US20200390877A1 - Formulations of dengue virus vaccine compositions - Google Patents

Formulations of dengue virus vaccine compositions Download PDF

Info

Publication number
US20200390877A1
US20200390877A1 US16/769,837 US201816769837A US2020390877A1 US 20200390877 A1 US20200390877 A1 US 20200390877A1 US 201816769837 A US201816769837 A US 201816769837A US 2020390877 A1 US2020390877 A1 US 2020390877A1
Authority
US
United States
Prior art keywords
formulation
dengue
live attenuated
sucrose
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/769,837
Other languages
English (en)
Inventor
Michael S. Ryan
Sherrie-Ann P. Martin
Morrisa Jones
Justin Stanbro
Akhilesh Bhambhani
Jeffrey Thomas Blue
Heidi Joanne Pixley
Erin J. Green-Trexler
Lynne Ann Isopi
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.)
Merck Sharp and Dohme LLC
Original Assignee
Merck Sharp and Dohme LLC
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 Merck Sharp and Dohme LLC filed Critical Merck Sharp and Dohme LLC
Priority to US16/769,837 priority Critical patent/US20200390877A1/en
Assigned to MERCK SHARP & DOHME CORP. reassignment MERCK SHARP & DOHME CORP. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: STANBRO, Justin, BHAMBHANI, AKHILESH, BLUE, Jeffrey Thomas, MARTIN, Sherrie-Ann P., PIXLEY, Heidi Joanne, RYAN, MICHAEL S., JONES, Morrisa, GREEN-TREXLER, Erin J., ISOPI, Lynne Ann
Publication of US20200390877A1 publication Critical patent/US20200390877A1/en
Assigned to MERCK SHARP & DOHME LLC reassignment MERCK SHARP & DOHME LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: MERCK SHARP & DOHME CORP.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K9/08—Solutions
    • 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
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to formulations of dengue virus vaccine comprising at least one live, attenuated dengue virus or live, attenuated chimeric flavivirus, a buffer, a sugar, a cellulose derivative and a sugar alcohol or glycol, and optionally an amino acid and an alkali or alkaline salt; and formulations of dengue virus vaccine comprising at least one live, attenuated dengue virus or live, attenuated chimeric flavivirus, a buffer, a sugar of at least 150 mg/ml, a carrier, and optionally an alkali or alkaline salt, or, alkali or alkaline salt and an amino acid.
  • Flaviviridae includes the prototype yellow fever virus (YF), the four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), Japanese encephalitis virus (JE), tick-borne encephalitis virus (TBE), West Nile virus (WN), Saint Louis encephalitis virus (SLE), and about 70 other disease causing viruses.
  • Flaviviruses are small, enveloped viruses containing a single, positive-strand RNA genome.
  • Ten gene products are encoded by a single open reading frame and are translated as a polyprotein organized in the order: capsid (C), “preMembrane” (prM, which is processed to “Membrane” (M) just prior to virion release from the cell), “envelope” (E), followed by non-structural (NS) proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (reviewed in Chambers, T. J. et al., Annual Rev Microbiol (1990) 44:649-688; Henchal, E. A. and Putnak, J. R., Clin Microbiol Rev . (1990) 3:376-396). Individual flaviviral proteins are then produced through precise processing events mediated by the host as well as virally encoded proteases.
  • the envelope of flaviviruses is derived from the host cell membrane and contains the virally-encoded membrane anchored membrane (M) and envelope (E) glycoproteins.
  • M membrane anchored membrane
  • E envelope glycoprotein
  • the E glycoprotein is the largest viral structural protein and contains functional domains responsible for cell surface attachment and intra-endosomal fusion activities. It is also a major target of the host immune system, inducing the production of virus neutralizing antibodies, which are associated with protective immunity.
  • Dengue viruses are transmitted to man by mosquitoes of the genus Aedes , primarily A. aegypti and A. albopictus . Infection by dengue viruses leads to a diverse clinical picture ranging from an inapparent or mild febrile illness, through classical dengue fever (DF), to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Dengue fever is characterized by high fever, headache, joint and muscle pain, rash, lymphadenopathy and leucopenia (Gibbons, R. V. and D. W. Vaughn, British Medical Journal (2002) 324:1563-1566).
  • DF dengue fever
  • DHF/DSS dengue hemorrhagic fever/dengue shock syndrome
  • DHF/DSS is a more severe form of infection more common in children, marked by vascular permeability and/or severe hemorrhagic manifestations ranging from the presence of petechiae and ecchymosis to spontaneous severe hemorrhage and profound shock. Without diagnosis and prompt medical intervention, the sudden onset and rapid progression of DHF/DSS can be fatal if untreated.
  • Dengue viruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality with an estimated one hundred million dengue infections occurring annually including at least 36 million cases of dengue fever and 250,000 to 500,000 cases of DHF/DSS (Gubler, D. J., Clin. Microbiol. Rev . (1998) 11:480-496; Gibbons, supra). With the global increase in population, urbanization of the population especially throughout the tropics, and the lack of sustained mosquito control measures, the mosquito vectors of dengue have expanded their distribution throughout the tropics, subtropics, and some temperate areas, bringing the risk of dengue infection to over half the world's population.
  • flavivirus vaccines have been met with mixed success.
  • live-attenuated, inactivated whole virus has been available for decades and more recently a live attenuated vaccine for Japanese encephalitis has been registered in various countries around the world.
  • inactivated whole virus vaccines has been demonstrated for TBE and JE viruses with several registered products available. Heinz et al. Flavivirus and flavivirus vaccines. Vaccine 30: 4301-06 (2012).
  • Ivy et al. U.S. Pat. No. 6,432,411 disclose a tetravalent subunit vaccine comprising DEN1-4 80% E (the peptide region of DEN1-4 corresponding to amino acids 1-395 of the DENV-2 envelope polypeptide) proteins. Ivy et al, supra, also report compositions comprising DENV 1-4 80% E and ISCOMATRIX® adjuvant. Coller et al. (WO 2012/154202) disclose tetravalent formulations comprising DEN1-4 80% E of DEN 1-4.
  • Inactivated viruses may also be used as potential vaccine candidates or as components of an effective vaccine (Putnak et al. Vaccine 23: 4442-4452 (2005), U.S. Pat. Nos. 6,190,859, 6,254,873 and Sterner et al. WO 2007/002470).
  • Compositions comprising a live attenuated dengue virus vaccine and a non-replicating dengue vaccine are disclosed in International Patent Application No. PCT/US14/042625 (WO2014/204892).
  • Whole viruses are one of the commonly used antigens in several vaccine products due to their ability to generate humoral and cellular immune responses.
  • Vaccine products containing whole viruses are challenging to stabilize as these are sensitive to heat, freeze/thaw and other processing stresses leading to significant potency losses.
  • These products are typically stored frozen (below ⁇ 20° C.) or as dried powder.
  • Frozen products are not easy to store and distribute as they need a stringent cold-chain requirement to prevent potency loss. Drying of whole viruses, especially enveloped viruses, often leads to significant loss of potency due to the freezing and drying stresses encountered during the drying process. Therefore, there is a need in the art to generate stable formulations of Dengue virus.
  • the current invention provides stable formulations of live attenuated dengue vaccine.
  • the addition of a cellulose derivative and a sugar alcohol or glycol improved stability and/or yield after drying.
  • the addition of sugar of at least 150 mg/ml improved stability and/or yield after microwave drying.
  • the invention provides a formulation comprising a live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus (LACV), a buffer at pH about 6.5 to 8.5, a sugar, a glycol or sugar alcohol, and a cellulose derivative selected from the group consisting of carboxymethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethyl cellulose (2-HEC), crosscarmellose, and methyl cellulose, or a pharmaceutically acceptable salt thereof optionally an alkali or alkaline salt, and optionally an amino acid selected from the group consisting of Ala, Asp, His, Leu, Lys, Gln, Pro or Glu, or a combination thereof.
  • LAV live attenuated dengue virus
  • LACV live attenuated chimeric flavivirus
  • the buffer is selected from the group consisting of succinate, histidine, phosphate, TRIS, Bis-Tris, MES, MOPS, HEPES, acetate and citrate, or a combination thereof.
  • the alkali or alkaline salt is magnesium chloride, calcium chloride, potassium chloride, sodium chloride or a combination thereof.
  • the sugar is trehalose or sucrose.
  • the cellulose derivative is a pharmaceutically acceptable salt of carboxymethyl cellulose.
  • the glycol is selected from the group consisting of propylene glycol, polypropylene glycol, ethylene glycol, polyethylene glycol, and polyethylene glycol monomethyl ethers.
  • the sugar alcohol is glycerol.
  • the formulation comprises a live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus at about 100-10,000,000 pfu/ml, a buffer at pH about 6.5 to 8.5, about 50-300 mg/ml sugar, about 2.5-10.0 mg/ml propylene glycol (PG) or glycerol, and about 0.3-10 mg/ml sodium carboxymethylcellulose (sodium CMC), optionally about 10-150 mM NaCl, and optionally about 10-100 mM amino acid selected from the group consisting of Ala, Asp, His, Leu, Lys, Gln, Pro or Glu, or a combination thereof a live attenuated dengue vaccine at about 100-100,000 pfu/ml, about 5-300 mM histidine, TRIS, Bis-Tris or phosphate buffer, or a combination thereof at pH about 7.0 to 8.0, about 50-300 mg/m
  • the formulation comprises a live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus at about 600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pH about 7.5-8, about 90 mg/ml sucrose, about 110 mg/ml trehalose, about 5 mg/ml propylene glycol, about 5 mg/ml sodium carboxymethylcellulose with average molecular weight of about 90,000, about 50 mM NaCl, and about 25 mM Leu.
  • the formulation further comprises a surfactant selected from poloxamer 188 and poloxamer 407 at about 0.0001 to 5% w/v.
  • the invention also provides a formulation that comprises a live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus at about 100-10,000,000 pfu/ml, a buffer at pH about 6.5 to 8.5, a sugar at about 150-300 mg/ml, a carrier selected from the group consisting of polyvinylpyrrolidone (PVP), carboxymethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethyl cellulose (2-HEC), crosscarmellose, methyl cellulose or a pharmaceutically acceptable salt thereof, Human Serum Albumin (HSA) and gelatin; optionally an alkali salt or alkaline salt at about 5-100 mM; and optionally an amino acid Gln, Pro or Glu, or a combination thereof.
  • LAV live attenuated dengue virus
  • chimeric flavivirus at about 100-10,000,000 pf
  • the buffer is selected from the group consisting of succinate, histidine, phosphate, TRIS, Bis-Tris, MES, MOPS, HEPES, acetate and citrate, or a combination thereof.
  • the alkali or alkaline salt is magnesium chloride, calcium chloride, potassium chloride, sodium chloride or a combination thereof.
  • the sugar is trehalose or sucrose, or a combination thereof. In one embodiment, the sucrose to trehalose ratio is between 1:1 to 1:4.
  • the carrier is a sodium carboxymethyl cellulose, HPMC, HSA or gelatin.
  • the invention provides formulations of a live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus at about 200-100,000 pfu/ml, a buffer at pH about 6.5-8.0, about 150-300 mg/ml sugar as a combination of sucrose and trehalose, about 0.3 to 40 mg/ml sodium CMC, HSA, HPMC or gelatin, optionally about 10-100 mM alkali or alkaline salt, and optionally about 5-25 mM glutamic acid; a live attenuated dengue vaccine at about 600-20,000 pfu/ml, about 5-300 mM histidine, TRIS or phosphate buffer, or a combination thereof at pH about 7.0 to 8.0, about 50-100 mg/ml sucrose, about 90-200 mg/ml trehalose, about 0.3-10 mg/ml sodium CMC or about 10-40 mg/ml gelatin
  • the formulation further comprises an aluminum adjuvant.
  • the above formulations can be frozen or lyophilized, or reconstituted in solution.
  • the reconstitution is performed with about 0.5-1.0 ml saline solution, water or Bacteriostatic Water for Injection (BWFI) and optionally a diluent comprising an aluminum adjuvant.
  • BWFI Bacteriostatic Water for Injection
  • the formulation is the aqueous solution prior to lyophilization or microwave vacuum drying.
  • the live attenuated dengue vaccine comprises tetravalent live attenuated dengue virus or live attenuated chimeric flavivirus.
  • the LAV or the LACV comprise a viral genome that contains a deletion of about 30 nucleotides corresponding to the TL-2 stem-loop structure of the 3′ untranslated (UTR) region; which reduces the replicative capacity of the virus.
  • the live attenuated dengue virus is an LAV that comprise a viral genome that contains a deletion of about 30 nucleotides corresponding to the TL-2 stem-loop structure of the 3′ untranslated (UTR) region, and is immunogenic against dengue serotype 3, wherein the viral genome of the LAV further contains a deletion of nucleotides upstream from the ⁇ 30 deletion corresponding to the TL-3 structure of the 3′UTR.
  • UTR untranslated
  • the live attenuated dengue vaccine is a live attenuated tetravalent vaccine comprising a DEN1 ⁇ 30 virus, a DEN2/4 ⁇ 30 virus (a DEN2 ⁇ 30LACV on a DEN4 backbone), a DEN3 ⁇ 30 virus and a DEN4 ⁇ 30 virus.
  • the live attenuated dengue virus is an LAV comprising rDEN1 ⁇ 30-1545, rDEN2/4 ⁇ 30 (ME)-1495,7163, rDEN3 ⁇ 30/31-7164, and rDEN4 ⁇ 30-7132,7163,8308.
  • FIG. 1 Effect of Sodium CMC, PG, amino acids on DENV4 lyophilization yield for DEN4 formulations.
  • FIG. 2 Effect of Sodium CMC, PG, amino acids on DENV4 stability for DEN4 formulations.
  • Formulation 26 (*) was not tested due to cake collapse after storage at 25° C.
  • FIG. 3 Effect of sugar alcohol on DENV4 lyophilization yield for DEN4 formulations.
  • FIG. 4 Effect of sugar alcohol on DENV4 stability for DEN4 formulations.
  • FIG. 5 Effect of pH on DENV4 lyophilization yield for DEN4 formulations.
  • FIG. 6 Effect of pH on DENV4 stability for DEN4 formulations.
  • FIG. 7 Effect of buffer on DENV4 lyophilization yield for DEN4 formulations.
  • FIG. 8 Effect of buffer on DENV4 stability for DEN4 formulations.
  • FIG. 9 Effect of NaCl concentration on DENV4 lyophilization yield. for DEN4 formulations.
  • FIG. 10 Effect of NaCl concentration on DENV4 stability for DEN4 formulations.
  • FIG. 11 Effect of propylene glycol and glyercol on lyophilization yields of Dengue serotypes.
  • FIG. 12 Effect of propylene glycol and glycerol on stability of Dengue serotypes.
  • FIG. 13 Effect of L-15 concentration on relative potency for frozen, microwave dried (MVD) and lyophilized (lyo) DEN1 formulations.
  • FIG. 14 Effect of L-15 concentration on relative potency for frozen, microwave dried (MVD) and lyophilized (lyo) DEN2 formulations.
  • FIG. 15 Effect of L-15 concentration on relative potency for frozen, microwave dried (MVD) and lyophilized (lyo) DEN3 formulations.
  • FIG. 16 Effect of L-15 concentration on relative potency for frozen, microwave dried (MVD) and lyophilized (lyo) DEN4 formulations.
  • FIG. 17 Relative potency for frozen, microwave dried (MVD) and lyophilized (lyo) DEN1 formulations.
  • FIG. 18A-B A) Stability of tetravalent formulations at 37° C. after one week. B) Stability of tetravalent formulations at 25° C. after one month.
  • FIG. 19A-D Stability of tetravalent formulations (DEN1-DEN4) at 2-8° C. tested every 3 months up to 18 months.
  • bulking agents comprise agents that provide the structure of the freeze-dried product.
  • Common examples used for bulking agents include mannitol, glycine, and lactose.
  • bulking agents may also impart useful qualities in regard to modifying the collapse temperature, providing freeze-thaw protection, and enhancing the protein stability over long-term storage. These agents can also serve as tonicity modifiers.
  • the “Dengue Virus reference sample” has the same dengue virus formulation components and ratios as the dengue virus formulation test sample, and refers to the solid composition immediately after drying the dengue virus formulation under the same conditions as the dengue virus formulation test sample (i.e. lyophilization, microwave dried, lyosphere dried), or the foregoing dried solid composition stored at conditions where there is no or minimal infectivity loss of the dengue virus (i.e. stored at or below ⁇ 70° C.); or the frozen solid dengue virus formulation at ⁇ 70° C.
  • “Glycol” refers to a chemical compound with two hydroxyl groups.
  • “Infectivity loss” refers to comparing the loss of viral replication of a dengue virus test sample to a dengue virus reference sample using methods known in the art. In one embodiment, the infectivity loss is measured using a dengue relative infectivity assay. In another embodiment, the infectivity loss is measured using a plaque assay.
  • “Lyosphere,” as used herein, refers to dried frozen unitary bodies comprising a therapeutically active agent which are substantially spherical or ovoid-shape.
  • the lyosphere diameter is from about 2 to about 12 mm, preferably from 2 to 8 mm, such as from 2.5 to 6 mm or 2.5 to 5 mm.
  • the volume of the lyosphere is from about 20 to 550 ⁇ L, preferably from 20 to 100 ⁇ L, such as from 20 to 50 ⁇ L.
  • the size of the lyosphere can be described with respect to its aspect ratio, which is the ratio of the longer dimension to the shorter dimension.
  • the aspect ratio of the lyospheres can be from 0.5 to 2.5, preferably from 0.75 to 2, such as from 1 to 1.5.
  • Microwave Vacuum Drying refers to a drying method that utilizes microwave radiation (also known as radiant energy or non-ionizing radiation) for the formation of dried vaccine products (preferably, ⁇ 6% moisture) of a vaccine formulation through sublimation.
  • microwave radiation also known as radiant energy or non-ionizing radiation
  • the microwave drying is performed as described in US2016/0228532.
  • the microwave radiation is in traveling wave format.
  • a “reconstituted solution”, as used herein, is one that has been prepared by dissolving dried virus in solid form (such as a lyophilized cake) in a diluent such that the virus is dispersed in the reconstituted solution.
  • the reconstituted solution is suitable for administration, (e.g. intramuscular administration), and may optionally be suitable for subcutaneous administration.
  • x % (w/v) is equivalent to x g/100 ml (for example 5% w/v equals 50 mg/ml).
  • live attenuated dengue virus also referred to as “LAV” herein, means the ability of the dengue virus to cause disease is reduced compared to wild-type dengue virus.
  • LAV live attenuated dengue virus
  • the LAV may contain these naturally occurring mutations, in addition to mutations introduced for cloning purposes.
  • the LAV may be a homogenous or heterogeneous population with none, or one or more of these mutations.
  • live attenuated chimeric virus (alternatively “live attenuated chimeric flavivirus”) or “LACV” refers to a live attenuated chimeric virus wherein the viral genome comprises a backbone of a first flavivirus (including C, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 genes) and the preMembrane (prM) and envelope (E) genes of a second flavivirus, wherein the second flavivirus is selected from DENV1, DENV2, DENV3 or DENV4.
  • the first flavivirus can be a different dengue serotype or another flavivirus, such as yellow fever virus.
  • ⁇ 30 LAV refers to a live attenuated DEN1, DEN2, DEN3, or DEN4 virus, wherein the LAV comprises a viral genome that contains a deletion of about 30 nucleotides (nt) corresponding to the TL2 stem-loop structure of the 3′ untranslated (UTR) region from about nt 143 to about nt 172, which reduces the replicative capacity of the virus (see WO 03/092592 and Whitehead et al., U.S. Pat. No. 8,337,860).
  • LACV refers to a live attenuated chimeric flavivirus (LACV) from DENV 1-4 wherein the LACV comprises a viral genome that contains a deletion of about 30 nt corresponding to the TL2 stem-loop structure of the 3′ UTR region from about nt 143 to about nt 172, which reduces the replicative capacity of the virus (see WO 03/092592 and Whitehead et al., U.S. Pat. No. 8,337,860).
  • ⁇ 30/ ⁇ 31 LAV refers to a live attenuated DEN1, DEN2, DEN3, or DEN4 virus, wherein the viral genome comprises a deletion of about 30 nt of the TL2 stem-loop structure of the 3′ UTR, and further comprises a separate, noncontiguous, upstream deletion of about 31 nt at about nt 258-228 of the 3′ UTR which removes sequence up to and including the TL-3 homologous structure so that the deletion extends as far as the 5′ boundary of the TL-3 homologous structure of the dengue 3′UTR. See Whitehead et al., U.S. Pat. No. 8,337,860.
  • the DEN3 LAV comprises the ⁇ 30/ ⁇ 31 mutations.
  • ⁇ 30/ ⁇ 31 LACV refers to a live attenuated chimeric DEN1, DEN2, DEN3, or DEN4 virus as described above, wherein the viral genome of the chimeric virus comprises a 30 nt deletion of the TL2 stem-loop structure of the 3′ UTR, and further comprises a separate, noncontiguous, upstream 31 nt deletion of the 3′ UTR, which deletes the TL-3 structure, as described above.
  • LATV live attenuated tetravalent dengue vaccine
  • LATV vaccine refers to a vaccine comprising an effective amount of a DEN1 LAV or LACV, a DEN2 LAV or LACV, a DEN3 LAV or LACV and a DEN4 LAV or LACV.
  • at least one of the dengue LAVs or LACVs comprises the ⁇ 30 mutation of the TL-2 structure in the 3′ UTR, as described above and in WO 03/092592.
  • the LATV comprises the following features: (1) rDEN1 ⁇ 30, which is a DENV1 LAV wherein the DENV1 viral genome comprises a 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR; (2) rDEN2/4 ⁇ 30, which is a DENV2 LACV comprising the DENV2 prM and E genes on a DENV4 backbone, wherein the DEN4 backbone comprises a 30-nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR; (3) rDEN3 ⁇ 30/ ⁇ 31, which is a DENV3 LAV wherein the DENV3 viral genome comprises a 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR and a separate, noncontiguous, upstream 31 nt deletion corresponding to the TL-3 structure of the 3′ UTR; and (4) rDEN4 ⁇ 30, which is a DENV4 LAV wherein the DENV4 viral genome comprises a 30 nt deletion
  • Non-replicating vaccine refers to a dengue virus vaccine for the prevention or treatment of dengue virus infection or the clinical symptoms thereof, selected from a recombinant subunit vaccine, an inactivated vaccine, a conjugate vaccine, or a DNA vaccine.
  • “Inactivated vaccine” refers to a vaccine comprising an effective amount of a killed or inactive whole dengue virus and a pharmaceutically acceptable carrier, wherein the virus is inactivated by any means, including with chemicals, heat or radiation.
  • An inactivated vaccine has a low residual infectivity following inactivation, e.g. ⁇ 5 plaque forming units (PFU's)/mL after inactivation.
  • PFU's plaque forming units
  • there is very low amount of residual infectivity following inactivation e.g. ⁇ 4 PFU's/mL, ⁇ 3 PFU's/mL, or ⁇ 2 PFU's/mL, ⁇ 1 PFU/mL, ⁇ 0.5 PFU/mL, or ⁇ 0.1 PFU/mL.
  • the PFU's of a particular vaccine may be determined, for example, by using a plaque assay, an immunostaining assay, or other method known in the art for detecting viral infectivity.
  • Conjugate vaccine refers to a vaccine comprising a dengue antigen covalently attached to a carrier protein.
  • a “DNA vaccine” is a vaccine comprising a sequence of nucleotides that encodes a dengue protein antigen, including dengue proteins, dengue protein fragments, and dengue fusion proteins, and variants thereof.
  • DNA vaccines comprise a plasmid (e.g. a DNA or viral plasmid) comprising a sequence of nucleotides that encode an antigen of interest, operably linked to a promoter.
  • Subunit vaccine refers to a vaccine that includes one or more dengue antigen components, but not complete dengue viruses, such as dengue immunogenic epitopes, dengue proteins, dengue antigen fusion proteins, including fusions of different dengue serotype antigens, or dengue protein fragments.
  • Subunit vaccines as used herein, can be monovalent (comprise a single dengue antigen) or multivalent (comprise more than one antigen component). In preferred embodiments, the subunit vaccine is tetravalent.
  • the term “prime-boost” refers to a therapeutic regimen comprising (1) administration to a patient in need thereof a first dengue virus vaccine composition, wherein the composition comprises (a) at least one live attenuated dengue virus (LAV) or live attenuated chimeric flavivirus (LACV), and (b) a pharmaceutically acceptable carrier; (2) waiting for a predetermined amount of time to pass; and (3) administration to the patient of a second dengue virus vaccine composition or non-replicating dengue vaccine.
  • the second dengue virus vaccine composition can be the same or different from the first dengue virus vaccine composition.
  • the second dengue virus vaccine is a live attenuated dengue vaccine or a recombinant dengue subunit vaccine.
  • the dengue virus vaccines used in the compositions of the invention are useful for inducing a virus neutralizing antibody response to the homologous dengue viruses in human patients.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. Individuals or patients “in need of” treatment include those already with a dengue infection, whether or not manifesting any clinical symptoms, as well as those at risk of being infected with dengue.
  • Treatment of a patient with the dengue vaccine compositions of the invention includes one or more of the following: inducing/increasing an immune response against dengue in the patient, inducing a virus neutralizing antibody response against one or more dengue viruses, preventing, ameliorating, abrogating, or reducing the likelihood of the clinical manifestations of dengue in patients who have been infected with dengue, preventing or reducing the likelihood of developing dengue fever, DHF, or DSS and/or other disease or complication associated with dengue infection, reducing the severity or duration of the clinical symptoms of dengue infection and/or other disease or complication associated with dengue, and preventing or reducing the likelihood of dengue infection.
  • pharmaceutically effective amount means sufficient vaccine composition is introduced to a patient to produce a desired effect, including, but not limited to: inducing/increasing an immune response against dengue in the patient, inducing/increasing a virus neutralizing antibody response against dengue in a patient, preventing or reducing the likelihood of dengue infection, preventing or reducing the likelihood of dengue recurrent infection, preventing, ameliorating or abrogating the clinical manifestations of dengue infection in patients who have been infected with dengue, preventing dengue fever, DHF and/or DSS, or reducing the severity or duration of disease associated with dengue.
  • this level may vary.
  • immune response refers to a cell-mediated (T-cell) immune response and/or an antibody (B-cell) response.
  • the term “patient” refers to a mammal capable of being infected with a dengue virus, such as DEN1, DEN2, DEN3, or DEN4, that is to receive the dengue vaccine compositions described herein, including both immunocompetent and immunocompromised individuals.
  • the patient is a human.
  • a “patient” includes those already infected with dengue, either through natural infection or vaccination or those that may subsequently be exposed.
  • ISCOM-like adjuvant is an adjuvant comprising an immune stimulating complex (ISCOM), which is comprised of a saponin, cholesterol, and a phospholipid, which together form a characteristic caged-like particle, having a unique spherical, caged-like structure that contributes to its function (for review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25 (1996)).
  • ISCOM immune stimulating complex
  • This term includes both ISCOMTM adjuvants, which are produced with an antigen and comprise antigen within the ISCOMTM particle and ISCOMTM matrix adjuvants, which are hollow ISCOM-type adjuvants that are produced without antigen.
  • the ISCOM-type adjuvant is an ISCOMTM matrix particle adjuvant, such as ISCOMATRIXTM, which is manufactured without antigen (ISCOMTM and ISCOMATRIXTM are registered trademarks of CSL Limited, Parkville, Australia).
  • rDEN1 ⁇ 30-1545 refers to a recombinant dengue 1 virus wherein the viral genome comprises (1) a 30 nt deletion of the TL2 stem-loop structure of the 3′ UTR and (2) a substitution at nucleotide position 1545 to G, which occurred after adaptation of the virus to growth in Vero cells.
  • rDEN2/4 ⁇ 30(ME)-1495,7163 refers to a recombinant chimeric dengue 2/4 virus, wherein the viral genome comprises: (1) a dengue 4 backbone (C, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 genes) comprising (i) a 30 nt deletion of the TL2 stem-loop structure of the 3′ UTR, and (ii) substitutions at nucleotide position 1495 to U and 7163 to C, which occurred after adaptation of the virus to growth in Vero cells, and (2) dengue 2 prM and E genes.
  • C dengue 4 backbone
  • NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 genes comprising (i) a 30 nt deletion of the TL2 stem-loop structure of the 3′ UTR, and (ii) substitutions at nucleotide position 1495 to U and 7163
  • rDEN3 ⁇ 30/31-7164 refers to a recombinant dengue 3 virus wherein the viral genome comprises: (1) a 30 nt deletion of the TL2 stem-loop structure of the 3′ UTR, (2) a separate, 31 nt deletion in the 3′UTR, upstream of the ⁇ 30 mutation, that deletes the TL-3 structure and (3) a substitution at nucleotide position 7164 to C, which occurred after adaptation of the virus to growth in Vero cells.
  • rDEN4 ⁇ 30-7132,7163,8308 refers to a recombinant dengue 4 virus wherein the viral genome comprises: (1) a 30 nt deletion of the TL2 stem-loop structure of the 3′ UTR and (2) substitutions at nucleotide position 7132 to U, 7163 to C and 8308 to G, which occurred after adaptation of the virus to growth in Vero cells.
  • V180 refers to a tetravalent subunit vaccine comprised of truncated envelope glycoproteins (DEN-80E) from each of the 4 dengue virus serotypes (DENV1, DENV2, DENV3, and DENV4), wherein the E proteins each constitute approximately 80% of the length of wild type E starting from amino acid residue 1 at its N-terminus, such that said E protein is secretable into growth medium when expressed recombinantly in a host cell. See Coller et al. WO 2012/154202.
  • C is the dengue capsid gene
  • DENV dengue virus
  • DF dengue fever
  • DHF dengue hemorrhagic fever
  • DSS dengue shock syndrome
  • h hours
  • GMT geometric mean titer
  • IM intramuscular
  • IMX is IscomatrixTM
  • JE Japanese encephalitis
  • LAV live attenuated virus
  • NS used in NS1-NS5
  • nt is nucleotide
  • PFU plaque forming units
  • prM is the dengue preMembrane gene
  • SC is subcutaneous
  • TBE tick-borne encephalitis
  • UTR untranslated region
  • WN alternately WNV
  • YF West Nile Virus
  • YF alternatively YFV
  • wt wild type.
  • the dengue virus vaccine compositions of the invention comprise a live attenuated dengue vaccine comprising at least one LAV, selected from the group consisting of dengue virus type 1 (DEN1), dengue virus type 2 (DEN2), dengue virus type 3 (DEN3) and dengue virus type 4 (DEN4), or LACV.
  • the LAV or LACV comprises a viral genome that comprises a TL-2 ⁇ 30 modification in the 3′UTR, and wherein the LAV or LACV: induces an immune response against dengue, induces a virus neutralizing antibody response against dengue, protects against or reduces the likelihood of infection or reduces the severity or duration of the clinical manifestations thereof.
  • the live attenuated dengue vaccine is monovalent, bivalent, trivalent or tetravalent, i.e. induces an immune response against or protects against one, two, three or four of DEN serotypes 1-4, respectively.
  • the live attenuated dengue vaccine is tetravalent, i.e. induces an immune response against or protects against DEN serotypes 1-4 and comprises a DEN1, a DEN2, a DEN3 and a DEN4 component, wherein each component is either an LAV or an LACV.
  • the live attenuated dengue vaccine is a tetravalent LAV or “LATV” (i.e. comprises live attenuated dengue viruses from DENV 1-4, or live attenuated chimeric flaviviruses from DENV 1-4, as defined herein, or a combination thereof, wherein at least one of the LAVs or LACVs is a ⁇ 30LAV or a ⁇ 30LACV).
  • the live attenuated dengue vaccine is tetravalent and comprises at least one chimeric flavivirus; wherein the chimeric flavivirus comprises a viral genome that contains nucleotide sequences encoding the prM and E proteins of a single dengue virus serotype and nucleotide sequences encoding the capsid and non-structural proteins of a different flavivirus, wherein the chimeric flavivirus is attenuated.
  • the capsid and nonstructural proteins of the chimeric flavivirus is from a different dengue serotype than the prM and E proteins.
  • each LAV or LACV component of a LATV of the invention comprises a live attenuated virus which is independently either an attenuated chimeric flavivirus or an attenuated dengue virus comprising the TL-2 ⁇ 30 modification in the 3′UTR of the viral genome. Attenuation of the dengue virus is achieved through the TL-2 ⁇ 30 modification.
  • additional attenuating mutations may also be included in one or more components of the vaccine, including, but not limited to: mutations at positions 1495, 1545, 7132, 7163, 7164 and 8308. Attenuating mutations can be achieved by different techniques, including methods known in the art such as through serial passage on tissue culture or through more defined genetic manipulations.
  • compositions of the invention comprise a tetravalent live attenuated dengue vaccine (LATV).
  • LATV tetravalent live attenuated dengue vaccine
  • Such tetravalent live attenuated vaccine can comprise four attenuated dengue viruses (LAVs), three LAVs and one attenuated chimeric flavivirus strain (LACV), two dengue LAVs and two LACVs, one dengue LAV and three LACVs, or four LACVs.
  • the LATV comprises the following features: (1) rDEN1 ⁇ 30, which is a DENV1 LAV wherein the DENV1 viral genome comprises a 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR; (2) rDEN2/4 ⁇ 30, which is a DENV2 LACV comprising the DENV2 prM and E genes on a DENV4 backbone, wherein the DEN4 backbone comprises a 30-nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR; (3) rDEN3 ⁇ 30/ ⁇ 31, which is a DENV3 LAV wherein the DENV3 viral genome comprises a 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR and a separate, noncontiguous, upstream 31 nt deletion corresponding to the TL-3 structure of the 3′ UTR; and (4) rDEN4 ⁇ 30, which is a DENV4 LAV wherein the DENV4 viral genome comprises a 30 nt deletion
  • each chimeric flavivirus comprises a viral genome that comprises nucleotide sequences encoding the prM and E proteins of a single dengue virus serotype and nucleotide sequences that encode the capsid and non-structural proteins (i.e. “the backbone”) of a different flavivirus, wherein each of the chimeric flaviviruses are attenuated.
  • Methods for construction of a recombinant live attenuated flavivirus strain may comprise the use of a known attenuated strain as a base, wherein the method comprises substituting the appropriate genes (prM and E) from a related virus of interest for the equivalent genes of the base virus.
  • chimeric virus is an intertypic chimeric based on an attenuated DEN-4 strain comprising prM and E genes of WNV (Bray, M. et al., J. Virol . (1996) 70:4162-4166; Chen, W., et al., J Virol . (1995) 69:5186-5190; Bray, M. and Lai, C.-J., Proc. Natl. Acad. Sci. USA (1991) 88:10342-10346; Lai, C. J. et al., Clin. Diagn. Virol . (1998) 10:173-179).
  • a chimeric yellow fever vaccine can be constructed comprising a yellow fever backbone by replacing the genes coding for prM and E proteins from any yellow fever strain, for example, YFV 17D, with those of a Dengue serotype. After DNA cloning, RNA is transcribed and transfected into Vero cells to obtain chimeric viruses possessing the YFV 17D replication machinery and the external coat of the relevant Dengue virus.
  • the live attenuated dengue vaccine comprises (1) at least one chimeric flavivirus comprising the prM and E proteins of a single dengue serotype and a yellow fever backbone and (2) at least one LAV or LACV which comprises a viral genome comprising a 30-nucleotide deletion of the TL-2 stem-loop structure of the 3′UTR.
  • Chimeric live attenuated flaviviruses useful in the compositions of the invention may also comprise a dengue chimeric virus, wherein the viral genome comprises prM and E genes of a single dengue virus serotype and the capsid and nonstructural genes of a different dengue virus serotype.
  • the chimeric virus comprises a backbone from a second dengue serotype
  • the dengue backbone comprises a deletion of about 30-nucleotides of the 3′UTR that corresponds to the TL-2 stem-loop structure and may optionally comprise additional attenuating mutations.
  • any attenuated dengue virus or wild-type dengue virus can be used as the backbone of the chimeric virus, by introduction of a 30-nucleotide deletion of the TL-2 stem-loop structure to an attenuated dengue backbone or wild-type dengue viral backbone.
  • Attenuation of a dengue virus backbone can be achieved through serial passage, through the introduction of defined genetic mutations, or through the use of known attenuated dengue strains.
  • Dengue chimeric vaccines are described, for example, in Whitehead et al. WO 03/092592.
  • the live attenuated vaccine comprises a chimeric flavivirus wherein the capsid and nonstructural proteins are from a different dengue serotype than the prM and E proteins.
  • the dengue virus vaccine compositions of the invention comprise an effective amount of live attenuated virus vaccine.
  • the potency of the live attenuated dengue vaccine is from 10 to about 1 ⁇ 10 7 plaque forming units (PFU's).
  • the potency of the live attenuated dengue vaccine is from about 1 ⁇ 10 2 to about 1 ⁇ 10 6 PFU's.
  • the potency of the live attenuated dengue vaccine is from about 1 ⁇ 10 3 to about 1 ⁇ 10 5 PFU's.
  • Viral plaque assays determine the number of plaque forming units (pfu) in a virus sample. Briefly, in a dengue immunoplaque assay, a confluent monolayer of host cells (e.g., Vero cells) is infected with dengue virus at varying dilutions and covered with a semi-solid overlay medium, containing methylcellulose, to prevent the virus infection from spreading indiscriminately. The virus infected cell(s) will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated.
  • host cells e.g., Vero cells
  • the infected cells will form a plaque (a group of infected Vero cells surrounded by uninfected cells) which can be seen visually after fixing and immune-staining using anti dengue serotype specific monoclonal antibodies (mAb). Plaques are counted and the results, in combination with the dilution factors, are used to calculate the number of plaque forming units per mL (pfu/mL) in the samples.
  • the dengue potency result in pfu/mL represents the number of infectious particles within the sample and is based on the assumption that each plaque formed is representative of one infectious virus particle.
  • the formulations further comprise a recombinant dengue subunit vaccine which comprises one or more dengue antigen proteins.
  • the recombinant dengue subunit vaccine comprises one or more dengue proteins, fusion proteins, or a fragment or fragments thereof.
  • the recombinant dengue subunit vaccine comprises dengue envelope or E protein, or a fragment thereof.
  • the recombinant dengue subunit vaccine is tetravalent, i.e. targets an immune response against all four dengue serotypes.
  • a recombinant dengue subunit vaccine can comprise four recombinant dengue proteins or less than four, e.g. a recombinant DEN1 protein, a recombinant DEN2 protein, and a recombinant DEN3/4 fusion protein.
  • the recombinant dengue subunit vaccine comprises dengue virus envelope glycoprotein, or fragments thereof, of DEN1-4 (e.g.
  • Said subunit vaccine may optionally comprise an adjuvant, as described more fully below.
  • the recombinant dengue subunit vaccine comprises one or more purified dengue virus envelope (“E”) proteins, a pharmaceutically acceptable excipient, wherein the E proteins each constitute approximately 80% of the length of wild type E starting from amino acid residue 1 at its N-terminus, such that said E protein is secretable into growth medium when expressed recombinantly in a host cell and wherein the composition induces the production of neutralizing antibodies in human subjects.
  • the recombinant dengue subunit vaccine further comprises an effective amount of an adjuvant.
  • the DEN-4 E protein is dimeric (“DEN4-80EZip”), as described in U.S. Pat. No. 6,749,857 and WO 2012/154202.
  • the E proteins in the composition described above are recombinantly produced and expressed in insect host cells.
  • the E protein is recombinantly produced and expressed in Drosophila melanogaster Schneider 2 (S2) host cells.
  • the recombinant dengue virus E proteins of can be produced by means of a cell culture expression system that uses Drosophila Schneider 2 (S2) cells. This system has been demonstrated to produce recombinant dengue envelope proteins that maintain native-like structure (Cuzzubbo et al., Clin. Diagn. Lab. Immunol . (2001) 8:1150-55; Modis et al., Proc. Natl. Acad. Sci . (2003) 100:6986-91; Modis et al., Nature (2004) 427:313-9; Zhang et al., Structure (2004) 12 (9):1607-18).
  • S2 Drosophila Schneider 2
  • This expression system has also been shown to express other recombinant envelope proteins from other flaviviruses such as West Nile, Japanese Encephalitis, hepatitis C, and Tick Borne Encephalitis viruses.
  • the recombinant dengue envelope proteins may be truncated at the C-terminus, leaving 80% of the native envelope protein (“80E”).
  • 80E is defined as approximately the first 80% of consecutive amino acids of E protein starting at amino acid 1 of its N-terminus.
  • some embodiments of this aspect of the invention comprise truncated 80E proteins which consist of approximately 80% of the length of wild type E starting from amino acid residue 1 at its N-terminus.
  • the E proteins used in some embodiments of the invention delete the membrane anchor portion (approximately the last 10% of E at the carboxy end) of the protein.
  • truncated 80E proteins of use in specific embodiments of the invention consist of up to the first 90% of consecutive amino acids of E starting at amino acid 1 of its N-terminus, thus allowing it to be secreted into the extracellular medium, facilitating recovery.
  • the truncation may further delete the “stem” portion of the E protein that links the 80E portion with the membrane anchor portion; the stem portion does not contain notable antigenic epitopes and therefore is not included in the preferred antigens, DEN1-80E, DEN2-80E, DEN3-80E, DEN4-80E, or DEN4-80EZip. More than 90%, but less than 100%, of the E protein can be cloned and secreted, i.e., the protein can be 90%+ in length, carboxy truncated, and can include a portion of the membrane spanning domain so long as the truncated E protein is secretable.
  • “Secretable” means able to be secreted, and typically secreted, from the transformed cells in the expression system.
  • dengue E proteins that are useful in the compositions and methods of the present invention may vary from the 80% exemplified herein, as long as the protein is secretable.
  • the DEN E proteins are about 80% in length starting from the N-terminal amino acid of the envelope protein and ending at an amino acid in the range of the 393 rd to 401 st amino acid, for example, from amino acid 1 to amino acid 395 of dengue virus type 2.
  • the dengue E protein may be about 75%, about 85%, about 90%, about 95%, or about 98% of the consecutive amino acids of E starting at amino acid 1 of its N-terminus.
  • the DEN E protein is approximately 80% of consecutive amino acids of E protein starting at amino acid 1 of its N-terminus; such as DEN1-80E, as set forth in SEQ ID NO:1, DEN2-80E, as set forth in SEQ ID NO:2, DEN3-80E, as set forth in SEQ ID NO:3 and DEN4-80E, as set forth in SEQ ID NO:4.
  • the secreted E protein may further contain domains which facilitate dimerization, such as in the DEN4-80EZip protein, such that the immunogenicity of the recombinant protein is further enhanced.
  • An exemplary DEN4-80EZip protein comprises an amino acid sequence as set forth in SEQ ID NO:5.
  • the DEN1, DEN2, and DEN3 80E antigens included in the composition are monomeric and the DEN4 80E antigen is dimeric.
  • the DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E proteins in the composition are monomeric.
  • the DEN4 component is present in an amount that is about 1.5 to about 3 times the individual amounts of DEN1, DEN2, and DEN3 proteins, preferably about 2 times the amount of the DEN1, DEN2, and DEN3 components (proteins).
  • the ratio of DEN1:DEN2:DEN3:DEN4 antigens in the compositions is approximately 1:1:1:2.
  • the amount of each E protein in the composition is from about 0.5 ⁇ g to about 500 ⁇ g. In alternative embodiments, the amount of each E protein is from about 0.5 ⁇ g to about 450 ⁇ g, 0.5 ⁇ g to about 400 ⁇ g, 0.5 ⁇ g to about 350 ⁇ g, 0.5 ⁇ g to about 300 ⁇ g, 0.5 ⁇ g to about 250 ⁇ g, 0.5 ⁇ g to about 200 ⁇ g, 0.5 ⁇ g to about 150 ⁇ g, 0.5 ⁇ g to about 100 ⁇ g, 0.5 ⁇ g to about 50 ⁇ g, 5.0 ⁇ g to about 500 ⁇ g, 5.0 ⁇ g to about 450 ⁇ g, 5.0 ⁇ g to about 400 ⁇ g, 5.0 ⁇ g to about 350 ⁇ g, 5.0 ⁇ g to about 300 ⁇ g, 5.0 ⁇ g to about 250 ⁇ g, 5.0 ⁇ g to about 200 ⁇ g, 5.0 ⁇ g to about 150 ⁇
  • the amount of each E protein in the composition is from about 1.0 ⁇ g to about 100 ⁇ g. In still further embodiments, the amount of each E protein in the composition is selected from approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 ⁇ g.
  • Inactivated dengue vaccines herein comprise one or more whole inactivated dengue viruses and/or one or more inactivated dengue chimeric viruses.
  • the inactivated dengue vaccine is tetravalent and comprises whole inactivated DEN1, DEN2, DEN3 and DEN4.
  • the inactivated vaccine comprises four inactivated chimeric dengue viruses.
  • the inactivated vaccine is tetravalent and comprises one or more whole inactivated dengue viruses and one or more inactivated dengue chimeric viruses, e.g.
  • an inactivated whole DEN1 virus an inactivated whole DEN2 virus, an inactivated DEN3 chimeric virus and an inactivated DEN4 chimeric virus.
  • an inactivated whole or chimeric DEN viruses may be used in the tetravalent compositions and methods of the invention, as long as the vaccine composition targets all four dengue serotypes.
  • dengue vaccines useful in the compositions and methods of the invention are described in Putnak et al. Vaccine 23: 4442-4452 (2005), U.S. Pat. Nos. 6,190,859, 6,254,873 and Sterner et al. WO 2007/002470.
  • dengue virus strains and chimeric dengue strains/chimeric flavivirus strains can be inactivated for use in the compositions through methods known in the art, e.g., with chemicals, heat or radiation.
  • the present invention also relates to the above formulations comprising effective amounts of a live attenuated dengue vaccine and a non-replicating dengue vaccine
  • the live, attenuated dengue vaccine comprises at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus (LACV), wherein the LAV or LACV comprise a viral genome that comprises a 30-nucleotide deletion of the TL-2 stem-loop structure in the 3′UTR.
  • the non-replicating dengue vaccine of the dengue virus vaccine compositions of the invention are selected from a recombinant dengue subunit vaccine or an inactivated dengue vaccine.
  • the formulation is lyophilized, frozen, microwave dried or has lyospheres with effective amounts of a live attenuated dengue vaccine and a non-replicating dengue vaccine.
  • the formulation of live attenuated dengue vaccine is reconstituted with a liquid solution comprising the non-replicating dengue vaccine, for example V180.
  • the live attenuated and the non-replicating dengue vaccines are tetravalent (i.e. comprise DEN1, DEN2, DEN3, and DEN4 components or induce an immune response against DEN1, DEN2, DEN3, and DEN4).
  • adjuvants Co-administration of vaccines with compounds that can enhance the immune response against the antigen of interest, known as adjuvants, has been extensively studied. In addition to increasing the immune response against the antigen of interest, some adjuvants may be used to decrease the amount of antigen necessary to provoke the desired immune response or decrease the number of injections needed in a clinical regimen to induce a durable immune response and provide protection from disease.
  • the dengue virus vaccine formulations of the invention may employ an adjuvant.
  • the adjuvant of the formulations described herein can be any adjuvant that performs the desired function, as described above, and does not inactivate or significantly impact the titer of the LAV or LACV of the composition.
  • Aluminum-based compounds were determined to possess adjuvant activity over 60 years ago (for review, see Lindblad, E. B. Immunol. and Cell Biol. 82: 497-505 (2004); Baylor et al. Vaccine 20: S18-S23 (2002)). Aluminum adjuvants are generally regarded as safe when used at appropriate dosages. Many have been approved for administration into humans by regulatory agencies worldwide.
  • aluminum-based compounds such as aluminum hydroxide (Al(OH) 3 ), aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS), or so-called “alum” (KAl(SO 4 ).12H 2 O) (see Klein et al., Analysis of aluminum hydroxyphosphate vaccine adjuvants by Al MAS NMR., J. Pharm. Sci. 89(3): 311-21 (2000)), may be combined with the compositions provided herein.
  • the aluminum adjuvant is aluminum hydroxyphosphate or AAHS.
  • the aluminum adjuvant is an aluminum phosphate adjuvant, referred to herein as “APA”.
  • the adjuvant is aluminum hydroxide.
  • an effective and safe dose of aluminum adjuvant varies from 50 ⁇ g to 1.25 mg elemental aluminum per dose (100 ⁇ g/mL to 2.5 mg/mL concentration).
  • compositions comprising a live attenuated dengue virus vaccine and further comprising an aluminum adjuvant.
  • the dengue compositions comprise an adjuvant which comprises from about 50 ⁇ g to about 1.25 mg of elemental aluminum per dose of vaccine.
  • the aluminum adjuvant per dose of vaccine composition comprises an amount of elemental aluminum ranging from about 100 ⁇ g to about 1.0 mg, from about 100 ⁇ g to about 900 ⁇ g, from about 100 ⁇ g to about 850 ⁇ g, from about 100 ⁇ g to about 800 ⁇ g, from about 100 ⁇ g to about 700 ⁇ g, from about 100 ⁇ g to about 600 ⁇ g, from about 100 ⁇ g to about 500 ⁇ g, from about 100 ⁇ g to about 400 ⁇ g, from about 100 ⁇ g to about 300 ⁇ g, from about 100 to about 250 ⁇ g, from about 200 ⁇ g to about 1.25 mg, from about 200 ⁇ g to about 1.0 mg, from about 200 ⁇ g to about 900 ⁇ g, from about 200 ⁇ g to about 850 ⁇ g, from about 200 ⁇ g to about 800 ⁇ g, from about 200 ⁇ g to about 700 ⁇ g, from about 200 ⁇ g to about 600 ⁇ g, from about 200 ⁇ g to about 500 ⁇ g
  • adjuvants that may be used in conjunction with the dengue virus vaccine compositions of the invention, include, but are not limited to, adjuvants containing CpG oligonucleotides, or other molecules acting on toll-like receptors such as TLR4 and TLR9 (for reviews, see, Daubenberger, C. A., Curr. Opin. Mol. Ther. 9(1):45-52 (2007); Duthie et al., Immunological Reviews 239(1): 178-196 (2011); Hedayat et al., Medicinal Research Reviews 32(2): 294-325 (2012)), including lipopolysaccharide, monophosphoryl lipid A, and aminoalkyl glucosaminide 4-phosphates.
  • TLR4 and TLR9 for reviews, see, Daubenberger, C. A., Curr. Opin. Mol. Ther. 9(1):45-52 (2007); Duthie et al., Immunological Reviews 239(1): 178-196 (2011); Hedayat et al
  • Additional adjuvants useful in the compositions of the invention include immunostimulatory oligonucleotides (IMO's; see, e.g. U.S. Pat. Nos. 7,713,535 and 7,470,674); T-helper epitopes, lipid-A and derivatives or variants thereof, liposomes, calcium phosphate, cytokines, (e.g.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • HSP heat-shock protein
  • CD134 CD137
  • CoVaccine HT non-ionic block copolymers, incomplete Freund's adjuvant, chemokines, cholera toxin; E. coli heat-labile enterotoxin; pertussis toxin; muramyl dipeptide, muramyl peptide analogues, MF59, SAF, immunostimulatory complexes, biodegradable microspheres, polyphosphazene; synthetic polynucleotides.
  • adjuvants for use with the compositions described herein are adjuvants containing saponins (e.g. QS21), either alone or combined with cholesterol and phospholipid in the characteristic form of an ISCOM (“immune stimulating complex,” for review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25 (1996); and Skene and Sutton, Methods 40: 53-59 (2006)).
  • saponins e.g. QS21
  • Such adjuvants are referred to herein as “saponin-based adjuvants”.
  • the mutant toxins and/or toxin proteins are combined with an ISCOM-type adjuvant or “ISCOM”, which is an ISCOM matrix particle adjuvant, such as ISCOMATRIXTM, which is manufactured without antigen (ISCOMTM and ISCOMATRIXTM are the registered trademarks of CSL Limited, Parkville, Australia).
  • ISCOM ISCOM-type adjuvant
  • ISCOMATRIXTM an ISCOM matrix particle adjuvant, such as ISCOMATRIXTM, which is manufactured without antigen
  • the formulations or compositions of the invention comprise a live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus (LACV), a buffer at pH about 6.5 to 8.5, a sugar, a glycol or sugar alcohol, and a cellulose derivative selected from the group consisting of carboxymethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethyl cellulose (2-HEC), crosscarmellose, and methyl cellulose, or a pharmaceutically acceptable salt thereof optionally an alkali or alkaline salt, and optionally an amino acid selected from the group consisting of Ala, Asp, His, Leu, Lys, Gln, Pro or Glu, or a combination thereof.
  • LAV live attenuated dengue virus
  • LACV live attenuated chimeric flavivirus
  • a buffer at pH about 6.5 to 8.5 a sugar, a
  • the formulation comprises live attenuated dengue vaccine comprising at least one live attenuated dengue virus (LAV) or at least one live attenuated chimeric flavivirus at about 20-200,000,00 pfu/ml, a buffer at pH about 6.5 to 8.5, a sugar at about 150-300 mg/ml, a carrier selected from the group consisting of polyvinylpyrrolidone (PVP), carboxymethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethyl cellulose (2-HEC), crosscarmellose, methyl cellulose or a pharmaceutically acceptable salt thereof, Human Serum Albumin (HSA) and gelatin; optionally an alkali salt or alkaline salt at about 5-100 mM; and optionally an amino acid Gln, Pro or Glu, or a combination thereof.
  • LAV live attenuated dengue virus
  • chimeric flavivirus at about 20-200,000,00 pfu/ml
  • the live attenuated dengue vaccine is at a concentration of 100-10,000,000 pfu/ml, 100-100,000 pfu/ml, or 600-20,000 pfu/ml in the formulation. In another embodiment, the live attenuated dengue vaccine is at a concentration of 200-200,000 pfu/ml, 600-200,000 pfu/ml, or 600-100,000 pfu/ml in the formulation.
  • the cellulose derivative is anionic and forms a salt, for example carboxymethyl cellulose sodium or potassium at about 0.3-10 mg/ml, 1-10 mg/ml, 3-7 mg/ml or 5 mg/ml in the live attenuated dengue vaccine formulation.
  • Carboxymethyl cellulose salt is available in high viscosity type with average molecular weight of about 700,000; medium viscosity type with average molecular weight of about 250,000; and low viscosity type with average molecular weight of about 90,000.
  • the cellulose derivative is carboxymethyl cellulose salt with average molecular weight of about 700,000 at about 0.3-1.5 mg/ml in the live attenuated dengue vaccine formulation.
  • the cellulose derivative is carboxymethyl cellulose salt with average molecular weight of about 250,000 at about 1-4 mg/ml. In a further embodiment, the cellulose derivative is carboxymethyl cellulose salt with average molecular weight of about 90,000 at about 3-7 or 3-10 mg/ml. In yet a further embodiment, the cellulose derivative is carboxymethyl cellulose salt with average molecular weight of about 50,000 to 1000,000 at about 0.3-10 mg/ml.
  • the buffer is selected from the group consisting of phosphate, succinate, histidine, TRIS, MES, MOPS, HEPES, acetate and citrate at about 5-300 mM, 5-20 mM, 10-12 mM or 11 mM.
  • magnesium chloride the group consisting of magnesium chloride, calcium chloride, potassium chloride, and sodium chloride or a combination thereof at about 10-150 mM, 10-100 mM, 15-75 mM, 30-90 mM, 75 mM, 50 mM or 30 mM.
  • the amino acid can be selected from the group consisting of Val, Ile, Ala, Asp, His, Leu, Lys, Gln, Pro and Glu, or a combination thereof at 10-100, 10-75, 10-50, 20-30, or 25 mM. In another embodiment, the amino acid can be selected from the group consisting of Ala, Asp, His, Leu, Lys, Gln, Pro and Glu, or a combination thereof at 10-100, 10-75, 10-50, 20-30, or 25 mM. In one embodiment, the amino acid is Lys, Leu or Glu. In another embodiment, the amino acids are Leu and Glu. In another embodiment, the amino acid is Leu, Lys, Glu, or Ala. In another embodiment, the amino acid is Leu.
  • the sugar and glycol or sugar alcohol can act as a cryoprotectant or stabilizing excipient.
  • the sugar is at a concentration of 50-300 mg/ml.
  • the sugar is trehalose or sucrose or a combination thereof at about 60-120 mg/ml, 90-110 mg/ml, or 80-100 mg/ml.
  • the sucrose to trehalose ratio is between 1:1 to 1:4.
  • the sucrose is 90 mg/ml and the trehalose is 90-200 mg/ml, and preferably 110 mg/ml.
  • the glycol is propylene glycol
  • the sugar alcohol is glycerol or sorbitol at about 2.5-7.5 mg/ml, 3-7 mg/ml or 5 mg/ml.
  • compositions of the invention can be administered to a subject by one or more methods known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, intra-nasally, subcutaneously, intra-peritonealy, and formulated accordingly.
  • compositions of the present invention are administered via epidermal injection, intramuscular injection, intravenous, intra-arterial, subcutaneous injection, or intra-respiratory mucosal injection of a liquid preparation.
  • Liquid formulations for injection include solutions and the like.
  • the composition of the invention can be formulated as single dose vials, multi-dose vials or as pre-filled syringes.
  • compositions of the present invention are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation.
  • Solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the formulation is a solid dried formulation prepared from lyophilization, freezing, microwave drying or through the generation of lyospheres.
  • the solid dried formulation is obtainable by or produced from the microwave drying process described in example 7.
  • the formulations can be stored at ⁇ 70° C., ⁇ 20° C., 2-8° C. or at room temperature (25 or 37° C.).
  • the dried formulations can be expressed in terms of the weight of the components in a unit dose vial, but this varies for different doses or vial sizes.
  • the dried formulations of the present invention can be expressed in the amount of a component as the ratio of the weight of the component compared to the weight of the drug substance (DS) in the same sample (e.g. a vial). This ratio may be expressed as a percentage.
  • Such ratios reflect an intrinsic property of the dried formulations of the present invention, independent of vial size, dosing, and reconstitution protocol.
  • the formulation is in lyospheres.
  • the formulation is a reconstituted solution.
  • a dried solid formulation can be reconstituted at different concentrations depending on clinical factors, such as route of administration or dosing.
  • a dried formulation may be reconstituted at a high concentration (i.e. in a small volume) if necessary for subcutaneous administration. High concentrations may also be necessary if high dosing is required for a particular subject, particularly if administered subcutaneously where injection volume must be minimized. Subsequent dilution with water or isotonic buffer can then readily be used to dilute the drug product to a lower concentration. If isotonicity is desired at lower drug product concentration, the dried powder may be reconstituted in the standard low volume of water and then further diluted with isotonic diluent, such as 0.9% sodium chloride.
  • Reconstitution generally takes place at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be employed as desired.
  • the time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and virus or protein.
  • Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • the reconstitution volume can be about 0.5-1.0 ml, preferably 0.5 ml or 0.7 ml. In one embodiment, a single dose has a volume of 0.5 ml.
  • the invention provides a method of preparing a liquid formulation comprising the steps of reconstituting the formulations of the invention with a diluent as described above.
  • the formulation is the aqueous solution prepared before lyophilization, freezing, microwave drying or generation of lyospheres.
  • Processes for preparing lyospheres are disclosed in US patent publication US20140294872, the disclosure of which is herein incorporated by reference in its entirety.
  • the method comprises dispensing at least one liquid droplet having a substantially spherical shape onto a solid and flat surface (i.e., lacking any sample wells or cavity), freezing the droplet on the surface without contacting the droplet with a cryogenic substance and lyophilizing the frozen droplet to produce a dried pellet that is substantially spherical in shape.
  • U.S. Pat. No. 9,119,794 also discloses processes for forming lyospheres.
  • the unitary volumes containing the aqueous medium mixture are formed on a solid element containing cavities.
  • the solid element is cooled below the freezing temperature of the mixture, the cavities are filled with the mixture, and the mixture is solidified while present in the cavity to form the unitary forms.
  • the unitary forms are dried in a vacuum to provide the lyospheres.
  • the lyospheres are formed in a substantially spherical shape and are prepared by freezing droplets of a liquid composition of a desired biological material on a flat, solid surface, in particular, a surface that does not have any cavities, followed by lyophilizing the unitary forms.
  • U.S. Patent Application Publication No. US2014/0294872 the disclosure of which is herein incorporated by reference, discloses similar processes for forming lyospheres.
  • the process comprises dispensing at least one liquid droplet having a substantially spherical shape onto a solid and flat surface (i.e., lacking any sample wells or cavity), freezing the droplet on the surface without contacting the droplet with a cryogenic substance and lyophilizing the frozen droplet to produce a dried pellet that is substantially spherical in shape.
  • the process may be used in a high throughput mode to prepare multiple dried pellets by simultaneously dispensing the desired number of droplets onto the solid, flat surface, freezing the droplets and lyophilizing the frozen droplets.
  • Pellets prepared by this process from a liquid formulation may have a high concentration of a biological material (such as a protein therapeutic) and may be combined into a set of dried pellets.
  • the solid, flat surface is the top surface of a metal plate which comprises a bottom surface that is in physical contact with a heat sink adapted to maintain the top surface of the metal plate at a temperature of ⁇ 90° C. or below. Since the top surface of the metal plate is well below the freezing point of the liquid formulation, the droplet freezes essentially instantaneously with the bottom surface of the droplet touching the top surface of the metal plate.
  • the solid, flat surface is hydrophobic and comprises the top surface of a thin film that is maintained above 0° C. during the dispensing step.
  • the dispensed droplet is frozen by cooling the thin film to a temperature below the freezing temperature of the formulation.
  • the lyophilized formulations of the present invention are formed by lyophilization (freeze-drying) of a pre-lyophilization solution. Freeze-drying is accomplished by freezing the formulation and subsequently subliming water at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about ⁇ 50 to 25° C. (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 30 to 250 mTorr.
  • the formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will dictate the time required for drying, which can range from a few hours to several days (e.g.
  • a secondary drying stage may be carried out at about 0-40° C., depending primarily on the type and size of container and the type of protein employed.
  • the secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours.
  • the moisture content of a lyophilized formulation is less than about 5%, and preferably less than about 3%.
  • the pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation, vial size and lyophilization trays.
  • the container in which reconstitution is to be carried out may, for example, be a 2, 3, 5, 10 or 20 ml vial.
  • Embodiments of the invention also include one or more of the dengue vaccine compositions or formulations described herein (i) for use in, (ii) for use as a medicament or composition for, or (iii) for use in the preparation of a medicament for: (a) therapy (e.g., of the human body); (b) medicine; (c) inhibition of dengue virus replication, including DEN1, DEN2, DEN3 and/or DEN4; (d) induction of an immune response or a protective immune response against one or more of DEN1, DEN2, DEN3 and/or DEN4; (e) induction of a virus neutralizing antibody response against one or more types of dengue; (f) treatment or prophylaxis of infection by dengue virus; (g) prevention of recurrence of dengue virus infection; (h) reduction of the progression, onset or severity of pathological symptoms associated with dengue virus infection and/or reduction of the likelihood of a dengue virus infection or, (i) treatment, prophylaxis of, or delay in
  • the dengue vaccine compositions can optionally be employed in combination with one or more adjuvants (e.g., AAHS, aluminum phosphate, aluminum hydroxide such as Alhydrogel®, or other aluminum salt adjuvant, a saponin-based adjuvant such as ISCOMATRIXTM (CSL, Ltd.), a TLR-agonist, or lipid nanoparticles, described herein).
  • adjuvants e.g., AAHS, aluminum phosphate, aluminum hydroxide such as Alhydrogel®, or other aluminum salt adjuvant, a saponin-based adjuvant such as ISCOMATRIXTM (CSL, Ltd.), a TLR-agonist, or lipid nanoparticles, described herein.
  • Prophylactic treatment can be performed using a dengue virus vaccine composition of the invention, as described herein.
  • the composition of the invention can be administered to the general population or to those persons at an increased risk of dengue infection, e.g. those persons who live in or will be travelling to areas of the world in which mosquitoes of the genus Aedes are prevalent.
  • Those “in need of treatment” include those already with a dengue infection (e.g. infected with one or more of DEN1, DEN2, DEN3, or DEN4), as well as those prone to have an infection or any person in which a reduction in the likelihood of infection is desired.
  • a dengue infection e.g. infected with one or more of DEN1, DEN2, DEN3, or DEN4
  • Dengue virus vaccine compositions of the invention can be formulated and administered to a patient using techniques well known in the art. Guidelines for pharmaceutical administration in general are provided in, for example, Vaccines Eds. Plotkin and Orenstein, W.B. Sanders Company, 1999; Remington's Pharmaceutical Sciences 20 th Edition , Ed. Gennaro, Mack Publishing, 2000; and Modern Pharmaceutics 2 nd Edition , Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990.
  • the invention provides a method for inducing a protective immune response in a patient against a dengue infection comprising the step of administering to the patient an immunologically effective amount of any of the dengue virus vaccine compositions described herein.
  • the dengue virus vaccine composition is co-administered in combination with other vaccines for treating or preventing diseases from Zika, Measles Mumps and Rubella, or Varicella etc.
  • Also provided by the invention is a method for treating dengue infection, or for treating any pathological condition associated with dengue infection, such treatment including prophylaxis of infection, and reduction in the severity of clinical symptoms, delay or prevention of the progression of disease, and/or reduction in the likelihood of infection or the clinical symptoms thereof; the method comprising the step of administering to the patient an immunologically effective amount of any of the vaccine compositions as described herein.
  • Additional embodiments of the invention comprise the administration of two or more compositions of the invention to a patient in a prime/boost regime. Accordingly, the invention relates to a method of preventing or reducing the likelihood of dengue infection in a patient in need thereof, comprising the steps of:
  • step (b) waiting for a predetermined amount of time to pass after step (a);
  • the dengue virus vaccine compositions of the invention are in the form of a frozen liquid.
  • the dengue virus vaccine compositions are lyophilized, or microwaved dried and reconstituted with a sterile diluent prior to administration to the patient.
  • the amount of time between the first dose of a dengue virus vaccine composition of the invention and the second dose of a dengue virus vaccine composition of the invention, or any dose thereafter, is from about 2 weeks to about 2 years. In preferred embodiments of the invention, a time of 2 months to 12 months is allowed to pass between multiple administrations. In alternative embodiments of this aspect of the invention, the amount of time between each administration of each dose of vaccine composition is independently selected from the group consisting of 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, and 24 months.
  • the first and second dengue virus vaccine compositions are the same. In alternative embodiments, the first and second dengue virus vaccine compositions are not the same.
  • the dengue virus vaccine compositions of the invention can be administered by different routes.
  • the compositions of the invention are administered parenterally, i.e. by intradermal, subcutaneous or intramuscular injection.
  • Subcutaneous and intramuscular administration can be performed using, for example, needles or jet-injectors.
  • compositions described herein may be administered in a manner compatible with the dosage formulation, and in such amount as is immunologically-effective to treat and/or reduce the likelihood of dengue infection.
  • the dose administered to a patient should be sufficient to affect a beneficial response in a patient over time such as a reduction in the level of dengue virus, or to reduce the likelihood of infection by dengue.
  • the quantity of the dengue virus vaccines to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the vaccine required to be administered will depend on the judgment of the practitioner.
  • the physician may evaluate circulating plasma levels, progression of disease, and the production of anti-dengue antibodies.
  • suitable dosages of the immunogenic compositions of the invention may be readily determined by those of skill in the art.
  • Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the route of administration; the desired effect; and the particular composition employed.
  • the timing of doses depends upon factors well known in the art, and can range from 2 weeks to 24 months. After the initial administration one or more additional doses may be administered to maintain and/or boost antibody titers.
  • the invention also relates to methods for preventing dengue infection, or preventing or ameliorating the symptoms thereof, comprising the steps of: administering to a patient in which dengue infection or the symptoms thereof are to be prevented or ameliorated compositions of the dengue virus vaccine. Further embodiments of this aspect of the invention comprise allowing a predetermined amount of time to pass after administration of the dengue virus vaccine composition, and administering a second dose of a dengue virus vaccine composition.
  • the first dengue vaccine is preferably tetravalent and comprises a DEN1, DEN2, DEN3, and DEN 4 component, wherein each component comprises either a live attenuated dengue virus or a live attenuated chimeric flavivirus, as described herein.
  • the live attenuated dengue vaccine comprises four chimeric flaviviruses; wherein each of the chimeric flavivirus comprises the prM and E proteins of a single dengue virus serotype and the capsid and non-structural proteins of a different flavivirus, wherein the each of the chimeric flavivirus is attenuated.
  • the capsid and nonstructural proteins of the four chimeric flaviviruses are from yellow fever virus. In alternative embodiments, the capsid and nonstructural proteins of each of the four chimeric flaviviruses are from a different dengue serotype than the prM and E proteins.
  • the second dengue vaccine is a tetravalent recombinant dengue subunit vaccine comprising dengue E proteins, or fragments thereof, from DEN1, DEN2, DEN3, and DEN4.
  • Subunit vaccines useful in this method of the invention are described herein.
  • the E proteins each constitute about 80% of the length of wild type E of DEN1, DEN2, DEN3 and DEN4, starting from amino acid residue 1 at its N-terminus.
  • rDEN1 rDEN1 ⁇ 30-1545 PMVS (SEQ ID NO: 6); rDEN2—rDEN2/4 ⁇ 30(ME)-1495,7163 PMVS (SEQ ID NO: 7); rDEN3—rDEN3 ⁇ 30/31-7164 PMVS (SEQ ID NO: 8); and rDEN4—rDEN4 ⁇ 30-7132,7163,8308 PMVS (SEQ ID NO: 9).
  • DENV1, 2, 3 and 4 wild type and original cDNA clone in the above tables correspond to the dengue virus serotype described in Whitehead, S. S. et al., J Virol 77:1653-1657 (2003); Blaney, J. E. et al. The American journal of tropical medicine and hygiene 71:811-821 (2004); Blaney, J. E., Jr. et al., BMC Infect Dis 4:39 (2004); Durbin, A. P. et al., The American journal of tropical medicine and hygiene 65:405-413 (2001).
  • DENV1 or DEN1, DENV2 or DEN2, DENV3 or DEN3 and DENV4 or DEN4 below in the examples.
  • the formulations had a potency of 2 ⁇ 10 5 pfu/ml of each of DENV1, DENV2, DENV3 or DENV4.
  • the formulations had a potency of 1.5 ⁇ 10 5 pfu/ml of each of DEN1, DEN2, DEN3 or DEN4.
  • compositions Formulation Number Composition 1 11 mM potassium phosphate, 90 mg/mL sucrose, 30 mM sodium chloride pH 7.5 2 11 mM potassium phosphate, 90 mg/mL sucrose pH 7.5 3 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM sodium chloride pH 7.5 4 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM sodium chloride, 5 mg/mL sodium carboxymethylcellulose pH 7.5 5 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM sodium chloride, 5 mg/mL sodium carboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 13 11 mM potassium phosphate, 90 mg/mL sucrose, 25 mg/mL sorbitol, 75 mM sodium chloride, 5 mg/mL sodium carboxymethylcellulose pH 7.5 18 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM sodium chloride, 5
  • DENV4 was formulated in 11 mM potassium phosphate, 90 mg/mL sucrose, and 75 mM NaCl (formulation 3), with the addition of 5 mg/mL sodium carboxymethylcellulose (sodium CMC) (formulation 4) or addition of 5 mg/mL Sodium CMC and 5 mg/mL propylene glycol (formulation 5).
  • Study 2 Formulation 5 was tested against comparable formulations containing either 25 mM leucine (formulation 20) or 25 mM proline (formulation 21) as well as 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM NaCl, 5 mg/mL sodium CMC and 5 mg/mL propylene glycol.
  • Study 3 Formulation 20 was tested against a comparable formulation containing 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM NaCl, 5 mg/mL sodium CMC and 5 mg/mL propylene glycol and 25 mM glutamic acid (formulation 19) and the Dengvaxia® formulation (formulation 26), which consists of 37.5 mg/mL sorbitol, 75 mg/mL sucrose, 55 mg/mL trehalose, 25 mg/mL urea, 6 mM TRIS, 15 mg/mL of an amino acid mixture.
  • samples were frozen and a portion were stored at ⁇ 70° C. as frozen liquid controls and a portion were lyophilized. After lyophilization, some samples were stored at ⁇ 70° C. as control and the remainder were placed at 25° C. for 1 week. After incubation, the 25° C. samples were frozen and tested with a dengue relative infectivity assay (DRIA) along with the frozen liquid controls and frozen lyophilized controls. Two individual vials of each sample were tested.
  • DRIA dengue relative infectivity assay
  • DRIA is a cell-based relative infectivity assay used to measure infectivity of dengue virus formulation samples based on expression of envelope protein.
  • Vero cells were plated in 96-well micro-titer plates, incubated for 24 hours, and then infected with serial dilutions of DEN1, DEN2, DEN3 and/or DEN4 reference standard and positive control specific for the serotype being tested in addition to the test articles.
  • the infected cells were incubated for 48 hours and followed by fixation of the cells with a dilute formaldehyde solution.
  • the fixed cells were then permeabilized before primary antibody (rabbit anti-DEN serotype-specific MAb) was added to the plates and incubated overnight.
  • Lyophilization yields were calculated by dividing the lyophilized infectivity result by the frozen liquid control infectivity result. To calculate log loss after storage at 25° C. for one week, infectivity values were converted into log scale and the 1 week 25° C. log result was subtracted from the ⁇ 70° C. lyophilized control result for each formulation.
  • DENV4 was formulated in a base formulation of 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM NaCl, and 5 mg/mL sodium CMC pH 7.5 with 5 mg/mL propylene glycol (formulation 5), 5 mg/mL glycerol (formulation 22), or 25 mg/mL sorbitol (formulation 13) as sugar alcohols.
  • Samples were frozen and a portion were stored at ⁇ 70° C. as frozen liquid controls and a portion were lyophilized. After lyophilization, some samples were stored at ⁇ 70° C. and the remainder were placed at 25° C. for 1 week. After incubation, the 25° C. samples were frozen and tested with a dengue relative infectivity assay along with the frozen liquid controls and frozen lyophilized controls. Two individual vials of each sample were tested.
  • Lyophilization yields were calculated by dividing the lyophilized infectivity result by the frozen liquid control infectivity result. To calculate log loss after storage at 25° C. for one week, infectivity values were converted into log scale and the 1 week 25° C. log result was subtracted from the ⁇ 70° C. lyophilized control result for each formulation.
  • DENV4 was formulated in formulation 22 (11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL sodium CMC, 5 mg/mL glycerol at pH 7.0, 7.5 or 8.0).
  • Samples were frozen and a portion were stored at ⁇ 70° C. as frozen liquid controls and a portion were lyophilized. After lyophilization, some samples were stored at ⁇ 70° C. and the remainder were placed at 25° C. for 1 week. After incubation, the 25° C. samples were frozen and tested with a dengue relative infectivity assay along with the frozen liquid controls and frozen lyophilized controls. Two individual vials of each sample were tested.
  • Lyophilization yields were calculated by dividing the lyophilized infectivity result by the frozen liquid control infectivity result. To calculate log loss after storage at 25° C. for one week, infectivity values were converted into log scale and the 1 week 25° C. log result was subtracted from the ⁇ 70° C. lyophilized control result for each formulation.
  • FIGS. 5 and 6 demonstrate that DENV4 can be formulated in formulation 22 from pH 7.0 to pH 8.0.
  • DENV4 was formulated in a base formulation of 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL sodium CMC, and 5 mg/mL glycerol with alternative buffer systems adjusted to pH 7.5.
  • Formulation 22 contained 11 mM potassium phosphate and formulation 25 contained 11 mM TRIS in addition to the base formulation.
  • DENV4 was formulated in a base formulation of 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL sodium CMC, and 5 mg/mL propylene glycol with alternative buffer systems adjusted to pH 7.5.
  • Formulation 5 contained 11 mM potassium phosphate and formulation 57 contained a combination of 5.5 mM histidine and 5.5 mM TRIS in addition to the base formulation.
  • Samples were frozen and a portion were stored at ⁇ 70° C. as frozen liquid controls and a portion were lyophilized. After lyophilization, some samples were stored at ⁇ 70° C. and the remainder were placed at 25° C. for 1 week. After incubation, the 25° C. samples were frozen and tested with a dengue relative infectivity assay along with the frozen liquid controls and frozen lyophilized controls. Two individual vials of each sample were tested.
  • Lyophilization yields were calculated by dividing the lyophilized infectivity result by the frozen liquid control infectivity result. To calculate log loss after storage at 25° C. for one week, infectivity values were converted into log scale and the 1 week 25° C. log result was subtracted from the ⁇ 70° C. lyophilized control result for each formulation.
  • FIGS. 7 and 8 demonstrate that DENV4 can be formulated in a variety of buffer systems at pH 7.5 including potassium phosphate, TRIS, or a combination of histidine and TRIS.
  • DENV4 was formulated in a base formulation of 11 mM potassium phosphate, 90 mg/mL sucrose, 5 mg/mL sodium CMC, and 5 mg/mL propylene glycol with a concentration range of NaCl from 15-75 mM.
  • Samples were frozen and a portion were stored at ⁇ 70° C. as frozen liquid controls and a portion were lyophilized. After lyophilization, some samples were stored at ⁇ 70° C. and the remainder were placed at 25° C. for 1 week. After incubation, the 25° C. samples were frozen and tested with a dengue relative infectivity assay along with the frozen liquid controls and frozen lyophilized controls. Two individual vials of each sample were tested.
  • Lyophilization yields were calculated by dividing the lyophilized infectivity result by the frozen liquid control infectivity result. To calculate log loss after storage at 25° C. for one week, infectivity values were converted into log scale and the 1 week 25° C. log result was subtracted from the ⁇ 70° C. lyophilized control result for each formulation.
  • FIGS. 9 and 10 show that DENV4 lyophilization yield and stability for 1 week at 25° C. were similar when the NaCl concentration ranged from 15-75 mM.
  • DENV1, DENV2, DENV3 and DENV4 were prepared as monovalent drug products in formulation 5 (11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL CMC, 5 mg/mL propylene glycol), formulation 20 (11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM NaCl, 5 mg/mL CMC, 5 mg/mL propylene glycol, and 25 mM Leucine) and formulation 22 (11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL CMC, 5 mg/mL glycerol) at pH 7.5.
  • formulation 5 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL CMC, 5 mg/mL propylene glycol
  • formulation 20 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM NaCl, 5
  • Samples were frozen and a portion were stored at ⁇ 70° C. as frozen liquid controls and a portion were lyophilized. After lyophilization, some samples were stored at ⁇ 70° C. and the remainder were placed at 25° C. for 1 week. After incubation, the 25° C. samples were frozen and tested with a dengue relative infectivity assay along with the frozen liquid controls and frozen lyophilized controls. Two individual vials of each sample were tested.
  • Lyophilization yields were calculated by dividing the lyophilized infectivity result by the frozen liquid control infectivity result. To calculate log loss after storage at 25° C. for one week, infectivity values were converted into log scale and the 1 week 25° C. log result was subtracted from the ⁇ 70° C. lyophilized control result for each formulation.
  • FIGS. 11 and 12 show that propylene glycol and glycerol stabilize all four serotypes in combination with sucrose, NaCl, and Sodium CMC.
  • the formulations were filled into 2R glass vials at a 0.5 mL fill and frozen at ⁇ 115° C. for 15 minutes. Once frozen the vials were dried in the Microwave Vacuum Dryer (MVD). Once dried, some vials were place on stability at 25° C. for 1 week. The vials were then submitted for potency testing using the Dengue Relative Infectivity Assay (DRIA).
  • MWD Microwave Vacuum Dryer
  • Microwave radiation is applied in a travelling wave format
  • selected magnetrons e.g. 2 magnetrons out of 4 total magnetrons
  • Power was increased throughout the drying cycle to achieve a final terminal temperature of 30-45° C.
  • Freeze/thaw (F/T) yield was calculated by dividing the reported relative potency by the expected relative potency for the frozen controls at ⁇ 70° C.
  • Drying yield was calculated by dividing the relative potency of the dried material by the relative potency of the frozen control.
  • the log loss was calculated by converting the relative potency of the TO timepoint of the dried material and the 1 week 25° C. stability material into logs by a Log 10 calculation. Once the numbers are converted into log, the stability timepoint was subtracted from the TO timepoint to determine the log loss at 25° C. for 1 week.
  • Formulations 2, 4, 5, and 12 showed the best combination of F/T yield, drying yield, and log loss at 25° C. for one week. All four of these formulations contained ⁇ 25% disaccharide (sucrose and/or trehalose).
  • SPG Sematrose, Potassium Phosphate, Glutamic acid
  • 650 mg/mL Sucrose 650 mg/mL Trehalose
  • 5M Sodium Chloride NaCl
  • 10 mg/mL sodium Carboxymethyl Cellulose sodium CMC
  • the formulations were filled into 2R glass vials at a 0.5 mL fill and frozen at ⁇ 115° C. for 15 minutes. Once frozen the vials were dried in the Microwave Vacuum Dryer (MVD). Once dried, some vials were place on stability at 25° C. for 1 week. The vials were then submitted for potency testing using the Dengue Relative Infectivity Assay (DRIA).
  • MWD Microwave Vacuum Dryer
  • the log loss was calculated by converting the relative potency of the TO timepoint of the dried material and the 1 week 25° C. stability material into logs by a Log 10 calculation. Once the numbers were converted into log, the stability timepoint was subtracted from the TO timepoint to determine the log loss at 25° C. for 1 week.
  • Formulations were 1 ⁇ SPG (11 mM Potassium Phosphate, 6 mM L-glutamic acid, 0.22M Sucrose) with varying amounts of L-15 (90%, 45%, and 25%) and DEN1, DEN2, DEN3, or DEN4.
  • the major component in Leibovitz's L-15 is NaCl (137.39 mM). Therefore, 90% L-15 equals 123.65 mM NaCl, 45% L-15 equals 61.83 mM NaCl, and 25% L-15 equals 34.35 mM NaCl.
  • the formulations were filled into 2R glass vials at a 0.5 mL fill and frozen at ⁇ 115° C. for 15 minutes. Once frozen the vials were dried in the Microwave Vacuum Dryer (MVD). The vials were then submitted for potency testing using the Dengue Relative Infectivity Assay (DRIA).
  • DRIA Dengue Relative Infectivity Assay
  • Formulations that were dried fast under MVD were more stable than formulations dried slower under lyophilization (See FIGS. 13-16 ).
  • salt concentration of ⁇ 61.83 mM NaCl appears to improve DEN4 stability during drying that is not observed in the other three types (DEN1, DEN2, and DEN3).
  • the formulations were filled into 2R glass vials at a 0.5 mL fill and frozen at ⁇ 115° C. for 15 minutes. Once frozen the vials were dried in either a Microwave Vacuum Dryer (MVD) or Lyophilizer (Lyo). The vials were then submitted for potency testing using the Dengue Relative Infectivity Assay (DRIA).
  • MMD Microwave Vacuum Dryer
  • Lyophilizer Lyophilizer
  • FIG. 17 shows that relative potency of vials for the formulations dried in the Microwave Vacuum Dryer is greater than or equal to those dried in the Lyophilizer.
  • Tetravalent formulations (formulation 20) of DENV1, DENV2, DENV3 and DENV4 were lyophilized and stored at 37° C. for one week ( FIG. 18A ), 25° C. for one month ( FIG. 18B ), and 2-8° C. for 18 months ( FIGS. 19A-D ). Potency was analyzed by plaque assay (as described earlier in the text) at each time point. A control sample stored at ⁇ 70° C. was tested by plaque assay in the same assay run as each stability time point. A log loss for each time point was calculated by subtracting the log result of the stability sample from the ⁇ 70° C. control sample. FIGS.
  • 18A-B and 19 A-D show the log loss over time for each of the serotypes in the tetravalent formulation 20.
  • the error bars indicate two standard error of the mean of the log loss calculated at each time point.
  • Formulation 20 provides thermal stability to all four dengue serotypes in the tetravalent vaccine at 37° C., 25° C. and 2-8° C. as evidenced by the minimal potency loss observed at 1 week, 1 month and 18 months, respectively.
  • the high throughput plaque assay “microplaque (g)” assay is an automated, miniaturized dengue plaque assay run in a 96-well microplate. Briefly, Vero cells are seeded into black-walled, clear bottom tissue-culture plates in OptiPro SFM with 2% L-glutamine at 40,000 cells per well. Cells are allowed to attach overnight at 37° C., 5% pCO 2 , >90% rH. Virus is pre-diluted in OptiMEM reduced serum media and further serially diluted 1:2 in media in ultra-low attachment plates. The plant medium is removed from the cell plates using gentle aspiration, and 25 ⁇ L/well of incolum is transferred from the serial dilution plate to the cell plate.
  • Viral adsorption proceeds for 4 hours at 37° C., 5% pCO 2 , >90% rH. After the adsorption incubation, 175 ⁇ L/well overlay medium is added to all wells to inhibit viral secretion and spread. Depending on serotype, infection proceeds for 2 or 3 days at the aforementioned incubation conditions.
  • Viral ⁇ ⁇ Titer ⁇ ( PFU mL ) plaques ⁇ ⁇ counted volume ⁇ ⁇ of ⁇ ⁇ inoculum ⁇ ⁇ ( mL ) ⁇ total ⁇ ⁇ dilution

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Inorganic Chemistry (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Dermatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
US16/769,837 2017-12-07 2018-12-03 Formulations of dengue virus vaccine compositions Abandoned US20200390877A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/769,837 US20200390877A1 (en) 2017-12-07 2018-12-03 Formulations of dengue virus vaccine compositions

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762595842P 2017-12-07 2017-12-07
US16/769,837 US20200390877A1 (en) 2017-12-07 2018-12-03 Formulations of dengue virus vaccine compositions
PCT/US2018/063541 WO2019112921A1 (en) 2017-12-07 2018-12-03 Formulations of dengue virus vaccine compositions

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/063541 A-371-Of-International WO2019112921A1 (en) 2017-12-07 2018-12-03 Formulations of dengue virus vaccine compositions

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/815,037 Continuation US11883480B2 (en) 2017-12-07 2022-07-26 Formulations of dengue virus vaccine compositions

Publications (1)

Publication Number Publication Date
US20200390877A1 true US20200390877A1 (en) 2020-12-17

Family

ID=66750326

Family Applications (3)

Application Number Title Priority Date Filing Date
US16/769,837 Abandoned US20200390877A1 (en) 2017-12-07 2018-12-03 Formulations of dengue virus vaccine compositions
US17/815,037 Active US11883480B2 (en) 2017-12-07 2022-07-26 Formulations of dengue virus vaccine compositions
US18/530,421 Pending US20240131144A1 (en) 2017-12-07 2023-12-06 Formulations of dengue virus vaccine compositions

Family Applications After (2)

Application Number Title Priority Date Filing Date
US17/815,037 Active US11883480B2 (en) 2017-12-07 2022-07-26 Formulations of dengue virus vaccine compositions
US18/530,421 Pending US20240131144A1 (en) 2017-12-07 2023-12-06 Formulations of dengue virus vaccine compositions

Country Status (23)

Country Link
US (3) US20200390877A1 (ja)
EP (1) EP3720487A1 (ja)
JP (1) JP7042341B2 (ja)
KR (1) KR102494651B1 (ja)
CN (2) CN117752780A (ja)
AU (1) AU2018381213B2 (ja)
BR (1) BR112020011205A8 (ja)
CA (1) CA3083772A1 (ja)
CL (1) CL2020001463A1 (ja)
CO (1) CO2020006836A2 (ja)
CR (1) CR20200242A (ja)
DO (1) DOP2020000098A (ja)
EA (1) EA202091398A1 (ja)
EC (1) ECSP20032084A (ja)
GE (1) GEP20237539B (ja)
IL (1) IL274897A (ja)
JO (1) JOP20200140A1 (ja)
MA (1) MA51060A (ja)
MX (1) MX2020005857A (ja)
NI (1) NI202000039A (ja)
PE (1) PE20210654A1 (ja)
SG (1) SG11202005251VA (ja)
WO (1) WO2019112921A1 (ja)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023069123A1 (en) * 2021-10-20 2023-04-27 3M Innovative Properties Company Phosphate and arginine containing compositions and the delivery of such compositions for virulence suppression
EP4356925A2 (en) 2022-10-18 2024-04-24 Takeda Vaccines, Inc. Dengue vaccine formulation
EP4344699A3 (en) * 2022-09-29 2024-05-22 The Board of Regents of the University of Texas System Methods and compositions for transport, storage, and delivery of nucleic acids and other molecules

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MA51060A (fr) 2017-12-07 2021-03-17 Merck Sharp & Dohme Formulations de compositions de vaccin contre le virus de la dengue

Family Cites Families (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2254055T3 (es) 1991-09-19 2006-06-16 The United States Of America, As Represented By The Secretary, Departm. Of Health And Human Services Flavivirus quimericos y/o de crecimiento restringido.
FR2702660B1 (fr) * 1993-03-17 1995-05-24 Karl Simpson Compositions thérapeutiques stabilisées et leur procédé de préparation.
US6254873B1 (en) 1995-04-17 2001-07-03 The United States Of America As Represented By The Secretary Of The Army Inactivated dengue virus vaccine
US6190859B1 (en) 1995-04-17 2001-02-20 The United States Of America As Represented By The Secretary Of The Army Method and kit for detection of dengue virus
US6432411B1 (en) 1999-07-13 2002-08-13 Hawaii Biotechnology Group Recombinant envelope vaccine against flavivirus infection
US20030180352A1 (en) * 1999-11-23 2003-09-25 Patel Mahesh V. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
ES2533085T3 (es) 2001-05-22 2015-04-07 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Desarrollo de mutaciones útiles para atenuar virus del dengue y virus del dengue quiméricos
US7476390B2 (en) * 2002-02-26 2009-01-13 Maxygen, Inc. Flavivirus antigens
MXPA04009394A (es) * 2002-03-26 2005-01-25 Inmunex Corp Metodos para utilizar ligando flt3 en procedimientos de inmunizacion.
WO2003092592A2 (en) 2002-05-03 2003-11-13 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dengue tetravalent vaccine containing a common 30 nucleotide deletion in the 3'-utr of dengue types 1,2,3, and 4, or antigenic chimeric dengue viruses 1,2,3, and 4
US20050123550A1 (en) 2003-05-12 2005-06-09 Laurent Philippe E. Molecules enhancing dermal delivery of influenza vaccines
CN101128216A (zh) * 2005-01-05 2008-02-20 费城健康和教育公司 输送载体、生物活性物质和病毒疫苗
EP1896069B1 (en) 2005-06-24 2013-03-13 Intervet International BV Inactivated chimeric vaccines and related methods of use
KR20080059289A (ko) 2005-10-04 2008-06-26 알크-아벨로 에이/에스 고체 백신 제형
CN101296705A (zh) * 2005-10-04 2008-10-29 阿尔克-阿贝洛有限公司 固体疫苗制剂
US8337860B2 (en) 2006-08-15 2012-12-25 The United States Of America, As Represented By The Secretary Of The Department Of Health & Human Services Development of dengue virus vaccine components
US20080275030A1 (en) * 2007-01-19 2008-11-06 Sveinbjorn Gizurarson Methods and Compositions for the Delivery of a Therapeutic Agent
HUE031617T2 (en) 2007-04-06 2017-07-28 Takeda Vaccines Inc Methods and preparations for live attenuated viruses
EP2143440A1 (fr) 2008-07-09 2010-01-13 Sanofi Pasteur Agent stabilisant et composition vaccinale comprenant un ou plusieurs flavivirus vivants atténués
CN102215865B (zh) * 2008-09-24 2013-10-30 米迪缪尼有限公司 病毒纯化方法
WO2010037402A1 (en) 2008-10-02 2010-04-08 Dako Denmark A/S Molecular vaccines for infectious disease
JP2012520084A (ja) 2009-03-13 2012-09-06 レンチゲン コーポレイション 非組み込み型レトロウイルスベクターワクチン
GB201002419D0 (en) * 2010-02-12 2010-03-31 Isis Innovation Stable live vaccine formulations
JP6018575B2 (ja) 2010-10-29 2016-11-02 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． デングウイルス組換えサブユニットスワクチン
JP2014515367A (ja) 2011-05-26 2014-06-30 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム 不活性化デング熱ウイルスワクチン
ES2729967T3 (es) 2012-02-07 2019-11-07 Infectious Disease Res Inst Formulaciones de adyuvante mejoradas que comprenden agonistas de TLR4 y métodos para usar las mismas
US9314519B2 (en) * 2012-08-21 2016-04-19 Intervet Inc. Liquid stable virus vaccines
US9974850B2 (en) * 2013-01-25 2018-05-22 Board Of Regents, The University Of Texas System Immunogenic compositions and uses thereof
US9254332B2 (en) * 2013-03-15 2016-02-09 Arecor Limited Stable aqueous formulations of adenovirus vectors
SG11201510266SA (en) 2013-06-21 2016-01-28 Merck Sharp & Dohme Dengue virus vaccine compositions and methods of use thereof
US9782470B2 (en) 2013-10-16 2017-10-10 Merck Sharp & Dohme Corp. Method of obtaining thermostable dried vaccine formulations
EP3060247A1 (en) 2013-10-25 2016-08-31 Leukocare Ag A novel method for the production of stabile vaccines
WO2015093452A1 (ja) 2013-12-16 2015-06-25 武田薬品工業株式会社 マイクロニードル
US11801223B2 (en) * 2013-12-31 2023-10-31 Access To Advanced Health Institute Single vial vaccine formulations
WO2015130157A1 (en) 2014-02-28 2015-09-03 Universiti Malaya Enhancement of dengue virus production
AU2015369875A1 (en) 2014-12-22 2017-06-15 Merck Sharp & Dohme Corp. Dengue virus vaccine compositions and methods of use thereof
EP3303571A4 (en) * 2015-06-04 2018-10-17 The University of Hong Kong Live-attenuated virus and methods of production and use
US20180296663A1 (en) * 2015-06-17 2018-10-18 Curevac Ag Vaccine composition
US10004795B2 (en) 2015-09-08 2018-06-26 Fundacao Butantan Process for preparing an attenuated tetravalent dengue vaccine
EP3355916A4 (en) * 2015-09-30 2018-08-29 Panacea Biotec Limited Stable live attenuated recombinant dengue vaccine
BE1024160B9 (fr) 2015-12-22 2017-12-06 Glaxosmithkline Biologicals Sa Formulation immunogène
WO2017165736A1 (en) 2016-03-25 2017-09-28 Visterra, Inc. Formulation of antibody molecules to dengue virus
AU2017250696A1 (en) * 2016-04-13 2018-11-22 Takeda Vaccines, Inc. Compositions and methods of vaccination against dengue virus in children and young adults
TW202309276A (zh) 2016-08-03 2023-03-01 美商武田疫苗股份有限公司 用於與經改良之調配物一起穩定黃病毒的組成物及方法
CA3037337A1 (en) 2016-09-19 2018-03-22 Vaxess Technologies, Inc. Vaccine formulations with increased stability
WO2018183426A1 (en) 2017-03-30 2018-10-04 Merck Sharp & Dohme Corp. Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids
US11660333B2 (en) 2017-10-16 2023-05-30 Serum Institute Of India Private Limited Stable vaccine compositions comprising inter alia live attenuated recombinant flavivirus and process for preparation thereof
MA51060A (fr) 2017-12-07 2021-03-17 Merck Sharp & Dohme Formulations de compositions de vaccin contre le virus de la dengue

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023069123A1 (en) * 2021-10-20 2023-04-27 3M Innovative Properties Company Phosphate and arginine containing compositions and the delivery of such compositions for virulence suppression
EP4344699A3 (en) * 2022-09-29 2024-05-22 The Board of Regents of the University of Texas System Methods and compositions for transport, storage, and delivery of nucleic acids and other molecules
EP4356925A2 (en) 2022-10-18 2024-04-24 Takeda Vaccines, Inc. Dengue vaccine formulation
WO2024086605A1 (en) 2022-10-18 2024-04-25 Takeda Vaccines, Inc. Dengue vaccine formulation
EP4356925A3 (en) * 2022-10-18 2024-05-01 Takeda Vaccines, Inc. Dengue vaccine formulation

Also Published As

Publication number Publication date
CL2020001463A1 (es) 2020-09-25
IL274897A (en) 2020-07-30
MA51060A (fr) 2021-03-17
CN117752780A (zh) 2024-03-26
EA202091398A1 (ru) 2020-09-01
SG11202005251VA (en) 2020-07-29
GEP20237539B (en) 2023-09-11
EP3720487A1 (en) 2020-10-14
MX2020005857A (es) 2020-09-09
CR20200242A (es) 2020-07-17
CN111447947B (zh) 2024-01-12
AU2018381213A1 (en) 2020-06-11
JP2021505588A (ja) 2021-02-18
KR20200096272A (ko) 2020-08-11
CA3083772A1 (en) 2019-06-13
NI202000039A (es) 2020-09-24
US20240131144A1 (en) 2024-04-25
BR112020011205A2 (pt) 2020-11-17
US11883480B2 (en) 2024-01-30
AU2018381213B2 (en) 2022-03-17
DOP2020000098A (es) 2020-08-31
ECSP20032084A (es) 2020-08-31
KR102494651B1 (ko) 2023-01-31
PE20210654A1 (es) 2021-03-31
CO2020006836A2 (es) 2020-06-19
BR112020011205A8 (pt) 2023-02-07
US20230061673A1 (en) 2023-03-02
CN111447947A (zh) 2020-07-24
WO2019112921A1 (en) 2019-06-13
JP7042341B2 (ja) 2022-03-25
JOP20200140A1 (ar) 2022-10-30

Similar Documents

Publication Publication Date Title
US11883480B2 (en) Formulations of dengue virus vaccine compositions
US9198964B2 (en) Recombinant subunit dengue virus vaccine
JP5687836B2 (ja) 凍結乾燥によるワクチンの安定化
BRPI0614265A2 (pt) uso de uma vacina do vìrus da febre amarela e de uma vacina de flavivìrus quimérico e kit contendo referidas vacinas
US9861692B2 (en) Dengue virus vaccine compositions and methods of use thereof
US10449243B2 (en) Dengue virus vaccine compositions and methods of use thereof
JP2016513658A (ja) 弱毒生アルファウイルス製剤のための組成物および方法
EP3474891B1 (en) Vaccine compositions comprising an attenuated mutant zika virus
AU2018361217A1 (en) Stable formulations of cytomegalovirus
US11123420B2 (en) Stable live attenuated recombinant dengue vaccine
Roongaraya et al. Chikungunya vaccines: An update in 2023
EA041699B1 (ru) Составы вакцинных композиций от вируса денге
WO2017109698A1 (en) Immunogenic formulation
WO2024086605A1 (en) Dengue vaccine formulation

Legal Events

Date Code Title Description
AS Assignment

Owner name: MERCK SHARP & DOHME CORP., NEW JERSEY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RYAN, MICHAEL S.;MARTIN, SHERRIE-ANN P.;JONES, MORRISA;AND OTHERS;SIGNING DATES FROM 20180629 TO 20180712;REEL/FRAME:052840/0925

STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

AS Assignment

Owner name: MERCK SHARP & DOHME LLC, NEW JERSEY

Free format text: MERGER;ASSIGNOR:MERCK SHARP & DOHME CORP.;REEL/FRAME:061102/0145

Effective date: 20220407

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION