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  • 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
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  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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  • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
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  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
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  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
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  • A61K9/51—Nanocapsules; Nanoparticles
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  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
• • A—HUMAN NECESSITIES
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  • 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
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
  • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of vaccines, specifically nucleic acid vaccines (NAVs).
• NAVs nucleic acid vaccines
• the invention relates to compositions, methods, processes, kits and devices for the selection, design,
• RNA ribonucleic acid
• vaccines e.g., mRNA vaccines.
• Vaccination is an effective way to provide phrophylactic protection against infectious diseases, including, but not limited to, viral, bacterial, and/or parasitic diseases, such as influenza, AIDS, hepatotisis virus infection, cholera, malaria and tuberculosis, and many other diseases.
• infectious diseases including, but not limited to, viral, bacterial, and/or parasitic diseases, such as influenza, AIDS, hepatotisis virus infection, cholera, malaria and tuberculosis, and many other diseases.
• influenza infections are the seventh leading cause of death in the United States with 200, 000 hospitalizations and 40,000 deaths seen in the United States per year and cause about 3-5 million
• a typical vaccine contains an agent that resembles a weakened or dead form of the disease-causing agent, which could be a microorganism, such as bacteria, virus, fungi, parasites, or one or more toxins and/or one or more proteins, for example, surface proteins, (i.e., antigens) of such a microorganism.
• the antigen or agent in the vaccine can stimulate the body's immune system to recognize the agent as a foreign invader, generate antibodies against it, destroy it and develop a memory of it.
• the vaccine-induced memory enables the immune system to act quickly to protect the body from any of these agents that it later encounters.
• Vaccine production used in the art e.g.
• antigen vaccine production has several stages, including the generation of antigens, antigen purification and inactivation, and vaccine formulation.
• the antigen is generated through culturing viruses in cell lines, growing bacteria in bioreactors, or producing recombinant proteins derived from viruses and bacteria in cell cultures, yeast or bacteria.
• Recombinant proteins are then purified and the viruses and bacteria are inactivated before they are formulated with adjuvants in vaccines. It has been a challenge to drastically reduce the time and expense associated with current technologies in vaccine development.
• Viruses often mutate their surface proteins to generate new antigens which can help them skipping the active immune system that has been immunized by vaccines containing the viruses.
• bacteria acquire and mutate key proteins to evade host defense and effective antibiotic applications.
• influenza A, B and C viruses are the etiological agents of influenza.
• Hemagglutinin (HA) the major envelop glycoprotein of influenza A and B viruses, or its homologue, hemagglutinin-esterase (HE) in influenza C virus, is the natural reservoir of the viruses.
• the rapid evolution of the hemagglutinin (HA) protein of the influenza virus results in the constant emergence of new strains, rendering the adaptive immune response of the host only partially protective to new infections.
• the biggest challenge for therapy and prophylaxis against influenza and other infections using traditional vaccines is the limitation of vaccines in breadth, providing protection only against closely related subtypes.
• today' s length of the production process inhibits any fast reaction to develop and produce an adapted vaccine in a pandemic situation.
• compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of nucleic acid vaccines are described herein.
• compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of nucleic acid vaccines e.g. , RNA vaccines and mRNA vaccines.
• compositions e.g. , pharmaceutically acceptable salts, e.g., pharmaceutically acceptable salts, and pharmaceutically acceptable salts.
• compositions comprising one or more onucleic acid vaccines or NAVs.
• the NAVs or NAV compositions or the invention may be designed to comprise one or more nucleic acid molecules, e.g. , polynucleotides, which encode one or more wild type or engineered proteins, peptides or polypeptides (e.g. , antigens).
• the nucleic acid molecule, e.g. , polynucleotide is RNA.
• the nucleic acid molecule, e.g. , polynucleotide is an mRNA.
• the NAV or NAV composition comprises a nucleic acid (e.g., a RNA polynucleotide) which is chemically modified.
• the infectious agent from which the antigen is derived or engineered includes, but is not limited to viruses, bacteria, fungi, protozoa, and/or parasites.
• RNAVs inducing, eliciting, boosting or triggering an immune response in a cell, tissue or organism, comprising contacting said cell, tissue or organism with any of the RNAVs described or taught herein.
• nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, formulated within a cationic lipid nanoparticle.
• Some aspects provide NAVs comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, formulated in a carrier having a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol; and 0.5- 15% PEG-modified lipid.
• the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
• the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
• the cationic lipid nanoparticle has a molar ratio of about 20-60% cationic lipid: about 5- 25% non-cationic lipid: about 25-55% sterol; and about 0.5- 15% PEG-modified lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 50% cationic lipid, about 1.5% PEG-modified lipid, about 38.5% cholesterol and about 10% non-cationic lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 55% cationic lipid, about 2.5% PEG lipid, about 32.5% cholesterol and about 10% non-cationic lipid.
• the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.
• the cationic lipid nanoparticle has a molar ratio of 50:38.5: 10: 1.5 of cationic lipid: cholesterol: PEG2000- DMG:DSPC.
• the cationic lipid nanoparticle has a mean diameter of 50-150 nm. In some embodiments, the cationic lipid nanoparticle has a mean diameter of 80-100 nm. In some embodiments, the vaccine includes 1.5mg/mL of RNA polynucleotide and 35-45 mg/mL lipids. In some embodiments, the NAV includes about 2 mg/mL of RNA polynucleotide and about 40 mg/mL lipids.
• the open reading frame is codon-optimized.
• the first antigenic polypeptide is derived from an infectious agent.
• the infectious agent is selected from a member of the group consisting of strains of viruses and strains of bacteria.
• the one or more RNA polynucleotides encode a further antigenic polypeptide.
• the further RNA polynucleotide comprises at least one chemical modification and a codon-optimized open reading frame, said open reading frame encoding an antigenic polypeptide.
• the one or more antigenic polypeptide is selected from those proteins listed in Tables 6-16, Tables 29-30, or fragments thereof.
• the open reading frame of the one or more RNA polynucleotides and/or the open reading frame of the second RNA polynucleotide each, independently, encodes an antigenic polypeptide selected from Tables 6-16, Tables 29-30, or fragments thereof.
• each of the open reading frame of the one or more RNA polynucleotides is selected from any of the RNA sequences Table 28, or fragments thereof.
• the infectious agent is a strain of virus selected from the group consisting of adenovirus; Herpes simplex, type 1;
• the virus is a strain of Influenza A or Influenza B or combinations thereof.
• the strain of Influenza A or Influenza B is associated with birds, pigs, horses, dogs, humans or non-human primates.
• the antigenic polypeptide encodes a hemagglutinin protein or fragment thereof.
• the hemagglutinin protein is HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof .
• the hemagglutinin protein does not comprise a head domain (HA1).
• the hemagglutinin protein comprises a portion of the head domain (HA1). In some embodiments, the hemagglutinin protein does not comprise a cytoplasmic domain. In some embodiments, the hemagglutinin protein comprises a portion of the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein. In some embodiments, the truncated hemagglutinin protein comprises a portion of the transmembrane domain.
• the amino acid sequence of the hemagglutinin protein or fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identify with any of the amino acid sequences provided in Table 6-14.
• the virus is selected from the group consisting of H1N1, H3N2, H7N9, and H10N8.
• the antigenic polypeptide is selected from those proteins listed in Tables 6-14, or fragments thereof.
• the infectious agent is a strain of bacteria selected from
• Tuberculosis Mycobacterium tuberculosis
• clindamycin-resistant Clostridium difficile clindamycin-resistant Clostridium difficile
• fluoroquinolon-resistant Clostridium difficile methicillin-resistant
• MRSA multidrug-resistant Enterococcus faecalis
• multidrug-resistant Enterococcus faecium multidrug-resistance Pseudomonas aeruginosa
• multidrug-resistant Acinetobacter baumannii multidrug-resistant Acinetobacter baumannii
• VRSA vancomycin-resistant Staphylococcus aureus
• the bacteria is Clostridium difficile.
• the NAV is multivalent.
• the open reading frame of the one or more RNA polynucleotides encode at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigenic polypeptides.
• the open reading frame of the one or more RNA polynucleotides encode at least 10, 15, 20 or 50 antigenic polypeptides.
• the open reading frame of the one or more RNA polynucleotides encode 2-10, 10-15, 15-20, 20-50, 50-100 or 100-200 antigenic polypeptides.
• the RNA polynucleotide includes a chemical modification and the chemical modification is selected from any of those listed in Tables 22 and 23.
• the chemical modification is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, Dihydropseudouridine, 5-methoxyuridine and 2'-0-methylmethyl
• the nanoparticle has a polydiversity value of less than 0.4.
• the nanoparticle has a net neutral charge at a neutral pH.
• the nanoparticle has a mean diameter of 80-100nm.
• the nanoparticle is a cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
• the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
• DLin-KC2-DMA 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane
• DLin-MC3-DMA dilinoleyl-methyl-4- dimethylaminobutyrate
• L319 di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy
• NAVs comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is a Nl -methyl pseudouridine. In some embodiments, the nucleic acid vaccine is formulated within a cationic lipid complex or cationic lipid nanoparticle.
• NAVs comprising one or more RNA
• polynucleotides having an open reading frame encoding a first antigenic polypeptide, at least one 5' terminal cap and at least one chemical modification, formulated within a cationic lipid nanoparticle.
• the 5' terminal cap is
• the chemical modification is selected from any of those listed in Tables 22 and 23.
• the chemical modification is selected from the group consisting of pseudouridine, Nl- methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine,, 2-thio-l- methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio- 1 -methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, Dihydropseudouridine, 5- methoxyuridine and
• polynucleotide further comprises a second chemical modification wherein said second chemical modification is selected from any of those listed in Tables 22 and 23. In some embodiments, the combination of first and second chemical modification is selected from those listed in Table 25.
• the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
• the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
• the cationic lipid nanoparticle has a molar ratio of about 20-60% cationic lipid: about 5- 25% non-cationic lipid: about 25-55% sterol; and about 0.5-15% PEG-modified lipid.
• NAVs comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein and a
• the hemagglutinin protein is selected from HAl, HA7 and HA 10.
• the RNA polynucleotide does not encode F protein. In some embodiments, the RNA polynucleotide further encodes
• the hemagglutinin protein is derived from a strain of Influenza A virus or Influenza B virus or combinations thereof.
• the Influenza virus is selected from H1N1, H3N2, H7N9, and H10N8.
• the RNA polynucleotide includes a chemical modification and the chemical modification is selected from any of those listed in Tables 22 and 23.
• t he chemical modification is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine,, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, Dihydropseudouridine, 5-methoxyuridine and 2'-
• the RNA polynucleotide further comprises a second chemical modification wherein said second chemical modification is selected from any of those listed in Tables 22 and 23. In some embodiments, the combination of first and second chemical modification is selected from those listed in Table 25.
• the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
• the cationic lipid is selected from the group consisting of 2,2-dilinoleyl- 4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en- 1 -yl) 9-((4-)
• the cationic lipid nanoparticle has a molar ratio of about 20-60% cationic lipid: about 5- 25% non-cationic lipid: about 25-55% sterol; and about 0.5-15% PEG-modified lipid.
• the RNA polynucleotide comprises SEQ ID NOs 2459- 2621. In some embodiments, the RNA polynucleotide comprises a polynucleotide having at least 80% sequence identity to SEQ ID NOs 2459-2621.
• the RNA polynucleotide comprises a polynucleotide encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO 2254. In some embodiments, the RNA polynucleotide comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO 2254. In some embodiments, the RNA polynucleotide comprises a polynucleotide encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO 2259. In some embodiments, the RNA polynucleotide comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO 2259. In some embodiments, the RNA polynucleotide comprises a
• the RNA polynucleotide encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO 2192.
• the RNA polynucleotide comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO 2192.
• the RNA polynucleotide comprises a polynucleotide encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO 2200.
• the RNA polynucleotide comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO 2200.
• the RNA polynucleotide comprises SEQ ID NO 2046.
• the RNA polynucleotide comprises a polynucleotide having 80-98% sequence identity to SEQ ID NO 2046. In some embodiments, the RNA polynucleotide comprises SEQ ID NO 2119. In some embodiments, the RNA polynucleotide comprises a polynucleotide having 80-98% sequence identity to SEQ ID NO 2119. In some embodiments, the RNA polynucleotide comprises SEQ ID NO 2054. In some embodiments, the RNA polynucleotide comprises a polynucleotide having 80-98% sequence identity to SEQ ID NO 2054. In some embodiments, the RNA polynucleotide comprises SEQ ID NO 2127. In some embodiments, the RNA polynucleotide comprises a polynucleotide having 80-98% sequence identity to SEQ ID NO 2127.
• aspects of the invention provide nucleic acids comprising 80-95% sequence identity to SEQ ID NO 2127 or SEQ ID NO 2054. Other aspects provide a nucleic acid comprising SEQ ID NO: 2624.
• Yet other aspects provide a method of inducing an antigen specific immune response in a subject comprising administering any of the vaccines described herein to the subject in an effective amount to produce an antigen specific immune response.
• the antigen specific immune response comprises a T cell response.
• the antigen specific immune response comprises a B cell response.
• the method of producing an antigen specific immune response involves a single administration of the vaccine.
• the method further comprises administering a booster dose of the vaccine.
• the vaccine is administered to the subject by intradermal or intramuscular injection.
• the booster dose of the vaccine is administered to the subject on day twenty one. In some embodiments, a dosage of between 10 ug/kg and 400 ug/kg of the vaccine is administered to the subject. In some embodiments, a dosage of 25 micrograms of the RNA polynucleotide is included in the vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node.
• the antigen specific immune response comprises a T cell response.
• the antigen specific immune response comprises a B cell response.
• the method of producing an antigen specific immune response involves a single administration of the vaccine.
• the vaccine is administered to the subject by intradermal or intramuscular injection.
• nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.
• a dosage of between 10 ug/kg and 400 ug/kg of the nucleic acid vaccine is administered to the subject.
• the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection.
• the nucleic acid vaccine is administered to the subject on day zero.
• a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.
• a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node.
• nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and a pharmaceutically acceptable carrier or excipient, wherein an adjuvant is not included in the vaccine.
• the stabilization element is a histone stem-loop.
• the stabilization element is a nucleic acid sequence having decreased GC content relative to wild type sequence.
• aspects of the invention provide NAVs comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo
• the antibody titer is a neutralizing antibody titer.
• NAVs comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the
• RNA polynucleotide is present in a formulation for in vivo administration to a host for eliciting a longer lasting high antibody titer than an antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.
• the RNA polynucleotide is present in a formulation for in vivo administration to a host for eliciting a longer lasting high antibody titer than an antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.
• the RNA polynucleotide is present in a formulation for in vivo administration to a host for eliciting a longer lasting high antibody titer than an antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic
• polynucleotide is formulated to produce a neutralizing antibodies within one week of a single administration.
• the adjuvant is selected from a cationic peptide and an immuno stimulatory nucleic acid.
• the cationic peptide is protamine.
• polynucleotide is present in the formulation for in vivo administration to a host such that the level of antigen expression in the host significantly exceeds a level of antigen expression produced by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.
• NAVs comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer.
• the RNA polynucleotide is present in a dosage of 25-100 micrograms.
• aspects of the invention also provide a unit of use vaccine, comprising between lOug and 400 ug of one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject.
• the vaccine further comprises a cationic lipid nanoparticle.
• aspects of the invention provide methods of creating, maintaining or restoring antigenic memory to an influenza strain in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification and two or more codon- optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient.
• the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration and subcutaneous administration.
• the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.
• aspects of the invention provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 ug/kg and 400 ug/kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide in an effective amount to vaccinate the subject.
• NAVs comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein fragment, wherein the hemagglutinin protein includes only a portion of at least one of: a head domain (HAl), a cytoplasmic domain, or a transmembrane domain.
• the hemagglutinin protein is HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, H18.
• the hemagglutinin protein does not comprise the head domain (HAl).
• the hemagglutinin protein does not comprise the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein does not comprise the transmembrane domain. In some embodiments, the amino acid sequence of the hemagglutinin protein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identify with any of the amino acid sequences provided in Tables 6-16.
• the vaccine is formulated within a cationic lipid complex or cationic lipid nanoparticle.
• the polynucleotide comprises at least one 5' terminal cap and at least one chemical modification.
• aspects also provide any of the vaccines described herein for use in a method of inducing an antigen specific immune response in a subject.
• the method comprises administering the vaccine to the subject in an effective amount to produce an antigen specific immune response.
• aspects also provide for any of the vaccines described herein for use in a method of preventing or treating influenza viral infection, the method comprising administering the vaccine to a subject.
• nucleic acid vaccines for use in a method of vaccinating a subject wherein the nucleic acid vaccine comprises a first RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.
• the method further comprises administering the vaccine to the subject.
• nucleic acid vaccine in the manufacture of a medicament for use in a method of vaccinating a subject
• the nucleic acid vaccine comprises a first RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.
• the method further comprises administering the vaccine to the subject.
• aspects of the invention provide an antigenic memory booster nucleic acid vaccine for use in a method of creating, maintaining or restoring antigenic memory to an influenza strain in an individual or population of individuals.
• the antigenic memory booster nucleic acid vaccine comprises (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification and two or more codon- optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient; and wherein the method comprises administering to said individual or population the antigenic memory booster nucleic acid vaccine.
• an antigenic memory booster nucleic acid vaccine in the manufacture of a medicament for use in a method of creating, maintaining or restoring antigenic memory to an influenza strain in an individual or population of individuals
• the antigenic memory booster nucleic acid vaccine comprises (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification and two or more codon-optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient; and wherein the method comprises administering to said individual or population the antigenic memory booster nucleic acid vaccine.
• nucleic acid vaccine for use in a method of vaccinating a subject, wherein the nucleic acid vaccine comprises a first RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide, and wherein the method comprises administering to the subject a single dosage of between 25 ug/kg and 400 ug/kg of the nucleic acid vaccine in an effective amount to vaccinate the subject.
• nucleic acid vaccine in the manufacture of a medicament for use in a method of vaccinating a subject, wherein the nucleic acid vaccine comprises a first RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide, and wherein the method comprises administering to the subject a single dosage of between 25 ug/kg and 400 ug/kg of the nucleic acid vaccine in an effective amount to vaccinate the subject.
• the NAV polynucleotides may encode one or more polypeptides of an influenza strain as an antigen.
• antigens include, but are not limited to those antigens encoded by the polynucleotides listed in the Tables presented herein.
• GenBank Accession Number or GI Accession Number represents either the complete or partial CDS of the encoded antigen.
• the NAV polynucleotides may comprise a region of any of the sequences listed in the tables or entire coding region of the mRNA listed. They may comprise hybrid or chimeric regions, or mimics or variants.
• FIG. 1 is a schematic of a polynucleotide construct.
• FIG. 1A is a schematic of a polynucleotide construct taught in commonly owned co-pending US Patent Application 13/791,922 filed March 9, 2013, the contents of which are incorporated herein by reference.
• FIG. IB is a schematic of a linear polynucleotide construct.
• FIG. 2 is a schematic of a series of chimeric polynucleotides of the present invention.
• FIG. 3 is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications and showing regions analogous to those regions of an mRNA polynucleotide.
• FIG. 4 is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications based on Formula I.
• FIG. 5 is a is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications based on Formula I and further illustrating a blocked or structured 3' terminus.
• FIGs. 6A and 6B are schematics of circular constructs of the present invention.
• FIGs. 7A-7B are schematics of circular constructs of the present invention.
• FIGs. 8A-8B are schematics of a circular constructs of the present invention.
• FIG 8A shows a circular construct comprising at least one sensor region and a spacer region.
• FIG. 8B shows a non-coding circular construct.
• FIG. 9 is a schematic of a non-coding circular construct of the present invention.
• FIG. 10 shows HA neutralization titres of a chemically modified mRNA influenza vaccine in comparison with protein and unmodified mRNA vaccines.
• FIG. 11 shows hemagglutinin inhibition titers in mice following vaccination with different doses and formulations of mRNA encoding the
• FIG. 12A-AD shows percent survival of mice after vaccination and challenge with influenza A/PR/8/34 virus.
• FIG. 12A shows percent survival at 1 week post challenge.
• FIG. 12B shows percent survival at 2 weeks post challenge.
• FIG. 12C shows percent survival at 3 weeks post challenge.
• FIG. 12D shows percent survival at 4 weeks post challenge.
• FIG. 13 shows the mean hemaglutination inhibition titers of mice after vaccination and challenge with influenza A/PR/8/34 virus
• FIGs. 14A-14C shows CD4 T cell IFNy cytokine responses.
• FIG. 14A shows IFNy production upon HI protein/peptide stimulation.
• FIG. 14B shows IFNy production upon H7 protein/peptide stimulation.
• FIG. 14C shows IFNy production upon PMA+ionomycin stimulation.
• FIGs. 15A-15D shows IgG production following HI and H7
• FIG. 16 is a graph showing hemagglutinin inhibition titers (HAI) against H10 following administration of the H10N8/ Nl -methyl pseudouridine /CO formulation MC3 vaccine at the indicated dosages.
• HAI hemagglutinin inhibition titers
• FIG. 18 is a graph comparing hemagglutinin inhibition titers (HAI) against H7 following administration of 10 ⁇ g/dose of the H7N9/C0 formulation compared to the H7N9/C 1 formulation.
• HAI hemagglutinin inhibition titers
• FIG. 19 is a graph of the mean hemagglutinin inhibition titers (HAI) in serum samples from cynomolgus monkey at various time points prior to and after administration of the indicated formulations and dosages.
• HAI hemagglutinin inhibition titers
• FIG. 20 is a graph showing the H7N9 viral load in ferrets challenged at day 21 after receiving a single immunization.
• FIGs. 21A-21D present mouse survival and HAI titers in mice challenged with a lethal dose following administration of a single dose of mRNA NAV encoding H7N9.
• FIG. 21A shows survival at day 7 post challenge.
• FIG. 21B shows survival at day 21 post challenge.
• FIG. 21C shows survival at day 84 post challenge.
• FIG. 21D shows HAI titers.
• FIG. 22 shows an alignment of amino acid sequences of hemagglutinin proteins from influenza A H7N9 strains relative to a consensus sequence.
• FIG. 23 shows an alignment of amino acid sequences of hemagglutinin proteins from influenza A H10N8 strains relative to a consensus sequence.
• RNA ribonucleic acid
• mRNA messenger RNA
• One beneficial outcome is to cause intracellular translation of the nucleic acid and production of at least one encoded peptide or polypeptide of interest.
• RNA ribonucleic acid
• mRNA messenger RNA
• an antigen e.g., an antigen derived from an infectious microorganism
• compositions including pharmaceutical compositions and methods for the selection, design, preparation, manufacture, formulation, and/or use of nucleic acid vaccines (NAVs) where at least one component of the NAV is a nucleic acid molecule, e.g., a polynucleotide.
• NAVs nucleic acid vaccines
• compositions including pharmaceutical compositions and methods for the selection, design, preparation, manufacture, formulation, and/or use of nucleic acid vaccines (NAVs) where at least one component of the NAV is a polynucleotide.
• compositions including pharmaceutical compositions
• NAVs nucleic acid vaccines
• the invention relates to compositions (including pharmaceutical compositions) and methods for the selection, design, preparation, manufacture, formulation, and/or use of ribonucleic acid vaccines (RNAVs) where at least one component of the RNAV is a ribonucleic acid molecule, e.g., a mRNA which encodes an antigen, e.g., an antigen derived from an infectious microorganism.
• RNAVs ribonucleic acid vaccines
• the present invention is directed, in part, to polynucleotides, specifically in vitro transcription (IVT) polynucleotides, chimeric polynucleotides and/or circular polynucleotides which may function as a vaccine or component of a vaccine.
• IVTT in vitro transcription
• polynucleotides may be modified in a manner as to avoid the deficiencies of or provide improvements over other polynucleotide molecules of the art.
• RNA vaccines including mRNA vaccines and self-replicating RNA vaccines
• the therapeutic efficacy of these RNA vaccines have not yet been fully established.
• the inventors have discovered a class of formulations for delivering mRNA vaccines in vivo that results in significantly enhanced, and in many respects synergistic, immune responses including enhanced antigen generation and functional antibody production with neutralization capability. These results are achieved even when significantly lower doses of the mRNA are administered in comparison with mRNA doses used in other classes of lipid based formulations.
• the formulations of the invention have demonstrated significant unexpected in vivo immune responses sufficient to establish the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents.
• the invention involves, in some aspects, the surprising finding that lipid nanoparticle (LNP) formulations significantly enhance the effectiveness of mRNA vaccines, including chemically modified and unmodified mRNA vaccines.
• LNP lipid nanoparticle
• the efficacy of mRNA vaccines formulated in LNP was examined in vivo using several distinct viral antigens and in a variety of different animal models.
• the results presented herein demonstrate the unexpected superior efficacy of the mRNA vaccines formulated in LNP over other mRNA vaccines formulated in other lipid based carriers as well as over protein antigens.
• the formulations of the invention generate a more rapid immune response following a single dose of antigen than other mRNA or protein based vaccines tested.
• IV intravenous
• IM intramuscular
• ID intradermal
• a study described herein involved intravenous (IV), intramuscular (IM), or intradermal (ID) vaccination of mice, followed by challenge with a lethal dose of virus.
• I intradermal
• significantly stronger early immune responses were observed (anti-viral activity via virus neutralization assay and HA inhibition (HAI)) in comparison to protein antigen and other lipid based formulations (lipoplex).
• HAI titer of greater than 40 is deemed sufficient to protect from a lethal challenge of influenza.
• the rapid response was unexpected, particularly when compared to the response seen with protein antigen and mRNA vaccines formulated in other lipid carriers (lipoplex), which at one week and even at three weeks following vaccination continued to show ineffective HAI titers of less than 40.
• the formulations of the invention continued to provide significantly stronger therapeutic responses than did protein antigen.
• both chemically unmodified and modified mRNA-LNP formulation administered by IV route had enhanced HAI titers with respect to the protein antigen.
• all of the animals receiving an mRNA-LNP formulation by IM or ID administration displayed HAI activity over 40, as compared to protein antigen, which at one week and three weeks continued to show HAI titers of less than 40.
• a mRNA-LNP formulation administered by ID route had a surprising HAI activity of greater than 10,000, in contrast to the HAI titer of around 400 for protein antigen at that time point.
• mice receiving a mRNA-LNP formulation also displayed neutralizing activity of 79-250 (IM) and 250 (ID) by microneutralization assay, in comparison to protein antigen, which had undetectable neutralization activity at that time point.
• IM 79-250
• ID 250
• mice vaccinated with protein antigen displayed neutralizing activity in only 3 of 5 mice and ranging only between 79 and 250.
• the mRNA-LNP formulations of the invention also produced quantitatively and qualitatively better immune responses than did mRNA vaccines formulated in a different lipid carrier (lipoplex).
• the mRNA -lipoplex vaccine produced HAI titers of 197, in comparison to those achieved by the mRNA-LNP formulations of the invention (HAI titers of 635-10,152).
• HAI titers of 635-10,152.
• none of the HAI titers reached the critical level of greater than 40.
• the mRNA -lipoplex vaccines did not result in any detectable neutralizing activity in the microneutralization activity, even as late as five weeks after vaccination.
• the mRNA-LNP formulations of the invention were superior to other lipid formulations even when the dose of mRNA was significantly lower than in the other lipid formulations.
• the data described above was generated using 10 ⁇ g of mRNA in the mRNA-LNP formulations in contrast to 80 ⁇ g of mRNA in the mRNA-lipoplex formulation.
• the formulations of the invention also showed strong efficacy in several non- rodent animal models, including non-human primates. Highly effective vaccination was observed in cynomoglus monkeys and ferrets. Cynomoglus monkeys were vaccinated with various doses of mRNA-LNP formulations (50 ⁇ g/dose, 200 ⁇ g/dose, 400 ⁇ g/dose). Quite surprisingly, the vaccine formulations of the invention at all doses measured significantly reduced viral titers in the lungs of ferrets when exposed to virus just 7 days following single vaccination. Statistically significant increases in antibody titer as measured by HAI and microneutralization were detected as early as 7 days following vaccination and through the entire length of the study (84 days). A single vaccination was able to eliminate all virus in most animals.
• LNP used in the studies described herein has been used previously to deliver siRNA various in animal models as well as in humans.
• the fact that LNP is useful in vaccines is quite surprising. It has been observed that therapeutic delivery of siRNA formulated in LNP causes an undesirable inflammatory response associated with a transient IgM response, typically leading to a reduction in antigen production and a compromised immune response.
• the LNP-mRNA formulations of the invention are
• NAVs Nucleic Acid Vaccines
• Nucleic Acid Vaccines (NAVs) of the present invention comprise one or more polynucleotides, e.g. , polynucleotide constructs, which encode one or more wild type or engineered antigens.
• Exemplary polynucleotides, e.g., polynucleotide constructs include antigen-encoding RNA polynucleotides, e.g. , mRNAs.
• polynucleotides of the invention, e.g. , antigen-encoding RNA polynucleotides may include at least one chemical modification.
• NAV compositions of the invention may comprise other components including, but not limited to, tolerizing agents or adjuvants.
• Adjuvants or immune potentiators may also be administered with or in combination with one or more NAVs.
• an adjuvant acts as a co-signal to prime T-cells and/or B- cells and/or NK cells as to the existence of an infection.
• adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccinees. These may be co-administered by any route, e.g., intramusculary, subcutaneous, IV or intradermal injections.
• Adjuvants useful in the present invention may include, but are not limited to, natural or synthetic. They may be organic or inorganic.
• Aduvants may be selected from any of the classes (1) mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidised detergent stabilised oil-in-water emulsion, purified saponin, oil-in- water emulsion, stabilised water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and/or (6) inert vehicles, such as gold particles; (7) microorganism derived adjuvants; (8) tensoactive compunds; (9) carbohydrates; or combinations thereof.
• mineral salts
• Adjuvants for DNA nucleic acid vaccines have been disclosed in, for example, Kobiyama, et al Vaccines, 2013, 1(3), 278-292, the contents of which are incorporated herein by reference in their entirety. Any of the adjuvants disclosed by Kobiyama may be used in the RNAVs of the present invention.
• RNAVs of the present invention include any of those listed on the web-based vaccine adjuvant database, Vaxjo;
• adjuvants may include,without limitation, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM(s)TM, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine
• Adjuvant DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium-derb/ed P40 Vaccine Adjuvant, MPLTM Adjuvant, AS04, AS02, Lipopolysaccharide Vaccine Adjuvant, Muramyl Dipeptide Adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetella pertussis component Vaccine Adjuvant, Cationic Liposomal Vaccine Adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3 Adjuvant, Aluminum vaccine adjuvant, Polygen Vaccine Adjuvant, AdjumerTM, Algal Glucan, Bay R100
• nanoemulsion vaccine adjuvant AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G Vaccine Adjuvant, E. coli heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic Adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly(LC), ISCOMATRIX®, Abisco-100 vaccine adjuvant, Albumin- heparin microparticles vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes Containing Antibodies to Costimulatory Molecules, SAF-1, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Threonyl muramyl dipeptide (TMDP), Ty Particles vaccine adjuvant, Bupivacaine vaccine
• adjuvants which may be co-administered with the NAVs of the invention include, but are not limited to interferons, TNF-alpha, TNF-beta, chemokines such as CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD-40L, CD28 agonists, PD-1, soluble PD1, LI or L2, or interleukins such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-21, IL-23, IL-15, IL-17, and IL-18. These may be administered with the NAV on the same encoded polynucleotide, e.g., polycistronic, or as separate mRNA encoding the adjuvant and antigen.
• interferons such as CCL21, eotaxin, HMGB1,
• NAVs of the present invention may vary in their valency. Valency refers to the number of antigenic components in the NAV or NAV polynucleotide (e.g., RNA polynucleotide) or polypeptide.
• the NAVs are monovalent.
• the NAVs are divalent.
• the NAVs are trivalent.
• the NAVs are multi-valent. Multivalent vaccines may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12,13,14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.).
• the antigenic components of the NAVs may be on a single polynucleotide or on separate polynucleotides.
• the NAVs of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine and/or for the priming of immune effector cells, e.g., stimulate/transfect PBMCs ex vivo and re-infuse the activated cells.
• a NAV described herein can be administered to a subject, wherein the polynucleotides is translated in vivo to produce an antigen.
• the active therapeutic agents of the invention include NAVs, cells containing NAVs or polypeptides translated from the polynucleotides contained in said NAVs.
• a polypeptide e.g., antigen or immunogen
• Such translation can be in vivo, ex vivo, in culture, or in vitro.
• the cell, tissue or organism is contacted with an effective amount of a composition containing a NAV which contains a polynucletotide that has at least one a translatable region encoding the polypeptide of intereste (e.g., antigen or immunogen).
• an "effective amount" of the NAV composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the NAV, and other determinants.
• an effective amount of the NAV composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen.
• Increased antigen production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the NAV), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered innate immune response of the host cell.
• aspects of the invention are directed to methods of inducing in vivo translation of a polypeptide antigen in a mammalian subject in need thereof.
• an effective amount of a NAV composition containing a polynucleotide that has at least one structural or chemical modification and a translatable region encoding the polypeptide (e.g., antigen or immunogen) is administered to the subject using the delivery methods described herein.
• the polynucleotide is provided in an amount and under other conditions such that the polynucleotide is translated in the cell.
• the cell in which the polynucleotide is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of NAV administration.
• the administered NAVs comprising polynucleotides directs production of one or more polypeptides that provide a functional immune system-related activity which is substantially absent in the cell, tissue or organism in which the polypeptide is translated.
• the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
• the administered polynucleotides directs production of one or more polypeptides that increases (e.g., synergistically) a functional activity related to the immune system which is present but substantially deficient in the cell in which the polypeptide is translated.
• the polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell.
• antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a carbohydrate, a protein toxin such as shiga and tetanus toxins, or a small molecule toxin such as botulinum, cholera, and diphtheria toxins.
• the antagonized biological molecule may be an endogenous protein that exhibits an undesirable activity, such as a cytotoxic or cytostatic activity.
• the proteins described herein may be engineered for localization within the cell, potentially within a specific compartment such as the cytoplasms or nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
• polynucleotides of the NAVs and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to viral infections (e.g. , influenza, HIV, HCV, RSV), parasitic infentions or bacterial infections.
• viral infections e.g. , influenza, HIV, HCV, RSV
• parasitic infentions e.g., bacterial infections.
• the subject to whom the therapeutic agent may be administered suffers from or may be at risk of developing a disease, disorder, or deleterious condition.
• GWAS genome- wide association studies
• the agents can be administered simultaneously, for example in a combined unit dose (e.g. , providing simultaneous delivery of both agents).
• the agents can also be administered at a specified time interval, such as, but not limited to, an interval of minutes, hours, days or weeks. Generally, the agents may be concurrently
• the agents may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In other embodiments, the agents may be delivered in separate unit dosages.
• the agents may be
• At least one administration of one of the agents may be made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent.
• combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than additive results.
• the NAVs comprising the polynucleotides disclosed herein, e.g., comprising RNA polynucleotides may act as a single composition as a vaccine.
• a "vaccine” refers to a composition, for example, a substance or preparation that stimulates, induces, causes or improves immunity in an organism, e.g., an animal organism, for example, a mammalian organism (e.g., a human.)
• a vaccine provides immunity against one or more diseases or disorders in the organism, including prophylactic and/or therapeutic immunity.
• Exemplary vaccines includes one or more agents that resembles an infectious agent, e.g., a disease-causing microorganism, and can be made, for example, from live, attenuated, modified, weakened or killed forms of disease-causing microorganisms, or antigens derived therefrom, including combinations of antigenic components.
• a vaccine stimulates, induces causes or improves immunity in an organism or causes or mimics infection in the organism without inducing any disease or disorder.
• a vaccine introduces an antigen into the tissues, extracellular space or cells of a subject and elicits an immune response, thereby protecting the subject from a particular disease or pathogen infection.
• the polynucleotides of the present invention may encode an antigen and when the polynucleotides are expressed in cells, a desired immune reponse is achieved.
• NAVs may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms.
• the NAVs of the present invention may also be administered as a second line treatment after the standard first line treatments such as antibiotics and antivirals have failed to induce passive immunity.
• the NAVs of the present invention are useful in settings where resistance to first line treatments has developed and disease persists and induces chronic disease.
• NAVs may be administered as part of a treatment regimen for latent bacterial infections, such as MRSA or Clostridium infections.
• one or more polynucleotides are administered which ultimately produce proteins which result in the removal or alterations of the protective shield surrounding a bacterium making the bacterium more susceptible to antibiotic treatement.
• a polynucleotide encoding one or several generic or patient-specific antibiotic resistance genes is administered to a patient, e.g. NDM-1.
• the polunucleotide is then translated to produce the enzyme in vivo. This production may raise an antibody-mediated immune response to the secreted and/or the intracellular enzyme that neutralized the antibiotic resistance and provides the bacteria susceptibal to the clearance by available antibiotics again. .
• Given the broad range of mutantions and variants in antibiotic resistance genes it would be possible to sequence the specific bacteria genes hosted by the patients and design the exact vaccine for the specific variant in the infected patient.
• RNA molecules are considered to be significantly safer than DNA vaccines, as RNAs are more easily degraded. They are cleared quickly out of the organism and cannot integrate into the genome and influence the cell's gene expression in an
• RNA vaccines to cause severe side effects like the generation of autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), vol. 2010, article ID623687).
• Transfetion with RNA requires only insertion into the cell's cytoplasm, which is easier to achieve than into the nucleus. Howerver, RNA is susceptible to RNase degradation and other natural decomposition in the cytoplasm of cells.
• polynucleotides vaccines (NAVs) of the invention will result in improved stability and therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.
• certain modified nucleosides, or combinations thereof, when introduced into the polynucleotides of the NAVs of the invention will activate the innate immune response.
• Such activating molecules are useful as adjuvants when combined with polypeptides and/or other vaccines.
• the activating molecules contain a translatable region which encodes for a polypeptide sequence useful as a vaccine, thus providing the ability to be a self-adjuvant.
• the polynucleotides of the NAVs of the present invention may be used in the prevention, treatment and diagnosis of diseases and physical disturbances caused by infectious agents.
• the polynucleotide of the present invention may encode at least one polypeptide of interest (antigen) and may be provided to an individual in order to stimulate the immune system to protect against the disease- causing agents.
• the biological activity and/or effect from an antigen or infectious agent may be inhibited and/or abolished by providing one or more polynucleotides which have the ability to bind and neutralize the antigen and/or infectious agent.
• the polynucleotides encoding an immunogen may be delivered to cells to trigger multiple innate response pathways (see International Pub. No. WO2012006377 and US Patent Publication No. US20130177639; herein incorporated by reference in its entirety).
• the polynucleotides of the NAVs of the present invention encoding an immunogen may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see International Pub. No. WO2012006372 and WO2012006369 and US Publication No. US20130149375 and US20130177640; the contents of each of which are herein incorporated by reference in their entirety).
• infectious diseases that the polynucleotide vaccines may treat includes, viral infectious diseases such as AIDS (HIV), HIV resulting in mycobacterial infection, AIDS related Cacheixa, AIDS related Cytomegalovirus infection, HIV-associated nephropathy, Lipodystrophy, AID related cryptococcal meningitis, AIDS related neutropaenia, Pneumocysitis jiroveci (Pneumocystis carinii) infections, AID related toxoplasmosis, hepatitis A, B, C, D or E, herpes, herpes zoster (chicken pox), German measles (rubella virus), yellow fever, dengue fever etc.
• viral infectious diseases such as AIDS (HIV), HIV resulting in mycobacterial infection, AIDS related Cacheixa, AIDS related Cytomegalovirus infection, HIV-associated nephropathy, Lipodystrophy, AID related cryptococcal meningitis, AIDS
• encephalitis such as Japanese encephalitis, Wester equine encephalitis and Tick-borne encephalitis (TBE)
• fungal skin diseases such as candidiasis, onychomycosis, Tinea captis/scal ringworm, Tinea corporis/body ringworm, Tinea cruris/jock itch, sporotrichosis and Tinea pedis/ Athlete's foot
• Meningitis such as Haemophilus influenza type b (Hib), Meningitis, viral, meningococcal infections and pneumococcal infection, neglected tropical diseases such as Argentine haemorrhagic fever, Leishmaniasis, Nematode/roundworm infections, Ross river virus infection and West Nile virus (WNV) disease
• Non-HIV STDs such as Trichomoniasis, Human papillomavirus (HPV) infections, sexually transmitted chlamydial diseases, Chancroid and
• Cysticercosis Echinococcosis, Trematode/Fluke infections, Trichinellosis,
• Coli 0157:H7 Escherichia coli
• Salmonellosis Salmonellosis (Salmonella species), Shingellosis (Shingella), Vibriosis and Listeriosis
• bioterrorism and potential epidemic diseases such as Ebola haemorrhagic fever, Lassa fever, Marburg haemorrhagic fever, plague, Anthrax Nipah virus disease, Hanta virus, Smallpox, Glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Tularemia (Fancisella tularensis), rubella, mumps and polio.
• NAVs of the present invention may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need.
• the NAVs of the present invention may be utilized to treat and/or prevent influenza infection, i.e. diseases and conditions related to influenza virus infection (seasonal and pandemic).
• Symptoms of the influenza infection include dry cough, fever, chills, myalgias progressing to respiratory failure and the risk of secondary bacterial infections (e.g., MRS A).
• Seasonal influenza is ubiquitous and consists of three principal strains (A [H1N1], A [H3N2], and B), which are covered by the annual vaccine.
• Pandemic flu occurs because the viruses' unique reassortment ability allowing antigenic shift as well as transfer between avian and swine flu strains.
• pandemic potential of several new strains have a high mortality rate with few available treatments. Anti-virals only provide symptomatic relief and must be given in the first 48 hours.
• the NAVs of the present invention have superior properties in that they produce much larger antibody titers, produce responses early than commercially available anti-virals and may be administered after the critical 48 hour period while retaining efficacy.
• NAVs of the invention are better designed to produce the appropriate protein conformation on translation as the NAVs co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the NAVs are presented to the cellular system in a more native fashion. Adding to the superior effects may also involve the formulations utilized which may neither serve to shield nor traffic the NAVs.
• NAVs represent a tailored active vaccine that not only can prevent infection but can limit transmission of influenza.
• the NAVs may be used to prevent pandemic influenza by reacting to emerging new strains with the very rapid NAV-based vaccine production process.
• new NAV for treating or prophylactically preventing influenza outbreaks including for emerging strains (e.g., H10N8), may be produced in less than six weeks, from the time of antigen identification to available vaccine.
• a single injection of a single antigen encoding NAV polynucleotide may provide protection for an entire flu season.
• polynucleotides of the NAVs of the invention are not self-replicating RNA.
• Self-replicating RNA have been described, for instance in US Pub. No.
• the polynucleotides of the NAVs of the invention may encode amphipathic and/or immunogenic amphipathic peptides.
• a formulation of the polynucleotides of the NAVs of the invention may further comprise an amphipathic and/or immunogenic amphipathic peptide.
• the polynucleotides comprising an amphipathic and/or immunogenic amphipathic peptide may be formulated as described in US. Pub. No. US20110250237 and International Pub. Nos. WO2010009277 and
• the polynucleotides of the NAVs of the invention may be immunostimultory.
• the polynucleotides may encode all or a part of a positive-sense or a negative- sense stranded RNA virus genome (see International Pub No. WO2012092569 and US Pub No. US20120177701, each of which is herein incorporated by reference in their entirety).
• the immunostimultory polynucleotides of the present invention may be formulated with an excipient for administration as described herein and/or known in the art (see International Pub No. WO2012068295 and US Pub No. US20120213812, each of which is herein incorporated by reference in their entirety).
• polynucleotides may further comprise a sequence region encoding a cytokine that promotes the immune response, such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF- ⁇ , GM-CFS, LT-a, or growth factors such as hGH.
• a cytokine that promotes the immune response
• a monokine such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF- ⁇ , GM-CFS, LT-a, or growth factors such as hGH.
• the vaccine formulation may include a MHC II binding peptide or a peptide having a similar sequence to a MHC II binding peptide (see International Pub Nos. WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965, each of which is herein incorporated by reference in their entirety).
• the vaccine formulations may comprise modified nicotinic compounds which may generate an antibody response to nicotine residue in a subject (see International Pub No. WO2012061717 and US Pub No. US20120114677, each of which is herein incorporated by reference in their entirety).
• the effective amount of the polynucleotides of the NAVs of the invention provided to a cell, a tissue or a subject may be enough for immune prophylaxis.
• the polynucleotides of the NAVs of the invention may be administrated with other prophylactic or therapeutic compounds.
• the prophylactic or therapeutic compound may be an adjuvant or a booster.
• booster refers to an extra administration of the prophylactic composition.
• a booster or booster vaccine may be given after an earlier administration of the prophylactic composition. The time of administration between the intial
• administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years
• the polynucleotides of the NAVs of the invention may be administered intranasally similar to the administration of live vaccines.
• the polynucleotide may be administered intramuscularly or intradermally similarly to the administration of inactivated vaccines known in the art.
• the NAVs of the invention may be used to protect against and/or prevent the transmission of an emerging or engineered threat which may be known or unknown.
• the NAVs may be formulated by the methods described herein.
• the formulation may comprise a NAV or
• the formulation may comprise polynucleotides encoding an antigen, including but not limited to a protein from an infectious agent such as a viral protein, a parasite protein or a bacterial protein.
• NAV antibodies of the present invention may be used for research in many applications, such as, but not limited to, identifying and locating intracellular and extracellular proteins, protein interaction, signal pathways and cell biology.
• the NAV may be used in to reduce the risk or inhibit the infection of influenza viruses such as, but not limited to, the highly pathogenic avian influenza virus (such as, but not limited to, H5N1 subtype) infection and human influenza virs (such as, but not limited to, H1N1 subtype and H3N2 subtype) infection.
• influenza viruses such as, but not limited to, the highly pathogenic avian influenza virus (such as, but not limited to, H5N1 subtype) infection and human influenza virs (such as, but not limited to, H1N1 subtype and H3N2 subtype) infection.
• influenza viruses such as, but not limited to, the highly pathogenic avian influenza virus (such as, but not limited to, H5N1 subtype) infection and human influenza virs (such as, but not limited to, H1N1 subtype and H3N2 subtype) infection.
• the polynucleotide described herein which may encode any of the protein sequences described in US Patent No. 8470771, the
• the NAV may be used to as a vaccine or modulating the immune response against a protein produced by a parasite.
• Bergmann-Leitner et al. in US Patent No. 8470560 the contents of which are herein incorporated by reference in its entirety, describe a DNA vaccine against the circumsporozoite protein (CSP) of malaria parasites.
• the polynucleotide may encode the CR2 binding motif of C3d and may be used a vaccine or therapeutic to modulate the immune system against the CSP of malaria parasites.
• the NAV may be used as a vaccine and may further comprise an adjuvant which may enable the vaccine to elicit a higher immune response.
• the adjuvant could be a sub-micron oil-in-water emulsion which can elicit a higher immune response in human pediatric populations (see e.g., the adjuvanted vaccines described in US Patent Publication No.
• NAVs of the present invention may be used to protect, treat or cure infection arising from contact with an infectious agent, e.g., microorganism.
• infectious agents include bacteria, viruses, fungi, protozoa and parasites.
• a microbial infection e.g., a bacterial infection
• a disease, disorder, or condition associated with a microbial or viral infection, or a symptom thereof in a subject
• a NAV comprising one or more polynucleotide encoding an antimicrobial polypeptide.
• the administration may be in combination with an antimicrobial agent (e.g., an anti-bacterial agent), e.g., an anti-microbial polypeptide or a small molecule anti-microbial compound described herein.
• the anti-microbial agents include, but are not limited to, anti-bacterial agents, anti- viral agents, anti-fungal agents, anti-protozoal agents, anti-parasitic agents, and anti-prion agents.
• Diseases, disorders, or conditions which may be associated with bacterial infections which may be treated using the NAVs of the invention include, but are not limited to one or more of the following: abscesses, actinomycosis, acute prostatitis, aeromonas hydrophila, annual ryegrass toxicity, anthrax, bacillary peliosis, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterium-related cutaneous conditions, bartonellosis, BCG-oma, botryomycosis, botulism, Brazilian purpuric fever, Brodie abscess, brucellosis, Buruli ulcer, campylobacteriosis, caries, Carrion's disease, cat scratch disease, cellulitis, chlamydia infection, cholera, chronic bacterial prostatitis, chronic recurrent multifocal osteomyelitis, clostridial necrotizing enteritis, combined
• the bacterium described herein can be a Gram-positive bacterium or a Gram- negative bacterium.
• Bacterial pathogens include, but are not limited to, Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis,
• Chlamydophila psittaci Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, coagulase Negative Staphylococcus,
• Escherichia coli enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, E. coli 0157:H7, Enter obacter sp., Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Preteus mirabilis, Proteus sps., Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcesens, Shigella flexneri, Shigella sonnei,
• Bacterial pathogens may also include bacteria that cause resistant bacterial infections, for example, clindamycin-resistant Clostridium difficile, fluoroquinolon- resistant Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRS A), multidrug -resistant Enterococcus faecalis, multidrug-resistant Enterococcus faecium, multidrug-resistance Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus (VRSA).
• MRS A methicillin-resistant Staphylococcus aureus
• VRSA vancomycin-resistant Staphylococcus aureus
• the NAVs of the present invention e.g., NAVs comprising one or more antigen-encoding polynucleotides of the present invention, may be administered in conjunction with one or more antibiotics.
• Anti-bacterial agents include, but are not limited to, aminoglycosides (e.g., amikacin (AMIKIN®), gentamicin (GARAMYCIN®), kanamycin (KANTREX®), neomycin (MYCIFRADIN®), netilmicin (NETROMYCIN®), tobramycin
• aminoglycosides e.g., amikacin (AMIKIN®), gentamicin (GARAMYCIN®), kanamycin (KANTREX®), neomycin (MYCIFRADIN®), netilmicin (NETROMYCIN®), tobramycin
• NEBCIN® Paromomycin
• ansamycins e.g., geldanamycin, herbimycin
• carbacephem e.g., loracarbef (LORABID®)
• Carbapenems e.g., ertapenem (INVANZ®), doripenem (DORIBAX®)
• imipenem/cilastatin e.g., imipenem/cilastatin
• cephalosporins first generation (e.g., cefadroxil (DURICEF®), cefazolin (ANCEF®), cefalotin or cefalothin (KEFLIN®), cefalexin (KEFLEX®), cephalosporins (second generation) (e.g., cefaclor
• CECLOR® cefamandole
• MCDOL® cefoxitin
• MEFOXIN® cefoxitin
• CEFZIL® cefprozil
• CEFTIN® cefuroxime
• ZINNAT® cephalosporins
• cefixime SUPRAX®
• cefdinir OMNICEF®, CEFDIEL®
• cefditoren SPECTRACEF®
• cefoperazone CEFOBID®
• cefotaxime CLAFORAN®
• cefpodoxime VANTIN®
• ceftazidime FORTAZ®
• ceftibuten CEDAX®
• ceftizoxime CEFIZOX®
• ceftriaxone COCPEPHIN®
• cephalosporins fourth generation
• cefepime MAXIPIME®
• cephalosporins fifth generation
• ceftobiprole ZEFTERA®
• glycopeptides e.g.
• teicoplanin TARGOCID®
• vancomycin VANCOCIN®
• telavancin VIBATIV®
• lincosamides e.g. , clindamycin (CLEOCIN®), lincomycin (LINCOCIN®)
• lipopeptide e.g. , daptomycin (CUBICIN®)
• macrolides e.g. , azithromycin (ZITHROMAX®, SUMAMED®, ZITROCIN®), clarithromycin (BIAXIN®), dirithromycin
• DYNABAC® erythromycin
• ERYTHOCIN® ERYTHROPED®
• roxithromycin troleandomycin
• TAO® troleandomycin
• TEREK® telithromycin
• TROBICIN® monobactams
• monobactams e.g. , aztreonam (AZACTAM®)
• nitrofurans e.g. , furazolidone (FUROXONE®), nitrofurantoin (MACRODANTIN®, MACROBID®
• penicillins e.g.
• amoxicillin (NOVAMOX®, AMOXIL®), ampicillin (PRINCIPEN®), azlocillin, carbenicillin (GEOCILLIN® ) , cloxacillin (TEGOPEN®), dicloxacillin (DYNAPEN®), flucloxacillin (FLOXAPEN®), mezlocillin (MEZLIN®), methicillin (STAPHCILLIN® ) , nafcillin (UNIPEN®), oxacillin (PROSTAPHLIN®), penicillin G (PENTIDS®), penicillin V (PEN-VEE-K®), piperacillin (PIPRACIL®), temocillin (NEGABAN®), ticarcillin (TICAR®)), penicillin combinations (e.g. ,
• amoxicillin/clavulanate (AUGMENTIN®), ampicillin/sulbactam (UNASYN®), piperacillin/tazobactam (ZOSYN®), ticarcillin/clavulanate (TIMENTIN®)), polypeptides (e.g. , bacitracin, colistin (COLY-MYCIN-S®), polymyxin B, quinolones (e.g. , ciprofloxacin (CIPRO®, CIPROXIN®, CIPROBAY®), enoxacin
• PENETREX® gatifloxacin
• LEVAQUIN® levofloxacin
• MAXAQUIN® lomefloxacin
• AVELOX® moxifloxacin
• mafenide SULFAMYLON®
• sulfonamidochrysoidine PRONTOSIL®
• sulfacetamide SULAMYD®, BLEPH- 10®
• sulfadiazine MICRO-SULFON®
• silver sulfadiazine SILVADENE®
• sulfamethizole THIOSULFIL FORTE®
• sulfamethoxazole GANTANOL®
• sulfanilimide sulfasalazine
• AZULFIDINE® sulfisoxazole
• trimethoprim PROLOPREVI®
• TRIMPEX® trimethoprim- sulfamethoxazole (co- trimoxazole)
• TMP-SMX BACTRIM®, SEPTRA®
• tetracyclines e.g.
• demeclocycline DECLOMYCIN®
• doxycycline VIBRAMYCIN®
• minocycline MINOCIN®
• TERRAMYCIN® oxytetracycline
• tetracycline SUMYCIN®, ACHROMYCIN® V, STECLIN®
• drugs against mycobacteria e.g. , clofazimine (LAMPRENE®), dapsone (AVLOSULFON®), capreomycin (CAPASTAT®), cycloserine (SEROMYCIN®), ethambutol (MYAMBUTOL®), ethionamide
• TRECATOR® isoniazid (I.N.H.®), pyrazinamide (ALDIN AMIDE®), rifampin (RIFADIN®, RIMACTANE®), rifabutin (MYCOBUTIN®), rifapentine
• PRIFTIN® streptomycin
• others e.g., arsphenamine (SALVARSAN®), chloramphenicol (CHLOROMYCETIN®), fosfomycin (MONUROL®), fusidic acid (FUCIDIN®), linezolid (ZYVOX®), metronidazole (FLAGYL®), mupirocin
• BACTROBAN® platensimycin, quinupristin/dalfopristin (SYNERCID®), rifaximin (XIFAXAN®), thiamphenicol, tigecycline (TIGACYL®), tinidazole (TINDAMAX®, FASIGYN®)).
• RNAV comprising one or more polynucleotides encoding an anti- viral polypeptide, e.g., an anti- viral polypeptide described herein in combination with an anti- viral agent, e.g., an anti-viral
• polypeptide or a small molecule anti- viral agent described herein are polypeptide or a small molecule anti- viral agent described herein.
• Diseases, disorders, or conditions associated with viral infections which may be treated using the NAVs of the invention include, but are not limited to, acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie infections, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, Cytomegalic inclusion disease, Kaposi sarcoma, multicentric Castleman disease, primary effusion lympho
• viral infectious agents include, but are not limited to, adenovirus; Herpes simplex, type 1 ; Herpes simplex, type 2; encephalitis virus, papillomavirus, Varicella-zoster virus; Epstein-barr virus; Human cytomegalovirus; Human herpesvirus, type 8; Human papillomavirus; BK virus; JC virus; Smallpox; polio virus, Hepatitis B virus; Human bocavirus; Parvovirus B19; Human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; Severe acute respiratory syndrome virus; Hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; Rubella virus; Hepatitis E virus; Human immunodeficiency virus (HIV); Influenza virus, type A or B; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-
• Viral pathogens may also include viruses that cause resistant viral infections.
• anti- viral agents include, but are not limited to, abacavir
• abacavir/lamivudine/zidovudine (trizivir®), aciclovir or acyclovir (CYCLOVIR®, HERPEX®, ACIVIR®, ACIVIRAX®, ZOVIRAX®, ZOVIR®), adefovir (Preveon®, Hepsera®), amantadine (SYMMETREL®), amprenavir
• AGENERASE® ampligen, arbidol, atazanavir (REYATAZ®), boceprevir, cidofovir, darunavir (PREZISTA®), delavirdine (RESCRIPTOR®), didanosine (VIDEX®), docosanol (ABREVA®), edoxudine, efavirenz (SUSTINA®,
• SELZENTRY®, CELSENTRI® methisazone, MK-2048, moroxydine, nelfinavir (VIRACEPT®), nevirapine (VIRAMUNE®), oseltamivir (TAMIFLU®), peginterferon alfa-2a (PEGASYS®), penciclovir (DENAVIR®), peramivir, pleconaril, podophyllotoxin (CONDYLOX®), raltegravir (ISENTRESS®), ribavirin (COPEGUs®, REBETOL®, RIBASPHERE®, VILONA® AND VIRAZOLE®), rimantadine (FLUMADINE®), ritonavir (NORVIR®), pyramidine, saquinavir (INVIRASE®, FORTOVASE®), stavudine, tea tree oil (melaleuca oil), tenofovir (VIREAD®), tenofovir/emtricitabine (
• VALTREX® valganciclovir
• VALCYTE® valganciclovir
• vicriviroc vidarabine
• viramidine viramidine
• zalcitabine viramidine
• zanamivir RELENZA®
• zidovudine zidothymidine (AZT), RETROVIR®, RETROVIS®.
• Diseases, disorders, or conditions associated with fungal infections which may be treated using the NAVs of the invention include, but are not limited to,
• dermatophytic fungi and keratinophilic fungi cause a variety of conditions, of which ringworms such as athlete's foot are common. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxonomic groups can evoke allergic reactions in some people.
• Fungal pathogens include, but are not limited to, Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis/posadasii, Candida albicans), Basidiomycota (e.g., Filobasidiella neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bieneusi), and
• Mucoromycotina e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera.
• anti-fungal agents include, but are not limited to, polyene antifungals (e.g., natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, hamycin), imidazole antifungals (e.g., miconazole (MICATIN®, DAKTARIN®), ketoconazole (NIZORAL®, FUNGORAL®, SEBIZOLE®), clotrimazole (LOTRIMIN®, LOTRIMIN® AF, CANESTEN®), econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole (ERTACZO®), sulconazole, tioconazole), triazole antifungals (e.g., albaconazole fluconazole, itraconazole, isavuconazole, ravucon
• echinocandins e.g., anidulafungin, caspofungin, micafungin
• others e.g., polygodial, benzoic acid, ciclopirox, tolnaftate (TINACTIN®, DESENEX®, AFTATE®), undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, haloprogin, sodium bicarbonate, allicin).
• NAVs of the invention Diseases, disorders, or conditions associated with protozoal infections which may be treated using the NAVs of the invention include, but are not limited to, amoebiasis, giardiasis, trichomoniasis, African Sleeping Sickness, American Sleeping Sickness, leishmaniasis (Kala-Azar), balantidiasis, toxoplasmosis, malaria, acanthamoeba keratitis, and babesiosis.
• Protozoal pathogens include, but are not limited to, Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp., and Babesia microti.
• anti-protozoal agents include, but are not limited to, eflornithine, furazolidone (FUROXONE®, DEPEND AL-M®), melarsoprol, metronidazole (FLAGYL®), ornidazole, paromomycin sulfate (HUMATIN®), pentamidine, pyrimethamine (DARAPRIM®), and tinidazole (TINDAMAX®, FASIGYN®).
• NAVs of the invention Diseases, disorders, or conditions associated with parasitic infections which may be treated using the NAVs of the invention include, but are not limited to, acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, balantidiasis,
• baylisascariasis baylisascariasis, chagas disease, clonorchiasis, cochliomyia, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, katayama fever, leishmaniasis, lyme disease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis
• Parasitic pathogens include, but are not limited to, Acanthamoeba, Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbug, Cestoda, chiggers,
• anti-parasitic agents include, but are not limited to, antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin), anticestodes (e.g., niclosamide, praziquantel, albendazole),
• antinematodes e.g., mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin
• anticestodes e.g., niclosamide, praziquantel, albendazole
• antitrematodes e.g., praziquantel
• antiamoebics e.g., rifampin, amphotericin B
• antiprotozoals e.g., melarsoprol, eflornithine, metronidazole, tinidazole.
• NAVs of the present invention may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. Some applications of the NAVs of the invention are outlined in Table 1.
• HPV Human Papillomavirus
• HCV Hepatitis C Virus
• HEV Human Enterovirus
• MERS-CoV Middle East Respiratory Syndrom Corona Virus
• VZV Varicella-zoster Virus
• MRSA Methicillin-resistant Staph areus
• TB tuberculosis
• WNV West Nile Virus
• VEV vesicular exanthema
• EEE Eastern equine encephalitis
• JE Japanese encephalitis
• ETEC ETEC
• Symptoms of the flu include dry cough, fever, chills, myalgias progressing to respiratory failure and the risk of secondary bacterial infections (e.g., MRSA).
• MRSA secondary bacterial infections
• Pandemic flu occurs because the viruses' unique reassortment ability allowing antigenic shift as well as transfer between avian and swine flu strains.
• pandemic potential of several new strains have a high mortality rate with few available treatments.
• Anti-virals only provide symptomatic relief and must be given in the first 48 hours.
• the NAVs of the present invention have superior properties in that they produce much larger antibody titers, produce responses early than commercially available anti-virals and may be administered after the critical 48 hour period while retaining efficacy.
• NAVs of the invention are better designed to produce the appropriate protein conformation on translation as the NAVs co-opt natural cellular machinery. Unlike tranditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the NAVs are presented to the cellular system in amore native fashion. Adding to the superior effects may also involve the
• formulations utilized which may enither serve to shield or traffic the NAVs.
• NAVs represent a tailored active vaccine that not only can prevent infection but can limit transmission of influenza.
• the NAVs may be used to prevent pandemic influenza by reacting to emerging new strains with the very rapid NAV-based vaccine production process.
• new NAV for treating or prophylactically preventing influenza outbreaks including for emerging strains (e.g., H7N9 and H10N8), may be produced in less than six weeks, from the time of antigen
• a single injection of a single antigen encoding NAV polynucleotide may provide protection for an entire flu season.
• the NAV compositions of the present invention may also be used to maintain or restore antigenic memory in a subject or population as part of a vaccination plan. With the speed and versatility of the NAV technology of the present invention, it is now possible to create a vaccination plan that spans both temporal and viral strain space.
• NAV compositions may be created which include polynucleotides that encode one or more flu year antigens.
• a flu year antigen is an antigen which is selected from a strain of influenza used as a component of a flu vaccine from a particular year.
• influenza A strain, A/Port Chalmers/l/1973(H3N2)-like virus represents one strain component of the Northern Hemisphere vaccine from 1974-1975.
• a vaccination scheme or plan is developed which allows for not only ongoing vaccination in the current year but antigenic memory booster vaccinations across years, strains, or groups thereof to establish and maintain antigenic memory in a population. In this manner, a population is less likely to succumb to any pandemic or outbreak involving recurrence of older strains or the appearance of antigens from older strains.
• any combination of prior vaccine component strains utilized to create or design an antigenic memory booster vaccine is referred to here as a reference set.
• NAVs which are antigenic memory booster vaccines are administered to boost antigenic memory across a time period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more years.
• NAVs which are antigenic memory booster vaccines are administered to boost antigenic memory for alternating historic years including every other year from the past vaccine component strains relative to a current year.
• the selection of the vaccine components can be from every 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th or more years.
• NAVs which are antigenic memory booster vaccines are administered to boost antigenic memory over ten year periods.
• NAVs which are antigenic memory booster vaccines are administered to boost antigenic memory and are selected from a number of influenza type A strains as a first selection combined with a selection from a number of influenza type B strains or other strains listed herein.
• the number of selections of type A or type B may be independently, 1, 2, 1, 4, 5, 6, 7, 8, 9, 10 or more.
• the antigenic memory booster vaccine strains for antigen encoding in the NAVs may be selected from either the Northern or Southern hemisphere vaccine components independently.
• the NAV booster vaccine may be used in a population either once or periodically to create herd immunity. Such immunity is present when greater than 30% of a population is protected.
• the components or strains of influenza which may be utilized in the antigenic memory booster vaccines include, but are not limited to, those in Tables 2-5.
• the NAV polynucleotides may encode one or more polypeptides of an influenza strain as an antigen.
• antigens include, but are not limited to those antigens encoded by the polynucleotides listed in Tables 6-18.
• the GenBank Accession Number represents either the complete or partial CDS of the encoded antigen.
• the NAV polynucleotides may comprise a region of any of the sequences listed in the tables or entire coding region of the mRNA listed. They may comprise hybrid or chimeric regions, or mimics or variants.
• strains referred to in Tables 6-14 may also be used in an antigenic memory booster vaccine as described herein.
• Influenza A virus (A/Brevig 1,497 AY744935.1
• Influenza A virus (A/Brevig 2, 151 DQ208311.1
• PA polymerase linear GI 324931 mRNA, partial cds mRNA
• Influenza A Virus (A/camel/Mongolia/82 (HlNl ) ) 379 bp M73972.1 polymerase 2 (P2) mRNA, partial cds linear GI : 324993 mRNA
• Influenza A virus (A/chicken/Hong 1, 169 U46782.1
• Influenza A virus (A/Chonnam/07/2002 (HlNl ) ) 1, 452 AY297141.1 neuraminidase (NA) mRNA, complete cds bp GI : 31871990 linear
• Influenza A virus (A/Chonnam/07/2002 (HlNl ) ) 1,137 AY297154.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140347 linear
• Influenza A virus (A/Chonnam/18/2002 (HlNl ) ) 1, 458 AY297143.1 neuraminidase (NA) mRNA, complete cds bp GI : 31871994 linear
• mRNA Influenza A virus (A/Chonnam/18/ 2 0 02 (HlNl ) ) 1, 176 AY297156.1 hemagglutinin (HA) mRNA, partial cds bp GI :32140355 linear
• Influenza A virus (A/Chonnam/19/ 2 0 02 (HlNl ) ) 1, 458 AY310410.1 neuraminidase (NA) mRNA, complete cds bp GI : 31872389 linear
• Influenza A virus (A/Chonnam/19/ 2 0 02 (HlNl ) ) 1, 167 AY299502.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140392 linear
• Influenza A virus (A/Chonnam/51/ 2 0 02 (HlNl ) ) 1, 443 AY310412.1 neuraminidase (NA) mRNA, complete cds bp GI : 31873090 linear
• Influenza A virus (A/Chonnam/51/ 2 0 02 (HlNl ) ) 1, 161 AY299498.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140384 linear
• Influenza A virus (A/Chungbuk/50/2002 (HlNl ) ) 1, 425 AY297150.1 neuraminidase (NA) mRNA, partial cds bp GI:31872010 linear
• Influenza A virus (A/Chungbuk/50/2002 (HlNl ) ) 1, 161 AY299506.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140400 linear
• Influenza A virus (A/Denmark/40/2000 (HlNl ) ) 1, 458 AJ518095.1
• Influenza A virus (A/Denver/1/57 (HlNl ) ) 379 bp AF305216.1 neuraminidase mRNA, partial cds linear GI : 10732818 mRNA
• Influenza A virus (A/Denver/1/57 (HlNl ) ) 442 bp AF305217.1 matrix protein gene, partial cds linear GI : 10732820 mRNA
• Influenza A virus (A/Denver/1/57 (HlNl ) ) 215 bp AF305218.1 hemagglutinin gene, partial cds linear GI : 10732822 mRNA
• hemagglutinin linear GI 1912348 precursor (HA) mRNA, partial cds mRNA
• Influenza A virus (A/duck/Bavaria/1/77 1, 777 AF091313.1
• Influenza A virus (A/duck/Bavaria/2/77 (HlNl ) ) 981 bp U47308.1 hemagglutinin precursor (HA) mRNA, partial linear GI : 1912346 cds mRNA
• Influenza A virus (A/duck/Eastern 1, 458 EU429749.1
• Influenza A virus (A/Duck/Ohio/118C/93 1,410 AF250361.2
• Influenza A virus (A/Duck/Ohio/175/86 (HlNl)) 1,410 AF250358.2 neuraminidase (NA) gene, complete cds bp GI : 13260565 linear
• Influenza A virus (A/Duck/Ohio/194/86 (HlNl)) 1,410 AF250360.2 neuraminidase (NA) gene, complete cds bp GI : 13260573 linear
• Influenza A virus (A/Duck/Ohio/30/86 (HlNl)) 1,410 AF250359.2 neuraminidase (NA) gene, complete cds bp GI : 13260570 linear
• Influenza A Virus (A/Fi j i/ 15899 / 83 (HlNl ) ) 2,341 AJ564805.1 mRNA for PB2 protein bp GI : 31442134 linear
• Influenza A Virus (A/Fi j i/ 15899 / 83 (HlNl ) ) 2,113 AJ564807.1 partial mRNA for PBl protein bp GI : 31442138 linear
• Influenza A virus (A/FM/1/47 (HlNl)) 1,395 AF250357.2 neuraminidase (NA) gene, complete cds bp GI : 13260561 linear
• Influenza A virus (A/goose/Hong 261 bp U48284.1
• Influenza A virus (A/Gwangju/55/2002 (HlNl ) ) 1, 431 AY297151.1 neuraminidase (NA) mRNA, complete cds bp GI : 31872012 linear
• Influenza A virus (A/Gwangju/55/2002 (HlNl ) ) 1,179 AY299507.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140402 linear
• Influenza A virus (A/Gwangju/57/2002 (HlNl ) ) 1, 446 AY297152.1 neuraminidase (NA) mRNA, complete cds bp GI:31872014 linear
• Influenza A virus (A/Gwangju/57/2002 (HlNl ) ) 1, 167 AY299508.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140404 linear
• Influenza A virus (A/Gwangju/58/2002 (H1N1 ) ) 1, 176 AY299509.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140406 linear
• Influenza A virus (A/Gwangju/90/2002 (H1N1 ) ) 1, 446 AY297147.1 neuraminidase (NA) mRNA, complete cds bp GI : 31872002 linear
• Influenza A virus (A/Gwangju/90/2002 (H1N1 ) ) 1, 164 AY299499.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140386 linear
• Influenza A virus (A/Hong 1, 403 AJ518101.1
• Influenza A virus (A/Hong 1, 352 AJ518102.1
• Influenza A virus (A/London/1/1918 (H1N1 ) ) 563 bp AY184805.1 hemagglutinin (HA) mRNA, partial cds linear GI : 32395285 mRNA
• Influenza A virus (A/London/1/1919 (H1N1 ) ) 563 bp AY184806.1 hemagglutinin (HA) mRNA, partial cds linear GI : 32395287 mRNA
• Influenza A virus (A/Loygang/4/1957 (H1N1 ) ) 1, 565 M76604.1 nucleoprotein mRNA, complete cds bp GI : 324255 linear
• Influenza A virus (A/Lyon/651/2001 (H1N1 ) ) 1,318 AJ518103.1 partial NA gene for neuraminidase, genomic bp GI : 31096416
• Influenza A virus (A/mallard/Alberta/119/98 947 bp AY664487.1
• hemagglutinin linear GI 1912350 precursor (HA) mRNA, partial cds mRNA
• Influenza A virus 1 777 AF091309.1
• HA hemagglutinin precursor
• Influenza A virus (A/New Jersey/4/1976 (HlNl ) ) 1, 565 M76605.1 nucleoprotein mRNA, complete cds bp GI : 324581 linear
• Influenza A virus (A/New Jersey/8/1976 (HlNl ) ) 1, 565 M76606.1 nucleoprotein mRNA, complete cds bp GI : 324583 linear
• Influenza A virus (A/New_York/ 1 / 18 (HlNl ) ) 1,220 AF116576.1 hemagglutinin (HA) mRNA, partial cds bp GI : 4325019 linear
• Influenza A virus (A/Ohio/3523/1988 (HlNl ) ) 1, 565 M76602.1 nucleoprotein mRNA, complete cds bp GI : 324889 linear
• Influenza A virus (A/Pusan/22/2002 (HlNl ) ) 1, 455 AY310411.1 neuraminidase (NA) mRNA, complete cds bp GI : 31872391 linear
• Influenza A virus (A/Pusan/22/2002 (HlNl ) ) 1,149 AY299503.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140394 linear
• Influenza A virus (A/Pusan/23/2002 (HlNl ) ) 1, 440 AY297144.1 neuraminidase (NA) mRNA, complete cds bp GI : 31871996 linear
• Influenza A virus (A/Pusan/23/2002 (HlNl ) ) 1, 158 AY297157.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140357 linear
• Influenza A virus (A/Pusan/24/2002 (HlNl ) ) 1, 449 AY297145.1 neuraminidase (NA) mRNA, complete cds bp GI : 31871998 linear
• Influenza A virus (A/Pusan/24/2002 (HlNl ) ) 1,128 AY299494.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140376 linear
• Influenza A virus (A/Pusan/44/2002 (HlNl ) ) 1, 431 AY297148.1 neuraminidase (NA) mRNA, complete cds bp GI:31872004 linear
• Influenza A virus (A/Pusan/44/2002 (HlNl ) ) 1, 167 AY299504.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140396 linear
• Influenza A virus (A/Pusan/45/2002 (HlNl ) ) 1, 434 AY297146.1 neuraminidase (NA) mRNA, complete cds bp GI:31872000 linear
• Influenza A virus (A/Pusan/45/2002 (HlNl ) ) 1, 167 AY299496.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140380 linear
• Influenza A virus (A/Pusan/46/2002 (HlNl ) ) 1, 176 AY299497.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140382 linear
• Influenza A virus (A/Pusan/47/2002 (HlNl ) ) 1, 437 AY297149.1 neuraminidase (NA) mRNA, complete cds bp GI:31872008 linear
• Influenza A virus (A/Pusan/47/2002 (HlNl ) ) 1,170 AY299505.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140398 linear
• Influenza A virus (A/Saudi 789 bp AJ519463.1
• genomic RNA for non structural protein 2, genomic RNA
• Influenza A virus (A/Seoul/11/2002 (HlNl ) ) 1, 452 AY297142.1 neuraminidase (NA) mRNA, complete cds bp GI : 31871992 linear
• Influenza A virus (A/Seoul/11/2002 (HlNl ) ) 1, 176 AY297155.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140349 linear
• Influenza A virus (A/Seoul/13/2002 (HlNl ) ) 1, 452 AY310409.1 neuraminidase (NA) mRNA, complete cds bp GI:31872387 linear
• Influenza A virus (A/Seoul/13/2002 (HlNl ) ) 1, 167 AY299500.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140388 linear
• Influenza A virus (A/Seoul/15/2002 (HlNl ) ) 1, 449 AY297140.1 neuraminidase (NA) mRNA, complete cds bp GI : 31871988 linear
• Influenza A virus (A/Seoul/15/2002 (HlNl ) ) 1,149 AY299501.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140390 linear
• Influenza A virus (A/Seoul/33/2002 (HlNl ) ) 1, 437 AY310407.1 neuraminidase (NA) mRNA, complete cds bp GI:31872383 linear
• Influenza A virus (A/Seoul/33/2002 (HlNl ) ) 1, 167 AY299495.1 hemagglutinin (HA) mRNA, partial cds bp GI : 32140378 linear
• Influenza A virus (A/swine/Cotes 1, 116 AM490219.1 d 'Armor/0118/2006 (HlNl ) ) partial mRNA for bp GI:222062898 haemagglutinin precursor (HA1 gene) linear
• Influenza A virus (A/swine/Cotes 1, 043 AM490223.1 d'Armor/0136_18/2006 (HlNl) ) partial mRNA for bp GI:222062906 haemagglutinin precursor (HA1 gene) linear
• Influenza A virus (A/swine/Cotes 1,089 AM490220.1 d 'Armor/0184/2006 (HlNl ) ) partial mRNA for bp GI:222062900 haemagglutinin precursor (HA1 gene) linear
• Influenza A virus (A/swine/Cotes 1,068 AM490221.1 d 'Armor/0227/2005 (HlNl ) ) partial mRNA for bp GI:222062902 haemagglutinin precursor (HA1 gene) linear
• Influenza A virus (A/swine/Cotes 1,024 AM490222.1 d 'Armor/0250/2006 (HlNl ) ) partial mRNA for bp GI : 222062904 haemagglutinin precursor (HA1 gene) linear
• Influenza A virus (A/swine/Cotes 1,011 AJ517820.1 d 'Armor/736/2001 (HlNl ) ) partial HA gene for bp GI : 38422533
• Influenza A virus (A/Swine/England/195852/92 1,410 AF250366.2
• Influenza A virus 1 730 Z46434.1
• Influenza A virus (A/swine/Hokkaido/2/81 981 bp U47306.1
• HA hemagglutinin precursor
• Influenza A virus (A/swine/Hokkaido/2/81 1, 778 AF091306.1
• Influenza A virus (A/swine/Hong 416 bp U47817.1
• Influenza A virus (A/swine/Hong 286 bp U48286.1
• Influenza A virus (A/swine/Hong 379 bp U48283.1
• Influenza A virus (A/swine/Hong 308 bp U48850.1
• Influenza A virus (A/swine/Hong 1, 330 U45452.1
• neuraminidase (NA) mRNA linear GI : 1912356 partial cds mRNA
• Influenza A virus (A/swine/Hong 328 bp U48287.1
• Influenza A virus (A/swine/Hong 240 bp U48282.1
• Influenza A virus (A/swine/Hong 336 bp U48851.1
• Influenza A virus (A/swine/Iowa/15/30 (HlNl ) ) 981 bp U47305.1 hemagglutinin precursor (HA) mRNA, partial linear GI : 1912340 cds mRNA
• Influenza A virus (A/swine/Iowa/15/30 (HlNl)) 1, 778 AF091308.1 segment 4 hemagglutinin precursor (HA) mRNA, bp GI : 4585158 complete cds linear
• Influenza A virus (A/Swine/Iowa/30 (HlNl)) 1,410 AF250364.2 neuraminidase (NA) gene, complete cds bp GI : 13260586 linear
• Influenza A virus (A/swine/ Iowa/ 17672 / 88 981 bp U47304.1
• HA hemagglutinin precursor
• Influenza A virus (A/swine/ Italy- 1, 777 AF091315.1
• Influenza A virus (A/NJ/11/76 (HlNl)) 1,410 AF250363.2 neuraminidase (NA) gene, complete cds bp GI : 13260583 linear
• Influenza A virus (A/Swine/Quebec/ 192 / 81 1, 438 U86144.1
• Influenza A Virus (A/swine/Schleswig- 1, 554 Z46438.1
• Influenza A virus 1 565 M76607.1
• Influenza A virus (A/Taiwan/0016/2000 (H1N1)) 303 bp AY303752.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330993 partial cds mRNA
• Influenza A virus (A/Taiwan/0030/2000 (H1N1)) 303 bp AY303704.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330897 partial cds mRNA
• Influenza A virus (A/Taiwan/0032/2002 (H1N1 ) ) 494 bp AY604804.1 hemagglutinin mRNA, partial cds linear GI : 50727488 mRNA
• Influenza A virus (A/Taiwan/0061/2002 (H1N1 ) ) 494 bp AY604795.1 hemagglutinin mRNA, partial cds linear GI : 50727470 mRNA
• Influenza A virus (A/Taiwan/0069/2002 (H1N1 ) ) 494 bp AY604803.1 hemagglutinin mRNA, partial cds linear GI : 50727486 mRNA
• Influenza A virus (A/Taiwan/0078/2002 (H1N1 ) ) 494 bp AY604805.1 hemagglutinin mRNA, partial cds linear GI : 50727490 mRNA
• Influenza A virus (A/Taiwan/0094/2002 (H1N1 ) ) 494 bp AY604797.1 hemagglutinin mRNA, partial cds linear GI : 50727474 mRNA
• Influenza A virus (A/Taiwan/0116/2002 (H1N1 ) ) 494 bp AY604796.1 hemagglutinin mRNA, partial cds linear GI : 50727472 mRNA
• Influenza A virus (A/Taiwan/0130/96 (H1N1)) 303 bp AY303707.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330903 partial cds mRNA
• Influenza A virus (A/Taiwan/0132/96 (H1N1)) 303 bp AY303708.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330905 partial cds mRNA
• Influenza A virus (A/Taiwan/0211/96 (H1N1)) 303 bp AY303709.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330907 partial cds mRNA
• HA HA
• H1N1 H1N1
• PB1 polymerase basic protein 1
• H1N1 hemagglutinin linear GI : 14571939 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/0255/96 (H1N1)) 303 bp AY303711.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330911 partial cds mRNA
• H1N1 hemagglutinin linear GI : 14571941 (HA) mRNA, partial cds mRNA
• H1N1 hemagglutinin linear GI : 14571943 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/0342/96 (H1N1)) 303 bp AY303714.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330917 partial cds mRNA
• H1N1 hemagglutinin linear GI : 14571945 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/0562/95 (H1N1)) 303 bp AY303720.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330929 partial cds mRNA
• H1N1 hemagglutinin linear GI : 14571949 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/0563/95 (H1N1)) 303 bp AY303721.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330931 partial cds mRNA
• Influenza A virus (A/Taiwan/0657/95 (H1N1)) 303 bp AY303724.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330937 partial cds mRNA
• Influenza A virus (A/Taiwan/0859/2002 (H1N1 ) ) 494 bp AY604801.1 hemagglutinin mRNA, partial cds linear GI : 50727482 mRNA
• H1N1 hemagglutinin linear GI : 14571953 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/0983/2002 (H1N1 ) ) 494 bp AY604800.1 hemagglutinin mRNA, partial cds linear GI : 50727480 mRNA
• Influenza A virus (A/Taiwan/1007/2006 (H1N1 ) ) 507 bp EU068163.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452199 mRNA
• Influenza A virus (A/Taiwan/1015/2006 (H1N1 ) ) 507 bp EU068171.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452215 mRNA
• Influenza A virus (A/Taiwan/112/1996-1 (H1N1 ) ) 1, 176 AF026153.1 haemagglutinin (HA) mRNA, partial cds bp GI:2554950 linear
• Influenza A virus (A/Taiwan/117/1996-1 (HlNl ) ) 1, 176 AF026155.1 haemagglutinin (HA) mRNA, partial cds bp GI : 2554954 linear
• Influenza A virus (A/Taiwan/117/1996-2 (HlNl ) ) 1, 176 AF026156.1 haemagglutinin (HA) mRNA, partial cds bp GI:2554956 linear
• Influenza A virus (A/Taiwan/117/1996-3 (HlNl ) ) 1, 176 AF026157.1 haemagglutinin (HA) mRNA, partial cds bp GI:2554958 linear
• Influenza A virus (A/Taiwan/118/1996-1 (HlNl ) ) 1, 176 AF026158.1 haemagglutinin (HA) mRNA, partial cds bp GI:2554960 linear
• Influenza A virus (A/Taiwan/118/1996-2 (HlNl ) ) 1, 176 AF026159.1 haemagglutinin (HA) mRNA, partial cds bp GI : 2554962 linear
• Influenza A virus (A/Taiwan/118/1996-3 (HlNl ) ) 1, 176 AF026160.1 haemagglutinin (HA) mRNA, partial cds bp GI : 2554964 linear
• Influenza A virus (A/Taiwan/1184/99 (HlNl)) 303 bp AY303726.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330941 partial cds mRNA
• Influenza A virus (A/Taiwan/1190/95 (HlNl)) 303 bp AY303727.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330943 partial cds mRNA
• Influenza A virus (A/Taiwan/1523/2003 (HlNl ) ) 494 bp AY604808.1 hemagglutinin mRNA, partial cds linear GI : 50727496 mRNA
• Influenza A virus (A/Taiwan/1566/2003 (HlNl ) ) 494 bp AY604806.1 hemagglutinin mRNA, partial cds linear GI : 50727492 mRNA
• Influenza A virus (A/Taiwan/1769/96 (HlNl ) ) 875 bp AF138710.2 matrix protein Ml (M) mRNA, partial cds linear GI : 4996871 mRNA
• Influenza A virus (A/Taiwan/1906/2002 (HlNl ) ) 494 bp AY604799.1 hemagglutinin mRNA, partial cds linear GI : 50727478 mRNA
• Influenza A virus (A/Taiwan/1922/2002 (HlNl ) ) 494 bp AY604802.1 hemagglutinin mRNA, partial cds linear GI : 50727484 mRNA
• Influenza A virus (A/Taiwan/2069/2006 (HlNl ) ) 507 bp EU068168.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452209 mRNA Influenza A virus (A/Taiwan/2157/2001 (H1N1)) 303 bp AY303733.1 polymerase basic protein 1 (PB1) mRNA, linear GI :32330955 partial cds mRNA
• Influenza A virus (A/Taiwan/2175/2001 (H1N1)) 561 bp AY303734.1 hemagglutinin (HA) mRNA, partial cds linear GI : 32330957 mRNA
• Influenza A virus (A/Taiwan/2200/95 (H1N1)) 303 bp AY303737.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330963 partial cds mRNA
• Influenza A virus (A/Taiwan/2966/2006 (H1N1 ) ) 507 bp EU068170.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452213 mRNA
• Influenza A virus (A/Taiwan/3168/2005 (H1N1 ) ) 507 bp EU068174.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452221 mRNA
• Influenza A virus (A/Taiwan/3355/97 (H1N1)) 303 bp AY303739.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330967 partial cds mRNA
• Influenza A virus (A/Taiwan/3361/2001 (H1N1)) 303 bp AY303740.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330969 partial cds mRNA
• Influenza A virus (A/Taiwan/3361/2001 (H1N1)) 561 bp AY303741.1 hemagglutinin (HA) mRNA, partial cds linear GI : 32330971 mRNA
• Influenza A virus (A/Taiwan/3518/2006 (H1N1 ) ) 507 bp EU068169.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452211 mRNA
• Influenza A virus (A/Taiwan/3896/2001 (H1N1)) 303 bp AY303746.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330981 partial cds mRNA
• Influenza A virus (A/Taiwan/3896/2001 (H1N1)) 561 bp AY303747.1 hemagglutinin (HA) mRNA, partial cds linear GI : 32330983 mRNA
• Influenza A virus (A/Taiwan/4050/2003 (H1N1 ) ) 494 bp AY604807.1 hemagglutinin mRNA, partial cds linear GI : 50727494 mRNA
• Influenza A virus (A/Taiwan/4054/2006 (H1N1 ) ) 507 bp EU068160.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452193 mRNA
• Influenza A virus (A/Taiwan/4360/99 (H1N1)) 303 bp AY303748.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330985 partial cds mRNA
• Influenza A virus (A/Taiwan/4415/99 (H1N1)) 303 bp AY303749.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330987 partial cds mRNA Influenza A virus (A/Taiwan/4509/2006 (H1N1 ) ) 507 bp EU068165.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452203 mRNA
• H1N1 hemagglutinin linear GI : 14571969 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/4845/99 (H1N1)) 303 bp AY303750.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330989 partial cds mRNA
• H1N1 hemagglutinin linear GI : 14571971 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/5010/2006 (H1N1 ) ) 507 bp EU068167.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452207 mRNA
• H1N1 hemagglutinin linear GI : 14571973 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/5063/99 (H1N1)) 303 bp AY303751.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330991 partial cds mRNA
• Influenza A virus (A/Taiwan/5084/2006 (H1N1 ) ) 507 bp EU068166.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452205 mRNA
• Influenza A virus (A/Taiwan/511/96 (H1N1 ) ) 875 bp AF138708.2 matrix protein Ml (M) mRNA, partial cds linear GI : 4996867 mRNA
• Influenza A virus (A/Taiwan/557/2006 (H1N1 ) ) 507 bp EU068156.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452185 mRNA
• Influenza A virus (A/Taiwan/562/2006 (H1N1 ) ) 507 bp EU068159.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452191 mRNA
• H1N1 hemagglutinin linear GI : 14571925 (HA) mRNA, partial cds mRNA
• Influenza A virus (A/Taiwan/5779/98 (H1N1)) 303 bp AY303702.1 polymerase basic protein 1 (PB1) mRNA, linear GI : 32330893 partial cds mRNA
• Influenza A virus (A/Taiwan/6025/2005 (H1N1 ) ) 507 bp EU068172.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452217 mRNA
• Influenza A virus (A/Taiwan/607/2006 (H1N1 ) ) 507 bp EU068157.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452187 mRNA
• Influenza A virus (A/Taiwan/615/2006 (H1N1 ) ) 507 bp EU068162.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452197 mRNA
• Influenza A virus (A/Taiwan/645/2006 (H1N1 ) ) 507 bp EU068164.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452201 mRNA
• Influenza A virus (A/Taiwan/680/2005 (H1N1 ) ) 507 bp EU068173.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452219 mRNA
• Influenza A virus (A/Taiwan/719/2006 (H1N1 ) ) 507 bp EU068158.1 hemagglutinin (HA) mRNA, partial cds linear GI : 158452189 mRNA Influenza A virus 1,410 EU021285.1
• Influenza A virus 1 777 AF091310.1
• Influenza A virus (A/turkey/North 1, 565 M76609.1 Carolina/1790/1988 (HlNl ) ) nucleoprotein mRNA, bp GI : 325096 complete cds linear
• Influenza A virus (A/Wilson-Smith/1933 (HlNl ) ) 1,497 EU330203.1 nucleocapsid protein (NP) mRNA, complete cds bp GI : 167989512 linear
• Influenza A virus (A/WI/4754/1994 (HlNl) ) PB1 235 bp U53156.1 (PB1) mRNA, partial cds linear GI : 1399590 mRNA
• Influenza A virus (A/WI/4754/1994 (HlNl ) ) PB2 168 bp U53158.1 (PB2) mRNA, partial cds linear GI : 1399594 mRNA
• Influenza A virus (A/WI/4754/1994 (HlNl) ) PA 621 bp U53160.1 (PA) mRNA, partial cds linear GI : 1399598 mRNA
• Influenza A virus (A/WI/4754/1994 (HlNl) ) 1, 778 U53162.1 hemagglutinin (HA) mRNA, complete cds bp GI : 1399602 linear

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