CA2615372A1 - Influenza virus-like particles (vlps) comprising hemagglutinin - Google Patents

Influenza virus-like particles (vlps) comprising hemagglutinin Download PDF

Info

Publication number
CA2615372A1
CA2615372A1 CA002615372A CA2615372A CA2615372A1 CA 2615372 A1 CA2615372 A1 CA 2615372A1 CA 002615372 A CA002615372 A CA 002615372A CA 2615372 A CA2615372 A CA 2615372A CA 2615372 A1 CA2615372 A1 CA 2615372A1
Authority
CA
Canada
Prior art keywords
plant
influenza
virus
vlps
vlp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002615372A
Other languages
French (fr)
Inventor
Marc-Andre D'aoust
Manon Couture
Frederic Ors
Sonia Trepanier
Pierre-Olivier Lavoie
Michele Dargis
Louis-Philippe Vezina
Nathalie Landry
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medicago Inc
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to SI200831024T priority Critical patent/SI2173886T1/en
Priority to CA2693956A priority patent/CA2693956C/en
Priority to EA201000195A priority patent/EA018206B1/en
Priority to US12/669,033 priority patent/US20100239610A1/en
Priority to CN2008801070729A priority patent/CN101883856B/en
Priority to ES08783201T priority patent/ES2428384T3/en
Priority to NZ582360A priority patent/NZ582360A/en
Priority to PT87832010T priority patent/PT2173886E/en
Priority to EP08783201.0A priority patent/EP2173886B1/en
Priority to PL08783201T priority patent/PL2173886T3/en
Priority to DK08783201.0T priority patent/DK2173886T3/en
Priority to PCT/CA2008/001281 priority patent/WO2009009876A1/en
Priority to MYPI2010000142A priority patent/MY155236A/en
Priority to BRPI0813724-2A priority patent/BRPI0813724B1/en
Priority to KR1020107002538A priority patent/KR101541330B1/en
Priority to AU2008278222A priority patent/AU2008278222B2/en
Priority to MX2010000525A priority patent/MX2010000525A/en
Priority to HK10109048.1A priority patent/HK1142627B/en
Priority to JP2010516334A priority patent/JP5624465B2/en
Priority to DK12183854.4T priority patent/DK2610345T3/en
Priority to BRPI0906960-7A priority patent/BRPI0906960B1/en
Priority to JP2010542486A priority patent/JP2011509661A/en
Priority to SG2013004577A priority patent/SG187500A1/en
Priority to EP12183854.4A priority patent/EP2610345B1/en
Priority to NZ587108A priority patent/NZ587108A/en
Priority to AU2009202819A priority patent/AU2009202819B2/en
Priority to PCT/CA2009/000032 priority patent/WO2009076778A1/en
Priority to EP09700061A priority patent/EP2238253B1/en
Priority to MX2010007962A priority patent/MX2010007962A/en
Priority to ES12183854.4T priority patent/ES2554703T3/en
Priority to KR1020107018343A priority patent/KR20100120157A/en
Priority to PT121838544T priority patent/PT2610345E/en
Priority to EA201001198A priority patent/EA034733B1/en
Priority to CN201310021693.8A priority patent/CN103122354B/en
Priority to CA2707235A priority patent/CA2707235C/en
Priority to US12/863,772 priority patent/US20100310604A1/en
Priority to CN2009801097815A priority patent/CN101978066A/en
Priority to KR1020167010959A priority patent/KR101956910B1/en
Publication of CA2615372A1 publication Critical patent/CA2615372A1/en
Priority to US13/003,570 priority patent/US9492528B2/en
Priority to IL203018A priority patent/IL203018A/en
Priority to CR11209A priority patent/CR11209A/en
Priority to IS8874A priority patent/IS2920B/en
Priority to MA32604A priority patent/MA31681B1/en
Priority to ZA2010/00972A priority patent/ZA201000972B/en
Priority to IL206967A priority patent/IL206967A/en
Priority to CR11612A priority patent/CR11612A/en
Priority to IS8919A priority patent/IS2982B/en
Priority to MA33088A priority patent/MA32439B1/en
Priority to ZA2010/05917A priority patent/ZA201005917B/en
Priority to US13/734,886 priority patent/US9452210B2/en
Priority to US13/748,531 priority patent/US9458470B2/en
Priority to HK13113472.5A priority patent/HK1186207B/en
Priority to IL230708A priority patent/IL230708A/en
Priority to JP2014039035A priority patent/JP5990207B2/en
Priority to JP2015231583A priority patent/JP2016032477A/en
Priority to US15/256,119 priority patent/US10190132B2/en
Priority to US16/219,306 priority patent/US11434497B2/en
Priority to US17/815,503 priority patent/US20230044454A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
    • C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06F—ELECTRIC DIGITAL DATA PROCESSING
    • G06F9/00—Arrangements for program control, e.g. control units
    • G06F9/06—Arrangements for program control, e.g. control units using stored programs, i.e. using an internal store of processing equipment to receive or retain programs
    • G06F9/46—Multiprogramming arrangements
    • G06F9/50—Allocation of resources, e.g. of the central processing unit [CPU]
    • G06F9/5005—Allocation of resources, e.g. of the central processing unit [CPU] to service a request
    • G06F9/5027—Allocation of resources, e.g. of the central processing unit [CPU] to service a request the resource being a machine, e.g. CPUs, Servers, Terminals
    • G06F9/505—Allocation of resources, e.g. of the central processing unit [CPU] to service a request the resource being a machine, e.g. CPUs, Servers, Terminals considering the load
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/517—Plant cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16171—Demonstrated in vivo effect
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16223—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—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
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06F—ELECTRIC DIGITAL DATA PROCESSING
    • G06F2209/00—Indexing scheme relating to G06F9/00
    • G06F2209/50—Indexing scheme relating to G06F9/50
    • G06F2209/508—Monitor

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Pulmonology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Software Systems (AREA)
  • Theoretical Computer Science (AREA)
  • Oncology (AREA)
  • General Physics & Mathematics (AREA)

Abstract

A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA
in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plants lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors.
The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Description

1NrL.liENZA.V3RUS-LIKE r'AZI`I"ICI..Ef`a ('1LP5; ;30Jtf1Pt-115144 HEMAGGLL1TINfN

FIELD OF YNVENTION

(0001] The present invention relates to the production of virus-like particles. More specitically, the present invention is directed to the production of virus-like particles comprising influenza antigens.
(0002] BACKGROUND OF THE INVENTION
[0003] Influenza is the leading cause of death in humans due to a respiratory viras.
t0 Common symptoms include fever, sore throat, shor[ness of breath, and muscle soreness, among others. During flu season, influenza viruses infect 10-20% of the population worldwide, leading to 250-500,000 deaths annually
(0004] Tnfluenza viruses are enveloped virus that bud from the plasma membrane of infected mammalian cells. They are classified into types A, B, or C, based on the nucleoproteins and matrix protein antigens present. Lnfluenza type A viruses rr-.ay be further divided into subtypes according to the eombination of hemagglutinin (HA) and rteuraminidase (NA) surface glycoprateins presented. HA governs the ability of the virus to bind to and penetrate the host celL NA removes terminal sialic acid residues from glycan chains on host cell aad viral surface proteins, which prevents viral aggregation and facilitates virus mobility. Currently, 16 HA (H1-1116) and 9 NA (N1-N9) subtypes are recognized. Each type A influenza virus presents one type of HA
and one type of NA glycoprotein. Generally, cach ~.:btype exhi' is sfk:c:ies specificity;
for example, all HA and NA subtypes are known to infect birds, while only subtypes Hl, H2, H3, H5,1=17, H9. H10. Nl, N2, N3 and N7 have been shown to infact humans (Horimoto 2006; Suzuki 2005). Intluenza viruses eomprising H5, H7 and H9 are considered the most highly pathogenic forms of intluenza A viruses, and are most likely to cause future pandemics.
[0005] Influenza pandemics are usually caused by highly transnuttable and virulent influenza viruses, and can lead to elevated levels of illness and death globally. The A '.;43Lyi-.:S rLii,.
century. i i1e Spanisil t`3u, caused by an H1N1 viras, in 1918-1919 ied to the deaths of over 50 million people worldwide between 1917 and 1920. Presently, the risk of the emergence of a new subtype, or of the transntission to humans of a subtype endemic in animals, is always present. Of particular concem is a highly virulent form of avian int7uenza (also called "bird flu"), outbreaks of which have been reported in several countries around the world. In many cases, this bud flu can result in mortality rates approaching 100% within 48 bours. The spread of the avian influenza virus (H5N1), first identified in Hong Kong in 1997, to other Asian countries and Europe has been postulated to be linked to the rnigratory patterns of wild birt ls.

(0006j The current method of combating influenza in humans is by annual vaccination. The vaccine is usually a combination of several strains that are predicted to be the dominam strains for the coming "flu-season". The prediction is coordinated by the World Health Organization. Generally, the number of vaccine doses produced each year is not sufficient to vaccirnate the world's population. For example, Canada and the United-States obtain enough vaccines doses to immitnize about one third of their population, while only 17% of the population of the Furopean Union can be vaccirlated. It is evident that current worldwide production of influenza vaccine would be insufficient in the face of a worldwide flu pandemic. Even if the necessary annual production could somehow be met in a given year, the dominant strains change from year to year, thus stockpiling at low-need times in the year is not practicaL
Economical, large scale production of an effective influenza vaccine is of significant interest to govemment and private industry alike.

[00071 The viral stocks for use in vaccines are produced in fertilized eggs.
The virus particles are harvested, and for an inactivated viral vaccine, disrupted by detergent to inactivate. Live attenuated vaccines are made of influenza viruses that were adapted for growth at low temperature which means that at norrual body temperature, the vaccine is attenuated. Such a vaccine is licensed in USA for use in individuals from 5 to 49 years of age. Inactivated whole virus vaccines are rendered harmless by inactivation with chemical agents and they have been produced in embryonic eggs or mammalian cell culture. All these types of vaccine show some specific advantages and disadvantages. One advantage of vaccines derived from whole viruses is the type ;mtibody response wizile vaccines made of whole viruses induce both an antibody (humoral) and cellular response. Even though a functional antibody response is a criterion for Iicensure th.alt correlates with protection induced by a vaccine, there is increasing evidence that a T-cell response is also important in influenza immunity -this may also provide better protection in the elderly.

[00081 In order to induce a cellular immune response, vaccines made of whole viruses were developed. Due to the high pathogenicity of the influenza strain (e.g.
H5N1), these vaccines are produced in BL3+ facility. For highly pathogenic influenza strains such as H5N1, some manufacturers bave rnodified the hemagglutinin gene sequence in order to reduce the pathogenicity of the influenza strain and to make it avirulent and more easily produced in embryonic eggs or mammalian cell culture.
Others also use reassortant influenza strains in which the genetic sequences for the hemagglutinin and neuraminidase proteins are cloned in a$igh-yielding low l5 pathogenic influenza donor strain (A1PRJ8134; Quan F-S et al, 2007). While these methods may produce useful vaccines, they do not provide a solution to the need for high-volume, low cost and fast production of vaecines in the scale neeessary to meet the global need in a nomW year, and would almost certainly be insufficient in the face of a pandemic.

[0009] Using this reverse genetic technology, one might also need to mutate the genetic sequence of the HA protein to make it avirulent. For highly pathogenic influenza strains, the production of whole virus vaccines either requires confinement procedures or the resulting vaccines do not exactly match the genetic sequence of the circulating vin-s. In the case of live-attenuated vaceines, thet+e is still a risk that the administered vaccine can recombine with an influenza virus from the host, leading to a new influenza virus.

[0010] While this method maintains the antigenic epitope and post-translatRonal modifications, there are a number of drawbacks to this method, including the risk of contamination due to the use of whole virus and variable yields depending on vinis strain. Sub-optisnal levels of protection may result from genetic heterogeneity in the virus due to its introduction in[o eggs. Other disadvantages includes eztensive and long production times. Also, persons hypersensitive to egg proteins may not be eligible candidates for rec^iving the vaccine.

[0011] In the case of a pandemic, split vaccine production is limited by the need to adapt the strain for growth in eggs and the variable production yields achieved.
Although this technology has been used for years for the production of seasone]
vaccines, it can hardly respond in a reasonable timefrarne to a pandemic and worldwide manufacturing capacity is limited.

[0012] To avoid the use of eggs, influenza viruses have also been produced in to mammalian ceil culture, for example in MDCK or PERC.6 cells, or the like.
Another approach is reverse genetics, in w.iich viruses are produced by cell transformation with viral genes_ These methods, bowever, also requires the use of whole virus as well as elaborate methods and specific culture environme.nts.

[0013] Several reoombinant products have been developed as recombinant influenza vaccine candidates. These approaches have focused on the expression, production, and purification of influenza type A HA and NA proteins, including expression of these proteins using baculovirus infected insect cells (Crawford et al, 1999;
Johansson, 1999), viral vectors, and DNA vaccine construets (Olsen et al., 1997).

[0014] Specifics of an influenza virus infection are well known. Briefly, , the infectious cycle is initiated by the attachment of the virion surface HA
protein to a sialic acid-containing cellular receptor (glycoproteins and glycolipids). The NA
protein mediates processing of the sialie acid receptor, and vitus penetration into the cell depends on AA-dependent receptor-mediated endocytosis. In the acidic confines of internalized endosomes contauiing an influenza virion, the HA protein undergoes conformational changes that lead to fusion of viral and cell membranes and virus uncoating and M2-mediated release of MI proteins from nucleocapsid-associated ribonucleoproteins (RNPs), which migrate into the cell nucleus for viral RNA
synthesis. Antibodies to HA proteins prevent virus infection by neutrdlizing virus infectivity, whereas antibodies to NA proteins mediete their effect on'_he early steps of viral replication.

iiLtecced insect cells. The cxpressed proteins arz described as being capable of preventing lethal influenza disease caused by a-: iar. I;s a:;d :~I7 influenza _- ubtypes.
Jobansson et al_ (1999) teach that baculovirus-expressed influenza HA and NA
proteins induce immune responses in animals superior to those induced by a conventional vaccine. Immunogenieity and efficacy of bacalovirus- expressed hemagglutinin of equine influenza viras was compared to a homologous DNA
vaccine candidate (Olsen et al., 1997)_ Collectively, these data demonstrate that a high degree of protection against inftuenza virus Challenge can be induced with recombinant HA.
or NA proteins, using various experimental approaches and in different animal models.

[0016] Since previous research has shown that the surface influenza glycoproteins, HA and NA, are the primary targets for elicitation of protective immurtity against influenza virus and that Ml provides a conserved target for cellular immunity to influenza, a new vaccine candidate may include these viral antigens as a protein macromolecular particle, such as virus-Iflte parricles (VLPs). As vaccine products, VLPs offer the advantage of being more immunogenic than subunit or recombinant antigens and are able to stinnulate both humora] and cellular immune response (Grgacic and Anderson, 2006). Further, the particle with these inEluenza antigens may display conformatiorn.al epitopes that elicit neutralizing antibodies to multiple strains of influenza viruses.

[0017] Production of a non-infectious influenza virus strain for vaccine purposes is one way to avoid inadvertent infection. Alternatively, virus-like particles (VLPs) as substitutes for the cultured virus have been investigat.ed. VI Ps mimic the struc'ture of the viral capsid, but iaek a genotne, and thus cannot replicate or provide a means for a secondary infection.

[0018] Several studies have demonstrated that recombinant influenza proteins self-assemble into VLPs in cell culture using mammalian expression plasnaids or baculovirus vectors (Gomez-Puertas et a1.. 1999; Nenmann et aL, 2000; Latham and Galarza, 2001). Gomez-Puertu et al. (1999) discloses thai efficient formation of influenza VLP depends on the expression levels of several viral proteins.
Neumann et i~ui c.iuus iUi,uc_iza virus-like particles ent;.r~iy trorc; c:on~,d cUtdAs.
Latham and Ga'.=-: (2010 1; rcported t4e fornation of i::nuen;:a `Y"LZs in :nse:::L
::c::'.s infecteci recombinant baculovirus co-expressing HA,, NA, Ml, and M2 genes. These studies demonstrated that influenza virion proteins may self-assemble upon co-expression in eukatyotic cells.

[0019] Gomez-Puettas et al.(2000) teach that, in addition to the hemagglutinin (HA), the matrix protein (M1) of the influcnza virus is essential for VLP budding from insect cells. However, Chen et al. (2007) teach that Ml migbt not be required for VLP
to formation, and observed that efficient release of Ma and VLPs required the presence of HA, and sialidase activity provided by NA. The NA deaves the sialic acids of the glycoproteins at the surface of the cells producing the VLPs, and releasing the VLPs in the medium.

[0020] Quan et al 2007 teaches that a VLP vaccine produced in a baculovirus expression system (insect cell) induces a protective immzmity against some strains of influeaza virus (A/1?'1;8/34 (H1N1)). The VLPs studied by Quan were observed to bud from the plasma meinbrane. and were considered to be of tbe cor.rect size and morphology, similai to those obtained in a mammalian system (MDCK cells).

[0021] Enveloped viruses may obtain their lipid envelope when 'budding' out of the infected cell and obtain the membrane from the plasma membrane, or from that of an intcmal organelle. Influenza virus particles and VLPs bud from the plasma membrane of the host cell. In mammalian or baculovirus cell systems, for example, influenza buds from the plasma membrane (Quan et al 2007). Only a few enveloped viruses are lmown to infect plants (for example, members of the Topoviruses and Rhabdoviruses). Of the known plant enveloped viruses, they are characterized by budding from internal membranes of the host cell, and not from the plasma membrane. Although a small number of recombinant VLPs have been produced in plant hosts, none were derived from the plasma membrane, raising the question whether plasma membrane-lerived VLPs, including influenza VLPs can be produced in plants.
6 . . . ... . v J, .
:nuitiple virai i -oscir?s, a,-i:i u!is Llependence represents a ura=erb::tik of t.esc technologies since in case of a pandemic and of yearIy epidemics, response time is crucial for vaccination. A simpler VLP production system, relying on the expression of only one viral protein is desirable to accelerate the development of vaccine.

[0023] In order to protect the world.population from influenza and to stave off future pandemics, vaccine manufacttu-ers will need to develop effective, rapid methods producing vaccine doses. The cutrent use of fertilized eggs to produce vaccines is insufficient and involves a lengthy process.

SUNEAARY OF TM IlvVIINTION

(0024) It is an object of the invention to provide improved inflnenza virus like particles.

(0025J According to the present invention there is provided a nucleic acid comprising a nuclcotide sequence encoding an encoding an antigeA from an enveloped virus operatively linked to a regulatory region active in a plant. The antigen may be an influenza hemagglutinin (HA).

[0026] The present invention also provides a method of producing influenza virus like particles (VLPs) in a plant comprising:

a) introducing a nucleic acid encoding an nrrtigen from an enveloped virus, for eumple an influenza hemagglutinin (HA), operatively l=mked to a regulatory region active in the plant, into the plant, or portion thereof, and b) incubating the plant or a portion therefore under conditions that permit the expression of the nucleic acid, tttereby producing the VLPs.

[0027] The present invention includes the above method wherein, in the step of introducing (step a), the nucleic acid may be either transiently expressed in the plant, or stably expressed in the plant- Furthermore, the VLPs may be purified using size exclusion chromatography.
7 .i:iJLill`.1 virus 11t1 prui-.Il atitl UL::: C*r flUre than one plant llpil,i.
1'uS111elmore, the influenza virus HA protein may be H5lndonesia.

L0029] Also included in rhe present invention is a composition comprising an effective dose of a VLP comprising an influenza virus HA protein, one or more than one plant lipid, and a pharmaceutically acceptable carrier. The influenza virus HA
protein may be H5 Indonesia.

[0034] A method of inducing imrttunity to an influenza virus infection in a subject, is also provided, the method comprising administering the virus like particle comprising an influenza virus HA protein, one or m.ore than one plant lipid, and a pharmaceutically acceptable ca-rier. The virus like particle may be administered to a subject orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcuttu-eously.

(0031] The present invention also pertains to a virus like particle (VLP) comprising L5 one or more than one proteiu dcrived from a virus selected from the group consisting of Influenza, Measles. Elbola. Marburg, and HIV, and one or more than one lipid derived fi[om a non-sialylating host production cell.

(0032) Additionally the present invention relates to a virus like particle (VL.P) comprising an influenza virus HA protein and one or more than one host lipid.
For example if the host is insect, then the virus like pardcle (VLP) may comprise an influenza virus HA protein and one or more than one inseot lipid, or if the host is a yeast, then the virus like particle (VLP) may comprise an influenza virus HA
protein and one or more than one yeast lipid.

[0033] The present invention pertains to a method for inducing immunity to influenZa virus infectiott in an animal or target organism comprising administering an effective dose of a vaccine comprising one or more than one VLP, the VLF' produced using a non-sialyating host, for example a plant host, an insect bost, or a yeast host. The vaccine may be administered orally, intradermally, intranasally, intramusciarly, intraperitoneaily, intravenously, or subcutaneously. The target organism may be selected from the group comprising humans, primates, horses, pigs, birds (avian)
8 , = ;r , . .

tiG::t', leopard, Clv;:(y IItilu:., i:....; ferrets, IlouSe 1)C:(:;, 11Vf:Si'v.:r;, mice, rats, seat, whales and the like.

[0034) The present invention provides a method for producing VLPs coD.taining hemagglutinin (HA) from different influenza strains in a suitable host capable of producing a VL.P, for example, a plant, insect, or yeast VLPs that are produced in plants contain lipids of plant origin, VLPs produced in insect cells comprise lipids from the plasma membrane of insect cells (generally referred to as "insect lipids"), and VLPs produced in yeast comprise lipids from the plasma membrane of yeast cells to (generally referred to as "yeast lipids").

[0035] The production of VLPs in plants presents several advantages over the production of these particles in insect cell culture. Plant lipids can stimulate specific immune cells and enhance the immune response itnduced. Plant membranes are made of lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and also contain glycosphingolipids that are unique to plants and some bacteria and protozoa.
Sphingolipids are unusual in that they are noc esters of glycerol like PC or PF, b t rather consist of a long chain amino alcohol that forms an amide linkage to a fa:y acid c.hai.n containing more than 18 carbons. PC and PE as well as glycosphingolipids can bind to CD1 molecules expressed by mammalian immune cells such as antigen-presenting cells (APCs) like dentritic ceils and macrophagt,s z,.n,d other cells incluci.ag B and'1' lymphocytes in the thymus and liver ('Tsuji M,_ 2006). Furthermore, :n addition to the potential adjuvant effect of the presence of plant lipids, the ability of plant N-glycans to facilitate the capture of glycoprotein antigens by antigen presenting cells (Saint-Jore-Dupas, 2007), may be advantageous of the production of VLPs in plants.

[003 j tiVi:hout wishing to be bound by thc:.y, it is anticip3ted that plant-made VLPs will inriuce a stronger inunune reaction than VLPs made in other manufacturing systems and that the imrnune reaction induced by these plant-made VLPs will be stronger when compared to the immune reaction induced by live or attenuated whole virus vacc'ines.
9 issue as it would be working with a whole, infectious virus, -and is not required for production. Plant-made VLPs provide a further advantage again by allowing the expression system to be grown in a greenhouse or field, thus being significantly more economical and suitable for scaie-up.

[0038] Additionally, plants do not comprise the enzymes involved in synthesizingtiffl r`
adding sialic acid residues to proteins. VLPs may be produced in the absence of neuraminidase (NA), and there is no need to co-express NA, or to treat the producing cells or extract with sialidase (neurarninida,se), to ensure VLP production in planrs.
[0()39] The VLPs produced 'ul accordance with the present invention do not comprise Ml protein which is known to bind RNA. RNA is a contaminant of the VLP
preparetaon and is undesired when obtsining regulatory approval for the VLP
product.
[0040) This summary of the invention does not necessarily describe all features of the invention.

BR.IBF DESCRIPTiON OF TAE DRAWINGS

[00411 These and other features of the invention will become more apparent from the following description in which reference is made to tbe appended drawings wherein:
[0042] FIGLJRE 1A shows a sequence of an alfalfa plastocyanin-based expression cassette used for the expression of Hl in accordance with an embodiment of the present invention (SEQ ID NO:8). Protein disulfide isomerase (PDI) signal peptide is underlined. Bglii (AGATCI`) and SacI (GAGCTC) restriction sites used for cloning are shown in bold. Figure 1B shows a schematic diagram of functiottal domains of influenza hemagglutinin. After cleavage of HAO, HAl and HA2 fragments remain bound together by a disulfide bridge.

,, . . . _- .., G r . . . _ . :. . _, ..: ~: ._1_ P1~1~.J1\l'. ' . . ;._:.t=...,_ -_.._.. _ ~:
C:L:.il: 1.~..
.,.,. ..L ~__. . , .
assemblc(~ u,w expr:.ssion of HA subtype IU.

[00"] FIGURE 3 shows a size exclusion chromatography of protein extracts from leaves producing hernagglutinin XII or I15. FIGURE 3A show the elution profile of H1; Blue Dextran 2000 (triangles) and proteins (diamonds). FIGURE 3B shows immunodetection (western blot; anti H1) of Hl clution fractions following size exclusion chromatography (S500HR beads). FIGURE 3C show the elution profile of H5; Blue Dextrm 2000 (triangles) and proteins (diamonds). F[GLTRE 3D shows immunodeteetion (western blot; anti I15) of H5 elution fractions following size exclusion chromatography (S500HR beads).

[0045] FIGURE 4 sbows an electron microscopy phototnicrograph of large hemagglutinin Hl and H5 structures from elution fraction 9 from a size exclusion coiumn. FIGURE 4A sbows a 50 000-fold ettlargement of a VLP from Hishowing the presence of multiple similar structures (the bar represents 200 nm). FIGURE 4B
shows a 150 000-fold enlargement of a VLP from HI (the bar represents 100 nm).
FIGURE 4C shows a 50 000-fold enlargement of a VLP from H5 showing the presence of multiple similar structures (the bar represents 50 nm).

[0046] FIGURE 5A shows the sequence of the N terminal fragment of Hi (SEQ ID
NO:1). FIGURE 5B shows the C terminal fragment of H1(SEQ II) NO:2). FIGURE
5C shows the complete sequence encoding HAO of Hi (SEQ ID NO:28).

[0047] EIGURE 6 sbows the sequence encoding H5 flanked by a Hind1II site immediately upstream of the initial ATG, and a SacI site immediately downstream of the stop (TAA) codon (SEQ ID NO:3) 15 10048] FIGURE 7A shows the sequence of the primer Plasto-443c (SEQ ID
NO:4).
FIGURE 7B shows the sequence of primer SpHA(Ind)-Plasto.r (SEQ ID NO:5).
FIGU1tE 7C shows the sequence of primer Plasto-SpHA(Ind).c (SEQ ID NO:6).
FIGURE 7D shows the sequence of ptimer HA(Ind)-Sac.r (SEQ ID NO:7).

~1 ~ . - = , ... .:i _ =j; . _ _. . . . i .i .
sequence (SEQ ID i~~:i:10). Native signa: peptide is indica4~-d in boid.

[0050] FIGURE 9 shows the sequencx of HA of influenza A subtype H.7 (SEQ ID
No:
11).

[0051] FIGURE 10A shows the sequence of Influenza A HA, subtype H2 (SEQ ID
NO:12). FIGiJRE lOS shows the sequence of Influenza A HA subtype H3 (SEQ ID
N0:13). FIGURE IOC shows the sequence of Influenza A HA subtype 144 (SEQ ID
NO:14)_ FIGURE 10D shows the sequence of Influenza A HA subtype H5 (SEQ ID
t0 NO:15). FIGURE l0E shows the sequenca of Influenza A HA subtype H6 (SEQ ID
NO:16). FIGURE 10F shows the sequence of Influenza A HA subtype HS (SEQ fD
NO: 17). FIGURE 100 shows the sequence of Influenza A HA subtype H9 (SEQ iD
NO:18). FIGURE tOH shows the sequence of Influenza A HA subtype H10 (SEQ ID
NO: 19). FIGURE 101 shows the sequence of Influenza A HA subtype H11(SEQ ID
NO:20). FIGURE I0J shows the sequence of Influenza A HA subtype H12 (SEQ ID
NO:21). FIGURE 10K shows the sequence of Influenza A HA subtype H13 (SEQ ID
N0:22). FTG[TRE 10L shows the sequence of Influenza A HA subtype H14 (SEQ U) NO:23). FIGURE 10M shows the sequence of Infiuenza A HA subtype H15 (SEQ ID
NO:24). FIGURE 10N shows the sequence of Influenza A HA subtype H16 (SEQ ID
NO:25). FIGURE 100 shows the sequence of Influenza B HA (SEQ ID NO:26).
FIGURE lOP shows the sequence of Influenza C HA (SEQ m NO:27). FIGURE 1pQ
shows the sequence of primer Xmal-pPlas.c (SEQ ID NO: 29). FIGURE 1OR shows the sequenoe of primer SacI-ATG-pPlasa (SEQ ID NO: 30). FIGURE lOS shows the sequence of primer Sael-PlasTer.c (SEQ 1D NO: 31)_ FIGURE lOT shows the sequence of primer EeoRI-plasTer.r (SEQ 1D NO: 32).

[0052) FIGURE 11 shows a schematic representation of several constructs as used herein. Construct 660 comprises the nucleotide sequence to encode the HA
subtype HS under operatively linked to the plastocyanin promoter (plasto) and temiinator (Pter); construct 540 comprises the nucleotide sequence to encode the HA
subtype Hl in eombination with an alfalfa protein disulfide isomerase signal peptide (SP
pDI), and is operatively linked to a plastocyanin promoter (Plasto) and terminator (Pter).

_.-. _ . .i.i. , c. -, . . . . .... . . . . . . . . , .. ,1.~ .. . .. . ., ..
.i -1ti .:.:::, peptade (SP PDI) and an GCN4pTl leucine zipper (in place of the transmembrane domain and cytoplasmie tail of HI) and operatively linked to the plastocyanin s promoter (Plasto) and tenninator (Pter); and conshuct 750 for the expression of M1 coding region from influenza A/l}R!$/34 is combined to the tobacco etch virus (TEV) 5'VIR, and operatively linked with the double 35S promoter and Nos terminator.
[0053] FIGURE 12 shows immu,nodetection of H5, using anti-H5 (Vieatand) antibodies, in protein extracts from N. benthamiana leaves transfortned with consnvct 660 (lane 3). Commercial H5 from influenza AlVietnam/1203/2004 was used as positive control of detection (lane 1), and a protein extract from leaves transformed with an empty vector were used as negative control (lane 2).

[0054] FTGURE 13 shows characterization of hemagglutinin stiuctures by size exclusion chromatography. Protein extract from separate biomasses producing H5, Hl, soluble HI, or Hi and M1 were separated by gel filtration on S-500 HR.
Commercial Hi in the form of rosettes was also fractionated (Hl rosette).
FIGURE
13A shows elution fractions analyzed for relative protein content (Relative Protein Level - a standard protein elution profile of a biomass fractionation is shown). Blue Dextran 2000 (21YIDa reference standard) elution peak is indicated. FIGURE 13B
shows elution fractions analyzed for the presence of hemagglutinisa by immunoblotting with anti-HS (Vietnam) antibodies (for H5). FIGURE 13C shows elution fractions analyzed for anti-influenza A antibodies for Hi. FIGURE 13D
shows elution fractions analyzed for anti-influenza A antibodies for soluble HI.
FIGURE
13E shows elution fractions analyzed for anti-influenza A antibodies for Hl rosette.
FIGURE 13F shows elution ffractions analyzed for anti-influenza A aniibodies for H1+M1.

[0055] FIGURE 14 shows concentration of influenza H5 structures by sucrose gradient centrifugation and electrvn microscopy examination of hemaggiutinin-concentrated fractions. FYGURE 14A shows characterization of fractions from sucrose density gradient cenorifugation. Each fraction was analyzed for the presence of H5 by immunoblotting using anti-H5 (Vietnam) antibodies (upper panel), and for their . = -= ,.., ;li ~io;1 ~~. =.,. . . . . . . ~. ~:
.. ~ y _.... ._.r~ .. ,._.._ 17, 18 and 19 from sucrose gradient centrifugation. The bar represents 100 nm.
[0056] FIGURE 15 shows purification of influenza H5 VLPs. p'TGURE 15A shows Coomassie Blue stained SDS-PAGE analysis of protein content in the clarifinatiofl steps - lane 1, crude extract; lane 2, pH 6-adjusted extraet; lane 3, heat-treated extract;
lane 4, DE-filtrated extract; the fetuin affinity purification steps: lane 5, load; l'ane 6, wash; lane 7, elution (IOX concentrated). FIGURE 15B shows negative 5taining transmission electron microscopy examinstion of the purified H5 VI.P' sarnple.
The bar represents 100 nm. FIGURE 15 C shows isolated H5 VLP enlarged to show details of the structure. FIGURE 15D shows the H5 VLP product on a Coomassie-stained reducing SDS-PAGE (lane A) and Westem blot (lane B) using rabbit polyclonal antibody raised against HA from strain A/Vietnam/1203/2004 (H5N1).
[0057] FIGURE 16 shows a nucleotide sequence for Influenza A virus (A/New Caledonia/20/99(H1N1)) hetnagglutinin (HA) gene, complete cds. GenBank Accession No_ AY289929 (SEQ ID NO: 33) [0058] FIGURE 17 shows a nucleotide sequence for Medicago sativa mRNA for protein disulfide isomerase. GenBank Accession No. Z11499 (SEQ ID NO: 34).
[00591 FIGURE 18 shows a nucleotide sequence for Influenza A viivs (A/Puerto Rieo/8/34(HiNi)) segment 7, cornpletc sequence. GenBank Accession No.
NC002016.1 (SEQ ID NO: 35).

[0060] FIGURE 19 shows localization of VLP accumulation by positive staining transmission electron microscopy observation of H5 producing tissue. CW: cell wall, ch: chloroplast, pm: plasma membrane, VLP: virus-like particle. The bar represents 100 ntn.

[00611 FIGURE 20 shows induction of serum antibody responses 14 days after boost in Balb/c mice vaccinated with plant-made influenza H5 VLP or recombinant soluble HA. FFIGLJRE 20(A) Antibody responses of mice immunized through intramuscular injection. FIGURE 20(B) Antibody responses of mice immunized through intranasal of reeiprocal end-point titers of five mice per group. Bars represenc nwan deviation.
p¾ 0.05 compared to recombinant soluble HA.

s [0062] FIGURE 21 shows hemagglutination inhibition antibody response (HAl) days after boost in Salb/c mice vaccinated with plant-made influenza H5 VLP or recombinant soluble HA. FYGURE 21(A) Antibody responses of mice immvnized through intramuscular injection. FIGURE 21(B) Antibody responses of mice immunized thmugh intranasal administration. HAI antibody responses were measured using inactivated whole H5N1 viruses (Allndonesia/5705). GMT:
geometric mean titer. Values are the GMT (logz) of reciprocal end-point titers of five mice per group. Bars represent mean deviation. * p< 0.05 and ** p< 0.01 compared to recombinant soluble HA.

[0063] FIGURE 22 shows the effect of adjuvant on immunogenicity of the VLPs in mice. FIGURE 22(A) Effect of alutn on mice immunized through intramuseular injection FIGURE 22(13) Effect of Chitosan on rnice itnmunized tthrough intrnnAaai administration. HAI antibody responses were measured using inactivated whole viruses (A/lndonesia/5/05). GMT: geometric mean titer. Values ate the GMT
(logZ) of reciprocal end-point titers of five mice per group. Bars represent mean deviation.
p< 0.05 compared to the corresponding recombinant soluble HA.

[0064] FIGURE 23 shows antibody response to VLP administration. FIGURE 23(A) Anti-Indonesial5/05 iinmunoglobulin isotype in mice vaccinated with intramuscular injection, 30 days after boost. Values are the GMT (log2) of reciprocal end-point titers of five mice per group. ELISA performed using whole inactivated vituses as the coating agent. Bars represmt mean deviation. * p< 0.05, ** p< 0.001 compared to the corresponding reeombinant soluble HA. FIGURE 23(B) Antibody titers against whole inactivated viruses. All groups are statistically different to negative control.
[0065] FIGURE 24 shows antibody titer against homologous whole inactivated viruses (A/Indonesia/5/05), 2 weeks after first dose (week 2), 14 days after boost (week 5) or 30 days after boost (week 7). GMT: geometric mean titer. Values are the :;,_.,., ..... . . _~ . . .

[0066] FIGURE 25 shows in vitro cross-reactivity of serum antibodies. (A) Antibody titers against whole inactivated viruses. (B) Hetnagglutination-inhibition titers against various whole inactivated viruses. Values are the GMT (log2) of reciprocal end-point titers of five mice per group. Bars represent mean deviation. Ail groups are statistically different to negative control. * p< 0.05, ** p< 0.001 "''r conapared to the corresponding recombinant soluble HA.

[0067] FIGURE 26 shows effeacy of the plant tnade H5 VLP. (A) Survival rate of mice after challenge with 10 L.D5o (4.09x10s CCID5o) of the influenza stz-ain A/furkey/582106 (H5N1) (B) Body weight of immutnised mice after challenge.
Values are the mean body weight of surviving mice [0068] FIGURE 27 shows Origin of plant-derived influenza VI.Ps. (A) Polar lipid composition of purified influenza V'LPs. Lipids contained in an equivalent of 40 g of proteins, were extracted from VLP as described, separated by HP-TLC, and compared to the migraeion profile of lipids isolated from highly purified tobacco plasma membrane (PM). Lipid abbreviations are as following: DGDG, Digalactosyldiacylglycerol; g1uCER, glucosyl-ceramide; PA, phosphatic acid;
PC, phosphatidylcholine; PE, phosphatidylethanolamiuse; PG, phosphatidylglycerol;
Pi, zo phosphatidylinositol; PS, phosphatidylserine; SG, Steryl-glycoside. (B) Neutral lipid composition of purified influenza VLPs. Lipids contained in an equivalent of 20 g of proteins were extracted from VLP as described, separated by HP-TLC and compared to the migration of sitosterol. (C) Immunodetection of the plasma membrane marker proton pump ATPase (PMA) in purified VLPs and highly-purified PM from tobamo 2.5 leaves (PML) and BY2 tobacco cells (PMeY2). Eighteen micrograms of protein were loaded in each lane.

DETAII..ED DFSCRIPTION

[0069] The pre.sent invention relates to the production of virus-like particles. More specifically, the present invention is directed to the production of virus-like particles 30 comprising influenza antigens.

t6 [Q071] The present it;v: ,o n provides a n-ocl ": acid couipr:is:i; ; a sequence encoding an antigen from an enveloped virus, for example, the influenza hemagglutinin (HA), operatively linked to a regulatory region active in a plant.

[0072] Furthetmore, the present invention provides a method of producing virus like particles (VLPs) in a plant. The method involves introducing a nucleic acid encoding an antigen operatively linked to a regulatory region active in the plant, into the plant, or portion of the plant, and incubating the plant or a portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLPs.
to [0073] VLPs may be produced from influenza virus, however, VLPs may also be produced from other plasma membrane derived virus including but not limited to Measles, Ebola, Marburg, and HIV.

[0074) The invention includes all types of humAn influenza virus, including for example, but not limited to the very prevalent A(H1N1) sub-type, the A/Zndonesia/5/05 sub-type (H5NI) and the less common B type (for example SEQ
ID
NQ_26, Figure 100), and C type (SEQ ID NO:27, Figure 10P), and to HAs obtained from other influenza subtypes. The present invention also pertains to influenza viruses which infect other mammals or host animals, for example humans, primates, horses, pigs, birds, avian water fowl, migratory birds, quail, duck, geese, poultry, chicken, camel, canine, dogs, feline, cats, tiger, leopard, civet, mink, stone marten, ferrets, house pets, livestock, mice, rats, seal, wbale and the like.

[0075) Non limiting examples of other antigens that may be expressed in plasma membrane derived virvses include, the Capsid protein of HIV - p24; gp12p. gp41 -enveiope proteins, the structural proteins VP30 and VP35; Gp/SGP (a glycosylated integral membrane protein) of Filoviruses, for example Ebola or Marburg, or the H
protein. and F protein of Paramyznviruses, for example, Measles.

[Q075] The inveiltion also inclt:d,-s, Uist is not limited to, i.nfluenza derived VI-Ps that obtain a lipid envelope from the plasma membrane of the cell in which ta`t:
VLP

. . . . , = ..~ _ ~:,. . . .. ._: .. . _ _ _ = . . ~
.,.~ i..~ u,lj 11; -1i(1 1 !;PId G[1V 4-1 01 lt:,~ ;:~.il_ =

[0077] Generally, the term "lipid" refers 'to a fat-soluble (lipophilic), naturally-occurring molecules. The term is also used more specifically to refer to fatty-acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids), a`wtll as other fat-soluble sterol-containing metabolites or sterols. Phospholipids are a major component of all biological membranes, along with glycolipids, sterols alid=jiro4eins.
Examples of phospholipids include pl-osphatidylethanolamine, ph,osphatidylcholine, phosphatidylirmsitol, phosphatidylserine, and the like. Examples of sterol.s=
include zoosterols (e.g., cholesterol) and phytosterols. Over 200 phytosterols have been identified in various plant species, the most common being campesterol, stigmasterol, ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-ave,nasterol, daunosterol, sitosterol, 24-methylcholesterol, cholesterol or beta-sitosteroi. As one of sldll in the art would understand, the lipid composition of the plasma xnembrane of a cell may vary with the culture or growth conditions of the cell or organism from which the csll is obtained.

[0078] Cell membranes generally comprise lipid bilayers, as well as proteins for various functions. Localized concentrations of particular lipids may be found in the lipid bilayer, refeazed to as `lipid rafts'. Without wishing to be bound by theory, lipid rafts may have significant roles in endo and exocytosis, cntty or egress of viruses or other infectious agents, inter-cell signal transduction, interaction with other structucal components of the cell or organism, such as intraoclhxlar and extracellular matrices.
[00791 With reference to influenza viras, the term "hemagglutinin" or "HA" as used herein refers to a glycoprotein found on the outside of influenza viral particles. HA is a homotrimeric membrane type I glycoprotein; generally comprising a signal peptide, an HAl domain, and an HA2 domain comprising a membrane-spanning anchor site at the C-terminus and a small cytoplasmic tail (Figure IB). Nucleotide sequences encoding 1-iA are well known and are available - see, for example, the BioDefence Public Health base (Influenza Virus; see URL- biohealthbase.org) or National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov), both of which are incorporated herein by reference_ -;J:! "IaH]lotlYIi1CY" or '-Ito[(1(';C,., . . .. ::3r: an p~'.oQT.:GL ;ti monomeric precursor protein (HAO), which assembles at the surface into an elongated tzirneric protein. Before rrirnerization occurs, the precursor protein is cleaved at a conserved activation cleavage site (also referred to as fusion peptide) into 2 polypeptide chains, HA1 (328 amino acids) and HA2 (221 amino acids; comprising the transmembrrane region), linked by a disulfide bond. Although this step is central for virus infectivity, it is not essential for the trimerization of the protein. Insertion of HA within the endoplasmic reticulum (ER) membrane of the host cell, signal peptide to cleavage arnd protein glycosylation are co-transiational events. Correct refolding of HA requires glycosylation of the protein and formation of 6 intra-chain disulfide bonds. The HA trimer assembles within the cis- and trans-Golgi complex, the transmernbrane domain playing a role in the trimerization process. The crystal structures of bromel3in-treated HA proteins, which lack the transrnembrane domain, have shown a hipbly conserved structure amongst influenza strains. It has also been established that HA undergoes major conformational changes during the infection process, which requires the preeursor HAO to be cleaved into the 2 polypeptide chains HA1 and HA2. T'he HA protein may be processed (i_e., comprise HA1 and HA2 domains), or may be unprocessed (i.e. compcise the HAO domain).

[0081] The present invention pertains to the use of an HA protein comprising the transmembiane domain and Includes HAl and HA2 domains, for example the HA
protein may be HAO, or processed HA comprising HAl and H.A2.

[0082] The HA of the present invention may be obtained from any subtype. For example, the HA maybe of subtype H1, H2, H3, H4, H5, H6, H7. H8, H9, H10, Hll, H12, H13, H14, H15, or H16. The recombinant HA of the present invention may also comprise an amino acid sequence based on the sequence any hemagglutinin known in the art- see, for example, the BioDefence Public Health base (Influenza Virus;
see URL: biohealthba.se.org) or National Center for Biotechnology Information (see URL:
ncbi.nlm.nih.gov). Flirthermore, the HA may be based on the sequence of a hemagglutinin that is isolated from one or more emerging or newly-identified influenza viruses:

more thar, one iA front tie subtype Hl (encoded by SEQ ID NO:28), H2 (encoded by SEQ ID NO:12), H3 (encoded by SEQ ID NO:13), H4 (encoded by SEQ ID NO:14), H5 (encoded by SEQ ID NO:15), H6 (encoded by SEQ Ib NO:16), H7 (encoded by SEQ ID NO:l 2), H8 (encoded by SEQ ID N0:17), H9 (encoded by SEQ ID NO:18), H10 (encoded by SEQ m NO:19), H11(encoded by SEQ II) NO:20), H12 (encoded by SEQ ID NO:21), H13 (encoded by SEQ ID NO:27), H14 (encoded by SEQ ID
NO:23), H15 (encoded by SEQ ID N0:24),H 16 (encoflcd by SFQ ID NO:25), or a to combination thereof. One or more that one HA from the one or more than one influenza subtypes may be co-expressed within a plant or insect cell to ensure that the synthesis of the one or more than one HA results in the formation of VLPs comprising a combination of HAs obtained from one or more than one influenza subtype.
Selection of the eombination of HAs may be determined by the intended use of the 15 vaccine prepared from the VLP. For example a vaccine for use in inoculating birds may comprise any combination of HA subtypes, while VLPs useful for inoculating humans may comprise subtypes one or more than one of subtypes Hi, H2, H3, H5, H7, H9, H10, Nl, N2, N3 tuad N7. However, other HA subtype combinations may be prepared depending upon the use of the inoculum.

20 [0084] Therefore, the present invention is directed to a VLP comprising orne or more than one HA subtype.

(0085] The present invention includes nucleotide sequences SEQ ID NO:28; SEQ
ID
NO:3; SEQ TD NO:11, encoding HA from H1, H5 or H7, respectively, a nucleotide sequence SEQ ID NO:28; SEQ ID NO:3; SEQ ID NO: 11, that hybridizes under 25 stringent hybridisation conditions to a nucleic acid that encodes the HA
from Hl, H5 or H7, respectively, or a nucteotide sequence SEQ ID NO:28; SEQ ID NO:3; SEQ
Ip NO: 11, that hybridizes under stringent hybridisation conditions to a compliment of a nucleic acid encoding the HA from Hl, H5 or H7, respectively, wherein the nucieotide sequence encodes a hemagglutinin protein that when expressed forms a 30 VLP, and that the V LP induces the production of an antibody. For example, e7ipression of the nucleotide sequence within a plant celt forms a VLP, and the VLP
may be used to produce an antibody that is capable of binding HA. including mature [0086] Hybridization under stringent hybridization conditions are known in the art (see for example Current Protocols in Molecu.lar Biology, Ausubel et al., eds.

and supplements; Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A
Laboratory Manual, 3rd edition 2001; each of which is incorporated he.rein by reference). An example of one such stringent hybridization conditions may be about 16-20 hours hybridization in 4 X SSC at 65 C, followed by washing in 0.1 X SSC
at to 65 C for an hour, or 2 washes in 0.1 X SSC at 65 C each for 20 or 30 minutes.
Alternatively, an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4 X SSC at 42 C, followed by washing in 0.1 X SSC
at 65 C for an hour, or 2 washes in 0.1 X SSC at 65 C each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7%
SDS; 0SM NaPOa buffer pH 7.2; 10 mM EDTA) at 65 C, with 2 washes either at 50 C in 0.1 X SSC, 0.196 SDS for 20 os 30 minutes each, or 2 washes at 65 C in SSC, 0.1% SDS for 20 or 30 minutes each.

[0087] The present invention also includes one or more than one HA protein encoded by nucleotide sequences SEQ ID NO:28; SEQ ID NO:3; SFQ ID NO:11 (encoding HA from Hl, IT5 or H7, respectively), a nucleotide sequence SEQ ID NO:28; SEQ

NO:3; SEQ ID NO: 11, that hybridizes under stringent hybridisation conditions to a nu.eteic acid that encodes the HA from Hl, H5 or H7, respectively, or a nucleatide sequence SEQ ID NO:28; SEQ ID NO:3; SEQ ID NO:11, that hybridizes under stringent hybridisation conditions to a eompliment of a nucleic acid encoding the HA
from Hl, H5 or H7, respectively, wherein the nucleotide sequence encodes a hemaggiutinin protein that when expressed forms a VLP, and that the VLP
induces the production of an antibody. For example, expression of the nucleotide sequence within a plnnt cell fornis a VLP. and the VLP may be used to produce an antibody that is capable of binding HA, including mature HA, HAO, HA1, or HA2. The VLP, when administered to a subject, induces an immune response.

, .,.. , . . . .. . . , , 100% or any amount therebetween, sequence identity, or szquenct; similarity, with ine nucleotide sequence encoding HA from Hl (SEQ II) NO.28), H5 (SEQ ID NO:3) or H7 (SEQ II7 NO:I 1), wherein the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that the VLP induces rhe production of an antibody. For exatnple, expression of the nucleatide sequence within a plant cell forms a VLP, and the VLP may be used to produce an antibody that is capable of binding HA, includic,g mature HA, HAO, HA1, or HA2. The VLP, when administered to a subject, induces an immune response.

[0089] Similarly, the present inventiau includes HAs associated with the following subtypes H 1(encoded by SEQ ID NO:28), H2 (encoded by SEQ ID NO:12), H3 (encoded by SEQ ID NO: 13), H4 (encoded by SEQ ID NO:14), H5 (encoded by SEQ
II) NO:15), H6 (encoded by SEQ ]b NO:16), H7 (encoded by SEQ ID NO:11), H8 (encoded by SEQ Il) NO:17), H9 (encoded by SEQ ID NO:18), H10 (encoded by SEQ ID NO:19), HI I (encoded by SEQ ID NO:20), H12 (encoded by SEQ ID
NO:21), H13 (encoded by SEQ ID NO:27), H14 (encoded by SEQ ID NO:23), H15 (encoded by SEQ I:D NO:24), H16 (encoded by SEQ II) NO:25); see Figures t0A to lOP), and nucleotide sequences that are characterized as having from about 70 to 100% or any amount therebetween, 80 to 100% or any amount there between, 90-100% or any amount therebetwecn, or 95-100% or any amount therebetween, sequence identity with HI (SEQ ID NO:28), H2 (SEQ ID NO:12), i;i3 (SEQ ID
NO:13), H4 (SEQ ID NO:14), H5 (SEQ ID NO:15), H6 (SEQ ID NO:16), H7 (SEQ
ID NO:11), H8 (SEQ ID NO:17), H9 (SEQ ID NO:18), H10 (SEQ ID NO:19), H11 (SEQ ID NO:20), H12 (SEQ TI) N0:21), H13 (SEQ IL) NO:27), H14 (SEQ ID
NO:23), H15 (SEQ ID NO:24), H16 (SEQ ID NO:25), wherein the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that the VLP induces the production of an antibody. For example, expression of the nucltotide sequence within a plant cell forms a vY.,P, and the VLP may be used to produce an antibody that is capable of binding HA, including mature HA, HAO, HAl, or HA2. The VI.P, when administered to a subject, induces an immune response.

.. . . . , . . ~'aa!. .:j._.... 4.... ... ._ _ ....1:.:_.~i.~._;~. .,. , ....
. cell-meuiated response. The humortil tesponzit is the aspu:t of immunity ti,at is mediated by secreted antibodies, produced in the cells of the B lymphocyte lineage (B
cell)_ Secreted antibodies bind to antigens on the surfaces of invading microbes (such as viruses or bacteria), which flags them for destruction. Humoral imtnunity is used generally to refer to antibody production and the processes that accompany it, as well as the effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell generation, opsonin promotion of phagocytosis, pathogen to elimination and the like.

[4Q91] A cell-mediated response is an immune response that does not involve antibodies but rather involves the activation of macrophages, natural Idller cells (NK), antisen-specific cytotoxic T-lymphoCytes, and the release of various cytokines in response to an antigen. Cell-rnediated immunity is used generally to refer to some Th cell activation, Tc cell activation and T-r,eli mediated responses. Cell mediated immunity is of particular importance in responding to viral infections.

[0092] Sequence identity or sequence similarity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (for example, using, but not limited to, the following parameters: GAP penalty 5, #of top diagonals 5, fixed GAP penalty 10, k-tuple 2, floating gap 10, and window size 5).
However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Snzith & Waterr,nan (1981, Adv. AppL Math. 2:482), Needleman & Wunsch (J. Mol. Biol_ 48:443,1970), Pearson & Lipman (1988, Proc.
Nat1. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (e.g. GAP, BESTFTI', FASTA, and BLAST)., or by manual alignment and visual inspection.

[0093] The term "hemagglutinin domain" refers to a peptide comprising either the HAO domain, or the 1-IA1 and HA2 domains. The hemagglutinin domain does not include the signal peptide, transmembrane domain, or the cytoplasmie tail found in the naturally occurring protein.

... _.. . . . . . ...., .:.5 ..~.r..., . .. .. . .. t . .., .,,._:.t ., i:ii-l,. ..., ... , protein. VI..rPs are generally morphologically and antigenically similar to virions produced in an infection, but lack genetic information sufficient to replicate and thus are non-infeetious.ln some examples, VLPs may comprise a single protein species, or more than ane protein species. For VLPs comprising more than one protein species, the protein species may be from the same species of viius, or may comprise a ptotein from a different species, genus, subfamily or family of vinis (as designated by the ICTV nomenclature). In other examples, one or more of the protein species comprising a VLP may be modified from the naturally occ-rrring sequence. VLPs may be produced in suitable host cells including plant and insect host cells.
Following extraction from. the host cell and upon isolation and further purification under suitable conditions, VLPs may be purified as intact structures.

[0095] The VLPs produced from influen2a derived proteins, in accordance with the I5 present invention do not comprise Ml protein. The MI protein is known to bind RNA (Wakefield and Brownlee,1989) which is a contaminant of the VLP
preparation. The presence of RNA is undesired when obtainimg regulatory approval for the VLP product, therefore a VLP preparation lacking RNA may be advantageous.
[00961 The VLPs of the present invention may be produced in a host cell that is characterized by lacking the ability to sialylate proteins, for example lacking sialidase, such as a plant cell, an insect cell, fungi, and other orgaaisms including sponge, coelenterara, annelida, arthoropoda, xnollusca, n,etnathelm.inthea, trochelmintes, plathelminthes, chactognatha, tentaculate, chlamydia, spirochetes, gram-positive bacteria, cyanobacteria, arcihaebacteria, as identified in glycoforuin (see, for exaoiple, the URL glycofon..m.gr.jplscience/word/evolutionlES-A03E.html). The VLPs produced as desctibed herein do not typically comprise neuramindase (NA).
However, NA may be co-expressed with HA should VLPs comprising HA and NA be desired.

[0097) A VLP produced in a plant according to some aspects of the invention may be complexed with plant-derived lipids. The VLP may comprise an HAR.HAl or 1-IA2 peptide. The plant-derived lipids may be in the form of a lipid bilayer, and may t,!7t}1F... ,i, ~~tiil.-1~.. ~; . .. _ .._.. _, _.. ~?.CtJ7t1}.t I,il=~1aU[i..... _. ..... y ,.. _,~.. u... , ..i la produced, including,and one or more than one plant derived lipid, for example but not limited to phosphatidylcholine (PC), phosphatidyiethanolamine (PE), glycosphingolipids, phytosterols or a combination thereof. A plant-derived lipid may altemately be referred to as a`plant lipid'.

[00981 As used herein, a"protein" refers generally to a string of amino acids connected by a peptide bond, which may be folded into secondary; tertiary or quatenaary structure to achieve a particular morphology. Alternately, the terms polypeptide, peptide or peptide $agments may be used in a s imilar context.

[0099] The present invention describes, but is not ltiutited to, the cloning of a nucleic acid encoding HA, for example but not limited to, a human influenza A/New Caledonia/20l99 (H1N1) virus HA into a plant expression vector, and the production of influenza VLPs from the plant, suitable for vaccine production. The VLPs may also be used to produce reagents comprised of recombinant influenza stractrual proteins that self-assemble into functional and immunogenic hornotypie macromolecular protein structures, including subviral influenza particles and influenza VLP, in transformed hosts cells, for example plant cells or insect cells.
[001001 Therefore, the invention provides for VLPs, and a method for producing viral VLPs in a plant expression system, from the expression of a single envelope protein. The VLPs may be influenza VLPs, or VLPs produce from other plasma membrane derivcd virus including but not limited to Measles, Ebola, Marburg, and HIV. However, proteins from other enveloped viruses, for example but not limited to Filovirida.e (e.g. Ebola virus, Marburg virus, or the like), Pat'amyxoviridae (e.g. Measles virus, Mumps virus, Respiratory syncytial virus, pneumoviruses, or the like), Retrovirida.e (e.g. Human Immunodeficiency Virus-1, Heman Immunodeficiency V irus-2, Human T-Cell Leukemia V irus-1, or the like), Plaviviridae (e.g. West Nile Encephalitis, Dengue virus, Hepatitis C virus, yellow fever virus, or the like), Bunyaviridae (e.g. Hantavirus or the like), Coronaviridae (e.g.
coronavirus. SARS, or the like), as would be known to those of skill in the art, may also be used. Non lintitiug Examples of antigens that may be expressed in plsma and iISGr^ 'a ' os i:~tc:: u:te~'a.t l~tuer7br~e rotein ;ur Param ',.oviruseS
13 l a'J" y a l~ 1. y" +
for example, Measles the H protcin, and F protein. However, other coat proteins may be used within the methods of the present invention as would be know to one of skill in the art.

[001011 The present invention further provides the cloning of a nucleic acid encoding an HA, for example but not limited to, human influenza A/Indonesia/5/05 virus HA (H5N1)-into a plant or insect expression vector (e.g. baculovinu expression to vector) and production of influenza vaccine candidates or reagents comprised of recombinant influenza structural proteins that self-assemble into functional and immunogenic homotypic nmacromolecular protein structures, including subviral influenza particles and influenza VLP, in transformed plant cells or transformed insect cells.
[00102] The nucleic acid encoding the HA, for example but not linuted to, a human influenza AJNew Caledoniai20/99 (H1N'1) virus HA, or the human influenza A/lndonesial5l05 virus HA gene may be expressed, for example, using a Baculovirus Expression System in an appropriate cell line, for example, Spodopterafnigiperda cells (e.g. Sf-9 cell line; ATCC PTA-4047). Other insect cell lines may also be used.

[00103] The nucleic acid encoding the HA may, alternately, be expressed in a plant cell, or in a plant. The nucleic acid encocling HA may be synthesized by reverse transcription and polymerase chain reaction (PCR) using HA RNA. As an example, the RNA may be isolated from human Influenza AlNcw Caledonia/20199 (H1N1) virns or human influenza A/Irxlonesia/5/05 (H5N1) virus, or from cells infected with an influenza virus. For reverse transcription and PCR, oligonucleotide primers specific for HA RNA, for example but not limited to, human influenza A/New Caledonia/20/99 (HINI) virus HA genes or human influenza A/Zndonesia/5f05 (H5N 1) virus HAO genes can be used. Additionally, the nucleic acid encoding HA
may be chemically synthesized using methods as would known to one of skill in the art.

, __ .__ ...._. _.._. ....._. . .. 'j ._.i~. ~..~:~7~~,:.:.... _w-A't =_ C7~~ I y510i; .:J:s :OI pianlS i:'i:. .icSGilJf u C:iGw, uiL:illalivGiy, ba;
u~:, jil,js expression vector, for example, pFastBacl (InVitrogen), resulting in pFastBacl-based plasnaids, using known methods, and information provided by the manufacturer's instructions nay be used.

(00105] The present invention is further directed to a gene construct comprising a nucleic acid encoding HA, as described above, operativelylibitbe#
no a regulatory element that is operative in a plant. Examples of regulatory elements operative in a plant cell and that may be used in accordance with the present invention inelude but are not limited to a plastocyariin regalatory region (US
7,125,978; which is incorporated herein by reference), or a regulatory region obtained from Ribulose 1,5-bisphosphate carboxylaseloxygenase (RuBisCO: US 4,962,028;
which is incorporated herein by reference), chlorophyll a/b binding protein (CAB;
Leutwiler et al; 1986; which is incorporated herein by reference), ST-LS1 (associated with the oxygen-evolving complex of photosystem II, Stockhaus et a11989; which is incorporated herein by reference). If the construct is expressed in an insect cell, examples of regulatory elements operative in an insect cell include but are not limited to the polyhedron promoter, the gp64 promoter and the like.

(00106] In plants, influenza VLPs bud from the plasma membrane (see Example 5, and Figure 19) therefore the lipid composition of the VLPs reflects their origin. The VLPs produced according to the present invention comprise HA, complexed with plant derived lipids. Plant lipids can stimulate specific immune cells and enhance the immune response induced. Plant membranes are made of lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and also contain glycosphingolipids. saponins, and phytosterols. Additionally, lipid rafts are also found in plant plasma membranes - these naierodosnains are enriched in sphingolipids and sterols. In plants, a variety of phytosterols are known to occur.
including stigmasterol, sitostecol, 24-methylcholesterol and cholesterol (Mongrand et al.. 2004).

[00107] PC and PE as well as glycosphingolipids can bind to CD1 molecules expressed by rnammalian immtme cells such as antigen-presenting cells (APCs) like _. , . . ..._.. .. . - .. .._, _J~-i). ~a%. . . .. _ . ., .,. . ..-....,. . .
....,.,,e: Li.
11i~' colsipatiuiiity col:ipiex (MHC) ntoie4 ~iCS of cliaiii I i31id LI.c:lr L01G' is to pI'i:de14{
glycolipid antigens to NKT cells (Natural Killer T cells). Upon activation, NKT cells activate innate immune cells such as NK cells and dendritic cells and also acctivate adaptive immune cells like the antibody-producing B cells and T-cells.

[00108] A variety of phytosterols may be found in a plasma membrane - the specific coznplement may vary depending on the species, growth conditions, nutrient resources or pathogen state, to name a few factors. Generally, beta-sit.ost.erol is the most abundant phytosterol.

[00109] The phytosterols present in an influenza VLP complexed with a lipid bilayer, such as an plasma-membrane derived envelope may provide for an advantageous vaccine compositian. Without wisktuig to be bound by theory, plant-made VLPs complexed with a lipid bilayer, such as a plasma-membrane derived envelope, may induce a stronger imrnune reaction than VLPs made in other expression systems, and may be similar to the immune reaction induced by live or attenuated whole virus vaccines.

[00110] Therefore, in some embodiments, the invention provides for a VLP
eomplexed with a plant-ilexived lipid bilayer. In some embodiments the plant-derived lipid bilayer may comprise the envelope of the VLP.

[00111] The VLP produced within a plant may induce an HA. comprising plant-specific N-glycans. Thercfore, this invention also provides for a vLP
comprising HA
having plant specific N-glycans.

[00112] Furthermore, modification of N-glycan in plants is known (see for example U.S. 601944,344; which is incorporated herein by reference) and HA
having modified N-glycans may be produced. HA comprising a modified glycosylation pattern, for example with reduced fucosylated, xylosylated, or both, fucosylated and xylosylated, N-glycans may be obtained, or HA having a modified glycosylaiion pattem may be obtained, wherein the protein lacks fucosylation, xytosylation, or both, and comprises increased galatosylatiotl. kllrthennore, modulation of post-~.. .... ~._...... _ . .. y: _,yi~.:_,; v_ .__ . ~: __. . . .__ . .,__.... _ ,. . _ . .
a Wild-tyi;,: exprLasing Hr..

[00113] For example, which is not to be considered limiting, the synthesis of HA having a modified glycosylation pattern may be achieved by co-expressing the protcin of interest along with a nucleotide sequence encoding beta-1.4galaetosyltransferase (GaIT), for example, but not litnited to manvnalian GaIT, or human Ga1T however GaIT from another sources may also be used. The catalytic =
domain of GaIT may aiso be fused to a CTS domain (i.e. the cytoplasmic tail, 1o transmembrane doniain, stem region) of N-acetylglucosaminyl transferase (GNTl), to produce a GNT1-Ga1T hybrid enzyme, and the hybrid enzyme may be co-expressed with HA. The HA may also be co-expressed along with a nucleotide sequence encoding N-acetyiglucosaminyltrasnferase III (GnT-III), for ex<:.-nple but not limited to mammalian GnT-III or human GnT-III, GnT-III from other sources may also be used.
Additionally, a GNT1-GnT-IlI hybrid enzyme, oompising the CTS of GNT1 fused to GnT-lIl may also be used .

[00114] Therefore the present invention also includes VLP's comprising HA
having modified N-glycans.

[00115] Without wishing to be bound by theory, the presence of plant N-glycans on HA may stimulate the immune responsc by promoting the binding of HA
by antigen presenting cells. Stimulation of the irnmune response using plant N
glycan has been proposed by Saint-jore-Dupas et at. (2007). Furtbermore, the conformation of the VLP may be advantageous for the presentation of the antigen, and enhance the adjuvmt effect of VLP when complexed with a plant derived pilid layer.

[00116] By "regulatory region' -regulatory element" or "promote.r" it is meant a portion of nucleic acid typically, but not always, upstream of the protein coding region of a gene, which may be comprised of either DNA or RNA. or both DNA 9nd RNA. When a regulatory region is active, and in operative association, or operatively linked, with a gene of interest, this may result in expression of the gene of interest. A
regulatory element may be capable of mediating organ specificity, or controlling 3g . .. . ..... ... . . . , .....=......_ . . . ... ., _ . u ... . . . ....,. t, ..i . .-A

.......... , .... t.l.._... . ... . .. . . .... ...,... .J t .. .tlt ~ll'~a.._.. .r ..., l _.. ....._. ,~ vare inducible in respi,~.se to an exterrral stimulus, elements u:at uleciiate promocer activity such as negative regulatory elements or transcriptional enhancers.
"Regulatory region". as used here.in, also includes elements that are active following transcription, for example, regulatory olements that modulate gene expression such as translational and transeriptiorzal enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability deterrmi.nants. Several of these latter elements may be located proximal to the coding region.

j001171 In the context of this disclosure, the term "regulatory element" or regulatory region" typically refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a stnletttral gene, which controls the expression of the coding region by providing the recognition for RNA
polymera.se and/or other factors required for transcription to start at a particular site.
However, it is to be understood that other nucleotide sequences, located within introns, or 3' of the sequence may also contribttte to the regulation of expression of a coding region of interest, An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure itutiation at a particular site is a promoter element. Most, but not all, eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence comprised of adenosine and thymidine nucleatide base pairs usually situated approximately 25 base pairs upstream of a trauscriptional start site. A promoter element comprises a basal prornoter element, responsible for the initiation of transcription, as well as other regulatory elements (as listed above) that modify gene expression.

[40118] There are several types of regulacory regions, i.nclutti_i;< <.":ose that are developmentally regulated, inducible or coustitutive. A regulatory region that is developmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory regions that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or at a basal level in other organs or tissues within _... ...... .r,_õ ... ..... . _ ~. ,.. ;~ ~ . . , ..., ,..,_ -r- .:... ....~_....-.~-.e~,._ , . .. -. _... ,. ~.. i ~.,..-.... , .~-_ .a._ .....__._.. r_,.,..._._. (i a::k et al., 1993, J. Plant PhyJc;L 152: 595-599;
3ilodeau et al., Plant Ceti 14:

125-130). An example of a leaf-specific promoter includes the plastocyanin promoter (Figure lb or SEQ ID NO:23; US 7,125,978, which is incorporated herein by= -refe.rence).

[00119] An inducible regulatory region is one that is capable of directly or =
.
indirectly activating transcription of one or more DNA sequences or genes in response -= to an inducer. In the absence of an inducer the DNA sequences or genes will not be to transcribed. Typically the protein factor that bimds specifically to an inducible regulatory region to activate transcription may be present in an inactive forrn, which is then directly or indirectly converted to the active form by the indncer.
However, the protein factor may also be absent. The inducer cau be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible regulatory region may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. Inducible regulatory elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, I.R.P., 1998, Trends Plant Sci. 3, 352-358;
which is incorporated by reference). Examples, of potential inducible promoters include, but not limited to, tetracycline-inducible promoter (Gatz, C.,1997, Ann. Rev.
Plant Physiol. Plant Mol. Biol. 48, 89-108; which is incorporated by reference), steroid inducible promoter (Aoyama, T. and Chua, N-11,1997, Plant J. 2, 397-404;
which is incorporated by reference) and ethanol-inducible promoter (Salter, M.G., et al, .1998, Plant Journal lfi, 127-132; Caddick, M.X., et a1,1998, Nature Bioteeh. 16, 177-180, which are incorporated by reference) cytokinin inducible I.B6 and genes (Brandstatter, I. and Kieber, J.J.,1998, Plant Cell 10, 1009-1019;
Kakimoto, T., 1996, Science 274, 982-985; which are incorporated by reference) and the auxin inducible element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971;
which is incotporated by reference).

[00 IAconstitutiveree,+l7tor.

+.1C:vCI(1^i-r1S:IIt. EJLC.mples of knorv:t CO11SLItltt2`lP TZaLi(a. ~nJ
CICI71."..Ilts IIlClUi,C 1)rolnC+tt,Ss associated with the CaMV 35S transcript. (Odell et al., 1985, Natnre, 313: 810-812), the rice actin 1(Zhang et al, 1991, Plant Cell, 3: I 155-1165), actin 2 (An et al., 1996, Plant,T., 10: 107-121), or tms 2 (U.S. 5,428,147, which is incorporated herein by referertce), and triosephosphate isomerase 1(Xu et. a1., 1994, Plant Physiol.
106: 459-467) gestes, the maize ubiquitin 1 gene (Cornejo et al, 1993, Plant Mol. $iol.
29: 637-646), theArabidopsis ubiquitin I and 6 genes (Holtorf et al, 1995, Plant Mol.
Biol.
29: 637-646), and the tobacco txanslational initiation factor 4A gene (Mandel et al, 1995 Plant Mol. $iol. 29: 995-1004). The term "constitutive as used herein does not necessarily indicate that a gene under control of the constitutive regulatory region is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types even though variation in abundance is often observed.

ts [00121] By "operatively linked" it is meant that the particular sequences, for exarrtple a regulatory element and a coding region of interest, interact either directly or indirectly to carry out an intended function, such as mediation or modulation of geno expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences.

[00122] The one or more than one nucleotide sequence of the present invention may be expressed in any suitable plant host that is transformed by the nucleotide sequence, or constructs, or vectors of the present invention. Exainpi;s of suitable hosts include, but are not limited to, agricultural crops including alfalfa-canola, Brassica spp., maize, Nicotiana spp., alfalfa, potato, ginseng, pea, oai, rice, soybean, wheat, barley, sunflower, cotton and the like.

[00123] The otie or more chitnerie gernetic constntcts of the present invention can further cotrzprise a 3' untranslated region. A 3' untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation sign2l and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is ustially characterized by effecting the addition of polyadenytie acid tracks to the 3' end of the rtiRNA precursor.

of th-- ;'ynctic constructs of the pres,;-tt invenr,= can c`5,3 fnclude further enbancers, either transladon or transcription enhancers, as may be required.
These enhancer regions are well known to persons skilled in the art, and can include the ATO initiation codon and adjacent sequences. The initiation cadon must be in phase with the reading frame of the ooding sequence to ensure translation of the entire sequence.

[00124] Non-limiting examples of suitable 3' regions are the 3' transcribed non-translated regions containing a polyadenylation signal of Agrobaeterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes, the small subunit of the ribulose-1, 5-bisphosphate crarboxylase (ssRUBISCO; US 4,962,028; which is incorporated herein by reference) gene, the pronnoter used in regulating plastocyanin expression (Pwee and Gray 1993; which is incorporated herein by reference). An example of a plastocyanin promoter is described in US 7,125,978 (which is incorporated herein by reference) [001251 As described herein, promoters comprising enhancer sequences with dernonstrated efficiency m leaf expression, have been found to be effective in transient expression. Without wishing to bc bound by theory, attachment of upstream regulatory elements of a photosynthetic gene by attachment to the nuclear matrix may mediate strong expression. For example up to -784 from the translation start site of the pea plastocyanin gene may be used mediate strong reporter gene expression.
[00126] The use of a reguIatory region from a photosynthetic gene, for example but not limited to a plastocyanin regulatory region (US 7,125,978; which is incorporated herein by reference), or a regulatory region obtained fronn Ribulose 1,5-bisphosphate carboxylaseloxygenase (Ru$isCO; US 4,962,028; which is incorporated herein by reference), chlorophyll a/b binding protein (CAB; I.eutwiler et a;
1986;
which is incorporated herein by reference), ST-LS 1(associated with the oxygen-evolving complex of photosystem II, Stockhaus et a1.1989; which is incorporated herein by reference) may be used in accordance witb the present invention.

CIO ?''71 AIl selectable rna.` include etizy:ries Chat provide for C-:-sistance to cl:eniicais such as an antibiotic for example, gentamycin, hygromycin, kanamycin, or herbicides such as phosphinothrycin, glyphosate, chlorosulfuron, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS
(beta-gtucuronidase), or luminescence, such as luciferase or GFP, may be used.
[00228] Also considered part of this invention are transgenic plants, plant cells or seeds containing the chimeric gene constrnct of the present invention_ Methods of regenerating whole plants firom plant cells are also known in the art. In general, transformed plant cells are cultured in fln appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The ptants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques. Z'ransgenic plants can also be generated without using tissue cultures.

[00129] Also considered part of this invention are trFUtsgenic plants, trees, yeast, bacteria, fungi, insect and animal cells containing the chimeric gene construct comprising a nucleic acid encoding recombinant HAO for VLP production, in accordance with the present inventiorc [00130] The regttiatory elements of the present invention may also be combined with coding region of interest for expression within a range of host organisms that are amenable to transfozmation, or transient expression. Such organisms include, but are not limited to plants, both monocots and dicots, for example but not limited to corn, cereal plants, wheat, barley, oat, Nicorfana spp, Brdssfca spp, soybean, bean, pea, alfalfa, potato, tomato, ginseng, and Arabidopsis.

ani{i''4P.F1"Tai. ' "" . ... .
.. , i !~~ }, I :~ r#. -. . .. = ri . .

t:-azsforrrcd and regenrraccd piar,t; is not critical to the present invention.
[00132] By "transformation" it Is meant the stable interspecific transfer of genetic infomtation (nucleotide sequence) that is manifested genotypically, pltenotypically or both. The interspecific transfer of genetic information from a chimeric construct to a host may be heritable and the transfer of genetic information considered stable, or the transfer may be transient and the transfer of genetic information is not inheritabl--_ [00133] By the term "plant matter", it is meant any material derived from a plant. Plant matter may comprise an entire plant, tissue, cells, or any fraccion thereof_ Further, plant matter may comprise intracellular plant components, extracellular plant components, liquid or solid extracts of plants, or a oombinnrion thereQf.
Further, plant matter may comprise plants, plant cells, tissue, a liquid extract, or a combination I S thereof, from plant leaves, stems, fruit, roots or a combination thereof.
Plant matter may comprise a piant or portion thereof which has not been subjected to arty processing steps_ However, it is also contemplated that the plant material may be subjected to miniuial processing steps as defined below, or more rigorous processing, including partial or substantial protein purification using techniques comrrtonly Imown within the art including, but not limited to chramatography, electrophoresis and the like.

[00134] By the term "minimat processing" it is meant plant matter, for example, a plant or portion thereof comprising a protein of interest which is partialiv rnirified to ,iield a plant extract, homogenate. fraction of plant homogenate or the lLkn (i.e.
!:i =.imally processed). Partial purit cation may comprise, but is not {irnited,c :n:,-: ~pting plant cellular structures therc,=y creating a composition comprnsin,- :nuzble plant cornponents, and insoluble plajit components which may be separated for example, but noi limited to, by centrifugation, filtration or a combination thereof. In this regard, proteins scrreted witb.in the extracelluiar space of leaf or other tissues could be readily obtained using vacuum or centrifttgal extraction. or tissues could be extracted under pre.csure by passage through rollers or grinding or the likc to squeere :f ric tei;~ free `;

~~ ~ = . :. . , ..; ~ .,,...~, .._~~ [[lC.~c vrduId have negii6;'.;if: CrJIltai I:::;~:iptl ~:ut'1 SPCOrltialy ~71i1P_`L
prodllct5.
Further, minimal processing may involve aqueous extraction of soluble protein from leaves, followed by precipitation with any suitable salt. Other methods may include large scale maceration and juice extraction in order to permit the direct use of the extract.

[00135] The plant mattcr, in the form of plant material or tissue may be orally delivered to a subject. The plant matter may be administered as part of a dietary supplement, along wirth other foods, or eneapsulated. The plant matter or tissue may also be concentrated to improve or increase palatability, or provided along with other r.oa.terials, ingrcdients, or pharmaceutical excipients, as required.

[00136] Examples of a subject or target organism that the VLPs of the present invention may be administered to include, but are not limited to. humans, primates, birds, water fiowl, migratory birds, quail, duck, gecse, poultry, ehicken, swine, sheep, equine, horse, camel, canine, dogs, feline, cats, tiger, leopard, civet, mink, stone marten, ferrets, house pets, livestock, rabbits, mice, rats, guinea pigs or other rodents, seal, whale and the like. Such target organisms are exemplary, and are not to be considered timit'utg to the applications and uses of the present inven,tion.

[00137) It is contemplated that a plant comprising the protein of interest, or expressing the VLP comprising the protein of izuesest may be administered to a subject or target organism, in a variety of ways depend.ing upon the need and the situation. For example, the protein of interest obtained from the plant may be extracted prior to its use in either a crude, partially purified, or purified form. If the protein is to be purified, then it may be produced in either edible or non-edible plants.
Furthermore, if the protein is orally administered, the plant ussue may be harvested and directly feed to the subjec-t, or the harvested tissue may be dried prior to feeding, or an animal may be permitted to graze on the plant with no prior harvest taking place.
It is also considered within the scope of this invention for the harvested plant tissues 3o to be provided as a food supplement within animal feed. If the plant tissue is being i.. ~ T... , . -, .. .. ..:}1 liL[.:. o!" ^(tt ~uCI::::C';n. - . ' . .. '..
....:i:. . 4:_.. ,.~r ` , -.-;. . ... .. .= _ -....,,: = ,r, .

tuJi3$1 Yost-transcriiptional gene sileacing (PTGS) may be involved in limiting expression of transgenes in plants, and co-expression of a suppressor of silencing from the potato vinis Y(HcPro) may be used to counteract the specific degradation of transgene nzxtNAs (Brigneti et al.. 1998). Altexnate suppressors of silencing are well known in the art and may be used as described herein (Chiba et al., 2006, Virology 346:7-14; which is incorporated herein by reference), for example but not limited to, TEV -pl/HC-Pro (Tobacco etch virus-p1/HC-Pro), BYV -p21, p19 of Tomato bushy to stunt virus (TBSV p19), capsid protein of Tomato crinide virus (TCV -CP), 2b of Cucumber mosaic virus; CMV-2b), p25 of Potato vin,s X(PVX-p25), pl I of Potato virus M(PVM-pI1), p1I of Potato viru<c S(PVS-p11), p16 of Blueberry scorch virus, (BScV -p16), p23 of Citrus tristexa viais (CTV-p23), p24 of Grapevine leafroll-associated virus-2, (GLRaV-2 p24), p10 of Orapevine virus A, (GVA-plO). p14 of Grapevine viYl74 B(GVB-p14), p10 of Heracleum latent virus (HLV-plO). or p16 of Garlic common latent virus (GCLV-p16). Therefore, a suppressor of silencing, for example, but not limited to, HcPro, TEV -p1/HC-Pro, $YV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25, pVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2 p24, GBV-p14, HLV-plO, GCLV-p16 or GVA-plO, may be co-expressed along with the nucleic acid sequence encoding the protein of interest to further ensure high levels of protein production within a plant.

1001391 Furthettnore, VLPs may be produced that comprise a combination of HA subtypes_ For example, VLPs may comprise one or more than one HA from the subtype H1, H2, H3, H4, 115, H6, H7, H8, H9, H10, HI1, H12, H13, H14, H15, H16, or a combination thereof. Selection of the combination of HA.s may be determined by the intended use of tlte vaccine prepared from the VLP. For example a vaccine for use in inoculating birds may comprise any combination of HA subtypes, while VLPs useful for inoculating humans niay comprise subtypes one or more than one of subtypes H 1, H2, H3, H5. However. other HA subtype combinations may be prepared depending upon the use of the VLP. In order to produce VLPs comprising combinations of HA subtypes, the desired HA subtype may be co-expressed within the same cetl, for example a plant cell.

3?

. .. ... .

compriS i::Z :A at.:l Nr. be desited.

(00141] Therefore, the present invention fnrttter includes a suitable vector comprising the chimeric construct suitable for use with either stable or transient expression systems_ The genetic information may be also provided within one or more than one construct_ For example, a nucleotide sequence encoding a protein of interest may be introdueed in one construct, and a second nucleotide sequence encoding a protein that modifies glycosylation of the protein of interest may be introduced using a separate construct. These nucleatide sequences may then be co-expressed within a plant_ However, a construct comprising a nucleotide sequence encoding both the protein of interest and the protein that modifies glycosylation profile of the protein of interest may also be used. In this case the nucleotide sequence would comprise a first sequence comprising a first nucleic acid sequenCe encoding the protein of interest opexatively linked to a promoter or regulatory region, and a second sequence comprising a second nucleic acid sequence encoding the protein that modifies the glycosylation profile of the protein of interest, the second sequence operatively linked to a promoter or regulatory region.

j001421 By "co-expressed" it is tneant that two, or more than two, nucleotide sequences are expressed at about the same time within the plant, and within the same tissue of the plant. However, the nucteotide sequences need not be expressed at exactly the same time. Rather, the two or more nucleotide sequences are expressed in a manner such that the encoded products have a chance to interact_ For example, the protein that modifies glycosylation of the protein of interest may be expressed either before or during the period when the protein of interest is eapressed so that modification of the glycosylation of the protein of interest takes place. The two or more than two nucieotide sequences can be co-expressed using a transient expression system, where the two or more sequences are introduced within the plant at about the same time under conditions that both sequences are expressed_ Alternatively, a platform plant comprising one of the nudeotide sequences, for example the sequence encoding the protein that modifies the glycosylation profile of the protein of interest, may be transformed, either transiently or in a stable manner, with an additional nc r.. ~,i .. .

e:S-Ir--sseC1 w'1tfua c. ;siiW clsyl:a, during a desired stage of developsn,c:it, or its expression may be induceld using an inducible promoter, and the additional seyuence encoding the protein of interest may be expressed under similar conditions and in the same tissue, to ens-ffe that the nucleatide sequences are co-expressed, [00143] The comstructs of the present invention can be introduced into plant cells using Ti plasuaids, Ri plasmids, plant virus vectors, direct DNA
transforniation, micro-injection, eIectroporation, etc. For reviews of such techniques see for example Weissbach and Weissbacbõ Methods for Plant Molecular Biology, Academy Rrss, New York VIII, pp. 421-463 (1988); Geierson and Corey, Planr Molecular Biology, 2d Ed. (1988); and Milci and Iyer, Fundarnentals of Gene Transfer in Plants, In Plant Metabolisrn, 2d Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly, L.aagmar,s Ltd. London, pp. 561-579 (1997). Other methods include direct DNA uptake, the use of liposornes, electroporation, for example using protoplasts, micro-injection, microprojeetiles or whiskers, and vacuum inf"iltration. See, for example, Belang, et aI. (Gene 100: 247-250 (1991). Scheid et al. (Mol. Gen.
Genet.
228: 104-112, 1991), Guerche et al. (Plant Science 52: 111-116, 1987), Neuhause et al. (Theor. Appl Genet. 75: 30-36, 1987), Klein et al., Nature 327: 70-73 (1987);
Howell et al. (Science 208: 1265, 1980), Horsch et ai. (Science 227: 1229-1231, 1985), DeBlock et al., Plant Physiology 91: 694-701, 1989), Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.. Academic Press Inc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press Inc., 1989), Liu and Lomonossoff (J. VirollVleth, 105:343-348, 2002,)., U_S, Pat. Nos.
4,945,050; 5,036,006; and 5,100,792, U.S. patent application Ser. Nos.
0$1438,666, filed May 10, 1995, and 071951,715, filed Sep. 25, 1992, (all of which are hereby incorporated by reference).

[00144] Transient expression methods may be used to express the constructs of the present invention (see Liu and Lomonossoff, 2002, Jotunal of Virological Methods, 105:343-348; which is incorparated herein by reference).
Alternatively, a vacuum-based transient expression method, as desen'bed by Kapila et al. 1997 (incorporated herein by reference) may be used. These methods may include, for a Ilii ti:u lf tiyfOt]CIC lBrtQ COInpCiiL^.g (1'ie .'1.cSiIF'Cl nucleic acid enter the intercellular spaces of a tissue, for example the leaves, aerial portion of the plant (including stem, leaves and flower), other portion of the plant (ster;., root, flower), or the whole plan' AJI.er crossing the epidermis tho Agrvbacreriurn infect and txansfec t-DN.5 copies into the cells. The t-DNA is episomally transcribed and the mRNA tanslated, leading to the production of the protein of interest in infected cells, however, the passage of t-DNA inside the nucleus is transient.

[00145) If the nucleotide sequence of interest encodes a product that is directly or indirectly toxic to the plant, then by using the method of the present invention, such toxicity may be rcduced throughout the plant by selectively expressing the nucleotide sequence of interest within a desired tissue or at a desired stage of platrt development.
In addition, the limited period of expression resulting from transient expression may reduce the effect when producing a toxic product in the plant. An inducible promoter, a tissue-specific promoter, or a cell specific promoter, may be used to selectively direct expression of the sequenr.e of interest.

[00146) The recombinant HA VLPs of the present invention can be used in conjunction with existing influenza vaccines, to supplement the vaccines, render them more efficacious, and to reduoe the administration dosages necessary. As would be known to a person of skill in the art, the vaccine may be directed against one or more than one influenza virus. Examples of suitable vaccines include, but are not limited to those commercially available from Sanofi-Pasteur, ID Biomedical, Merial, Sinovac, C'hiron, Roche, Medirmnune, G1axoSmithKline, Novartis, Sanofi-Aventis, Serono, Shire Pharmaceuticals and the like.

[001471 If desired, the VLPs of the present invention may be admixed with a suitable adjuvant as would bc known to one of skill in the art. Furthermore, the VLP
may be used in a vaccine composition comprising an effective dose of the VLP
for the treatment of a target organism, as defined abave. Furehormore, the VLP
produced ..- . . , n~_ ..'.r... .. . ,.... ,; . . . ,,.,.. ..

[0014aj Therefore, the preseiit invention provides a method for inducing immunity to influenza virus infection in an animal or target organism comprising administering an effective dose of a vaccine comprising one or more than one VLP.
The vaccine may be administered orally, intraderr,ally, intranasally, intramuscularly, intraneritoneally, intravenously, or subcutaneously.

[00145] Administration of VLPs produced according to the present invention is described in Example 6. Adxninstration of plant-made H5 VLP resulted in a significantly ttigher response when compared to administration of soluble HA
(see Figures 21A and 21B).

[00150] As shown in Figares 26A aad 26 B a subject admirdstered Allndonesia/5/05 H5 VLPs provided cross-protection to a challen.ge with influenza A/I'urkey/582/06 (H5N1; "Turkey H5N1r'). Adrninistration of Indonesia H5 VLPs before challenge did not result in any loss of body mass. However in subject not administered H5VLPs, but challenged with Turkey H5N1, exhibited significant loss of body mass, and several subject died.

[00151] These data, therefore, demonstrate that plant-made influenza VLPs comprising the H5 hemagglutinin viral protein induce an immune response specific for pathogenic influenza strains, and that virus-like particles may bud from a plant plasma membrane.

[00152] Therefore, the present invention provides a composition comprising an effective dose of a VLP comprising an influenza virus HA protein, one or more than one pla.nt lipid, and a plzamaceutiGally acceptable carrier. The influeatza virus HA
protein may be H5 Indonesia. Also provided is a method of inducing im.munity to an influenza virus infection in a subject. The method comprising administering the virus like particle comprising an influenza virus HA protein, one or more than one plant lipid, and a pharmaceutically acceptable carrier. The virus like particle may be administered to a subject orally, intradermally, intranasally, intrarnusclarly, intraperitoneally, intravenously, or subcuianeously.

_ = . , . ... _ _ . . .. , .. ~ . , .. ~, cozt:p:.;._ -r;t t}.. ,~i-iy iiave isiununostimuiatory etfects_ Ta ittvtsti aate this possibiiitv, plant-made H5 VLf's were administered to aaimals in the presence or absence of an adjuvant, and the HA1(klemagglutination inhibition antibody response) determined (Figures 22A, 22B). In the absence of an added adjuvant plant-made H5 Y1Ps demonstrate a significant HAI, intiicative of a systemic irnmune response to administration of the ar]tigen. Furthermore, the antibody isotype profiles of VLPs administered in the present or absence of adjuvant are similar (Figure 23A).

Table 1: SeÃ;ue]a.-ne description for seqUencti idesrtifie_s.

SFQ ID No Secmence Eltycriptku In Disclpsul e 1 N terminal H[ 1ia Ft ure 5a 2 C ternrinal Hi fra t Fi ure 5b 3 H5 codin uence Figure 6 4 prinw Plato-443c Fi 7a 5 pnmer S HA(Xr1d)-PIasto.r r-imue 7b 6 rimer Plasto-S )-c F urc 7c 7 primer HA(Ind)-Sac_r Fi e 7d 8 Sequence of the alfalfa plastocyanin-based Figure 1 oz ession ca,asette u.sed for the ez tEss ion of H t 9 HA1 peptide se ueacc Figure 8a
10 HAS pepu& se uence F ure $b
11 influenza A Sub e H7 sequence F' ure 9
12 Influenza A S H2 uenoe F re 108
13 Influenza A Subt H3 ftquence Figum I Ob
14 influenza A S2tgpe H4 se ucnce Fi ure I Oc Inftuenza A Sv H5 s ence Figure 10d 16 Influeeza A Subtype H6 se uence Fi ure 10e 17 Influenza A Sub e H8 sequence F'i IOf 18 Influenza A Subt3W H9 uence Fi ure 10 19 Influetua A SubWpe H10 se uence Fil!LM ldb Tafluettza A Subtype H l l uetx.e ri ute 10i 21 Xnfluenzn A Subtype H12 s uence I.7t ure 1 22 Influenza A Sub FI13 sequence Fi e 10k 23 7nfluenza A Subrfpe H]4 sequence Fri e 101 24 [n1'luetua A Subtype H1S se uence Fi ure LOrn Influeoza A Sub~gM H16 se uence Figure lOn 26 Influenza B liA se uec~ec i i e 1 Oo 27 Influenza C HA seqpqme Fi l 28 Co ete HAO Si ueme PS urc 5c 29 Priraer 7tmal- Plas.c Figure I
Primer SacI-ATG Pias.r Fi ure l Or 31 Primer Sacl-P1as7er.c Fi ure 105 32 PrimerEcoRl-P1asTer.r Fi ure [(]t Z; ~ r..?., ~ _ ..__......_ _ _ _ .
....--= , _ ; !
_{ ~l:ccssrooNo. 71 1499 Figure IS
35 'lis'uer;oitico/~/34 (I~INI) I GenBanlc Accession No. NC_002016.1 [00154] The invention will now be described in detail by way of reference only to the following non-li.miting examples.

Methods and Materials 1. Assembly of eRpression cassettes [00155] All manipulations were done using the general molecular biology protocols of Sambrook and Russell (2001; which is incorporated herein by reference).
The first cloning step consisted in assembling a receptor plasmid containing upstream and downstream regulatory elements of the alfalfa plastocyanin gene. The pl.astocyanin prontoter and 5'UTR sequences were amplified from alfalfa genomic DNA using oligonucleotide primers XmaI-pPlas.c (SEQ ID NO: 29; Figure 10Q) and Sat -ATG-pPlas.r (SEQ ID NO: 30; Figure 10R)- The resulting amplification product was digested with Xmal and Sacl and ligated into pCANI$1A2300 (Cambia, Canberra, Australia), previously digested with the same enzymes, to create pCAMBIApromo Plasto. Similarly, the 3'UTR sequences and temtinator of the plastocyanin gene was amplif,ed from alfalfa genomic DNA using the following primers: SacI-PlasTer.c (SEQ I) NO: 31; Figure lOS) and EcoRI-PlasTer.r (SEQ ID NO: 32; Figura lOT), and the product was digested with SacI and EcoRI before being inserted into the same sites of pCAMBIApromoPlasto to create pCAMB7APlasto.

[00156] The open reading frame from the H1 gene of influenza strain AINew Caledonia/20/99 (H1N1) was synthesized in two fragments (Plant Biotechnology Institute, National Research Council, Saskatoon, Canada). A first fragment synthesized corresponds to the wild-type Hl coding sequence (GenBank acc. No.
AY289929: SEQ ID NO: 33; Figure 16) lacking the signal peptide coding sequence at the 5'end and the transmembran.e domain coding sequence at the 3'end_ A BgII[
restriction site was added at the 5' end of the coding sequence and a dual Sacl/Stul . , . _ ;~ in.., . . 1.. , ~ ~ - - . -=,.1., ... ....: ~ i.. L . lAll 0 t . .., ,, , ..... _ . . . . . _ .., . _,._ õ. .... . . _ ..._ :.:,: : .
.: ~u^smeribrane dcrnu.in and tytopiasmic tail) from the ICpnI site to the stop codon, and flanked in 3' by SacI and Stul =,. . . .
restriction sites was also synthesized (SEQ ID NO. 2; Figure 5B).

[00157] The first Hl fragment was digested with Bg1II and SacI and clocted into the same sites of a binary vector (pCANBIf#Plasto) containing the plastc,cyanin promoter and 5'U7R fused to the signal peptide of alfalfa protein disulfide isomerase (PDI) gene (nucleotides 32-103; Accession No. ZI1499; SEQ ID NO: 34; Figure 17) resulting in a PDI-Hl chiun.aric gene downstream of the plastocyanin regulatory elements. The seqtsence of the plastocyanin-based cassetta containing the PDI
signal peptide is presented in Figure 1(SEQ ID NO:8). The resulting plasmid contained Hi coding region fused to the PDI signal peptide and flanked by plastocyanin iegulatory clements. The addition of the C-terminal end coding region (encoding the transmembrane domain and the cytoplasmic tail) was obtained by inserting the synthesized fragment (SEQ ID NO: 2; Figure 5B) previously digested with Kpnl and Sacl, into the Hl expression plasmi.d. The resulting plasmid, named 540, is presented in Figure 11 (also see Figure 2A)_ 2. Assemblv of H5 exrnression cassette [00158] A fragment encoding hemagglutinin from influenza strain AlIndonesia/5/05 (H5NI; Acc. No. LANL ISDN125873) was syntbesized by Epoch Biotabs (Sugar Land, 'I'X, USA). The frfagment produced, containiuag the complete H5 coding region inciudibg the native signal peptide flanked by a HindIII site immediately upstream of the initial ATG, and a SacI site immediately downstream of the stop (TAA) codon, is presented in SEQ ID NO: 3 (Figure 6). The H5 coding region was cloned into a plastocyanin-based expression cassette by the PCR-based ligation method presented in Darveau et al. (1995). Briefly, a fsrst PCR
amplif'ication was obtained using primers Plato-443e (SEQ II) NO: 4; Figure 7A) and SpHA(Ind)-Plasto.r (SEQ ID NO:5; Figure 7B) and pCAMBIA pronnoPlasto as template. In parallel, a second acnplifieation was performed with primers Plasto-SpHA(Ind).c (SEQ I) NO: 6; Figure 7C) and HA(Ind)-Sac.r (SEQ ID NO:7; Figure 7D) with H5 ~ .. . . . . .. ._ ... :.Iii uuW rL~ac.iuW werc 37:uiV 4=t3c (SEQ ;vU: 4; i-iSa;,.'Jty} and Hfi(Irld)-Sac.r (SEQ I1.7 NO: 7; Figure 7D) as primers. The resulting fragment was digested with BamHl (in the plastocyanin promoter) and SacI (at the 3' end of the fragment) and cloned into pCAMBIAPlasto previously digested with the same enzymes. Ttte resulting plasmid, named 660, is presented in figure 2B (also see Figure 11).

[00] 59] The cassette encoding the soluble form of Hl was prepared by replacing the region coding for the transmetnbrane domain and the cytoplasmic tail in 540 by a fragment encoding the leucine zipper GCN4 pII variant (Harbury et al, 1993, Science 1993; 262: 1401-1407). This fragment was synthesized with flanking Kpnl and SacI sites to facilitate cloning. The plasmid resultin from this replacement was named 544 and the expression cassette is illustrated in figure 11.

[00160] A fusion between the tobacco etch virus (TEV) 5'UTR and the open reading frame of the influenza AIPRI8l34 MI gene (Acc. # NC_0b2016) was synthesized with a tlanking SaeI site added downstream of the stop codon. The fragment was digested with Swal (in the TEV 5'CT1R) and SacI, and cloned into a 2X35S/TEV based expression cassette in a pCAMBIA binary plasmid. The resulting plasmid bore the M1 coding region under the control of a 2X35S/TEV promioter and 5'UTR and the NOS terminator (construct 750; figure 11).

(001611 An HePro construct (35HcPro) was prepared as described in Hanaitton et al. (2002). All clones were sequenced to confirm the integrity of the constructs.
The plasmids were used to transform Agrobacteium rurnefaciens (AGLI; ATCC, Manassas, VA 20108. USA) by electroporation (Mattanovich et al., 1989). The integrity of all A. turnefaciens strains were confirmed by restriction mapping.
3. Preparation of glant biomass inoculum, agroinfiltration, and harvesting [00162] Nicoriana benrhamiana plants were grown from seeds in flats filled with a commercial peat moss substrate. The plants were allowed to grow in the greenhouse under a 16/8 photoperiod and a temperature regime of 25 C day/20 C
night. Three weeks after seeding, individual plantlets were picked out, transplanted in xt. tirt ., wi~t:s the sa[ne envlfRemcnz ,l ~~nr~jtinnc ~;r= =n + ..., r '.... . _ "-, .
., ious tunc3 as indicated v, by piacuu:!5- the buds frorn riie plant, or by chemically treating the plant [00163] Agrobacteria transfected with 660, 540, 544, 750 or 35SHcPro were grown in a YED medinm supplemented with 10 mM 2-[N-morpholinolethanesulfonic acid (MES), 20 M acetosyringone. 50 g{rni kanamycin and 25 itg/ml of carbenicillin pH5.6 until they reached an QD(;Co between 0,6 and 1.6.
Agrobacter-ium suspensions were centrifuged before use and resuspended in infiltration medium (10 to mM MgC12 and 10 mM IvfES pH 5.6). Syringe-infiitration was performed as described by Liu and Lomonossoff (2002, Journal of Virological Methods, 105:343-348). For vacuum-infiltrntian, A. tumefaciens suspensions were centrifuged, resuspended in the infiltration medium and stored ove,rnight at 4 C. On the day of iafiltration, c,ulture batches were diluted in 2.5 culture volumes and allowed to wazTn 25 before use. Whole plants of Nicodana benthamiana were placed upside down in the bacterial suspension in an air-tight stainless steel tank under a vacuum of 20-40 Torr for 2-min. Following syringe or vacuum infiltration, plants were retumed to the greenhouse for a 4-5 day incubation period until harvest.

4_ Leaf saznpling and total ymtGin extraction 2U [00164] Following incubation, t,he aerial part of plants was harvested, frozen at -804C, crushed into pieces. Total soluble proteins were extracted by honmogenizing (Polytron) each sample of frozen-crushed plant material in 3 volumes of cold 50 mM
Tris pH 7.4, 0.15 M NaC1, and 1 mM phe.nylmethanesulfonyl fluoride. After homogenization, the slurries were centrifuged at 20.000 g for 20 min at 44C
and these 25 clarified crude extracts (supernatant) kept for anaJyses. The totnl protein content of clarified cnide extracts was determined by the Bradford assay (Bio-Rad, Hercules, CA) using bovine serum albumin as the reference standard.

5. Size exclusion chromatoarayhy of pMein extract [001651 Size exclusion chromatography (SEC) columns of 32 ml SephacrylT14 30 S-500 high resolution beads (S-500 HR : GE Healthcare, Uppsala, Sweden, Cat. No.

17.;~~iZ-=(1` -, Tris Dl-IR, 15() mlA;! Fr('ll rlno ~ 1 n? ir .- ..,_ , . . .... . ..... . . ,... . .. .,,,_ ..l.J 1. ili....onto the co-, - .. by an elution step witl: cf equi.:5rati.ir.:eiuuon buffer. The elution was collected in fractions of 1.5 ntL relative protein content of eluted fractions was monitored by mixing 10 L of the fraction with 200 pL of diluted Bia-Rad protein dye reagent ($io-Rad, Hercules, CA The column was washed with column volumes of 0.2N NaOH followed by 10 column volumes of 50 mM 'I'ris pH8, 150 mM NaCI, 20% ethanol. Each separation was followed by a calibration of the column wiLh Blue Dextran 2000 (GE Healthcare Bio-Science Corp., Piscataway, NJ.
USA). Elution profiles of Blue Dextran 2000 and host soluble proteins were compared between each separation to enstur uniformity of the elution profiles between the columns used.

6. Protein Analysis and Imrnunoblotting (001661 Protein concentrations were determined by the BCA protein assay (Pierce Biochemicsls, Rockport IL). Proteins were separated by SDS-PAGE under reducing conditions and stained with Coomassie Blue. Stained gels were scanned and densitometry analysis performed using IirtageJ Software (NIH).

[00167] Proteins from elution fraction from SEC werc precipitated with acetone (Bollag et al., 1996), resuspended in 115 volume in equitibration/elution buffer and separated by SDS-PAGE under reducing conditions and electrotransferced onto polyvinytene difluoride (PVDF) membranes (Roche Diagnostics Corporation, Indianapolis, IN) for irnmtmodetection. Prior to im[nunoblotting, the membranes were blocked with 5% skim milk and 0.14b Tween-20 in Tris-buf,Fered saline (TBS-T) for 16-1 8h at 4 C.

[00168] Immunobl,otting was performed by incubation with the following antibodies: for the detection of H1, a mouse anti-influenza A monoclonal antibody (Fitzgerald Industrie,s InternationaI, Concord, MA. USA. Cat_ No. 10-L50) (2 g/ml in 2% skira milk in TBS-Tween 20 0.196), and for the detection of H5, a rabbit anti-H5 (Vietnam) antibody (Immune Technology, Woodside, NY, USA, Cat No.1T-003-005V) diluted 1/4000 in 2% skim milk in TBS-Tween 20 0.M A peroxidase-:J -l;, t~=,' - , ' LlIIQT3tnCit$. ~'L~`r,c7 ~*n,r *, , r.( ~ ~, _ . . . _. .. - .. _. . . .,..,._.i!_.r~
s;.im Tnii t 20 t1.1:o)was used as secondary antibody. hnmunoreactive complexes were detected by chemiluminescence using luminol as the substrate (Roche Diagnostics Corporation). Horseradish peroxidase-euzyme conjugation of human 1gG antibody was camed out by using the EZ-Link Plus Activated Peroxidase con}ugation kit (Pierce, Rockford,1T.).

[00169] Heinagglutination assay for I=15 was based on a method described by Nayak. and Reichl (2004). Briefly, serial double dilutions of the test samples (100 L) were made in V-bottomed 96-well microtiter plates containing 100 L PBS, leaving 100 L of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link lnc., Sytacuse, NY) were added to each well, and plates were incubated for 2h at room temperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as HA activity. In parallel.
arecombinant HA standard (AlVietnaml1203/2004ILSN1) (Protein Science Corporation, Meriden, CT) was diluted in PBS and run as a control on each plate.
7. Sucrose mdienE ultracentrifuaation (001701 One milliliter of fractions 9, 10 and 1 I eluted from the gel filtration chromatography on H5-containing biomass were pooled, loaded onto a 20-60%
(w/v) discontinuous sucrose density gradient, and centrifaged 17,5 h at 125 000 g(4 C).
The gradient was fractionated in 19 3-mL fractions starting from the top, and dialyzed to remove sucrose prior to immunological analysis and hemagglutination assays.

8. Electron microspW

[001711 Elution fractions from SEC to be observed by electron microscopy (EM) were first concentrated using 30 MWCO ultrafiltration urtits (Millipore, Billerica, MA, USA). The concentrated fraeuons were fixed in PBS pH 7.4 containing 2% giuteraldehyde for 24 h at 4 C. Once fixed the samples were adsorbed onto Formvar-coated 200-mesh nickel grids (Caraemcp. Lakefield, Canada) for 2 min, and the grids were washed twice with deionized water before being stained in I9b phosphotungstic acid. Observation was perfortned under tran,cmission electron . . ! ~'~ _J~> ~:~. .... _. ...
[00172] Afteznately, one hundred microIiters oE the samptes to be examined were placed in an Airfuge ultracxntrifugation tube (Beckman Instruments, Palo Alto, CA, USA). A grid was placed at the bottom of the tube which was then centtifuged 5 min at 120 000 S. The grid was removed, gently dried, and placed on a drop of 3%
phosphotungstie acid at pH 6 for staining. Grids were examined on a Hitachi transnrission electron microscopc (TEM) (for images in Figures 14B, 15B L7d 15C).
(00173] For images in Figure 19, Leaf blocks of approximately I mm3 were fixed in PBS containing 2,5% g]utaraldehyde and washed in PBS containing 3%
sucrose before a post-fntation step in 1,33% osraium terroxide. Fixed samples were imbedded in Spurr resin and ultrathin layers were laid on a grid. Samples were positively stained with 5% uranyl acetate and 0,2% lead citrate before observation.
Grids were examined on a Hitachi 7100 transmission electron microscope (TEM).

9. Plasma membrane lipid anaty5is [001741 Plasma membranes (PM) were obtained from tobacco leaves and cultured BY2 cells after cell fractionation according to Mongrand et aLby partitioning in an aqueous polymer two-phase system with polyethylene glyco13350/dextran T-500 (6.6% each). All steps were performed at 4 C.

[00175] Lipids were extracted and purified from the different fractions according to Bligh and Dyer. Polar and neutrai lipids were separated by monodimensional HP'-TLC using the solvent systems described in I.efebvre et at..
Lipids of PM fractions were detected after staining with copper acetate as described by Macala et al. I.apids were identified by comparison of their migration time with those of standards (all standards were obtained from Sigma-Aldrich, St-l.ouis, MO, USA, except for SQ which was obtained from Matreya, Pleasant Gap, PA, USA).
(001761 10. H,S VLP purification ... . _ .a .ti=e,e acu;u~a~ti~:;~:, ; , i.5' . . . -.l t.,+~' T ,~7 r= T.r -G5 ~. ( a eo.aiuereia] blender. The was sttpplemented with 1r0s PMSF and adjusted to pH 6 with 1 M acetic acid before being heated at 42bC for min. Diatornaceous earth (DE) was added to the heat-treated extract to adsorb the contaminants precipitated by the pH shift and heat treatment, and the slurry was filtered through a Whatman paper filter. The resulting clarified extract was centrifuged at 10,000 x g for 10 minutes at RT to renwve residual DE, passed througb 0.8/0.2 p m Acropacti 20 filters and loaded onto a fetu.inragarose affinity colummn (Sigma-Aldrich, St-Louis, MO, USA). Following a wash step in 400 rnlVl NaC1, 25 mM Tris pH 6, bound proteins were eluted with 1.5 M NaCl, 50 mM MES pH C. Eluted VLP were supplemented with Tween-80 to a final concentration of 0.0005% (vlv). VLP were concentrated on a 100 kDa MWCO Amicon membrane, centrifuged at 10,000 x g for 30 minutes at 4C and resu,cpended in PBS pH 7.4 witti. 0.01% Tween-80 and 0.01%
thimerosAl. Suspended VLPs werC filter-sterilized before use.

11. Animal studies [00178] Studies on the immune response to influenza VLP administration were performed with 6-8 week old female BALB/c mice (Charles River Laboratories).
Seventy mice were randomly divided into fourteen groups of five anitnals.
Eight 2o groups were used for intramuscular immuaixation and six groups were used to test intranasal route of administration. All groups were immunized in a two-dose regiment, the boost immunization being done 3 weeks following the first immunization.

[001791 For intramuscular administration in hind legs, unanaesthetized mice were immunized with either the plant-made VLP H5 vaccine (0.1, 1, 5 or 12 g), or a control hernagglutii,<in (HA) antigen. The control HA com.prised recombittant soluble hemagglutinin produced based on strain A/Irxdonesia/5/05 H5N1 and purified from 293 ee[l culture (Immune Technology Corp., New York. USA) (used at 3 Ng per injection unless otherwise indicated). Buffer control was PBS. This antigen consists of amino acids 18-530 of the HA protein, and has a His-tag and a modified cleavage c.i [00180] To measure the effect of adjuvant, two groups of animals were immunized with 5 pg plant-made VLP H5 vaccine plus one volume Alhydrogel2%
(alum, Accurate Chemical & Scientific Corporation, Westbury, NY, US) or with 5 jig recombinant hemagglutinin purified from 293 cell culture plus I volume alum.
Seventy mice were randornly divided into fourteen groups of five aninaals_ Eight groups were used far intramuscular imrnunization and six groups were used to test intranasal route of administration. All groups were inununized according to a prime-to boost regimen, the boost immunization performed 3 weeks following the first immunization.

[00181} For intiamuscular administration in hind legs, unanaesthetized mice were immunized with the plant-made H5 YL.P (0_1, 1, 5 or 12 pg), or the control hemagglutinin (HA) antigen (5 g) or PBS. All antigen preparations were mixed with Alhydrogel 1% (alum, Accurate ChemicaI & Scientific Corporatiqn, Westbury, NY, US) in a 1:1 volume ratio prior to immuni2ations . To measure the effect of adjuvant, two groups of anirnals were immunized with eitb= 5 g plant-made VLP
HS vaccine or with 5 g of control HA antigen without any ad1uvant.

[001821 For intranasal administrauon, mice were briefly anaesthetized by inhalation of isoflurane using an automated induction chamber. They were then immunized by addition of 4 N1 drop/nostril with the plant-made VLP vaccine (0.1 or I
pg), or with control HA antigen (1 Itg) or with PBS. All antigen preparations were maxed with chitosan glutamate 1% (Protosan, Novamatrix/ FMC BioPolymer, Norway) prior to inntnunizations. The mice then breathed in the solutions. To verify the effect of adjuvant with the intranasal route of administration, two groups of animais were unmunized with 1 g plant-made VI.P H5 vaccine or witlt ] g control HA antigen.

Fxperimental designfor lethal challenge [001831 One hundred twenty eight mice were randomly divided into sixteen groups of eight animals, one group being unimmunized and not challenged (negative S) fP'.,.. .., ='_, r... ,_ ,..ar.,},,,iil,! ~L',.._ . .-...,..._ 1 .1..~
. . . ~ u ~~ iy immunizaticn.

[00184i For intramuscular administration in hind legs, unanaesthetized mice were immunized with the plant-made N5 VLP (1, 5 or 15 pg), or 15 jig of control HA
antigen or PBS. All antigen preparations were mixed with one volume of Alhydrogel 1% prior to immunizations (alum, Accurate Chemical & Scientific Corporation, Westbury, NY, US)_ [00185] During the immunization period, mice were weighted once a week and observation and monitored for local reactions at the injection site.

[00186] Twenty two days following the second immunization, anesthetized mice were challenged intranasally (i.n.) into a BTA containment laboratory (P4-Jean Merieux-IlVSERM, Lyon, France) with 4-09 x 106 50% celt culture infective dose (CCID50) of iufluenza AlTurkeyl582l06 vixus (kindly provided by Dr. Brunio Lina, Lyon University, Lyon, France). Following challenge, mice were observed for ill clinical symptoms and weighed daily, over a fourteen day period. Mice with severe infection symptoms and weight loss of ~:25R6 were euthanized after anaesthesia.
Blood collectiort, lung and nasal washes and spleen collection [00187] Lateral saphenous vein blood collection was perfarmed fourteen days after the first ixnmunization and fourtoen days after second immunization on unanaesthetized animal. Serum was collected by centrifuging at 8000 g for 10 man.
(00188] Four weeks after second immunisation, mice were anaesthetized with COZ gas and immediately upon termination, cardiac puncture was used to coliect blood-[00189] After final bleeding, a catheter was inserted into the trachea towards the lungs and one ml of cold PBS-protease inhibitor cocktail solution was put into a 1 ce syringe attached to the catheter and injected into the lungs and then removed for analysis. This wash procedure was performed two times- The lung washes were centrifuged to remove celluiar debris. For nasal washes, a catheter was inserted ~ . . a? -i-, .. ,_ ... . _, -...... ., _.,. . õ us Spleen Co?leCt:Or1 'N'S pCrfPt'?it, ~
on micx immunized intramuscularly with 5pg of adjuvanted plant-made vaccine ar g adjuvanted recombinant H5 antigen as well as on mice immunized intranasaly with 1 g of adjuvanted plant-made vaccine or 1 g adjuvanted recombinant H5 antigen.
Collected spleens were placed in RPMI supplemented with gentamycin and mashed in a50 ml conical tube with plunger from a 10 mi syringe. Mashed spleens were rinsed 2 times and centrifuged at 2000 rpm for 5 min and resuspended in ACK lysing buffer to for 5 min at room temperature. The splenocytes were washed in PBS-gentamycin, resuspended in 59'o RPMI and counted. Splenocytes were used for proliferation assay.
Antibody titers [00190] Anti-influenza antibody titers of sera were measured at 14 days after the first immunization as well as 14 and 28 days after the second immunisation. The titer were deteimined by enzyme-linked inamunosorbent assay (ELISA) using the inactivated virus A/Indonesia/5/05 as the coating antigen. The end-point titers were expressed as the reciprocal value of the highest dilution that reached an OI) value of at least 0.1 higher than that of negative control samples.

[00191) For antibody class determination (IgGI, IgG2a, IgG2b,1gG3, IgM), the tit.ers were evaluated by EI.ISA as previously described.

Hemagglutination inhibition (HI) titers [00192] Hennagglutination inhibition (HI) titers of sera were measured at 14 and 28 days after the second immunisation as previously deseribed (WHO 2002;
Kendal 1982). Inactivated virus preparations from strains A/lndrniesial5/05 or A/Vietnarn/1203/2004 were used to test mouse serum samples for HI activity.
Sera were pre-treated with receptor-destroying enzyme lI (RDE II) (Denka Seiken Co..
Tokyo, Japan) prepared from Vibrio cholerae (Kendal 1982). HI assays were performed with 0.5% turkey red blood cells. HI antibody titre9 were defined as the reciprocal of the highest dilution causing cornplete Inhibition of agglutination.

t:xampte 1:Tra-rsient expression of influenza virus A/Indonesia/5/05 (H5N1) hemagglutinin by agroinfiltration in N. beRtitarniana plants [00193] The ability of the transient expression system to produce influenza hemzgglutinin was determined through the expression of the H5 subtype from straini A/indonesia/5/05 (H5NI). As presented in Figure 11, the hemagglutinin gene coding sequence (Ace. #EF541394), with its native signal peptide and transmembrane domain, was first assembled in the plastocyanin expression cassette -promoter, 5'UTR, 3'UTR and transctiption termination sequences from the alfalfa plastocyanin gene - and the assembled cassette (660) was inserted into to a pCAMBIA binary plasmid. This plasmid was then transfected into Agrobacteriurn (AGL1), creating the recombinant strain AGLI/660, which was used for transient expression.

[00194] N. benthrtmianu plants were i.nf'iltrated with AGLI/660, and the leaves were harvested after a six-day i.ncubation period. To detennine whether H5 accumulated in the agroinfiltrated leaves, protein were first extracted from infiltrated leaf tissue and analyzed by Westem blotting using anti-H5 (Vietnarn) polyclonal antibodies. A unique band of approximately 72 kDa was detected in extracts (Figure 12), corresponding in size to the uncleaved HAO form of influenza hemagglutinin.
The commercial HS used as positive control (AlVietnarn/1203/2004: Protein Science Corp., Meriden, CT, USA) was detected as two bands of approximately 48 and 28 kDa, corresponding to the moleculnr weight of HAl and HA2 fragments, respectively.
This demonstrated that expression of H5 in infiltrated leaves results in the accumutation of the uncleaved translation product.

[001951 The forrnation of active HA trimers was demonstrated by the capacity of crude protein extracts from AGT..1/660-transformed leaves to agglutinate turkey red blood eells (data not shown).

Example 2: Characteriaation of hema.gglutinin=containing structures In plant extracts using size exclusfon chromatography was F:SSCSSi Cl G :i:i diiol2. Crude protein eX[raclS frOm AGLI/660-infiltrated plants (1.5 mL) were fractionated by size exclusion chromatography (SEC) on SephacrylTm S-500 HR columns (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). Elution fractions were assayed for their total protein content and for HA abundance using immunodetection with anti-HA
antibodies (Figure 13A).As shown in Figure 13A, Blue Dextran (2 M7a) elution peaked early in fraction 10 while the bulk of host proteins was retained in the column and elut.ed between fractions 14 and 22. When proteins from 200 L of each SEC
elution fraction were concentrated (5-fold) by acetone-precipitation and analyzed by Western blotting (Figure 15A, 145), henvagglutinin (H5) was primarily found in fractions 9 to 14 (Figure 13B). Without wishing to be bound by theory, this suggests that the HA protein had either assembled into a large superstructure or that it has attached to a high motecutar weight structure.

100197) A second expression cassette was assembled with the H1 nucleic acid sequence from A/New Caledcnia/20/99 (H1N1) (SEQ ID NO: 33; Figure 16;
GenHank Accession No. AY289929) to produce consttuct 540 (Figure 11). A
chimeric gene construct was designed so as to produce a soluble trimeric form of H1 in which the signal peptide originated from a plant protein disulfide isomerase gene, and the transmembrane domain of Hl was replaced by the pIZ variant of the GCN4 lencine zipper, a peptide shown to self-assemble into rrimers (Harbury et al., 1993) (cassette 544, figure 11). Ahhougb lacking the transmembrane domain, this soluble trimeric form was capable of hemagglutiriation (data not shown).

[00198] Protein extracts from plants infiltrated with AGLI/540 or AGLt/544 were fractionated by SEC and the presence of Hl eluted fractions was examined by Western blotting with anti-influenza A antibodies (Fitzgeraid, Concord, MA, USA).
In AGL1/540-inFiltrated leaves, Hl accumulated mainly as a very high molecular weight structure, with the peak was skewed toward smaller size structures (HI;
Figure 13C). In AGL1I544-infiltrated leaves, the soluble form of Hi accumulated as isolated trimers as demonstrated by the elution pattem from gel filtration which parallels the host protein elution profile (soluble Hl; Figure 13D). In comparison, H1 rosettes (Protein Science Corp., Meriden, CT, USA), consisting in micelles of 5-6 trimers of " . , - . .. . .._ S~!- ...:L .v-...

[oa1991 't-~i -~ti-a?uaFe the impact of Mt cv-Cxpressio;i on hernagglutinin assembly into structure, a Ml expression cassette was assembled using the nucleic acid corresponding to the coding sequence of the A/PR/8/34 (H1N!) MI (SEQ ID
NO: 35; Figure 18; GenBank Accession No. NC_002016). The construct was named 750 and is presented in Figure 11. For the co-expression of M1 and H1, svspensions of AGLI/540 and AGL1r750 were mixed in equal volumE before infiltration. Co-infiltration of mnltiple Agrobacteritsm suspensions permits co-expression of multiple iQ transgenes. The Western blot analysis of SEC elution fractions shows that the co-expression of Mi did not modify the elution profile of the HT structures, but resulted in a decrease in HI accumulation in the agroinfiltrated leaves (see Figure 13F).
Example 3: Isolation of H5 structures by centrifugation in sucrose gradient and observation under electron m;croseopy [00200J The observation of hemagglutinin structure under electron microscopy (EM) required a higher concentration and purity level than that obtained from SEC on ciude leaf protein extracts. To allow EM observation of H5 structures, a crude leaf protein extract was ftrst concentrated by PEG precipitation (20% PEG) followed by resuspension in 1/10 volumes of extrection buffer. The concentrated protein extraet was fractionated by S-500 Hlt gel filtration and elution fractions 9, 10, and (corresponding to the void volume of the column) were pooled and further isolated from host proteins by ultracentrifugation on a 20-60% sucrose density gradient The sucrose gradient was fractionated starting from, the top and the fractions were dialysed and concentrated on a 100 NMWL centrifugal filter unit pr;or to analysis. As shown on the Westem blots and hemagglutination results(Figure 14A), H5 accumulated mainly in fractions 16 to 19 which contained *,6096 sucrose, whereas most of the host proteins peaked at fraction 13. Fractions 17, 18, and 19 were pooled, negatively stained, and observed under EM. Examination of the sample clearly demonstrated the presence of spiked spheric stxuctures ranging in size from 80 to 300 rim which matched the morphological characteristics of influenza VLPs (Figure 14B).

[00201] Tr. z:ddition to an abundant content of soluble proteins, plant leaf extracts contaii:. .com?lex naixtnire of soluble sugars, nucleic acids and lipids. The crude extract was clarified by a pH shift and heat treatment followed by filtration on diatomaceous earth (see Material and method section for a detailed description of the claxif'ieation method)_ Figure 15A (lanes 1-4) presents a Coomassie Blue stained gel comparing protein content at the various steps of clarification. A comparison of protein content in the crude extract (lane I) and in the clarified extract (lane 4) reveals the capacity of the clarification steps to reduce the global protein content and remove most of the major contaminant visible at 50 kDa in crude leaf extracts. The 50 kDa band corresponds to the RuBisCO large subunit, representing up to 30% of total leaf proteins-(00202) lnfluenza H5 VLFs were purified from these clarified extracts by affinity chromatography on a fetuin colurnn. A comparison of the load fraction (Figure ts 15A, lane 5) with ihe flowthrou,gh (Figure 15A, lane 6) and the cluted VLPs (Ffigure 15A, lane 7) demonstrates the specificity of the fetuin affinity coltunn for influenza 145 VLPs in plant clarified extract.

[00203] The purification procedure resulted in over 75% purity in H5, as determined by densitometry on the Coomassie Blue stained SDS-PAGE gel (Figure 15A, lane 7). In order to assess the structural quality of the purified product, the purified H5 was concentrated on a 1001VMWL (nominal molecular weight limit) eantrifugal filter unit and examined under EM after negative staining. Figure shows a representative sector showing the presence of profuse VLPs. A closer examination conf'umed the presence of spikes on the VLPs (figure 15C)_ [00204] As shown in Figure 15D, H5 VLPs were purified to approx. 89%
purity from clarified leaf extract by affinity chromatography on a fetuin coltunn, based on the density of the Coomassie Blue stained H5 hemagglutinin and on total protein content determination by the BCA method.

j002051 The bioactivity of HA VLPs was confirmed by their capacity to agglutinate turkey red blood cells (data not shown)_ f(11 ri Fl r.;,., rj 1-,. .

a.:ld inu;luttoc;eLCG~G;; '`; iCti an a.rili-H;1 polyCloaal serUIT1 (ANietnam/1 20312004). A unique band of appreximately 72 kDa is detected and corresponds in size to the uncleaved HAO form of irifluenza hemagglutinin.
Figure 15c shows the VLP structure of the vaccine with the hemagglutinin spikes covering its structure.

[00207] VLPs were fonmtiated for immunization of mice by filtering through a 0.22 pm filter, endotoxin content was measured using the endotoxin LAL
(Lirnulus Amebocyte Lysate) detection kit (Lonza, Wallcserville, MS, USA)_ The filtered vaccine contained 105,8 t11,6% ECJ/mI (endotoxin units/ml).
Example 5: Localization otinflnenza VLPs in plants To localize the VLPs and confirm their plasma membrane origin, thin leaf sections of H5-producing plants were fixed and exarnined under TEM after positive staining.
Observation of leaf cells indicated the presence of VLPs in extracellular cavities formed by the invagination of the plasma membrane (Figure 19). The shape and position of the VLPs obscrved demonstrated that despite the apposition of their plasma membranes on the cell wall, plant cclls have the plasticity required to produce influenza VLPs derived from their plasma membrane and accumulate them in the apoplastic space.Exmnple 6: Plasma Membrane Lipid analysis Further eonfitination of the composition and origin of the plant influenza VI.Ps was obtained from analyses of the lipid content, Lipids were extracted from purified VLPs and their composition was compared to that of highly purified tobacco plasmri membranes by high perforsnance tttin layer chromatography (HP-TLC). The migration pat.terns of polar and neutral lipids from VLPs and control plasma membranes were similar. Purified VLPs cont,ained the major phospholipids (phosphatidyicholine and phosphatidylethanolaniine) and sphin.gotipids (glucosyl-ceramide) found in the plasma membrane (Figure 27A), and both contained free sterols as the sole neutral lipids (Figure 27B). However, inununodetection of a plasma membrane protein marker (ATPase) in purified VLP extracts showed that the VLP lipid bilayer does not contain one of the major proteins associated with plant plasrna membranes, suggesting that :._.. a, . r=~__~.. ~.
!i.:Lli;to froi.i the plant cel',s (F'sgure' 7L '_ h:.;arsslrie 7:
Irnmunogenicity of the HS VLPs and effect of route of administration [00208] Mice were administered plattt-made 1-15 VI Ps by intramuscular injection, or intranasal (inhalation). 0.1 to 12 ug of VL.Ps were injected intramuscularly into mice, with alum as an adjuvant, according to the described method,s_ Peak antibody titers were observed with the lowest antigen quantity, in a similar magnitude to that of 5 ug recombinant, soluble hetnagglutinin (HA) (F'igure 20A).

[00209] 0.1 to 1 ug plant-made H5 VLPs were administered intranasally with a chitosan adjuvant provided for an antibody response greater than that of the recombinant soluble HA with an alum adjuvant (Figure 20B).

[002101 For both administration routes, and over a range of antigen quantities, scroconversion was observed in all of the mice tested. Recotnbinant H5 soluble antigen conferred low (t1/40) or negligible (1<1/10 for the non-adjuvanted recombinant H5) HI titres.

Example 8:$emagglutination-inhibition antibody titer (HAI) H5 VLP
[002113 Figure 21 A, B illustrates the hemagglutination inhibition (HA!) antibody response 14 days following a "boost" with plant-made H5 VLP, or recombinant soluble HA. The lowest dose of antigen (0.1 ug) when administered intramuscularly produced a superior HAI response to a 10-fold greater administration (5 ug) of recombinant soluble HA. Increasing doses of 115 VLP provided a modest increase in HA1 over the lowest dose.

[002121 HAI response following intranasal administration was significantly increased in mice administered plant-made H5 VLPs (1.0 or 0.1 ug) compared to those administered 1 ug recombinant soluble HA, which was sitnilar to the negative control. All mice immunized by intramuscular injection of HS VLPs (from 0.1 to Ng) had higher HAI titers than mice immunised with the control HA antigen (Figure 4a - now 21A)_ For the same dose of 5 g, VLPs induced HAI titers 20 times higher rti M: .. .. . . . . _ . i c = -. . . r', . . .. . _. . ... .. . . ... ` . ..,,. . .aai~l,~., 1;AI [iters than the cont:ol EIA a.ntigeu wben deiivered through the intra.-lasal rou[e (Figure 21b). For a given dose of H5 VLP the levels of HAI
titers were iower in mice imrnunised intranasally than for mice immunised intramuscularly;
1 g VLP induced a mean HAI titer of 210 when adrninistered i.m. while the same dose induced a mean HAI titer of 34 administered i.n..

[00213] When adrninistered intramusculariy, all doses of VLPs induced high level of antibodies capable of binding homologous whole inactivated viruses (Figures 20b and 24). No significant difference was found between the plant-made VLP
vaecine and the control HA antigen (except the 12 g VLP group 14 days after boost), as both antigen preparations induce high binding antibody titers against the homologous strain. However, when adrninistered intranasally, VLPs induced higher binding antibody titers in than did the con.trot HA antigen (Figure 20b). When mixed with Chitosan, immwni7ation with one microgram VLP induced a reciprocal mean Ab titer of 5 500, 8.6 times higher than the level found in mice immunized with 1 g of the control HA antigen (reciprocal mean Ab titer of 920).

[00214] The immunogenicity of the plant-derived influenaa VLPs was then investigated through a dose-ranging study in mice. Groups of five BALB/c mice were immuniied intramuscularly twice at 3-week intervals with 0.1 g to 121sg of VI-.Ps containing HA from influenza A/Indonesia/5105 (H5N 1) formulated in alum (1:1 ratio). Hemagglutinacion-inhibition titers (I3n, using whole inactivated virus antigen (Allndonesia/5/05 (H5N I)), were measured on sera collected 14 days after the second immunization. Immunization with doses of VLP as low as 0.1 g induced the production of antibodies that inhibited viruses from agglutinating erythrocytes at high dilutions (Figure 21A). Parallel immunization of mice with 5 g of non-VLP a(um-adjuvanted conuol H5 antigen (also from A/Indonesia/5105) induce an HI
response that was 2-3 logs lower than that achieved with the lowest VLP dose.

[00215] For both administration routes, and over a range of aruigen quantities, the HAI response is superior in mice administered VLPs.

Example 9: Effect of adjuvant on immnnogenicity of H5 VLPs (n/Y?tt;~ r-. ... , .. _ =- n. .,, . . ~.::.ui_t:. .,,..._.. :,._;: {,~'ivlu'c ~ , - = , ,..., . , -- y hxwrnptc to be bound by tneory, cnr',~~oNcu viruses or VLPs of enveloped vir-.:s: sucr.;;rally acquire iheir envelope from the mernbrane they bud through. Plant plasma membranes h.ave a phytosterol complement that is tarely, if ever found in animal cells, and se'veral of these sterols have been demonstrated to exhibit immunostirnulatory effects.

[00217] Plant-made H5 VLPs were administered intramuscularly (Figure 22A) or intranasally (Figuze 22B) to mice in the presenee or absence of an adjuvant, and the HAI (hemagglutination inbibition antibody response) determined. VLPs, in the to presence or absence of an added adjuvant (alum or chitosan, as in these examples) in either system of administration demonstrated a significantly greater HAI
hemagglutinin inhibition than recombinant soluble HA. Even in the absence of an added adjuvant (i.e. alum or chitosan), plant-made H5 VLPs demonstrate a signifieant HAI, indicative of a systemic immune response to administration of the antigen.

(00218] Alum enhanced the mean level of HAX titers by a factor of 5 for intramuscular administration of VLP (Figure 22a) and by a factor of 3.7 for the control HA antigen. When administered i.m., 5 g VLPs induced a mean HAI titer 12 times higher than the corresponding dose of control HA antigen. Clutosan did not boost the mean HAI level of the control HA antigen (Figure 22b) while it increased the mearl HAI level of mice itmmunised with I pg VLP administered i.n. by a factor of 5-fold.
Example 10: Antibody isotypes (00219] Mice administered plant-tnade 145 VLPs or recombinant soluble HA in the presenee or absence of alum as an added adjuvant demonstrate a variety of immunoglobulin isotypes (Figure 23A).

[00224] In the presence of an added adjuvant, the antibody isotype profiles of VLPs and ehe HA are similar, with IgGi being the dominant isotype. When VI.,Ps or HA are administered without an addcd adjuvant, IgGl response is reduced, but remains the dominant isotype response to VLPs, with IgM, IgG2a, IgG2$ and IgG3 maintaining similar titers as in the presence of an added adjuvant. IgGi, IgG2a, and r,f~ , . . . . . ,.._.. .,i_'.......... ...........-...,.,.,~.Gil aC1j u V1nt.

[00221; Thes. ..:_: c.t:L.,;;urc, dcmuasuate that planc-made VLPs do not require an added adjuvant to elicit a antibody response in a host.

[00222] Antibody titers against whole inactivated influenza virus strains (A/Indonesia/5/05; A/Vietnarn/1203/04)1 in mice administered plant-made VLPs or soluble recombinant HA intramuscularly in the presence of an added antigen are illustrated in Figure 23B. No significant difference is observed in the antibody titers for these influenza strains in mice administered I ug or 5 ug of VLPs or 5 ug of soluble HA.

Example 11: Cross-reactivity of serum antibodies induced by the H5 VLP vaccine [Q0223] Cross-reactivity of serum antibodies induced by H5 VLP was assessed against whole inactivated influenza virases of different strains. All VLP
doses (from 0.1 to 12 pg) as well as 5 g of control HA antigen induced high binding antibody titers against a clade 1 strain (AlVictnam/1194/04), the homologous strain A/Indonesia/5/05 of clade 2.1, and a clade 2.2 stra.i.n A/turkey/Turkey/1/05 (Figure 25A).

[00224] However, only the plant-made VLP induced HAI titer against the A/hurkey/Turkey/1/05 strain (Figure 25b). HAI titers for the A/Indonesial5/05 were taigh for VLPs.

Example 12: Cross-protection conferred by immunization with plant-made H5 vLP

[00225] Mice that previously had been administered a two-dose regimen of A/Iudonesia/5105 H5 VLPs as described, were subsequently challenged intranasatly with influenza AlTurkey/582/06 (H5N1) ("Turkey H5N1") infectious virus, and observed. The dose administered, per animal. was 10 LDso (4.09 X 10S CCIDSO).
(00226] By 7 days post-challenge, only 37.5g'o of the mice adrnininstered the PBS vaccine control had survived exposure to Turkey H5N1 (Figure 26A). 100% of ,. . _,, i.. .

survived up to 17 days post-cha]lenge, when the experiment was terminated.

j00227] Body mass of the mice was also monitored during the experiment, and the average mass of the surviving mice plotted (Figure 26B). Mice administered 1, 5 .:,,;...._...., r~.
or 15 ug of the Indonesia H5 VLPs before challenge did not lose any appreciable mass during the course of the experiment, and in particular mice administered 5 ug of the VLPs appear to have gained significant mass. Negative control mice (no Turkey H5N1 challenge) did not appreciably gain or lose body mass. Positive cantrol mice (not administered VLPs, but challenged with Turkey H5Nl) exhibited significant loss of body mass during the course of the experiment, and three of these mice died. As body mass is an average of a11 mice in the cohort, removal of the `sickest' mice (the 3 that died) may lead to an apparent overall inemase in mass, however note t}tat the average body mass of the positive control cohort is still significantly below that of the negative or the VLP-treated cohorts.

[00228] These data, therefore, demonstrate that plant-made influenza VLPs comprising the HS hemagglutinin viral protein induce an unmune response specific for pathogenic influenza strains, and that virus-Ii.ke particles may bud fiom a plant plasma membrane. .

[00229] These data, therefore, demonstrate that plnn,ts are capable of producing i afluenza virus-like particles, and also for the first time, that virus-like particles can bud fronn a plant plasma membrane.

[00230] Further, using the current transient expression technology, a first antigen lot was produced only 16 days after the sequence of the target HA was obtained. Under the current yields for H5 VLPs, and at an exemplary dose of 5 g per subject, each kg of infittrated leaf may produce -20,000 vaccine doses. This unique combination of platform simplicity, surge capacity and powerful immunogenicity provides for, aimong other embodiments, a new method response in the context of a pandemic.

100231 j All citations are hereby incorporated by referenee.

emboclimcnts. However. it will be apparent to persons skilled in the art that a number of variations and rnodifications can be made without depart:n; from the scope of the invention as defined in the claims.

References:

Boliag, D.M., Rozycki, M.D., and Edelstein, S.J. (1996) Protein methods (2"d edition).
Wiley-Liss, New York, USA.

Bligh,, E.G., & Dyer, W.J. Cam J. Med. Sci. 37, 911-917 (1959).

Chen, B.J., L.eser, G.P., Morita, E., and Lamb R.A. (2007) Influenza virus hemagglutinin and neurdtninidase, but not the matrix protein, are required for assembly and budding of plasrnid-derived virus-like particles. J. Virol. 81, 7111-7123.
Crawford, J. , Witlcinson, B. , Vosnesensky, A. , Smith, G. , Garcia, M. , Stone, Ii. , and Perdue, M_ L. (1999). Baculovires-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and i17 subtypes. Vaccine 17,2265-2274.

Darveau, A., Pelletier. A. & Pe,rreault, J. PCR-mediated synthesis of chimeric molecules. Methods Neurosc. 26, 77-85 (1995).

Grgacic EVI., Anderson DA. Virus-Iike particles: passport to immune recognition.
Methods 2006; 40: 60-65.

Gillinn Ross, L, and Subbarao, K. (2006) Emerging respiratory viruses:
chanllenges and vaccine strategies. Clin. Microbiol. Rev. 19, 614-636.

Gomez-Puertas, P., Mena, L, Castillo, M., Vivo, A., Perez-Pastrana, E. and Portela, A.
(1999) Efficient formation of influenza virus-like partieles: dependence on the expression lcvel of viral proteins. J. Gen. Virol. 80, 1635-1645.

Gomez-Puertas, P., Albo, C., Perez-Pastraila, E., Vivo, A_, and Portela, A.
(2000) 2s Influenza Vinu protein is the major driving force in viius budding. J
Virol. 74, 11538-11547.

Hamilton, A., Voinnet, 0., Chappell, L. & Baulcombe, D. Two classes of shott interfering RNA in RNA silencing. EMBO J. 21, 4671-4679 (2002).

HtSfgen, R. & Willmitzer, L_ Storage of competent cells for Agrobacterium transformation. Nucleic Acid Res. 16, 9877 (1988).

Harbury PB, 7.hang T, Kim 1'S; Alber T. (1993) A switch between two-, three-, and four-atranded coiled coils in GCN4leucine zipper mutants. Science; 262: 1401-1407) Horiinoto T_, Kawaoka Y. Strategies for developing vaccines against hSNl influenza a viruses. Trends in Mol. Med. 2006; 12(11):506-514.

liJ, rsGutuier N, ArnlzeIl (:.1, l ii,`:;l:i'.=Cli21 Y, Nl:j:ion HJ.
Virus-lce partic.e expression and assembly in planis: hepatitis B and Norwalk viruses, Vaccine_ 2005 Mar 7;23(15):1851-8.

Johansson, B. E. (099). Immunization with influenza A virus hernagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine. Vaccine 17, 2073-2080.

L.atham, T. , and Galarza, J. M. (2001). Formation of wild-type and cbimeric influenza virus-like particles following simultaneous expression of only four structural proteins.
J. Virol. 75,6154- 6165.

L.efebvre, B. et a1. Ptant Physiol. 144, 402-418 (2007).
Liu, L & Lomonossoff, G.P. Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs. J. ViraL Methods 105,343-348 (2002).

Macala, L.J_. Yo, R.K. & Ando, S. JLipid Res. 24, 1243-1250 (1983) Mattanovich, D., Ruker, F_, da Camara Maclaado, A., Laimer, M_, Regaer, F., Steinkellner, H., Himmler, G., and Katinger, H. (1989) Efficient transformation of Agrobacteriwn spp. By eiectroporation. Nue1. Ac. Rps. 17, 6747_ Mena,1., Vivo, A., Perez, E., and Portela. A. (1996) Rescue of synthetic chlorarnphenicol acetyltransferase RNA into influenza vinu-like particles obtained from recombinant plasmids. J. Virol. 70, 5016-5024, Mongrand S, Morel J, L.aroche J. Claverol S, Carde JP, Hartmann MA et al_.
Lipid rafts in higher plant cells. The Jaumal of Biological Chemistry 2004; 279(35):

36286.

Neumann,, G., Watanabe, T., and Kawaoka, Y. (2000) Plasniid-driven formation of virus-like particles. J. Virol. 74, 547-551.

Nayak DP. Reichl U. (2004) Netuaminidase activity assays for monitoring NIDCK
cell culture derived influenza vizus. J Virul Metbods 122(I):9-15.

Olsen, C. W. , McGregor, M. W. , Dybdabl-Sissoko, N. , Schram, B. R. , Nelson, K.
M. , Lunn, D., Macklin, M. D. , and Swain, W. F. (1997). Immunogenicity and efficacy of baculovirus- exprassed and DNA-based equine influenza virus hemagglutinin vaccines in mice. Vaccine 15, 1149-1156.

Quan FS, Huang C, Compans RW, Kang SM. Virus-like particle vaccine induces protective immunity against homologous and beterologous strains of influenza virus.
Journal of Virology 2007; 87 (7): 3514-3524.

Sambrook J, and Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Prc.ss, 2001 _ Suzula, Y. (2005) Sialobiology of influenza Molecular mechanism of host range vaziation of influenza viruses. Biol. PharnL Bull 28, 399-408.

6J (2006), ~~G1-Ei7jJ

Wakefield L., G.G. Brownlee Nuc Acid Res. 17 (1989); 8569-8580.

Kendal, AP, Pereira MS, Sbehel J. Concepts and procedures for laboratory-based influenza surveillance. Atlanta:CDC; 1982. p.B17-B35 WHO. Manual on animal influenza diagnosis and surveillance. .Departement of communicable disease surveillance and response. World Health Organisation Global Influenza Psogram. 2002.

Claims (19)

WHAT IS CLAIMED IS:
1. A nucleic acid comprising a nucleotide sequence encoding an influenza hemagglutinin (HA) operatively linked to a regulatory region active in a plant.
2. The nucleic acid of claim 1, wherein the influenza hemagglutinin is H5.
3. The nucleic acid of claim 1, wherein the influenza hemagglutinin is H5 Indonesia.
4. A method of producing influenza virus like particles (VLPs) in a plant comprising:
a) introducing the nucleic acid of claim 1 into the plant, or portion thereof, and b) incubating the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLPs.
5. The method of claim 4, wherein in the step of introducing (step a), the nucleic acid is transiently expressed in the plant.
6. The method of claim 4, wherein the nucleic acid is stably expressed in the plant.
7. The method of claim 4 further comprising a step of c) harvesting the host and purifying the VLPs.
8. A virus like particle (VLP) comprising an influenza virus HA protein and one or more than one lipid derived from a plant.
9. The virus like particle (VLP) of claim 8, wherein the influenza HA protein is H5 Indonesia.
10. A composition comprising an effective dose of the VLP of claim 8 and a pharmaceutically acceptable carrier.
11. A composition comprising an effective dose of the VLP of claim 9 and a pharmaceutically acceptable carrier.
12. A method of inducing immunity to an influenza virus infection in a subject, comprising administering the virus like particle of claim 8.
13. A method of inducing immunity to an influenza virus infection in a subject, comprising administering the virus like particle of claim 9.
14. The method of claim 12, wherein the virus like particle is administered to a subject orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
15. A virus like particle (VLP) comprising an influenza virus HA bearing plant-specific N-glycans, or modified N-glycans.
16. A composition comprising an effective dose of the VIP of claim 15 and a pharmaceutically acceptable carrier.
17. A method of inducing immunity to an influenza virus infection in a subject, comprising administering the composition of claim 16.
13. The method of claim 14, wherein the composition is administered to a subject orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
19. A virus like particle (VLP) comprising one or more than one protein derived from a virus selected from the group consisting of influenza, Measles, Elbola, Marburg, and HIV, and one or more than one lipid derived from a non-sialylating host production cell.
CA002615372A 2007-07-13 2008-01-21 Influenza virus-like particles (vlps) comprising hemagglutinin Abandoned CA2615372A1 (en)

Priority Applications (58)

Application Number Priority Date Filing Date Title
SI200831024T SI2173886T1 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
CA2693956A CA2693956C (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
EA201000195A EA018206B1 (en) 2007-07-13 2008-07-11 INFLUENZA VIRUS-LIKE PARTICLES (VLPs) COMPRISING HEMAGGLUTININ PRODUCED WITHIN A PLANT
US12/669,033 US20100239610A1 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
CN2008801070729A CN101883856B (en) 2007-07-13 2008-07-11 Influenza virus-like particles (VLPs) comprising hemagglutinin produced within plant
ES08783201T ES2428384T3 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (VLP) containing hemagglutinin produced within a plant
NZ582360A NZ582360A (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
PT87832010T PT2173886E (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
EP08783201.0A EP2173886B1 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
PL08783201T PL2173886T3 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
DK08783201.0T DK2173886T3 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (VLPS) comprising hemagglutinin produced in a plant
PCT/CA2008/001281 WO2009009876A1 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
MYPI2010000142A MY155236A (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
BRPI0813724-2A BRPI0813724B1 (en) 2007-07-13 2008-07-11 METHOD OF PRODUCTION OF INFLUENZA VIRUS-LIKE PARTICLES (VLPS) IN A PLANT, VIRUS-LIKE PARTICLES (VLPS) AND COMPOSITION
KR1020107002538A KR101541330B1 (en) 2007-07-13 2008-07-11 INFLUENZA VIRUS-LIKE PARTICLES(VLPs) COMPRISING HEMAGGLUTININ PRODUCED WITHIN A PLANT
AU2008278222A AU2008278222B2 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (VLPs) comprising hemagglutinin produced within a plant
MX2010000525A MX2010000525A (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant.
HK10109048.1A HK1142627B (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
JP2010516334A JP5624465B2 (en) 2007-07-13 2008-07-11 Influenza virus-like particles (VLPs) containing hemagglutinin produced in plants
DK12183854.4T DK2610345T3 (en) 2007-11-27 2009-01-12 RECOMBINANT INFLUENZA VIRUS SIMULAR PARTICULARS (VLPS) MADE IN TRANSGENE PLANTS THAT EXPRESS HEMAGGLUTININ
BRPI0906960-7A BRPI0906960B1 (en) 2008-01-21 2009-01-12 NUCLEIC ACID COMPRISING A SEQUENCE OF NUCLEOTIDES CODING AN INFLUENZA VIRUS HEMAGLUTININ (HA), VIRUS-LIKE PARTICLES (VLPS), THEIR METHOD OF PRODUCTION, COMPOSITION AND USE THEREOF
JP2010542486A JP2011509661A (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (VLP) produced in transgenic plants expressing hemagglutinin
SG2013004577A SG187500A1 (en) 2008-01-21 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
EP12183854.4A EP2610345B1 (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (VLPS) produced in transgenic plants expressing hemagglutinin
NZ587108A NZ587108A (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
AU2009202819A AU2009202819B2 (en) 2007-07-13 2009-01-12 Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
PCT/CA2009/000032 WO2009076778A1 (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
EP09700061A EP2238253B1 (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
MX2010007962A MX2010007962A (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin.
ES12183854.4T ES2554703T3 (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (VLP) produced in transgenic plants that express hemagglutinin
KR1020107018343A KR20100120157A (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles(vlps) produced in transgenic plants expressing hemagglutinin
PT121838544T PT2610345E (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
EA201001198A EA034733B1 (en) 2008-01-21 2009-01-12 Nucleic acid for increased expression of hemagglutinin of influenza virus in a plant and use thereof
CN201310021693.8A CN103122354B (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
CA2707235A CA2707235C (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
US12/863,772 US20100310604A1 (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
CN2009801097815A CN101978066A (en) 2007-11-27 2009-01-12 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
KR1020167010959A KR101956910B1 (en) 2008-01-21 2009-01-12 RECOMBINANT INFLUENZA VIRUS-LIKE PARTICLES(VLPs) PRODUCED IN TRANSGENIC PLANTS EXPRESSING HEMAGGLUTININ
US13/003,570 US9492528B2 (en) 2007-07-13 2009-07-02 Influenza virus-like particles (VLPS) comprising hemagglutinin
IL203018A IL203018A (en) 2007-07-13 2009-12-29 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
CR11209A CR11209A (en) 2007-07-13 2010-01-11 PARTICLES SIMILAR TO THE INFLUENZA VIRUS (VLPS) THAT INCLUDE HEMAGLUTININ PRODUCED WITHIN A PLANT
IS8874A IS2920B (en) 2007-07-13 2010-01-27 Influenza-like particles (VLPs) containing erythropoietin produced in plant.
MA32604A MA31681B1 (en) 2007-07-13 2010-02-08 Semi - particles of influenza virus (VLP) a blood ligament (HA) produced in a plant
ZA2010/00972A ZA201000972B (en) 2007-07-13 2010-02-10 Influenza virus-like particles (vlps) comprising hemagglutinin produces produced within a plant
IL206967A IL206967A (en) 2007-11-27 2010-07-13 Nucleic acid comprising a nucleotide sequence encoding influenza hemagglutinin and a method of producing an influenza virus- like particle comprising the same
CR11612A CR11612A (en) 2007-11-27 2010-07-29 RECOMBINANT INFLUENZA VIRUS TYPE PARTICLES (VLP) PRODUCED IN TRANSGENIC PLANTS EXPRESSING HEMAGLUTININE
IS8919A IS2982B (en) 2007-11-27 2010-08-04 Variety of influenza-like particles (VLPs) produced in genetically modified plants expressing erythrocytes.
MA33088A MA32439B1 (en) 2007-11-27 2010-08-13 RECOMBINANT INFLUENZA PSEUDOVIRAL PARTICLES (VLPs) PRODUCED IN TRANSGENIC PLANTS EXPRESSING HEMAGGLUTININ
ZA2010/05917A ZA201005917B (en) 2007-11-27 2010-08-19 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
US13/734,886 US9452210B2 (en) 2007-07-13 2013-01-04 Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant
US13/748,531 US9458470B2 (en) 2007-11-27 2013-01-23 Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
HK13113472.5A HK1186207B (en) 2008-01-21 2013-12-04 Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
IL230708A IL230708A (en) 2007-07-13 2014-01-29 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
JP2014039035A JP5990207B2 (en) 2007-11-27 2014-02-28 Recombinant influenza virus-like particles (VLP) produced in transgenic plants expressing hemagglutinin
JP2015231583A JP2016032477A (en) 2007-11-27 2015-11-27 Recombinant influenza virus-like particles (VLP) produced in transgenic plants expressing hemagglutinin
US15/256,119 US10190132B2 (en) 2007-11-27 2016-09-02 Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
US16/219,306 US11434497B2 (en) 2007-11-27 2018-12-13 Recombinant influenza virus-like particles (VLPS) produced in transgenic plants
US17/815,503 US20230044454A1 (en) 2007-07-13 2022-07-27 Recombinant influenza virus-like particles (vlps) produced in transgenic plants

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US95941407P 2007-07-13 2007-07-13
US60/959,414 2007-07-13
US99060307P 2007-11-27 2007-11-27
US60/990,603 2007-11-27
US1327207P 2007-12-12 2007-12-12
US61/013,272 2007-12-12

Publications (1)

Publication Number Publication Date
CA2615372A1 true CA2615372A1 (en) 2009-01-13

Family

ID=40255124

Family Applications (2)

Application Number Title Priority Date Filing Date
CA002615372A Abandoned CA2615372A1 (en) 2007-07-13 2008-01-21 Influenza virus-like particles (vlps) comprising hemagglutinin
CA2693956A Active CA2693956C (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant

Family Applications After (1)

Application Number Title Priority Date Filing Date
CA2693956A Active CA2693956C (en) 2007-07-13 2008-07-11 Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant

Country Status (19)

Country Link
US (4) US20100239610A1 (en)
EP (1) EP2173886B1 (en)
JP (1) JP5624465B2 (en)
KR (1) KR101541330B1 (en)
CN (1) CN101883856B (en)
AU (1) AU2008278222B2 (en)
CA (2) CA2615372A1 (en)
CR (1) CR11209A (en)
DK (1) DK2173886T3 (en)
EA (1) EA018206B1 (en)
ES (1) ES2428384T3 (en)
MX (1) MX2010000525A (en)
MY (1) MY155236A (en)
NZ (1) NZ582360A (en)
PL (1) PL2173886T3 (en)
PT (1) PT2173886E (en)
SI (1) SI2173886T1 (en)
TN (1) TN2009000557A1 (en)
WO (1) WO2009009876A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010151159A2 (en) 2009-06-26 2010-12-29 Instytut Biochemii I Biofizyki Virus-like particle vector as a polyvalent platform for intracellular delivery of high-molecular-weight therapeutic substances, method for generating a virus like particle vector and use of a virus-like particle vector and a pharmaceutical composition containing said virus-like particle vector
US8771703B2 (en) 2008-07-08 2014-07-08 Medicago Inc. Soluble recombinant influenza antigens
US9452210B2 (en) 2007-07-13 2016-09-27 Medicago Inc. Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant
US9458470B2 (en) 2007-11-27 2016-10-04 Medicago Inc. Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
US9546375B2 (en) 2008-07-18 2017-01-17 Medicago Inc. Influenza virus immunizing epitope
US9815873B2 (en) 2011-03-23 2017-11-14 Medicago Inc. Method for recovering plant-derived proteins
US10272148B2 (en) 2009-06-24 2019-04-30 Medicago Inc. Chimeric influenza virus-like particles comprising hemagglutinin
US11826419B2 (en) 2009-09-22 2023-11-28 Medicago Inc. Method of preparing plant-derived VLPs

Families Citing this family (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI324181B (en) 2001-04-16 2010-05-01 Martek Biosciences Corp Product and process for transformation of thraustochytriales microorganisms
WO2005018539A2 (en) 2003-06-16 2005-03-03 Medimmune Vaccines, Inc. Influenza hemagglutinin and neuraminidase variants
ES2527503T3 (en) 2005-03-08 2015-01-26 Medimmune, Llc Reordering influenza viruses
JP5683811B2 (en) * 2006-07-27 2015-03-11 タケダ ヴァクシーンズ， インコーポレイテッド Chimera influenza virus-like particles
RU2523587C2 (en) * 2008-07-11 2014-07-20 МЕДИММЬЮН, ЭлЭлСи Versions of hemagglutinin and neuraminidase of influenza virus
AU2015202195B2 (en) * 2008-07-18 2017-10-12 Medicago Inc. New influenza virus immunizing epitope
TWI504749B (en) 2009-03-16 2015-10-21 Dsm Ip Assets Bv Protein production in microorganisms of the phylum labyrinthulomycota
JP2012521786A (en) 2009-03-30 2012-09-20 モウント シナイ スクール オフ メディシネ Influenza virus vaccine and use thereof
US8933200B2 (en) 2009-04-09 2015-01-13 Alfa Biogene International B.V. Filtration of a liquid comprising a plant stress protein
AU2010266129B2 (en) 2009-07-02 2016-04-14 Massachusetts Institute Of Technology Compositions and methods for diagnosing and/or treating influenza infection
IN2012DN06277A (en) * 2009-12-28 2015-09-25 Dsm Ip Assets Bv
AR079767A1 (en) * 2009-12-28 2012-02-15 Merial Ltd ANTIGEN NDV (NEWCASTLE DISEASE VIRUS) RECOMBINANT AND USES OF THE SAME
EP2519636B8 (en) 2009-12-28 2017-08-09 DSM IP Assets B.V. Production of hemagglutinin-neuraminidase protein in microalgae
AU2015202267B2 (en) * 2010-01-26 2017-03-23 The Trustees Of The University Of Pennsylvania Influenza nucleic acid molecules and vaccines made therefrom
US8298820B2 (en) 2010-01-26 2012-10-30 The Trustees Of The University Of Pennsylvania Influenza nucleic acid molecules and vaccines made therefrom
US9701723B2 (en) 2010-02-18 2017-07-11 Icahn School Of Medicine At Mount Sinai Vaccines for use in the prophylaxis and treatment of influenza virus disease
MX2012011300A (en) 2010-03-30 2012-11-29 Sinai School Medicine Influenza virus vaccines and uses thereof.
CA2811023A1 (en) 2010-09-21 2012-03-29 Massachusetts Institute Of Technology Influenza treatment and/or characterization; human-adapted ha polypeptides; vaccines
MX2013003451A (en) 2010-09-30 2013-09-02 Franvax S R L Generation of virosome particles.
WO2012047941A2 (en) 2010-10-04 2012-04-12 Massachusetts Institute Of Technology Hemagglutinin polypeptides, and reagents and methods relating thereto
EP2632562B1 (en) * 2010-10-27 2020-08-05 Philip Morris Products S.a.s. Methods for capturing virus like particles from plants using expanded bed chromatography
ES2614527T3 (en) 2010-11-04 2017-05-31 Medicago Inc. Plant expression system
TWI526539B (en) * 2010-12-22 2016-03-21 苜蓿股份有限公司 Method for producing viroid-like particles (VLP) in plants and VLP produced by the method
EP2665817A1 (en) * 2011-01-17 2013-11-27 Philip Morris Products S.a.s. Vectors for nucleic acid expression in plants
US20140227322A1 (en) * 2011-06-13 2014-08-14 Medicago Inc. Rabies virus like particle production in plants
JP6113155B2 (en) 2011-06-20 2017-04-12 ユニバーシティ オブ ピッツバーグ − オブ ザ コモンウェルス システム オブ ハイヤー エデュケイション H1N1 influenza antigen with broad reactivity optimized by calculation
HK1200102A1 (en) 2011-09-20 2015-07-31 西奈山医学院 Influenza virus vaccines and uses thereof
US11390878B2 (en) 2011-09-30 2022-07-19 Medicago Inc. Increasing protein yield in plants
TWI700368B (en) 2011-09-30 2020-08-01 苜蓿股份有限公司 Increasing virus like particle yield in plants
WO2013068593A1 (en) * 2011-11-11 2013-05-16 Philip Morris Products S.A. Influenza virus -like particles (vlps) comprising hemagglutinin produced nicotiana tabacum
US9234008B2 (en) 2012-02-07 2016-01-12 University of Pittsburgh—of the Commonwealth System of Higher Education Computationally optimized broadly reactive antigens for H3N2, H2N2, and B influenza viruses
US9566327B2 (en) 2012-02-13 2017-02-14 University of Pittsburgh—of the Commonwealth System of Higher Education Computationally optimized broadly reactive antigens for human and avian H5N1 influenza
US9364530B2 (en) 2012-03-22 2016-06-14 Fraunhofer Usa, Inc. Virus-like particles comprising a matrix protein from a plant enveloped virus and uses thereof
PL3597757T3 (en) * 2012-05-11 2021-05-31 Medicago Inc. Rotavirus-like particle production in plants
AU2013312971B2 (en) * 2012-09-05 2019-04-18 Medicago Inc. Picornavirus-like particle production in plants
NZ627796A (en) 2012-12-18 2017-07-28 Icahn School Med Mount Sinai Influenza virus vaccines and uses thereof
US9908930B2 (en) 2013-03-14 2018-03-06 Icahn School Of Medicine At Mount Sinai Antibodies against influenza virus hemagglutinin and uses thereof
JP2016516415A (en) * 2013-03-28 2016-06-09 メディカゴ インコーポレイテッド Generation of influenza virus-like particles in plants
US12467058B2 (en) 2013-03-28 2025-11-11 Aramis Biotechnologies Inc. Influenza virus-like particle production in plants
WO2015034042A1 (en) 2013-09-06 2015-03-12 三菱化学株式会社 Method for producing protein using plant
AU2015234595B2 (en) 2014-03-27 2021-03-25 Medicago Inc. Modified CPMV enhancer elements
WO2016004536A1 (en) 2014-07-11 2016-01-14 Medicago Inc. Modifying protein production in plants
HK1245333A1 (en) 2015-01-23 2018-08-24 Medicago Inc. Rotavirus-like particle production in plants
EP3247389A4 (en) 2015-01-23 2019-10-30 Icahn School of Medicine at Mount Sinai VACCINATION REGIMES FOR INFLUENZA VIRUSES
KR101637955B1 (en) * 2015-05-18 2016-07-08 한국생명공학연구원 Universal influenza virus vaccine composition
RU2728472C2 (en) 2015-07-02 2020-07-29 Медикаго Инк. Activator of jasmonic acid signaling pathway
MX2018015755A (en) 2016-06-15 2019-08-29 Icahn School Med Mount Sinai Influenza virus hemagglutinin proteins and uses thereof.
CA3058652A1 (en) 2017-04-07 2018-10-11 Icahn School Of Medicine At Mount Sinai Anti-influenza b virus neuraminidase antibodies and uses thereof
JP7423875B6 (en) 2018-03-14 2024-03-15 メディカゴ インコーポレイテッド plant expression enhancer
KR101964044B1 (en) * 2018-03-14 2019-04-02 인제대학교 산학협력단 Multi-valent live-attenuated influenza vaccine platform using recombinant adenovirus
US12364746B2 (en) 2018-06-21 2025-07-22 Icahn School Of Medicine At Mount Sinai Mosaic influenza virus hemagglutinin polypeptides and uses thereof
AU2019295495B2 (en) 2018-06-27 2025-05-22 Aramis Biotechnologies Inc. Influenza virus hemagglutinin mutants
CA3104392A1 (en) * 2018-07-04 2020-01-09 Probiogen Ag Method for purifying an enveloped virus
BR112021017899A2 (en) 2019-03-14 2021-12-21 Medicago Inc Endogenous plant expression enhancer
CA3137160A1 (en) 2019-04-24 2020-10-29 Icahn School Of Medicine At Mount Sinai Anti-influenza b virus neuraminidase antibodies and uses thereof
CA3145200A1 (en) 2019-07-03 2021-01-07 Aillis Inc. Pharmaceutical composition for treating influenza virus infections
CN111122417B (en) * 2020-02-22 2022-06-21 太原理工大学 A device and method for measuring the total volume expansion rate of open and closed pores of gas-containing coal
EP4351320A1 (en) 2021-06-09 2024-04-17 ImmunityBio, Inc. Methods and systems for producing a protein of interest in a plant
CN115992101B (en) * 2023-03-22 2023-07-28 深圳市卫光生物制品股份有限公司 Preparation method of influenza virus split vaccine stock solution

Family Cites Families (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0203177A4 (en) 1984-11-29 1987-04-28 Scripps Clinic Res Polypeptides and antibodies related to deglycosylated viral glycoproteins.
US5232833A (en) * 1988-09-14 1993-08-03 Stressgen Biotechnologies Corporation Accumulation of heat shock proteins for evaluating biological damage due to chronic exposure of an organism to sublethal levels of pollutants
ATE142883T1 (en) * 1991-03-28 1996-10-15 Rooperol Na Nv COMPOSITIONS OF PHYTOSTEROLS WITH PHYTOSTEROLINS AS IMMUNE MODULATORS
US6326470B1 (en) 1997-04-15 2001-12-04 The Penn State Research Foundation Enhancement of accessibility of cellulose by expansins
US5762939A (en) * 1993-09-13 1998-06-09 Mg-Pmc, Llc Method for producing influenza hemagglutinin multivalent vaccines using baculovirus
GB9414118D0 (en) 1994-07-13 1994-08-31 Axis Genetics Ltd Modified plant viruses as vectors of heterologous peptides
US6020169A (en) 1995-07-20 2000-02-01 Washington State University Research Foundation Production of secreted foreign polypeptides in plant cell culture
US6042832A (en) 1996-08-28 2000-03-28 Thomas Jefferson University Polypeptides fused with alfalfa mosaic virus or ilarvirus capsid proteins
US20010006950A1 (en) 1998-02-11 2001-07-05 Juha Punnonen Genetic vaccine vector engineering
US6489537B1 (en) 1998-08-07 2002-12-03 The Trustees Of The University Of Pennsylvania Phytochelatin synthases and uses therefor
MXPA01001445A (en) 1998-08-11 2002-11-29 Large Scale Biology Corp Method for recovering proteins from the interstitial fluid of plant tissues.
US6392121B1 (en) 1998-10-07 2002-05-21 Boyce Thompson Institute For Plant Research Gemini virus vectors for gene expression in plants
US6287570B1 (en) * 1998-11-23 2001-09-11 Patricia L. Foley Vaccine against swine influenza virus
FR2791358B1 (en) 1999-03-22 2003-05-16 Meristem Therapeutics CHEMICAL EXPRESSION PROMOTERS, EXPRESSION CASSETTES, PLASMIDS, VECTORS, PLANTS AND TRANSGENIC SEEDS CONTAINING THEM AND METHODS OF OBTAINING THEM
ES2262514T3 (en) 1999-04-29 2006-12-01 Syngenta Limited HERBICID RESISTANT PLANTS.
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
US7449188B2 (en) 2001-01-18 2008-11-11 Vlaams Interuniversitair Instituut Voor Biotechnologie Recombinant oligometric protein complexes with enhanced immunogenic potential
CN1333370A (en) * 2001-08-16 2002-01-30 深圳市三方圆信息技术有限公司 Crops capable of resisting virus diseases of poultry and production method thereof
AU2003219745B8 (en) * 2002-02-13 2008-10-09 Wisconsin Alumni Research Foundation Signal for packaging of influenza virus vectors
US7041500B2 (en) 2002-02-14 2006-05-09 Novavax, Inc. Insect cell line
EP2319935B1 (en) * 2002-03-19 2016-04-27 Stichting Dienst Landbouwkundig Onderzoek GnTIII (UDP-N-acetylglucosamine:Beta -D mannoside Beta (1,4)-N-acetylglucosaminyltransferase III) expression in plants.
ES2224792B1 (en) 2002-06-28 2007-02-16 Era Plantech, S.L. PRODUCTION OF PEPTIDES AND PROTEINS BY ACCUMULATION OF PROTEIN BODIES DERIVED FROM ENDOPLASMIC RETICLES IN PLANTS.
CU23202A1 (en) * 2003-01-31 2007-05-18 Ct Ingenieria Genetica Biotech RECOMBINANT ANTIGENS OF HEPATITIS VIRUS TO OBTAINED IN VEGETABLE CELLS
CN1460718A (en) * 2003-04-11 2003-12-10 浙江大学 Production method of transgenic potato for expressing infectious bronchitis virus fibre mutein
NZ542933A (en) 2003-05-05 2009-08-28 Dow Agrosciences Llc Stable immunoprophylactic and therapeutic compositions derived from transgenic plant cells and methods for production
AU2004235813B2 (en) * 2003-05-05 2010-07-29 Boyce Thompson Institute For Plant Research Vectors and cells for preparing immunoprotective compositions derived from transgenic plants
WO2005018539A2 (en) * 2003-06-16 2005-03-03 Medimmune Vaccines, Inc. Influenza hemagglutinin and neuraminidase variants
US8592197B2 (en) 2003-07-11 2013-11-26 Novavax, Inc. Functional influenza virus-like particles (VLPs)
CN1926238A (en) * 2004-02-27 2007-03-07 陶氏化学公司 High efficiency peptide production in plant cells
WO2006016380A2 (en) 2004-08-13 2006-02-16 Council Of Scientific And Industrial Research A chimeric g protein based rabies vaccine
ES2662118T3 (en) 2005-04-29 2018-04-05 University Of Cape Town Expression of viral proteins in plants
CN100410378C (en) 2005-05-09 2008-08-13 中国农业科学院生物技术研究所 Gene encoding avian influenza hemagglutinin and its plant expression vector and application
CA2615658A1 (en) 2005-07-19 2007-01-25 Dow Global Technolgies Inc. Recombinant flu vaccines
US7871626B2 (en) 2005-08-04 2011-01-18 St. Jude Children's Research Hospital Modified influenza virus for monitoring and improving vaccine efficiency
EP1945250A4 (en) 2005-08-16 2010-05-19 Hawaii Biotech Inc RECOMBINANT SUBUNIT VACCINE FOR INFLUENZA VIRUS
RU2483751C2 (en) * 2005-10-18 2013-06-10 Новавакс, Инк. FUNCTIONAL INFLUENZA VIRUS-LIKE PARTICLES (VLPs)
AU2006335256B2 (en) 2005-11-22 2012-10-18 Novartis Vaccines And Diagnostics, Inc. Norovirus and sapovirus antigens
EP1984405A4 (en) 2006-02-13 2010-06-30 Fraunhofer Usa Inc INFLUENZA ANTIGENS, VACCINE COMPOSITIONS AND ASSOCIATED METHODS
KR20080109094A (en) 2006-04-21 2008-12-16 다우 아그로사이언시즈 엘엘씨 Avian Influenza Vaccine and How to Use
US8778353B2 (en) 2006-05-01 2014-07-15 Technovax, Inc. Influenza virus-like particle (VLP) compositions
WO2008054540A2 (en) 2006-05-18 2008-05-08 Pharmexa Inc. Inducing immune responses to influenza virus using polypeptide and nucleic acid compositions
US20070286873A1 (en) * 2006-05-23 2007-12-13 Williams John V Recombinant Influenza H5 Hemagluttinin Protein And Nucleic Acid Coding Therefor
US7730950B2 (en) 2007-01-19 2010-06-08 Halliburton Energy Services, Inc. Methods for treating intervals of a subterranean formation having variable permeability
CN104962579A (en) 2007-06-15 2015-10-07 麦迪卡格公司 Modifying glycosylation production in plants
KR100964462B1 (en) 2007-07-10 2010-06-16 성균관대학교산학협력단 Avian Influenza Virus Vaccine Derived from Transgenic Plant and Its Manufacturing Method
CA2615372A1 (en) * 2007-07-13 2009-01-13 Marc-Andre D'aoust Influenza virus-like particles (vlps) comprising hemagglutinin
CA2696764A1 (en) 2007-08-20 2009-02-26 Fraunhofer Usa, Inc. Prophylactic and therapeutic influenza vaccines, antigens, compositions, and methods
NZ587108A (en) * 2007-11-27 2013-04-26 Medicago Inc Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
CN101909454A (en) 2007-12-28 2010-12-08 荷兰联合利华有限公司 Process for recovering aroma compounds from tea
GB0800272D0 (en) 2008-01-08 2008-02-13 Plant Bioscience Ltd Protein expression systems
SG187500A1 (en) 2008-01-21 2013-02-28 Medicago Inc Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
DK2294202T3 (en) 2008-07-08 2015-08-31 Medicago Inc SOLUBLE RECOMBINANT INFLUENZA ANTIGENES
KR101745925B1 (en) 2008-07-18 2017-06-12 메디카고 인코포레이티드 New influenza virus immunizing epitope
CA2736796A1 (en) 2008-08-27 2010-03-04 Arizona Board Of Regents For And On Behalf Of Arizona State University A dna replicon system for high-level rapid production of vaccines and monoclonal antibody therapeutics in plants
WO2010025235A1 (en) 2008-08-29 2010-03-04 Regents Of The University Of Michigan Selective ligands for the dopamine 3 (d3) receptor and methods of using the same
WO2010077712A1 (en) 2008-12-09 2010-07-08 Novavax, Inc. Bovine respiratory syncytial virus virus-like particle (vlps)
SI2445928T1 (en) * 2009-06-24 2018-05-31 Medicago Inc. Chimeric influenza virus-like particles comprising hemagglutinin
WO2011011390A1 (en) 2009-07-20 2011-01-27 Novavax, Inc. Purified recombinant influenza virus ha proteins
SG178918A1 (en) 2009-09-22 2012-04-27 Medicago Inc Method of preparing plant-derived vlps
CA2789945A1 (en) 2010-02-18 2011-08-25 Technovax, Inc. Universal virus-like particle (vlp) influenza vaccines
ES2614527T3 (en) 2010-11-04 2017-05-31 Medicago Inc. Plant expression system
CA2817005C (en) 2010-11-05 2018-09-11 Novavax Inc. Rabies glycoprotein virus-like particles (vlps)
TWI526539B (en) 2010-12-22 2016-03-21 苜蓿股份有限公司 Method for producing viroid-like particles (VLP) in plants and VLP produced by the method

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9452210B2 (en) 2007-07-13 2016-09-27 Medicago Inc. Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant
US9492528B2 (en) 2007-07-13 2016-11-15 Medicago Inc. Influenza virus-like particles (VLPS) comprising hemagglutinin
US9458470B2 (en) 2007-11-27 2016-10-04 Medicago Inc. Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
US10190132B2 (en) 2007-11-27 2019-01-29 Medicago Inc. Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
US11434497B2 (en) 2007-11-27 2022-09-06 Medicago Inc. Recombinant influenza virus-like particles (VLPS) produced in transgenic plants
US8771703B2 (en) 2008-07-08 2014-07-08 Medicago Inc. Soluble recombinant influenza antigens
US9546375B2 (en) 2008-07-18 2017-01-17 Medicago Inc. Influenza virus immunizing epitope
US10272148B2 (en) 2009-06-24 2019-04-30 Medicago Inc. Chimeric influenza virus-like particles comprising hemagglutinin
WO2010151159A2 (en) 2009-06-26 2010-12-29 Instytut Biochemii I Biofizyki Virus-like particle vector as a polyvalent platform for intracellular delivery of high-molecular-weight therapeutic substances, method for generating a virus like particle vector and use of a virus-like particle vector and a pharmaceutical composition containing said virus-like particle vector
US11826419B2 (en) 2009-09-22 2023-11-28 Medicago Inc. Method of preparing plant-derived VLPs
US11833200B2 (en) 2009-09-22 2023-12-05 Medicago Inc. Method of preparing plant-derived proteins
US9815873B2 (en) 2011-03-23 2017-11-14 Medicago Inc. Method for recovering plant-derived proteins
CN110041397A (en) * 2011-03-23 2019-07-23 麦迪卡格公司 The method for recycling the protein of plant origin

Also Published As

Publication number Publication date
US20230044454A1 (en) 2023-02-09
US9452210B2 (en) 2016-09-27
CN101883856A (en) 2010-11-10
PT2173886E (en) 2013-09-03
CR11209A (en) 2010-03-23
US20130183341A1 (en) 2013-07-18
CA2693956C (en) 2013-09-24
KR20100032920A (en) 2010-03-26
EP2173886B1 (en) 2013-06-26
SI2173886T1 (en) 2013-11-29
EA201000195A1 (en) 2010-10-29
CA2693956A1 (en) 2009-01-22
MX2010000525A (en) 2010-04-27
KR101541330B1 (en) 2015-08-05
DK2173886T3 (en) 2013-09-30
TN2009000557A1 (en) 2011-03-31
PL2173886T3 (en) 2013-12-31
AU2008278222B2 (en) 2014-03-06
WO2009009876A1 (en) 2009-01-22
EP2173886A1 (en) 2010-04-14
EA018206B1 (en) 2013-06-28
NZ582360A (en) 2012-04-27
CN101883856B (en) 2013-10-30
AU2008278222A1 (en) 2009-01-22
EP2173886A4 (en) 2010-10-13
JP2010533001A (en) 2010-10-21
US20100239610A1 (en) 2010-09-23
MY155236A (en) 2015-09-30
ES2428384T3 (en) 2013-11-07
JP5624465B2 (en) 2014-11-12
US9492528B2 (en) 2016-11-15
US20110293650A1 (en) 2011-12-01

Similar Documents

Publication Publication Date Title
US20230044454A1 (en) Recombinant influenza virus-like particles (vlps) produced in transgenic plants
US11434497B2 (en) Recombinant influenza virus-like particles (VLPS) produced in transgenic plants
EP2570484B1 (en) Influenza virus-like particles (VLPS) comprising hemagglutinin
EP2318530A1 (en) New influenza virus immunizing epitope
IL203018A (en) Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
HK1142627B (en) Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant
HK1186207B (en) Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin
HK1183324A (en) Influenza virus-like particles (vlps) comprising hemagglutinin

Legal Events

Date Code Title Description
EEER Examination request
FZDE Discontinued

Effective date: 20150121