WO2010148386A1 - Particules du type virus de la grippe a porcine (h1n1) et leurs procédés d'utilisation - Google Patents

Particules du type virus de la grippe a porcine (h1n1) et leurs procédés d'utilisation Download PDF

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WO2010148386A1
WO2010148386A1 PCT/US2010/039325 US2010039325W WO2010148386A1 WO 2010148386 A1 WO2010148386 A1 WO 2010148386A1 US 2010039325 W US2010039325 W US 2010039325W WO 2010148386 A1 WO2010148386 A1 WO 2010148386A1
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influenza
protein
vlp
virus
strain
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PCT/US2010/039325
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Gale Smith
Peter Pushko
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Novavax, Inc.
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is generally related to virus-like particles (VLPs) comprising influenza A proteins and methods of their use. Specifically, the invention relates to VLPs comprising swine-origin influenza A (HlNl) proteins. The invention further provides methods of producing such VLPs. Also provided are methods of using the VLPs for immunizing animals against the influenza virus of interest.
  • VLPs virus-like particles
  • HlNl swine-origin influenza A
  • Influenza virus is a member of Orthomyxoviridae family (for review, see Murphy and Webster, 1996). There are three subtypes of influenza viruses designated A, B, and C.
  • the influenza virion contains a segmented negative-sense RNA genome.
  • the influenza virion includes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PBl), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2) proteins.
  • the HA, NA, Ml, and M2 are membrane associated, whereas NP, PBl, PB2, PA, and NS2 are nucleocapsid associated proteins.
  • the NSl is the only nonstructural protein not associated with virion particles but specific for influenza-infected cells.
  • the Ml protein is the most abundant protein in influenza particles.
  • the HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell, and the sources of the major immunodominant epitopes for virus neutralization and protective immunity. Both HA and NA proteins are considered the most important components for prophylactic influenza vaccines because they are highly immunogenic.
  • a new vaccine candidate may include these viral antigens as a protein macromolecular particle, such as virus-like particles (VLPs).
  • VLPs mimic the overall structure of a virus particle without the requirement of containing infectious material.
  • VLPs lack a viral DNA or RNA genome, but retain the three-dimensional structure of an authentic virus.
  • VLPs have the ability to stimulate B-cell mediated responses, CD4 proliferative responses and cytotoxic T lymphocytes responses (see, Schirmbeck et al. (1996) Eur. J.
  • the present inventors have observed surprisingly high immunogenicity responses, particularly among elderly patients, following injection with influenza A (HlNl) 2009 virus- like particle (VLP) vaccines.
  • the present invention provides virus-like particles (VLPs) comprising an Ml protein, an HA protein, and an NA protein, wherein the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine-origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein derived from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25.
  • the present invention provides a virus-like particles (VLPs) consisting of an Ml protein, an HA protein, and an NA protein, wherein the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine-origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein derived from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25.
  • the HA and/or NA proteins may have hemagglutinin and/or neuraminidase respectively.
  • the present invention provides a method of making an influenza VLP, comprising expressing nucleic acids encoding an Ml protein, an HA protein, and an NA protein in a cell culture expression system and purifying said VLPs from the cell culture supernatent.
  • the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine- origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the nucleic acids encoding the Ml protein, the HA protein, and the NA protein are expressed in a eukaryotic cell under conditions that permit the formation of VLPs.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein derived from A/Califomia/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25..
  • the eukaryotic cell utilized for expression is be selected from the group consisting of yeast, insect, amphibian, avian, and mammalian cells.
  • the eukaryotic cell is an insect cell.
  • the cell culture expression system is the baculovirus expression system.
  • the present invention provides an antigenic formulation comprising a VLP described herein.
  • the present invention provides a vaccine comprising a VLP described herein.
  • the VLP is formulated with an adjuvant or immune stimulator.
  • the present invention provides a method of inducing immunity in a vertebrate comprising administering to said vertebrate a VLP described herein.
  • the immune response is a humoral immune response.
  • the immune response is a cellular immune response.
  • the present invention provides a method of preventing and/or reducing a viral infection or symptom thereof, comprising administering to a vertebrate a
  • the present invention provides a method of reducing the severity of influenza in a population, comprising administering a VLP described herein to enough individuals in said population in order to prevent or decrease the chance of influenza virus transmission to another individual in said population.
  • Figure 1 depicts a purified HlNl VLP.
  • Lane 1 is a coomassie blue protein molecular weight standard.
  • Lane 2 is a western blot detecting the presence of Ml and HA.
  • Figure 2 depicts the expression of HlNl influenza VLPs generated by co-infection of recombinant baculoviruses encoding individual influenza proteins.
  • Figure 3 depicts an electron microscope (EM) image of swine-origin
  • FIG. 4 depicts a baculovirus vector containing coding sequences for swine-origin
  • Figure 5 depicts an electron microscope (EM) image of swine-origin
  • A/California/04/09 influenza A (HlNl) VLPs generated by infection using a single recombinant baculovirus encoding three influenza proteins.
  • Figure 6 depicts the geometric mean titer as assessed by the hemagglutination inhibition (HAI) assay of three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the
  • Figure 7A depicts the geometric mean titer as assessed by the hemagglutination inhibition (HAI) assay of three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the
  • A/California/04/2009 strain 14 days post- vaccination in subjects 18-49 years of age.
  • Figure 7B depicts the geometric mean titer as assessed by the hemagglutination inhibition (HAI) assay of three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the
  • Figure 8 depicts the proportion of subjects showing a 4-fold or greater rise in titer 14 days post-vaccination with three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the
  • Figure 9 depicts the proportion of subjects showing a 4-fold or greater rise in titer 14 days post-vaccination with three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the
  • Figure 10 depicts the proportion of subjects showing a 4-fold or greater rise in titer by baseline 14 days post-vaccination with three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g
  • Figure 11 depicts the proportion of subjects showing a hemagglutination inhibition result that is greater than or equal to the 1 :40 ratio pre- and post-vaccination.
  • Figure 12A depicts the proportion of subjects showing a hemagglutination inhibition result that is greater than or equal to the 1 :40 ratio pre- and post- vaccination in subjects 18-49 years of age.
  • Figure 12B depicts the proportion of subjects showing a hemagglutination inhibition result that is greater than or equal to the 1 :40 ratio pre- and post- vaccination in subjects 18-49 years of age.
  • Figure 13A depicts the percentage of respondents reporting adverse systemic events within 10 days of the first vaccination with three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g
  • Figure 13B depicts the percentage of respondents reporting adverse local events within 10 days of the first vaccination with three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g
  • Figure 14 depicts the percentage of respondents reporting adverse oculorespiratory events within 10 days of the first vaccination with three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the A/California/04/2009 strain).
  • Figure 15 depicts the percentage of respondents reporting selected adverse systemic and local events within 10 days of the first vaccination with three dose levels of VLPs (5 ⁇ g,
  • antigenic formulation or “antigenic composition” refers to a preparation which, when administered to a vertebrate, especially a bird or a mammal, will induce an immune response.
  • adjuvant refers to a compound that, when used in combination with a specific immunogen (e.g. a VLP) in a formulation, augments or otherwise alters or modifies the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.
  • a specific immunogen e.g. a VLP
  • Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.
  • avian influenza virus refers to influenza viruses found chiefly in birds but that can also infect humans or other animals. In some instances, avian influenza viruses may be transmitted or spread from one human to another. An avian influenza virus that infects humans has the potential to cause an influenza pandemic, i.e., morbidity and/or mortality in humans. A pandemic occurs when a new strain of influenza virus (a virus in which human have no natural immunity) emerges, spreading beyond individual localities, possibly around the globe, and infecting many humans at once.
  • an "effective dose” generally refers to that amount of the VLP of the invention sufficient to induce immunity, to prevent and/or ameliorate influenza virus infection or to reduce at least one symptom of influenza infection and/or to enhance the efficacy of another dose of a VLP.
  • An effective dose may refer to the amount of the VLP sufficient to delay or minimize the onset of an influenza infection.
  • An effective dose may also refer to the amount of the VLP that provides a therapeutic benefit in the treatment or management of influenza infection.
  • an effective dose is the amount with respect to the VLPs of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an influenza viral infection.
  • An effective dose may also be the amount sufficient to enhance a subject's (e.g., a human's) own immune response against a subsequent exposure to influenza virus.
  • Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay.
  • an "effective dose" is one that prevents disease or reduces the severity of symptoms.
  • influenza VLP refers to a VLP comprising at least one influenza protein. Said VLPs can comprise additional influenza and/or non-influenza proteins.
  • hemagglutinin activity refers to the ability of HA- containing proteins, VLPs, or portions thereof to bind and agglutinate red blood cells
  • neuroaminidase activity refers to the enzymatic activity of
  • infectious agent refers to microorganisms that cause an infection in a vertebrate. Usually, the organisms are viruses, bacteria, parasites and/or fungi.
  • the term also refers to different antigenic variations of the same infectious agent.
  • immune stimulator refers to a compound that enhances an immune response via the body's own chemical messengers (cytokines). These molecules comprise various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-I , IL-2,
  • IL-3, IL-4, IL-12, IL-13 growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
  • the immune stimulator molecules can be administered in the same formulation as the influenza VLPs, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.
  • Immunity refers to induction of the immune system of a vertebrate wherein said induction results in the prevention, amelioration, and/or reduction of at least one symptom of an infection in said vertebrate. Immunity may also refer to a hemagglutination inhibition (HI) titer of > 40 when VLPs of the invention have been administered to a vertebrate and said VLPs have induced an immune response against a HA of an influenza virus.
  • HI hemagglutination inhibition
  • non-avian influenza protein or “non-avian source” refers to a protein that is heterologous to an avian influenza virus. Said non-avian influenza protein may be recombinantly expressed from an expression vector and may be heterologous to the expression vector.
  • the term "vaccine” refers to a preparation of dead or weakened pathogens, or of derived antigenic determinants that is used to induce formation of antibodies or immunity against the pathogen.
  • a vaccine is given to provide immunity to the disease, for example, influenza, which is caused by influenza viruses.
  • the term “vaccine” also refers to a suspension or solution of an immunogen (e.g. VLP) that is administered to a vertebrate to produce protective immunity, i.e., immunity that prevents or reduces the severity of disease associated with infection.
  • the present invention provides for vaccine compositions that are immunogenic and may provide protection against a disease associated with infection.
  • vertebrate or “subject” or “patient” refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species.
  • Farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like are also non-limiting examples .
  • the terms "mammals” and “animals” are included in this definition. Both adult and newborn individuals are intended to be covered.
  • virus-like particle refers to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious.
  • Virus- like particle in accordance with the invention do not carry genetic information encoding for the proteins of virus-like particles.
  • virus-like particles lack a viral genome and, therefore, are noninfectious.
  • virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified. Swine Influenza and a Novel Influenza A Virus Strain HlNl
  • Swine influenza is known to be caused by influenza A subtypes HlNl, H1N2, H3N1,
  • H3N2, and H2N3 In pigs, three influenza A virus subtypes (HlNl, H3N2, and H1N2) are the most common strains world- wide (Kothalawala et ai, 2006, Vet Q 28(2): 46-53). In the
  • HlNl subtype was the only subtype found among swine populations until
  • H3N2 sub-types have also been isolated from pigs.
  • influenza A Of the three genera of influenza viruses that cause human flu, two also cause influenza in pigs: influenza A and influenza C. Although the strains found in pigs and humans are largely distinct, genes may transfer among strains during reassortment, allowing for strains to cross species boundaries.
  • HlNl a novel influenza A virus strain, of swine-origin has been detected in humans in Mexico and the United States.
  • the virus strain is spread via person-to-person contact and is transmitted in a similar fashion as is typically seen in seasonal influenza viruses: mainly through the coughs and sneezes of individuals who are infected with the virus.
  • Isolates of the HlNl antigenic subtypes from a recent outbreak include influenza
  • the HA protein is unglycosylated on top of the HA globular head within the receptor binding domain (RBD).
  • the RBD is a 148 amino acid domain which harbors the sialic acid-binding site.
  • the HA from the pandemic influenza A/California/04/09 lacks glycosylation at amino acid positions
  • the present invention describes the cloning of the HlNl strain's HA, NA, and Ml genes into a single baculovirus expression vector alone or in tandem, allowing for the production of functional and immunogenic virus-like particles (VLPs).
  • VLPs represent promising influenza vaccine candidates for the prevention of influenza infection with the
  • VLPs of the invention and methods of making VLPs
  • VLPs virus-like particles
  • the HA and/or NA proteins is derived from an influenza A virus HlNl strain of swine-origin.
  • the HA and NA proteins are derived from influenza A/California/04/09.
  • the HA and NA proteins may be derived from influenza A/California/08/09 or influenza A/Mexico/4108/09.
  • the HA and/or NA proteins may have hemagglutinin and/or neuraminidase activity, respectively.
  • the VLPs of the present invention may comprise an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from an H9N2 influenza strain.
  • influenza Ml can be isolated from any one of the H9N2 influenza viruses selected from the group consisting of A/quail/Hong Kong/Gl/97, A/Hong Kong/1073/99, A/Hong Kong/2108/03, Duck/HK/Y280/97, CK/HK/G9/97, Gf/HK/SSP607/03, Ph/HK/CSWl 323/03, WDk/ST/4808/01, CK/HK/NT 142/03, CK/HK/WF 126/03, SCk/HK/WF285/03, CK/HK/YU463/03, CK/HK/YU577/03, SCk/HK/YU663/03, Ck/HK/CSWl 61/03.
  • H9N2 influenza viruses selected from the group consisting of A/quail/Hong Kong/Gl/97, A/Hong Kong/1073/99, A/Hong Kong/2108/03, Duck/HK/Y280/97, CK/HK/
  • the H9N2 influenza strain is A/Hong Kong/1073/99.
  • the avian influenza Ml protein is derived from an H5N1 influenza strain.
  • the influenza Ml can be isolated from any one of the H5N1 influenza viruses selected from the group consisting of A/Vietnam/ 1194/04, A/Vietnam/I 203/04, A/Hongkong/213/03, A/Indonesia/2/2005, A/Bar headed goose/Quinghai/lA/2005, A/Anhui/1/2005, and A/Indonesia/5/05.
  • the H5N1 strain is A/Indonesia/5/05.
  • the VLPs of the present invention may comprise an influenza Ml protein from a non-avian source, such as from an HlNl strain of swine-origin.
  • the Ml protein is derived from influenza A/California/04/09.
  • the Ml proteins may be derived from influenza A/California/08/09 or influenza A/Mexico/4108/09.
  • influenza Ml protein comprising the YKKL L-domain sequence at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25 increases the efficiency of influenza VLP production.
  • the VLPs of the present invention comprise an influenza Ml protein that contains the YKKL L-domain sequence.
  • influenza Ml protein comprising the YKKL L-domain sequence at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25 is an avian influenza Ml protein.
  • influenza Ml protein comprising the YKKL L-domain sequence at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25 is a non-avian influenza Ml protein.
  • the non-avian influenza Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein derived from A/California/04/09 is comprised of SEQ ID NO: 20.
  • influenza Ml proteins identified herein may be readily identified in other influenza Ml proteins by one of skill in the art.
  • one with skill in the art can identify an influenza Ml protein in nature exhibiting an increased ability to mediate VLP formation.
  • one with skill in the art would be able to make one or more modifications which would result in an increased ability to mediate VLP formation, in any influenza Ml protein of interest.
  • influenza Ml proteins that occur in nature with the YKKL amino acids at positions corresponding to residues 100-103 of SEQ ID NOs: 20 and 25, as well as those that have been mutated either naturally or through genetic engineering to contain the YKKL L-domain at positions corresponding to residues 100-103 of SEQ ID NOs: 20 and 25 fall within the scope of this invention.
  • the VLPs may comprise proteins from at least two different influenza viruses.
  • the VLPs may comprise an HA from influenza A/California/04/09 and an NA from influenza A/Mexico/4108/09.
  • the VLPs may comprise an HA from both influenza A/California/04/09 and influenza A/Mexico/4108/09.
  • said VLPs are multivalent VLPs capable of inducing an immune response to several proteins and therefore, several strains of influenza virus.
  • said multivalent VLPs comprise an influenza Ml protein.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein derived from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25.
  • the invention also encompasses variants of the said proteins expressed on or in the VLPs of the invention.
  • the variants may contain alterations in the amino acid sequences of the constituent proteins.
  • the term "variant" with respect to a protein refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
  • the variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a variant can have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
  • Natural variants can occur due to mutations in the proteins. These mutations may lead to antigenic variability within individual groups of infectious agents, for example influenza. Thus, a person infected with an influenza strain develops antibody against that virus, as newer virus strains appear, the antibodies against the older strains no longer recognize the newer virus and reinfection can occur.
  • the invention encompasses all antigenic and genetic variability of proteins from infectious agents for making the VLPs.
  • General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, cell culture and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152 Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M.
  • the invention also encompasses using known methods of protein engineering and recombinant DNA technology to improve or alter the characteristics of the proteins expressed on or in the VLPs of the invention.
  • mutagenesis can be used to produce and/or isolate variant nucleic acids that encode for protein molecules and/or to further modify/mutate the proteins in or on the VLPs of the invention.
  • mutagenesis include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction- purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, is also included in the present invention.
  • mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.
  • the invention further comprises protein variants which show substantial biological activity, e.g., able to elicit an effective antibody response when expressed on or in VLPs of the invention.
  • Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • the gene encoding a specific virus protein can be isolated by RT-PCR from polyadenylated mRNA extracted from cells which had been infected with a virus (DNA or RNA virus) or PCR from cells which had been infected with a DNA virus.
  • the resulting product gene can be cloned as a DNA insert into a vector.
  • vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome- conjugated DNA, or the like, that is not autonomously replicating.
  • the vectors of the present invention are plasmids or bacmids.
  • the invention comprises nucleotides that encode proteins cloned into an expression vector that can be expressed in a cell that induces the formation of VLPs of the invention.
  • An "expression vector” is a vector, such as a plasmid that is capable of promoting expression, as well as replication of a nucleic acid incorporated therein.
  • the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.
  • the vector comprises nucleotides that encode for HA, NA, and Ml.
  • the vector comprises nucleotides that encode HA and/or NA, wherein said HA and/or NA are from a swine-origin influenza A virus.
  • the vector comprises nucleotides that encode HA and/or NA, wherein said HA and/or NA are derived from a swine-origin influenza A virus such as HlNl.
  • the HlNl strain is influenza A/California/04/09. In other embodiments, the HlNl strain may be selected from influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the nucleotides encoding Ml encode an Ml protein comprising an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the nucleotides encoding Ml encode a swine influenza Ml protein.
  • the nucleotides encoding Ml encode the swine influenza Ml protein derived from influenza strain A/California/04/09.
  • the nucleotides encoding Ml encode an Ml protein derived from A/California/04/09 that is comprised of SEQ ID NO: 20.
  • nucleotides encoding Ml encode an avian influenza Ml protein. In a further embodiment, the nucleotides encoding Ml encode an avian influenza Ml protein derived from influenza strain A/Indonesia/5/05. In an exemplary embodiment, the nucleotides encoding Ml encode an Ml protein that is comprised of SEQ ID NO: 25.
  • said proteins may comprise, mutations containing alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.
  • Nucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by insect cells such as Sf9 cells). See U.S. patent publication 2005/0118191, herein incorporated by reference in its entirety for all purposes.
  • nucleotides can be sequenced to ensure that the correct coding regions were cloned and do not contain any unwanted mutations.
  • the nucleotides can be subcloned into an expression vector (e.g. baculovirus) for expression in any cell.
  • an expression vector e.g. baculovirus
  • the above is only one example of how the influenza proteins can be cloned. A person with skill in the art understands that additional methods are available and are possible.
  • the invention also provides for constructs and/or vectors that comprise nucleotides that encode for the influenza proteins described above.
  • the constructs and/or vectors that comprise influenza proteins should be operatively linked to an appropriate promoter, such as the AcMNPV polyhedrin promoter (or other baculovirus), phage lambda PL promoter, the E. coli lac, phoA and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs are non-limiting examples.
  • an appropriate promoter such as the AcMNPV polyhedrin promoter (or other baculovirus), phage lambda PL promoter, the E. coli lac, phoA and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs are non-limiting examples.
  • Other suitable promoters will be known to the skilled artisan depending on the host cell and/or the rate of expression desired.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome-binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • Expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin, or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • virus vectors such as baculovirus, poxvirus ⁇ e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canine adenovirus), herpesvirus, and retrovirus.
  • vectors for use in bacteria comprise vectors for use in bacteria, which comprise pQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540. pRIT5.
  • preferred eukaryotic vectors are pFastBacl pWINEO, pSV2CAT, pOG44, pXTl, and pSG, pSVK3, pBPV, pMSG, and pSVL.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • the recombinant constructs mentioned above could be used to transfect, infect, or transform and can express influenza proteins, into eukaryotic cells and/or prokaryotic cells.
  • the invention provides for host cells that comprise a vector (or vectors) that contain nucleic acids which code for influenza proteins, and permit the expression of said constructs in said host cell under conditions which allow the formation of VLPs.
  • yeast yeast
  • insect avian
  • plant C. elegans (or nematode)
  • mammalian host cells include yeast, insect, avian, plant, C. elegans (or nematode), and mammalian host cells.
  • insect cells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, SfZl, Trichoplusia ni cells, e.g. High Five cells, and Drosophila S2 cells.
  • fungi including yeast
  • yeast S. cerevisiae
  • Kluyveromyces lactis Kluyveromyces lactis
  • species of Candida including C. albicans and C. glabrata
  • Aspergillus nidulans Schizosaccharomyces pombe
  • pombe Pichiapastoris
  • Yarrowia lipolytica examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, and African green monkey cells, CVl cells, HeLa cells, MDCK cells, Vero, and Hep-2 cells. Xenopus laevis oocytes, or other cells of amphibian origin, may also be used.
  • Prokaryotic host cells include bacterial cells, for example, E. coli, B. subtilis, and mycobacteria.
  • Vectors e.g., vectors comprising polynucleotides of influenza proteins
  • introducing nucleic acids into eukaryotic cells can be by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents.
  • said vector is a recombinant baculovirus.
  • said recombinant baculovirus is transfected into a eukaryotic cell.
  • said cell is an insect cell.
  • said insect cell is a Sf9 cell.
  • said vector and/or host cell comprise nucleotides that encode Ml, HA, and NA proteins.
  • said vector and/or host cell consists essentially of Ml, HA, and NA proteins.
  • said vector and/or host cell consists of Ml, HA, and NA proteins.
  • the invention also provides for constructs and methods that will further increase the efficiency of VLPs production.
  • the addition of leader sequences to the Ml, HA, and/or NA proteins can improve the efficiency of protein transporting within the cell.
  • a heterologous signal sequence can be fused to Ml, HA, and/or NA.
  • the signal sequence can be derived from the gene of an insect preprotein and fused to Ml, HA, and/or NA.
  • the signal peptide is the chitinase signal sequence, which works efficiently in baculovirus expression systems.
  • the invention also provides for methods of producing VLPs of the invention, said methods comprising expressing nucleic acids encoding an Ml protein, an HA protein, and an NA protein in a cell culture expression system and purifying said VLPs from the cell culture supernatent.
  • the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine-origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the nucleic acids encoding the Ml protein, the HA protein, and the NA protein are expressed in a eukaryotic cell under conditions that permit the formation of VLPs.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25..
  • the eukaryotic cell utilized for expression is be selected from the group consisting of yeast, insect, amphibian, avian, and mammalian cells.
  • the eukaryotic cell is an insect cell.
  • the cell culture expression system is the baculovirus expression system.
  • VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the recombinant proteins are expressed and VLPs are formed.
  • the selection of the appropriate growth conditions is within the skill or a person with skill of one of ordinary skill in the art.
  • Methods to grow cells engineered to produce VLPs of the invention include, but are not limited to, batch, batch-fed, continuous and perfusion cell culture techniques.
  • Cell culture means the growth and propagation of cells in a bioreactor (a fermentation chamber) where cells propagate and express protein (e.g. recombinant proteins) for purification and isolation.
  • cell culture is performed under sterile, controlled temperature and atmospheric conditions in a bioreactor.
  • a bioreactor is a chamber used to culture cells in which environmental conditions such as temperature, atmosphere, agitation and/or pH can be monitored.
  • said bioreactor is a stainless steel chamber.
  • said bioreactor is a pre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech, Bridgewater, NJ). In other embodiment, said pre-sterilized plastic bags are about 50 L to 1000 L.
  • VLPs are then isolated using methods that preserve the integrity thereof, such as by gradient centrifugation, e.g., cesium chloride, sucrose and iodixanol, as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • gradient centrifugation e.g., cesium chloride, sucrose and iodixanol
  • standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • Production of VLPs of the invention can start by seeding Sf9 cells (non-infected) into shaker flasks, allowing the cells to expand and scaling up as the cells grow and multiply (for example from a 125 ml flask to a 50 L Wave bag).
  • the medium used to grow the cell is formulated for the appropriate cell line (preferably serum free media, e.g. insect medium ExCell-420, JRH).
  • said cells are infected with recombinant baculovirus at the most efficient multiplicity of infection (e.g. from about 1 to about 3 plaque forming units per cell).
  • the Ml such as avian influenza Ml and at least one influenza heterologous protein (i.e.
  • VLPs of the invention can be harvested approximately 48 to 96 hours post infection, when the levels of VLPs in the cell culture medium are near the maximum but before extensive cell lysis.
  • the Sf9 cell density and viability at the time of harvest can be about 0.5 x 10 6 cells/ml to about 1.5 x 10 6 cells/ml with at least 20% viability, as shown by dye exclusion assay.
  • the medium is removed and clarified.
  • NaCl can be added to the medium to a concentration of about 0.4 to about 1.0 M, preferably to about 0.5 M, to avoid VLP aggregation.
  • the removal of cell and cellular debris from the cell culture medium containing VLPs of the invention can be accomplished by tangential flow filtration (TFF) with a single use, pre-sterilized hollow fiber 0.5 or 1.00 ⁇ m filter cartridge or a similar device.
  • VLPs in the clarified culture medium can be concentrated by ultrafiltration using a disposable, pre-sterilized 500,000 molecular weight cut off hollow fiber cartridge.
  • the concentrated VLPs can be diafiltrated against 10 volumes pH 7.0 to 8.0 phosphate- buffered saline (PBS) containing 0.5 M NaCl to remove residual medium components.
  • PBS phosphate- buffered saline
  • the concentrated, diafiltered VLPs can be further purified on a 20% to 60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaCl by centrifugation at 6,500 x g for 18 hours at about 4°C to about 10 0 C.
  • VLPs will form a distinctive visible band between about 30% to about 40% sucrose or at the interface (in a 20% and 60% step gradient) that can be collected from the gradient and stored.
  • This product can be diluted to comprise 200 mM of NaCl in preparation for the next step in the purification process.
  • This product contains VLPs and may contain intact baculovirus particles.
  • Further purification of VLPs can be achieved by anion exchange chromatography, or 44% isopycnic sucrose cushion centrifugation. In anion exchange chromatography, the sample from the sucrose gradient (see above) is loaded into column containing a medium with an anion (e.g.
  • Matrix Fractogel EMD TMAE Matrix Fractogel EMD TMAE
  • a salt gradient from about 0.2 M to about 1.0 M of NaCl
  • the sample comprising the VLPs is added to a 44% sucrose cushion and centrifuged for about 18 hours at 30,000 g.
  • VLPs form a band at the top of 44% sucrose, while baculovirus precipitates at the bottom and other contaminating proteins stay in the 0% sucrose layer at the top.
  • the VLP peak or band is collected.
  • the intact baculovirus can be inactivated, if desired. Inactivation can be accomplished by chemical methods, for example, formalin or ⁇ -propyl lactone (BPL). Removal and/or inactivation of intact baculovirus can also be largely accomplished by using selective precipitation and chromatographic methods known in the art, as exemplified above. Methods of inactivation comprise incubating the sample containing the VLPs in 0.2% of BPL for 3 hours at about 25 0 C to about 27°C. The baculovirus can also be inactivated by incubating the sample containing the VLPs at 0.05% BPL at 4°C for 3 days, then at 37°C for one hour.
  • BPL ⁇ -propyl lactone
  • the product comprising VLPs can be run through another diafiltration step to remove any reagent from the inactivation step and/or any residual sucrose, and to place the VLPs into the desired buffer (e.g. PBS).
  • the solution comprising VLPs can be sterilized by methods known in the art (e.g. sterile filtration) and stored in the refrigerator or freezer.
  • the above techniques can be practiced across a variety of scales. For example, T- flasks, shake-flasks, spinner bottles, up to industrial sized bioreactors.
  • the bioreactors can comprise either a stainless steel tank or a pre-sterilized plastic bag (for example, the system sold by Wave Biotech, Bridgewater, NJ). A person with skill in the art will know what is most desirable for their purposes.
  • Expansion and production of baculovirus expression vectors and infection of cells with recombinant baculovirus to produce recombinant influenza VLPs can be accomplished in insect cells, for example Sf9 insect cells as previously described.
  • the cells are Sf9 infected with recombinant baculovirus engineered to produce VLPs of the invention.
  • the invention comprises an antigenic formulation comprising VLP comprising an Ml protein, an HA protein, and an NA protein.
  • the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine-origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25.
  • the formulations of the invention may comprise one or more VLPs as described herein in combination with, or formulated with, one or more purified or partially purified HA antigens.
  • a formulation comprises a VLP comprising HA and NA for influenza A/California/04/09 in combination with purified HA (and or NA) from that same virus.
  • the VLP is combined with a homologous or heterologous HA or NA.
  • formulations of the at least one VLP and at least one HA or NA are made and administered to a patient or subject concurrently or separately.
  • antibody production as measured by HA antibody titers or other measure is superior when the VLP and HA are administered in a single or separate formulation to the same patient or subject (or animal).
  • Said formulations of the invention comprise a formulation comprising VLPs of the invention and a pharmaceutically acceptable carrier or excipient.
  • Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • compositions useful herein contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition, and which may be administered without undue toxicity and a VLP of the invention.
  • compositions can be useful as a vaccine and/or antigenic compositions for inducing a protective immune response in a vertebrate.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the invention also comprises a vaccine comprising a VLP comprising an Ml protein, an HA protein, and an NA protein.
  • the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine- origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25..
  • the invention also provides for a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention.
  • the kit comprises two containers, one containing VLPs and the other containing an adjuvant.
  • the kit comprises two containers, one containing freeze dried VLPs and the other containing a solution to resuspend said VLPs.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the VLP formulation be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition.
  • the VLP composition is supplied as a liquid, in another embodiment, as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to the appropriate concentration for administration to a subject.
  • said container comprises at least about 50 ⁇ g/ml, more preferably at least about 100 ⁇ g/ml, at least about 200 ⁇ g/ml, at least 500 ⁇ g /ml, or at least 1 mg/ml of an antigen associated with VLPs of the invention.
  • the VLP composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the VLP composition.
  • the liquid form of the VLP composition is supplied in a hermetically sealed container at least about 50 ⁇ g/ml, more preferably at least about 100 ⁇ g/ml, at least about 200 ⁇ g/ml, at least 500 ⁇ g /ml, or at least 1 mg/ml of an antigen associated with VLPs of the invention.
  • VLPs of the invention are administered in an effective amount or quantity (as defined above) sufficient to stimulate an immune response against one or more infectious agents.
  • administration of the VLP of the invention elicits immunity against an infectious agent.
  • the dose can be adjusted within this range based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors.
  • the prophylactic vaccine formulation is systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needle-less injection device.
  • the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract.
  • the invention also comprises a method of formulating a vaccine or antigenic composition that induces immunity to an infection or at least one symptom thereof to a mammal, comprising adding to said formulation an effective dose of VLPs of the invention.
  • compositions comprising VLPs include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppositories).
  • parenteral administration e.g., intradermal, intramuscular, intravenous and subcutaneous
  • epidural e.g., epidural and mucosal
  • mucosal e.g., intranasal and oral or pulmonary routes or by suppositories.
  • compositions of the present invention are administered intramuscularly, intravenously, subcutaneously, transdermally or intradermally.
  • compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • epithelial or mucocutaneous linings e.g., oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.
  • intranasal or other mucosal routes of administration of a composition comprising VLPs of the invention may induce an antibody or other immune response that is substantially higher than other routes of administration.
  • intranasal or other mucosal routes of administration of a composition comprising VLPs of the invention may induce an antibody or other immune response that will induce cross protection against other strains or organisms that cause infection.
  • a VLP comprising influenza protein when administered to a vertebrate, can induce cross protection against several influenza strains. Administration can be systemic or local.
  • the vaccine and/or antigenic formulation is administered in such a manner as to target mucosal tissues in order to elicit an immune response at the site of immunization.
  • mucosal tissues such as gut associated lymphoid tissue (GALT) can be targeted for immunization by using oral administration of compositions which contain adjuvants with particular mucosal targeting properties.
  • Additional mucosal tissues can also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and bronchial- associated lymphoid tissue (BALT).
  • Vaccines and/or antigenic formulations of the invention may also be administered on a dosage schedule, for example, an initial administration of the vaccine composition with subsequent booster administrations.
  • a second dose of the composition is administered anywhere from two weeks to one year, preferably from about 1 , about 2, about 3, about 4, about 5 to about 6 months, after the initial administration.
  • a third dose may be administered after the second dose and from about three months to about two years, or even longer, preferably about 4, about 5, or about 6 months, or about 7 months to about one year after the initial administration.
  • the third dose may be optionally administered when no or low levels of specific immunoglobulins are detected in the serum and/or urine or mucosal secretions of the subject after the second dose.
  • a second dose is administered about one month after the first administration and a third dose is administered about six months after the first administration.
  • the second dose is administered about six months after the first administration.
  • said VLPs of the invention can be administered as part of a combination therapy.
  • VLPs of the invention can be formulated with other immunogenic compositions, antivirals and/or antibiotics.
  • the dosage of the pharmaceutical formulation can be determined readily by the skilled artisan, for example, by first identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of virus specific immunoglobulins or by measuring the inhibitory ratio of antibodies in serum samples, or urine samples, or mucosal secretions. Said dosages can be determined from animal studies. A non-limiting list of animals used to study the efficacy of vaccines include the guinea pig, hamster, ferrets, chinchilla, mouse and cotton rat. Most animals are not natural hosts to infectious agents but can still serve in studies of various aspects of the disease.
  • any of the above animals can be dosed with a vaccine candidate, e.g. VLPs of the invention, to partially characterize the immune response induced, and/or to determine if any neutralizing antibodies have been produced.
  • a vaccine candidate e.g. VLPs of the invention
  • many studies have been conducted in the mouse model because mice are small size and their low cost allows researchers to conduct studies on a larger scale.
  • the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and as such, adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.
  • MPL trehalose dimycolate
  • CWS cell wall skeleton
  • MF-59, Novasomes ® , MHC antigens may also be used.
  • the adjuvant is a paucilamellar lipid vesicle having about two to ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a large amorphous central cavity free of lipid bilayers.
  • Paucilamellar lipid vesicles may act to stimulate the immune response several ways, as non-specific stimulators, as carriers for the antigen, as carriers of additional adjuvants, and combinations thereof.
  • Paucilamellar lipid vesicles act as non-specific immune stimulators when, for example, a vaccine is prepared by intermixing the antigen with the preformed vesicles such that the antigen remains extracellular to the vesicles.
  • the vesicle acts both as an immune stimulator and as a carrier for the antigen.
  • the vesicles are primarily made of nonphospholipid vesicles.
  • the vesicles are Novasomes ® .
  • Novasomes ® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They comprise Brij 72, cholesterol, oleic acid and squalene. Novasomes ® have been shown to be an effective adjuvant for influenza antigens (see, U.S. Patents 5,629,021, 6,387,373, and 4,911,928, herein incorporated by reference in their entireties for all purposes).
  • Immune stimulators include, but not limited to, various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte- macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
  • interleukins e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13
  • growth factors e.g., granulocyte- macrophage (GM)-colony stimulating factor (CSF)
  • CSF colonny stimulating factor
  • other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
  • the immunostimulatory molecules can be administered in the same formulation as the RSV VLPs, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.
  • the invention comprises antigenic and vaccine formulations comprising an adjuvant and/or an immune stimulator.
  • one embodiment of the invention comprises a formulation comprising a VLP and adjuvant and/or an immune stimulator.
  • said adjuvant are Novasomes ® .
  • said formulation is suitable for human administration.
  • the formulation is administered to a vertebrate orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.
  • different VLPs are blended together to create a multivalent formulation. These VLPs may comprise VLPs HA and/or NA from different strains of influenza virus.
  • stimulation of immunity with a single dose is preferred, additional dosages can be administered by the same or different route to achieve the desired effect.
  • multiple administrations may be required to elicit sufficient levels of immunity.
  • Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against infections.
  • adults who are particularly susceptible to repeated or serious infections such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses.
  • Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.
  • the VLPs of the invention are useful for preparing compositions that stimulate an immune response that confers immunity to infectious agents. Both mucosal and cellular immunity may contribute to immunity to infectious agents and disease. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. Secretory immunoglobulin A (slgA) is involved in protection of the upper respiratory tract and serum IgG in protection of the lower respiratory tract. The immune response induced by an infection protects against reinfection with the same virus or an antigenically similar viral strain. For example, influenza undergoes frequent and unpredictable changes; therefore, after natural infection, the effective period of protection provided by the host's immunity may only be a few years against the new strains of virus circulating in the community.
  • slgA Secretory immunoglobulin A
  • VLPs of the invention can induce on immunity in a vertebrate (e.g. a human) when administered to said vertebrate.
  • the immunity results from an immune response against VLPs of the invention that protects or ameliorates infection or at least reduces a symptom of infection in said vertebrate.
  • said infection will be asymptomatic.
  • the response may be not a fully protective response.
  • said vertebrate is infected with an infectious agent, the vertebrate will experience reduced symptoms or a shorter duration of symptoms compared to a non-immunized vertebrate.
  • the invention comprises a method of inducing immunity in a vertebrate comprising administering to said vertebrate a VLP comprising an Ml protein, an HA protein, and an NA protein.
  • the HA protein and/or the NA protein is derived from a swine- origin influenza A virus.
  • the swine-origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25.
  • the immune response induced is a humoral immune response.
  • the immune response induced is a cellular immune response.
  • an "antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • the invention comprises a method of inducing a protective cellular response to an infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of VLPs of the invention, wherein said VLPs comprise an Ml protein, an HA protein, and an NA protein.
  • the HA protein and/or the NA protein is derived from a swine-origin influenza A virus.
  • the swine-origin influenza A virus is an HlNl strain.
  • the HlNl strain is influenza A/California/04/09.
  • the HlNl strain may be selected from the influenza strains influenza A/California/08/09 and influenza A/Mexico/4108/09.
  • the Ml protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 25.
  • the Ml protein is a swine influenza Ml protein.
  • the swine influenza Ml protein is derived from influenza strain A/California/04/09.
  • the Ml protein from A/California/04/09 is comprised of SEQ ID NO: 20.
  • the Ml protein is an avian influenza Ml protein.
  • the avian influenza Ml protein is derived from influenza strain A/Indonesia/5/05.
  • the Ml protein derived from influenza strain A/Indonesia/5/05 is comprised of SEQ ID NO: 25..
  • the VLPs of the invention can prevent or reduce at least one symptom of influenza in a subject when administered to said subject. Most symptoms of influenza are well known in the art.
  • the method of the invention comprises the prevention or reduction of at least one symptom associated with an influenza.
  • a reduction in a symptom may be determined subjectively or objectively, e.g., self assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement (e.g.
  • the objective assessment comprises both animal and human assessments.
  • the invention comprises a method of preventing and/or reducing an infection with influenza or symptom thereof, comprising administering to said vertebrate a VLP of the invention.
  • a strategy for the control of infectious diseases during an outbreak is the universal vaccination of healthy individuals, including children.
  • vaccination with current influenza vaccines of approximately 80% of schoolchildren in a community has decreased respiratory illnesses in adults and excess deaths in the elderly (Reichert et al, 2001).
  • This concept is known as community immunity or "herd immunity” and is thought to play an important part of protecting the community against diseases.
  • vaccinated people have antibodies that neutralize and infectious agent, e.g. influenza virus, they are much less likely to transmit said agent to other people.
  • the invention also comprises a method of reducing the severity of influenza in a population, comprising administering a VLP of the invention to enough individuals in said population in order to prevent or decrease the chance of transmission to another individual in said population.
  • the invention also encompasses a method of inducing immunity to an influenza virus to a population or a community in order to reduce the incidence of infections among immunocompromised individuals or non-vaccinated individual buy administering VLPs of the invention to a population in a community.
  • most school- aged children are immunized by administering the VLPs of the invention.
  • most healthy individuals in a community to are immunized by administering the VLPs of the invention.
  • VLPs of the invention are part of a "dynamic vaccination" strategy. Dynamic vaccination is the steady production of a low-efficacy vaccine that is related to an emerging pandemic strain, but due to an antigenic drift may not provide complete protection in a mammal (see Germann et al, 2006).
  • the primary antibodies used were anti-NA (an A/Indonesia/5/05 H5N1 antibody), IgG Rabbit (1:100), anti-influenza Ml (1 :20000) (Serotec), and Influenza A/New Caledonia/20/99 HlNl anti-rabbit (1 :500).
  • the secondary antibodies used were goat anti-rabbit (1 :5000) and goat anti-mouse (1:20000).
  • the HlNl VLPs were also analyzed using negative staining transmission electron microscopy (Figure 3). The HlNl VLPs show characteristic influenza morphology.
  • the HlNl VLPs were analyzed using negative staining transmission electron microscopy
  • HAI hemagglutinin inhibition
  • Figures 7 A and 7B show the geometric mean titer as assessed by the hemagglutination inhibition (HAI) assay using three dose levels of VLPs (5 ⁇ g, 15 ⁇ g, and 45 ⁇ g HA from the A/California/04/2009 strain) 14 days post-vaccination in subjects 18-49 years of age and 50-64 years of age, respectively.
  • HAI hemagglutination inhibition
  • One additional measure of the immunogenic response is the rise in titer among groups with different baseline antibody titers.
  • Figure 10 illustrates, a significant percentage of respondents having a baseline titer of greater than or equal to 10 showed a 4-fold or greater rise in titer with the three dose levels of VLPs.
  • subjects that had a baseline titer of less than 10 a significant percentage of respondents showed a 4-fold or greater rise in titer with the three dose levels of VLPs.
  • Figures 1 1 and 12A-12B demonstrate that the hemagglutination inhibition result is greater than or equal to a ratio of 1 :40 for pre- and post-vaccination levels 14 days after vaccination in subjects aged 18-49.

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Abstract

La présente invention concerne des particules du type virus de la grippe (VLP) qui comportent des protéines grippales dérivées d'un virus de la grippe A d'origine porcine. L'invention concerne des procédés de fabrication de VLP de la grippe, des procédés de fabrication de formulations vaccinales et antigéniques comportant lesdites VLP, et des procédés d'utilisation des VLP pour induire une immunité chez les vertébrés.
PCT/US2010/039325 2009-06-19 2010-06-21 Particules du type virus de la grippe a porcine (h1n1) et leurs procédés d'utilisation WO2010148386A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110081367A1 (en) * 2008-06-14 2011-04-07 Veredus Laboratories Pte Ltd Influenza sequences
WO2014078674A1 (fr) * 2012-11-16 2014-05-22 Medimmune, Llc Variants d'hémagglutinine et de neuraminidase de la grippe porcine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070184526A1 (en) * 2003-07-11 2007-08-09 Gale Smith Functional influenza virus like particles (VLPs)
US20080031895A1 (en) * 2006-05-01 2008-02-07 Techno Vax, Inc. Influenza virus-like particle (VLP) compositions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070184526A1 (en) * 2003-07-11 2007-08-09 Gale Smith Functional influenza virus like particles (VLPs)
US20080031895A1 (en) * 2006-05-01 2008-02-07 Techno Vax, Inc. Influenza virus-like particle (VLP) compositions

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of a Novel Swine -Origin Influenza A (H1N1) Virus in Humans.", THE NEW ENGLAND JOURNAL OF MEDICINE., vol. 360, 18 June 2009 (2009-06-18), pages 2605 - 2615 *
DATABASE GENBANK Database accession no. FJ966954 *
RANJAN ET AL.: "5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication.", VIROL J., vol. 7, no. 102, 21 May 2010 (2010-05-21), pages 4 *
TRIANA-BALTZER ET AL.: "Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein.", PLOS ONE., vol. 4, no. 11, 6 November 2009 (2009-11-06), pages E7788 *
ZHENG ET AL.: "Genome evolution of novel influenza A (H1N1) viruses in humans.", CHINESE SCIENCE BULLETIN., vol. 54, no. 13, 25 May 2009 (2009-05-25), pages 2159 - 2163 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110081367A1 (en) * 2008-06-14 2011-04-07 Veredus Laboratories Pte Ltd Influenza sequences
WO2014078674A1 (fr) * 2012-11-16 2014-05-22 Medimmune, Llc Variants d'hémagglutinine et de neuraminidase de la grippe porcine

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