EP2254554A1 - Sugar glassified virus like particles (vlps) - Google Patents

Sugar glassified virus like particles (vlps)

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Publication number
EP2254554A1
EP2254554A1 EP09713904A EP09713904A EP2254554A1 EP 2254554 A1 EP2254554 A1 EP 2254554A1 EP 09713904 A EP09713904 A EP 09713904A EP 09713904 A EP09713904 A EP 09713904A EP 2254554 A1 EP2254554 A1 EP 2254554A1
Authority
EP
European Patent Office
Prior art keywords
composition
vlp
powder
sugar glass
sugar
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.)
Withdrawn
Application number
EP09713904A
Other languages
German (de)
English (en)
French (fr)
Inventor
Dinesh Shenoy
James Robinson
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 EP2254554A1 publication Critical patent/EP2254554A1/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • C12N2760/16023—Virus like particles [VLP]

Definitions

  • VLPS SUGAR GLASSIFIED VIRUS LIKE PARTICLES
  • This invention relates to stabilizing virus like particles (VLPs) by associating said VLPs with a sugar glass.
  • VLPs virus like particles
  • VLPs Virus like particles
  • diseases e.g. influenza
  • Virus- like particles closely resemble mature virions, but they do not contain viral genomic material (i.e., viral genomic RNA). Therefore, VLPs are nonreplicative in nature, which make them safe for administration in the form of an immunogenic composition (e.g., vaccine).
  • VLPs can express envelope glycoproteins on the surface of the VLP, which is the most physiological configuration.
  • VLPs may be more effective in inducing neutralizing antibodies to the envelope glycoprotein than soluble envelope antigens.
  • VLPs can be administered repeatedly to vaccinated hosts, unlike many recombinant vaccine approaches.
  • VLPs are sensitive to temperature changes and this must be maintained in a controlled environment.
  • the ideal VLP formulation would resist high (greater that about 25°C) and low temperatures (less than about 2°C), to facilitate distribution.
  • Current methods for avoiding temperature associated degradation of vaccines are inadequate. For example, live-attenuated vaccines (and some non-live vaccines) are often lyophilized because of their intrinsic instability. The lyophilized products are reconstituted with diluent immediately before administration.
  • lyophilized vaccines are usually presented in multi- dose vials. Some global guidelines require that unused vaccines in a multi-dose vial be discarded within six hours of reconstitution due to the concerns of potential contamination and potency loss. This results in vaccine wastage, which can account for losses of 50% or more of the vaccine doses distributed.
  • compositions and methods for stabilizing temperature-sensitive vaccines and specifically for compositions and methods for stabilizing vaccines.
  • the present invention comprises a composition comprising at least one virus like particle (VLP) and a sugar glass.
  • said sugar glass is composed of a monosaccharide and/or disaccharide.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • VLP and said sugar glass is a powder.
  • said powder is made by spray drying, freeze drying, milling or a combination thereof.
  • said powder has a mean particle diameter between about 0.1 nm to about 100 microns.
  • said powder is suspended in a nonaqueous solvent that will not dissolve said sugar glass.
  • said nonaqueous solution is selected from the group consisting of triacetin, isopropyl myristate, medium chain triglycerides, short, medium, long-chain mono-, di-, tri-, glycerides, aliphatic and aromatic alcohols or a combination thereof.
  • said powder is administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, subcutaneous Iy, or mucosally (e.g. sublingually or buccally).
  • said VLP comprises at least one viral protein.
  • said vial protein is from influenza, RSV and/or VZV.
  • said VLP has increased stability when compared to VLP that is not associated with a sugar glass.
  • the present invention also comprises, a composition comprising at least one VLP and a sugar glass, wherein said VLP has increased stability when compared said composition without sugar glass.
  • said increased stability is increase thermal stability.
  • said sugar glass is composed of a monosaccharide or disaccharide. In another embodiment, wherein said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.
  • said stabilized sugar glass VLP is a powder.
  • the present invention also comprises, a dry powder formulation comprising a VLP and a sugar glass.
  • said sugar glass is composed of a monosaccharide or disaccharide.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.
  • said dry powder is made by spray drying, milling or a combination thereof.
  • said powder is administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
  • said powder is administered to an animal via inhalation using an inhaler and/or subcutaneously using a jet of high-pressure air.
  • the present invention also comprises, a method of delivering a sugar glass stabilized VLP comprising reconstituting a solid form of sugar glassified VLPs in a solvent and administering said reconstituted VLPs into an animal.
  • said powder is administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
  • the present invention also comprises, a method of delivering a sugar glass stabilized VLP comprising administering a solid form of sugar glassified VLPs via inhalation and/or injection.
  • Figure IA SDS PAGE and western blot of VLPS before the glassification of VLPs
  • Figure IB SDS PAGE and western blot of VLPS after the glassification of VLPs
  • Figure 2 SDS PAGE and western blot of VLPS before and after the glassification of VLPs
  • adjuvant refers to a compound that, when used in combination with a specific immunogen (e.g. a VLP) in a formulation, will augment or otherwise alter or modify 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.
  • ambient temperatures or conditions are those at any given time in a given environment. Typically, ambient room temperature is approximately 22°C, ambient atmospheric pressure, and ambient humidity are readily measured and will vary depending on the time of year, weather conditions, altitude, etc.
  • buffer refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components.
  • the pH of the buffer will generally be chosen to stabilize the active material of choice, and will be ascertainable by those in the art. Generally, this will be in the range of physiological pH, although some proteins, can be stable at a wider range of pHs, for example acidic pH. Thus, preferred pH ranges are from about 1 to about 10, with from about 3 to about 8, from about 6.0 to about 8.0, from about 7.0 to about 7.4, and from about 7.0 to about 7.2.
  • Suitable buffers include a pH 7.2 phosphate buffer and a pH 7.0 citrate buffer.
  • Suitable buffers include, but are not limited to, amino acids, potassium phosphate, sodium phosphate, sodium acetate, histidine-HCl, sodium citrate, sodium succinate, ammonium bicarbonate and carbonate.
  • buffers are used at molarities from about 1 mM to about 2 M, with from about 2 mM to about 1 M being preferred, and from about 10 mM to about 0.5 M being especially preferred, and 25 to 50 mM being particularly preferred.
  • an effective dose generally refers to that amount of VLPs sufficient to induce immunity, to prevent and/or ameliorate an infection or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of a VLP.
  • An effective dose may refer to the amount of VLPs sufficient to delay or minimize the onset of an infection.
  • An effective dose may also refer to the amount of VLPs that provides a therapeutic benefit in the treatment or management of an infection.
  • an effective dose is the amount with respect to VLPs of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an 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 an infectious agent.
  • Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay.
  • an "effective dose" is one that prevents disease and/or reduces the severity of symptoms.
  • excipients generally refer to compounds or materials that are added to increase the stability of a therapeutic agent during glassification (by, e.g. , the spray freeze dry process) and afterwards, for long-term storage.
  • Suitable excipients can be, e.g., agents that do not thicken or polymerize upon contact with water, are basically innocuous when administered to a patient and does not significantly interact with the therapeutic agent in a manner that alters its biological activity.
  • Suitable excipients are described below and include, but are not limited to, proteins such as human and bovine serum albumin, gelatin, carbohydrates, sugar alcohols, e.g.
  • Excipients can be multifunctional constituents of solutions or suspensions of invention.
  • the term "effective amount” refers to an amount of VLPs necessary or sufficient to realize a desired biologic effect.
  • An effective amount of the composition would be the amount that achieves a selected result, and such an amount could be determined as a matter of routine by a person skilled in the art.
  • an effective amount for preventing, treating and/or ameliorating an infection could be that amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to VLPs of the invention.
  • the term is also synonymous with "sufficient amount.”
  • the term "infectious agent” refers to microorganisms that cause an infection in a vertebrate.
  • the term "glass” or “glassy state” or “glassy matrix,” refers to a liquid that has a markedly reduced ability to flow, i.e. it is a liquid with a very high viscosity, wherein the viscosity ranges from 10 10 to 10 14 pascal-seconds. It can be viewed as a metastable amorphous system in which the molecules have vibrational motion but have very slow (almost immeasurable) rotational and translational components. As a metastable system, it is stable for long periods of time when stored well below the glass transition temperature.
  • glass transition temperature is represented by the symbol T g and is the temperature at which a composition changes from a glassy or vitreous state to a syrup or rubbery state.
  • T g is determined using differential scanning calorimetry (DSC) and is standardly taken as the temperature at which onset of the change of heat capacity (Cp) of the composition occurs upon scanning through the transition.
  • DSC differential scanning calorimetry
  • Cp change of heat capacity
  • the definition of T g is always arbitrary and there is no present international convention.
  • the T g can be defined as the onset, midpoint or endpoint of the transition; for purposes of this invention we will use the onset of the changes in Cp when using DSC and DER. See the article entitled “Formation of Glasses from Liquids and Biopolymers" by C. A. Angell: Science, 267, 1924-1935 (Mar. 31, 1995) and the article entitled “Differential Scanning Calorimetry Analysis of Glass Transitions" by Jan P.
  • stable formulation or composition is one in which the biologically active material therein (e.g. VLPs) essentially retains its physical stability and/or chemical stability and/or biological activity upon storage.
  • Stability can be measured at a selected temperature for a selected time period.
  • Trend analysis can be used to estimate an expected shelf life before a material has actually been in storage for that time period.
  • the term "vaccine” refers to a formulation which contains VLPs which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of VLPs.
  • the vaccine Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.
  • VLPs vaccines are associated with sugar glass and/or were stabilized with sugar glass.
  • virus-like particle refers to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious.
  • Virus-like particles in accordance with the invention do not carry genetic information encoding for the proteins of the virus-like particles. In general, virus-like particles lack a viral genome and, therefore, are noninfectious. In addition, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.
  • Vaccines or drugs in solution ready for injections are inherently unstable. Methods to tackle this instability are known in the art (e.g. freeze drying). However, this is an inconvenient and inherently dangerous, since incorrect reconstitution or dried vaccines or drugs can result in wrong doses or contaminated solution and freezing and/or thawing of a vaccine or drug can also result in wrong dosing.
  • new improved vaccine formulations that makes vaccine delivery easier and safer, decrease dependency on the cold chain and/or increase thermal stability of vaccines and/or reduce the number of immunizations interventions. Such vaccine formulations would make distribution of vaccines worldwide more efficient and economical.
  • carbohydrates can protect various types of drug substances like proteins and vaccines during freezing, drying and storage. If dried properly, a proteinaceous drug can be incorporated in a matrix consisting of carbohydrate in the amorphous glassy state (sugar glass).
  • a proteinaceous drug can be incorporated in a matrix consisting of carbohydrate in the amorphous glassy state (sugar glass).
  • the stabilizing effect of these sugar glasses has been explained by the formation of a matrix that strongly reduces diffusion and molecular mobility and acts as a physical barrier between particles or molecules. Both the lack of mobility and physical barrier provided by the glass matrix, prevent aggregation and degradation of the dried material (Amorij, JP et al. (2007) Vaccine, 25, 6447-6457).
  • the sugar must be dried below the glass transition temperature of a carbohydrate, e.g. a sugar.
  • the glass transition temperature is the temperature below which the physical properties of amorphous materials vary in a manner similar to those of a solid phase (glassy state), and above which amorphous materials behave like liquids (rubbery state).
  • a material's glass transition temperature, T g is the temperature below which molecules have little relative mobility. T g is usually applicable to wholly or partially amorphous phases such as glasses and plastics. Above T g , the secondary, non-covalent bonds between the polymer chains become weak in comparison to thermal motion, and the polymer becomes rubbery and capable of elastic or deformation without fracture.
  • Sugar glass is made when drying a sugar mixture below the T g temperature. As the sugar solution containing an active molecule is dried, it can either crystallize when the solubility limit of the sugar is reached or can become a supersaturated syrup. The ability of the sugar to resist crystallization is a crucial property of a good stabilizer. Trehalose is good is known to make glass (Green J L. & Angel C A. Phase relations and vitrification in saccharide water solutions and the trehalose anomaly J. Phys. Chem. 93 2880-2882 (1989)) but is not unique. Further drying progressively solidifies the syrup, which turns into a glass at low residual water content. Imperceptibly, the active molecules change from liquid solution in the water to solid solution in the dry sugar glass. Chemical diffusion is negligible in a glass and therefore chemical reactions virtually cease. Since denaturation is a chemical change it cannot occur in the glass and the molecules are stabilized.
  • the invention comprises a composition comprising at least one virus like particle (VLP) associated and a sugar glass.
  • VLP virus like particle
  • said VLP is encased by said sugar glass.
  • said VLP associates with said sugar glass.
  • said sugar glass is composed of a monosaccharide and/or disaccharide.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.
  • sugars include galactose, mannose, xylose, sorbose, lactose, palactose, raffinose, maltodextrins, melezitose, maltose, fructose, arabinose, xylose, ribose, rhamnose, xylitol, erythritol, threitol, gluconate, and/or the like.
  • the suspension or solution can also include, e.g., a polymer, such as starch, starch derivatives, carboxymethyl starch, hydroxyethyl starch (HES), and/or dextran.
  • VLPs are stable in ambient temperatures, when said VLPs are associated with a sugar glass.
  • Said sugar is preferably one which does not crystallize at freezing temperatures such that it destabilizes said VLPs in a glassy formulation.
  • the amount of sugar used in the suspension or solution can vary depending on the nature of the VLP, the type of sugar, and the intended use. However, generally, the final concentration of the sugar is between about 1% and 40%; more preferably, between about 1% and 20% by weight.
  • the suspension or solution comprises about 60 mg/ml Trehalose. In another embodiment, the suspension or solution comprises about 50 mg/ml of Mannitol.
  • the suspension or solution comprises about 30 mg/ml of Trehalose and about 25 mg/ml of Mannitol. In another embodiment, the suspension or solution comprises about 30 mg/ml of Trehalose and about 30 mg/ml of Mannitol.
  • said VLP and sugar glass composition may be a powder. In another embodiment, said powder is made by spray drying, freeze-drying, milling or a combination thereof. In other embodiment, said powder has a mean particle diameter between about 0.1 nm to about 100 microns.
  • the formulation of the invention comprise virus-like particles (VLPs) and a sugar glass.
  • Said VLPs comprise at least a viral core protein (e.g. Influenza Ml , retrovirus gag, RSV M, Newcastle disease M etc.) and at least one viral surface envelope protein (e.g. influenza HA and/or NA, HIV gpl20, RSV F, Newcastle disease F).
  • Chimeric VLPs are VLPs having at least two proteins in the VLP, wherein one protein can drive VLP formation (e.g.
  • infectious agent proteins may have antigenic variations of the same protein.
  • infectious agent protein is from an unrelated agent.
  • said chimeric VLPs comprise a chimeric protein (fusion protein) comprising the antigenic portion of one protein fused to the transmembrane and/or cytoplasmic region of a different (heterologous) protein (See U.S. applications 60/902,337, filed February 21, 2007, and 60/970,592, filed September 7, 2007, both of which are incorporated herein by reference in their entireties for all purposes. [037] Infectious agents can be viruses, bacteria and/or parasites.
  • a protein that may be expressed on the surface of VLPs can be derived from viruses, bacteria and/or parasites.
  • the proteins derived from viruses, bacteria 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.
  • viruses from which said infectious agent proteins can be derived from are the following: influenza (A and B, e.g. HA and/or NA), coronavirus (e.g. SARS), hepatitis viruses A, B, C, D & E3, human immunodeficiency virus (HIV), herpes viruses 1, 2, 6 & 7, cytomegalovirus, varicella zoster, papilloma virus, Epstein Barr virus, parainfluenza viruses, adenoviruses, bunya viruses (e.g.
  • hanta virus coxsakie viruses, picoma viruses, rotaviruses, rhinoviruses, rubella virus, mumps virus, measles virus, Rubella virus, polio virus (multiple types), adeno virus (multiple types), parainfluenza virus (multiple types), avian influenza (various types), shipping fever virus, Western and Eastern equine encephalomyelitis, Japanese encephalomyelitis, fowl pox, rabies virus, slow brain viruses, rous sarcoma virus, Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retro viridae (HTLV-I, HTLV-II, Lentivirus), Togaviridae (e.g., Rubivirus), Newcastle disease virus, West Nile fever virus, Tick borne encephalitis, yellow fever
  • the specific proteins from viruses may comprise: HA and/or NA from influenza virus (including avian), S protein from coronavirus, gpl60, gpl40 and/or gp41 from HIV, gp I to IV and Vp from varicella zoster, E and preM/M from yellow fever virus, Dengue (all serotypes) or any flavivirus. Also included are any protein from a virus that can induce an immune response (cellular and/or humoral) in a vertebrate that can prevent, treat, manage and/or ameliorate an infectious disease in said vertebrate.
  • said VLP comprises at least one influenza protein.
  • said influenza protein is HA or NA.
  • said VLP comprises at least one RSV protein.
  • said RSV protein is RSV M, F and/or G.
  • said VLP comprises a VZV protein.
  • said VZV protein is gE and/or at least one tegument protein.
  • 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. botulinum, C. perfringens, C.feseri and other gas gangrene bacteria, B. anthracis, P. pestis, P. multocida, Neisseria meningitidis, N.
  • gonorrheae 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 E. coli and Enterohemorrhagic E. col ⁇ ), Enterobacter (e.g.
  • Enterobacter aerogenes Enterobacter aerogenes
  • Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella), Erysipelothrix, 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), Vibrio (e.g., Vibrio cholerae), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Trepone
  • Non-limiting examples of parasites from which said infectious agent proteins can be derived which are the causative agent for following: leishmaniasis (Leishmania tropica mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagasi), trypanosomiasis ⁇ Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense) , toxoplasmosis ⁇ Toxoplasma gondii) , schistosomiasis ⁇ Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum) , malaria ⁇ Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and
  • Another embodiment of the invention comprises a composition comprising at least one VLP in a sugar glass, wherein said VLP has increased stability when compared to VLP that is not in a sugar glass.
  • said increased stability is increase thermal stability and increased stability in ambient temperature.
  • VLPs are "stable" in a pharmaceutical composition if, e.g. , said VLP shows no significant increase in aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, as measured by UV light scattering or by size exclusion chromatography, or any biological assay.
  • a “stable" formulation or composition is one in which the biologically active material therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage.
  • Various analytical techniques for measuring stability are described below and available in the art and are reviewed, e.g., in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).
  • Stability can be measured at a selected temperature for a selected time period.
  • Trend analysis can be used to estimate an expected shelf life before a material has actually been in storage for that time period.
  • the composition can be stable at room temperature (about 25°C) for at least 3 months, and/or stable at about 2-8°C for at least 1 year. Furthermore, the composition can be stable following freezing (to, e.g., -70 0 C) and thawing of the composition.
  • said sugar glass is composed of a monosaccharide or disaccharide.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.
  • said stabilized sugar glass VLP is a powder.
  • the VLP associated sugar glass composition may have additional exceptions that help stabilize said VLPs.
  • the present invention includes compositions, such as suspensions or solutions of VLPs, a polymer additive, an amino acid additive, and/or a surfactant, to help improved stability.
  • the suspensions or solutions can include other ingredients (i.e. excipients) such as buffers, carriers, preservatives, colloidal stabilizers, fillers, diluents, lubricants, amphiphiles, and/or stabilizers.
  • polymers can be included in the suspensions or solutions of the method, e.g., to provide protective and structural benefits.
  • the linear or branching strands of polymers can provide, e.g., increased structural strength to the particle compositions of the invention.
  • Polymers can be applied as a protective and/or time release coat to the outside or powder particles of the invention.
  • Many polymers such as polyvinyl pyrrolidone, polyethylene glycol, poly amino acids, such poly L-lysines, can significantly enhance reconstitution rates in aqueous solutions.
  • Polymer protective agents, in the methods of the invention can include, e.g., starch and starch derivatives, such as oxidized starch, carboxymethyl starch and hydroxy ethyl starch (HES).
  • Others include hydro lyzed gelatin, unhydrolyzed gelatin, ovalbumin, collagen, chondroitin sulfate, a sialated polysaccharide, actin, myosin, microtubules, dynein, kinetin, human serum albumin, and/or the like.
  • Other excipients can be included in the formulation.
  • amino acids such as arginine and methionine can be constituents of the formulation and compositions.
  • the amino acids can, e.g., act as zwitterions that block charged groups on processing surfaces and storage containers preventing nonspecific binding of bioactive materials.
  • the amino acids can increase the stability of compositions by, e.g., scavenging oxidation agents, scavenging deamidation agents, and stabilizing the conformations of proteins.
  • glycerol can be included in the formulations of the invention, e.g., to act as a plasticizer in the powder particle compositions.
  • EDTA can be included in the composition, e.g., to reduce aggregation of formulation constituents and/or to scavenge metal ions that can initiate destructive free radical chemistries.
  • VLP and sugar glass composition of the invention may also include, e.g., a surfactant compatible with the particular bioactive material involved.
  • a surfactant can enhance solubility of other formulation components to avoid aggregation or precipitation at higher concentrations.
  • Surface active agents can, e.g., lower the surface tension of the suspension or solution so that bioactive materials are not denatured at gas-liquid interfaces, and/or so that finer droplets can be formed during spraying.
  • the suspensions or solutions according to the invention comprise between about 0.001 and 5%; and preferably, between about 0.05 and 1%, or about 0.2%, of a nonionic surfactant, an ionic surfactant, or a combination thereof.
  • Buffers can be added to the formulations of the invention, e.g., to provide a suitable stable pH to the formulations of the method and compositions of the invention.
  • Typical buffers of the invention include, e.g., amino acids, potassium phosphate, sodium phosphate, sodium acetate, sodium citrate, histidine, glycine, sodium succinate, ammonium bicarbonate, and/or a carbonate.
  • the buffers can be adjusted to the appropriate acid and salt forms to provide, e.g., pH stability in the range from about pH 3.0 to about pH 10.0, from about pH 4.0 to about pH 8.0.
  • said VLP associated sugar glass comprises a phosphate buffer at pH 7.2 with 0.5 M NaCl.
  • said VLP and sugar glass composition is a powder suspended in a non-aqueous solvent that will not dissolve said sugar glass.
  • said nonaqueous solution is selected from the group consisting of triacetin, isoprppyl myristate, medium chain triglycerides, short, medium, and/or long-chain monoglycerides, dimonoglycerides, trimonoglycerides, aliphatic and aromatic alcohols, hydrofluoroether, perfluoroether, hyrofiuoroamine, perfluoroamine, hydro fluorothioether, perfluorothioether, and hydro fluoropolyether or a combination thereof (see WO 2005/099669, herein incorporated by reference in its entirety).
  • said powder is administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
  • said powder is made by spray drying, milling or a combination thereof.
  • said powder has a mean particle diameter between about 0.1 nm to about 100 microns.
  • said powder is suspended in a non-aqueous solvent that will not dissolve said sugar glass.
  • said powder is administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
  • VLPs can have the protein from a bacteria, virus, fungal, parasite, as described above.
  • said VLP comprises at least one influenza protein.
  • said influenza protein is HA or NA.
  • said VLP comprises at least one RSV protein.
  • said VLP comprises a VZV protein.
  • said VZV protein is gE.
  • said composition further comprises an adjuvant.
  • Another embodiment of the invention comprises a dry powder formulation comprising a VLP in a sugar glass.
  • said sugar glass is composed of a monosaccharide or disaccharide.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.
  • said dry powder is made by spray drying, freeze drying, milling or a combination thereof.
  • said powder has a mean particle diameter between about 0.1 nm to about 100 microns.
  • said powder is administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
  • said powder is reconstituted in a solvent before administration.
  • said powder is administered to an animal via inhalation using an inhaler and/or subcutaneously using a jet of high-pressure air.
  • the composition may comprise any of the excipients described above.
  • Another embodiment of the invention comprises a method of delivering a sugar glass stabilized VLP comprising reconstituting a solid form of sugar glassified VLPs in a solvent and administering said reconstituted VLPs into an animal.
  • said VLPs are administered to an animal orally, via inhalation, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or subcutaneously.
  • Another embodiment of the invention comprises a method of delivering a sugar glass stabilized VLP comprising administering a solid form of sugar glassified VLPs via inhalation and/or injection. In one embodiment, said injection is administered via a jet of high-pressure air.
  • Another embodiment of the invention comprises a method of enhancing thermal stability of a VLP, comprising formulating said VLP into a sugar glass.
  • said sugar glass is composed of a monosaccharide and/or disaccharide.
  • said monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.
  • said disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.
  • said sugar glass comprising the VLP is a powder.
  • said powder is made by spray drying, freeze drying, milling or a combination thereof.
  • said powder has a mean particle diameter between about 0.1 nm to about 100 microns.
  • said powder is suspended in an organic solvent that will not dissolve said sugar glass.
  • said VLP comprises at least one influenza protein.
  • said influenza protein is HA or NA.
  • said VLP comprises at least one RSV protein.
  • said VLP comprises a VZV protein.
  • 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.
  • said VLP complexed with a sugar glass formulation comprises at least one adjuvant.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.
  • the adjuvant is a paucilamellar lipid vesicle having about two to ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a large amorphous central cavity free of lipid bilayers.
  • Paucilamellar lipid vesicles may act to stimulate the immune response several ways, as non-specific stimulators, as carriers for the antigen, as carriers of additional adjuvants, and combinations thereof.
  • Paucilamellar lipid vesicles act as non-specific immune stimulators when, for example, a vaccine is prepared by intermixing the antigen with the preformed vesicles such that the antigen remains extracellular to the vesicles.
  • the vesicle acts both as an immune stimulator and a carrier for the antigen.
  • the vesicles are primarily made of nonphospholipid vesicles.
  • the vesicles are Novasomes. Novasomes are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm.
  • an adjuvant effect is achieved by use of an agent, such as alum, used in about 0.05 to about 0.1% solution in phosphate buffered saline.
  • the VLPs can be made as an admixture with synthetic polymers of sugars (Carbopol ® ) used as an about 0.25% solution.
  • Some adjuvants for example, certain organic molecules obtained from bacteria; act on the host rather than on the antigen.
  • An example is muramyl dipeptide (N-acetylmuramyl-L- alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan.
  • hemocyanins and hemoerythrins may also be used with VLPs of the invention.
  • the use of hemocyanin from keyhole limpet (KLH) is preferred in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.
  • polysaccharide adjuvants may also be used.
  • various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et ciL, 1989).
  • Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.
  • a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide which is described for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol.
  • Amphipathic and surface active agents e.g., saponin and derivatives such as QS21 (Cambridge Biotech) form yet another group of adjuvants for use with the VLPs of the invention.
  • Nonionic block copolymer surfactants Roskowich et ah, 1994
  • Oligonucleotides are another useful group of adjuvants (Yamamoto et al, 1988).
  • Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present invention.
  • Another group of adjuvants are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in vertebrates.
  • the detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant formulation.
  • combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Pat. No. 4,435,386.
  • Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No.
  • CWS cell wall skeleton
  • trehalose dimycolate as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900.
  • Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Pat. No. 4,520,019.
  • adjuvants that can be conjugated to vaccines in accordance with this invention and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others.
  • ALP alkyl lysophosphilipids
  • BCG BCG
  • biotin including biotinylated derivatives
  • Certain adjuvants particularly contemplated for use are the teichoic acids from Gram-cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the invention (Takada e ⁇ ⁇ /., 1995).
  • Various adjuvants may still be employed in other vertebrates, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells.
  • Another method of inducing an immune response can be accomplished by formulating the VLPs of the invention with "immune stimulators.” These are the body's own chemical messengers (cytokines) to increase the immune system's response.
  • Immune stimulators include, but not limited to, various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
  • the immunostimulatory 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.
  • the VLP associated with sugar glass of the invention can be administered, e.g., to a mammal.
  • Said VLP and sugar glass composition of the invention can include, influenza, HIV, RSV, VZV VLPs.
  • Said VLP and sugar glass composition can be administered to a patient by topical application.
  • the powder particles can be mixed directly into a salve, carrier ointment, pressurized liquid, gaseous propellants, and/or penetrant, for application to the skin of a patient.
  • the powder particles can, e.g., be reconstituted in an aqueous solvent before admixture with other ingredients before application.
  • Said VLPs and sugar glass composition can be administered by inhalation.
  • Dry powder particles less than about 10 um in aerodynamic diameter can be inhaled into the lungs for pulmonary administration.
  • powder particles of about 20 um, or greater, in aerodynamic diameter can be administered intranasally, or to the upper respiratory tract, where they are removed from the air stream by inertial impact onto the mucus membranes of the patient.
  • the powder particles can alternately be reconstituted to a suspension or solution for inhalation administration as an aqueous mist.
  • Said VLP and sugar glass composition can be administered by injection.
  • the powder particles can be administered directly under the skin of a patient using, e.g., a jet of high pressure air. More commonly, the powder particles can be, e.g., reconstituted with a sterile aqueous buffer for injection through a hollow syringe needle.
  • Such injections can be, e.g., intramuscular, intra venous, subcutaneous, intrathecal, intraperitoneal, and the like, as appropriate.
  • Powder particles of the invention can be reconstituted to a solution or suspension with a bioactive material concentration, e.g., from less than about 0.1 ng/ml to from less than about 1 mg/ml to about 500 mg/ml, or from about 5 mg/ml to about 400 mg/ml, as appropriate to the dosage and handling considerations.
  • Reconstituted powder particles can be further diluted, e.g., for multiple vaccinations, administration through IV infusion, and the like.
  • the appropriate dosage ("therapeutically effective amount") of the VLPs material will depend, for example, on the condition to be treated, the severity and course of the condition, whether the biologically active material is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the biologically active material, the type of biologically active material used, and the discretion of the attending physician.
  • the VLPs are suitably administered to the patent once, or over a series of administrations, and may be administered to the patient at any time.
  • the VLPs may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
  • the therapeutically effective amount of VLPs administered will be in the range of about 0.00001 to about 50 mg/kg of patent body weight whether by one or more administrations, with the typical range of protein used being from less than about 0.01 ng/kg to about 20 mg/kg, more preferably about 0.1 mg/kg to about 15 mg/kg, administered daily, for example (as measured by the antigenic protein on said VLP).
  • the typical range of protein used being from less than about 0.01 ng/kg to about 20 mg/kg, more preferably about 0.1 mg/kg to about 15 mg/kg, administered daily, for example (as measured by the antigenic protein on said VLP).
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.
  • the invention also encompasses methods of increasing the "shelf-life" or storage stability of VLPs stored at elevated temperatures. Increased storage stability can be determined by recovery of biological activity in accelerated aging trials.
  • the dry particle compositions produced by methods of the invention can be stored at any suitable temperature. Preferably, the compositions are stored at about 0 0 C. to about 80 0 C. More preferably, the compositions are stored at about 20 0 C. to about 60 0 C. Most preferably, the compositions are stored at ambient temperatures.
  • Example 1 Freeze-drying of VLPs [069] H5N1 Clade 2 Influenza VLPs were made and purified according to the methods described in co-pending application 11/582,540, filed October 18, 2006, herein incorporated by reference in its entirety.
  • the VLPs were purified and suspending in phosphate buffered saline, pH 7.2 with the desired concentration of NaCl in a PETG bottle with the desired concentration of sugar.
  • the formulations are described in Table 1.
  • SRID Single Radial Immunodiffusion
  • total protein was measured by the BCA method (using a commercial kit that employs bicinchoninic acid).
  • Particle size was measured using the Malvern zeta sizer (dynamic light scattering principle).
  • HA activity was measured using haemagglutination assay employing Turkey RBCs and NA activity was measured by fluorimetry using Munana reagent).
  • SDS PAGE and western blot analysis were conducted. Tables 3 to Table 7 and Figure 1 summarize the results of these analysis.
  • Figures 1 A and B compares VLPs comprising HA and NA via SDS and western blots before glassification and after glassification. As shown, after glassification of the VLPs there is no difference in the amount HA and NA as shown in the gels. Thus, glassification does not reduce or chew up the VLPs or antigen expressed on the VLPs.
  • the recommended storage temperature for an aqueous suspension containing VLPs is A- 8°C.
  • the freeze-dried vials control, cryo-II and cryo-V for 5 or 12 weeks) where stored at 25°C and 50 0 C to investigate the protection effect of sugars on VLP stability.
  • H5N1 Clade 2 Influenza VLPs were made and purified according to the methods described in co-pending application 11/582,540, filed October 18, 2006, herein incorporated by reference in its entirety.
  • Placebo was prepare in 6% w/v solution of Trehalose by dissolving 24 grams of
  • VLPs were collect in Phosphate buffered saline, pH 7.2 with 0.15 M NaCl (potency of about 45 ⁇ g HAJmL) in a PETG bottle. Trehalose was then added to constitute 6% w/v solution.
  • the solution was mixed gently (hand shaking) until all Trehalose is dissolved.
  • Run #1 Placebo formulation
  • Run #2 VLP-sugar formulation
  • Figures IA SDS-PAGE and Western blot gels indicate the integrity of the three principal proteins (HA, NA and Ml) with no band shifting or broadening for any of the formulations, before and after freeze-drying.

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CN102014873A (zh) 2011-04-13
BRPI0908861A2 (pt) 2018-02-06
CA2716546A1 (en) 2009-09-03
RU2010139478A (ru) 2012-05-20
JP2011514337A (ja) 2011-05-06
MX2010009351A (es) 2011-03-04
WO2009108689A1 (en) 2009-09-03

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