[go: up one dir, main page]

WO2011011390A1 - Protéines ha recombinantes purifiées du virus de la grippe - Google Patents

Protéines ha recombinantes purifiées du virus de la grippe Download PDF

Info

Publication number
WO2011011390A1
WO2011011390A1 PCT/US2010/042568 US2010042568W WO2011011390A1 WO 2011011390 A1 WO2011011390 A1 WO 2011011390A1 US 2010042568 W US2010042568 W US 2010042568W WO 2011011390 A1 WO2011011390 A1 WO 2011011390A1
Authority
WO
WIPO (PCT)
Prior art keywords
purified
protein
influenza
virus
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.)
Ceased
Application number
PCT/US2010/042568
Other languages
English (en)
Inventor
Gale Smith
Steven Pincus
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.)
Novavax Inc
Original Assignee
Novavax Inc
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 Novavax Inc filed Critical Novavax Inc
Publication of WO2011011390A1 publication Critical patent/WO2011011390A1/fr
Anticipated expiration legal-status Critical
Ceased 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
    • 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
    • C12N2760/16211—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

Definitions

  • This invention relates to generally to the purification of influenza HA proteins and to methods of use thereof.
  • 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.
  • HA hemagglutinin
  • NA neuraminidase
  • Ml matrix
  • M2 proton ion-channel protein
  • NP nucleoprotein
  • NP nucleoprotein
  • PBl polymerase basic protein 1
  • PA polymerase acidic protein
  • NS2 nonstructural protein 2
  • 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 M 1 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 ceil, 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.
  • Influenza virus infection 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 sialic acid receptor, and virus penetration into the cell depends on HA-dependent receptor-mediated endocytosis.
  • the HA protein undergoes conformational changes that lead to fusion of viral and host cell membranes followed by virus uncoating and M2-mediated release of Ml proteins from nucleocapsid-associated ribonucleoproteins (RNPs), which migrate into the cell nucleus for viral RNA synthesis.
  • RNPs nucleocapsid-associated ribonucleoproteins
  • Antibodies to HA molecule can prevent virus infection by neutralizing virus infectivity, whereas antibodies to NA proteins mediate their effect on the early steps of viral replication.
  • Prior art processes for producing recombinant HA can generate HA proteins with conformational structures that are not recognized by the immunized host as native, resulting in poor antigenicity in assays such as the single radial immunodiffusion assay (SRlD assay).
  • the present inventors have developed improved purification methods that produce purified HA proteins with surprising and unexpected antigenic properties. When administered to a host animal such as a sheep, the purified HA proteins are recognized as native and the host animal produces antibodies that recognize the virus. In contrast, prior art methods produce denatured HA proteins that are poorly antigenic in a host animal.
  • the present invention provides purified HA proteins that demonstrate increased antigenicity as compared to prior art HA proteins.
  • the present invention provides a purified viral protein.
  • the purity of the viral protein is at least about 95%.
  • the viral protein is a recombinant or chimeric protein derived from an influenza virus.
  • the influenza virus is selected from the group consisting of avian influenza virus and mammalian influenza virus.
  • the mammalian influenza virus is a human influenza virus strain.
  • the mammalian influenza virus is a swine influenza virus.
  • said viral protein may be derived from a pandemic strain or a seasonal strain of influenza virus.
  • the purified viral protein is an HA protein.
  • the HA protein is selected from the group consisting of Hl, H2, H3, H4, H5, H ⁇ , 11,7 H8, H9, H10, H11 1, HI2, H13, H 14 , H15 and H16.
  • the HA is derived from an HlNl influenza virus strain.
  • the HlNl influenza virus strain is selected from the group consisting of A/Califomi a/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.
  • the purified HA protein exhibits enhanced or improved antigenicity in a single radial immunodiffusion (SRlD) assay as compared to partially denatured or denatured HA proteins.
  • SRlD single radial immunodiffusion
  • the present invention provides a method of purifying an HA protein, comprising steps of:
  • step (iv) Purifying said HA from said pool in step (iii) using affinity chromatography, wherein a second pool of fractions containing said ItA is produced;
  • the host cells in step are insect cells.
  • the insect cells are Sf9 insect cells.
  • the ion exchange chromatography in step (iii) is anion exchange chromatography.
  • said anion exchange chromatography is a TMAE anion exchange chromatography.
  • the affinity chromatography in step (iv) is a lentil lectin affinity chromatography.
  • the desalting step (v) is conducted with desalting chromatography.
  • the desalting chromatography is performed using a desalting column packed with SephadexTM 0-25.
  • the desalting step (v) is conducted with dialysis.
  • the dialysis is performed using a dialysis bag, tubing, or a stirred cell comprising a selectively permeable membrane.
  • the selectively permeable membrane has a molecule weight cut off (MWCC)) of about 20 to about 30 KD.
  • the hydroxyapatite chromatography in step (vi) is performed using a column packed with synthetic hydroxyapatite.
  • the synthetic hydroxyapatite is ceramic hydroxyapatite (e.g., CHTTM hydroxyapatite Type I).
  • the viral protein is concentrated and dialyzed in a stirred cell with selectively permeable membrane in step (vii).
  • the viral protein is steriled by passing through a micron filter (e.g., a 0.2 micron SFCA (or PVDF) filter).
  • the present invention provides VLPs comprising a purified HA.
  • the HA protein is selected from the group consisting of Hl, H2, H3, H4, H5, H6, H,7 H8, H9, HlO, HI l, H12, H13, H14 , H15 and H16.
  • the HA is derived from an Hl Nl influenza virus strain.
  • the HlNl influenza virus strain is selected from the group consisting of A/Ca]ifornia/04/09, A/New Caledonia/20/ 1999, A/Solomon ls/3/2006, and A/Brisbane/59/2007.
  • the VLPs further comprises at least one influenza matrix protein (Ml).
  • Ml protein is derived from an HlNl influenza virus strain.
  • Hl Nl influenza virus strain is selected from the group consisting of A/Califoraia/04/09, A/New Caledonia/20/ 1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.
  • the Ml is derived from an avian influenza virus strain.
  • the VLPs further comprise at least one influenza neuraminidase protein.
  • the NA protein is derived from an Hl Nl influenza virus strain.
  • the H 1 N 1 influenza virus strain is selected from the group consisting of A/California/04/09, A/New Caledonia/20/ 1999, A/Solomon ⁇ s/3/2006, and A/Brisbane/59/2007.
  • the VLPs further comprise at least one influenza M2 protein. In another embodiment, the VLPs further comprise at least one nucleocapsid (NP) protein.
  • NP nucleocapsid
  • the present invention also comprises methods of purifying said VLPs, wherein said methods comprise steps of :
  • VLPs further purifying VLPs by anion exchange chromatography, or isopycnic sucrose cushion centrifugation.
  • said anion exchange chromatography is a -(MAE anion exchange chromatography.
  • said discontinuous sucrose gradient is a 20% to 60% discontinuous sucrose gradient.
  • said isopycnic sucrose cushion centrifugation is a about 44% isopycnic sucrose cushion centrifugation.
  • the present invention also provides a method of using the purified HA protein and/or purified VLPs comprising a purified HA as a standard in a vaccine potency assay.
  • the potency assay has several applications, and may be useful for industrial firms, governmental institutions (e.g., N1H), non-profit organizations (e.g., WHO) and regulatory bodies (e.g., FDA).
  • the vaccine potency assay is a single radial immunodiffusion (SRID) assay.
  • a purified RA protein is administered to a host animal.
  • a purified VLl' comprising a purified HA is administered to a host animal.
  • both purified HA protein and purified VLPs comprising a purified HA are administered to a host animal.
  • the host animal mounts an immune response to the purified HA protein and/or purified VLP comprising a purified HA protein.
  • the host animal produces antibodies against the purified HA protein and/or purified VLP comprising a purified HA protein.
  • said antibodies are used as a standard in the single radial immunodiffusion (SRlD) assay.
  • the purified HA protein and/or purified VLPs comprising a purified HA are used as a standard in a direct challenge of a vaccinated subject with infectious vims.
  • the direct challenge of a vaccinated subject with infectious virus is evaluated with percentage of subjects showing infection symptoms, virus titer in the serum, or EDso-
  • the purified HA protein and/or purified VLPs comprising a purified HA are used as a standard in an immunoassay, wherein the immunoassay is selected from the group consisting of radioimmunoassay, enzyme immunoassay, fluorescence polarization immunoassay (FPlA), Kinetic Interaction of Microparticles in solution (KIMS), lateral flow technology, or T-cell responses assay.
  • the immunoassay is selected from the group consisting of radioimmunoassay, enzyme immunoassay, fluorescence polarization immunoassay (FPlA), Kinetic Interaction of Microparticles in solution (KIMS), lateral flow technology, or T-cell responses assay.
  • monoclonal and/or polyclonal antibodies are utilized in the immunoassay.
  • said enzyme immunoassay is selected from the group consisting of EMIT, Cloned Enzyme Donor Immunoassay (CEDI), and Enzyme- [.inked Immunosorbent
  • the invention provides immunogenic compositions comprising one or more purified HA proteins as described herein.
  • the invention provides a micelle comprised of one or more purified HA proteins (e.g. an HA micelle).
  • the purified HA proteins may be used for the prevention and/or treatment of influenza infection.
  • the invention provides a method for eliciting an immune response against influenza. The method involves administering an immunologically effective amount of a composition containing a purified HA protein to a subject, such as a human or animal subject.
  • the present invention provides pharmaceutically acceptable vaccine compositions comprising a purified HA protein, an HA micelle comprising a purified
  • HA protein or a VLP comprising a purified HA protein.
  • the invention comprises an immunogenic formulation comprising at least one effective dose of a purified HA protein. In another embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of an
  • the invention comprises an immunogenic formulation comprising at least one effective dose of a VLP comprising a purified HA protein.
  • the invention 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 invention provides a method of formulating a vaccine or antigenic composition that induces immunity to an infection or at least one disease symptom thereof to a mammal, comprising adding to the formulation an effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the infection is an influenza infection.
  • the purified HA proteins of the invention are useful for preparing compositions that stimulate an immune response that confers immunity or substantial immunity to infectious agents.
  • the invention provides a method of inducing immunity to infections or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention provides a method of inducing substantial immunity to an influenza vims infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • compositions of the invention can induce substantial immunity in a vertebrate (e.g. a human) when administered to the vertebrate.
  • a vertebrate e.g. a human
  • the invention provides a method of inducing substantial immunity to an influenza virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA proteinu an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention provides a method of vaccinating a mammal against influenza comprising administering to the mammal a protection-inducing amount of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention comprises a method of inducing a protective antibody response to an infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention comprises a method of inducing a protective cellular response to an influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein.
  • the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of an HA micelle comprising a purified HA protein.
  • the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a VLP, wherein the VXP comprises a purified HA protein.
  • Figure 1 depicts a TMAE chromatogram ( Figure Ia) of purifying HA derived from influenza B/Brisbane/60 and SDS-PAGE gel stained with Coomassie blue, loaded with fractions from the TMAE chromatography ( Figure 1 b).
  • Figure 2 depicts a lentil lectin chromatogram ( Figure 2a) of purifying HA derived from influenza B/Brisbane/60 and SDS PAGB gel stained with Coomassie blue, loaded with fractions from the lentil lectin chromatography ( Figure 2b)
  • Figure 3 depicts a CHTTM Hydroxyapatite chromatogram ( Figure 3a) of purifying
  • Figure 4 depicts SDS-PAGE gel analysis and Western Blot analysis of purified HA derived from influenza A/Califomia/04/09 with high purity (>97%). The yield was about 1 1 mg ' '1iter of cell culture.
  • Figure 5 depicts a vaccine potency assay using SRID.
  • baculovirus also known as baculoviridae, refers to a family of enveloped DNA viruses of arthropods, members of which may be used as expression vectors for producing recombinant proteins in insert cell cultures.
  • the virion contains one or more rod-shaped nucleocapsids containing a molecule of circular s ⁇ percoiled double-stranded DNA (Mr 54 x 10 6 - 154 x 10 6 ).
  • the virus used as a vector is generally
  • NTP Autographa californica nuclear polyhedrosis virus
  • the term "derived from” refers to the origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules.
  • the proteins and molecules of the present invention may be derived from influenza or non-influenza molecules.
  • 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
  • heterotypic refers to one or more different types or strains of virus.
  • homotypic refers to one type or strain of virus.
  • Macromolecular protein structure refers to the construction or arrangement of one or more proteins.
  • the term ''multivalent vaccine refers to a vaccine against multiple types or strains of influenza virus.
  • non-influenza refers to a protein or molecule that is not derived from influenza virus.
  • 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 present invention provides vaccine compositions that are immunogenic and provide protection.
  • die term “vaccine” also refers to a suspension or solution of an imm ⁇ nogen (e.g. VLP) that is administered to a vertebrate to produce protective immunity, i.e., immunity that reduces the severity of disease associated with infection.
  • the Venn "substantial immunity” refers to an immune response in which when VLPs of the invention are administered to a vertebrate there is an induction of the immune system in said vertebrate which results in the prevention of influenza infection, amelioration of influenza infection or reduction of at least one symptom related to influenza virus infection in said vertebrate.
  • Substantial immunity may also refer to a haemaggiutination inhibition (Hl) titer of ⁇ 40 in a mammal wherein the VLPs of the invention have been administered and have induced 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.
  • 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, 1L-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.
  • an "effective dose” generally refers to that amount of the VLP of the invention sufficient to induce immunity, to prevent and/or ameliorate influenza vims 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 scrum 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.
  • 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 immuniiy) emerges, spreading beyond individual localities, possibly around the globe, and infecting many humans at once.
  • influenza pandemic occurs when a new strain of influenza virus (a virus in which human have no natural immuniiy) emerges, spreading beyond individual localities, possibly around the globe, and infecting many humans at once.
  • seasonal influenza virus refers to the influenza viral strains that have been determined to be passing within the human population for a given influenza season based on epidemiological surveys conducted by National Influenza Centers worldwide. These epidemiological studies, and some isolated influenza viruses, are sent to one of four World Health Organization (WHO) reference laboratories, one of which is at the Centers for Disease Control and Prevention (CDC) in Atlanta for detailed testing. These laboratories test how well antibodies made to the current vaccine react to the circulating virus and new flu viruses. This information, along with information about flu activity, is summarized and presented to an advisory committee of the U.S. Food and Drug Administration (FDA) and at a WHO meeting.
  • WHO World Health Organization
  • substantially protective antibody response refers to an immune response mediated by antibodies against an influenza virus, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates influenza infection or reduces at least one symptom thereof.
  • VLPs of the invention can stimulate the production of antibodies that, for example, neutralizing antibodies that block influenza viruses from entering cells, blocks replication of said influenza virus by binding to the virus, and/or protect host cells from infection and destruction.
  • substantially protective cellular response refers to an immune response that is mediated by T-lymphocytes and/or other white blood cells against influenza virus, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates influenza infection or reduces at least one symptom thereof.
  • a vertebrate e.g., a human
  • One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTL”s).
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes.
  • MHC major histocompatibility complex
  • helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
  • a "cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
  • the term "substantial immunity in a population- wide basis” refers to immunity as a result of VLPs of the invention administered to individuals in a population.
  • the immunity in said individual in said population results in the prevention, amelioration of influenza infection, or reduction of at least one symptom related to influenza virus infection in said individual, and prevents the spread of said influenza virus to others in the population.
  • the term population is defined as group of individuals (e.g. schoolchildren, elderly, healthy individuals etc.) and may comprise a geographic area (e.g. specific cities, schools, neighborhoods, workplace, country, state, etc.).
  • 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.
  • 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 it. this definition. Both adult and newborn individuals are intended to be covered.
  • the present inventors have observed that prior art recombinant HA proteins, when administered to a host animal, fail to produce antibodies which recognize viral antigens in their native conformation (e.g. HA from the HlNl virus). In general, the failure of the prior art recombinant HA proteins can be attributed to the fact that they produced in a denatured form.
  • the present inventors have developed an improved purification process that is less denaturing (e.g. produces HA proteins that closely resemble their native conformation). When administered to an animal, the purified HA proteins stimulate the production of antibodies that recognize viral HA antigens in their native conformation.
  • the purified HA proteins produced by the methods described herein have a number of diagnostic, prophylactic, and therapeutic applications.
  • antibodies produced against the purified HA proteins are useful in potency assays such as the single radial immunodiffusion assay (SRlD).
  • the purified HA proteins of the present invention may be administered to a subject for the generation of an antibody response, and thus find utility in the treatment and prevention of influenza virus.
  • the present invention provides purified viral proteins.
  • said purified viral proteins are recombinant proteins or chimeric proteins.
  • said purified viral protein is a recombinant or chimeric HA protein of influenza virus.
  • the HA protein is a recombinant or chimeric protein derived from an influenza virus.
  • the influenza virus is selected from the group consisting of avian influenza virus and mammalian influenza virus.
  • the mammalian influenza virus is a human influenza virus strain.
  • the mammalian influenza virus is a swine influenza virus.
  • said HA protein may be derived from a pandemic strain or a seasonal strain of influenza virus.
  • the HA protein is selected from the group consisting of HI , m, H3, H4, HS 5 H6, H7 H8, H9, HlO, HI 1, H 12, H 13, H 14, H 15, and H 16.
  • the HA is derived from an Hl N 1 influenza virus strain.
  • the HlNl influenza virus strain is selected from the group consisting of A/California/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.
  • the present invention comprises purified recombinant or chimeric influenza HA with high purity.
  • the purity of said recombinant or chimeric influenza HA is at least about 60%, about 70%, about 80%, about 90%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%.
  • the HA proteins of the present invention are found in the form of micelles (e.g. rosettes).
  • the micelles obtainable in accordance with the invention consist of aggregates of the immunogenically active HA proteins having a rosette-like structure.
  • the rosettes are visible in the electron microscope.
  • the micelles are generally comprised of 20- 1 (M) HA trimers ' particle.
  • the particle size of the micelle range from 20-40 nanometers (nm) in length.
  • the HA micelles of the present invention are useful for stimulating an immune response both in vitro and in vivo.
  • the present invention provides a method of purifying a viral protein.
  • the viral protein is an HA protein.
  • the method of purifying the HA protein comprises the steps of:
  • step (iv) Purifying said HA from said pool in step (iii) using affinity chromatography, wherein a .second pool of fractions containing said HA is produced;
  • the HA protein is selected from the group consisting of Hl , H2, H3, H4, H5, H6, H,7 H8, H9, H 10, H 11 H12, H 13, H 14 , H I S and H 16.
  • the HA is derived from an Hl Nl influenza virus strain.
  • the H1N1 influenza virus strain is selected from the group consisting of A/CaKfornia/04/09, A/New Caledonia/20/1999, A/Solomon ls/3/200 ⁇ , and
  • the fractions el ⁇ ted from columns in steps (iii), (Iv), (vi) are analyzed with SDS-PAGE gel stained by Coomassie blue and Western Blot by antibody against said viral protein, respectively, to determine fractions contain majority of said viral protein, wherein said fractions are subsequently combined together to form a pool.
  • yield of said purified viral protein can be sacrificed in a reasonable range.
  • the purity of finally purified viral protein is at least about 60%, about 70%, about 80%, about 90%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%. In an exemplary embodiment, the purity of said viral protein is at least about 95%.
  • Ion Exchange Chromatography relies on charge-charge interactions between the proteins in your sample and the charges immobilized on the resin of your choice. Ion exchange chromatography can be subdivided into cation exchange chromatography, in which positively charged ions bind to a negatively charged resin; and anion exchange chromatography, in which the binding ions are negative, and the immobilized functional group is positive. Once the solutes are bound, the column is washed to equilibrate it in your starting buffer, which should be of low ionic strength, then the bound molecules are eluted off using a gradient of a second buffer which steadily increases the ionic strength of the elute solution.
  • the pH of the elute buffer can be modified as to give your protein or the matrix a charge at which they will not interact and your molecule of interest elutes from the resin.
  • protein sequence can be inputted into software to predict the charge status of the protein. If it is negatively charged at the pH, use an anion exchanger; if it is positive, use a cation exchanger. In many cases it may be more advantageous to actually select conditions at which the protein will flow through while the contaminants will bind. This mode of binding is often referred to as "flow through mode". Tins is a particularly good mode to use in the case of anion exchange.
  • Non-limiting examples of ion exchanger resins used for packing columns are, Q-resin, Di Ethyl AminoEthane (DEAE) resin, dimethylaminoethyl (DMAE) resin, Trimethylaminoethyl (TMAE) resin, SOf resin, COO ⁇ resin, and those described in U.S. Patent Nos.
  • said ion exchange chromatography in step (iii) is an anion exchange chromatography (AEC), wherein the surface charge of the solutes (proteins, nucleic acids, endotoxin) which bind will be net negative, thus to get binding of a specific protein one should be above the pi of that protein.
  • AEC anion exchange chromatography
  • Common anion exchange resins used to pack anion exchange columns are Q-resin (a Quaternary amine), DEAE resin (DiEthylAminoEthane), dimethylamraoethyl (DMAE) resin, Trimethylaminoethyl (TMAB) resin, SCV resin, and COO " resin.
  • Anion exchange chromatography is often used as a primary chromatography step due to its high capacity, (Matrices can bind from 10 to 100 mg of protein per ml) and ability to bind up and separate fragmented nucleic acids and lipopolysaccharides from the initial slurry.
  • anion exchange chromatography is performed using buffers at a pH between 7 and 10 and running a gradient from a solution containing just this buffer to a solution containing this buffer with NaCl. The salt in the solution competes for binding to the immobilized matrix and releases the protein from its bound state at a given concentration. Proteins separate because the amount of salt needed to compete varies with the external charge of the protein.
  • Uses of anion exchange chromatography include initial clean up of a crude slurry, separation of proteins from each other, concentrating a protein, and the removal of negatively charged endotoxin from protein preparations.
  • the ion exchange chromatography in said step (iii) uses a anion exchange column packed with Trimethylaminoethyl (TMAE) anion exchanger resins (e.g., Fractoprep® EMD TMAE resins).
  • TMAE Trimethylaminoethyl
  • Affinity chromatography is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.
  • Affinity chromatography combines the size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. Due to its interdisciplinary' nature, affinity chromatography has been the means by which many scientists from different disciplines have been introduced to the exciting fields of modem biology.
  • Affinity chromatography can be used to purify and concentrate a molecule from a mixture into a buffering solution, reduce the amount of a molecule in a mixture, discern what biological compounds bind to a particular molecule, such as drugs, and purify and concentrate an enzyme solution.
  • affinity chromatography usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum.
  • the molecule of interest w ⁇ l have a well known and defined property which can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in solution will not become trapped as they do not possess this property.
  • the solid medium can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Binding to the solid phase may be achieved by column chromatography, whereby the solid medium is packed onto a chromatography column, the initial mixture run through the column to allow binding, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure (as opposed to HPLC or FPLC).
  • binding may be achieved using a batch treatment, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase (by eentrirugation for example), removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the eluate.
  • a hybrid method is employed, the binding is done by the batch method, then the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.
  • a third method, expanded bed adsorption which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top.
  • Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and antibody purification from blood serum.
  • the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are tagged in order to aid their purification. The protein may have been genetically modified so as to allow it to be selected for affinity binding, this is known as a fusion protein.
  • Non-limiting exemplary tags include His-tags and GST (glutathione-S-transferase) tags.
  • affinity chromatography is, antibody affinity chromatography, immobilized metal ion affinity chromatography, and lectin affinity chromatography.
  • said affinity chromatography in step (iv) in the purification method mentioned above is lectin affinity chromatography ⁇ e.g., Lentil lectin Sepharose 4B chromatography, GH).
  • Solution containing soluble protein needs to be desalted before it is loaded on certain type of column (e.g., hydroxyapatite column).
  • certain type of column e.g., hydroxyapatite column.
  • desalting methods are, desalting column, desalting tips, and dialysis.
  • said pool containing viral protein is desalted it by passing through a desalting column, a desalting tip, or by dialysis.
  • a desalting column can be selected from the group consisting of Pierce desalting column, HttrapTM Desalting Column (GE Health), HiPrepTM Desalting Column (GE Health), PD-10 Desalting column (GE Health), and Uptima desalting column.
  • said desalting column is packed with SephadexTM G-25.
  • Dialysis is one type of diffusion, or osmosis. It allows the passage of small molecules such as salt but not larger ones such as protein to pass through a semi or partially permeable membrane bag or tubing made from regenerated cellulose or cellophane. It can be used in clinical circumstances to ensure a filtered flow of molecules, preventing the flow of larger solute molecules, or used in purification field to desalt. Small molecules such as salt can be washed out of a solution which is contained in a bag or pumped through the tubing into a solvent, usually water, which surrounds the bag or tubing and in which small molecules can be flushed away.
  • a solvent usually water
  • the sealed dialysis bag is placed in a container of a different exchange solution, or water.
  • Molecules small enough to pass through the tubing water, salts, monosaccharides, and other small molecules
  • Larger molecules such as proteins, or polysaccharides
  • a hypotonic solution crosses the semipermeable membrane into the hypertonic solution in an attempt to reach equilibrium.
  • the exchange solution in the container can be replaced for several times to reach desired desalting goal.
  • a semi or partial permeable membrane also termed selectively permeable membrane, or differentially permeable membrane, is a membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized facilitated diffusion.
  • the rate of passage depends on the pressure, concentration, and temperature of the molecules or solutes on either side, as well as the permeability of the membrane to each solute.
  • permeability may depend on solute size, solubility, properties, or chemistry.
  • Non-limiting exemplar ⁇ ' semi-permeable membranes are, thin film composite membranes (TFC or TFM), cellulose ester membrane (CEM), charge mosaic membrane (CMM), bipolar membrane (BPM), anion exchange membrane (AEM), alkali anion exchange membrane (AAEM) and proton exchange membrane (PEM).
  • TFC or TFM thin film composite membranes
  • CEM cellulose ester membrane
  • CCM charge mosaic membrane
  • BPM bipolar membrane
  • AEM anion exchange membrane
  • AAEM alkali anion exchange membrane
  • PEM proton exchange membrane
  • Selectively permeable membrane can be classified by a perimeter called molecular cut-off (MWCO), which refers to the molecular weight of the biggest molecule that can pass through the membrane.
  • MWCO molecular cut-off
  • dialysis bag, tubing, cassette or a stirred cell with selectively permeable membrane can be used to desalt a pool containing viral protein to be purified in step (v) mentioned above.
  • said dialysis bag, tubing, cassette, or stilted cell comprises a selective permeable membrane having a MVVCO of about 5 kD, about 1OkD, about 15 kD, about 20 kD > about 25 kD, about 30 kD, about 35kD, about 35kD, or about 4OkD.
  • the bag/tubing comprises a selective permeable membrane having a MWCO of about 20 to about 30 kD (e.g., slide-A-lyzer dialysis cassette or Ami con Stir Cells).
  • Hydroxyapatite also called hydroxyapatite, is a naturally occurring mineral form of calcium apatite with the formula Ca S (POt) 3 (OH) (a.k.a. Ca 10 (P(M) 6 (OH) 2 ). Hydroxyapatite is the hydroxyl end member of the complex apatite group. The OH- ion can be replaced by fluoride, chloride or carbonate. It crystallizes in the hexagonal crystal system.
  • Hydroxyapatite can be used in chromatography for purification.
  • the mechanism of hydroxyapatite chromatography is complicated and has been described as "mixed-mode" ion exchange. It involves nonspecific interactions between positively charged calcium ions and negatively charged phosphate ions on the stationary phase hydroxyapatite resin with protein negatively charged carboxyl groups and positively charged amino groups. It may be difficult to predict the effectiveness of hydroxyapatite chromatography based on physical and chemical properties of the desired protein to be purified. For elution, a buffer with increasing phosphate concentration is typically used.
  • Non-limiting exemplary hydroxyapatite that can be used to pack column for chromatography are, natural hydroxyapatite and synthetic hydroxyapatite (e.g., crystalline hydroxyapatite, ceramic hydroxyapatite).
  • solution containing desired viral protein can be further purified in step (iv) mentioned above by a hydroxyapatite column, wherein said hydroxyapatite column is packed with natural hydroxyapatite or synthetic hydroxyapatite, wherein the synthetic hydroxyapatite is crystalline hydroxyapatite or ceramic hydroxyapatite.
  • the column is packed with spherical, macroporous form of CHTTM hydroxyapatite, Type I (ceramic hydroxyapatite).
  • the present invention further comprises method of concentrating, dialyzing and sterilizing viral protein product purified in above mentioned purification method.
  • the viral protein product is further concentrated and dialyzed using a stirred cell with selectively permeable membrane as described above.
  • the viral protein product is concentrated and dialyzed using a Millipore Stirred Cell with selectively permeable membrane against DPBS buffer.
  • the selective membrane has a MWCO is about 30 KD.
  • a minimum of three additions of 10-fold buffer volume is needed for dialysis.
  • Sterile Filtration is performed using a micron filter (e.g., a 0.2 micron SFCA (or PVDF) filter).
  • virus-like particles lack a viral genome and, therefore, are noninfectious.
  • virus-like particles can often be produced by heterologous expression and can be easily purified.
  • Most VLPs comprise at least a viral core protein. This core protein usually drive* budding and release of particles from a host cell. Examples of such proteins comprise RSV M, influenza Ml, HIV gag and vesicular stomatis virus (VSV) M protein.
  • VLPs are useful for preparing antigenic formulation and/or vaccines against infectious agents, e.g. influenza.
  • HA hemagglutinin
  • NA neuraminidase
  • Wild birds are the primary natural reservoir for all types of influenza A viruses and are thought to be the source of all types of influenza A viruses in all other vertebrates. These subtypes differ because of changes in the hemagglutinin (HA) and neuraminidase (NA) on their surface. Many different combinations of HA and NA proteins are possible. Each combination represents a different type of influenza A virus. In addition, each type can be further classified into strains based on different mutations found in each of its 8 genes.
  • the present invention describes the production of influenza vaccine candidates or reagents comprised of influenza proteins that self-assemble into functional VLPs. AU combinations of viral proteins must be co-expressed with a matrix protein 1 (Ml).
  • Ml matrix protein 1
  • the invention described herein provides VLPs comprising a purified influenza HA protein.
  • the purity of the HA protein is at least about 95%.
  • the HA protein is a recombinant or chimeric protein derived from an influenza virus.
  • the influenza virus is selected from the group consisting of avian influenza virus and mammalian influenza virus.
  • the mammalian influenza virus is a human influenza virus strain.
  • the mammalian influenza virus is a swine influenza virus.
  • said HA protein may be derived from a pandemic strain or a seasonal strain of influenza virus.
  • the HA protein is selected from the group consisting of Hl, H2, H3, H4, HS, 116, H7 H8, H9, HlO, HI l, H 12, H 13, H 14, H 15, and H 16.
  • the HA is derived from an HlN 1 influenza virus strain. Jn one embodiment, the HlNl influenza virus strain is selected from the group consisting of A/California/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.
  • the VLPs further comprises at least one influenza matrix protein (Ml).
  • Ml influenza matrix protein
  • the Ml protein is derived from an H lNl influenza virus strain.
  • the HlNl influenza virus strain is selected from the group consisting of A/Califomia/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.
  • the Ml protein is derived from an avian influenza virus strain.
  • the VLPs further comprise an influenza NA protein.
  • said NA is derived from an avian or mammalian influenza virus, and is selected from the group consisting of Nl , N2, N3, N4, N5, N6, N7, N8 and N9.
  • the NA protein is derived from an HlNl influenza virus strain.
  • the H lNl influenza virus strain is selected from the group consisting of A/California/04/09, A/New CaIedonia/20/1999, A/Solomon ls/3/2006, and A/Brisbane/59/2007.
  • the invention comprises VLPs that consist essentially of HA, NA and Ml.
  • Said HA and NA can be from the above list of HA and NA.
  • These VLPs may comprise additional influenza proteins and/or protein contaminates in negligible concentrations.
  • These VLPs contain HA, NA and Ml and may contain additional cellular constituents such as cellular proteins, baculovirus proteins, lipids, carbohydrates etc., but do not contain additional influenza proteins (other than fragments of Ml, HA and/or NA).
  • the HA and/or the NA may exhibit hemagglutinin activity and/or neuraminidase activity, respectively, when expressed on the surface of VLPs.
  • the invention also encompasses variants of the said influenza 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 polypeptide refers to an amino acid sequence that is altered by one or more amino acids wilh 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 ait, for example, DNASTAR software.
  • Natural variants can occur due to antigenic drifts. Antigenic drifts are small changes in the viral proteins that happen continually over time. Thus, a person infected with a particular flu virus 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. This is why there is a new vaccine for influenza each season.
  • some changes in an influenza virus can cause influenza virus to cross species. For example, some avian influenza viruses developed genetic variations associated with the capability of crossing the species barrier. Such a virus is capable of causing a pandemic because people have no natural immunity to the virus and the virus can easily spread from person to person. These naturally occurring variations of the influenza proteins are an embodiment of the invention.
  • the invention also encompasses using known methods of protein engineering and recombinant DNA technology to improve or alter the characteristics of the influenza proteins expressed on or in the VLPs of the invention.
  • Various types of mutagenesis can be used to produce and/or isolate variant HA, NA and/or Ml molecules and/or to further modify/mutate the polypeptides 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. In one embodiment, 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.
  • influenza gene encoding a specific influenza protein can be isolated by RT-PCR from polyadenylated mRNA extracted from cells which had been infected with an influenza 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 ⁇ v ⁇ ru9es, 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 also provides for methods of producing VLPs, said methods comprising expressing an avian, pandemic and/or seasonal influenza proteins under conditions that allow VLP formation.
  • the 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.
  • VLPs can expressed in eukaryotic cells and/or prokaryotic cells.
  • eukaryotic host cells are yeast, insect, avian, plant. C. elegam (or nematode), and mammalian host cells.
  • Non limiting examples of insect cells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21, Trichoplusia ni cells, e.g. High Five cells, and Drosophila S2 cells.
  • yeast yeast
  • yeast yeast
  • insect avian
  • C. elegam or nematode
  • mammalian host cells are examples of insect cells.
  • Spodoptera frugiperda (Sf) cells e.g. Sf9, Sf21, Trichoplusia ni cells, e.g. High Five cells
  • Drosophila S2 cells examples of fungi (including yeast) host cells are S. cerevisiae, Kluyreromyces lact
  • mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (MEK) cells, and African green monkey cells.
  • CVI 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. suhtilis, and mycobacteria.
  • a preferred host cell is a SrP insect cell.
  • 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 fe ⁇ nentation chamber) where cells propagate and express protein (e.g. recombinant proteins) for purification and isolation.
  • protein e.g. recombinant proteins
  • cell culture is performed under sterile, controlled ternperaiure 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 bags.
  • VLPs are then isolated using methods that preserve the integrity thereof, such as by gradient centrif ⁇ gation, e.g., cesium chloride, sucrose and iodixanol, as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • gradient centrif ⁇ gation e.g., cesium chloride, sucrose and iodixanol
  • standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • VLPs of the invention can be made, isolated and purified.
  • VLPs are produced from recombinant cell lines engineered to create a VLP when said cells are grown in cell culture (see above). Production of VLPs may be accomplished as illustrated in Examples. A person of skill in the art would understand that there are additional methods that can be utilized to make and purify VLPs of the invention, thus the invention is not limited to the method described.
  • 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).
  • influenza HA, NA and Ml proteins are expressed from the virus genome, self assemble into VLPs and are secreted from the cells approximately 24 to 72 hours post infection. Usually, infection is most efficient when the cells are in mid-log phase of growth (4-8 * IO 6 cells/ml) and are at least about 90% viable.
  • the present invention also comprises methods of purifying VLPs, wherein the purified VLPs have high purity and high potency as vaccine.
  • Said methods of purifying VLPs comprises steps consisting of:
  • the anion exchange chromatography in step b is a TMAE anion exchange chromatography
  • VLPs of trie 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 0.5x H) 6 cells/ml to about 1.5 x K) 6 cell.s/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 ⁇ rn filter cartridge or a similar device.
  • TMF tangential flow filtration
  • 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 furthered purified on a 20% to 60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaCl by centrifugarion at 6,500 x g for 18 hours at about 4° C to about 10° 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,
  • VLPs Further purification of VLPs can be achieved by anion exchange chromatography, or 44% isopycnic sucrose cushion centrifugation.
  • 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) and eluded via a salt gradient (from about 0.2 M to about 1.0 M of NaCl) that can separate the VLP from other contaminates (e.g. baculovirus and DNA/R.NA).
  • the sucrose cushion method the sample comprising the VLPs is added to a 44% sucrose cushion and centrif ⁇ ged 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 °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 diaf ⁇ ltration 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 tecliniques 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.
  • said VLPs comprise more than one protein from an infectious agent.
  • said VLPs are multivariant VLPs capable of inducing an immune response to several proteins from infectious agents.
  • said VLPs comprise proteins from at least two different influenza viruses.
  • said multivariant VLPs can comprise a HA and/or NA from a seasonal influenza virus A and/or B and/or from an avian influenza virus.
  • This embodiment also comprises the presentation of HA and/or NA of the three influenza viruses (two subtypes of influenza A viruses and one influenza B virus) that are chosen by WHO and the CDC to be in the flu vaccines for the fall and winter in a single VLP.
  • said multivariant VLPs comprise proteins from several viruses, bacteria and/or parasites.
  • said VLPs comprise proteins from influenza and RSV, influenza, RSV and parainfluenza.
  • said chimeric proteins comprise a fusion between the influenza HA with the protein, or a portion thereof, from an infectious agent.
  • said chimeric proteins comprise a fusion between the proteins, or a portion thereof, of two infectious agents or antigenic variations of the same agent.
  • Said fusion protein will comprise antigenic agents from each protein from said infectious agent.
  • said chimeric protein comprises an amino acid linker between the proteins.
  • An example of this embodiment is a fusion between the influenza HA and the RSV F protein.
  • a protein that may be expressed on the surface of chimeric VLPs of the invention can be derived from viruses, bacteria, rungi and/or parasites.
  • the proteins expressed on the surface of said VLPs may be tumor or cancer antigens.
  • the proteins derived from viruses, bacteria, fungi and/or parasites can induce an immune response (cellular and/or humoral) in a vertebrate that which will prevent, treat, manage and/or ameliorate an infectious disease in said vertebrate.
  • Non-limiting examples of viruses from which said infectious agent proteins can be derived from are the following: Orthopoxvirus (e.g., Cowpoxvirus, Monkeypox virus, Vaccinia virus. Variola virus, Parapoxvirus, Bovine papular stomatitis vims, Orf virus, Pseudocowpox virus, MoHuscipoxvims, Molluscum contagiosuin virus, Yatapoxvirus, Tanapox virus, Yaba monkey tumor virus), Simplexvirus (e.g., Human herpesvirus 1 (Herpes simplex virus 1), Human herpesvirus 2 (Herpes simplex virus 2), Human herpesvirus 3 (Varicella-zoster vims)), Cytomegalovirus (e.g.
  • Orthopoxvirus e.g., Cowpoxvirus, Monkeypox virus, Vaccinia virus. Variola virus, Parapoxvirus, Bovine papular stomatitis vims
  • Human herpesvirus 5 Human cytomegalovirus
  • Roseolvirus e.g., Human herpesvirus 6 > Human herpesvirus 7
  • Lymphocryptovirus e.g., Huam herpesvirus 4
  • Rhadinovirus e.g., Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus)
  • Mastadenovirus e.g., Human adenovirus A, Human adenovirus B, Human adenovirus C. Human adenovirus D, Human adenovirus E, Human adenovirus F
  • Polyomavirus e.g. BK polyomavirus.
  • Human polyomavirus JC polyomavirus
  • Alphapapillomavirus e.g., Human papillomavirus 2, Human papillomavirus 10, Human papillomavirus 6, Human papillomavirus 7, Human papillomavirus 16, Human papillomavirus 18, Human papillomavirus 26, Human papillomavirus 32, Human papillomavirus 34, Human papillomavirus 53, Human papillomavirus 54, Human papillomavirus 61, Human papillomavirus 71 , Human papillomavirus cand90), Betapapillomavir ⁇ s (e.g., Human papillomavirus 2, Human papillomavirus 10, Human papillomavirus 6, Human papillomavirus 7, Human papillomavirus 16, Human papillomavirus 18, Human papillomavirus 26, Human papillomavirus 32, Human
  • Human papillomavirus 5 Human papillomavirus 9, Human papillomavirus 49, Human papillomavirus cand92, Human papillomavirus cand96.
  • Gammapapillomavirus e.g., Human papillomavirus 4, Human papillomavirus 48, Human papillomavirus 50, Human papillomavirus 60, Human papillomavirus 88
  • Mupapillomavirus e.g..
  • Human papillomavirus 1 Human papillomavirus 1 , Human papillomavirus 63 ),Erythro virus (e.g., BI 9 virus), Orthohepadnavirus (e.g., Hepatitis B virus), Deltaretrovirus (e.g., Primate T-lymphotropic virus 1, Primate T-lymphotropic virus 2), Lentivirus (e.g., Human immunodeficiency vims 1 , Human immunodeficiency virus 2), Orthoreovirus (e.g., Mammalian orthore ⁇ virus), Orbivirus (e.g... African horse sickness virus, Changuinola virus, Corriparta virus, Orungo virus).
  • Orthohepadnavirus e.g., Hepatitis B virus
  • Deltaretrovirus e.g., Primate T-lymphotropic virus 1, Primate T-lymphotropic virus 2
  • Lentivirus e.g., Human immunodeficiency vims 1
  • Rotavirus e.g., Rotavirus A, Rotavirus B
  • Marburgvirus e.g., Lake Victoria marburgvirus
  • Ebolvirus e.g.. Ivory Coast ebolavirus, Resto ⁇ ebolavirus, Sudan ebolavirus, Zaire ebolavirus
  • Respirovirus e.g., Human parainfluenza virus 1 , Human parainfluenza virus 3
  • Morbillivirus e.g., Measles virus (Edmonston virus)
  • Rubulavirus e.g., Human parainfluenza virus 2, Human parainfluenza virus 4, Mumps virus
  • Henipavirus e.g.,
  • Hendravirus e.g., Nipahvirus
  • Pneumovirus e.g., Human respiratory syncytial virus
  • Metapneumovirus e.g., Human metapneurnovirus
  • Vesiculovirus e.g., Chandipura virus
  • Influenzavirus A e.g., Influenza A virus
  • Influenzavirus B e.g., Influenza B virus
  • Influenzavirus C e.g., Influenza C virus
  • Bunyavirus e.g., Bunyamwera virus
  • Bwamba virus California encephalitis virus, Guama virus, Oriboca virus, Oropouche virus,
  • Phlebovirus e.g., Rift
  • Arenavirus e.g., JLassa virus, Lymphocytic choriomeningitis virus, Guanarito vims, Junin virus, Machupo virus, Sabia virus
  • Deltavirus e.g., JLassa virus, Lymphocytic choriomeningitis virus, Guanarito vims, Junin virus, Machupo virus, Sabia virus
  • Coronavirus e.g., Human coronavirus 229E, Human coronavirus
  • OC43 Human enteric coronavirus, Severe acute respiratory syndrom coronavirus
  • Torovims Enterovirus (e.g., Human enterovirus A, Human enterovirus B, Human enterovirus
  • Human enterovirus D Poliovirus
  • Rhinovirus e.g., Human rhinovirus A, Human rhinovirus B
  • Hepatovims e.g., Hepatitis A virus
  • Parechovirus e.g., Human parechovirus
  • Norwalk virus e.g., Sapovirus
  • Sapporo virus Hepatitis E virus
  • Mamastrovirus e.g., Human astrovims
  • Alphavims e.g., Chikungunya virus, O'nyong- nyong virus, Mayaro virus, Ross River vims, Barmah Forest virus, Sindbis vims, Ockelbo virus, Venezuelan equine encephalitis vims, Western equine encephalitis virus, Eastern equine encephalitis vims
  • Rubivirus e.g., Rubella virus
  • Flavivirus e.g., Kyasanur Forest disease vims, Omsk hemorrhagic fever virus, Powassan virus, I ⁇ uping ill virus, Tick-borne encephalitis virus, Dengue virus, Japanese encephalitis vims, Murray Valley encephalitis virus, St.
  • Non-limiting examples of bacteria from which said infectious agent proteins can be derived from are the following: B. pertussis, Leptospira pomona, S. paratyphi A and B, C. diphtheriae, C. tetani, C. botulimim, C, perfnngens, C. feseri and other gas gangrene bacteria, ⁇ . anthracis, P, pestis, P. multocida, Neisseria meningitidis, N.
  • gonorrhea Hemophilus influenzae, Actinomyces ⁇ e.g., Norcardia), Acinetobacter, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e .g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Campylobacter, Chlamydia, Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), E. coli (e.g., Enterotoxigenic B. coli and Enterohemorrhagic E. coli), Enterobacter (e.g.
  • Enterobacter aerogems Enterobacter aerogems
  • Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella), ErysipeJothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis).
  • Salmonella e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella), ErysipeJothrix
  • Haemophilus e.g., Haemophilus influenza type B
  • Helicobacter Legionella (
  • Vibrio e.g., Vibrio cholerae
  • Paste ⁇ rellacea Proteus
  • Pseudomonas e.g., Psetidomonas aeruginosa
  • Rickettsiaceae Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp.
  • Meningococcus, Pneumococcus and Streptococcus e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci
  • Ureaplasmas e.g., Vibrio cholerae
  • Paste ⁇ rellacea Proteus
  • Pseudomonas e.g., Psetidomonas aeruginosa
  • Rickettsiaceae e.g., Spirochetes (e.
  • Non-limiting examples of parasites from which said infectious agent proteins can be derived from are the following: leishmaniasis (teishmania tropica mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, i ⁇ iishmania chagasi), trypanosomiasis (Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense), toxoplasmosis (Toxoplasma gondii) , schistosomiasis (Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma inter calatum), malaria (Plasmodium virax, Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale) Am
  • Non-limiting examples of fungi from which said glycoproteins can be derived are from the following: Absidia (e.g. Absidia corymbifera), Ajcllomyces (e.g. Ajellomyces capsulatus, Ajellomyces dermatilidis), Arthroderma (e.g. Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gyjxseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii), Aspergillus (e.g. Aspergillus ⁇ tmigatus, Aspergillus niger), Candida (e.g. Candida albicans, Candida albicans var.
  • Absidia e.g. Absidia corymbifera
  • Ajcllomyces e.g. Ajellomyces capsulatus, Ajellomyces dermatilidis
  • Arthroderma e.g. Arthroderma benhamia
  • Candida stellatoidea Candida dublinensis, Candida glabrata, Candida guilliermondii (Pichia guilliermondii), Candida lcrusei (Issatschenkia orientalis), Candida parapsilosis, Candida pelliculosa (Pichia anomala), Candida tropicalis, Coccidioides ⁇ e.g. Coccidi ⁇ ides immitis), Cryptococcus (e.g. Cryptococcus neoformans (Filobasidiella neoformans), Histoplasma (e.g. Histoplasma capsulatum (Ajellomyces capsidalus), Microsporum (e.g.
  • Microsporum canls ( ⁇ rthroderma otae), Microsporum fttlvurn (Arthroderma fulvum), Microsporum gypseum, Genus Pichia (e.g. Pichia anomala, Pichia guilliermondi ⁇ ), Pneumocystis (e.g. Pneumocystis jirovecii), Cryptosporidium, Malassezia furfur, Paracoccidiodes.
  • the present invention provides purified viral protein and/or purified VLPs that can be used as a standard in a vaccine potency assay.
  • said viral protein is a recombinant viral protein or a chimeric viral protein.
  • said purified recombinant viral protein is a recombinant or chimeric HA.
  • said recombinant or chimeric HA is selected from the group consisting of Hl, H2, H3, H4, H5, H ⁇ , 11,7 H8, H9, H10, HI l, H 12, H 13, H14 , H15 and H 16.
  • said VLPs comprises or essentially comprises an influenza HA, an matrix protein, and optionally an influenza NA.
  • said purified recombinant or chimeric HA has a purity of at least about 95%.
  • said purified recombinant or chimeric HA and/or said purified VLl 3 S can be used in a potency assay of a vaccine produced by industry, governmental institutions (e.g., N1H), no-profit organizations (e.g., WHO) or regulatory bodies (e.g., FDA).
  • Commonly used vaccine potency assays include, but are not limited to, direct challenge of vaccinated subject with infectious virus (e.g., ED 50 (vaccine dosage capable of protecting 50% of the population)), immunoassay (e.g., radio-linked immunoassay, enzyme- linked immunoassay), and single radio immunodiffusion assay.
  • infectious virus e.g., ED 50 (vaccine dosage capable of protecting 50% of the population)
  • immunoassay e.g., radio-linked immunoassay, enzyme- linked immunoassay
  • single radio immunodiffusion assay e.g., single radio immunodiffusion assay.
  • Methods of direct challenge of vaccinated subject with infectious virus are known in the art.
  • groups of subjects e.g., vertebrates
  • different dosages of said vaccines to be tested and said standard vaccine can be tested in one comparison test.
  • said administrating can be intravenous, intraarterial, intramuscular, intracerebral, intracerebroventricular, intracardiac, subcutaneous, intraosseous infusion, intradermal, intrathecal, intraperitoneal, intravesical, intracavernosal, intranasal, transdermal, transmucosal, inhalational, intracisternal, epidural, or intravitreal.
  • Groups of subjects e.g, vertebrates, such as humans
  • a second round immunization is performed to boost immune responses in the subjects.
  • Said groups of subjects are then challenged with infectious virus, and the effects are analyzed after a certain period of time.
  • results can be evaluated in various ways.
  • percentage of subjects showing infection symptoms can be compared between said vaccine to be tested and said standard vaccine.
  • virus titer in the serum can be compared between subjects immunized with said vaccine to be tested and said standard vaccine.
  • ED 50 (vaccine dosage capable of protecting 50% of the population) can be compared between said vaccine to be tested and said standard vaccine.
  • Immunoassays are well known to one skilled in the art. Immunoassays are a diverse group of analytical techniques based on specific antibody/antigen interactions producing a measurable signal that can be related to the concentration of a compound in solution.
  • the antibody is monoclonal antibody.
  • the antibody is polyclonal antibody.
  • said immunoassays are competitive immunoassays.
  • said immunoassays are non-competitive immunoassays.
  • said immunoassays are heterogeneous assays wherein separation of bound and unbound components is required after the reaction has taken place (e.g., ELlSA).
  • said immunoassays are homogeneous assays wherein the binding reaction is measured in place without the separation of reaction components.
  • immunoassays include, but are not limited to, radioimmunoassay (wherein one or more radioactive labels such as I !2$ , H 3 , C 14 is utilized to emits radiation measured with a beta or gammacounter), enzyme immunoassay (wherein one or more enzymes are used to provide a measurable signal, e.g., glucose-6-phosphate dehydrogenase or ⁇ -galactosidase), Enzyme Linked Immunosorbent Spot (ELISI 5 OT) assay, fluorescence polarization immunoassay (FPIA), Kinetic Interaction of Microparticles in solution (KIMS), and lateral flow technology.
  • radioimmunoassay wherein one or more radioactive labels such as I !2$ , H 3 , C 14 is utilized to emits radiation measured with a beta or gamma
  • Non-limiting examples of said enzyme immunoavssays are EMIT, Cloned Enzyme Donor Immunoassay (CEDl), and Enzyme-Linked Immunosorbent Assay (ELlSA). More detailed infoi ⁇ nation of immunoassays is described in David Wild, The immunoassay handbook, 2005, 3 rd Edition, Gulf Professional Publishing, ISBN 0080445268, 9780080445267.
  • serum of groups of subjects immunized with a vaccine to be test, at least one standard vaccine, and optionally at least one negative control respectively are analyzed in one or more immunoassay to decide the potency of said vaccine to be tested compared to one or more Standard vaccine.
  • responses of T-cells expressing antibody in immunized subjects can be compared to determine vaccine potency using Fluorescence-Activated Cell Sorter (FACS).
  • FACS Fluorescence-Activated Cell Sorter
  • Single radial immunodiffusion is used extensively for the quantitative estimation of antigens.
  • the antigen-antibody precipitation is made more sensitive by the incorporation of antiserum in the agarose.
  • Antigen (Ag) is then allowed to diffuse from wells cut in the gel in which the antiserum is uniformly distributed. Initially, as the antigen diffuses out of the well, its concentration is relatively high and soluble antigen-antibody adducts are formed. However, as Ag diffuses farther from the well the Ag-Ab complex reacts with more amount of antibody resulting in a lattice that precipitates to form a precipitin ring.
  • a calibration graph is plotted. Antigen concentrations of unknown samples, run on the same gel can be found by measuring the diameter of precipitin rings and extrapolating this value on the calibration graph.
  • serum of groups of subjects immunized with a vaccine to be test, at least one standard vaccine, and optionally at least one negative control respectively are analyzed in a SRlD assay to decide the potency of said vaccine to be tested compared to one or more standard vaccine.
  • 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 purified HA protein, an HA micelle comprising a purified HA protein, or a VLl' comprising a purified HA protein of the invention.
  • 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 purified HA protein, an HA micelle comprising a purified HA protein, or a VLl' comprising a purified HA protein of the invention.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pha ⁇ nacopia, European Pharmacopia or other generally recognized pharma
  • the invention also encompasses a pharmaceutically acceptable vaccine composition comprising a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein as described above.
  • a kit for immunizing a vertebrate such as a human subject comprising a purified HA protein, an HA micelle comprising a purified HA protein, OT a VLP comprising a purified HA protein as described above.
  • the invention comprises an immunogenic formulation comprising at least one effective dose of a purified HA protein.
  • the invention comprises an immunogenic formulation comprising at least one effective dose of an HA micelle comprising a purified HA protein.
  • the invention comprises an immunogenic formulation comprising at least one effective dose of a VLP comprising a purified HA protein as described above.
  • the immunogenic formulation of the invention comprises a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein, 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.
  • saline buffered saline
  • dextrose dextrose
  • water glycerol
  • sterile isotonic aqueous buffer and combinations thereof.
  • the formulation should suit the mode of administration.
  • the formulation is suitable for administration to humans, preferably is sterile, non-particulate and/or non-pyrogenic.
  • the composition 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 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 a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein, and the other containing an adjuvant.
  • Associated with such containers 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 invention also provides that the formulation be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition.
  • the 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.
  • the composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the composition.
  • the liquid form of the 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/mi, at least 500 ⁇ g/ml, or at least 1 mg/ml.
  • the invention also comprises a method of formulating a vaccine or antigenic composition that induces immunity to an infection or at least one disease symptom thereof to a mammal, comprising adding to the formulation an effective dose of a purified MA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the infection is an influenza infection.
  • While stimulation of immunity with a single dose is possible, 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, e.g. influenza infection.
  • 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.
  • compositions of administering a composition comprising a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein 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 epidural
  • mucosal ⁇ e.g., intranasal and oral or pulmonary routes or by suppositories e.g., intranasal and oral or pulmonary routes or by suppositories.
  • compositions of the present invention are administered intramuscularly, intravenously, subcutaneously, transdermally or inrradermally.
  • 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.
  • intranasal or other mucosal routes of administration of a composition 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 of the invention may induce an antibody or other immune response that will induce cross protection against other strains of influenza. Administration can be systemic or local.
  • the vaccine and/or immunogenic 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 immunogenic 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.
  • compositions of the invention can be administered as part of a combination therapy.
  • compositions of the invention can be formulated with other immunogenic compositions, antiviral s and/or antibiotics.
  • the dosage of the pharmaceutical composition 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.
  • the 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. The inclusion of any adjuvant described in Vogel ei al., "A Compendium of Vaccine Adjuvants and Excipients (2 nd Edition)," herein incorporated by reference in its entirety for all purposes, is envisioned within the scope of this invention.
  • 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, CCJP (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/IVeen 80 emulsion also is contemplated.
  • MF-59, Novasomes ® , MHC antigens may also be used.
  • the purified ILA protein or HA micelle comprising a purified HA can be used as an adjuvant to boost a vaccine comprising a VXP.
  • the VLP comprises an HA protein.
  • 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.
  • Pauciiamellar 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 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 ran 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 4,91 1,928, herein incorporated by reference in their entireties for all purposes).
  • compositions of the invention can also be formulated with ''immune stimulators.”
  • cytokines the body's own chemical messengers (cytokines) to increase the immune system's response.
  • Immune stimulators include, but not limited to, various cytokines, Iymphokines and chemokines with immunostimulatory > immunopotenliating, and pro-inflammatory activities, such as interleukins (e.g., IL-I , IL-2, IL-3, 1L-4, IL- 12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other imrnunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1 ; B7.2, etc.
  • interleukins e.g., IL-I , IL-2, IL-3, 1L-4, IL- 12, IL-13
  • growth factors e.g., granulocyte-
  • the immunostimulatory molecules can be administered in the same formulation as the compositions of the invention, 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.
  • the purified HA proteins of the invention are useful for preparing compositions that stimulate an immune response that confers immunity or substantial 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 (actor in resistance to natural infection. Secretory immunoglobulin A (slgA) is involved in the 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 effective for a few years against the new strains of virus circulating in the community.
  • the invention encompasses a method of inducing immunity to infections or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention comprises a method to induce immunity to influenza infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein.
  • the invention comprises a method to induce immunity to influenza infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of an HA micelle comprising a purified HA protein.
  • the invention comprises a method to induce immunity to influenza infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of influenza VLP, wherein the VLPs comprise a purified HA protein.
  • the invention also encompasses inducing immunity to an infection, or at least one symptom thereof, in a subject caused by an infectious agent, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • compositions of the invention can induce substantial immunity in a vertebrate (e.g. a human) when administered to the vertebrate.
  • the substantial immunity results from an immune response against compositions of the invention that protects or ameliorates infection or at least reduces a symplom of infection in the vertebrate.
  • the infection will be asymptomatic.
  • the response may not be a folly protective response.
  • 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 substantial immunity to influenza virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention comprises a method of vaccinating a mammal against influenza comprising administering to the mammal a protection-inducing amount of purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention also encompasses a method of inducing substantial immunity to an infection, or at least one disease symptom in a subject caused by an infectious agent, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.
  • the invention comprises a method of inducing a protective antibody response to an infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein as described above.
  • an "antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG. IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light-' (about 25 kD) and one "heavy" chain (about 50-70 kD).
  • the N -terminus of each chain defines a variable region of about 100 to UO 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. [00155]
  • the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein.
  • the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose an HA micelle comprising a purified HA protein.
  • the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose a VLP, wherein the VLP comprises a purified HA protein as described above.
  • Hemagglutinin, HA from Influenza A virus (A/CaJifo ⁇ iaia/04/2009 HlNl)
  • influenza HA protein purification method of influenza HA protein was demonstrated by purifying HA protein derived from influenza B/Brisbane/60. Tubes at -20°C containing Sf9 cells transfected with baculovirus expressing influenza HA was thawed at room temperature. Since HA proteins are expressed intracell ⁇ larly, cell lysis and extraction for recovery are required. Bac ⁇ iovirus protein gp64 is the most common contaminant that was observed to c ⁇ -purirled with HA protein. Varied results were observed among different HA when apply the same chromatography procedure. The purification method can be evaluated and, if necessary, modified for each individual HA due to differences in physical/chemical characteristics.
  • CaC12 CaJcium chloride dihydrate, granular, USP, FCC, Mallinckrodt, Cat# 4616-04, FW 147.01
  • MnC12 Manganese chloride tetrahydrate, ACS grade, Sigma-Aldrich, Cat# 221279, FW 197.91
  • NP-9 Tergitol NP-9, Aidrich, Cat# 521078
  • Tergitol NP-9 (Tergilol NP-9 density 1.06 g/mL) to water, QS 100 ml.
  • TMAE Equilibration Buffer (30 mM Tris, pH 8.0, 100 mM NaCI, 0.02% NP-9) (3L)
  • TMAE EIution buffer (30 niM Tris, pH 8.0, IM NaCl, 0.02% NP-9) (IL)
  • Lentil lectin equilibration buffer (30 mM Tris, pH 8, 500 mM NaCl, 0.02 % NP- 9, 1 mM CaCIi, 1 niM MnCh) (1000ml)
  • Lentil lectin elution buffer (30 mM Tris, pH 8 S 500 mM NaCl, 0.02 % NP-9, 1 mM CaCl 2 , 50OmM Methyl- ⁇ -D-mannopyranoside) (250ml)
  • Pelleted Sfl cells containing recombinant influenza HA were suspended in approximately 800-1000 ml of extraction buffer (see above). The suspension was then allowed to stir for 1 hour at room temperature using a magnetic mixer, and centrifuged at 8000 rpm in GS-4 rotor for 30 nuns. The supernatant containing recombinant influenza HA was then filtered using 0.45 micron cellulose acetate or other equivalent filters. The supernatant was first run though Fractogel® EMD TMAE (M) column.
  • M Fractogel® EMD TMAE
  • Fractogel EMD TMAE (M) column (150-40OmI) column was sanitized before use as described below:
  • the HA pool from TMAE column was further purified by lentil lectin (LL) affinity chromatography.
  • a 10-20 ml Ll., column was equilibrated with sufficient (>3 CV) LL EQ buffer at flow rate of 50 cm/hr through the entire run.
  • the hA pool to be loaded was adjusted to 1 mM calcium and manganese by adding 1 ml of 1 M MnCl 2 and 1 mL of 1 M CaCl? per liter.
  • the HA pool from TMAB column was then loaded onto the LL column.
  • the column was washed with 2-3 CV of EQ buffer.
  • HA was the eluted from the column with lentil lectin elution buffer collecting 5ml fractions.
  • Figure 2 showed the lentil lectin chromatography ( Figure 2a) and SDS PAGE gel scan ( Figure 2b) for influenza HA of B/Brisbane 60. There were two bands observed on the gel, one was HA, another one is gp64 from baculovirus.
  • the HA pool from lentil lectin affinity chromatography was desalted with a G25 Desalting Chromatography or Dialysis before further purified with CHTTM column.
  • the pool of HA fractions from the LL column must be buffer exchanged to the EQ buffer of CHTTM column prior to loading on to CHTTM column. Either Sephadex G25 chromatography or dialysis can be used.
  • CHT EQ buffer was used for G25 desalting step.
  • G25 column was equilibrated with CHTTM EQ buffer first.
  • HA pool was then loaded to G25 column at flow rate of 100 cm/hr. 50 ml fractions were collected and pooled at the protein peak (A280).
  • Desalted protein peak containing influenza HA was further purified with CHTTM column.
  • a CHT column (Type 1, 5-10 ml) was equilibrated with CHT rM EQ buffer. Flow rate through EQ, load and wash was at 0.5-1.0 CV/min (2.5-5.0 ml/min for a 5 ml column). The protein peak from the G-25 column or solution after dialysis was loaded onto the column. The cohimn was washed with 2-3 CV of EQ buffer. Protein was eluted with 10-15 CV gradient from 0 to 100% of elution buffer at flow rate of 0.5CV/min with fraction size at ⁇ !/2 CV. The flow through and elution fractions were analyzed on SDS-PAGE.
  • Figure 3 showed B/Bris HA CHTTM chromatogram (Figure 3a) and SDS-PAGE ( Figure 3b).
  • H5N1 HA protein was observed in the FT of CHTTM chromatography, while others, including H3N2 HA, HlNl HA and B strain HA, were observed in late fractions of the gradient elution.
  • Impurity gp64
  • Fraction pooling must be performed carefully, and yield is typically sacrificed for good purity.
  • HA protein can be concentrated and dialyzed extensively using a Millipore Stirred Cells with 20-30K MWCO membrane against DPBS buffer. A minimum of three additions of 10-fold buffer volume is needed for dialysis. Final H ⁇ protein concenration at >0.5 mg/ml is preferred. Sterile Filtration is performed using a 0.2 micron SFCA (or PVDF) filter. HA yield is approximately 5-10 mg (from 2L cell culture).
  • influenza HA protein purification method of influenza HA protein was demonstrated by purifying HA protein derived from influenza A/Cal/04/09. Tubes at -20°C containing Sf9 cells transfected with baculovirus expressing influenza HA derived from influenza A/Cal/04/09 was thawed at room temperature. It was estimated that the wet cells weight was 24.9 gram. Cell pellet was re-suspended with 400 ml 25 mM TrisCl pH 8.0, 50 mM NaCl, 1% NP9, 2 ug-'ml Le ⁇ peptin, 16 mi lysis buffer per gram cell, and stirred at room temp for 70 min. The solution was then centrifuged at 10,000 g, SLA 1500 rotor for 30 min at 15 C. 400 ml supernatant was taken for purification.
  • sequences below are recombinant HA, recombinant NA, and recombinant Ml derived from influenza A/Californai/04/09, which were expressed to assemble influenza A/Californai/04/09 VUPs. These sequences are co-expressed in a baculovirus expression system to produce chimeric VLPs that express influenza HA antigens on the surface of VLPs.
  • Hemagglutinin, HA from Influenza A virus (A/Caiifomata/04/2009 Hl Nl )
  • VLPs were harvested and isolated from the supernatant by centrifugation and by a discontinuous sucrose step gradient. The fraction comprising the VLPs was collected from the top of the gradient. The VLPs isolated from the sucrose gradient were analyzed by SDS-PAGE and western blot. VLPs were turther purified as described below.
  • VLPs were furthered purified on a 20% to 60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaCl by centrif ⁇ gation at 6,500 x g for 18 hours at about 4° C to about 10° 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 was diluted to comprise 200 mM of NaCl in preparation for the next step in the purification process.
  • This product contained VLPs and might contain intact baculovirus particles.
  • VLPs Further purification of VLPs was achieved by anion exchange chromatography, or 44% isopycnic sucrose cushion ceritrifugation.
  • anion exchange chromatography the sample from the sucrose gradient (see above) was loaded into column containing a medium with an anion (e.g. Matrix Fractogel EMD TMAE) and eluded via a salt gradient (from about 0.2 M to about 1.0 M of NaCl) that can separate the VLP from other contaminates (e.g. baculovirus and DNA/RNA).
  • a medium with an anion e.g. Matrix Fractogel EMD TMAE
  • a salt gradient from about 0.2 M to about 1.0 M of NaCl
  • the sample comprising the VLPs was added to a 44% sucrose cushion and centrifuged for about 18 hours at 30,000 g.
  • VLPs formed a band at the top of 44% sucrose, while baculovirus precipitated at the bottom and other
  • VLPs (Lot 747.3, 0.35 mg/ml) were used in a vaccine potency assay.
  • HA was harvested and used to make agarose gel for SRID assay.
  • a non-limiting exemplary protocol of SRID assay is demonstrated below: 1. Prepare 10 ml of 1.0% agarose (0.1 g/ 1 OmI) in 1 X assay buffer (Phosphate buffered saline) by heating slowly till agarose dissolves completely. Take care not to scorch or troth the solution.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur une protéine virale purifiée et/ou des pseudoparticules virales purifiées avec une pureté élevée et une efficacité vaccinale élevée, et sur des procédés de purification de celles-ci. L'invention porte également sur des utilisations de la protéine virale purifiée et/ou des pseudoparticules virales purifiées comme étalon dans un essai d'efficacité vaccinale.
PCT/US2010/042568 2009-07-20 2010-07-20 Protéines ha recombinantes purifiées du virus de la grippe Ceased WO2011011390A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US22699409P 2009-07-20 2009-07-20
US61/226,994 2009-07-20

Publications (1)

Publication Number Publication Date
WO2011011390A1 true WO2011011390A1 (fr) 2011-01-27

Family

ID=43499377

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/042568 Ceased WO2011011390A1 (fr) 2009-07-20 2010-07-20 Protéines ha recombinantes purifiées du virus de la grippe

Country Status (1)

Country Link
WO (1) WO2011011390A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013044390A1 (fr) * 2011-09-30 2013-04-04 Medicago Inc. Augmentation du rendement en particules de type viral dans des plantes
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
WO2018161858A1 (fr) 2017-03-06 2018-09-13 Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. Procédés de production et de caractérisation de vaccin antiviral et composition de vaccin antiviral
US10358652B2 (en) 2013-03-28 2019-07-23 Medicago Inc. Influenza virus-like particle production in plants
EP3420076A4 (fr) * 2017-03-06 2020-01-08 Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. Procédés de production et de caractérisation de vaccin antiviral et composition de vaccin antiviral
US11390878B2 (en) 2011-09-30 2022-07-19 Medicago Inc. Increasing protein yield in plants
US12467058B2 (en) 2013-03-28 2025-11-11 Aramis Biotechnologies Inc. Influenza virus-like particle production in plants

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6245532B1 (en) * 1993-09-13 2001-06-12 Protein Sciences Corporation Method for producing influenza hemagglutinin multivalent vaccines
US20050220854A1 (en) * 2004-04-01 2005-10-06 Yuh-Fun Maa Apparatus and method for transdermal delivery of influenza vaccine
US20060159700A1 (en) * 1998-12-17 2006-07-20 Cates George A Multivalent immunogenic composition containing RSV subunit composition and influenza virus preparation
US20070224205A1 (en) * 2006-03-07 2007-09-27 Powell Thomas J Compositions that include hemagglutinin, methods of making and methods of use thereof
US20080207487A1 (en) * 2006-11-02 2008-08-28 Neose Technologies, Inc. Manufacturing process for the production of polypeptides expressed in insect cell-lines

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6245532B1 (en) * 1993-09-13 2001-06-12 Protein Sciences Corporation Method for producing influenza hemagglutinin multivalent vaccines
US20060159700A1 (en) * 1998-12-17 2006-07-20 Cates George A Multivalent immunogenic composition containing RSV subunit composition and influenza virus preparation
US20050220854A1 (en) * 2004-04-01 2005-10-06 Yuh-Fun Maa Apparatus and method for transdermal delivery of influenza vaccine
US20070224205A1 (en) * 2006-03-07 2007-09-27 Powell Thomas J Compositions that include hemagglutinin, methods of making and methods of use thereof
US20080207487A1 (en) * 2006-11-02 2008-08-28 Neose Technologies, Inc. Manufacturing process for the production of polypeptides expressed in insect cell-lines

Cited By (15)

* 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
US10190132B2 (en) 2007-11-27 2019-01-29 Medicago Inc. Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin
US9458470B2 (en) 2007-11-27 2016-10-04 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
US11155581B2 (en) 2011-09-30 2021-10-26 Medicago Inc. Increasing virus-like particle yield in plants
WO2013044390A1 (fr) * 2011-09-30 2013-04-04 Medicago Inc. Augmentation du rendement en particules de type viral dans des plantes
US11390878B2 (en) 2011-09-30 2022-07-19 Medicago Inc. Increasing protein yield in plants
US10358652B2 (en) 2013-03-28 2019-07-23 Medicago Inc. Influenza virus-like particle production in plants
US11085049B2 (en) 2013-03-28 2021-08-10 Medicago Inc. Influenza virus-like particle production in plants
US12467058B2 (en) 2013-03-28 2025-11-11 Aramis Biotechnologies Inc. Influenza virus-like particle production in plants
EP3420076A4 (fr) * 2017-03-06 2020-01-08 Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. Procédés de production et de caractérisation de vaccin antiviral et composition de vaccin antiviral
US11319529B2 (en) 2017-03-06 2022-05-03 Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. Methods of producing and characterizing virus vaccine and virus vaccine composition
WO2018161858A1 (fr) 2017-03-06 2018-09-13 Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. Procédés de production et de caractérisation de vaccin antiviral et composition de vaccin antiviral
US12297461B2 (en) 2017-03-06 2025-05-13 Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. Methods of producing and characterizing virus vaccine and virus vaccine composition

Similar Documents

Publication Publication Date Title
DK2540312T3 (en) Chimeric avian influenza VLPs
US8697088B2 (en) VLPs derived from cells that do not express a viral matrix or core protein
KR102399854B1 (ko) 면역원성 중동호흡기증후군 코로나바이러스 (ΜＥＲＳ-ＣｏＶ) 조성물 및 방법
US8551756B2 (en) Avian influenza chimeric VLPS
JP6195965B2 (ja) 狂犬病糖タンパク質ウイルス様粒子（ｖｌｐ）
US10544399B2 (en) Highly efficient influenza matrix (M1) proteins
WO2011011390A1 (fr) Protéines ha recombinantes purifiées du virus de la grippe
US20100143406A1 (en) Methods of enhancing protein incorporation into virus like particles
WO2008061243A2 (fr) Particules de type viral (vlp) apparentées au virus respiratoire syncytial
WO2013044203A2 (fr) Nouveaux vaccins à base de protéine hémagglutinine de la grippe
NZ567164A (en) Functional influenza virus like particles (VLPS)
EP2953642A1 (fr) Vaccin combiné pour le virus respiratoire syncytial et la grippe
CA2678966A1 (fr) Pseudo-particules virales (vlp) chimeriques de la maladie de newcastle
RU2575800C2 (ru) Вирусоподобные частицы (vlp) из гликопротеина вируса бешенства

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10802763

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10802763

Country of ref document: EP

Kind code of ref document: A1