above-listed protein fragments, polypeptides, or peptides.
  • the vaccine comprises at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21- 28, and 32-66.
  • a fourth non-limiting aspect of the present invention provides a composition comprising one or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the composition comprises one or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32- 66.
  • the composition comprises at least one of the peptides of SEQ ID NO(s): 1-12.
  • the composition comprises a mixture of peptides, wherein the mixture comprises isolated or synthesized peptides of SEQ ID NO(s): 1- 12.
  • the composition comprises at least one of the peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the composition comprises a mixture of peptides, wherein the mixture comprises isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the composition comprises an approximately equal molar mixture of the isolated or synthesized peptides of SEQ ID NO(s): 1- 12 or an approximately equal molar mixture of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the composition comprises approximately equal weight of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 or approximately equal weight of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the composition comprises about 10% by weight SEQ ID NO: 1, about 9% by weight SEQ ID NO: 2, about 10% by weight SEQ ID NO: 3, about 6 % by weight SEQ ID NO: 4, about 8% by weight SEQ ID NO: 5, about 8% by weight SEQ ID NO: 6, about 7% by weight SEQ ID NO: 7, about 6% by weight SEQ ID NO: 8, about 10% by weight SEQ ID NO: 9, about 8% by weight SEQ ID NO: 10, about 7% by weight SEQ ID NO: 11, and about 11% by weight SEQ ID NO: 12.
  • a fifth aspect of the present invention provides a vaccine comprising one or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the vaccine comprises one or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the vaccine comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the vaccine comprises at least one of the peptides of SEQ ID NO(s): 1-12.
  • the vaccine comprises a mixture of peptides, wherein the mixture comprises isolated or synthesized peptides of SEQ ID NO(s): 1-12.
  • the vaccine comprises at least one of the peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the vaccine comprises a mixture of peptides, wherein the mixture comprises isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21- 28.
  • the vaccine comprises an approximately equal molar mixture of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 or an approximately equal molar mixture of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the vaccine comprises approximately equal weight of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 or approximately equal weight of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.
  • the vaccine comprises about 10% by weight SEQ ID NO: 1, about 9% by weight SEQ ID NO: 2, about 10% by weight SEQ ID NO: 3, about 6 % by weight SEQ ID NO: 4, about 8% by weight SEQ ID NO: 5, about 8% by weight SEQ ID NO: 6, about 7% by weight SEQ ID NO: 7, about 6% by weight SEQ ID NO: 8, about 10% by weight SEQ ID NO: 9, about 8% by weight SEQ ID NO: 10, about 7% by weight SEQ ID NO: 11, and about 11% by weight SEQ ID NO: 12.
  • the vaccine comprises a pharmaceutically acceptable carrier and/or adjuvant.
  • the vaccine is for the treatment or prevention of influenza virus infection.
  • the vaccine is directed against HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A.
  • a sixth non-limiting aspect of the invention provides an antibody, antibody fragment, or binding agent that binds to at least a portion of an amino acid sequence of at least one protein fragment, polypeptide, or peptide comprising a peptide A, wherein the peptide A is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the antibody, antibody fragment, or binding agent binds to at least a portion of an amino acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the antibody, antibody fragment, or binding agent binds to at least a portion of an amino acid sequence of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a seventh non- limiting aspect of the present invention provides an isolated or synthesized polypeptide or peptide comprising a peptide A that has about the same number of amino acid residues as a peptide B, where peptide B is one of the peptides of SEQ ID NO: 1-28, and where the lysine residues and histidine residues in peptide A are conserved as compared to the lysine residues and histidine residues in peptide B, wherein said isolated or synthesized polypeptide or peptide further comprises up to 100 more amino acid residues than does peptide A, and wherein said up to 100 more amino acid residues of said isolated or synthesized polypeptide or peptide are positioned to the amino-terminus and/or carboxy-terminus of the lysine or histidine termini of peptide A.
  • the up to 100 more amino acid residues is up to one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, or fifty more amino acid residues.
  • the isolated or synthesized polypeptide or peptide consists of peptide A.
  • a further non- limiting embodiment of the seventh aspect of the present invention provides an isolated or synthesized peptide consisting of:
  • a peptide consisting of about 26 amino acid residues with a histidine residue within 5 residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 8, a histidine residue at position 10, a lysine residue at position 13, a lysine residue at position 18, and a lysine residue at position 26, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 26;
  • a peptide consisting of about 19 amino acid residues with a lysine residue within 5 residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1, and wherein relative to position 1 there is a histidine residue at position 3, a lysine residue at position 6, a lysine residue at position 11, and a lysine residue at position 19, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 19;
  • a peptide consisting of about 29 amino acids residues with a lysine residue within 5 residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 2, a lysine residue at position 10, a histidine residue at position 28, and a histidine residue at position 29, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the histidine residue at position 29;
  • a peptide consisting of about 27 amino acid residues with a histidine residue within 5 residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a histidine residue at position 2, a lysine residue at position 14, a lysine residue at position 19, and a lysine residue at position 27, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 27.
  • a peptide consisting of about 21 amino acid residues with a histidine residue within 5 residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1 and wherein relative to position 1 there is a lysine residue at position 6, a lysine residue at position 11, and a lysine residue at position 21, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 21;
  • a peptide consisting of about 22 amino acid residues with a lysine residue within 5 residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1 , and wherein relative to position 1 there is a lysine residue at position 11 , and a histidine residue at position 22, and wherein up to five additional residues may be present on the carboxy- terminus of the peptide after the histidine residue at position 22;
  • a peptide consisting of about 17 amino acids with a lysine residue within 5 residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 9, and a histidine residue at position 17, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the histidine residue at position 17;
  • a peptide consisting of about 15 amino acid residues with a histidine residue within 5 residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 5, a lysine residue at position 14, and a lysine residue at position 15, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 15;
  • a peptide consisting of about 35 amino acid residues with a histidine residue within 5 residues of the amino terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 6, a lysine residue at position 28, and a lysine residue at position 35, and wherein up to five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 35.
  • the isolated or synthesized peptide has an amino-terminus at position 1 and has a carboxy-terminus at the amino acid residue for which a position is expressly numbered that is the farthest to the carboxy-terminus of the peptide.
  • An eighth non- limiting aspect of the present invention provides a method of making a vaccine comprising: selecting at least one isolated or synthesized protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 as a component of a vaccine; and making said vaccine.
  • the method of making a vaccine comprises: selecting at least one isolated or synthesized peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 as at least one component; and making said vaccine with the at least one component.
  • the method of making a vaccine comprises selecting at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 as the at least one component of said vaccine.
  • the at least one isolated or synthesized protein fragment, polypeptide, or peptide has the same amino acid sequence as at least one protein fragment, polypeptide or peptide identified in an emerging strain of influenza virus up to six months, one year, two years, or three years prior to making said vaccine.
  • a ninth non- limiting aspect of the present invention provides a method for preventing or treating influenza virus infection comprising administering at least one isolated or synthesized protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 to an animal or human.
  • the at least one isolated or synthesized protein fragment, polypeptide, or peptide consists of at least one peptide A at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides SEQ ID NO(s): 1-12, 13-20, 21-28, and 32- 66.
  • the at least one isolated or synthesized peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 is administered to an animal or human.
  • At least one agent is capable of binding at least a portion of said peptide A that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a tenth non- limiting aspect of the present invention provides an isolated or synthesized nucleic acid sequence that encodes a protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the isolated or synthesized nucleic acid sequence encodes for a peptide consisting of 7 to about 50 amino acid residues and comprising any one or more of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the nucleic acid sequence encodes for a peptide that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In another non-limiting embodiment, the nucleic acid sequence encodes for a peptide that consists of at least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the isolated or synthesized nucleic acid sequence is comprised in an immunogenic compound. In another non-limiting embodiment, the isolated or synthesized nucleic acid sequence is comprised in a vaccine. [00037] Another non- limiting embodiment of the tenth aspect of the present invention provides an isolated or synthesized nucleic acid sequence that is antisense to a nucleic acid that encodes for a peptide that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • Another non-limiting embodiment provides a small interfering nucleic acid sequence that is about 10 to about 50 nucleic acids in length and is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that encodes for any portion of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or is 30%, 40%, 50%, 60%, 70%, 80%, 90% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any portion of one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the small interfering nucleic acid sequences is about 15 to about 45, about 20 to about 30, or about 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleic acids in length.
  • An eleventh non- limiting aspect of the present invention provides for a vaccine comprising at least one protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a hemagglutinin protein area of influenza virus, or a synthesized version thereof, and at least one protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a protein or peptide encoded by a pBl gene area of influenza virus, or a synthesized version thereof.
  • the at least one protein fragment, polypeptide, or peptide is isolated from an isolate of influenza virus predicted to have a greater infectivity than at least one other isolate of influenza virus and the at least one protein fragment, polypeptide, or peptide isolated from the pBl gene area, or synthesized version thereof, is isolated from an isolate of influenza virus predicted to have a greater lethality than at least one other isolate of influenza virus.
  • the at least one protein fragment, polypeptide, or peptide isolated from the hemagglutinin protein area, or synthesized version thereof is a plurality of protein fragments, polypeptides, and/or peptides isolated from the hemagglutinin protein area and the at least one protein fragment, polypeptide, or peptide isolated from the pBl gene area, or synthesized version thereof, is a plurality of protein fragments, polypeptides, and/or peptides isolated from the pBl gene area.
  • the at least one protein fragment, polypeptide, or peptide isolated from the hemagglutinin protein area, or synthesized version thereof is at least one Replikin peptide isolated from the hemagglutinin protein area and the at least one protein fragment, polypeptide, or peptide isolated from the pBl gene area, or synthesized version thereof, is at least one Replikin peptide isolated from the pBl gene area.
  • the at least one Replikin peptide isolated from a hemagglutinin protein area, or synthesized version thereof is a plurality of Replikin peptides isolated from a hemagglutinin protein area and the at least one Replikin peptide isolated from a pB 1 gene area, or synthesized version thereof, is a plurality of Replikin peptides isolated from a pBl gene area.
  • the plurality of Replikin peptides isolated from a hemagglutinin protein area, or synthesized version thereof is a plurality of the shortest Replikin peptides identified in an influenza virus isolate or a plurality of influenza virus isolates predicted to have a greater infectivity than at least one other isolate of influenza virus and said plurality of Replikin peptides isolated from a pBl gene area, or synthesized version thereof, is a plurality of the shortest Replikin peptides identified in an influenza virus isolate or a plurality of influenza virus isolates predicted to have a greater lethality than at least one other isolate of influenza virus.
  • the vaccine is directed against influenza A, influenza B, or influenza C.
  • the vaccine is directed against HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A virus.
  • a twelfth non- limiting aspect of the present invention provides a method of making a vaccine comprising: selecting at least one peptide A, wherein said peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a hemagglutinin protein area (or a synthesized version thereof) as a component of said vaccine; and selecting at least one peptide B, wherein said peptide B is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a pBl gene area (or a synthesized version thereof) as a component of said vaccine and making said vaccine comprising said components.
  • a method of making a vaccine comprises: identifying (1) at least one protein, protein fragment, polypeptide, or peptide of a hemagglutinin protein area in or derived from an isolate of influenza virus having relatively greater infectivity than another isolate of influenza virus or a plurality of isolates of influenza viruses, and (2) at least one protein, protein fragment, polypeptide, or peptide of a pBl gene area in or derived from an isolate of influenza virus having relatively greater lethality than another isolate of influenza virus or a plurality of isolates of influenza virus; and combining said at least one protein, protein fragment, polypeptide, or peptide of a hemagglutinin protein area and said at least one protein, protein fragment, polypeptide, or peptide of a pBl gene area to form a vaccine.
  • a method of differentiating the relative infectivity of isolate A of influenza virus or a plurality of isolates A of influenza virus from the relative infectivity of isolate B of influenza virus or a plurality of isolates B of influenza virus and the relative lethality of isolate A of influenza virus or a plurality of isolates A of influenza virus from the relative lethality of isolate B of influenza virus or a plurality of isolates B of influenza virus comprising: comparing the Replikin Count of the hemagglutinin protein area of isolate A or the mean Replikin Count of the hemagglutinin protein areas of a plurality of isolates A to the Replikin Count of the hemagglutinin protein area of isolate B or the mean Replikin Count of the hemagglutinin protein area of a plurality of isolates B; comparing the Replikin Count of the pBl gene area of isolate A or the mean Replikin
  • the isolate A or the plurality of isolates A is from a different region or time from the isolate B or the plurality of isolates B.
  • Another non-limiting embodiment provides a method of differentiating a predicted future relative infectivity of at least one strain A of influenza virus as compared to a time T 0 from a predicted future relative lethality of said at least one strain A of influenza virus as compared to time To comprising: comparing a trend of Replikin Counts in the hemagglutinin protein area of a plurality of isolates of strain A ending at time T 0 , wherein said isolates are isolated at different time periods including time To, to a trend of Replikin Counts in the pBl gene area of a plurality of isolates of strain A ending at time To, wherein said isolates are isolated at different time periods including time T 0 , and differentiating the future relative infectivity of said at least one strain A from the future relative lethality of said at least one
  • Figure 1 illustrates an immune response with protective effect following administration of a vaccine comprising a mixture of peptides of SEQ ID NO(s): 1-12 to chickens later challenged with Low-Path H5N1 virus.
  • Eighty chickens were divided into four groups of twenty chickens each on a first day after hatch.
  • Group 1 was a negative control subjected to neither vaccination nor infection with the Low-Path H5N1 virus.
  • Group 2 was a vaccine control subjected to vaccination intranasally on day 1 after hatch, intraocularly on day 7 after hatch, and via spray inhalation on day 14 after hatch.
  • Group 2 was not subject to infection with the Low- Path H5N1 virus.
  • Group 3 was subjected to vaccination on the same schedule as Group 2 and Low-Path H5N1 was introduced to the soft palate of the chickens on day 28.
  • Group 4 was a challenged control that was not vaccinated but was infected with H5N1 on day 28 via the soft palate.
  • the data from the serum antibody tests are contained in Table 1 and illustrated in Figure 1.
  • Figure 1 illustrates that only one of seven (14%) chickens tested in Group 3 (vaccinated and challenged with virus) was observed to produce antibody in serum seven days after challenge while four of seven chickens (57%) tested in Group 4 (not vaccinated but challenged) was observed to produce antibody in serum seven days after challenge.
  • Figure 1 further illustrates that only three of six chickens (50%) tested in Group 3 were observed to produce antibody in serum fourteen days after challenge while seven of nine (78%) chickens tested in Group 4 were observed to produce antibody in serum fourteen days after challenge.
  • Figure 1 further illustrates that two of seven (29%) chickens tested in Group 3 were observed to produce antibody in serum twenty-one days after challenge while three of nine (33%) of chickens tested in Group 4 were observed to produce antibody in serum twenty-one days after challenge.
  • Example 2 In combination with data provided in Table 2 (in Example 1 below), which demonstrates that no H5N1 virus was observed by PCR detection in feces or saliva for chickens in Groups 1, 2, and 3 (negative control, vaccine control, a vaccine/challenge groups, respectively) and that H5N1 virus was observed by PCR detection in feces and saliva for all chickens in Group 4 (challenge control), one of ordinary skill in the art concludes that chickens in the vaccinated and challenged group (Group 3) were provided a measure of protection from the challenge with Low-Path H5N1 on day 28 following hatch.
  • Figure 2 illustrates a double differentiation between the infectivity and the lethality of isolates of H5N1 isolated between 2004 and 2008.
  • the black columns represent the mean annual Replikin Count for hemagglutinin protein area sequences of isolates of H5N1 influenza virus publicly available at www.pubmed.com for a given year between 2004 and 2008. Standard deviation is denoted by the capped line on top of the black columns.
  • the hemagglutinin protein area is associated with infectivity in influenza.
  • the gray columns represent the mean annual Replikin Count for sequences from the pBl gene area of isolates of H5N1 influenza virus publicly available at www.pubmed.com for a given year between 2004 and 2008.
  • the pBl gene area of influenza is associated with lethality in influenza.
  • the data for Figure 2 is disclosed in Table 3 in Example 2 below.
  • Figure 2 illustrates that Replikin Count for the hemagglutinin protein area is differentiable from Replikin Count for the pBl gene area and that infectivity properties in H5N1 are differentiable from lethality properties in H5N1.
  • the data in Figure 2 corresponds to epidemiological data in H5N1. Human mortality (related to the lethality property of the pBl gene area) has increased in H5N1 from 1997 through at least 2007, when mortality rates reached as high as 80% in Indonesia.
  • Figure 3 illustrates a double differentiation between the infectivity and the lethality of isolates of HlNl isolated between 2004 and May 18, 2009.
  • the black columns represent the mean annual Replikin Count for hemagglutinin protein area sequences (associated with infectivity) publicly available at www.pubmed.com for isolates of HlNl influenza in a given year between 2004 and 2009. Standard deviation is denoted by the capped line on top of the black columns.
  • the gray columns represent the mean annual Replikin Count for sequences from the pBl gene area (associated with lethality) publicly available at www.pubmed.com for isolates of HlNl influenza in a given year between 2004 and 2009. Standard deviation is denoted by the capped line on top of the gray columns.
  • Figure 3 illustrates that Replikin Count for the hemagglutinin protein area is differentiable from Replikin Count for the pBl gene area and that infectivity properties in HlNl are differentiable from lethality properties in HlNl .
  • the data in Figure 3 corresponds to epidemiological data in HlNl. Infectivity in HlNl has increased dramatically in 2009 resulting in a global outbreak of HlNl influenza apparently beginning in or near Mexico or the soiled United States around the spring of 2009. The increase in Replikin Count in the hemagglutinin protein area of isolates of HlNl in the winter of 2008 allowed for an April 2008 prediction of the current global outbreak in 2009.
  • Figure 4 illustrates a double differentiation between the infectivity and the lethality of isolates of HlNl isolated between 2001 and June 8, 2009.
  • black columns represent the mean annual Replikin Count for hemagglutinin protein area sequences (associated with infectivity) of isolates of HlNl influenza publicly available at www.pubmed.com for a given year between 2001 and 2009 (the 2009 column represents the mean annual Replikin Count for hemagglutinin protein area sequences of isolates of HlNl influenza publicly available from January 1, 2009 through June 8, 2009). Standard deviation is denoted by the capped line on top of the black columns.
  • Gray columns represent the mean annual Replikin Count for sequences from the pBl gene area (associated with lethality) of isolates of HlNl influenza publicly available at www.pubmed.com for a given year between 2001 and 2009 (the 2009 column represents the mean annual Replikin Count for the pBl gene area sequences of isolates of HlNl influenza publicly available from January 1, 2009 through June 8, 2009). Standard deviation is denoted by the capped line on top of the gray columns.
  • the data for Figure 4 is disclosed in Tables 5 and 6 in Example 4 below.
  • Figure 4 illustrates that Replikin Count for the hemagglutinin protein area is differentiable from Replikin Count for the pB 1 gene area and that infectivity properties in HlNl are differentiable from lethality properties in HlNl.
  • the data in Figure 4 corresponds to epidemiological data in HlNl. As described above, infectivity in HlNl increased dramatically in 2009 resulting in an HlNl pandemic. Additionally, lethality in HlNl has been observed to remain generally low between 2004 and 2008 with a spike in lethality in 2009 based on data analyzed between May 18 and June 8, 2009. The spike in lethality in 2009 is observed as statistically significant with a p-value of less than 0.001. See Table 6 in Example 4 below. An earlier rise in the Replikin Count in the pBl gene area in 2005 is not statistically significant with a p-value of less than 0.40. See Table 6 below.
  • Figure 5 illustrates a double differentiation between the infectivity and the lethality of isolates of HlNl isolated between 2001 and September 23, 2009.
  • the white columns represent the mean annual Replikin Count for hemagglutinin protein area sequences (publicly available at www.pubmed.com) of isolates of HlNl influenza isolated in a given year for years 2001 through 2007 and represent the mean Replikin Count from the beginning of a given year through to the given date for years 2008 and 2009. Standard deviation is denoted by a capped line on top of each white column.
  • the hemagglutinin protein area is associated with infectivity in influenza.
  • the black columns represent the mean annual Replikin Count for sequences from the pBl gene area (publicly available at www.pubmed.com) of isolates of HlNl influenza isolated in a given year for years 2001 through 2007 and represent the mean Replikin Count from the beginning of a given year through to the given date for years 2008 and 2009. Standard deviation is denoted by a capped line on top of each black column.
  • the pBl gene area of influenza is associated with lethality in influenza.
  • the data for Figure 5 is disclosed in Table 7 in Example 5 below.
  • Figure 5 illustrates that Replikin Count for the hemagglutinin protein area is differentiable from Replikin Count for the pBl gene area and that infectivity properties in HlNl are differentiable from lethality properties in HlNl.
  • the data in Figure 5 corresponds to epidemiological data. As described above, infectivity in HlNl increased dramatically in 2009 resulting in a global outbreak of HlNl influenza apparently beginning in or near Mexico or the soiled United States around the spring of 2009. In further correspondence to Figure 5, the lethality of the 2009 HlNl outbreak has been fairly low with the proportion of deaths in the United States attributable to pneumonia and influenza below the epidemic threshold.
  • a “protein fragment” as used in this specification is any portion of an expressed whole protein.
  • a protein fragment may reflect an expressed whole protein with one or more amino acids removed from the amino acid sequence of the expressed whole protein.
  • a protein fragment may also reflect an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with any portion of an expressed whole protein.
  • a "polypeptide,” as used in this specification, is any portion of a protein fragment and is less than an expressed whole protein.
  • a "whole protein” or an "expressed whole protein” as used in this specification reflect a protein that is expressable from an intact gene of an influenza virus from a start codon to a stop codon.
  • a whole protein or expressed whole protein may also reflect a whole protein or expressed whole protein that has been subject to cellular processing to create a protein that is capable of functioning within the virus replication system in a proper manner for virus replication.
  • a protein fragment, polypeptide, or peptide "partially matches" the amino acid sequence of an expressed whole protein when the protein fragment, polypeptide, or peptide shares substantially homology with the expressed whole protein but at least one of the amino acids of the expressed whole protein are not present in the protein fragment, polypeptide, or peptide.
  • “Homologous” or “homology” or “sequence identity” as used in this specification indicate an amino acid sequence or nucleic acid sequence exhibits substantial structural equivalence with another sequence, namely any one of SEQ ID NO(s): 1-66 (for purposes of this paragraph, the basis sequences) or any nucleotide sequence encoding SEQ ID NO(s): 1-66 (a redundancy in a coding sequence may be considered identical to a sequence encoding the same amino acid).
  • the sequence is aligned for optimal comparison purposes with any one of the basis sequences. Where gaps are necessary to provide optimal alignment, gaps may be introduced in the identified sequence or in the basis sequence.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared between the identified sequence and the basis sequence.
  • the total number of amino acid residues or nucleotides in the identified sequence that are identical with amino acid residues or nucleotides in the basis sequence is divided by the total number of residues or nucleotides in the basis sequence (if the number of residues or nucleotides in the basis sequence is greater than the total number of residues or nucleotides in the identified sequence) or by the total number of amino acid residues or nucleotides in the identified sequence (if the number of residues or nucleotides in the identified sequence is greater than the total number of residues or nucleotides in the basis sequence).
  • the final number is determined as a percentage.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps (where a gap must be introduced for optimal alignment of the two sequences) and the length of each gap. Any structural or functional differences between sequences having sequence identity or homology will not affect the ability of the sequence to function as indicated in the desired application.
  • SEQ ID NO: 1 HAQDILEKEHNGKLCSLKGVRPLILK
  • SEQ ID NO: 29 is the identified sequence.
  • SEQ ID NO: 1 is the basis sequence.
  • the 26 aligned identical residues are divided by the total number of residues in SEQ ID NO: 29, namely 30 residues, giving 0.867 or more than 86% homology.
  • SEQ ID NO: 1 is more than 86% homologous with HAQDXILEKEHNGKLCXSLKGVRXXPLILK (SEQ ID NO: 30) because it is identical to SEQ ID NO: 30 in all residues except for the residues represented by the four X residues.
  • SEQ ID NO: 2 (KEHNGKLC SLKGVRPLILK) is more than 68% homologous with KEHNGKLCSLKGK (SEQ ID NO: 31).
  • SEQ ID NO: 2 is the basis sequence and has 19 residues.
  • SEQ ID NO: 31 is the reference sequence and has 13 residues that are identical to SEQ ID NO: 2 but VRPLIL is not present between the glycine at position 12 and the terminal lysine at position 13 (all of the other residues are identical). To determine percent homology, then, the 13 aligned identical residues are divided by the total number of residues in SEQ ID NO: 2, namely 19 residues, giving 0.684 or more than 68% homology. [00056] To determine homology between an identified sequence that is contained in a larger polypeptide, protein fragment, or protein, and a basis sequence, the polypeptide, protein fragment, or protein must first be optimally aligned with the basis sequence.
  • the residue in the identified sequence that is farthest to the amino-terminus of the polypeptide, protein fragment, or protein and identical to a residue in the basis sequence that is farthest to the amino-terminus of the basis sequence is considered the amino-terminal residue of the identified sequence.
  • the residue in the identified sequence that is farthest to the carboxy-terminus of the polypeptide, protein fragment, or protein and identical to a residue in the basis sequence that is farthest to the carboxy-terminus of the basis sequence is considered the carboxy-terminal residue of the identified sequence.
  • An amino acid sequence of a protein fragment, polypeptide, or peptide is "derived from" an identified protein or gene area of an influenza virus (such as a hemagglutinin protein area or a pBl gene area) if one of ordinary skill in the art would understand from the structure, history, or other relevant information of the amino acid sequence that it originated from an amino acid sequence of the identified protein or gene area of influenza.
  • an influenza virus such as a hemagglutinin protein area or a pBl gene area
  • one of ordinary skill may employ analysis of the homology of the amino acid sequence with the identified protein or gene area.
  • One of ordinary skill may also employ the history of research used in developing the amino acid sequence to determine that the amino acid sequence is derived from an original sequence of the identified protein or gene area.
  • a protein fragment, polypeptide, or peptide is derived from an identified protein, polypeptide, or peptide if it is traceable to the identified protein, polypeptide, or peptide, if it is deducible or inferable from the identified protein, polypeptide, or peptide, if the identified protein, polypeptide, or peptide is the source of the peptide, or if the protein fragment, polypeptide, or peptide is derived from the identified protein, polypeptide, or peptide as understood by one of skill in the art.
  • One of ordinary skill may employ any method known now or hereafter for determining whether an amino acid sequence is derived from an identified protein or gene area of an influenza virus.
  • transmission means, the movement of a pathogen by any means from one animal host to any neighboring animal host.
  • reservoir means, a collection of animals, one or all of which are infected with a particular infectious agent, wherein the collection of animals continues to provide a source of infection outside of the collection of animals.
  • a reservoir is self-perpetuating and permits time for modification of viruses within the reservoir and passing of viruses, including modified viruses, to hosts outside of the reservoir.
  • “concomitant” or “concomitantly” or related words reflect a difference between the change in infectivity and the change in lethality in a strain of influenza or in different strains or isolates of influenza within a particular time period or at a particular time point or within a particular region. For example, if the relative infectivity of a first isolate from a given time period or time point or from a particular region is greater than the relative infectivity of a second isolate from the same time period or same time point or same particular region and the relative lethality of the first isolate is not greater than the relative lethality of the second isolate, then the lethality of the first isolate is not concomitantly greater than the relative lethality of the second isolate.
  • an increase in the relative infectivity over time in a group of isolates from a particular time period or region that is not accompanied by, attended by, or does not correspond with an increase in the relative lethality over time in the same group of isolates is an increase in infectivity that is not concomitant with an increase in lethality in the same group of isolates.
  • Changes in infectivity that are not concomitant with changes in lethality in a strain of influenza virus allow for the differentiation of the properties of infectivity and lethality in a strain of influenza over a particular time period or across different regions.
  • vaccine is any substance, compound, composition, mixture, or other therapeutic substance that, when administered to a human or animal via any method of administration known to the skilled artisan now or hereafter, produces an immune response, an antibody response, or a protective effect in the human or animal.
  • a protein area or a gene area of an influenza protein or gene is the protein or gene of influenza as known to one of skill in the art. Because one skilled artisan may choose to identify a first terminus of a protein or gene in influenza at a different starting point than another skilled artisan and one skilled artisan may choose to identify a second terminus of a protein or gene in influenza at a different ending point than another skilled artisan based on research conditions, one of skill in the art understands that the hemagglutinin protein and the pBl gene may be considered as a protein area or a gene area.
  • a "Replikin sequence” is an amino acid sequence of 7 to about 50 amino acids comprising or consisting of a Replikin motif wherein the Replikin motif comprises:
  • Replikin sequence For the purpose of determining Replikin concentration, a Replikin sequence must have a lysine residue at one terminus and a lysine or a histidine residue at the other terminus. For diagnostic, therapeutic, and preventive purposes, a Replikin sequence may or may not have defined termini.
  • the term "Replikin sequence" can also refer to a nucleic acid sequence encoding an amino acid sequence having 7 to about 50 amino acids comprising: (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
  • amino acid sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine.
  • peptide or "protein” refers to a compound of two or more amino acids in which the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond.
  • an "isolated" peptide may be synthesized by organic chemical methods.
  • An isolated peptide may also be synthesized by biosynthetic methods.
  • An isolated peptide also may refer to a peptide that is, after purification, substantially free of cellular material or other contaminating proteins or peptides from the cell or tissue source from which the peptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized by any method, or substantially free from contaminating peptides when synthesized by recombinant gene techniques or a protein or peptide that has been isolated in silico from nucleic acid or amino acid sequences that are available through public or private databases or sequence collections.
  • An isolated peptide may be synthesized by biosynthetic or organic chemical methods.
  • Protein fragments, polypeptides, or peptides in this specification may be chemically synthesized by any method known to one of skill in the art now and hereafter.
  • isolated protein fragment, polypeptides, or peptides may be synthesized by solid phase synthesis.
  • the production of these materials by chemical synthesis avoids the inclusion of (or the need to remove by purification) materials that are byproducts of other production methods such as recombinant expression or isolation from biological material.
  • Such byproducts may include, for example, avian proteins associated with vaccines produced using birds' eggs or bacterial proteins associated with recombinant production in bacteria.
  • An "encoded” or “expressed” protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of ordinary skill in the art now or hereafter. It should be noted that it is well-known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence.
  • a method of identifying a Replikin amino acid sequence also encompasses a method of identifying a nucleic acid sequence that encodes a Replikin amino acid sequence wherein the Replikin amino acid sequence is encoded by the identified nucleic acid sequence.
  • “conserved” or “conservation” refers to conservation of particular amino acids due to lack of substitution. Conservation may occur at a specific position in a protein or polypeptide or may occur at a position that is close to a specific position in a protein or polypeptide but not the exact specific position. This type of conservation occurs because additional amino acid residues may be substituted in a protein or polypeptide such that the numbering of residue positions may shift toward either terminus of the protein or polypeptide.
  • “Replikin Count” or “Replikin Concentration” refers to the number of Replikin sequences per 100 amino acids in a protein, protein fragment, virus, or organism.
  • Replikin concentration is determined by counting the number of Replikin sequences in a given sequence, wherein a Replikin sequence is a peptide of 7 to about 50 amino acid residues with a lysine residue on one end and a lysine residue or a histidine residue on the other end wherein the peptide comprises (1) a lysine residue six to ten residues from another lysine residue, (2) a histidine residue, (3) and 6% or more lysine residues, or wherein a Replikin sequence is a nucleic acid that encodes a Replikin peptide sequence.
  • An aspect of the present invention provides compounds for diagnostic, therapeutic, and/or preventive purposes in influenza, methods of differentiating infectivity and lethality in influenza, and methods of designing therapies against influenza based on compounds of the invention and differentiation of infectivity and lethality in influenza.
  • Compounds of the invention include Replikin peptides and homologues of Replikin peptides identified in and isolated from different strains of influenza and conserved over time in the same and different strains of influenza. These Replikin peptides have been shown to be useful when comprised in immunogenic compounds and have provided a protective effect against influenza infection including antagonism of both the infectivity of strains of influenza and the replication and lethality of strains of influenza.
  • a homologue may be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more or 100% homologous with a peptide against which the homologue is compared.
  • the methods have provided peptides for a vaccine that may be applied for prevention or treatment of any strain of influenza virus.
  • the vaccine is known as TransFluTM
  • the applicants have now additionally developed another vaccine that comprises eight additional peptides identified in the hemagglutinin protein area and pBl gene area of the HlNl virus. Homologues of any one of these eight peptides may also be used in an immunogenic compound against any strain of influenza virus that contains a homologue of one of the eight peptides. A homologue of one these eight peptides may likewise be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more or 100% homologous with a peptide against which the homologue is compared.
  • the peptides disclosed in the vaccines herein described are peptides that are conserved over time in specific strains and shared between strains (also over time), one of skill in the art expects such peptides (and peptides that are similar in structure and function) to also be useful in immunogenic compounds for influenza infections of various strains. This expectation is based on, for example, the function of the peptides identified herein and the commonality of structure and position of the peptides and their homologues as described herein as well as the functionality of the peptides and the homologues in the hemagglutinin protein area and pBl gene area in different strains of influenza. See, e.g., Figures 2-5.
  • Replikin peptides have been shown to be broadly antigenic, to be conserved, and to be related to rapid replication and outbreaks across many different strains of influenza virus. See, e.g.
  • Replikin peptides and homologues disclosed herein are shown to be structurally and functionally related to the infectivity and lethality of influenza virus based on the positions in the hemagglutinin protein area or pBl gene area of influenza, respectively.
  • the peptides and their homologues described herein are, among other things, antigenic, common to various strains of influenza virus in both position and function, conserved in various strains of influenza over time, conserved in specific positions in the hemagglutinin protein area and pBl gene areas over time, conserved in their lysines and histidines within the Replikin structure, and associated with mechanisms of infectivity and/or lethality.
  • Replikin peptides and their homologues described herein would expect the Replikin peptides and their homologues described herein to be useful in immunogenic compounds for therapies against influenza virus within strains, across strains, and across time.
  • Replikins sequences and their homologues provided by an aspect of the invention may be identified in strains of influenza virus including any strain of influenza virus known now or identified or known hereafter.
  • Compounds of the invention may be conserved within strains of influenza virus, across types within strains of influenza virus, and across strains of influenza virus.
  • the compounds, because they are Replikin sequences, related to Replikin sequences, derived from Replikin sequences, identified as comprising Replikin sequences, or designed to comprise Replikin sequences, are related to rapid replication, virulence, and lethality in influenza. See Figures 2-5.
  • Compounds of the invention are useful as immunogenic compounds to stimulate the immune system of a subject to produce an immune response, which may include production of antibodies or other binding molecules.
  • Compounds of the invention are also useful in therapies such as vaccines.
  • Compounds of the invention are likewise useful in producing antibodies, antibody fragments, or other binding or antagonizing agents, which may be used, among other things, for diagnostic and therapeutic purposes, including passive immunity.
  • the immunogenic compounds, antibodies (and other binding or antagonizing agents) and vaccines of the invention are useful against any strain of influenza virus including influenza A, B, or C strains. Within strains of influenza A, they are useful against any strain of influenza A including, but not limited to, HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, and H10N7. They are useful in any organism that is capable of producing an immune response.
  • the compounds of the invention are also useful for diagnostic purposes, including identifying rapidly replicating, virulent, or lethal strains of virus.
  • the compounds of the invention may be conserved in the H5N1 strain of virus including low-pathogenic (Low-Path) strains of H5N1 and high-pathogenic (High-Path) strains of H5N1.
  • the compounds may also be conserved in other strains of influenza including HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, and H10N7.
  • the following twenty-eight peptides and homologues of the following twenty-eight peptides are provided as an aspect of the invention as isolated or synthesized peptides, as immunogenic compounds, as vaccines, and as targets for antibodies and binding agents of the invention, among other things: H AQDILEKEHNGKLC SLKG VRPLILK (SEQ ID NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4), HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRL
  • SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 are sequences that were initially identified in HlNl or H5N1 as related to infectivity or lethality in those strains of influenza virus. Further investigation of the conservation of certain of those sequences in other strains of influenza virus provided identical sequences or homologues of those sequences conserved in other strains of influenza virus including H9N2, H3N2, H5N1, and HlNl where the conserved homologues shared the same amino acid residue position in the functional protein of the other strain of influenza virus. As a result, the conserved homologues would be expected to share the same functional characteristics in those other influenza viruses where they are conserved.
  • the conserved homologues are further identified in positions in the hemagglutinin protein area and pBl gene areas of various strains of influenza where these genes are directly associated with infectivity and lethality, respectively. Further, a vaccine based on these homologues has provided successful results in chickens in antagonizing both the infectivity of influenza virus and the replication (or lethality) of influenza virus once it has entered a host system. See, e.g. , Example 1 below.
  • TransFluTM a vaccine that offers cross-strain protection for a variety of strains of influenza.
  • SEQ ID NO(s) : 1 - 12 were initially identified in a strain of Low-Path H5N1. These peptides have since that time been identified in a series of highly pathogenic (High-Path) strains of H5N1 influenza, including a lethal strain of H5N1 isolated in Vietnam, among others.
  • SEQ ID NO(s): 13-20 have now also been identified and isolated as homologues of at least one amino acid sequence of SEQ ID NO(s): 1-12. Certain of these homologues have been identified not only in strains of H5N1 but also in other strains of influenza virus such as H5N2, H3N2, and HlNl. Additionally, SEQ ID NO(s): 21-28 are also provided as a vaccine against HlNl . Homologues of these sequences in other strains of influenza are expected to provide cross-strain protection.
  • Replikin peptides in general are seen to be conserved across strains of influenza.
  • amino acid residues that provide for the Replikin sequence structure of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 is conserved widely across strains and time in influenza.
  • the key amino acid residues that provide for the Replikin sequence structure are the lysine and histidine residues wherein a Replikin sequence has at least one lysine on one terminus and at least one lysine or one histidine on the other terminus, at least one lysine that is six to ten residues from at least one other lysine, at least one histidine, and at least six percent lysines in total between the terminal lysine and the terminal lysine or histidine.
  • One aspect of the present invention provides a protein, a protein fragment, a polypeptide, or a peptide that comprises at least one peptide A homologous with at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • Peptide A may be 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous or 100% homologous with any of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the protein, protein fragment, or peptide may likewise be a peptide that consists of a peptide A that is homologous with any of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a peptide consisting of any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 is also provided.
  • amino acid sequence of the provided isolated or synthesized protein, protein fragment, polypeptide, or peptide may partially match an amino acid sequence of an expressed whole protein. At least one, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred, five hundred and fifty or more amino acid residues of the amino acid sequence of the expressed whole protein may not be present in the protein, protein fragment, polypeptide, or peptide.
  • the amino acid sequence of the isolated or synthesized protein, protein fragment, polypeptide, or peptide may also partially match the amino acid sequence of an expressed whole protein where at least one, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of at least one terminus of the amino acid sequence of the expressed whole protein is(are) not present at at least one terminus of said protein fragment, polypeptide, or peptide. Any additional number of amino acids may be situated on one or the other terminus or on both termini of the protein, protein fragment, polypeptide, or peptide.
  • Replikin peptide such as SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66
  • SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 is associated with rapid replication, infectivity, and/or lethality, in functional proteins in influenza viruses
  • inclusion of any Replikin peptide in a protein, protein fragment, polypeptide, or peptide does not negate the functional nature of the Replikin peptide.
  • antagonism of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 (with homology of 30% or greater) within a protein, protein fragment, polypeptide, or peptide would be expected to antagonize the replication, infectivity, and/or lethality of the protein, protein fragment, polypeptide, or peptide.
  • a provided peptide may further be a peptide B of 7 to about 50 amino acid residues where peptide B contains a peptide A that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous or 100% homologous with any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a non-limiting peptide may further be a peptide A that is a Replikin peptide wherein the Replikin peptide has a lysine residue on one end and a lysine residue or a histidine residue on the other end wherein the Replikin peptide comprises: (1) a lysine residue six to ten amino acids from another lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
  • An isolated or synthesized protein, protein fragment, polypeptide, or peptide may consist of a peptide that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 where the length of the peptide is no more than one, five, ten, twenty, thirty, forty, or fifty amino acid residues longer than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 with which it is homologous.
  • An isolated or synthesized protein fragment, polypeptide, or peptide may likewise be no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues longer than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 with which it is homologous.
  • An isolated or synthesized protein fragment, polypeptide, or peptide may likewise consist of any one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • An isolated or synthesized polypeptide or peptide may comprise a peptide A that has about the same number of amino acid residues as a peptide B, where peptide B is one of the peptides of SEQ ID NO: 1-28 and where the lysine residues and histidine residues in peptide A are conserved as compared to the lysine residues and histidine residues in peptide B.
  • An isolated or synthesized polypeptide or peptide comprising peptide A may have up to 100 additional amino acid residues as compared to peptide B.
  • Some or all of the up to 100 additional amino acid residues may be positioned toward the amino-terminus and/or carboxy -terminus of the lysine or histidine termini of peptide A. Some of the additional amino acid residues may be positioned within the lysine or histidine termini of peptide A so long as a level of homology is maintained between peptide A and peptide B that retains at least some of the functionality of the Replikin peptide of peptide B. Functionality may include, but is not limited to, antigenicity, rate of replication, antagonizability of a protein containing said peptide A or said peptide B, binding capacity of binding agents to peptides A or B, etc.
  • An isolated or synthesized polypeptide or peptide may also comprise up to about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, about 4, about 3, about 2, or about 1 additional amino acid residues.
  • the residues may be entirely outside of the Replikin structure or entirely within the Replikin structure or partially within and partially outside the Replikin structure. A level of homology should be maintained between peptides B and A when additional residues are present or are added.
  • Residues outside of the Replikin structure are those residues on the amino-terminus or carboxy-terminus of the polypeptide or peptide as compared to the lysine or histidine termini of peptide A.
  • Residues within the Replikin structure are those residues that are between the lysine or histidine termini of peptide A.
  • An isolated or synthesized polypeptide or peptide may also consist of peptide A and peptide A may consist of peptide B.
  • An isolated or synthesized peptide may consist of a peptide of about 26 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino- terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 8, a histidine residue at position 10, a lysine residue at position 13, a lysine residue at position 18, and a lysine residue at position 26, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 26.
  • the isolated or synthesized peptide will consist of about 36 amino acids.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 1 and may be used as an immunogenic compound or as a component of a vaccine against infectivity in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 19 amino acid residues with a lysine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1, and wherein relative to position 1 there is a histidine residue at position 3, a lysine residue at position 6, a lysine residue at position 11, and a lysine residue at position 19, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 19. If five residues are present on each end of the peptide, it will consist of about 29 amino acids.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 2 and may be used as an immunogenic compound or as a component of a vaccine against infectivity in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 29 amino acids residues with a lysine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 2, a lysine residue at position 10, a histidine residue at position 28, and a histidine residue at position 29, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the histidine residue at position 29. If five residues are present on each end of the peptide, it will consist of about 39 amino acids.
  • An isolated or synthesized peptide is a homologue of SEQ ID NO: 3 and may be used as an immunogenic compound or as a component of a vaccine against infectivity in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 27 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1 , and wherein relative to position 1 there is a histidine residue at position 2, a lysine residue at position 14, a lysine residue at position 19, and a lysine residue at position 27, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 27.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 4 and may be used as an immunogenic compound or as a component of a vaccine against infectivity in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 21 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1 and wherein relative to position 1 there is a lysine residue at position 6, a lysine residue at position 11, and a lysine residue at position 21, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 21. If five residues are present on each end of the peptide, it will consist of about 31 amino acids.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 5 and may be used as an immunogenic compound or as a component of a vaccine against infectivity in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 22 amino acid residues with a lysine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 11, and a histidine residue at position 22, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy- terminus of the peptide after the histidine residue at position 22. If five residues are present on each end of the peptide, it will consist of about 32 amino acids.
  • An isolated or synthesized peptide is a homologue of SEQ ID NO: 6 and may be used as an immunogenic compound or as a component of a vaccine against infectivity in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 17 amino acids with a lysine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1 , and wherein relative to position 1 there is a lysine residue at position 9, and a histidine residue at position 17, and wherein up to one, two, three four, or five additional residues may be present on the carboxy -terminus of the peptide after the histidine residue at position 17.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 7 and may be used as an immunogenic compound or as a component of a vaccine against lethality in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 15 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1 , and wherein relative to position 1 there is a lysine residue at position 5, a lysine residue at position 14, and a lysine residue at position 15, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 15. If five residues are present on each end of the peptide, it will consist of about 25 amino acids.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 8 and may be used as an immunogenic compound or as a component of a vaccine against lethality in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 18 amino acid residues with a lysine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the lysine residue is considered to reside at position 1 , and wherein relative to position 1 there is a lysine residue at position 2, a histidine residue at position 5, a lysine residue at position 6, a lysine residue at positions 11, 12, and 13, and a lysine residue at position 18, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 18.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 9 and may be used as an immunogenic compound or as a component of a vaccine against lethality in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 14 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 2, a lysine residue at positions 7, 8, and 9, and a lysine residue at position 14, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 14. If five residues are present on each end of the peptide, it will consist of about 24 amino acids.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 10 and may be used as an immunogenic compound or as a component of a vaccine against lethality in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 26 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino-terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 16, and a lysine residue at positions 24, 25, and 26, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 26. If five residues are present on each end of the peptide, it will consist of about 36 amino acids.
  • An isolated or synthesized peptide is a homologue of SEQ ID NO: 11 and may be used as an immunogenic compound or as a component of a vaccine against lethality in any strain of influenza virus.
  • An isolated or synthesized peptide may consist of about 35 amino acid residues with a histidine residue within zero, one, two, three, four, or five residues of the amino terminus of the peptide wherein the histidine residue is considered to reside at position 1, and wherein relative to position 1 there is a lysine residue at position 6, a lysine residue at position 28, and a lysine residue at position 35, and wherein up to one, two, three, four, or five additional residues may be present on the carboxy-terminus of the peptide after the lysine residue at position 35.
  • Such an isolated or synthesized peptide is a homologue of SEQ ID NO: 12 and may be used as an immunogenic compound or as a component of a vaccine against lethality in any strain of influenza virus.
  • Any one of the above-listed isolated or synthesized peptides may have an amino- terminus at position 1 and a carboxy-terminus at the amino acid residue for which a position is expressly numbered where that expressly-numbered position is the farthest numbered position toward the carboxy-terminus of the peptide.
  • a homologue of SEQ ID NO: 7 will have a terminal lysine at position 1 and a terminal histidine at position 17
  • a homologue of SEQ ID NO: 4 (HHSNEQGSGYAADKESTQKAIDGITNK) will have a terminal histidine at position number 1 and a terminal lysine at position number 27.
  • the at least one isolated or synthesized protein, protein fragment, or peptide may also comprise at least one peptide A and at least one peptide C where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 and where peptide C is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • Peptide C may be homologous with a different peptide from among SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 than the peptide that peptide A is homologous with.
  • the at least one isolated or synthesized protein, protein fragment, or peptide may comprise three or more peptides homologous with at least three different peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • All of the above-discussed proteins, protein fragments, polypeptides, and peptides comprise the functional unit of a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. These proteins, protein fragments, polypeptides, and peptides share a functional role in either infectivity or lethality. Antagonism of any of the homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 will likewise antagonize either the infectivity function or lethality function in any strain of influenza that share a homologue of any one of the sequences.
  • proteins, protein fragments, polypeptides, and peptides are useful as immunogenic compounds, therapeutic compounds, vaccines, and for other therapies directed at antagonizing the infectivity and/or lethality of a strain of influenza.
  • disclosed proteins, protein fragments, polypeptides, and peptides are expected to be capable of limiting the excretion or shedding of influenza virus such that the virus is limited in its spread from host to host or from host to reservoir to host, etc..
  • disclosed compounds are effective at limiting sources of influenza infection.
  • any binding agent that binds one of the proteins, protein fragments, polypeptides, and peptides discussed above will antagonize the infectivity and/or lethality of a strain of influenza and limit sources of influenza infection such as transmission from host to host or from host to reservoir to host.
  • a non-limited protein, protein fragment, or peptide of the invention may be comprised in an immunogenic compound.
  • the proteins, protein fragment, polypeptides, and peptides provided by an aspect of the invention comprise at least a portion that is homologous with a Replikin peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • These homologues are expected by one of ordinary skill in the art to stimulate the immune system of a subject upon sufficient exposure to produce antibodies against at least the homologous portion of the protein, protein fragment, polypeptide, or peptide.
  • antibodies or other binding agents arrayed against a protein or protein fragment comprising one of the antigenic homologues disclosed herein would be antagonized.
  • One of ordinary skill would also expect an antagonist of one of these homologues to antagonize any influenza virus that comprises a homologue within its hemagglutinin protein area or pBl gene area since an immune response against SEQ ID NO(s): 1-12 has been shown to antagonize both the infectivity and replication (including excretion) of H5N1. Because homologues of SEQ ID NO(s): 1-12 have been shown to be conserved across strains of influenza in the hemagglutinin protein area and the pBl gene area, one of ordinary skill would expect antagonism of such homologues to result in antagonism of influenza replication similar to what was observed in SEQ ID NO(s): 1-12 in chickens. One of ordinary skill would further expect particular antagonism of the infectivity and lethality mechanisms of influenza when an immune system is stimulated against a homologue of SEQ ID NO(s): 1-6 and SEQ ID NO(s): 7-12, respectively.
  • the applicants disclose herein a series of homologues of SEQ ID NO(s): 1-12 identified in the hemagglutinin and pBl gene areas of a wide range of strains of influenza.
  • Each of these homologues is provided as a component that may be used in an immunogenic compound to stimulate the immune system of a subject against influenza infection.
  • other homologous sequences are likewise provided as immunogenic compounds to stimulate the immune system of a subject against influenza infection.
  • Any homologue that shares 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homology with any one of SEQ ID NO(s): 1-12 is disclosed as a peptide that may be used in an immunogenic compound against influenza infection.
  • any protein, protein fragment, polypeptide, or peptide comprising such a homologue may be used as an immunogenic compound or be comprised within an immunogenic compound.
  • An immune response against such compounds would be understood by one of ordinary skill in the art to be useful in stimulating the immune system against an influenza infection.
  • any homologue that shares 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homology with any one of SEQ ID NO(s): 21-28 is disclosed as a peptide that may be used in an immunogenic compound against influenza infection.
  • Any protein, protein fragment, polypeptide, or peptide comprising such a homologue may also be used as an immunogenic compound or may be comprised within an immunogenic compound.
  • An immune response against such compounds would be understood by one of ordinary skill in the art to be useful in stimulating the immune system against an influenza infection.
  • Vaccines Comprising Peptides Homologous to Influenza Replikin Peptides
  • An immunogenic compound provided as an aspect of the invention may be used as a component of a non- limiting vaccine against any strain of influenza.
  • a vaccine comprising one or more homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 may be used against influenza infection.
  • a vaccine comprising one or more homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 derived from a hemagglutinin protein area and one or more homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 derived from a pBl gene area may be used against influenza infection and may antagonize the infectivity and/or replication and lethality of an influenza infection.
  • mixtures of homologues of SEQ ID NO(s): 1-6 and SEQ ID NO(s): 7-12 are provided as vaccines to antagonize both the infectivity and replication and lethality of an influenza infection. Such vaccines are useful for antagonizing infectivity, replication, lethality, and excretion or spread of influenza virus.
  • a non-limiting vaccine comprising: at least one protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%,
  • peptide A is isolated from a hemagglutinin protein area of influenza virus, or a synthesized version thereof; and at least one protein fragment, polypeptide, or peptide comprising at least one peptide B, where peptide B is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 and wherein peptide B is isolated from a protein or peptide encoded by a pBl gene area of influenza virus, or a synthesized version thereof.
  • the peptide A may be identified in, isolated from, derived from, or synthesized from an isolate of influenza virus predicted to have a greater infectivity than at least one other isolate of influenza virus and the peptide B may be identified in, isolated from, derived from, or synthesized from an isolate of influenza virus predicted to have a greater lethality than at least one other isolate of influenza virus.
  • a vaccine may further comprise a plurality of protein fragments, polypeptides, and/or peptides from the hemagglutinin protein area and a plurality of protein fragments, polypeptides, and/or peptides from the pBl gene area.
  • a vaccine may further comprise at least one Replikin peptide from the hemagglutinin protein area and at least one Replikin peptide from the pBl gene area.
  • a vaccine may further comprise a plurality of Replikin peptides from a hemagglutinin protein area where the at least one Replikin peptide from a pBl gene area is a plurality of Replikin peptides from a pBl gene area.
  • a vaccine may comprise a plurality of the shortest Replikin peptides from a hemagglutinin protein area and a plurality of the shortest Replikin peptides from a pBl area.
  • a vaccine may comprise the shortest Replikin peptides from a hemagglutinin protein area identified in an influenza virus isolate or a plurality of influenza virus isolates predicted to have a greater infectivity than at least one other isolate of influenza virus and may comprise the shortest Replikin peptides from a pBl gene area identified in an influenza virus isolate or a plurality of influenza virus isolates predicted to have a greater lethality than at least one other isolate of influenza virus.
  • a vaccine may further comprise a plurality of the longest Replikin peptides from a hemagglutinin protein area and a plurality of the longest Replikin peptides from a pBl area.
  • a vaccine may comprise the longest Replikin peptides from a hemagglutinin protein area identified in an influenza virus isolate or a plurality of influenza virus isolates predicted to have a greater infectivity than at least one other isolate of influenza virus and may comprise the longest Replikin peptides from a pBl gene area identified in an influenza virus isolate or a plurality of influenza virus isolates predicted to have a greater lethality than at least one other isolate of influenza virus.
  • a vaccine may also comprise a mixture of the shortest and longest Replikin peptides in the hemagglutinin protein area and/or pBl gene area.
  • a vaccine may be directed against any influenza virus including, influenza A, influenza B, or influenza C.
  • a vaccine may be directed against HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A virus. Any of these vaccines may be synthesized in seven days or less, which allows for administration of vaccines that are a best fit for a particular virulent strain of virus.
  • a vaccine may be formulated with a pharmaceutically acceptable excipient, carrier, or adjuvant.
  • One pharmaceutically acceptable carrier or excipient is water.
  • Excipients, carriers, or adjuvants may include, but are not limited to, excipients, carriers and adjuvants known to those of skill in the art now or hereafter.
  • compositions of the invention may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal or any other routes.
  • parenteral e.g., intravenous
  • intradermal subcutaneous, oral, nasal, bronchial
  • ophthalmic transdermal (topical)
  • transmucosal any other routes.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • Solutions or suspensions used for intranasal, intraocular, spray inhalation, parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water (for dermal, nasal, or ocular application, spraying, or injection), saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water (for dermal, nasal, or ocular application, spraying, or
  • compositions suitable for injectable use typically include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition should be sterile and should be fluid to the extent that easy syringability exists.
  • Preferred pharmaceutical formulations are stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • Administration of the vaccine via any method may produce an immune response in the animal or human, it may further produce an antibody response in the animal or human. In a further non-limiting embodiment, the vaccine may produce a protective effect in the animal or human.
  • the vaccine of the present invention may be administered to a rabbit, a chicken, a shrimp, a pig, a ferret, a human, or any animal capable of an immune resposne. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a range of strains of influenza.
  • the vaccines of the present invention can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • antiviral drugs such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • the dosage of peptides is in the range of from about 0.01 ⁇ g to about 500 mg, from about 0.05 ⁇ g to about 200 mg, about 0.075 ⁇ g to about 30 mg, about 0.09 ⁇ g to about 20 mg, about 0.1 ⁇ g to about 10 mg, about 10 ⁇ g to about 1 mg, and about 50 ⁇ g to about 500 ⁇ g.
  • the skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
  • compositions Comprising Any of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66
  • a non-limiting composition comprising one or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a composition is likewise provided comprising one or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12 or SEQ ID NO(s): 21-28.
  • a composition comprising one or more isolated or synthesized peptides consisting of at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or at least one peptide of SEQ ID NO(s): 1-12 or at least one peptide of SEQ ID NO(s): 21-28.
  • a composition is further provided comprising two, three, four five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a composition comprising a mixture of peptides is provided wherein the mixture comprises at least each of the isolated or synthesized peptides of SEQ ID NO(s): 1-12 and/or at least each of the isolated or synthesized peptides of SEQ ID NO(s): 21-28.
  • a mixture is provided that is equimolar.
  • a mixture is also provided that is equal by weight.
  • Such a composition may comprise about 10% by weight the peptide of SEQ ID NO: 1, it may comprise about 9% by weight the peptide of SEQ ID NO: 2, it may comprise about 10% by weight the peptide of SEQ ID NO: 3, it may comprise about 6% by weight the peptide of SEQ ID NO: 4, it may comprise about 8% by weight the peptide of SEQ ID NO: 5, it may comprise about 8% by weight the peptide of SEQ ID NO: 6, it may comprise about 7% by weight the peptide of SEQ ID NO: 7, it may comprise about 6% by weight the peptide of SEQ ID NO: 8, it may comprise about 10% by weight the peptide of SEQ ID NO: 9, it may comprises about 8% by weight the peptide of SEQ ID NO: 10, it may comprise about 7% by weight the peptide of SEQ ID NO: 11, and/or it may comprise about 11% by weight the peptide of SEQ ID NO: 12.
  • the composition may further comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more isolated or synthesized peptides of SEQ ID NO(s): 1-12 or two, three, four, five, six, seven, eight, or more isolated or synthesized peptides of SEQ ID NO(s): 21- 28.
  • the composition may also comprise any number of peptides of SEQ ID NO(s): 13-20.
  • composition may also comprise one or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of peptides SEQ ID NO(s): 1-12, SEQ ID NO(s) 13-20, and/or SEQ ID NO(s): 21-28.
  • the composition may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of peptides SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28.
  • the composition may further comprise a mixture of peptides comprising isolated or synthesized peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28.
  • Identification of conserved Replikin peptides across strains of influenza virus has provided for the development of vaccines that may be directed across strains of influenza virus. Identification of conserved Replikin peptides in isolates of influenza of any strain may be accomplished in any way known to one of skill in the art now or hereafter. One method is by review of in silico sequences provided at www.pubmed.com. Peptides that share exact identity or 100% homology with earlier identified Replikin peptides may be tracked using computer searching methods. Peptides that share 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homology with an earlier identified Replikin peptide may also be tracked by computer methods.
  • a vaccine has now been developed for prevention and treatment of infection of H5N1 virus. See, e.g., Example 1 below.
  • the sequences that are used in the vaccine in Example 1 have now been identified as conserved across many different strains.
  • SEQ ID NO: 8 (HFQRKRRVRDNMTKK), which was originally identified in the pBl gene area of H5N1, shares homology with SEQ ID NO: 13 (HFQRKRRVRDNVTK), which has been identified as conserved in the pBl-F2 gene area of HlNl.
  • SEQ ID NO: 8 is homologous with SEQ ID NO: 13 in that the valine at position 12 in SEQ ID NO: 13 is substituted with a methionine in SEQ ID NO: 8.
  • SEQ ID NO: 8 also has one additional lysine on its C-terminus.
  • a vaccine comprising SEQ ID NO: 8 or SEQ ID NO: 13 may be used against either HlNl or H5N1 or any other strain expressing a homologue of these sequences. If such a homologue is expressed in the pBl gene area or the pBl-F2 gene area of a strain, the vaccine will be particularly useful against such a strain.
  • a vaccine containing SEQ ID NO(s): 1-12 is available as a vaccine against HlNl strains as well as H5N1 strains of influenza virus since such a vaccine comprises the peptide of SEQ ID NO: 8.
  • Sequences that are homologues of SEQ ID NO(s): 1-12 are appropriate sequences for inclusion in a vaccine directed against influenza virus including, HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A virus and against any strain of influenza B or influenza C virus.
  • sequences that are homologues of SEQ ID NO(s): 13-20 are appropriate sequences for inclusion in a vaccine directed against influenza virus including, HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A virus and against any strain of influenza B or influenza C virus.
  • Sequences that are homologues of SEQ ID NO(s): 21- 28 are also appropriate sequences for inclusion in a vaccine directed against influenza virus including, HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A virus and against any strain of influenza B or influenza C virus.
  • homologues are expected by one of ordinary skill in the art to provide antigenicity that is comparable to any one of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13- 20, SEQ ID NO(s): 21-28. Further, because these homologues are often conserved in the hemagglutinin and pBl gene areas of different strains of influenza virus, these homologues are useful for developing antagonists against influenza infections, including for vaccinating a subject with the homologous peptides to stimulate the immune system of the subject against the peptides and in-turn against influenza virus proteins harboring such peptides or other homologues of such peptides.
  • Homology that is sufficient to produce a useful target for antagonism includes peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100% homologous with any of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. Homology may be determined with peptides wherein gaps exists in the sequence that is being compared to any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 between amino acids that are identical to those of the peptide chosen from SEQ ID NO(s): 1-12.
  • SEQ ID NO: 1 HAQDILEKEHNGKLCSLKGVRPLILK
  • SEQ ID NO: 29 SEQ ID NO: 29
  • SEQ ID NO: 30 SEQ ID NO: 30
  • SEQ ID NO(s) 29 and 30 are 86% homologous with SEQ ID NO: 1, SEQ ID NO(s): 29 and 30 are available as peptides for inclusion in a vaccine directed against infectivity in H5N1 or in any influenza virus strain wherein homologues to SEQ ID NO: 1 are conserved.
  • gaps the number of gaps in either the basis sequence or the identified sequence should be limited to the number of gaps allowable without significantly compromising the function of the identified sequence as compared to the basis sequence.
  • many gaps in the sequence of the basis peptide or in the sequence of the identified peptide are allowed based on homology as defined herein. Relatively more gaps are allowed if the lysines and histidines that create the definition of the Replikin peptide are identically shared between the basis peptide and the identified peptide.
  • lysines and histidines that create the definition of the Replikin peptide are shared at least in close position (for example within ten, nine, eight, seven, six, five, four, three, two, or one amino acid residue). If some of the lysines and histidines that create the definition of the Replikin peptide are not present in the identified peptide, fewer gaps may be allowed. Nevertheless, if the identified peptide functions similarly to the basis peptide, any number of gaps are allowed.
  • three or more gaps are allowed in the sequence of the basis peptide or in the sequence of the identified peptide within ten amino acid residues of the basis peptide if no lysines or histidines are present in the identified peptide. Two or more gaps or one or more gaps are also allowed. Nevertheless, if the identified sequence provides the same or a similar function to the basis sequence, more gaps are allowed up to the number of gaps that will provide a homology of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more homology. Additionally, where the lysines and histidines of the Replikin definition are present in both the identified peptide and the basis peptide, there should be no limit on how many gaps are allowed.
  • One aspect of the invention therefore, provides conserved sequences in the hemagglutinin protein area and conserved sequences in the pB 1 gene area for diagnostic, predictive, and therapeutic purposes, including vaccines that provide cross-strain influenza protection.
  • Replikin sequences that are shared across strains and Replikin sequence homologues that are shared across strains provide targets for diagnostic, predictive, and therapeutic purposes.
  • Replikin sequences are associated by the applicants with mechanisms of rapid replication and because Replikin sequences in the hemagglutinin protein area and the pBl gene area are associated by the applicants with infectivity and lethality, respectively.
  • Replikin sequences that are shared across influenza strains or homologues of Replikin sequences that are shared across influenza strains provide excellent targets for diagnostics and therapeutics directed at these shared sequences. Identifying these targets provides for therapies such as a vaccine or a binding agent (e.g., an antibody or antibody fragment) that may be directed at Replikin sequences or their homologues in an array of influenza types and strains.
  • Replikin sequences have been identified by the applicants that are shared among, for example, HlNl, H5N1, H3N2, and H9N2. Such conserved sequences provide targets for vaccines that provide cross-strain protection in these strains of influenza A and provide cross- strain protection in strains that share the sequences or homologues of the sequences. For example, the applicants have identified five Replikin sequences in the HlNl pBl-F2 gene area that are conserved within HlNl and are shared with H5N1 or H3N2.
  • SEQ ID NO: 13 One such sequence is HFQRKRRVRDNVTK (SEQ ID NO: 13).
  • SEQ ID NO: 13 has been observed to be conserved at position 184 in the pBl-F2 gene area in isolates of HlNl since at least 1948 and shares homology with SEQ ID NO: 8 (HFQRKRRVRDNMTKK), which was originally identified by the applicants in the pBl gene area of H5N1 and has been observed to be conserved from 2000 through 2009 in isolates of H5N1 virus. See, e.g., Accession No. AAF74314.
  • SEQ ID NO: 8 is homologous with SEQ ID NO: 13 in that the valine at position 12 in SEQ ID NO: 13 is substituted with a methionine in SEQ ID NO: 8.
  • SEQ ID NO: 8 also has one additional lysine on its C-terminus.
  • a vaccine comprising SEQ ID NO: 8 or SEQ ID NO: 13 may be used against either HlNl or H5N1 or any other strain expressing a homologue of these sequences. If such a homologue is expressed in the pBl gene area or the pBl-F2 gene area of a strain, the vaccine will be particularly useful against such a strain.
  • a vaccine containing SEQ ID NO(s): 1-12 is available as a vaccine against HlNl strains as well as H5N1 strains of influenza virus since such a vaccine comprises the peptide of SEQ ID NO: 8.
  • a vaccine comprising SEQ ID NO(s): 1-12 provides an example of a vaccine to be used as a cross-strain vaccine.
  • SEQ ID NO: 14 is further conserved in HlNl with a substitution of the cysteine residue at position 2 by a tyrosine residue.
  • the resulting sequence is HYQKTMNQ VVMPK (SEQ ID NO: 15), which has been observed as conserved at position 41 in the pBl-F2 gene area of HlNl isolates of HlNl from at least 1951 through 1983.
  • SEQ ID NO: 15 has also been observed as conserved in H5N1.
  • a vaccine comprising a peptide of SEQ ID NO(s): 14 or 15 or homologues of one of those sequences is available as a vaccine against HlNl, H5N1, and H3N2 strains of influenza virus or any strain expressing a homologue of the peptides.
  • SEQ ID NO: 16 The sequence KRWRLFSKH (SEQ ID NO: 16) has been observed to be conserved at position 78 of the pBl-F2 gene area of HlNl in isolates from 1918 through 2008. SEQ ID NO: 16 is also conserved at position 78 of the pBl-F2 gene area of H3N2 in at least 1968 and 2008. See, e.g. Accession Nos. ABI92289, ACK99430, ACI26481, ACI26437, ACI26294.
  • a vaccine comprising SEQ ID NO: 16 is, therefore, available against both HlNl and H3N2 or any other influenza strain expressing one or more homologues of SEQ ID NO: 16.
  • sequence KKKHKLDK (SEQ ID NO: 17) is also conserved at position 207 of the pBl-F2 gene area of isolates of HlNl in isolates from at least 1991 through 2009. SEQ ID NO: 17 is also conserved in H5N1.
  • a homologue of SEQ ID NO: 17, namely, sequence KKKQRLTKX n H (SEQ ID NO: 18) (where n any amino acid from 1 to 41 residues), is conserved in the pBl gene area of isolates of HlNl and H5N1 at position 207.
  • a vaccine comprising SEQ ID NO(s): 17 or 18 is available against HlNl and H5N1 or any influenza strain expressing homologues of these sequences.
  • sequence HFQRKRRVRDNMTK (SEQ ID NO: 19) is also conserved at position 184 in the pBl gene area of H5N1 in isolates from at least 2000 through 2009. See, e.g., Accession No. AAF74314.
  • SEQ ID NO: 19 is a 93% homologue with SEQ ID NO: 8 with only one additional lysine on the c-terminus.
  • SEQ ID NO: 19 is also a homologue of SEQ ID NO: 13 with about 93% homology.
  • SEQ ID NO: 19 is conserved in HlNl at position 184 as well as in H9N2.
  • HFQRKRRVRDNMTKKMVTQRTIGKKKQRLNK (SEQ ID NO: 20), which is conserved at position 184 in the pBl gene area of H5N1 isolates from at least 2000 through 2005. See Accession No. AAF74314.
  • SEQ ID NO: 20 is 48% homologous with SEQ ID NO: 8 and 45% homologous with SEQ ID NO: 19. All of these sequences share homology with SEQ ID NO: 13, which has been observed to be conserved at position 184 in the pBl-F2 gene area in isolates of HlNl since at least 1948.
  • a vaccine comprising SEQ ID NO(s): 8, 13, 19, or 20, or any combination thereof, is available against HlNl, H5N1, H9N2 or any influenza strain expressing homologues of these sequences.
  • the invention also provides methods of designing and making vaccines.
  • the invention provides a method of making a vaccine comprising selecting at least one or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28 as a component of a vaccine and making said vaccine.
  • the method may comprise selecting from 1 to up to 12 or more isolated or synthesized peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28 as a component of a vaccine.
  • the method may comprise identifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virus up to about 3 years before the vaccine is made.
  • the method may comprise identifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virus up to about 1 year before the vaccine is made.
  • the method may comprise identifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virus up to about 6 months before the vaccine is made.
  • the method may comprise identifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virus up to about 7 days before the vaccine is made.
  • An emerging strain may be any strain of influenza virus identified by one of skill in the art as a strain of influenza virus that is predicted to expand in a population in hosts or that is predicted to increase in virulence, morbidity, and or mortality in its hosts.
  • An emerging strain may likewise be a strain of influenza virus wherein Replikin concentration is observed to be increasing over time.
  • An emerging strain may likewise be a strain of influenza virus identified within a rising portion of Replikin cycle, following a peak in a Replikin cycle, following a stepwise rise in a Replikin cycle, or identified by a Replikin Count Virus Expansion Index as an emerging strain of virus. See U.S. Appln. Ser. No. 12/429,044, filed April 23, 2009, which is incorporated herein by reference.
  • a method of making a vaccine comprising: selecting at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 as a component of a vaccine; and making said vaccine.
  • An isolated or synthesized protein, protein fragment, polypeptide, or peptide may comprise a peptide that is 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • At least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 may be selected. Also, at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 may be selected.
  • the isolated or synthesized protein, protein fragment, polypeptide, or peptide may have the same amino acid sequence as at least one protein, protein fragment, polypeptide or peptide identified in an emerging strain of influenza virus up to one, two, or three or more years prior to making said vaccine.
  • the at least one protein, protein fragment, polypeptide or peptide may be identified in an emerging strain of influenza virus one week, one month, two months, three months, four months, five months, or six months prior to making said vaccine.
  • a method of making a vaccine comprising: selecting as a component of the vaccine at least one protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a hemagglutinin protein area (or a synthesized version thereof), and selecting as a component of the vaccine at least one protein fragment, polypeptide, or peptide comprising at least one peptide B, where peptide B is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a pBl gene area (or a synthesized version thereof); and making a vaccine comprising the components.
  • a method of making a vaccine comprising: identifying (1) at least one protein, protein fragment, polypeptide, or peptide of a hemagglutinin protein area in or derived from an isolate of influenza virus having relatively greater infectivity than another isolate of influenza virus or a plurality of isolates of influenza viruses, and (2) at least one protein, protein fragment, polypeptide, or peptide of a pBl gene area in or derived from an isolate of influenza virus having relatively greater lethality than another isolate of influenza virus or a plurality of isolates of influenza virus; and making a vaccine comprising the at least one protein, protein fragment, polypeptide, or peptide of a hemagglutinin protein area and the at least one protein, protein fragment, polypeptide, or peptide of a pBl gene area.
  • the invention also provides a kit for making a vaccine where the kit includes at least one isolated or synthesized peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the kit may also include two, three, four, and up to twelve or more peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the new vaccine technology has been tested and demonstrated to work in independent trials against influenza H5N1 virus in chickens, and against lethal Taura Syndrome virus in shrimp. Both TransFluTM (the first synthetic cross-strain Pan Flu vaccine) and Taura Syndrome Virus vaccines have been manufactured in 7 days. Kilogram amounts of these vaccines may be manufactured in a few weeks, rather than 6 to 12 months by biological methods. The cost is far less than the cost of vaccines by current biological methods.
  • the invention further provides preventing or treating influenza in a human or animal by methods comprising administering at least one isolated or synthesized peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 to the animal or human.
  • the at least one isolated or synthesized peptide is administered intravenously, intramuscularly, orally, intranasally, intraocularly, via spray inhalation, or by any method of administration known to one of ordinary skill in the art now or hereafter.
  • the vaccine may be administered intranasally, intraocularly, or via spray inhalation.
  • the vaccine may be administered to a human, a bird, a horse, a ferret, or a pig.
  • the bird may be a domestic bird or a wild bird and may include a chicken, a duck, a goose, or any other domestic or wild bird.
  • the vaccine may be administered to a chicken including to a chicken at 7, 14, and 21 days after hatch.
  • a non-limiting vaccine of the invention is provided for, among other things, treatment or prevention of all strains of influenza virus.
  • a non- limiting vaccine of the invention may contain sequences that are conserved in strains of Low-Path H5N1, strains of High-Path H5N1, and across other strains of influenza virus including HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza A virus, influenza B virus, or influenza C virus.
  • SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 of the invention have been observed to be conserved across many strains of influenza with particular conservation noted in the lysine and histidine residues of the sequences.
  • the lysine and histidine residues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 are the key amino acid residues that provide the Replikin structure of the sequences.
  • SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 have likewise been observed to be conserved in both High-Path and Low-Path H5N1 and are useful in both treatment and prevention for outbreaks of these strains of influenza as well as all other strains of influenza.
  • One non-limiting aspect of the present invention provides methods of differentiating the infectivity of an influenza virus isolate or strain of influenza virus from the lethality of the influenza virus isolate or strain of influenza virus.
  • Compounds for diagnostic, therapeutic, and/or preventive purposes in influenza and therapies for the prevention and treatment of influenza are provided based on the disclosed methods of differentiation.
  • a method of differentiating the relative infectivity of isolate A of influenza virus from the relative infectivity of isolate B of influenza virus and the relative lethality of isolate A of influenza virus from the relative lethality of isolate B of influenza virus comprising: comparing the Replikin Count of the hemagglutinin protein area of isolate A to the Replikin Count of the hemagglutinin protein area of isolate B; and comparing the Replikin Count of the pBl gene area of isolate A to the Replikin Count of the pBl gene area of isolate B.
  • the relative infectivity of isolate A may be greater than, less than, or about the same as the relative infectivity of isolate B if the Replikin Count of the hemagglutinin protein area of isolate A is greater than, less than, or about the same as the Replikin Count of the hemagglutinin protein area of isolate B, respectively, and the relative lethality of isolate A may be greater than, less than, or about the same as the relative lethality of isolate B if the Replikin Count of the pBl gene area of isolate A is greater than, less than, or about the same as the Replikin Count of the pBl gene area of isolate B, respectively.
  • the relative infectivity of isolate A may also be greater than, less than, or about the same as the relative infectivity of isolate B and the relative lethality of isolate A may not be concomitantly greater than, less than, or about the same as the relative lethality of isolate B.
  • the relative infectivity of isolate A may likewise be greater than the relative infectivity of isolate B and the relative lethality of isolate A may be less than or about the same as the relative lethality of isolate B.
  • the relative infectivity of isolate A may also be less than the relative infectivity of isolate B and the relative lethality of isolate A may be greater than or about the same as the relative lethality of isolate B.
  • the relative lethality of isolate A may also be greater than, less than, or about the same as the relative lethality of isolate B and the relative infectivity of isolate A may be not concomitantly greater than, less than, or about the same as the relative infectivity of isolate B.
  • a method of differentiating the relative infectivity and relative lethality of a plurality of isolates A of influenza from a given region or time period from the relative infectivity and relative lethality of an isolate B from a different region or different time period or from the relative infectivity and relative lethality of a plurality of isolates B from a different region or different time period comprising: comparing the mean Replikin Count of the hemagglutinin protein area and the mean Replikin Count of the pBl gene area of the plurality of isolates A to the Replikin Count of the hemagglutinin protein area of isolate B or the mean Replikin Count of the hemagglutinin protein area of the plurality of isolates B, and to the Replikin Count of the pBl gene area of isolate B or to the mean Replikin Count of the pBl gene area of the plurality of isolates B.
  • a plurality of isolates A of influenza from a given region or time period may have a relative infectivity that is greater than, less than, or about the same as the relative infectivity of isolate B or the plurality of isolates B if the mean Replikin Count of the hemagglutinin protein area of the plurality of isolates A is greater than, less than, or about the same as the Replikin Count of the hemagglutinin protein area of isolate B or is greater than, less than, or about the same as the mean Replikin Count of the hemagglutinin protein area of the plurality of isolates B, and the relative lethality of the plurality of isolates A is greater than, less than, or about the same as the relative lethality of isolate B or the plurality of isolates B if the mean Replikin Count of the pBl gene area of the plurality of isolates A is greater than, less than, or about the same as the Replikin Count of the pBl gene area of isolate B or is greater than
  • the relative infectivity of the plurality of isolates A may also be greater than, less than, or about the same as the relative infectivity of isolate B or the relative infectivity of the plurality of isolates B, and the relative lethality of the plurality of isolates A may be not concomitantly greater than, less than, or about the same as the relative lethality of isolate B or the relative lethality of the plurality of isolates B.
  • the relative infectivity of the plurality of isolates A may also be greater than the relative infectivity of the plurality of isolates B and the relative lethality of isolate A may be less than or about the same as the relative lethality of isolate B or the relative lethality of the plurality of isolates B.
  • the relative infectivity of the plurality A of isolates may also be less than the relative infectivity of isolate B or the relative infectivity of the plurality of isolates B, and the relative lethality of plurality of isolates A may be greater than or about the same as the relative lethality of isolate B or the relative lethality of the plurality of isolates B.
  • the relative lethality of the plurality of isolates A may also be greater than, less than, or about the same as the relative lethality of isolate B or the relative lethality of the plurality of isolates B and the relative infectivity of the plurality of isolates A may be not concomitantly greater than, less than, or about the same as the relative infectivity of isolate B or the relative infectivity of the plurality of isolates B.
  • a method of differentiating the future relative infectivity of at least one strain A of influenza virus as compared to a time To from the future relative lethality of said at least one strain A of influenza virus as compared to time T 0 comprising: comparing a trend of Replikin Counts in the hemagglutinin protein area of a plurality of isolates of strain A ending at time To, wherein said isolates are isolated at different time periods including time To, to a trend of Replikin Counts in the pBl gene area of a plurality of isolates of strain A ending at time T 0 , wherein said isolates are isolated at different time periods including time T 0 .
  • the future relative infectivity of strain A may be predicted to be greater than, less than, or about the same as the relative infectivity of strain A at time To if the trend of Replikin Counts in the hemagglutinin protein area of said plurality of isolates of strain A is rising, falling, or remaining about the same and the future relative lethality of strain A may be predicted to be greater than, less than, or about the same as the relative lethality of strain A at time To if the trend of Replikin Counts in the pBl gene area of said plurality of isolates of strain A is rising, falling, or remaining about the same.
  • the future relative infectivity of strain A may also be predicted to be greater than, less than, or about the same as the relative infectivity of strain A at time T 0 and the future relative lethality of strain A may be not concomitantly greater than, less than, or about the same as the relative lethality of strain A at time To.
  • the future relative infectivity of strain A may also be predicted to be greater than the relative infectivity of strain A at time To and the relative lethality of strain A may be predicted to be less than or about the same as the relative lethality of strain A at time To.
  • a vaccine comprising at least one Replikin amino acid sequence from the hemagglutinin protein area of an isolate of influenza virus and at least one Replikin amino acid sequence from the pBl gene area of an isolate of influenza virus.
  • the at least one Replikin amino acid sequence from the hemagglutinin protein area may be from an isolate of influenza virus predicted to have a greater infectivity than at least one other isolate of influenza virus and the at least one Replikin amino acid sequence from the pB 1 gene area may be from an isolate of influenza virus predicted to have a greater lethality than at least one other isolate of influenza virus.
  • a vaccine may also comprise at least one Replikin amino acid sequence from the hemagglutinin protein area and at least one Replikin amino acid sequence from the pBl gene area isolated from (or a synthesized version of) an isolate of influenza predicted to have a greater infectivity and a greater lethality than at least one other isolate of influenza.
  • a vaccine may also comprise a plurality of Replikin peptides from the hemagglutinin protein area and a plurality of peptides from the pBl gene area.
  • a computer readable storage medium having stored thereon instructions which, when executed, cause a processor to perform a method of differentiating the relative infectivity of an influenza virus from the relative lethality of an influenza virus.
  • a processor may report the differentiation of the relative infectivity of the influenza virus from the relative lethality of the influenza virus to a display, user, researcher, or other machine or person. The reported differentiation may report whether the relative infectivity of the virus is greater than, less than, or about equal to the relative infectivity of another virus and whether the relative lethality of the virus is greater than, less than, or about equal to the relative lethality of another virus.
  • a method of preventing or treating influenza virus infection across different strains comprising administering at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising a homologue of SEQ ID NO(s): 1-12, 13-20, 21- 28, and 32-66 to an animal or human.
  • the isolated or synthesized protein, protein fragment, polypeptide, or peptide may comprise a peptide that is 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a method may comprise administering at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more peptide(s) that is (are) 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or may comprise administering at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more peptide(s) that consist(s) of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a method may further comprise administering at least one isolated or synthesized protein fragment, polypeptide, or peptide that consists of at least one peptide A, which is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a method of preventing or treating influenza virus infection across strains may also comprise administering at least one agent that is capable of antagonizing a protein comprising a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 wherein said agent is capable of binding at least a portion of said homologue that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • isolated Replikin peptides may be used to generate antibodies, antibody fragments, or to generate or identify other binding agents, which may be used, for example for diagnostic purposes or to provide passive immunity in an individual. See, e.g., U.S. Appln. Ser. No. 11/355,120, filed February 16, 2006 and U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008 (each incorporated herein by reference in their entirety).
  • Various procedures known in the art may be used for the production of antibodies to Replikin sequences or to proteins, protein fragments, polypeptides, or peptides comprising Replikin sequences.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Antibodies that are linked to a cytotoxic agent may also be generated.
  • Antibodies may also be administered in combination with an antiviral agent.
  • combinations of antibodies to different Replikins may be administered as an antibody cocktail.
  • a Replikin peptide or a combination of Replikin peptides including, but not limited to, rabbits, mice, rats, and larger mammals.
  • Monoclonal antibodies to Replikins may be prepared using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and
  • Antibody fragments that contain binding sites for a Replikin may be generated by known techniques.
  • fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be generated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Binding agents are provided including an antibody, antibody fragment, or binding agent that binds to at least a portion of an amino acid sequence of at least one protein, protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the amino acid sequence of a protein fragment, polypeptide, or peptide may partially match the amino acid sequence of an expressed whole protein where at least one, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred or more amino acid residues of the amino acid sequence of the expressed whole protein are not present in the protein fragment, polypeptide, or peptide.
  • the amino acid sequence of the protein fragment, polypeptide, or peptide may also partially match the amino acid sequence of an expressed whole protein where at least one, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of the amino acid sequence of at least one terminus of the expressed whole protein are not present at at least one terminus of said protein fragment, polypeptide, or peptide.
  • Binding agents are also provided including an antibody, antibody fragment, or binding agent that binds to at least a portion of an amino acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the length of peptide A may be no more than one, five, ten, twenty, thirty, forty, or fifty amino acid residues longer than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 with which it is homologous.
  • Binding agents are also provided that bind to at least a portion of an amino acid sequence of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • Binding agents may specifically bind to the target protein, protein fragment, polypeptide, or peptide. Binding agents may specifically bind to a homologue of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. Binding agents may likewise specifically bind to a peptide consisting of any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • Binding agents may also specifically bind to a portion of a peptide consisting of any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 including a single amino acid within a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66, two amino acids, three amino acids, four amino acids, five amino acids, or any number of amino acids spread within or outside a homologue.
  • An isolated or synthesized nucleic acid sequence is also provided that encodes a protein, protein fragment, polypeptide, or peptide comprising at least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • a nucleic acid sequence may also encode a protein, a protein fragment, a polypeptide, or a peptide where the amino acid sequence of the protein, protein fragment, polypeptide, or peptide partially matches the amino acid sequence of an expressed whole protein and at least one, two, three, four, five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, three hundred, four hundred, five hundred or more amino acid residues of the amino acid sequence of the expressed whole protein are not present in the protein fragment, polypeptide, or peptide.
  • amino acid sequence of the protein, protein fragment, polypeptide, or peptide may partially match the amino acid sequence of an expressed whole protein where at least one, two, three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred fifty, two hundred, two hundred fifty, three hundred, three hundred fifty, four hundred, four hundred fifty, five hundred, five hundred fifty or more amino acid residues of the amino acid sequence of at least one terminus of the expressed whole protein may not be present at at least one terminus of the protein, protein fragment, polypeptide, or peptide
  • An isolated or synthesized nucleic acid sequence may also encode a peptide consisting of 7 to about 50 amino acid residues comprising at least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. It may also encode a peptide that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with at least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. It may also encode a peptide consisting of at least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the invention further provides an immunogenic composition that comprises an isolated or synthesized nucleic acid provided above.
  • the invention further provides a vaccine against influenza comprising an isolated or synthesized nucleic acid provided above.
  • the invention further provides a nucleic acid sequence that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or a small interfering nucleic acid sequence that interferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the nucleotide sequence of the invention may be used in hybridization assays of biopsied tissue or blood, e.g., Southern or Northern analysis, including in situ hybridization assays, to diagnose the presence of a particular influenza strain in a tissue sample or an environmental sample, for example.
  • the present invention also provides kits containing antibodies specific for particular Replikins that are present in a particular pathogen of interest, or containing nucleic acid molecules (sense or antisense) that hybridize specifically to a particular Replikin, and optionally, various buffers and/or reagents needed for diagnosis.
  • oligoribonucleotide sequences that include antisense RNA and DNA molecules and ribozymes that function to inhibit the translation of Replikin-containing mRNA.
  • Both antisense RNA and DNA molecules and ribozymes may be prepared by any method known in the art.
  • the antisense molecules can be incorporated into a wide variety of vectors for delivery to a subject. The skilled practitioner can readily determine the best route of delivery, although generally intravenous or intramuscular delivery is routine. The dosage amount is also readily ascertainable.
  • the invention further provides antisense nucleic acid molecules that are complementary to a nucleic acid of the invention, wherein the antisense nucleic acid molecule is complementary to a nucleotide sequence encoding a peptide of the invention.
  • the nucleic acid sequence may be anti-sense to a nucleic acid sequence that has been demonstrated to be conserved over a period of six months to one or more years and/or which are present in a strain of influenza virus shown to have an increase in concentration of Replikins relative to Replikin concentration in other influenza virus strains.
  • the invention also provides compositions comprising RNAi-inducing entities used to inhibit or reduce influenza virus infection or replication including small interfering RNA, which is a class of about 10 to about 50 and often about 20 to about 25 nucleotide-long double- stranded RNA molecules.
  • siRNA is involved in the RNA interference pathway, where it interferes with the expression of a specific genes such as the hemagglutinin gene or the pBl gene area of influenza. siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism.
  • An effective amount of an RNAi-inducing entity is delivered to a cell or organism prior to, simultaneously with, or after exposure to influenza virus.
  • compositions of the invention may comprise a single siRNA species targeted to a target transcript or may comprise a plurality of different siRNA species targeting one or more target transcripts.
  • the invention provides a small interfering nucleic acid sequence that is about 10 to about 50 nucleic acids in length and is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes for any portion of SEQ ID NO(s): 1-12, 13-20, 21- 28, and 32-66 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any portion of one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.
  • the nucleic acid sequence is about 15 to about 30 nucleic acids. In a further non-limiting embodiment, the nucleic acid sequence is about 20 to about 25 nucleic acids. In a further non-limiting embodiment, the nucleic acid sequence is about 21 nucleic acids.
  • Taura syndrome virus is an often lethal rapidly- replicating pathogen that has a significant negative impact on the shrimp industry.
  • Synthetic Replikin vaccines have also been demonstrated in the H5N1 strain of influenza virus in chickens.
  • a protective effect was observed at both the entry site of influenza (diminished antibody production in the serum was observed as compared to a control) and at excretion sites of influenza (influenza virus was not observed excreted in feces or saliva from treated chickens as compared to a control). See Example 1 below.
  • Example 1 For example, as may be seen in Example 1 below, three of six vaccinated chickens, when inoculated with H5N1 virus, produced no measurable amount of antibodies against H5N1 in their serum. Instead, the virus was apparently blocked by mucosal immunity from even entering the chickens' system. For those three chickens in which a serum immune response was measured (that is, virus did enter their system), the vaccine additionally provided a protective effect against replication of the virus in the chickens' system (no virus was excreted in the feces or saliva of the chickens). As such, mucosal immunity, in addition to other immunities, is an important aspect of the immunity imparted by Replikin-based vaccines.
  • One aspect of the present invention provides a method of differentiating the infectivity of a strain of virus from the lethality of the strain of virus.
  • This double differentiation of infectivity and lethality provides advance warning of the future course of strains of influenza virus.
  • double differentiation of the infectivity and lethality in the HlNl virus from 2004 through 2009 and the H5N1 virus from 2004 through 2008 provides advance warning of the future course of HlNl and H5N1 (see Figures 2-5) and provides for the production of synthetic influenza Replikin vaccines.
  • Synthetic influenza Replikin vaccines include vaccines such as the H5N1 vaccine described in Example 1 herein.
  • Synthetic influenza Replikin vaccines include vaccines that comprise at least one protein, protein fragment, or peptide comprising, consisting of, homologous with, or derived from a Replikin peptide identified in a hemagglutinin protein area (the hemagglutinin protein area may be isolated from an isolate of influenza virus identified as associated with higher infectivity than another isolate of influenza virus) and/or at least one protein, protein fragment, or peptide comprising, consisting of, homologous with, or derived from a Replikin peptide identified in the pBl gene area of an isolate of influenza virus (the pB 1 gene area may be associated with higher lethality than another isolate of influenza virus).
  • influenza virus genes By isolating separate influenza virus genes in silico that differentiate infectivity from lethality, the applicants have now provided a method of differentiating the infectivity and lethality of influenza viruses.
  • the hemagglutinin protein area in influenza virus is now associated with infectivity.
  • high Replikin Counts are associated with outbreaks of various strains of influenza A virus ⁇ e.g., HlNl, H2N2, H3N2, H5N1, etc.) in the 20 th century.
  • the pBl gene area of influenza virus is likewise now associated with lethality.
  • infectivity was observed to change over time in a manner different from that observed in lethality.
  • infectivity was observed to increase in HlNl from 2004 through 2009 while lethality was observed to remain steady or decrease. See Figures 3-5.
  • infectivity was observed to remain about the same in H5N1 from 2004 through 2008 while lethality was observed to increase. See Figure 2.
  • the data in Figure 5 is derived from the most recent sequence data available on PubMed. A review of the data in Figure 5 reveals that HlNl infectivity is predicted to remain high in humans in the immediate future and HlNl lethality is predicted to remain low in humans for the immediate future.
  • the initial increase in HlNl lethality in 2005 may be related to an initial high lethality observed in the first cases in the outbreak of HlNl in or near Mexico in the spring of 2009. This increase in Replikin Count was not sustained, however, as may be seen from the Replikin Count data from 2006 through 2009. In agreement with this data, the mortality rate of subsequent cases has been observed to decline.
  • One vaccine was initially engineered from sequences identified in the Low-Path H5N1 isolated from the black duck in North Carolina, USA and confirmed to be conserved in both Low-Path and High-Path H5N1 strains as well as across influenza strains with conservation particularly noted in the key amino acid residues of the Replikin sequence, namely, lysine and histidine amino acid residues.
  • the vaccine was designed to deliver an approximately equal- parts-by- weight mixture of twelve Replikin peptides to the immune system of an animal or human. Six of the peptides were isolated in silico from the hemagglutinin protein area and six of the peptides were isolated in silico from the pBl gene area.
  • a vaccine has now successfully protected chickens from low-pathogenic H5N1 infection and has successfully blocked excretion of low- pathogenic H5N1 virus from infected chickens.
  • the vaccine was developed from influenza Replikin peptides shared between influenza strains and conserved for decades within influenza strains and was engineered as a mixture of twelve Replikin peptides identified as expressed from the genome of H5N1 virus.
  • Six of the Replikin peptides were synthesized according to sequences isolated from the hemagglutinin protein area of H5N1, which is involved in attachment and entry of influenza virus into a cell.
  • Six of the Replikin peptides were synthesized according to sequences isolated from the pBl gene area of H5N1, which has been identified as involved in replication of influenza virus in a host cell.
  • Another exemplary vaccine has been designed from sequences identified as conserved in HlNl isolates. The sequences are likewise conserved across strains with conservation particularly noted in the key amino acid residues of the Replikin sequence, namely, lysine and histidine amino acid residues.
  • the vaccine was designed to deliver an approximately equal-parts-by- weight mixture of eight Replikin peptides to the immune system of an animal or human. All eight peptides are synthesized, and combined in a vaccine.
  • the peptide mixture may be administered in any manner known to one of skill in the art including with a pharmaceutically acceptable carrier. Administration may be intraocularly, intranasally, transdermally, intramuscularly, or via any method of administration known now or hereafter to one of skill in the art. Because the vaccine is based on influenza Replikin peptides shared between influenza strains and conserved for decades within influenza strains, the vaccine may be administered as a therapy against infection by any influenza virus infection harboring conserved Replikin peptides sharing homology with at least one peptide of the vaccine. Such strains include any strain of influenza A, B, or C.
  • the vaccine may be administered, for example, against HlNl, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, or H10N7 strains of influenza virus.
  • the vaccine may be further administered against H5N1, H5N2, H3N2, H9N2, or HlNl. Therapies against possible combination of HlNl and H5N1
  • Figures 2-5 provide examples of the association of Replikin Count in hemagglutinin with infectivity and Replikin Count in the pBl gene area with lethality.
  • the principle that Replikin Count in a genome could be associated with lethality has been quantitatively measured in a predictive study of the relative lethality of four strains of Taura Syndrome virus. See U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008. The applicability of this principle to antiviral therapies was demonstrated when Taura Syndrome virus was blocked by a specific synthetic Replikin sequence vaccine, protecting 91% of challenged shrimp from pathogenic mortality.
  • an increase in the lethality of H5N1 in human cases in 2007 and 2008 was predicted in advance by the strain-specific Replikin Count of the Lethality Gene of H5N1. See id.
  • Replikin Counts of the pBl gene area of H5N1 Genes in eight Asian countries in 2006 the geographic site which would be first and worst struck in 2007 was correctly predicted as Indonesia. See id.
  • a synthetic Replikin vaccine containing an approximately equal-parts-by- weight mixture of twelve H5N1 Replikin peptides was tested in chickens against a low pathogenic strain of H5N1 isolated from a black duck in North Carolina, USA.
  • Low-Path H5N1 strains infect migratory birds and impair health and productivity of commercial flocks of U.S. chickens, usually with little mortality in the commercial flocks.
  • These Low-Path H5N1 strains are very closely related in virus structure to their more lethal High-Path H5N1 relatives in Eurasia.
  • a mutation from a Low-Path to a High-Path strain has so far not been observed but mutations of this type over time may be expected by one of skill in the art.
  • the tested vaccine was engineered from sequences identified in the Low-Path H5N1 and confirmed to be conserved in both Low-Path and High-Path H5N1 strains over decades as well as across influenza strains with conservation particularly noted in the key amino acid residues of the Replikin sequence, namely, lysine and histidine amino acid residues.
  • the tested vaccine was engineered to block both the entry site of H5N1 virus and the replication site of those H5N1 viruses that manage to enter into host cells. As such, the vaccine is called the TWO-PUNCH vaccine.
  • the vaccine comprises a mixture of twelve Replikin peptides. Six of the Replikin peptides are synthesized according to sequences isolated from the hemagglutinin protein area of H5N1, which is involved in attachment and entry of influenza virus into a cell. Six of the Replikin peptides are synthesized according to sequences isolated from the pBl gene area of H5N1, which has been identified as involved in replication of influenza virus in a host cell. [000214] The following six Replikin sequences contained in the vaccine were isolated from the hemagglutinin protein area:
  • HAQDILEKEHNGKLCSLKGVRPLILK SEQ ID NO: 1
  • HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5);
  • KDVMESMDKEEMEITTH SEQ ID NO: 7
  • the vaccine comprises an approximate equal-parts-by- weight mixture of the twelve peptides.
  • the following peptide amounts were combined to create an initial mixture of the vaccine:
  • HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5) 170.8 mg
  • KDVMESMDKEEMEITTH (SEQ ID NO: 7) 161.9 mg
  • KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9) 217.8 mg
  • HKRTIGKKKQRLNK (SEQ ID NO: 10) 178.0 mg
  • the peptide mixture was then divided into three equal parts for administration of the vaccine on three different days following hatch (days 1, 7, and 28). After dissolution with water, the three equal parts were administered to individual birds in two groups of 20 birds each for a total administration on each day of 40 birds. The total amount of active peptide ingredient administered to each bird at the time of administration (either intranasally and intraocularly or via spray inhalation) was about 18.6 mg per bird per administration.
  • the vaccine solution was administered to chickens intranasally at a first administration on day 1 after hatch, intraocularly at a second administration on day 7 after hatch, and via fine spray inhalation at a third administration on day 14 after hatch.
  • Chickens on the first day of life were separated into four groups with twenty chickens per group.
  • the first group was a control group not vaccinated and not challenged with Low-Path H5N1.
  • the second group was vaccinated and not challenged with Low-Path H5N1.
  • the third group was vaccinated and subsequently challenged with Low-Path H5N1 on day 28 after hatch.
  • the fourth group was not vaccinated and was challenged with Low-Path H5N1 on day 28 after hatch.
  • Vaccinated chickens were subject to the vaccine on days 1, 7, and 21 after hatch as described above.
  • Challenged chickens were inoculated with Low-Path H5N1 virus in the soft palate on day 28 after hatch.
  • Serum from selected chickens was analyzed in all groups for antibodies against the H5N1 virus on days 7, 14, and 21 following challenge (days 35, 42, and 49 after hatch).
  • PCR for virus fecal excretion was also analyzed for all groups.
  • Unvaccinated control chickens demonstrated both an expected high virus entry (as indicated by a high titer of antibodies against H5N1) and an expected high virus replication (as indicated by high fecal and salival excretion of the virus detected by PCR).
  • the vaccinated chickens demonstrated lower virus entry (as indicated by a low titer of antibodies against H5N1 or by the observation of no antibodies against H5N1 in serum) and an absence of fecal or salival excretion of virus indicating low or no virus replication in the vaccinated chickens.
  • the data suggest, therefore, that the virus was partially blocked on entry by the chickens' immune response to the vaccine and the limited amount of virus that did enter the chickens' system was blocked from sufficient replication in the chickens' host cells to excrete virus in the feces or saliva.
  • Table 1 The data in Table 1 below provide the numbers of chickens tested in each of the four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day and the numbers of chickens in which production of antibodies to H5N1 was detected with a serum titer.
  • Table 2 The data in Table 2 below provide the number of chickens tested for H5N1 virus in their saliva and feces in each of the four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day and the numbers of chickens in which H5N1 was detected in their feces and saliva based on PCR analysis.
  • Table 2 demonstrates the absence of detectable influenza in the feces and saliva of vaccinated birds. That viral excretion was blocked by this influenza Replikins vaccine is particularly significant because it is generally acknowledged that the maintenance of reservoirs of H5N1 virus in flocks of migratory birds and domestic chickens in both Asia and the U.S. (and the regional spread of H5N1 virus from these reservoirs) is dependent on viral excretions picked up by neighboring chickens and birds. Regardless of the level of lethality of a strain of H5N1 virus, absent excretion of virus, there is expected to be no spread of the virus.
  • the vaccine may likewise be administered against H5N2, HlNl, H9N2, H3N2 or any other influenza strain having Replikin sequences that share homology with the peptides of the vaccine.
  • Replikin Counts in the pBl gene area are associated with lethality and Replikin Counts in the hemagglutinin protein area are associated with infectivity.
  • Table 3 as illustrated in Figure 2 demonstrate that lethality was increasing from 2004 through 2008 while infectivity was fairly steady.
  • the data correlate with epidemiological data in H5N1.
  • H5N1 has continued to cause high rates of mortality in humans.
  • the highest presently recorded lethality was predicted by the applicants following analysis of publicly available H5N1 pBl sequences from isolates in 2005 and 2006. In 2006 the applicants predicted that mortality rates would increase in H5N1 infections in response to increasing Replikin Counts in the pBl gene area of the virus.
  • H5N1 influenza virus has apparently remained steady over the years from 2004 through 2008 with very low rates of infection and very low rates of possible transmission between humans.
  • the H5N1 virus produced less than 300 World Health Organization confirmed deaths over the 10 years through the spring of 2008 even though the virus killed as many as 60% of those infected.
  • the high human mortality rate in combination with a low infectivity, appear to limit the ability of H5N1 to presently produce an influenza pandemic.
  • Replikin Counts in the hemagglutinin protein area are associated with infectivity and Replikin Counts in the pB 1 gene area are associated with lethality.
  • the data illustrated in Figure 3 demonstrate that infectivity was on the increase from 2004 through 2009 while lethality was fairly steady with a noticeable increase around 2005.
  • the data correlate with epidemiological data from the global 2009 outbreak of HlNl.
  • the global outbreak of the HlNl strain of influenza virus in the spring of 2009 has been observed to have high infectivity with effective transmission from host to host and the potential for an efficient and rapid spread of the virus internationally. See www.cdc.gov/mmwr/preview/mmwrhtml/mm5817al.htm.
  • HlNl influenza virus has apparently remained generally steady over the years from 2004 through 2009. There appears to have been a spike in lethality in Mexico, however, just at the beginning of the spring 2009 outbreak. This spike in lethality appears to have waned as the outbreak spread in Mexico and globally. This initial spike may be related to HlNl isolates carrying a high Replikin Count in the pBl gene area as reflected in the 2005 isolates disclosed in Table 4 above. However, as the outbreak spread, the 2005 increase in Replikin Count in the pBl gene area was apparently lost and the mortality rate of subsequent cases also declined.
  • the infectivity and lethality data for 2009 differs in Table 5 above from the data in Table 4 above in that the data in Table 5 represent the most recent genomic sequences published at www.pubmed.com as of June 8, 2009.
  • the data in Table 4 above represent a much smaller number of genomic sequences published at www.pubmed.com only through May 18, 2009.
  • the data in Table 5 demonstrate an increase in mean annual Replikin Count for the pBl gene area from 2 (+/- 0.2) in 2008 to 3.2 (+/- 3.7) in 2009
  • the data in Table 4 demonstrate a steady mean annual Replikin Count for the pBl gene area from 2 (+/- 0) in 2008 to 2 (+/-0) in 2009.
  • Figure 5 illustrates analysis of all data published on PubMed for concentrations of Replikin sequences in the virus genomes.
  • the dates in Figure 5 represent the publication dates of each specimen sequence at www.pubmed.com.
  • Publication dates generally reflect one to four months delay from the date a specimen is actually collected. This time difference represents time taken for sequence analysis, review, and publication.
  • more real-time analysis of concentrations of Replikin sequences would be expected to improve the predictive and analytical capacity of known Replikin concentrations. Closer-in- time analysis could be realized if the time for sequence analysis and publication were shortened.
  • greater numbers of publicly reported isolates would be expected to provide improved analysis of mean Replikin Counts.
  • FIG. 5 shows that for the HlNl Infectivity Gene (hemagglutinin protein area in white), the Mean Replikin Count increased from 4.3 (+1-2) in 2001 to 6.7 (+/-1.2) in 2008 (p ⁇ 0.001). At that time, the applicants published their warning that HlNl was the leading candidate for a pandemic. The Mean Replikin Count then continued to increase 43% to a mean count of 10 by April 2009. At that point, the clinical HlNl outbreak in Mexico and California was reported. By June 2009, the World Health Organization stated that the outbreak had sufficiently spread globally to be declared a pandemic.
  • Figure 5 also shows that for the HlNl Lethality Gene (pBl gene area, in black), the Mean Replikin Count between 2001 to 2008, despite some activity, did not increase significantly (in contrast to the Infectivity Gene — hemagglutinin protein area). However, the Standard Deviation of the Mean (SD) in the pBl gene area (represented by capped lines) increased fivefold between 2001 and December 2008 and forty-five fold between 2001 and April 2009. An increase in standard deviation of mean Replikin Count indicates that some viruses in a virus population have high Replikin Counts and are engaging in high replication rates.
  • SD Standard Deviation of the Mean
  • a synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of eight HlNl Replikin peptides is tested in pigs.
  • the tested vaccine is engineered from sequences identified in HlNl in humans from 1918 to the present and confirmed to be conserved in HlNl over decades as well as across influenza strains with conservation particularly noted in the key amino acid residues of the Replikin sequence, namely, lysine and histidine amino acid residues.
  • the tested vaccine is engineered to block both the entry site of HlNl virus and the replication site of those HlNl viruses that manage to enter into host cells. As such, the vaccine is called the TWO-PUNCH vaccine.
  • the vaccine comprises a mixture of the following twenty Replikin peptides in sterile water:
  • HAQDILEKEHNGKLCSLKGVRPLILK SEQ ID NO: 1
  • KSQLKNNAKEIGNGCFEFYH (SEQ ID NO: 27)
  • KHSNGTVK (SEQ ID NO: 28).
  • the first group is a control group which is neither vaccinated nor inoculated with HlNl influenza virus.
  • the second group is vaccinated.
  • the third group is vaccinated and inoculated with influenza virus.
  • the fourth group is not vaccinated but is nevertheless inoculated with HlNl influenza virus.
  • the vaccine is administered to all pigs in groups 2 and 3 on days 7, 14, and 21. All pigs in groups 3 and 4 are inoculated with HlNl on day 28. Thereafter, antibody production is monitored in the serum of selected pigs in each group. Additionally, the pigs are monitored for symptoms of influenza infections. External body fluids are also tested via PCR for shedding of HlNl influenza. The pigs in group 2, 3, and 4 produce antibodies to HlNl. The pigs in group 2 demonstrate no symptoms of influenza and shed no influenza virus detected by PCR. The pigs in group 4 demonstrate significant symptoms of influenza and shed influenza virus detected by PCR. The pigs in group 3 demonstrate reduced symptoms of influenza and shed considerably less influenza virus detected by PCR than do the pigs in group 4.
  • Table 9 provides examples of Replikin peptides that have been identified as conserved in various strains of influenza.
  • KFEIFPKTSSWPNH (SEQ ID NO: 35) observed as conserved in isolates from 1918, 1930, 1991, and 2009
  • HPVTIGECPKYVRSTK (SEQ ID NO: 37) observed as conserved in isolates from 1918, 1933, 1942, 1943, 1945, 1948, 1949, 1950, 1951, 1954, 1957, 1977-1984, 1986- 1989, 1991, 1994-1997, 1999-2001, 2003, 2004, 2006-2009 on the C-terminal portion of the protein
  • KEFNHLEK (SEQ ID NO: 38) observed as conserved in isolates from 1976, 1988, 1991, 1997, 1998, 2003, 2004, 2009
  • HLEKRIENLNKK (SEQ ID NO: 39) observed as conserved in isolates from 1976, 1988, 1991, 1997, 1998, 2003, 2004, 2009
  • KHSNGTVK (SEQ ID NO : 41 ) observed as conserved in isolates from 2009
  • HKCDNTCMESVK (SEQ ID NO: 46) observed as conserved in isolates from 2009
  • HSVNILEDKHNGKLCK (SEQ ID NO: 48) observed as conserved in isolates from 2009
  • KHSNGTVK (SEQ ID NO: 49) observed as conserved in isolates from 1918, 1933, 1934, 1940, 1947, 1977, 1978, 1979, 1980, 1983, 1985
  • HNGKLCKLKGIAPLQLGK SEQ ID NO: 50 observed as conserved in isolates from 1918, 1933, 1934, 1982-1984, 2009
  • HNGKLCKLKGIAPLQLGK (SEQ ID NO : 51 ) observed as conserved in isolates from 1918, 1933, 1934, 1982, 1983, 1984, 2009
  • HPVTIGECPKYVKSTK (SEQ ID NO: 53) observed as conserved in isolates from 1930-2008
  • HDSNVKNLYEKVK (SEQ ID NO: 54) observed as conserved in isolates from 1934-2008 24) HDSNVKNLYEKVKSQLK (SEQ ID NO: 55) observed as conserved in isolates froml 934-2008
  • HPVTIGECPKYVRSAK (SEQ ID NO: 56) observed as conserved in isolates from 1934-2008
  • HKCNNECMESVK (SEQ ID NO: 57) observed as conserved in isolates from 1940-2008
  • HPITIGECPKYVKSTK (SEQ ID NO: 59) observed as conserved in isolates from 1976- 2008
  • HKCDDECMESVK SEQ ID NO: 60
  • HNGKSSFYKNLLWLTGK (SEQ ID NO: 61) observed as conserved in isolates from 1996-2008
  • HKCNDECMESVK (SEQ ID NO : 62) observed as conserved in isolates from 1996, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008
  • HNGESSFYRNLLWLTGKNGLYPNLSK SEQ ID NO: 65
  • ABI97312 position 184 ABI94786 position 103.
  • ABV48012 position 177 ABV48001 position 184 , ABV47968 position 184 , ABV47957 position 184 , ABV47924 position 184 , ABV47913 position 184 , ABV47891 position 184 , ABV47880 position 184 , ABV47858 position 184 , ABV47825 position 184 , ABV47814 position 184 , ABV47792 position 184 , ABV47704 position 184 , ABB58993 position 184 , ABB58989 position 184 , ABK00142 position 184 , ACA42426 position 184 , ABB19963 position 184 , ABB58999 position 184 , ABB58998 position 184 , ABB58997 position 184 , ABB58996 position 184 , ABB58995 position 184 , ABB58994 position 184 , ABB58992 position 184 , ABB58991 position 184
  • 2004 id 83744850 position 207 .

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Abstract

La présente invention porte sur des procédés pour différencier l'infectiosité et la létalité d'isolats de virus de la grippe et porte sur des composés pour diagnostiquer, prévenir et traiter des épidémies de virus de la grippe comprenant des composés pour diagnostiquer, prévenir et traiter différentes souches du virus de la grippe.
PCT/US2009/061108 2009-04-23 2009-10-16 Séquences de réplikine pour la prévention/le traitement de la grippe et la détermination de l'infectiosité/la létalité WO2010123519A1 (fr)

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EP20090743995 EP2421546A1 (fr) 2009-04-23 2009-10-16 Séquences de réplikine pour la prévention/le traitement de la grippe et la détermination de l'infectiosité/la létalité
AU2009344843A AU2009344843A1 (en) 2009-04-23 2009-10-16 Replikin-sequences for preventing/treating influenza and determining infectivity/lethality
CA2759325A CA2759325A1 (fr) 2009-04-23 2009-10-16 Sequences de replikine pour la prevention/le traitement de la grippe et la determination de l'infectiosite/la letalite
SG2011076536A SG175275A1 (en) 2009-04-23 2009-10-16 Replikin-sequences for preventing/treating influenza and determining infectivity/lethality
NZ595812A NZ595812A (en) 2009-04-23 2009-10-16 Replikin-sequences for preventing/treating influenza and determining infectivity/lethality
IL215770A IL215770A0 (en) 2009-04-23 2011-10-23 Replikin-sequences for preventing/treating influenza and determining infectivity/ lethality

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USPCT/US2009/041565 2009-04-23
PCT/US2009/041565 WO2009132209A2 (fr) 2008-04-23 2009-04-23 Procédés et composés pour atténuer les flambées pathogènes au moyen des cycles de la réplikine
US12/429,044 US20090269367A1 (en) 2008-04-23 2009-04-23 Methods and compounds for mitigating pathogenic outbreaks using replikin count cycles
US17968609P 2009-05-19 2009-05-19
US61/179,686 2009-05-19
US18516009P 2009-06-08 2009-06-08
US61/185,160 2009-06-08
US12/538,027 2009-08-07
US12/538,027 US20100144589A1 (en) 2008-08-08 2009-08-07 Methods of predicting cancer lethality using replikin counts
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WO2013151662A2 (fr) 2012-03-09 2013-10-10 Samuel Bogoch Thérapies, vaccins et procédés de prédiction, relatifs au virus de l'anémie infectieuse du saumon
WO2014165274A3 (fr) * 2013-03-13 2014-12-11 Samuel Bogoch Procédés de diagnostic, de prévention et de traitement d'infections pathogéniques résistantes aux médicaments au moyen de séquences de réplikine
US9320784B2 (en) 2009-08-07 2016-04-26 Samuel Bogoch Peptides shared among lethal cancers and therapeutic compositions comprising said peptides
CN110680912A (zh) * 2019-09-26 2020-01-14 中国农业大学 H3n2和h3n8亚型犬流感二价灭活苗及其制备方法与应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9320784B2 (en) 2009-08-07 2016-04-26 Samuel Bogoch Peptides shared among lethal cancers and therapeutic compositions comprising said peptides
WO2013151662A2 (fr) 2012-03-09 2013-10-10 Samuel Bogoch Thérapies, vaccins et procédés de prédiction, relatifs au virus de l'anémie infectieuse du saumon
WO2014165274A3 (fr) * 2013-03-13 2014-12-11 Samuel Bogoch Procédés de diagnostic, de prévention et de traitement d'infections pathogéniques résistantes aux médicaments au moyen de séquences de réplikine
CN110680912A (zh) * 2019-09-26 2020-01-14 中国农业大学 H3n2和h3n8亚型犬流感二价灭活苗及其制备方法与应用
CN110680912B (zh) * 2019-09-26 2021-08-17 中国农业大学 H3n2和h3n8亚型犬流感二价灭活苗及其制备方法与应用

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