WO2011008974A2 - Rsv f protein compositions and methods for making same - Google Patents

Rsv f protein compositions and methods for making same Download PDF

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Publication number
WO2011008974A2
WO2011008974A2 PCT/US2010/042161 US2010042161W WO2011008974A2 WO 2011008974 A2 WO2011008974 A2 WO 2011008974A2 US 2010042161 W US2010042161 W US 2010042161W WO 2011008974 A2 WO2011008974 A2 WO 2011008974A2
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Prior art keywords
seq
rsv
protein ecto
domain
protein
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PCT/US2010/042161
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French (fr)
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WO2011008974A3 (en
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Kurt Swanson
Philip R. Dormitzer
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Novartis AG
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Novartis AG
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Priority to CA2768186A priority Critical patent/CA2768186A1/en
Priority to RU2012105308/10A priority patent/RU2585227C2/ru
Priority to EP21199223.5A priority patent/EP3988115A3/en
Priority to EP22200670.2A priority patent/EP4183412B1/en
Priority to CN201080040594.9A priority patent/CN102639147B/zh
Priority to MX2012000667A priority patent/MX2012000667A/es
Priority to EP22214608.6A priority patent/EP4218800A1/en
Priority to EP16190004.8A priority patent/EP3178490B1/en
Application filed by Novartis AG filed Critical Novartis AG
Priority to EP22214601.1A priority patent/EP4218799A1/en
Priority to SG2012002481A priority patent/SG178026A1/en
Priority to US13/383,897 priority patent/US20120164176A1/en
Priority to JP2012520789A priority patent/JP5829210B2/ja
Priority to EP10734875.7A priority patent/EP2453918B1/en
Priority to ES10734875.7T priority patent/ES2563730T3/es
Priority to BR112012001666A priority patent/BR112012001666A2/pt
Publication of WO2011008974A2 publication Critical patent/WO2011008974A2/en
Publication of WO2011008974A3 publication Critical patent/WO2011008974A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Respiratory syncytial virus is an enveloped non-segmented negative- strand RNA virus in the family Paramyxoviridae, genus Pneumovirus. It is the most common cause of bronchiolitis and pneumonia among children in their first year of life. RSV also causes repeated infections including severe lower respiratory tract disease, which may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems.
  • RSV conserved fusion protein
  • RSV F conserved fusion protein
  • the RSV F protein is translated from mRNA into an approximately 574 amino acid protein designated F 0 .
  • Post-translational processing of F 0 includes removal of an N-terminal signal peptide by a signal peptidase in the endoplasmic reticulum.
  • Fo is also cleaved at two sites (approximately 109/110 and approximately 136/ 137) by cellular proteases (in particular furin) in the trans-Golgi. This cleavage results in the removal of a short intervening sequence and generates two subunits designated Fi ( ⁇ 50 kDa; C- terminal; approximately residues 137-574) and F2 ( ⁇ 20 kDa; N- terminal;
  • Fi contains a hydrophobic fusion peptide at its N-terminus and also two amphipathic heptad-repeat regions (HRA and HRB). HRA is near the fusion peptide and HRB is near the transmembrane domain. Three F 1 -F 2 heterodimers are assembled as homotrimers Of F 1 - F 2 in the virion.
  • a vaccine against RSV infection is not currently available, but is desired.
  • One potential approach to producing a vaccine is a subunit vaccine based on purified RSV F protein.
  • the purified RSV F protein is in a single form and conformation that is stable over time, consistent between vaccine lots, and conveniently purified.
  • the RSV F protein can be truncated, for example by deletion of the
  • transmembrane domain and cytoplasmic tail to permit its expression as an ectodomain, which may be soluble.
  • RSV F protein is initially translated as a monomer, the monomers are cleaved and assemble into trimers. When RSV F protein is in the form of cleaved trimers, the hydrophobic fusion peptide is exposed. The exposed hydrophobic fusion peptides on different trimers, e.g., soluble ecto-domain trimers, can associate with each other, resulting in the formation of rosettes.
  • the hydrophobic fusion peptides can also associate with lipids and lipoproteins, for example from cells that are used to express recombinant soluble RSV F protein. Due to the complexity of RSV F protein processing, structure and refolding, purified,
  • the invention relates to immunogenic compositions that contain one or more RSV F polypeptides, and to certain engineered RSV F proteins and nucleic acids that encode the engineered RSV F proteins.
  • the RSV F protein is soluble.
  • the RSV F protein can have the transmembrane region and cytoplasmic tail deleted.
  • the soluble RSV F contains one or more of 1) one or more mutations to one or both furin- cleavage sites, 2) one or more mutations to the fusion peptide, 3) one or more mutations to the p27 linker, 4) contains an added oligomerization sequence, and 5) contains an added amino acid sequence that provides a protease cleavage site.
  • the RSV F protein is a monomer, a trimer, or a combination of monomers and trimers. The trimer can be monodispered or in the form of a rosette.
  • the RSV F protein can be in a prefusion conformation, an intermediate conformation or a postfusion conformation.
  • the immunogenic composition contains one or more respiratory syncytial virus F (RSV F) polypeptides in which amino acids 100-150 are replaced with the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7; SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:91 or SEQ ID NO:92.
  • RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition contains an RSV F polypeptide in which amino acids 100-150 of the RSV F are replaced with the amino acid sequence of SEQ ID NO: 12.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition contains an RSV F polypeptide in which amino acids 100-150 of the RSV F are replaced with the amino acid sequence of SEQ ID NO:9, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7; SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:92.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition contains an RSV F polypeptide in which amino acids 100-150 are replaced with the amino acid sequence of SEQ ID NO:9.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition contains an RSV F polypeptide in which RSV F contains amino acids 23-99 and 151-524 of SEQ ID NO: 1 or SEQ ID NO:2.
  • RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition contains a polypeptide selected from the group consisting of SEQ ID NO:49, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID - A -
  • the signal peptide and/or HIS tag is omitted.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition contains SEQ ID NO: 68 or alternatively, SEQ ID NO: 68 in which the signal peptide, and optionally the HIS tag, is omitted.
  • the immunogenic composition contains a polypeptide selected from the group consisting of SEQ ID NO:49, SEQ ID NO:71, and any of the foregoing sequences in which the signal peptide, and optionally the HIS tag, is omitted.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the immunogenic composition will include an adjuvant.
  • the adjuvant is preferably an aluminum salt, a squalene-in-water emulsion (such as MF59), a benzonaphthyridine compound, a phospholipid compound (such as E6020), a small molecule immunepotentiator or a combination of any two or more of any of the foregoing.
  • Yet another aspect of the invention includes recombinant RSV F polypeptides.
  • the RSV F may be in the form of a monomer, trimer, rosette of trimers, or combination of monomers and trimers.
  • the recombinant polypeptide may include a heterologous oligomerization domain, an epitope or a signal peptide.
  • the heterologous oligomerization domain may include a heterologous oligomerization domain, an epitope or a signal peptide.
  • oligomerization domain is preferably a trimerization domain from influenza hemagglutinin, from SARS spike, or from HIV gp41, NadA, modified GCN4, or ATCase.
  • the recombinant RSV F polypeptide has amino acids 100-150 replaced with the amino acid sequence of SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:91 or SEQ ID NO: 92.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the recombinant RSV F polypeptide has amino acids 100-150 of the RSV F replaced with the amino acid sequence of SEQ ID NO: 12.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the recombinant RSV F polypeptide has amino acids 100-150 of the RSV F replaced with the amino acid sequence of SEQ ID NO:9, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7; SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:92.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the recombinant RSV F polypeptide has amino acids 100- 150 of the RSV F replaced with the amino acid sequence of SEQ ID NO:9.
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • the recombinant polypeptide is selected from the group consisting of SEQ ID NO:49, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO
  • the RSV F polypeptide is soluble (e.g., an ectodomain).
  • nucleic acids encoding any of the foregoing polypeptides.
  • the nucleic acid may be a self-replicating RNA molecule.
  • Another aspect of the invention is an immunogenic composition
  • an immunogenic composition comprising a self-replicating RNA that encodes an RSV F polypeptide.
  • the immunogenic composition can include a delivery system.
  • Another aspect of the invention includes methods of inducing an immune response to RSV F by administering any of the immunogenic compositions.
  • the invention relates to methods for preparing compositions and to
  • compositions that contain RSV F protein such as soluble RSV F ecto-domain polypeptides, including immunogenic compositions.
  • the RSV F ecto-domain polypeptides can be in a single form, such as uncleaved monomers, uncleaved trimers, cleaved trimers, or rosettes of cleaved trimers.
  • the RSV F ectodomain polypeptides can also be in two or more forms, for example two or more forms that exist in equilibrium, such as equilibrium between uncleaved monomers and uncleaved trimers.
  • the invention provides several advantages. For example, the presence of a single desired form of RSV F in an immunogenic composition provides a more predictable immune response when the composition is administered to a subject, and more consistent stability and other physical and chemical characteristics when formulated into a vaccine.
  • the invention is a method for producing a composition comprising cleaved RSV F protein ecto-domain polypeptides.
  • the method includes a) providing uncleaved RSV F protein ecto-domain polypeptides containing one or more protease cleavage sites that, when cleaved, produce Fi and F 2 fragments, and b) cleaving the uncleaved RSV F protein ecto-domain polypeptides with a protease or proteases that recognize the protease cleavage site or sites.
  • the amino acid sequence of the uncleaved RSV F protein ecto-domain polypeptides contains altered furin cleavage sites, and the RSV F protein ecto-domain polypeptides are secreted from a host cell that produces them uncleaved at a position from amino acid 101 to amino acid 161, (e.g., is not cleaved at the furin cleavage sites at positions 106-109 and 131-136).
  • the uncleaved RSV F protein ecto-domain polypeptides provided in a) are purified.
  • the uncleaved RSV F protein ecto-domain polypeptides provided in a) can comprise an intact fusion peptide or an altered fusion peptide (e.g., a deleted fusion peptide or mutated fusion peptide).
  • an altered fusion peptide e.g., a deleted fusion peptide or mutated fusion peptide.
  • the cleaving in step b) results in the formation of rosettes of trimers.
  • the uncleaved RSV F protein ecto- domain polypeptides provided in a) comprise an altered fusion peptide
  • the cleaving in step b) results in the formation of trimers.
  • the method can further comprise the optional step of purifying the rosettes or trimers produced by cleaving the uncleaved RSV F protein ecto-domain polypeptides.
  • the cleaved RSV F protein ecto-domain polypeptides produced according to the method are substantially free of lipids and lipoproteins.
  • the invention is a method for producing a composition comprising uncleaved RSV F protein ecto-domain polypeptide monomers, trimers or a combination of monomers and trimers.
  • the method includes a) providing a biological material that contains uncleaved RSV F protein ecto-domain polypeptides; and b) purifying uncleaved RSV F protein ecto-domain polypeptide monomers or trimers from the biological material.
  • the amino acid sequence of the uncleaved RSV F protein ecto-domain polypeptides contains altered furin cleavage sites, and the RSV F protein ecto-domain polypeptides are secreted from a host cell that produces them uncleaved at a position from amino acid 101 to amino acid 161, (e.g., is not cleaved at the furin cleavage sites at positions 106-109 and 131-136).
  • the amino acid sequence of the uncleaved RSV F protein ecto-domain polypeptides further contain altered trypsin cleavage sites, and the RSV F protein ecto-domain polypeptides are not cleaved by trypsin at a site between amino acid 101 and amino acid 161.
  • the amino acid sequence of the uncleaved RSV F protein ecto- domain polypeptides further contain an altered fusion peptide.
  • uncleaved RSV F protein ecto-domain polypeptide trimers are purified.
  • uncleaved RSV F protein ecto-domain polypeptide monomers are purified.
  • a mixture of uncleaved RSV F protein ecto-domain monomers and trimers, which may be in a dynamic equilibrium are purified.
  • the cleaved RSV F protein ecto- domain polypeptides produced according to the method are substantially free of lipids and lipoproteins.
  • the invention is a method for producing a composition comprising cleaved RSV F protein ecto-domain polypeptide monomers, trimers or a combination of monomers and trimers.
  • the method includes a) providing a biological material that contains cleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide; and b) purifying cleaved RSV F protein ecto-domain polypeptides from the biological material.
  • cleaved RSV F protein ecto-domain polypeptide trimers are purified.
  • cleaved RSV F protein ecto-domain polypeptide monomers are purified.
  • a mixture of cleaved RSV F protein ecto-domain monomers and trimers, which may be in a dynamic equilibrium are purified.
  • the cleaved RSV F protein ecto-domain polypeptides produced according to the method are preferably substantially free of lipids and lipoproteins.
  • a cleaved RSV F protein ectodomain trimer containing an altered fusion peptide is purified.
  • the invention provides compositions, including immunogenic compositions, produced using the methods of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the schematic of the wild type RSV F (FIG IA) and of a ectodomain construct in which the transmembrane domain and cytoplasmic tail have been removed and an optional HIS6-tag has been added to the C-terminus (FIG IB).
  • residue numbering is related to the wild type A2 strain RSV F beginning at the N-terminal signal peptide and is not altered in constructs containing amino acid deletions. Labeled in the schematics is the signal sequence or signal peptide (sp).
  • sp signal sequence or signal peptide
  • IA is a schematic of RSV F protein showing the signal sequence or signal peptide (SP), p27 linker region, fusion peptide (FP), HRA domain (HRA), HRB domain (HRB), transmembrane region (TM), and cytoplasmic tail (CT).
  • SP signal sequence or signal peptide
  • FP fusion peptide
  • HRA HRA
  • HRB HRB domain
  • TM transmembrane region
  • CT cytoplasmic tail
  • FIG IB is a general schematic of the RSV F ectodomain construct depicting the shared features with the schematics in FIG. IA and including an optional HlS ⁇ -tag (HIS TAG).
  • HlS ⁇ -tag HlS ⁇ -tag
  • 1C also shows the amino acid sequence of amino acids 100 - 150 of RSV F (wild type) (SEQ ID NO: 108) and several proteins (Furmt-SEQ ID NO:3; Furdel-SEQ ID NO:4; Furx-SEQ ID NO:6; Furx Rl 13Q, K123N, K124N-SEQ ID NO:5; Furx Rl 13Q, K123Q, K124Q-SEQ ID NO:92; Delp21 furx-SEQ ID NO:7; Delp23 furx-SEQ ID NO: 8; Delp23 furdel-SEQ ID NO:9; N-Term Furin-SEQ ID NO: 10; C-term Furin-SEQ ID NO: 11; Fusion Peptide Deletionl-SEQ ID NO: 12; and Factor Xa-SEQ ID NO: 13) in which the one or both furin cleavage sites and/or fusion peptide region were mutated or deleted.
  • F wild type
  • FIG. 1C the symbol “-” indicates that the amino acid at that position is deleted.
  • FIG. 2 shows the amino acid sequence of the carboxy terminus from amino acid position 488 to the start of the TM region of RSV F (wild type) (SEQ ID NO:94) and several proteins (SEQ ID NOS:95-100) that contain added protease cleavage sites.
  • the symbol "-" indicates that there is no amino acid at that position.
  • FIG. 3 is a chromatogram and image of an electrophoresis gel showing the purification of RSV F monomers (3) using size exclusion chromatography.
  • FIGS. 4A-4F shows the nucleotide sequence (SEQ ID NO: 101) of the plasmid encoding the pT7-TC83R-FL.RSVF (A317) self-replicating RNA molecule which encodes the respiratory syncytial virus F glycoprotein (RSV-F).
  • the nucleotide sequence encoding RSV-F is underlined.
  • FIG. 5 is an alignment of the amino acid sequences of F proteins from several strains of RSV. The alignment was prepared using the algorithm disclosed by Corpet, Nucleic Acids Research, 1998, 16(22): 10881-10890, using default parameters
  • FIG. 6 shows relevant regions of size exclusion (SEC) chromatograms from select RSV F antigen purifications.
  • the principle peak containing the indicated antigen is indicated by an asterisk with the retention time of the Superdex P200 16/60 column (GE Healthcare) is indicated in milliliters.
  • the approximate retention times of 47 mis, 65 mis and 77 mis correspond to the column void volume, the retention of the RSV F trimer and the retention of the RSV F monomer, respectively.
  • the uncleaved Delp23 Furdel ( ⁇ p23 Furdel) construct is purified from the monomer peak at approximately 77 mis.
  • FIG. 7 shows representative EM images of select RSV F antigens.
  • FIG. 7A shows an EM image of RSV F ⁇ p23 (Delp23) before trypsin treatment.
  • the crutch shapes in FIG. 7A consistent with a postfusion trimer conformation, are not always observed in the uncleaved ⁇ p23 (Delp23) Furdel construct.
  • the ⁇ p23 (Delp23) Furdel contruct is treated with trypsin and purified from the void volume of an SEC column and observed by EM the proteins are found to have adopted rosette
  • FIG. 7B When the RSV F fusion peptide deletion construct is purified from the trimer peak on an SEC column a monodispersed crutch shape is observed, consistent with the a postfusion trimer (FIG. 7C). Shown in FIG. 7D are three preparations of either ⁇ p21 (Delp21) furx RSV F (labeled Monomer), Fusion peptided deletion RSV F (lanes labeled Trimer) and purified RSV F rosettes (labeled Rosettes).
  • the gel contains several lanes of GE Full Range Standard (molecular weights standard are labeled to the left of the gel) while approximate retention times of RSV F fragments are indicated on the right of the gel.
  • FIGS. 8A-8C are graphs showing that monomers (uncleaved ⁇ p21 (Delp21) furx), rosettes of trimers (cleaved ⁇ p23 (Delp23) Furdel), and trimers (fusion peptide deletion) of RSV F ecto-domain polypeptides are immunogenic in cotton rats.
  • Serum titers of anti-RSV F IgG and neutralizing anti-RSV antibodies were measured 2 weeks after the 1 st vaccination (2wpl), 3 weeks after the 1 st vaccination (3wpl), and/or 2 weeks after the 2 nd vaccination (2wp2).
  • the invention relates to respiratory syncytial virus F (RSV F) polypeptides and/or proteins, immunogenic compositions comprising RSV F polypeptides and/or proteins, methods for producing RSV F polypeptides and/or proteins and compositions comprising RSV F polypeptides and/or proteins, and nucleic acids that encode RSV F polypeptides and/or proteins.
  • RSV F respiratory syncytial virus F
  • the immunogenic compositions comprise RSV F polypeptides and/or proteins that contain mutations (e.g., amino acid replacements, deletions or additions) which provide beneficial characteristics, such as one or more of 1) stabilized prefusion or intermediate (non-post fusion) conformation, 2) reduced or eliminated exposure of the fusion peptide, 3) improved stability (e.g., reduced aggregation and/or degradation, and 4) more closely resemble active F1/F2 viral protein.
  • mutations e.g., amino acid replacements, deletions or additions
  • beneficial characteristics such as one or more of 1) stabilized prefusion or intermediate (non-post fusion) conformation, 2) reduced or eliminated exposure of the fusion peptide, 3) improved stability (e.g., reduced aggregation and/or degradation, and 4) more closely resemble active F1/F2 viral protein.
  • non-post fusion conformations of RSV F protein i.e., prefusion conformation, intermediate conformations
  • Reducing or eliminating the exposure of the fusion peptide will reduce the hydrophobicity of the polypeptide and facilitate purifications, and also reduce or eliminate the RSV F protein from associating with cell membranes in a subject to whom the protein is administered.
  • Improved stability of the protein facilitates producing immunogenic compositions in which the protein has a decreased tendency to aggregate or degrade, which provides a more predictable immune response when the composition is administered to a subject.
  • mutant RSV F polypeptides or proteins that resemble F 1/F2 viral protein for example by deletion of all or part of the p27 linker region, may elicit a better neutralizing antibody response.
  • the invention also relates to methods for preparing compositions that contain RSV F protein, in particular RSV F ecto-domain polypeptides, and to compositions including immunogenic compositions comprising RSV F protein.
  • RSV F ecto-domain polypeptides are in a single form or in a dynamic equilibrium between known forms. Definitions
  • population refers to more than one RSV F polypeptide or protein that is present in a composition.
  • the population can be substantially homogenous, in which substantially all RSV F polypeptides or proteins are substantially the same (e.g., same amino acid sequence, same conformation), heterogenous, or have a desired degree of homogenicity (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the RSV F polypeptides or proteins are prefusion conformation, are postfusion conformation, are monomers, are trimers).
  • the "post fusion conformation" of RSV F protein is a trimer characterized by the presence of a six-helix bundle comprising 3 HRB and 3HRA regions.
  • the "pre- fusion conformation" of RSV F protein is a conformation
  • trimer that contains a triple helix comprising 3 HRB regions.
  • RSV F ecto-domain polypeptide refers to an RSV F protein polypeptide that contains substantially the extracellular portion of mature RSV F protein, with our without the signal peptide (e.g., about amino acids 1 to about amino acid 524, or about amino acid 22 to about amino acid 524) but lacks the transmembrane domain and cytoplasmic tail of naturally occurring RSV F protein.
  • cleaved RSV F ecto-domain polypeptide refers to a RSV F ectodomain polypeptide that has been cleaved at one or more positions from about 101/102 to about 160/161 to produce two subunits, in which one of the subunits comprises Fi and the other subunit comprises F 2 .
  • C-terminal uncleaved RSV F ecto-domain polypeptide refers to an RSV F ectodomain polypeptide that is cleaved at one or more positions from about 101/102 to about 131/132, and is not cleaved at one or more positions from about 132/133 to about 160/161, to produce two subunits, in which one of the subunits comprises Fi and the other subunit comprises F 2 .
  • uncleaved RSV F ecto-domain polypeptide refers to an RSV F ectodomain polypeptide that is not cleaved at one or more positions from about 101/102 to about 160/161.
  • An uncleaved RSV F ecto-domain polypeptide can be, for example, a monomer or a trimer.
  • fusion peptide refers to amino acids 137-154 of RSV F protein.
  • altered fusion peptide refers to a fusion peptide in which one or more amino acids are independently replaced or deleted, including replacement or deletion of all amino acids from positions 137-154.
  • cleaved RSV F ecto- domain polypeptides that contain an "altered fusion peptide” do not form rosettes.
  • a "purified" protein or polypeptide is a protein or polypeptide which is recombinantly or synthetically produced, or produced by its natural host, and has been isolated from other components of the recombinant or synthetic production system or natural host such that the amount of the protein relative to other
  • a purified protein will be at least about 50% homogeneous and more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogeneous.
  • substantially free of lipids and lipoproteins refers to compositions, proteins and polypeptides that are at least about 95% free of lipids and lipoproteins on a mass basis when protein and/or polypeptide (e.g., RSV F polypeptide) purity is observed on an SDS PAGE gel and total protein content is measured using either UV280 absorption or BCA analysis, and lipid and lipoprotein content is determined using the Phospholipase C assay (Wako, code no. 433-36201).
  • polypeptide e.g., RSV F polypeptide
  • altered furin cleavage site refers the amino acid sequence at about positions 106-109 and at about positions 133-136 in naturally occurring RSV F protein that are recognized and cleaved by furin or furin-like proteases, but in an uncleaved RSV F protein ecto-domain polypeptide contains one or more amino acid replacements, one or more amino acid deletions, or a combination of one or more amino acid replacement and one or more amino acid deletion, so that an RSV F ecto- domain polypeptide that contains an altered furin cleavage site is secreted from a cell that produces it uncleaved at the altered furin cleavage site.
  • RSV F protein ecto-domains suitable for use in this invention are described herein with reference to particular amino acids that are identified by the position of the amino acid in the sequence of RSV F protein from the A2 strain (SEQ ID NO: 1).
  • RSV F protein ecto-domains can have the amino acid sequence of the F protein from the A2 strain or any other desired strain.
  • the amino acids of the F protein are to be numbered with reference to the numbering of the F protein from the A2 strain, with the insertion of gaps as needed.
  • sequence alignments are preferably produced using the algorithm disclosed by Corpet, Nucleic Acids Research, 1998, 16(22): 10881-10890, using default parameters (Blossum 62 symbol comparison table, gap open penalty: 12, gap extension penalty: 2).
  • the invention provides soluble RSV F polypeptides and proteins, and immunogenic compositions comprising the soluble RSV F polypeptides and proteins, as well as compositions comprising nucleic acids (e.g., self-replicating RNA molecules) that encode the soluble RSV F polypeptides and proteins.
  • nucleic acids e.g., self-replicating RNA molecules
  • the RSV F polypeptides can be in any desired form, such as in a single form, such as uncleaved monomers, uncleaved trimers, cleaved trimers, or rosettes of cleaved trimers.
  • the RSV F ectodomain polypeptides can also be in two or more forms, for example two or more forms that exist in equilibrium, such as equilibrium between uncleaved monomers and uncleaved trimers.
  • the invention provides several advantages. For example, the presence of a single desired form of RSV, or a dynamic equilibrium between known forms, in an immunogenic
  • composition provides for more predictable formulation, solubility and stability, and for a more predictable immune response when the composition is administered to a subject.
  • the RSV F ecto-domain polypeptides are in a single form, such as uncleaved monomers, uncleaved trimers, cleaved trimers, rosettes of cleaved trimers, or in a dynamic equilibrium between a subset of such forms (e.g., equilibrium between uncleaved monomers and uncleaved trimers).
  • the RSV F polypeptides and proteins are in pre- fusion conformation.
  • the epitopes of the pre-fusion conformation may be better able to elicit antibodies that can recognize and neutralize natural virions.
  • an immunogenic composition comprises a population of respiratory syncytial virus F glycoproteins in pre-fusion conformation.
  • an immunogenic composition comprises a population of respiratory syncytial virus F glycoproteins which disfavor the post- fusion conformation as compared to a population of isolated RSV F glycoproteins.
  • the invention also provides an immunogenic composition comprising a polypeptide that displays an epitope present in a pre-fusion or an intermediate fusion conformation of respiratory syncytial virus F glycoprotein but absent the glycoprotein's post-fusion conformation.
  • the F glycoprotein is a polypeptide that displays an epitope present in a pre-fusion or an intermediate fusion conformation of respiratory syncytial virus F glycoprotein but absent the glycoprotein's post-fusion conformation.
  • the F glycoprotein of RSV directs viral penetration by fusion between the virion envelope and the host cell plasma membrane. It is a type I single-pass integral membrane protein having four general domains: N-terminal ER-translocating signal sequence (SS), ectodomain (ED), transmembrane domain (TM), and a cytoplasmic tail (CT). CT contains a single palmitoylated cysteine residue.
  • SS N-terminal ER-translocating signal sequence
  • ED ectodomain
  • TM transmembrane domain
  • CT cytoplasmic tail
  • CT contains a single palmitoylated cysteine residue.
  • the sequence of F protein is highly conserved among RSV isolates, but is constantly evolving (7). Unlike most paramyxoviruses, the F protein in RSV can mediate entry and syncytium formation independent of the other viral proteins (FTN is usually necessary in addition to F in other paramyxoviruses).
  • the hRSV F mRNA is translated into a 574 amino acid precursor protein designated F 0 , which contains a signal peptide sequence at the N-terminus that is removed by a signal peptidase in the endoplasmic reticulum.
  • F 0 is cleaved at two sites (a.a. 109/110 and 136/137) by cellular proteases (in particular furin) in the trans-Golgi, removing a short glycosylated intervening sequence and generating two subunits designated F 1 ( ⁇ 50 kDa; C-terminus; residues 137-574) and F 2 (-20 kDa; N- terminus; residues 1-109) (See, e.g., FIG. 1).
  • Fi contains a hydrophobic fusion peptide at its N- terminus and also two hydrophobic heptad-repeat regions (HRA and HRB). HRA is near the fusion peptide and HRB is near to the transmembrane domain (See, e.g., FIG. 1). The F 1 -F 2 heterodimers are assembled as homotrimers in the virion.
  • RSV exists as a single serotype but has two antigenic subgroups: A and B.
  • the F glycoproteins of the two groups are about 90% identical.
  • the A subgroup, the B subgroup, or a combination or hybrid of both can be used in the invention.
  • An example sequence for the A subgroup is SEQ ID NO: 1 (A2 strain; GenBank GI: 138251; Swiss Prot P03420), and for the B subgroup is SEQ ID NO: 2 (18537 strain; GI: 138250; Swiss Prot P13843).
  • SEQ ID NO: 1 and SEQ ID NO:2 are both 574 amino acid sequences.
  • the signal peptide in A2 strain is a.a. 1-21, but in 18537 strain it is 1-22.
  • the TM domain is from about a.a. 530-550, but has alternatively been reported as 525-548.
  • the invention may use any desired RSV F amino acid sequence, such as the amino acid sequence of SEQ ID NO: 1 or 2, or a sequence having identity to SEQ ID NO: 1 or 2. Typically it will have at least 75% identity to SEQ ID NO: 1 or 2 e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%,identity to SEQ ID NO: 1 or 2.
  • the sequence may be found naturally in RSV.
  • an ectodomain of F protein in whole or in part, it may comprise:
  • polypeptide comprising about amino acids 23-525 of SEQ ID NO: 2.
  • polypeptide comprising an amino acid sequence having at least 75% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity) to (i) or (ii).
  • a polypeptide comprising a fragment of (i), (ii) or (iii), wherein the fragment comprises at least one F protein epitope.
  • the fragment will usually be at least about 100 amino acids long, e.g., at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450 amino acids long.
  • the ectodomain can be an Fo form with or without the signal peptide, or can comprises two separate peptide chains (e.g., an Fi subunit and a F 2 subunit) that are associated with each other, for example, the subunits may be linked by a disulfide bridge. Accordingly, all or a portion of about amino acid 101 to about 161, such as amino acids 110-136, may be absent from the ectodomain.
  • the ectodomain in whole or in part, can comprise:
  • a first peptide chain and a second peptide chain that is associated with the first polypeptide chain where the first peptide chain comprises an amino acid sequence having at least 75% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity) to about amino acid 22 to about amino acid 101 of SEQ ID NO: 1 or to about amino acid 23 to about amino acid 101 of SEQ ID NO: 2, and the second peptide chain comprises an amino acid sequence having at least 75% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% identity) to about amino acid 162 to about 525 of SEQ ID NO: 1 or to about amino acid 162 to 525 of SEQ ID NO: 2.
  • the first peptide chain comprises an amino acid sequence having at least 75% identity (e.g., at least 80%, at least 85%
  • first peptide chain and a second peptide chain that is associated with the first polypeptide chain
  • first peptide chain comprises an amino acid sequence comprising a fragment of about amino acid 22 to about amino acid 101 of SEQ ID NO: 1 or of about amino acid 23 to about amino acidlO9 of SEQ ID NO: 2
  • second peptide chain comprises a fragment of about amino acid 162 to about amino acid 525 of SEQ ID NO: 1 or of about amino acid 161 to about amino acid 525 of SEQ ID NO: 2.
  • One or both of the fragments will comprises at least one F protein epitope.
  • the fragment in the first peptide chain will usually be at least 20 amino acids long, e.g., at least 30, at least 40, at least 50, at least 60, at least 70, at least 80 amino acids long.
  • the fragment in the second peptide chain will usually be at least 100 amino acids long, e.g., at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450 amino acids long.
  • an amino acid sequence used with the invention may be found naturally within RSV F protein (e.g., a soluble RSV F protein lacking TM and CT, about amino acids 522-574 of SEQ ID NOS: 1 or 2), and/or it may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) single amino acid mutations (insertions, deletions or substitutions) relative to a natural RSV sequence. For instance, it is known to mutate F proteins to eliminate their furin cleavage sequences, thereby preventing intracellular processing.
  • the RSV F protein lacks TM and CT (about amino acids 522-574 of SEQ ID NOS: 1 or T) and contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) single amino acid mutations (insertions, deletions or substitutions) relative to a natural RSV sequence.
  • RSV F polypeptides or proteins may contain one or more mutations that prevent cleavage at one or both of the furin cleavage sites (i.e., amino acids 109 and 136 of SEQ ID NOS: 1 and T). These mutations can prevent aggregation of the soluble polypeptides or proteins and thereby facilitate purifications, can prevent cell-cell fusion if the RSV F protein is expressed on the surface of a cell, such as by expression from a viral replicon (e.g., alphavirus replicon particles), or if the RSV F protein is a component of a virus-like particle. These mutations, alone or in combination with other mutations described herein, may also stabilize the protein in the pre-fusion
  • furin cleavage mutations include replacement of amino acid residues 106 - 109 of SEQ ID NO: 1 or 2 with RARK (SEQ ID NO: 77), RARQ (SEQ ID NO:78), QAQN (SEQ ID NO:79), or IEGR (SEQ ID NO:80).
  • amino acid residues 133 - 136 of SEQ ID NO: 1 or 2 can be replaced with RKKK (SEQ ID NO: 81), ⁇ R, QNQN (SEQ ID NO: 82), QQQR (SEQ ID NO:83) or IEGR (SEQ ID NO:80).
  • Suitable trypsin cleavage mutations include deletion of any lysine or arginine residue between about position 101 and position 161 of SEQ ID NO: 1 or 2, or replacement of any such lysine or arginine residue with an amino acid other than lysine or arginine.
  • lysine and/or arginine residues in the p27 region can be substituted or deleted, including deletion of the p27 region in whole or in part.
  • RSV F polypeptides or proteins may contain one or more mutations in the fusion peptide region (amino acids 137 and 153 of SEQ ID NOS: 1 or T). For example, this region can be deleted in whole or in part.
  • sequence of amino acid residue 100 - 150 of the RSV F polypeptide or protein such as SEQ ID NO: 1, SEQ ID NO:2, or the soluble ecto domains thereof, is
  • soluble RSV F polypeptides or proteins may contain one or more oligomerization sequences.
  • an oligomerization sequence is present, it is preferably a trimerization sequence.
  • Suitable oligomerization sequences are well known in the art and include, for example, the coiled coil of the yeast GCN4 leucine zipper protein, trimerizing sequence from bacteriophage T4 fibritin ("foldon"), and the trimer domain of influenza HA. These and other suitable oligomerization sequences are described in greater detail herein.
  • sequence of the carboxy terminus of the RSV F polypeptide or protein, starting from position 480 is
  • GCN PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNDKIEEILSKIYHIENEIARIKKLIGE
  • RSV F polypeptides or proteins that contain a transmembrane region may contain an added amino acid sequence that provides a protease cleavage site.
  • This type of RSV F polypeptide or protein can be produced by expression on the surface of a cell, and recovered in soluble form after cleavage from the cell surface using an appropriate protease.
  • the amino acid sequence that provides a protease cleavage site will be located within about 60 amino acids, about 50 amino acids, about 40 amino acids, about 30 amino acids, about 20 amino acids, about 10 amino acids, or substantially adjacent to the amino terminus of the transmembrane domain (amino acid 525 of SEQ ID NO: 1 or 2).
  • Many suitable amino acid sequences that are cleaved by commercially available proteases are well-known in the art. For example, thrombin cleaves the sequence LVPR (SEQ ID NO:75), factor Xa cleaves the sequence IEGR and enterokinase cleaves the sequence DDDDK (SEQ ID NO:76).
  • These amino acid sequences can be introduced into an RSV F polypeptide.
  • the sequence of the RSV F polypeptide or protein, starting from position 488 to the TM region is a sequence shown in FIG. 2.
  • Immunogenic polypeptides used according to the invention will usually be isolated or purified. Thus, they will not be associated with molecules with which they are normally, if applicable, found in nature.
  • an F protein used with the invention will not be in the form of a RSV virion (although it may be in the form of an artificial virion, such as a virosome or VLP).
  • Polypeptides will usually be prepared by expression in a recombinant host system. Generally, they (e.g., RSV ecto-domains) are produced by expression of recombinant constructs that encode the ecto-domains in suitable recombinant host cells, although any suitable methods can be used.
  • Suitable recombinant host cells include, for example, insect cells (e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster), avian cells (e.g., chicken, duck, and geese), bacteria (e.g., E.
  • insect cells e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni
  • mammalian cells e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., ham
  • yeast cells e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis,
  • Tetrahymena cells e.g., Tetrahymena thermophila
  • suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)).
  • Suitable mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH- 3T3 cells, 293 -T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL- 160), Madin-Darby bovine kidney (“MDBK”) cells, Madin-Darby canine kidney (“MDCK”) cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), baby hamster kidney (BHK) cells, such as BHK21-F, HKCC cells, and the like.
  • CHO Chinese hamster ovary
  • HEK293 cells human embryonic kidney cells
  • Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, duck cells (e.g., AGEl. CR and AGEl.CR.pIX cell lines (ProBioGen) which are described, for example, in Vaccine 27:4975-4982 (2009) and WO2005/042728), EB66 cells, and the like.
  • chicken embryonic stem cells e.g., EBx® cells
  • chicken embryonic fibroblasts e.g., chicken embryonic germ cells
  • duck cells e.g., AGEl. CR and AGEl.CR.pIX cell lines (ProBioGen) which are described, for example, in Vaccine 27:4975-4982 (2009) and WO2005/042728
  • EB66 cells e.g., EB66 cells, and the like.
  • Suitable insect cell expression systems such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas
  • baculovirus/insert cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA.
  • Avian cell expression systems are also known to those of skill in the art and described in, e.g., U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent No. EP 0787180B; European Patent Application No. EP03291813.8 ;WO 03/043415; and WO 03/076601.
  • bacterial and mammalian cell expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et ah, eds., 1989) Butterworths, London.
  • Recombinant constructs encoding RSV F protein ecto-domains can be prepared in suitable vectors using conventional methods.
  • a number of suitable vectors for expression of recombinant proteins in insect or mammalian cells are well-known and conventional in the art.
  • Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a
  • transcriptional control element e.g., a promoter, an enhancer, a terminator
  • a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species.
  • a suitable baculovirus expression vector such as pFastBac (Invitrogen) is used to produce recombinant baculovirus particles.
  • the baculovirus particles are amplified and used to infect insect cells to express recombinant protein.
  • a vector that will drive expression of the construct in the desired mammalian host cell e.g., Chinese hamster ovary cells
  • RSV F protein ecto-domain polypeptides can be purified using any suitable methods.
  • methods for purifying RSV F ecto-domain polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et at, J. Gen. Virol, 55:3677-3687 (2004).
  • Suitable methods for purifying desired proteins including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are well-known in the art.
  • Suitable purification schemes can be created using two or more of these or other suitable methods.
  • the RSV F protein ecto-domain polypeptides can include a "tag" that facilitates purification, such as an epitope tag or a HIS tag.
  • tagged polypeptides can conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography.
  • the RSV F polypeptides may also be produced in situ by expression of nucleic acids that encode them in the cells of a subject. For example, by expression of a self- replicating RNA described herein.
  • Polypeptides may include additional sequences in addition to the RSV sequences.
  • a polypeptide may include a sequence to facilitate purification (e.g., a poly-His sequence).
  • the natural leader peptide of F protein may be substituted for a different one.
  • reference 6 used a honeybee melittin leader peptide in place of the natural one.
  • the invention includes immunogenic compositions that include any of the forms and conformations of RSV F polypeptides and proteins disclosed herein, including any desired combination of the forms and conformations of RSV F polypeptides and proteins disclosed herein.
  • the RSV F polypeptide can be a monomer, or the RSV F protein can be a trimer comprising three monomer polypeptides. Trimers can be monodispersed or can be in the form of a rosette, for example, due to interactions between the fusion peptides of individual timers.
  • Immunogenic compositions may comprise polypeptides that are monomers, trimers, a combination of monomers and trimers (e.g., in dynamic equilibrium), rosettes of trimers, and any combination of the foregoing.
  • the RSV F protein can be in a post- fusion conformation, a pre-fusion conformation, or intermediate conformation.
  • the RSV F protein can be in a pre-fusion conformation, a post-fusion conformation or an intermediate conformation.
  • the "post fusion conformation" of RSV F protein is believed to be the low energy conformation of native RSV F, and is a trimer characterized by the presence of a six-helix bundle comprising 3 HRB and 3HRA regions.
  • the post- fusion conformation has a characteristic "crutch” or "golf tee” shape by electron microscopy.
  • the "pre-fusion conformation" of RSV F protein is a conformation characterized by a trimer that contains a coiled coil comprising 3 HRB regions.
  • the fusion peptide is not exposed in the pre-fusion conformation and, therefore, prefusion conformations generally do not form rosettes, and have a a "lollipop" or "ball and stem” shape by electron microscopy.
  • the RSV F protein is in the post-fusion conformation.
  • the RSV F protein can be in the form of a monodisperse trimer in the post- fusion conformation, or in the form of a rosette comprised of post-fusion trimers.
  • the RSV F polypeptide is a monomer. In some embodiments, the RSV F polypeptide is a trimer.
  • the RSV F protein is in the pre-fusion conformation.
  • the pre-fusion conformation or intermediate forms of RSV F protein may contain epitopes that are the same as those on the RSV F protein expressed on natural RSV virions, and therefore provide advantages for eliciting neutralizing antibodies.
  • polypeptides that disfavors the post-fusion conformation of the F protein.
  • the polypeptides (in whole or in part) will display an epitope of the pre-fusion F protein or an epitope of an intermediate conformation in the conversion from the pre-fusion conformation to the post-fusion conformation.
  • These polypeptides may be native or mutated F proteins in a pre-fusion state, may be native or mutated F proteins in an intermediate conformation, or may be a population of native or mutated proteins where the post-fusion conformation has been disfavored or preferentially excluded.
  • the native or mutated protein may be combined with one or more additional molecules that assist in maintaining the polypeptides in one of the foregoing states such as a monoclonal antibody that preferentially binds the pre-fusion conformation or an intermediate conformation.
  • the polypeptides may be derivatives of native F proteins. Such derivatives include polypeptides comprising one or more fragments of a native F protein, fusion polypeptides comprising a native F protein (or fragment thereof) and a heterologous sequence, and polypeptides comprising a native F protein sequence having one or more mutations. These (or other) modifications may disfavor the post-fusion conformation. Exemplary approaches to disfavor the post-fusion conformation include stabilizing the pre-fusion conformation, stabilizing an intermediate conformation, destabilizing the post- fusion conformation or increasing the activation barrier of one or more steps leading to the post fusion conformation.
  • the invention is a polypeptide that displays at least one epitope that is specific to the pre-fusion conformation F protein or an intermediate conformation F protein.
  • An epitope that is specific to the pre-fusion conformation F protein or an intermediate conformation F protein is an epitope that is not presented in the post- fusion conformation. It is preferred that the at least one epitope is stably presented, e.g., the epitope is stably presented in solution for at least twelve hours, at least one day, at least two days, at least four days, at least six days, at least one week, at least two weeks, at least four weeks, or at least six weeks.
  • Such polypeptides may be native or mutated F proteins in the pre-fusion state, an intermediate state or a population of states where the post- fusion state is
  • polypeptides comprising one or more fragments of a native F protein include polypeptides comprising one or more fragments of a native F protein, fusion polypeptides comprising a native F protein (or fragment thereof) and a heterologous sequence, and polypeptides comprising a native F protein sequence having one or more mutations.
  • modifications may stabilize an F protein amino acid sequence in its pre-fusion conformation, stabilize an F protein amino acid sequence in an intermediate conformation, destabilize the post fusion conformation of an F protein amino acid sequence, increase the energy barrier in a transition leading to the post-fusion conformation of an F protein amino acid sequence, or a combination of two or more of the foregoing.
  • TM and/or CT domains of F proteins are important for the stability of the pre-fusion conformation (8). Thus these domains may usefully be retained in immunogens of the invention. As it may be desirable not to include transmembrane domains in soluble immunogens, though, the functional effect of TM may be achieved by other means. For instance, the pre- and post- fusion behavior of F protein of parainfluenza virus 5 has been studied in some detail (6), and the authors stabilized the ED' s pre-fusion structure by fusing a heterologous trimerization domain to the C- terminus of the ED.
  • the composition may comprise a polypeptide (e.g., recombinant polypeptide) that comprises a first domain and a second domain, wherein (i) the first domain comprises the RSV F protein (e.g. RSV ectodomain, in whole or part), and (ii) the second domain comprises a heterologous oligomerization domain.
  • the second domain permits oligomerization of the polypeptide, thereby facilitating the first domain to adopt a pre-fusion state or an intermediate state.
  • the polypeptide preferably will be present as an oligomer, and in particular as a trimer.
  • oligomerization domains are available to the skilled person. These are sequences of amino acids that can form a structure that can interact with
  • trimerization domains (whether the same or different) in other polypeptides (whether the same or different) such that the multiple polypeptides can associate (usually non- covalently) to form oligomers, e.g., trimers.
  • trimerization of the F protein of HIV i.e., gpl60
  • ATCase catalytic subunit of is. coli aspartate transcarbamoylase
  • trimerization of the F proteins of HIV (10) and PIV5 (6) has been achieved by fusing their ectodomains to GCNt.
  • the oligomerization domain used with the present invention may comprise the coiled coil of the yeast GCN4 leucine zipper protein (11). Trimerization of the ectodomain of HA protein from influenza A virus has been achieved by using a trimerizing sequence ('foldon') from the bacteriophage T4 fibritin
  • oligomerization domain used with the present invention may comprise such a foldon.
  • Naturally-occurring protein oligomers (both hetero-oligomers and homo- oligomers) associate in a variety of different ways, e.g., by association of ⁇ -sheets in different monomers, by association of ⁇ -helices in different monomers, by association of hydrophobic surface patches, etc.
  • One common structural motif involved in protein oligomerization is the coiled-coil domain.
  • the coiled ⁇ -helix structural motif can itself form coils, and two, three, four or five ⁇ -helices can wrap around each other to form a left-handed super-helix known as the "coiled coil” though artificial right-handed super helices have been designed (13-19).
  • the simplicity of the coiled-coil domain has made it a popular choice for designing chimeric proteins with defined oligomerization states (16).
  • the ⁇ -helices interact through hydrophobic residues that form an apolar stripe along one side of each helix, and there may also be stabilizing electrostatic interactions between side chains on either side of this stripe.
  • the apolar stripe is defined by hydrophobic side chains at residues a and d, with any electrostatic interactions being primarily at residues e and g.
  • Position a is most frequently Leu, He or Ala and position d is usually Leu or Ala.
  • Residues e and g are often GIu or GIn, with Arg and Lys also prominent at position g.
  • oligomerization domain to allow refolding and rearrangement such as occurs in the F protein.
  • coiled-coil domain sequences are known in the art, and any suitable sequence can be used as an oligomerization domain with the invention, provided that it retains the ability to oligomerize with other coiled-coil domains and that it does not destroy the function of the other domains within the polypeptide. It is preferred to use a coiled-coil domain which is found extracellularly (20) and which naturally acts as an oligomerization domain. As an alternative to using a natural coiled-coil domain, artificial coiled-coil domains can be used (21, 22).
  • Domain (b) may include a leucine zipper sequence or an alanine zipper sequence (23).
  • the coiled-coil domain used in the polypeptide of the invention is preferably one which forms a trimer, such that the polypeptide of the invention can also assemble into a trimer.
  • Preferred coiled-coil domains are those taken from bacterial
  • transmembrane proteins A preferred subset of transmembrane proteins is the adhesins (i.e., cell-surface proteins that mediate adhesion to other cells or to surfaces), and particularly non-fimbrial adhesins (e.g., in the oligomerization coiled-coil adhesins, or 'Oca', family).
  • adhesins i.e., cell-surface proteins that mediate adhesion to other cells or to surfaces
  • non-fimbrial adhesins e.g., in the oligomerization coiled-coil adhesins, or 'Oca', family.
  • sequences for use with the invention include those disclosed in reference 24 from Yersinia enterocolitica adhesin YadA, Neisseria meningitidis adhesin NadA, Moraxella catarrhalis surface protein UspA2, and other adhesins, such as the HadA adhesin from Haemophilus influenzae biogroup aegyptius etc (SEQ ID NOs 28- 31 & 42-58 of ref. 24).
  • the eukaryotic heat-shock transcription factor has a coiled-coil trimerization domain that can be separately expressed and therefore used with the invention.
  • the heptad-repeat nature of the ⁇ -helices means that the boundary of the coiled-coil domain can be determined with some precision, but the precise residue where a coiled-coil arrangement can be said to end may not be known with absolute accuracy. This lack of absolute precision is not a problem for practicing the invention, however, as routine testing can reveal whether the coiled-coil requires any particular amino acid residue for which there might be doubt.
  • the invention does not require the boundaries to be known with absolute precision, as the only basic requirement for the invention is that the coiled-coil domain should function in a way that allows the polypeptide to oligomerize with other coiled-coil domains without destroying the function of the other domains within the polypeptide.
  • oligomerization domain that can be used with the invention is found in the left-handed triple helix known as the collagen helix (25). These triple helix-forming sequences involve a basic tripeptide repeat sequence of 1 Gly- 2 Xaa- 3 Xaa, where 2 Xaa is often Pro, and 3 Xaa is often 4-hydroxyproline. Although this motif is known as the "collagen" helix, it is found in many proteins beyond just collagen.
  • the oligomerization domain may thus be a sequence comprising multiple repeats of the sequence motif 1 Gly- 2 Xaa- 3 Xaa, which motif folds to form a helical structure that can oligomerize with corresponding helical structures in other polypeptide chains.
  • Collagen also provides another class of oligomerization domain.
  • Reference 26 describes a motif found in the non-collagenous domain 1 (NCl) of type X collagen, and this motif can be used for trimer and higher order multimer formation without a triple helix. This trimeric association is highly thermostable without intermolecular disulfide bonds.
  • the oligomerization domain may thus comprise an NCl sequence.
  • oligomerization domains may be derived from the transmembrane domains of oligomeric TM proteins. As these are usually lipophilic, hydrophobic residues positioned on the outside of their TM regions may be substituted with charged residues, to provide a soluble domain.
  • Such methods of solubilizing transmembrane domains by protein engineering are known in the art, for example from reference 27. This method has also been used for GCN4, where the "a" and "d" heptad repeat positions were replaced with isoleucine (11):
  • KQIEDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 20).
  • Suitable coiled coil sequences for use within the oligomerization domain will usually be between 20 and 35 amino acids long, e.g., 23 to 30 amino acid residues long.
  • Oligomerization domains used with the invention can generally maintain an oligomeric structure without the need for the formation of inter-monomer disulfide bridges, but oligomers containing disulfide-linked monomers are not excluded from the invention.
  • mutation can be used.
  • reference 28 reports that mutation in a conserved region of the F2 subunit of the F proteins in simian virus 5 or hendra virus can influence the stability of the pre-fusion conformation.
  • a low pH may also be used to favor the pre-fusion conformation.
  • conformation of the F protein may be disfavored by stabilization of the HRB domain trimer.
  • the HRB domain forms a triple stranded coiled coil in the pre-fusion and likely the intermediate forms.
  • coiled-coils have been extensively studied as model systems for intermolecular interactions between proteins and as model systems for longer range intra-molecular interactions (i.e., tertiary folding interactions). These studies are useful in teaching methods that may be used to stabilize the HRB domain in the trimeric coiled-coil form.
  • one or more residues at the a and/or d positions of the heptad repeat may be replaced with residues that favor formation of stable trimeric coiled-coils such as He residues.
  • disfavorable ionic interactions at the e and g positions may be deleted or favorable ionic interactions at the e and g positions may be added.
  • the preferred region of the HRB domain for manipulation is the heptad repeat between P484-N517.
  • Preferred examples of a and d residues to target for mutations are F488, 1492, V495, 1499, S502, 1506, S509, L512, and V516.
  • the serine residues are especially preferred as replacement of the hydrophilic residues with hydrophobic residues would stabilize the hydrophobic core of the coiled-coil.
  • Another preferred target would be the phenylalanine with a smaller hydrophobic residue that would pack better in the core such as an isoleucine.
  • conformation of the F protein may be disfavored by destabilization of the HRA domain trimer.
  • the HRA domain forms a triple stranded coiled coil in the post-fusion and possibly one or more intermediate forms.
  • one or more residues at the a and/or d positions of the heptad repeat may be replaced with residues that disfavor formation of stable trimeric coiled-coils.
  • favorable ionic interactions at the e and g positions may be deleted or disfavorable ionic interactions at the e and g positions may be added.
  • such mutations will be selected that have minimal impact on the stability of the HRA domain in the pre-fusion conformation as may be modeled based upon the available crystal structures of the PIV5 F protein in the pre-fusion and post fusion forms.
  • modifications can further be designed based upon molecular modeling of the hRSV F proteins based upon the available crystal structures of the PIV5 F protein in the pre-fusion and post fusion forms. Mutations may be made that destabilize the post-fusion conformation such as the 6HB fold of the HRA and HRB domains or that stabilize the pre-fusion
  • amino acids N-terminal to the HRB domain amino acids N-terminal to the HRB domain
  • tether (approximately a.a. 449-482, preferably V459-F483) act as a "tether" that allows the HRB domain to shift from one side of the F protein trimer to the other side so that the HRB domain can participate in the 6HB of the post-fusion conformation. Deletion of one or more of these amino acids will impair or outright prevent the HRB domain from participating in the 6HB fold of the post-fusion conformation of the F protein (see Figure 3).
  • the interaction between the tether and the F protein in the pre- fusion conformation can be stabilized to prevent the tether from pulling away to allow the HRB domain to participate in the 6HB fold. Examples of stabilizing mutations that could be made are cysteine bridges between the tether and the portion of the F protein which the tether contacts in the pre-fusion conformation.
  • the hydrophobic core may be stabilized by replacing buried hydrophilic or ionic residues with similarly sized hydrophobic residues.
  • cysteine bridges may be introduced at the surface or within the core.
  • the hydrophobic cores or proteins are relatively rigid and therefore introducing holes predictably destabilized the lysozyme mutants.
  • repacking the core of the F protein in the pre-fusion conformation to eliminate any natural holes can stabilize the F protein in the pre-fusion or intermediate forms, thus disfavoring the post-fusion conformation.
  • the invention relates to methods for preparing compositions and to
  • compositions that contain RSV F protein in particular soluble RSV F ecto-domain polypeptides, including immunogenic compositions.
  • the RSV F ecto- domain polypeptides are in a single form, such as uncleaved monomers, uncleaved trimers, cleaved trimers, rosettes of cleaved trimers, or in a dynamic equilibrium between a subset of such forms (e.g., equilibrium between uncleaved monomers and uncleaved trimers).
  • the invention provides methods for producing compositions that contain a predominate desired form of RSV F protein, or a single desired form of RSV F protein, such as uncleaved monomers, uncleaved trimers, cleaved trimers, rosettes of cleaved trimers, a dynamic equilibrium between a subset of such forms (e.g., equilibrium between uncleaved monomers and uncleaved trimers), or a mixture of desired form of RSV F protein.
  • a predominate desired form of RSV F protein or a single desired form of RSV F protein
  • a single desired form of RSV F protein such as uncleaved monomers, uncleaved trimers, cleaved trimers, rosettes of cleaved trimers, a dynamic equilibrium between a subset of such forms (e.g., equilibrium between uncleaved monomers and uncleaved trimers), or a mixture of desired form of RSV F protein.
  • These types of compositions can be used for a variety of purposes, such
  • RSV F protein ecto-domain polypeptides When RSV F protein ecto-domain polypeptides are produced by conventional recombinant expression in host cells, the polypeptides are cleaved at the furin cleavage sites at about position 109/110 and at about position 136/137 during production in the host cell before they are secreted into the culture media. Cleavage of the polypeptides by the host cells is permissive for RSV F protein ecto-domain polypeptide refolding, which results in exposure of the hydrophobic fusion peptide.
  • the cleaved RSV F protein ecto-domain polypeptides due to the presence of an exposed fusion peptide, form rosettes and associate with lipids and lipoproteins that are derived from the host cells and culture media.
  • electron microscopy of cleaved RSV F ectodomain that are poduced in insect cells and purified by virtue of a HIS 6 -tag showed that the polypeptides had a crutch shape consistent with a post- fusion form and were bound to what appeared to residual cell debris. Accordingly, high purity preparations of rosettes and other forms and confirmations of RSV F protein ecto-domain polypeptides cannot be readily obtained by conventional recombinant expression in host cells.
  • the invention is a method for preparing a composition that contains cleaved RSV F protein ecto-domain polypeptides.
  • the method involves providing uncleaved RSV F protein ecto-domain polypeptides and then cleaving them to produce a Fi subunit and a F 2 subunit.
  • uncleaved RSV F protein ecto-domain polypeptides can be readily purified and separated from contaminating lipids and lipoproteins using suitable methods, such as size exclusion chromatography.
  • uncleaved RSV F protein ecto-domain polypeptides can be cleaved to produce Fi and F 2 subunits, which can be purified as trimers, rosettes of trimers, or a mixture of trimers and rosettes of trimers.
  • Uncleaved RSV F protein ecto-domain polypeptides can be produced using any suitable method. For example, by recombinant production in host cells that do not contain active furin or furin-like proteases at the time the RSV F protein ecto-domain polypeptides are being produced.
  • a variety of methods can be used to achieve this method of production, such as, production in recombinant host cells that are mutated to prevent expression of furin or furin-like protease (conditionally or complete "knock- out"), and various methods that reduce or prevent expression of furin or furin-like proteases in the host cells, for example, using RNA interference or other similar methods, or inhibiting furin or furin-like protease activity in host cells using inhibitors of the proteases.
  • RSV F protein ecto-domain polypeptides are preferably produced by recombinant expression of constructs that encode a RSV F protein ecto-domain in which the amino acid sequence of the furin cleavage sites are altered, so that the RSV F protein ecto-domain polypeptides are secreted by a host cell that produces the polypeptides uncleaved.
  • the uncleaved RSV F protein ecto-domain polypeptides can be produced using any suitable host cell, such as insect cells (e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster), avian cells (e.g., chicken, duck, and geese, bacteria (e.g., E.
  • insect cells e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni
  • mammalian cells e.g., human, non-human primate, horse, cow, sheep
  • yeast cells e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris , Schizosaccharomyces pombe and Yarrowia lipolytica
  • Tetrahymena cells e.g., Tetrahymena thermophila
  • suitable insect cells and mammalian cells are well-known in the art. Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line
  • Suitable mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293 -T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCC CCL- 171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL-160), Madin- Darby bovine kidney (“MDBK”) cells, Madin-Darby canine kidney (“MDCK”) cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), baby hamster kidney (BHK) cells, such as BHK21-F, HKCC cells, and the like.
  • CHO Chinese hamster ovary
  • HEK293 cells human embryonic kidney cells
  • Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, duck cells (e.g., AGEl. CR and AGEl.CR.pIX cell lines (ProBioGen) which are described, for example, in Vaccine 27:4975-4982 (2009) and WO2005/042728), EB66 cells, and the like.
  • chicken embryonic stem cells e.g., EBx® cells
  • chicken embryonic fibroblasts e.g., chicken embryonic germ cells
  • duck cells e.g., AGEl. CR and AGEl.CR.pIX cell lines (ProBioGen) which are described, for example, in Vaccine 27:4975-4982 (2009) and WO2005/042728
  • EB66 cells e.g., EB66 cells, and the like.
  • Suitable insect cell expression systems such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas
  • baculovirus/insert cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA.
  • Avian cell expression systems are also known to those of skill in the art and described in, e.g., U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent No. EP 0787180B; European Patent Application No. EP03291813.8 ;WO 03/043415; and WO 03/076601.
  • bacterial and mammalian cell expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et ah, eds., 1989) Butterworths, London.
  • the amino acid sequence of an uncleaved RSV F protein ecto-domain is altered to prevent cleavage at the furin cleavage sites at about position 109/110 and about position 136/137, but contains a naturally occurring or introduced protease cleavage site, that when cleaved produce a F 1 subunit and a F 2 subunit.
  • the uncleaved RSV F protein ecto-domain polypeptide can have an amino acid sequence that is altered to prevent cleavage at the furin cleavage sites at about position 109/110 and about position 136/137, but contain one or more naturally occurring or introduced protease cleavage sites from about position 101 to about position 161.
  • a variety of particular amino acid sequences that will allow uncleaved RSV F protein ecto-domain polypeptides to be produced and expressed by host cells including amino acid sequences that are not cleaved at the furin cleavage sites at about position 109/110 and about position 136/137 can be readily designed and envisioned by a person of ordinary skill in the art.
  • one or more amino acids that are part of, or are located near by, the furin cleavage sites at about position 109/110 and about position 136/137 are independently replaced or deleted.
  • Some amino acid substitutions and deletions that are suitable to prevent cleavage of RSV F protein ecto-domain polypeptides are known. For example, the substitutions R108N, R109N,
  • R108N/R109N which inhibit cleavage at 109/110, and the substitution K13 IQ or the deletion of the amino acids at positions 131-134, which inhibit cleavage at 136/137, have been described
  • Gonzalez-Reyes et al Proc. Natl. Acad. ScL USA, 98:9859-9864 (2001).
  • An uncleaved RSV F ecto-domain polypeptide that contains the amino acid substitutions R108N/R109N/ K131Q/R133Q/ R135Q/R136Q has been described. Ruiz- Arguello et al, J. Gen. Virol. 55:3677687 (2004).
  • the altered furin cleavage sites contain at least one amino acid substitution or deletion at about positions 106-109, and at least one amino acid substitution or deletion at about positions 133-136.
  • amino acid sequences of uncleaved RSV F protein ecto-domain polypeptides that contain a protease cleavage site (e.g., naturally occurring or introduced) that when cleaved produce a first subunit that comprises an Fi and a second subunit that comprises F 2 , are possible and can be readily designed and envisioned.
  • the amino acid sequence of RSV F protein from about position 101 to about position 161 contains trypsin cleavage sites, and one or more of the trypsin cleavage sites can be cleaved by trypsin to generate Fi and F 2 subunits.
  • one or more suitable protease recognition sites can be introduced into the uncleaved RSV F protein ecto-domain polypeptide, for example, between about positions 101 to about position 161.
  • the introduced protease recognition sites can be cleaved using the appropriate protease to generate Fi and F 2 subunits.
  • a protease recognition site is introduced into the amino acid sequence of an uncleaved RSV F protein ecto-domain polypeptide, it is preferred that the site is recognized by a protease that does not cleave the ecto-domain of naturally occurring RSV F protein.
  • the method of this aspect of the invention includes: a) providing uncleaved RSV F protein ecto-domain polypeptides containing a protease cleavage site that, when cleaved, produces F 1 and F 2 subunits, and b) cleaving the uncleaved RSV F protein ecto-domain polypeptides with a protease that recognizes the protease cleavage site.
  • the amino acid sequence of the uncleaved RSV F protein ecto-domain polypeptides contains altered furin cleavage sites, and the RSV F protein ecto-domain polypeptides are secreted from a host cell that produces them uncleaved at the furin cleavage sites at about positions 106-109 and about positions 131-136.
  • the provided uncleaved RSV F protein ecto-domain polypeptides can be purified to the desired degree.
  • the provided uncleaved RSV F protein ecto-domain polypeptides can be provided as a cell lysate, cell homogenate or cell culture conditioned media that is substantially unprocessed (e.g., unprocessed, or clarified only), or in partially or substantially purified form.
  • the provided uncleaved RSV F protein ecto-domain polypeptides are provided in cell culture conditioned media selected from the group consisting of insect cell conditioned media, mammalian cell conditioned media, avian cell conditioned media, yeast cell conditioned media, Tetrahymena cell conditioned media, and combinations thereof.
  • uncleaved RSV F protein ecto-domain polypeptides are purified, for example, purified to be at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogenous.
  • uncleaved RSV F protein ecto-domain polypeptides can be readily purified from lipids and lipoproteins, while conventionally produced cleaved forms of RSV F protein co-purify with lipid and lipoprotein contaminants.
  • the method can be used to readily produce a composition containing cleaved RSV F protein ecto-domains that are substantially free of lipids or lipoproteins.
  • Suitable methods for cleaving polypeptides using a protease are well-known and conventional in the art. Generally, the polypeptides to be cleaved are combined with a sufficient amount of protease under conditions (e.g., pH, polypeptide and protease concentration, temperature) suitable for cleavage of the polypeptide. Many suitable proteases are commercially available, and suitable conditions for performing polypeptide cleavage are well-known for many proteases. If desired, the cleaved RSV F protein ecto-domain polypeptides can be purified following cleavage with protease.
  • uncleaved RSV F protein ecto-domain polypeptides are provided that contain an intact fusion peptide, such as an uncleaved RSV F protein ecto-domain polypeptide in which none of the amino acids from positions 137-154 are substituted or deleted.
  • the provided uncleaved RSV F protein ecto-domain polypeptides are purified.
  • the provided uncleaved RSV F protein ecto-domain polypeptides that contain an intact fusion peptide are cleaved, and cleavage results in the formation of rosettes of cleaved RSV F protein ecto-domain polypeptide trimers. If desired, the rosettes can be purified further using any suitable methods, such as size exclusion chromatography.
  • uncleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide, such as an uncleaved RSV F protein ecto-domain polypeptide in which about amino 137-152, about amino acids 137-153, about amino acids 137-145 or about amino acids 137-142 are deleted.
  • Other suitable fusion peptide deletions have also been described, such as the deletion of the amino acids at positions 137-146. Ruiz-Arguello et al, J. Gen. Virol, 85:3677- 3687 (2004).
  • the provided uncleaved RSV F protein ecto-domain polypeptides are purified.
  • the provided uncleaved RSV F protein ecto-domain polypeptides are cleaved, and cleavage results in the formation of trimers of cleaved RSV F protein ecto-domain polypeptides.
  • the trimers can be purified further using any suitable methods, such as size exclusion chromatography.
  • the provided uncleaved RSV F protein ecto-domain polypeptides contain at least one polypeptide selected from the group consisting of furdel and delp23 furdel (e.g., homogenous trypsin-cleavable furdel, homogenous trypsin-cleavable delp23 furdel, or a mixture of trypsin-cleavable furdel and trypsin-cleavable delp23 furdel).
  • furdel and delp23 furdel e.g., homogenous trypsin-cleavable furdel, homogenous trypsin-cleavable delp23 furdel, or a mixture of trypsin-cleavable furdel and trypsin-cleavable delp23 furdel.
  • the provided uncleaved RSV F protein ecto- domain polypeptides are cleaved, for example with trypsin, and cleavage results in the formation of cleaved trimers, rosettes of cleaved trimers, or a combination of cleaved trimers and rosettes of cleaved trimers of RSV F protein ecto-domain polypeptides.
  • the cleaved trimers and/or rosettes of cleaved trimers can be purified further using any suitable methods, such as size exclusion chromatography.
  • the invention is a method for preparing a composition that contains uncleaved RSV F protein ecto-domain polypeptides.
  • the method involves providing a biological material that contains uncleaved RSV F protein ecto- domain polypeptides , such as a cell lysate, cell homogenate or cell culture conditioned medium, and then purifying the uncleaved RSV F protein ecto-domain polypeptides.
  • uncleaved RSV F protein ecto- domain polypeptide monomers can self associate to form uncleaved trimers, and that there is a mixture of uncleaved monomers and uncleaved trimers or an equilibrium between the uncleaved monomers and uncleaved trimers. Without wishing to be bound by any particular theory, it is believed that the equilibrium favors the monomer, but that the equilibrium will shift toward the trimer in concentrated solutions.
  • the method of this aspect of the invention includes: a) providing a biological material that contains uncleaved RSV F protein ecto-domain polypeptides , such as a cell lysate, cell homogenate or cell culture conditioned medium; and b) purifying uncleaved RSV F protein ecto-domain polypeptide monomers, trimers or a combination of monomers and trimers from the biological material.
  • uncleaved RSV F protein ecto-domain polypeptide monomers are purified, or uncleaved RSV F protein ecto-domain polypeptide trimers are purified, or monomers and trimers are purified.
  • the amino acid sequence of the uncleaved RSV F protein ecto- domain polypeptides contains altered furin cleavage sites, and the RSV F protein ecto- domain polypeptides are secreted from a host cell that produces them uncleaved between about position 101 to about position 161 (including at the furin cleavage sites at positions 106-109 and 131-136).
  • the biological material that contains uncleaved RSV F protein ecto-domain polypeptides includes at least one polypeptide selected from the group consisting of furmt, furdel, delp21 furx, delp23 furx, delp21 furdel, delp23 furdel, and the factor Xa construct, which can be cleaved using factor Xa.
  • the amino acid sequence of the RSV F protein ecto- domain polypeptide contains altered furin cleavage sites, and other protease cleavage sites (e.g., trypsin cleavage sites) between about position 101 and about position 161 are altered or deleted to prevent protease (e.g., trypsin) cleavage.
  • protease e.g., trypsin
  • trypsin is well-known to cleave after lysine and arginine residues.
  • the amino acid sequence of the uncleaved RSV F protein ecto-domain polypeptide contains altered furin cleavage sites, one or more lysine and/or arginine residues (e.g., all lysine and arginine residues) present between about position 101 and about position 161 are deleted or replaced with an amino acid that is not lysine or arginine, the RSV F protein ecto-domain polypeptides are secreted from a host cell that produces them uncleaved between about position 101 and about position 161, and the RSV F protein ecto-domain polypeptides are not cleaved by trypsin between about position 101 and about position 161.
  • one or more lysine and/or arginine residues e.g., all lysine and arginine residues
  • the RSV F protein ecto-domain polypeptides are not cleaved by trypsin when a 1 mg/ml solution of RSV F protein ecto-domain polypeptide (diluted in 25 mM Tris pH 7.5, 300 mM NaCl) is treated with one-one thousandth volume of trypsin solution (trypsin from bovine plasma diluted to a 1 mg/ml concentration in 25 mM Tris pH 7.5, 300 mM NaCl; final mass ratio in digestion reaction is 0.001: 1 trypsin:RSV F ecto-domain; trypsin used at 10-15 BAEE units per mg protein) for 1 hour at 37°C.
  • the uncleaved RSV F protein ecto-domain polypeptides can further contain an altered fusion peptide, such as an uncleaved RSV F protein ecto-domain polypeptide in which, for example, about amino acids 137-152 are deleted, about amino acids 137-154 are deleted, about amino acids 137-145 are deleted or about amino acids 137-142 are deleted.
  • an altered fusion peptide such as an uncleaved RSV F protein ecto-domain polypeptide in which, for example, about amino acids 137-152 are deleted, about amino acids 137-154 are deleted, about amino acids 137-145 are deleted or about amino acids 137-142 are deleted.
  • Other suitable fusion peptide deletions have also been described, such as the deletion of the amino acids at positions 137-146. Ruiz-Arguello et ah, J. Gen. Virol, 85:3677-3687 (2004).
  • the method includes: a) providing a biological material that contains uncleaved RSV F protein ecto-domain polypeptides , such as a cell lysate, cell homogenate or cell culture conditioned medium, wherein the amino acid sequence of the uncleaved RSV F protein ecto-domain polypeptide contains altered furin cleavage sites, the lysine and arginine residues present between about position 101 and about position 161 are deleted or replaced with an amino acid that is not lysine or arginine, the RSV F protein ecto-domain polypeptides are secreted from a host cell that produces them uncleaved between about position 101 and about position 161, and the RSV F protein ecto-domain polypeptides are not cleaved by trypsin between about position 101 and about position 161; and b) purifying uncleaved RSV F protein ecto- domain polypeptide monomers, trimers or a combination of monomers and trimers from
  • the biological material that contains uncleaved RSV F protein ecto-domain polypeptides includes at least one polypeptide selected from the group consisting of Furx, Furx Rl 13Q K123N K124N, delp21 furx and delp23 furx.
  • the method includes: a) providing a biological material that contains uncleaved RSV F protein ecto-domain polypeptides in which the fusion peptide is mutated (e.g., at least a portion of the fusion peptide is deleted) , such as a cell lysate, cell homogenate or cell culture conditioned medium; and b) purifying uncleaved RSV F protein ecto-domain polypeptides from the biological material.
  • a biological material that contains uncleaved RSV F protein ecto-domain polypeptides in which the fusion peptide is mutated (e.g., at least a portion of the fusion peptide is deleted) , such as a cell lysate, cell homogenate or cell culture conditioned medium.
  • the uncleaved RSV F protein ecto-domain polypeptide can contain altered furin cleavage sites, and the RSV F protein ecto-domain polypeptides are secreted from a host cell that produces them uncleaved between about position 101 to about position 161 (including at the furin cleavage sites at positions 106-109 and 131-136). If desired, the uncleaved RSV F protein ecto-domain polypeptide with altered furin cleavage sites further contains altered or deleted sites for other proteases (e.g., trypsin cleavage sites) between about position 101 and about position 161 to prevent protease (e.g., trypsin) cleavage.
  • proteases e.g., trypsin cleavage sites
  • one or more lysine and/or arginine residues present between about position 101 and about position 161 are deleted or replaced with an amino acid that is not lysine or arginine, and the RSV F protein ecto- domain polypeptides are not cleaved by trypsin between about position 101 and about position 161.
  • the uncleaved RSV F protein ecto-domain polypeptide monomers, trimers and combinations of monomers and trimers can be purified to the desired degree. It is generally preferred that the uncleaved RSV F protein ecto-domain polypeptide monomers or trimers are purified, for example, to be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogenous. As described herein, uncleaved RSV F protein ecto-domain polypeptides can be readily purified from lipids and lipoproteins, for example, by size exclusion chromatography.
  • the method can be used to readily produce a composition containing cleaved RSV F protein ecto-domain polypeptide monomers, trimers, or a combination of monomers and trimers that are substantially free of lipids and lipoproteins.
  • the method includes providing insect cell culture conditioned medium, mammalian cell culture conditioned medium, avian cell conditioned medium, yeast cell conditioned medium, Tetrahymena cell conditioned medium, or a combination thereof.
  • uncleaved RSV F protein ecto-domain polypeptide trimers are purified.
  • uncleaved RSV F protein ecto-domain polypeptide monomers are purified.
  • uncleaved RSV F protein ecto-domain polypeptide monomers and trimers are purified.
  • the invention is a method for preparing a composition that contains cleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide.
  • RSV F protein ecto-domain polypeptides that do not contain altered furin cleavage sites are expressed in host cells, the host cells process the polypeptides, in part by cleaving the polypeptide at the furin sites at about positions 109/110 and about positions 136/137 to produce Fi and F 2 subunits.
  • the processed polypeptides are secreted into the culture and can be recovered as associated F 1 -F 2 subunits (e.g., disulphide bonding Fi and F 2 subunits), which can form rosettes of trimers through aggregation of exposed fusion peptides.
  • RSV F protein ecto-domain polypeptides that contain altered fusion peptides can be produced in and secreted from host cells as associated Fi-F 2 subunits, and preferably do not aggregate into rosettes or with lipids or lipoprotein contaminants. Without wishing to be bound by any particular theory, it is believed that the polypeptides do not form rosettes or associate with lipid and lipoprotein contaminants because the altered fusion peptide does not mediate aggregation.
  • the method of this aspect of the invention includes: a) providing a biological material that contains cleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide (e.g., at least a portion of the fusion peptide is deleted), such as a cell lysate, cell homogenate or cell culture conditioned medium; and b) purifying cleaved RSV F protein ecto-domain polypeptides from the biological material.
  • the purified cleaved RSV F protein ecto-domain polypeptides can be purified as cleaved trimers, rosettes of cleaved trimers, or a mixture of cleaved trimers and rosettes of cleaved trimers.
  • Suitable RSV F protein ecto-domain polypeptides that contain altered fusion peptides contain cleavable furin cleavage sites at about 109/110 and about 136/137 and further contain an altered fusion peptide as described herein.
  • an RSV F protein ecto-domain polypeptide in which about amino acids 137-152 are deleted, about amino acids 137-153 are deleted, about amino acids 137-145 are deleted, about amino acids 137-146 are deleted or about amino acids 137-142 are deleted can be used in the method.
  • the biological material that contains uncleaved RSV F protein ecto-domain polypeptides includes at least the fusion peptide deletion 1.
  • the cleaved RSV F protein ecto-domain polypeptides can be purified to the desired degree. It is generally preferred that the cleaved RSV F protein ecto-domain polypeptides are purified, for example, to be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogenous.
  • cleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide can be readily purified from lipids and lipoproteins, for example, by size exclusion chromatography. Accordingly, the method can be used to readily produce a composition containing cleaved RSV F protein ecto-domain polypeptide trimers, rosettes of cleaved trimers, or a combination of cleaved trimers and rosettes of cleaved trimers that are substantially free of lipids and lipoproteins.
  • the invention is a method for preparing a composition that contains C-terminal uncleaved RSV ecto-domain polypeptides and a method for preparing cleaved RSV F protein ecto-domain polypeptides.
  • C-terminal uncleaved RSV F protein ecto-domain polypeptides are cleaved by cells that produce the proteins at the furin cleavage site at about postion 109/110, but not at the furin cleavage site at about position 136/137, and are secreted into the media as an Fi subunit that is associated with an F 2 subunit.
  • C-terminal uncleaved RSV F protein ecto-domains can be cleaved further to produce a Fi subunit, in which the amino terminus is from position 110 to about position 161, that is associated with a F 2 subunit.
  • Fi and F 2 subunits which can be purified as trimers, rosettes of trimers, or a mixture of trimers and rosettes of trimers.
  • the amino acid sequence of a C-terminal uncleaved RSV F protein ecto-domain is altered to prevent cleavage at the furin cleavage site at about position 136/137, but contains a naturally occurring or introduced protease cleavage site, that when cleaved produces a Fi subunit, in which the amino terminus is from position 110 to about position 161, and a F 2 subunit.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptide can have an amino acid sequence that is altered to prevent cleavage at the furin cleavage sites at about position 136/137, but contain one or more naturally occurring or introduced protease cleavage sites from about position 101 to about position 161.
  • the amino acid sequence of a C- terminal uncleaved RSV F protein ecto-domain is altered to prevent cleavage at the furin cleavage site at about position 136/137, but contains a naturally occurring furin cleavage site at about position 109/110.
  • a variety of particular amino acid sequences that will allow C-terminal uncleaved RSV F protein ecto-domain polypeptides to be produced and expressed by host cells including amino acid sequences that are not cleaved at the furin cleavage sites at about position 136/137, can be readily designed and envisioned by a person of ordinary skill in the art.
  • one or more amino acids that are part of, or are located near by, the furin cleavage sites at about position 136/137 are independently replaced or deleted. Suitable amino acid substitutions and deletions that prevent cleavage at about position 136/137 are described herein.
  • C-terminal uncleaved RSV F protein ecto-domain polypeptides comprise at least one amino acid substitution or deletion at about positions 133-136.
  • amino acid sequences of C-terminal uncleaved RSV F protein ecto-domain polypeptides that contain a protease cleavage site that when cleaved produce a first subunit that comprises an Fi and a second subunit that comprises F 2
  • a protease cleavage site e.g., naturally occurring or introduced
  • the amino acid sequence of RSV F protein from about position 101 to about position 161 contains trypsin cleavage sites, and one or more of the trypsin cleavage sites can be cleaved by trypsin to generate Fi and F 2 subunits.
  • one or more suitable protease recognition sites can be introduced into the C-terminal uncleaved RSV F protein ecto-domain polypeptide, for example, between about positions 101 to about position 161.
  • the introduced protease recognition sites can be cleaved using the appropriate protease to generate Fi and F 2 subunits.
  • a protease recognition site is introduced into the amino acid sequence of a C-terminal uncleaved RSV F protein ecto-domain polypeptide, it is preferred that the site is recognized by a protease that does not cleave the ecto-domain of naturally occurring RSV F protein.
  • C-terminal uncleaved RSV F protein ecto-domain polypeptides can be produced using any suitable method.
  • a preferred method is by recombinant expression of constructs that encode a RSV F protein ecto-domain in which that amino acid sequence of the furin cleavage site at about positions 136/137 is altered, so that the C-terminal uncleaved RSV F protein ecto-domain polypeptides are secreted by a host cell that produces the polypeptides uncleaved at the furin cleavage site at about position 136/137.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptide is secreted by a host cell that produces it as an Fi subunit that is associated with an F 2 subunit, wherein the amino terminus of the Fi subunit is from position 132 to about position 161, but not position 137.
  • the C-terminal uncleaved RSV F protein ecto- domain polypeptides can be produced using any suitable host cell, as described herein.
  • One method of this aspect of the invention includes: a) providing C-terminal uncleaved RSV F protein ecto-domain polypeptides that comprise an altered furin cleavage site at position 136/137, and said C-terminal uncleaved RSV F protein ecto- domain polypeptides are secreted from a cell that produces them in the form of an F 2 fragment that is associated with a subunit that comprises Fi but is uncleaved at position 136/137, and b) cleaving the provided C-terminal uncleaved RSV F protein ecto- domain polypeptides with a protease that cleaves RSV F protein ecto-domain at a site between positions 101 and 161, thereby producing said composition.
  • step b) comprises cleaving the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides with a protease that cleaves RSV F protein ecto- domain at a site between about positions 101 and 132, or about positions 132 and 161, or about positions 110 and 132.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptides comprise an altered furin cleavage site at position 136/137, with the proviso that the altered furin cleavage site is not deletion of amino acids 131-134.
  • the biological material that contains C-terminal uncleaved RSV F protein ecto-domain polypeptides includes at least the N-term Furin polypeptide.
  • the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides can be purified to the desired degree.
  • the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides can be provided in a cell lysate, cell homogenate, or cell culture conditioned media that is substantially unprocessed (e.g., unprocessed, or clarified only), or in partially or substantially purified form.
  • the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides are provided in cell culture conditioned media selected from the group consisting of insect cell conditioned media, mammalian cell conditioned media, avian cell conditioned media, yeast cell conditioned media, Tetrahymena cell conditioned media, and combinations thereof. It is generally preferred that the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides are purified, for example, purified to be at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogenous.
  • C-terminal uncleaved RSV F protein ecto-domain polypeptides can be readily purified from lipids and lipoproteins, while conventionally produced cleaved forms of RSV F protein co-purify with lipid and lipoprotein contaminants. Accordingly, when purified C-terminal uncleaved RSV F protein ecto-domain polypeptides are provided, the method can be used to readily produce a composition containing cleaved RSV F protein ecto-domains that are substantially free of lipids or phospholipids.
  • Suitable methods for cleaving polypeptides using a protease are well-known and conventional in the art. Generally, the polypeptides to be cleaved are combined with a sufficient amount of protease under conditions (e.g., pH, polypeptide and protease concentration, temperature) suitable for cleavage of the polypeptide. Many suitable proteases are commercially available, and suitable conditions for performing polypeptide cleavage are well-known for many proteases. If desired, the RSV F protein ecto-domain polypeptides can be purified following cleavage with protease.
  • C-terminal uncleaved RSV F protein ecto- domain polypeptides that contain an intact fusion peptide, such as a C- terminal uncleaved RSV F protein ecto-domain polypeptide in which none of the amino acids from positions 137-154 are substituted or deleted.
  • C-terminal uncleaved RSV F protein ecto-domain polypeptides are provided that contain an altered fusion peptide, such as a C-terminal uncleaved RSV F protein ecto-domain polypeptide in which about amino 137-152, about amino acids 137-153, about amino acids 137-145 or about amino acids 137-142 are deleted.
  • Other suitable fusion peptide deletions have also been described, such as the deletion of the amino acids at positions 137-146. Ruiz-Arguello et al, J. Gen. Virol, 85:3677-3687 (2004).
  • the provided C-terminal uncleaved RSV F protein ecto- domain polypeptides are purified.
  • the provided uncleaved RSV F protein ecto-domain polypeptides are cleaved, and cleavage results in the formation of trimers of cleaved
  • the trimers can be purified further using any suitable methods, such as size exclusion chromatography.
  • the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides contain at least the N-terminal Furin polypeptide (FIG. 1).
  • the provided C-terminal uncleaved RSV F protein ecto-domain polypeptides are cleaved, for example with trypsin, and cleavage results in the formation of cleaved trimers, rosettes of cleaved trimers, or a combination of cleaved trimers and rosettes of cleaved trimers of RSV F protein ecto-domain polypeptides.
  • the cleaved trimers and/or rosettes of cleaved trimers can be purified further using any suitable methods, such as size exclusion chromatography.
  • Another method of this aspect of the invention includes: a) providing a biological material, such as a cell lysate, cell homogenate or cell culture conditioned medium, that contains a C-terminal uncleaved RSV F protein ecto-domain polypeptides that comprise an altered furin cleavage site at position 136/137, and said soluble RSV F protein ecto-domain polypeptides are secreted from a cell that produces them in the form of an F 2 fragment that is associated with a subunit that comprises Fi but is uncleaved at position 136/137, with the proviso that the altered furin cleavage site is not deletion of amino acids 131-134; and b) purifying the C-terminal uncleaved RSV F protein ecto-domain polypeptides from the biological material, thereby producing the composition.
  • a biological material such as a cell lysate, cell homogenate or cell culture conditioned medium
  • the amino terminus of the Fi subunit is from about position 110 to about position 132. More preferably, the amino terminus of the Fi subunit is about position 110. It is particulary preferred that the amino terminus of the Fi subunit is not position 137.
  • the biological material that contains C- terminal uncleaved RSV F protein ecto-domain polypeptides includes at least the N- term Furin polypeptide.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptide further contains altered or deleted sites for other proteases (e.g., trypsin cleavage sites) between about position 101 and about position 161 to prevent protease (e.g., trypsin) cleavage.
  • proteases e.g., trypsin cleavage sites
  • one or more lysine and/or arginine residues present between about position 101 and about position 161 are deleted or replaced with an amino acid that is not lysine or arginine, and the C-terminal uncleaved RSV F protein ecto-domain polypeptides are not cleaved by trypsin between about position 101 and about position 161.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptides can contain an intact fusion peptide or an altered fusion peptide, as described herein.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptides can be purified to the desired degree. It is generally preferred that the C-terminal uncleaved RSV F protein ecto-domain polypeptide monomers or trimers are purified, for example, to be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or substantially homogenous. As described herein, C-terminal uncleaved RSV F protein ecto-domain polypeptides can be readily purified from lipids and lipoproteins, for example, by size exclusion chromatography.
  • the method can be used to readily produce a composition containing C-terminal uncleaved RSV F protein ecto- domain polypeptides, e.g., monomers, trimers, or a combination of monomers and trimers, that are substantially free of lipids and lipoproteins.
  • the C-terminal uncleaved RSV F protein ecto-domain polypeptides contain at least the N-terminal Furin polypeptide (FIG. 1).
  • the method includes providing insect cell culture conditioned medium, mammalian cell culture conditioned medium, avian cell conditioned media, yeast cell conditioned media, Tetrahymena cell conditioned media or a combination thereof.
  • C-terminal uncleaved RSV F protean ecto-domain polypeptide trimers are purified.
  • C-terminal uncleaved RSV F protein ecto-domain polypeptide monomers are purified.
  • C- terminal uncleaved RSV F protein ecto-domain polypeptide monomers and trimers are purified.
  • the RSV-F polypeptides described herein can be produced by expression of recombinant nucleic acids that encode the polypeptides in the cells of a subject.
  • Prefereed nucleic acids that can be administered to a subject to cause the production of RSV-F polypeptides are self-replicating RNA molecules.
  • the self-replicating RNA molecules of the invention are based on the genomic RNA of RNA viruses, but lack the genes encoding one or more structural proteins.
  • the self-replicating RNA molecules are capable of being translated to produce non-structural proteins of the RNA virus and heterologous proteins encoded by the self-replicating RNA.
  • the self-replicating RNA generally contains at least one or more genes selected from the group consisting of viral replicase, viral proteases, viral helicases and other nonstructural viral proteins, and also comprise 5'- and 3 '-end cis-active replication sequences, and if desired, a heterologous sequences that encode a desired amino acid sequences (e.g., a protein, an antigen).
  • a subgenomic promoter that directs expression of the heterologous sequence can be included in the self-replicating RNA.
  • the heterologous sequence may be fused in frame to other coding regions in the self- replicating RNA and/or may be under the control of an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • Self-replicating RNA molecules of the invention can be designed so that the self-replicating RNA molecule cannot induce production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary for the production of viral particles in the self- replicating RNA.
  • an alpha virus such as Sinebis virus (SIN), Semliki forest virus and Venezuelan equine encephalitis virus (VEE)
  • one or more genes encoding viral structural proteins, such as capsid and/or envelope glycoproteins can be omitted.
  • self-replicating RNA molecules of the invention can be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.
  • a self-replicating RNA molecule can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (or from an antisense copy of itself).
  • the self-replicating RNA can be directly translated after delivery to a cell, and this translation provides a RNA- dependent RNA polymerase which then produces transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These transcripts are antisense relative to the delivered RNA and may be translated themselves to provide in situ expression of a gene product, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the encoded RSV-F polypeptide.
  • RNA replicon One suitable system for achieving self-replication is to use an alphavirus-based RNA replicon. These + stranded replicons are translated after delivery to a cell to give of a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein which auto cleaves to provide a replication complex which creates genomic - strand copies of the + strand delivered RNA. These - strand transcripts can themselves be transcribed to give further copies of the + stranded parent RNA and also to give a subgenomic transcript which encodes the RSV-F polypeptide. Translation of the subgenomic transcript thus leads to in situ expression of the RSV-F polypeptide by the infected cell. Suitable alphavirus replicons can use a replicase from a Sindbis virus, a semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc.
  • a preferred self-replicating RNA molecule thus encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an RSV-F polypeptide.
  • the polymerase can be an alphavirus replicase e.g. comprising alphavirus protein nsP4.
  • an alphavirus based self-replicating RNA molecule of the invention does not encode alphavirus structural proteins.
  • the self replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing alphavirus virions.
  • the inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in infectious form.
  • alphavirus structural proteins which are necessary for perpetuation in wild- type viruses are absent from self replicating RNAs of the invention and their place is taken by gene(s) encoding the desired gene product, such that the subgenomic transcript encodes the desired gene product rather than the structural alphavirus virion proteins.
  • a self-replicating RNA molecule useful with the invention may have two open reading frames.
  • the first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an RSV-F polypeptide.
  • the RNA may have additional (downstream) open reading frames e.g. that encode further desired gene products.
  • a self-replicating RNA molecule can have a 5' sequence which is compatible with the encoded replicase.
  • the self-replicating RNA molecule is derived from or based on an alphavirus.
  • the self-replicating RNA molecule is derived from or based on a virus other than an alphavirus, preferably, a positive-stranded RNA viruses, and more preferably a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.
  • a virus other than an alphavirus preferably, a positive-stranded RNA viruses, and more preferably a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.
  • Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md.
  • alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR- 1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR- 1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR- 1242), Fort Morgan (ATCC VR- 924), Getah virus (ATCC VR-369, ATCC VR- 1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR- 1277), Middleburg (ATCC VR- 370), Mucambo virus (ATCC VR-580, ATCC VR- 1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR- 1245), Ross River virus (ATCC VR-373, ATCC VR- 1246), Semliki Forest (ATCC VR-67, ATCC VR- 1247), Sindbis virus
  • the self-replicating RNA may be associated with a delivery system.
  • the self- replicating RNA may be administered with or without an adjuvant.
  • the self-replicating RNA of the invention are suitable for delivery in a variety of modalities, such as naked RNA delivery or in combination with lipids, polymers or other compounds that facilitate entry into the cells.
  • Self-replicating RNA molecules of the present invention can be introduced into target cells or subjects using any suitable technique, e.g., by direct injection, microinjection, electroporation, lipofection, biolystics, and the like.
  • the self-replicating RNA molecule may also be introduced into cells by way of receptor-mediated endocytosis. See e.g., U.S. Pat. No. 6,090,619; Wu and Wu, J. Biol. Chem., 263: 14621 (1988); and Curiel et al, Proc.
  • U.S. Pat. No. 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety (e.g., poly-L-lysine having 3-100 lysine residues), which is itself coupled to an integrin receptor-binding moiety (e.g., a cyclic peptide having the sequence Arg-Gly-Asp).
  • a polycation moiety e.g., poly-L-lysine having 3-100 lysine residues
  • an integrin receptor-binding moiety e.g., a cyclic peptide having the sequence Arg-Gly-Asp
  • the self-replicating RNA molecule of the present invention can be delivered into cells via amphiphiles. See e.g., U.S. Pat. No. 6,071,890.
  • a nucleic acid molecule may form a complex with the cationic amphiphile. Mammalian cells contacted with the complex can readily take it up.
  • the self-replicating RNA can be delivered as naked RNA (e.g. merely as an aqueous solution of RNA) but, to enhance entry into cells and also subsequent intercellular effects, the self-replicating RNA is preferably administered in combination with a delivery system, such as a particulate or emulsion delivery system.
  • a delivery system such as a particulate or emulsion delivery system.
  • delivery systems include, for example liposome-based delivery (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No.
  • Three particularly useful delivery systems are (i) liposomes (ii) non-toxic and biodegradable polymer microparticles (iii) cationic submicron oil-in-water emulsions.
  • RNA-containing aqueous core can have an anionic, cationic or zwitterionic hydrophilic head group. Formation of liposomes from anionic phospholipids dates back to the 1960s, and cationic liposome- forming lipids have been studied since the 1990s. Some phospholipids are anionic whereas other are zwitterionic.
  • Suitable classes of phospholipid include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols, and some useful phospholipids are listed in Table 20.
  • Useful cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane
  • DOTAP l,2-distearyloxy-N,N-dimethyl-3-aminopropane
  • DODMA 1,2-dioleyloxy- N,Ndimethyl-3 -aminopropane
  • DLinDMA 1,2-dilinoleyloxy-N,N-dimethyl-3 - aminopropane
  • DLenDMA 1,2-dilinolenyloxy-N,N-dimethyl-3 -aminopropane
  • Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are DPPC, DOPC and dodecylphosphocholine. The lipids can be saturated or unsaturated.
  • Liposomes can be formed from a single lipid or from a mixture of lipids.
  • a mixture may comprise (i) a mixture of anionic lipids (ii) a mixture of cationic lipids (iii) a mixture of zwitterionic lipids (iv) a mixture of anionic lipids and cationic lipids (v) a mixture of anionic lipids and zwitterionic lipids (vi) a mixture of zwitterionic lipids and cationic lipids or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids.
  • a mixture may comprise both saturated and unsaturated lipids.
  • a mixture may comprise DSPC (zwitterionic, saturated), DHnDMA (cationic, unsaturated), and/or DMPG (anionic, saturated).
  • a mixture of lipids not all of the component lipids in the mixture need to be amphiphilic e.g. one or more amphiphilic lipids can be mixed with cholesterol.
  • the hydrophilic portion of a lipid can be PEGylated (i.e. modified by covalent attachment of a polyethylene glycol). This modification can increase stability and prevent non-specific adsorption of the liposomes.
  • lipids can be conjugated to PEG using techniques such as those disclosed in Heyes et al. (2005) J Controlled Release 107:276-287.
  • a mixture of DSPC, DHnDMA, PEG-DMPG and cholesterol is used in the examples.
  • a separate aspect of the invention is a liposome comprising DSPC, DHnDMA, PEG-DMG and cholesterol.
  • This liposome preferably encapsulates RNA, such as a self-replicating RNA e.g. encoding an immunogen.
  • Liposomes are usually divided into three groups: multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles (LUV).
  • MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments.
  • SUVs and LUVs have a single bilayer encapsulating an aqueous core; SUVs typically have a diameter ⁇ 50nm, and LUVs have a diameter >50nm.
  • Liposomes useful with of the invention are ideally LUVs with a diameter in the range of 50-220nm.
  • compositions comprising a population of LUVs with different diameters: (i) at least 80% by number should have diameters in the range of 20-220nm, (H) the average diameter (Zav, by intensity) of the population is ideally in the range of 40-200nm, and/or (Hi) the diameters should have a polydispersity index ⁇ 0.2.
  • Liposomes Methods and Protocols, Volume 1 : Pharmaceutical Nanocarriers: Methods and Protocols, (ed. Weissig). Humana Press, 2009. ISBN 160327359X; Liposome Technology, volumes I, II & III. (ed. Gregoriadis). Informa Healthcare, 2006; and Functional Polymer Colloids and Microparticles volume 4 (Microspheres,
  • microcapsules & liposomes (eds. Arshady & Guyot). Citus Books, 2002.
  • One useful method involves mixing (i) an ethanolic solution of the lipids (H) an aqueous solution of the nucleic acid and (Hi) buffer, followed by mixing, equilibration, dilution and purification (Heyes et al. (2005) J Controlled Release 107:276-87.).
  • RNA is preferably encapsulated within the liposomes, and so the liposome forms a outer layer around an aqueous RNA-containing core. This encapsulation has been found to protect RNA from RNase digestion.
  • the liposomes can include some external RNA (e.g. on the surface of the liposomes), but at least half of the RNA (and ideally all of it) is encapsulated.
  • RNA molecules can form microparticles to encapsulate or adsorb RNA.
  • the use of a substantially non-toxic polymer means that a recipient can safely receive the particles, and the use of a biodegradable polymer means that the particles can be metabolised after delivery to avoid long-term persistence.
  • Useful polymers are also sterilisable, to assist in preparing pharmaceutical grade formulations.
  • Suitable non-toxic and biodegradable polymers include, but are not limited to, poly( ⁇ -hydroxy acids), polyhydroxy butyric acids, polylactones (including
  • polycaprolactones polydioxanones, polyvalerolactone, polyorthoesters,
  • polyanhydrides polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl- pyrrolidinones or polyester-amides, and combinations thereof.
  • the microparticles are formed from poly( ⁇ -hydroxy acids), such as a poly(lactides) ("PLA”), copolymers of lactide and glycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), and copolymers of D,L-lactide and caprolactone.
  • PLG polymers include those having a lactide/glycolide molar ratio ranging, for example, from 20:80 to 80:20 e.g. 25:75, 40:60, 45:55, 55:45, 60:40, 75:25.
  • Useful PLG polymers include those having a molecular weight between, for example, 5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000, 40,000- 50,000 Da.
  • microparticles ideally have a diameter in the range of 0.02 ⁇ m to 8 ⁇ m.
  • a composition comprising a population of microparticles with different diameters at least 80% by number should have diameters in the range of 0.03-7 ⁇ m.
  • a microparticle may include a cationic surfactant and/or lipid e.g. as disclosed in O'Hagan et al.
  • Microparticles of the invention can have a zeta potential of between 40-100 mV.
  • RNA can be adsorbed to the microparticles, and adsorption is facilitated by including cationic materials (e.g. cationic lipids) in the microparticle.
  • Oil-in-water emulsions are known for adjuvanting influenza vaccines e.g. the MF59TM adjuvant in the FLUADTM product, and the AS03 adjuvant in the
  • RNA delivery according to the present invention can utilise an oil-in-water emulsion, provided that the emulsion includes one or more cationic molecules.
  • a cationic lipid can be included in the emulsion to provide a positive droplet surface to which negatively-charged RNA can attach.
  • the emulsion comprises one or more oils.
  • Suitable oil(s) include those from, for example, an animal (such as fish) or a vegetable source.
  • the oil is ideally biodegradable (metabolisable) and biocompatible.
  • Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils.
  • Jojoba oil can be used e.g. obtained from the jojoba bean.
  • Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.
  • corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used.
  • 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and so may be used. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
  • cod liver oil cod liver oil
  • shark liver oils and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
  • a number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids.
  • Squalane the saturated analog to squalene
  • Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art.
  • oils are the tocopherols, particularly in combination with squalene.
  • the oil phase of an emulsion includes a tocopherol
  • any of the ⁇ , ⁇ , ⁇ , ⁇ , ⁇ or ⁇ tocopherols can be used, but ⁇ -tocopherols are preferred.
  • DL- ⁇ -tocopherol can both be used.
  • a preferred ⁇ -tocopherol is DL- ⁇ -tocopherol.
  • An oil combination comprising squalene and a tocopherol (e.g. DL- ⁇ -tocopherol) can be used.
  • the oil in the emulsion may comprise a combination of oils e.g. squalene and at least one further oil.
  • the aqueous component of the emulsion can be plain water (e.g. w.f.i.) or can include further components e.g. solutes. For instance, it may include salts to form a buffer e.g. citrate or phosphate salts, such as sodium salts.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer.
  • a buffered aqueous phase is preferred, and buffers will typically be included in the 5-2OmM range.
  • the emulsion also includes a cationic lipid.
  • this lipid is a surfactant so that it can facilitate formation and stabilisation of the emulsion.
  • Useful cationic lipids generally contains a nitrogen atom that is positively charged under physiological conditions e.g. as a tertiary or quaternary amine. This nitrogen can be in the hydrophilic head group of an amphiphilic surfactant.
  • Useful cationic lipids include, but are not limited to: l,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-
  • DC Cholesterol dimethyldioctadecyl- ammonium
  • DDA dimethyldioctadecyl- ammonium
  • DMTAP l,2-Dimyristoyl-3-Trimethyl-AmmoniumPropane
  • DPTAP dipalmitoyl(C16:0)trimethyl ammonium propane
  • DSTAP distearoyltrimethylammonium propane
  • Other useful cationic lipids are: benzalkonium chloride (BAK), benzethonium chloride, cetramide (which contains tetradecyltrimethylammonium bromide and possibly small amounts of
  • dedecyltrimethylammonium bromide and hexadecyltrimethyl ammonium bromide cetylpyridinium chloride (CPC), cetyl trimethylammonium chloride (CTAC), N,N',N- polyoxyethylene (10)-N-tallow-l,3 -diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecyl- ammonium chloride, benzyltrimethylammonium methoxide,
  • cetyldimethylethylammonium bromide dimethyldioctadecyl ammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl trioctylammonium chloride), N,N-dimethyl-N-[2 (2-methyl-4-(l,l,3,3tetramethylbutyl)- phenoxy]-ethoxy)ethyl]-benzenemetha-naminium chloride (DEBDA), dialkyldimetylammonium salts, [l-(2,3-dioleyloxy)-propyl]-
  • acyl group dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), l,2-dioleoyl-3-(4'-trimethyl- ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl 3-succinyl-sn- glycerol choline ester, cholesteryl (4'-trimethylammonio) butanoate), N-alkyl pyridinium salts (e.g.
  • cetylpyridinium bromide and cetylpyridinium chloride N- alkylpiperidinium salts, dicationic bolaform electrolytes (C12Me6; C12BU6), dialkylglycetylphosphorylcholine, lysolecithin, L- ⁇ dioleoylphosphatidylethanolamine, cholesterol hemisuccinate choline ester, lipopolyamines, including but not limited to dioctadecylamidoglycylspermine (DOGS), dipalmitoyl phosphatidylethanol- amidospermine (DPPES), lipopoly-L (or D)- lysine (LPLL, LPDL), poly (L (or D)- lysine conjugated to N- glutarylphosphatidylethanolamine, didodecyl glutamate ester with pendant amino group (C ⁇ GluPhCnN ), ditetradecyl glutamate ester with pendant amino group (
  • the cationic lipid is preferably biodegradable (metabolisable) and
  • an emulsion can include a non-ionic surfactant and/or a zwitterionic surfactant.
  • surfactants include, but are not limited to: the polyoxy ethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWF AXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypoly ethoxy ethanol) being of particular interest;
  • octylphenoxy polyethoxyethanol
  • phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.
  • Preferred surfactants for including in the emulsion are polysorbate 80 (T ween 80; polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-IOO.
  • ком ⁇ онент can be included in the emulsion e.g. Tween 80/Span 85 mixtures, or Tween 80/Triton-X100 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxy-polyethoxyethanol (Triton X-IOO) is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • Useful mixtures can comprise a surfactant with a HLB value in the range of 10-20 (e.g. polysorbate 80, with a HLB of 15.0) and a surfactant with a HLB value in the range of 1-10 (e.g. sorbitan trioleate, with a HLB of 1.8).
  • Preferred amounts of oil (% by volume) in the final emulsion are between 2- 20% e.g. 5-15%, 6-14%, 7-13%, 8-12%.
  • a squalene content of about 4-6% or about 9- 11% is particularly useful.
  • Preferred amounts of surfactants (% by weight) in the final emulsion are between 0.001% and 8%.
  • polyoxyethylene sorbitan esters such as polysorbate 80
  • polysorbate 80 0.2 to 4%, in particular between 0.4-0.6%, between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%, between 1.9-2.1%, about 2%, or 0.85- 0.95%, or about 1%
  • sorbitan esters such as sorbitan trioleate
  • 0.02 to 2% in particular about 0.5% or about 1%
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100
  • polyoxyethylene ethers such as laureth 9 0.1 to 8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
  • the absolute amounts of oil and surfactant, and their ratio, can be varied within wide limits while still forming an emulsion.
  • a skilled person can easily vary the relative proportions of the components to obtain a desired emulsion, but a weight ratio of between 4: 1 and 5: 1 for oil and surfactant is typical (excess oil).
  • the oil droplet size (diameter).
  • the most effective emulsions have a droplet size in the submicron range.
  • the droplet sizes will be in the range 50-750nm.
  • the average droplet size is less than 250nm e.g. less than 200nm, less than 150nm.
  • the average droplet size is usefully in the range of 80-180nm.
  • at least 80% (by number) of the emulsion's oil droplets are less than 250 nm in diameter, and preferably at least 90%. Apparatuses for determining the average droplet size in an emulsion, and the size distribution, are commercially available.
  • the distribution of droplet sizes has only one maximum i.e. there is a single population of droplets distributed around an average (mode), rather than having two maxima.
  • Preferred emulsions have a polydispersity of ⁇ 0.4 e.g. 0.3, 0.2, or less.
  • Suitable emulsions with submicron droplets and a narrow size distribution can be obtained by the use of microfluidisation.
  • This technique reduces average oil droplet size by propelling streams of input components through geometrically fixed channels at high pressure and high velocity. These streams contact channel walls, chamber walls and each other. The results shear, impact and cavitation forces cause a reduction in droplet size.
  • Repeated steps of microfluidisation can be performed until an emulsion with a desired droplet size average and distribution are achieved.
  • thermal methods can be used to cause phase inversion. These methods can also provide a submicron emulsion with a tight particle size distribution.
  • Preferred emulsions can be filter sterilised i.e. their droplets can pass through a 220nm filter. As well as providing a sterilisation, this procedure also removes any large droplets in the emulsion.
  • the cationic lipid in the emulsion is DOTAP.
  • the cationic oil-in-water emulsion may comprise from about 0.5 mg/ml to about 25 mg/ml DOTAP.
  • the cationic oil-in-water emulsion may comprise DOTAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6 mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, from about 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25 mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml to about 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3 mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, from about 1.5 mg/ml to about 25 mg/ml, from about
  • the cationic oil-in-water emulsion comprises from about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8 mg/ml, 1.2 mg/ml, 1.4 mg/ml or 1.6 mg/ml.
  • the cationic lipid is DC Cholesterol.
  • the cationic oil- in-water emulsion may comprise DC Cholesterol at from about 0.1 mg/ml to about 5 mg/ml DC Cholesterol.
  • the cationic oil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/ml to about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5 mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml,
  • the cationic lipid is DDA.
  • the cationic oil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/ml DDA.
  • the cationic oil-in-water emulsion may comprise DDA at from about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.5 mg/ml, from about 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml to about 5 mg/ml, from about 0.4 mg/m
  • the cationic oil-in-water emulsion may comprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about 21.6 mg/ml, about 25 mg/ml.
  • the cationic oil-in-water emulsion comprises from about 0.73 mg/ml to about 1.45 mg/ml DDA, such as 1.45 mg/ml.
  • RNA molecules of the invention may be used to deliver the self-replicating RNA molecules of the invention, as naked RNA or in combination with a delivery system, into a target organ or tissue.
  • Suitable catheters are disclosed in, e.g., U.S. Pat. Nos. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and 6,129,705, all of which are incorporated herein by reference.
  • the present invention includes the use of suitable delivery systems, such as liposomes, polymer microparticles or submicron emulsion microparticles with encapsulated or adsorbed self-replicating RNA, to deliver a self-replicating RNA molecule that encodes an RSV-F polypeptide, for example, to elicit an immune response alone, or in combination with another macromolecule.
  • suitable delivery systems such as liposomes, polymer microparticles or submicron emulsion microparticles with encapsulated or adsorbed self-replicating RNA, to deliver a self-replicating RNA molecule that encodes an RSV-F polypeptide, for example, to elicit an immune response alone, or in combination with another macromolecule.
  • the invention includes liposomes, microparticles and submicron emulsions with adsorbed and/or encapsulated self-replicating RNA molecules, and combinations thereof.
  • the self-replicating RNA molecules associated with liposomes and submicron emulsion microparticles can be effectively delivered to the host cell, and can induce an immune response to the protein encoded by the self-replicating RNA.
  • the immunogenic composition is an immunogenic composition
  • the invention provides immunogenic compositions.
  • the immunogenic compositions may include a single active immunogenic agent, or several immunogenic agents.
  • the immunogenic composition can comprise RSV F polypeptides that are in a single form (e.g., monomer, trimer, or rosettes) or in two or more forms (e.g., a mixture of monomer and trimer or a dynamic equilibrium between monomer and trimer).
  • the immunogenic composition can comprise a self-replicating RNA encoding an RSV-F polypeptide, and preferably also comprises a suitable delivery system, such as liposomes, polymeric microparticles, an oil-in-water emulsion and combinations thereof.
  • Immunogenic compositions of the invention may also comprise one or more immunoregulatory agents.
  • one or more of the immunoregulatory agents include one or more adjuvants, for example two, three, four or more adjuvants.
  • the adjuvants may include a THl adjuvant and/or a TH2 adjuvant, further discussed below.
  • an immunogenic composition of the invention comprises a polypeptide that displays an epitope present in a pre-fusion or an intermediate conformation of RSV-F glycoprotein, but does not display the
  • glycoprotein's post-fusion conformation is glycoprotein's post-fusion conformation.
  • an immunogenic composition of the invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an RSV F protein, in whole or in part, and the second polypeptide comprises a heterologous oligomerization domain.
  • the first polypeptide can comprise an RSV F protein ectodomain.
  • the second polypeptide can be a trimerization domain from influenza hemagglutinin, a trimerization domain from SARS spike, a trimerization domain from HIV gp41, NadA, modified GCN4, or ATCase.
  • the invention is a composition comprising cleaved RSV F protein ecto-domain polypeptides produced by providing uncleaved RSV F protein ectodomain polypeptides, or C-terminal uncleaved RSV F protein ecto-domain
  • polypeptides and cleaving them to produce Fi and F 2 subunits, as described herein.
  • the invention is a composition comprising uncleaved RSV F protein ecto-domain polypeptide trimers and/or monomers produced by providing a biological material that contains uncleaved RSV F protein ecto-domain polypeptides, and purifying uncleaved RSV F protein ecto-domain polypeptides monomers, uncleaved trimers, or a combination of uncleaved monomers and uncleaved trimers (e.g., a mixture or a dynamic equilibrium) from the biological material, as described herein.
  • a biological material that contains uncleaved RSV F protein ecto-domain polypeptides
  • purifying uncleaved RSV F protein ecto-domain polypeptides monomers, uncleaved trimers, or a combination of uncleaved monomers and uncleaved trimers e.g., a mixture or a dynamic equilibrium
  • the RSV F protein ecto-domain polypeptide contains altered furin cleavage sites at about positions 106-109 and at about positions 133-136, and if desired can further contain an altered fusion peptide. In other embodiments, the RSV F protein ecto-domain contains altered furin cleavage sites about positions 106- 109 and at about positions 133-136, and altered trypsin cleavage sites between about position 101 and about position 161, and if desired can further contain an altered fusion peptide.
  • the invention is a composition comprising C-terminal uncleaved RSV F protein ecto-domain polypeptide trimers and/or monomers produced by providing a biological material that contains C-terminal uncleaved RSV F protein ecto-domain polypeptides, and purifying uncleaved RSV F protein ecto-domain polypeptides monomers, uncleaved trimers, or a combination of uncleaved monomers and uncleaved trimers (e.g., a mixture or a dynamic equilibrium) from the biological material, as described herein.
  • the invention is a composition comprising cleaved RSV F protein ecto-domain polypeptides produced by providing a biological material that contains cleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide (e.g., at least a portion of the fusion peptide is deleted) and purifying cleaved RSV F protein ecto-domain polypeptide trimers from the biological material, as described herein.
  • the invention is a composition comprising uncleaved RSV F protein ecto-domain polypeptides produced by providing a biological material that contains uncleaved RSV F protein ecto-domain polypeptides that contain an altered fusion peptide (e.g., at least a portion of the fusion peptide is deleted) and purifying uncleaved RSV F protein ecto-domain polypeptide monomers from the biological material, as described herein.
  • an altered fusion peptide e.g., at least a portion of the fusion peptide is deleted
  • compositions of the invention are preferably suitable for administration to a mammalian subject, such as a human, and include one or more pharmaceutically acceptable carrier(s) and/or excipient(s), including adjuvants.
  • a mammalian subject such as a human
  • compositions will generally be in aqueous form. When the composition is an immunogenic composition, it will elicit an immune response when administered to a mammal, such as a human.
  • immunogenic composition can be used to prepare a vaccine formulation for immunizing a mammal.
  • the immunogenic compositions may include a single active immunogenic agent, or several immunogenic agents.
  • the RSV F protein ecto-domain polypeptide can be in a single form (e.g., uncleaved monomer, cleaved monomer, uncleaved trimer, cleaved trimer, or rosettes of cleaved trimers) or in two or more forms (e.g., a mixture of uncleaved monomer and uncleaved trimer or a dynamic equilibrium between uncleaved monomer and uncleaved trimer).
  • compositions can contain an RSV F protein ecto-domain polypeptide and one or more other RSV proteins (e.g., a G protein and/or an M protein) and/or it may be combined with immunogens from other pathogens.
  • RSV F protein ecto-domain polypeptide e.g., a G protein and/or an M protein
  • the composition may include preservatives such as thiomersal or 2- phenoxyethanol. It is preferred, however, that the vaccine should be substantially free from (i.e., less than 5 ⁇ g/ml) mercurial material, e.g., thiomersal-free. Immunogenic compositions containing no mercury are more preferred. Preservative-free
  • immunogenic compositions are particularly preferred.
  • a physiological salt such as a sodium salt.
  • Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml.
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, and the like.
  • Compositions will generally have an osmolality of between 200 mOsm/kg and
  • 400 m ⁇ sm/kg preferably between 240-360 m ⁇ sm/kg, and will more preferably fall within the range of 290-310 m ⁇ sm/kg.
  • Compositions may include one or more buffers.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer.
  • Buffers will typically be included in the 5-2OmM range.
  • the pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0, e.g., between 6.5 and 7.5, or between 7.0 and 7.8.
  • a process of the invention may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging.
  • the composition is preferably sterile.
  • the composition is preferably non-pyrogenic, e.g., containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • the composition is preferably gluten free.
  • Human vaccines are typically administered in a dosage volume of about 0.5ml, although a half dose (i.e., about 0.25ml) may be administered to children.
  • Adjuvants are typically administered in a dosage volume of about 0.5ml, although a half dose (i.e., about 0.25ml) may be administered to children.
  • compositions of the invention that contain RSV-F polypeptids, or nucleic acids that encode RSV-F polypeptids, may also include one or more adjuvants, for example two, three, four or more adjuvants, which can function to enhance the immune responses (humoral and/or cellular) elicited in a patient who receives the composition.
  • the adjuvants may include a THl adjuvant and/or a TH2 adjuvant.
  • Adjuvants which may be used in compositions of the invention include, but are not limited to:
  • Mineral-containing compositions suitable for use as adjuvants in the invention include mineral salts, such as calcium salts and aluminum salts (or mixtures thereof).
  • the invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc., or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred.
  • Calcium salts include calcium phosphate (e.g., the "CAP" particles disclosed in ref. 38).
  • Aluminum salts include hydroxides, phosphates, sulfates, and the like.
  • the mineral containing compositions may also be formulated as a particle of metal salt (39). Aluminum salt adjuvants are described in more detail below.
  • Oil emulsion compositions suitable for use as adjuvants in the invention include squalene- water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80 and 0.5% Span, formulated into submicron particles using a microfluidizer).
  • squalene- water emulsions such as MF59 (5% Squalene, 0.5% Tween 80 and 0.5% Span, formulated into submicron particles using a microfluidizer).
  • Cytokine-inducing agents suitable for use in the invention include toll-like receptor 7 (TLR7) agonists (e.g. benzonaphthyridine compounds disclosed in WO 2009/111337.
  • TLR7 toll-like receptor 7
  • Saponins (chapter 22 of ref. 74), which are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla),
  • Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
  • QS21 is marketed as STIMULON (TM).
  • Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C.
  • the saponin is QS21.
  • a method of production of QS21 is disclosed in ref. 40.
  • Saponin formulations may also comprise a sterol, such as cholesterol (41). Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs) (chapter 23 of ref. 74).
  • ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA & QHC. ISCOMs are further described in refs. 41-43.
  • the ISCOMS may be devoid of additional detergent (44).
  • a review of the development of saponin based adjuvants can be found in refs. 45 & 46.
  • Fatty adjuvants including oil-in-water emulsions, modified natural lipid As derived from enterobacterial lipopolysaccharides, phospholipid compounds (such as the synthetic phospholipid dimer, E6020) and the like.
  • Bacterial ADP-ribosylating toxins e.g., the E. coli heat labile enterotoxin "LT”, cholera toxin “CT”, or pertussis toxin “PT”
  • LT heat labile enterotoxin
  • CT cholera toxin
  • PT pertussis toxin
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 48 and as parenteral adjuvants in ref. 49.
  • Bioadhesives and mucoadhesives such as esterified hyaluronic acid microspheres (50) or chitosan and its derivatives (51).
  • Microparticles i.e., a particle of -100 nm to -150 ⁇ m in diameter, more preferably -200 nm to -30 ⁇ m in diameter, or -500 nm to -10 ⁇ m in diameter
  • materials that are biodegradable and non-toxic e.g., a poly( ⁇ - hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, and the like
  • poly(lactide-co-glycolide) being preferred, optionally treated to have a negatively-charged surface (e.g., with SDS) or a positively-charged surface (e.g., with a cationic detergent, such as CTAB).
  • Liposomes Choapters 13 & 14 of ref. 74). Examples of liposome formulations suitable for use as adjuvants are described in refs. 52-54.
  • P olyoxy ethylene ethers and polyoxyethylene esters (55). Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (56) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (57).
  • Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9- steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • Muramyl peptides such as N-acetylmuramyl-L-threonyl-D-isoglutamine (“thr- MDP"), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N- acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (“DTP-DPP", or "TheramideTM), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-( 1 '-2'dipalmitoyl-sn-glycero-3 - hydroxyphosphoryloxy)-ethylamine (“MTP-PE").
  • thr- MDP N-acetylmuramyl-L-threonyl-D-isoglutamine
  • An outer membrane protein proteosome preparation prepared from a first Gram- negative bacterium in combination with a liposaccharide preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteosome and liposaccharide preparations form a stable non-covalent adjuvant complex.
  • Such complexes include "IVX-908", a complex comprised of Neisseria meningitidis outer membrane and lipopolysaccharides.
  • a polyoxidonium polymer (58, 59) or other N-oxidized polyethylene-piperazine derivative is added.
  • MIMP Methyl inosine 5 '-monophosphate
  • R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g., cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof.
  • alkyl e.g., cycloalkyl
  • alkenyl alkynyl and aryl groups
  • examples include, but are not limited to: casuarine, casuarine-6- ⁇ -D-glucopyranose, 3-epz-casuarine, 7-epz-casuarine,
  • a CDId ligand such as an ⁇ -glycosylceramide (62-69) (e.g., ⁇ - galactosylceramide), phytosphingosine-containing ⁇ -glycosylceramides, OCH,
  • KRN7000 [(2S,3S,4R)-l-O-( ⁇ -D-galactopyranosyl)-2-(N-hexacosanoylamino)- 1,3,4-octadecanetriol], CRONY-101, 3"-O-sulfo-galactosylceramide, etc.
  • Virosomes and virus-like particles These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses.
  • viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q ⁇ -phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi).
  • influenza virus such as HA or NA
  • Hepatitis B virus such as core or capsid proteins
  • Hepatitis E virus measles virus
  • Sindbis virus Rotavirus
  • Foot-and-Mouth Disease virus Retrovirus
  • Norwalk virus Norwalk virus
  • human Papilloma virus HIV
  • RNA-phages Q ⁇ -phage (such as coat proteins)
  • GA-phage such as fr-phage,
  • compositions may include two, three, four or more adjuvants.
  • compositions of the invention may advantageously include both an oil-in-water emulsion and a cytokine-inducing agent, or both a mineral-containing composition and a cytokine-inducing agent, or two oil-in-water emulsion adjuvants, or two
  • Antigens and adjuvants in a composition will typically be in admixture.
  • Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer).
  • squalene-water emulsions such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer).
  • CFA incomplete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Various oil-in-water emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolizable) and biocompatible.
  • the oil droplets in the emulsion are generally less than 5 ⁇ m in diameter, and may even have a sub-micron diameter, with these small sizes being achieved with a microfluidizer to provide stable emulsions. Droplets with a size less than 220 nm are preferred as they can be subjected to filter sterilization.
  • the invention can be used with oils such as those from an animal (such as fish) or vegetable source.
  • Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils.
  • Jojoba oil can be used, e.g., obtained from the jojoba bean.
  • Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used.
  • 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils.
  • Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention.
  • the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
  • Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
  • a number of branched chain oils are synthesized biochemically in 5- carbon isoprene units and are generally referred to as terpenoids.
  • Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23- hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred herein.
  • Squalane the saturated analog to squalene
  • Fish oils, including squalene and squalane are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.
  • Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance).
  • Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16.
  • the invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX (TM) tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypoly ethoxy ethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such
  • TERGITOL NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.
  • Brij surfactants polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols
  • SPANs sorbitan esters
  • Non-ionic surfactants are preferred.
  • Preferred surfactants for including in the emulsion are TWEEN 80 (TM) (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-IOO.
  • surfactants can be used e.g., TWEEN 80 (TM)/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (TWEEN 80 (TM)) and an octoxynol such as t- octylphenoxypolyethoxyethanol (Triton X-IOO) is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as TWEEN 80 (TM)) 0.01 to 1%, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-IOO, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.
  • polyoxyethylene sorbitan esters such as TWEEN 80 (TM)
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-IOO, or other detergents in the Triton series
  • polyoxyethylene ethers such as laureth 9
  • oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
  • a submicron emulsion of squalene, TWEEN 80 (TM), and Span 85 The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85.
  • This adjuvant is known as 'MF59' (71-73), as described in more detail in Chapter 10 of ref. 74 and chapter 12 of ref. 75.
  • the MF59 emulsion advantageously includes citrate ions, e.g., 1OmM sodium citrate buffer.
  • the emulsion may include phosphate buffered saline. It may also include Span 85 (e.g., at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% TWEEN 80 (TM), and the weight ratio of squalene:tocopherol is preferably ⁇ 1 as this provides a more stable emulsion. Squalene and TWEEN 80 (TM) may be present volume ratio of about 5:2.
  • One such emulsion can be made by dissolving TWEEN 80 (TM) in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL- ⁇ -tocopherol and 5ml squalene), then microfluidizing the mixture.
  • the resulting emulsion may have submicron oil droplets, e.g., with an average diameter of between 100 and 250nm, preferably about 180nm.
  • An emulsion of squalene, a tocopherol, and a Triton detergent e.g., Triton X- 100.
  • the emulsion may also include a 3d-MPL (see below).
  • the emulsion may contain a phosphate buffer.
  • An emulsion comprising a polysorbate (e.g., polysorbate 80), a Triton detergent (e.g., Triton X-100) and a tocopherol (e.g., an ⁇ -tocopherol succinate).
  • the emulsion may include these three components at a mass ratio of about 75: 11 : 10 (e.g., 750 ⁇ g/ml polysorbate 80, 110 ⁇ g/ml Triton X-100 and 100 ⁇ g/ml ⁇ - tocopherol succinate), and these concentrations should include any contribution of these components from antigens.
  • the emulsion may also include squalene.
  • the emulsion may also include a 3d-MPL (see below).
  • the aqueous phase may contain a phosphate buffer.
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 (“PLURONIC (TM) L121").
  • the emulsion can be formulated in phosphate buffered saline, pH 7.4.
  • This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-I” adjuvant (76) (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF” adjuvant (77) (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).
  • Microfluidization is preferred.
  • An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or 'Span 80').
  • the emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm.
  • the emulsion may also include one or more of: alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. Such emulsions may be lyophilized.
  • An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant.
  • preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
  • Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 79, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N- dioctadecyl-N,N-bis (2 -hydroxy ethyl)propanediamine.
  • a saponin-lipophile conjugate such as GPI-0100, described in reference 79, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid
  • dimethyidioctadecylammonium bromide and/or N,N- dioctadecyl-N,N-bis (2 -hydroxy ethyl)propanediamine.
  • An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer).
  • An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer).
  • a saponin e.g., QuilA or QS21
  • a sterol e.g., a cholesterol
  • the emulsions may be mixed with antigen extemporaneously, at the time of delivery.
  • the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use.
  • the antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids.
  • the volume ratio of the two liquids for mixing can vary (e.g., between 5: 1 and 1 :5) but is generally about 1 : 1.
  • Cytokine-inducing agents for inclusion in compositions of the invention are able, when administered to a patient, to elicit the immune system to release cytokines, including interferons and interleukins.
  • Preferred agents can elicit the release of one or more of: interferon- ⁇ ; interleukin- 1 ; interleukin-2; inter leukin- 12; TNF- ⁇ ; TNF- ⁇ ; and GM-CSF.
  • Preferred agents elicit the release of cytokines associated with a Thl-type immune response, e.g., interferon- ⁇ , TNF- ⁇ , interleukin-2. Stimulation of both interferon- ⁇ and interleukin-2 is preferred.
  • a patient will have T cells that, when stimulated with a RSV F protein, will release the desired cytokine(s) in an antigen-specific manner.
  • T cells purified from their blood will release ⁇ -interferon when exposed in vitro to F protein.
  • Methods for measuring such responses in peripheral blood mononuclear cells (PBMC) are known in the art, and include ELISA, ELISPOT, flow-cytometry and real-time PCR.
  • reference 81 reports a study in which antigen-specific T cell-mediated immune responses against tetanus toxoid, specifically ⁇ -interferon responses, were monitored, and found that ELISPOT was the most sensitive method to discriminate antigen-specific TT-induced responses from spontaneous responses, but that intracytoplasmic cytokine detection by flow cytometry was the most efficient method to detect re-stimulating effects.
  • Suitable cytokine-inducing agents include, but are not limited to:
  • An immunostimulatory oligonucleotide such as one containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine), or a double-stranded RNA, or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.
  • a benzonaphthyridine compound such as: (a) a compound having the formula:
  • R 4 is selected from H, halogen, -C(O)OR 7 , -C(O)R 7 , -C(O)N(R 11 R 12 ),
  • Ci-C ⁇ haloalkyl C 2 -Csalkene, C 2 -Csalkyne, Ci-C ⁇ alkoxy, Ci-C ⁇ haloalkoxy, aryl, heteroaryl, C 3 -Cscycloalkyl, and C3-C8heterocycloalkyl, wherein the Ci-C ⁇ alkyl, Ci-C ⁇ heteroalkyl, Ci-C ⁇ haloalkyl, C 2 -Csalkene, C 2 -Csalkyne, Ci-C ⁇ alkoxy, Ci-C ⁇ haloalkoxy, aryl, heteroaryl, C 3 -Cscycloalkyl, and
  • C 3 -Csheterocycloalkyl groups of R 4 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, NO 2 , -R 7 , -OR 8 , -C(O)R 8 , -OC(O)R 8 , -C(O)OR 8 , -N(R 9 ) 2 , - P(O)(OR 8 ) 2 , -OP(O)(OR 8 ) 2 , -P(O)(OR 10 ) 2 ,
  • each L is independently selected from a bond, -(0(CH 2 ) m ) r , Ci-C ⁇ alkyl, CrC ⁇ alkenylene and C 2 -C6alkynylene, wherein the Ci-C ⁇ alkyl, C 2 -C6alkenylene and C 2 -Cealkynylene of L are each optionally substituted with 1 to 4 substituents independently selected from halogen, -R 8 , -OR 8 , -N(R 9 ) 2 , -P(O)(OR 8 ) 2 , -OP(O)(OR 8 ) 2 ,
  • R 7 is selected from H, Ci-C 6 alkyl, aryl, heteroaryl, Cs-Cscycloalkyl, Ci-C 6 heteroalkyl, Ci-C 6 haloalkyl, C 2 -C 8 alkene, C 2 -C 8 alkyne, Ci-C 6 alkoxy, Ci-C 6 haloalkoxy, and Cs-Csheterocycloalkyl, wherein the Ci-C 6 alkyl, aryl, heteroaryl, C 3 -Cscycloalkyl,
  • Ci-C ⁇ heteroalkyl Ci-C 6 haloalkyl, C 2 -C 8 alkene, C 2 -Csalkyne,
  • Ci-C 6 alkoxy, Ci-C 6 haloalkoxy, and Cs-Csheterocycloalkyl groups of R 7 are each optionally substituted with 1 to 3 R 13 groups;
  • each R 8 is independently selected from H, -CH(R 10 ) 2 , d-Cgalkyl,
  • Ci-C ⁇ heteroalkyl Ci-C ⁇ heteroalkyl, Cs-Cscycloalkyl, C 2 -C 8 heterocycloalkyl,
  • Ci-C 6 hydroxyalkyl and Ci-C 6 haloalkoxy wherein the Ci-Csalkyl, C 2 -Csalkene, C 2 -Csalkyne, Ci-C 6 heteroalkyl, Ci-C 6 haloalkyl, Ci-C 6 alkoxy, Cs-Cscycloalkyl, -Csheterocycloalkyl,
  • Ci-C ⁇ hydroxyalkyl and Ci-C ⁇ haloalkoxy groups of R 8 are each optionally substituted with 1 to 3 substituents independently selected from -CN, R 11 , -OR 11 , -SR 11 , -C(O)R 11 , -OC(O)R 11 ,
  • each R 9 is independently selected from H, -C(O)R 8 , -C(O)OR 8 , -C(O)R 10 , -C(O)OR 10 , -S(O) 2 R 10 , -Ci-C 6 alkyl, Ci-C 6 heteroalkyl and C 3 -C 6 cycloalkyl, or each R 9 is independently a Ci-C 6 alkyl that together with N they are attached to form a Cs-Csheterocycloalkyl, wherein the C 3 -C 8 heterocycloalkyl ring optionally contains an additional heteroatom selected from N, O and S, and wherein the Ci-C 6 alkyl, Ci-C 6 heteroalkyl, C 3 -C 6 cycloalkyl, or
  • C 3 -Csheterocycloalkyl groups of R 9 are each optionally substituted with 1 to 3 substituents independently selected from
  • each R 10 is independently selected from aryl, C3-Cscycloalkyl,
  • C 3 -Cscycloalkyl, Cs-Csheterocycloalkyl and heteroaryl groups are optionally substituted with 1 to 3 substituents selected from halogen,
  • R 11 and R 12 are independently selected from H, Ci-C ⁇ alkyl,
  • Ci-C ⁇ heteroalkyl Ci-C ⁇ haloalkyl, aryl, heteroaryl,
  • Ci-C 6 alkyl, Ci-C 6 heteroalkyl, Ci-C 6 haloalkyl, aryl, heteroaryl, C 3 - Cscycloalkyl, and Cs-Csheterocycloalkyl groups of R 11 and R 12 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, R 8 , -OR 8 , -C(O)R 8 ,
  • R 11 and R 12 are each independently Ci-C ⁇ alkyl and taken together with the N atom to which they are attached form an optionally substituted Cs-Csheterocycloalkyl ring optionally containing an additional heteroatom selected from N, O and S;
  • each R 13 is independently selected from halogen, -CN, -LR 9 , -LOR 9 ,
  • each R A is independently selected from -R 8 , -R 7 , -OR 7 , -OR 8 , -R 10 ,
  • n is, independently at each occurrence, O, 1, 2, 3, 4, 5, 6, 7 or 8;
  • each m is independently selected from 1, 2, 3, 4, 5 and 6, and
  • t 1, 2, 3, 4, 5, 6, 7 or 8;
  • R 4 is selected from H, halogen, -C(O)OR 7 , -C(O)R 7 , -C(O)N(R 11 R 12 ), -N(R 11 R 12 ), -N(R 9 ) 2 , -NHN(R 9 ) 2 , -SR 7 , -(CH 2 ) n OR 7 , -(CH 2 ) n R 7 , -LR 8 , -LR 10 , -OLR 8 , -OLR 10 , Ci-C 6 alkyl, Ci-C 6 heteroalkyl,
  • Ci-C ⁇ haloalkyl C 2 -Csalkene, C 2 -Csalkyne, Ci-C ⁇ alkoxy, Ci-C ⁇ haloalkoxy, aryl, heteroaryl, C 3 -Cscycloalkyl, and C 3 - Csheterocycloalkyl, wherein the Ci-C ⁇ alkyl, Ci-C ⁇ heteroalkyl, C 1 - Cghaloalkyl, C 2 -C 8 alkene, C 2 -C 8 alkyne, Ci-C 6 alkoxy, Ci-C ⁇ haloalkoxy, aryl, heteroaryl, C 3 -Cscycloalkyl, and
  • C 3 -Csheterocycloalkyl groups of R 4 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, NO 2 , -R 7 , -OR 8 , -C(O)R 8 , -OC(O)R 8 , -C(O)OR 8 , -N(R 9 ) 2 , - P(O)(OR 8 ) 2 , -OP(O)(OR 8 ) 2 , -P(O)(OR 10 ) 2 ,
  • each L is independently selected from a bond, -(O(CH 2 ) m ) r , Ci-C 6 alkyl, C 2 -
  • Ci-C 6 alkyl, C 2 - C 6 alkenylene and C 2 -C 6 alkynylene of L are each optionally substituted with 1 to 4 substituents independently selected from halogen, -R 8 , -OR 8 , -N(R 9 ) 2 , -P(O)(OR 8 ) 2 , -OP(O)(OR 8 ) 2 ,
  • R 7 is selected from H, Ci-C 6 alkyl, aryl, heteroaryl, C 3 -CsCyC loalkyl,
  • Ci-C ⁇ heteroalkyl Ci-C 6 haloalkyl, C 2 -Csalkene, C 2 -Csalkyne, Ci-C 6 alkoxy, Ci-C 6 haloalkoxy, and Q-Csheterocycloalkyl, wherein the Ci-C 6 alkyl, aryl, heteroaryl, Q-Cscycloalkyl,
  • Ci-C ⁇ heteroalkyl Ci-C 6 haloalkyl, C 2 -Csalkene, C 2 -Csalkyne,
  • Ci-C ⁇ alkoxy, Ci-C ⁇ haloalkoxy, and Cs-Csheterocycloalkyl groups of R 7 are each optionally substituted with 1 to 3 R 13 groups;
  • each R 8 is independently selected from H, -CH(R 10 ) 2 , d-Cgalkyl,
  • Ci-C ⁇ heteroalkyl Ci-C ⁇ heteroalkyl, Cs-Cscycloalkyl, C 2 -C 8 heterocycloalkyl,
  • Ci-C ⁇ hydroxyalkyl and Ci-C ⁇ haloalkoxy wherein the Ci-Cgalkyl, C 2 -C8alkene, C 2 -Csalkyne, Ci-C ⁇ heteroalkyl, Ci-C ⁇ haloalkyl, Ci-C ⁇ alkoxy, Cs-Cscycloalkyl, C 2 -C 8 heterocycloalkyl,
  • Ci-C ⁇ hydroxyalkyl and Ci-C ⁇ haloalkoxy groups of R 8 are each optionally substituted with 1 to 3 substituents independently selected from -CN, R 11 , -OR 11 , -SR 11 , -C(O)R 11 , -OC(O)R 11 ,
  • each R 9 is independently selected from H, -C(O)R 8 , -C(O)OR 8 , -C(O)R 10 , - C(O)OR 10 , -S(O) 2 R 10 , -Ci-C 6 alkyl, Ci-C 6 heteroalkyl and C 3 -C 6 cycloalkyl, or each R 9 is independently a Ci-C ⁇ alkyl that together with N they are attached to form a Cs-Csheterocycloalkyl, wherein the C 3 -Csheterocycloalkyl ring optionally contains an additional heteroatom selected from N, O and S, and wherein the C 1 -Ce alkyl, C 1 -Ce heteroalkyl, C3-C6 cycloalkyl, or
  • Cs-Csheterocycloalkyl groups of R 9 are each optionally substituted with 1 to 3 substituents independently selected from
  • each R 10 is independently selected from aryl, C 3 -Cscycloalkyl,
  • C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl and heteroaryl groups are optionally substituted with 1 to 3 substituents selected from halogen,
  • R 11 and R 12 are independently selected from H, Ci-C ⁇ alkyl,
  • Ci-C ⁇ heteroalkyl Ci-C ⁇ haloalkyl, aryl, heteroaryl,
  • Ci-C ⁇ alkyl, Ci-Ceheteroalkyl, Ci-C ⁇ haloalkyl, aryl, heteroaryl, C 3 - Cscycloalkyl, and Cs-Csheterocycloalkyl groups of R 11 and R 12 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, R 8 , -OR 8 , -C(O)R 8 ,
  • R 11 and R 12 are each independently Ci-C ⁇ alkyl and taken together with the N atom to which they are attached form an optionally substituted C 3 -Csheterocycloalkyl ring optionally containing an additional heteroatom selected from N, O and S;
  • each R 13 is independently selected from halogen, -CN, -LR 9 , -LOR 9 , -OLR 9 , -LR 10 , -LOR 10 , -OLR 10 , -LR 8 , -LOR 8 , -OLR 8 , -LSR 8 , -LSR 10 , -LC(O)R 8 , -OLC(O)R 8 , -LC(O)OR 8 , -LC(O)R 10 , -LOC(O)OR 8 , -LC(O)NR 9 R 11 , -LC(O)NR 9 R 8 , -LN(R 9 ) 2 , -LNR 9 R 8 , -LNR 9 R 10 , -LC(O)N(R 9 ) 2 , -LS(O) 2 R 8 , -LS(O)R 8 , -LC(O)NR 8 OH, -LNR 9 C(O)R 8
  • each R A is independently selected from -R 8 , -R 7 , -OR 7 , -OR 8 , -R 10 ,
  • n is, independently at each occurrence, O, 1, 2, 3, 4, 5, 6, 7 or 8;
  • each m is independently selected from 1, 2, 3, 4, 5 and 6, and
  • t is 1, 2, 3, 4, 5, 6, 7 or 8; or (c) a pharmaceutically acceptable salt of any of (a) or (b).
  • Other benzonaphthyridine compounds, and methods of making benzonaphthyridine compounds, are described in WO 2009/111337.
  • a benzonaphthyridine compound, or a salt thereof, can be used on its own, or in combination with one or more further compounds. For example, a
  • benzonaphthyridine compound can be used in combination with an oil-in-water emulsion or a mineral-containing composition.
  • a benzonaphthyridine compound is used in combination with an oil-in-water emulsion (e.g. a squalene-water emulsion, such as MF59) or a mineral- containing composition (e.g., a mineral said such as an aluminum salt or a calcium salt).
  • an oil-in-water emulsion e.g. a squalene-water emulsion, such as MF59
  • a mineral- containing composition e.g., a mineral said such as an aluminum salt or a calcium salt.
  • a thiosemicarbazone compound such as those disclosed in reference 94. Methods of formulating, manufacturing, and screening for active compounds are also described in reference 94.
  • the thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear
  • a tryptanthrin compound such as those disclosed in reference 95. Methods of formulating, manufacturing, and screening for active compounds are also described in reference 95.
  • the thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ .
  • a nucleoside analog such as: (a) Isatorabine (ANA-245; 7-thia-8- oxoguanosine):
  • Ri and R2 are each independently H, halo, -NR 3 Rb, -OH, Ci-6 alkoxy, substituted Ci_6 alkoxy, heterocyclyl, substituted heterocyclyl, C ⁇ -io aryl, substituted C ⁇ -io aryl, Ci_6 alkyl, or substituted Ci_6 alkyl;
  • R3 is absent, H, Ci_6 alkyl, substituted Ci_6 alkyl, C ⁇ -io aryl, substituted C ⁇ -io aryl, heterocyclyl, or substituted heterocyclyl;
  • R 4 and R5 are each independently H, halo, heterocyclyl, substituted heterocyclyl, -C(O)-Rd, C 1-6 alkyl, substituted C 1-6 alkyl, or bound together to form a 5 membered ring as in R 4 _ 5 :
  • Xi and X 2 are each independently N, C, O, or S;
  • R 8 is H, halo, -OH, Ci_ 6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, -OH, -NR a R b ,
  • R9 is H, Ci-6 alkyl, substituted C 1-6 alkyl, heterocyclyl, substituted 10 heterocyclyl or R 9a , wherein R 9a is:
  • Rn are each independently H, halo, C 1-6 alkoxy, substituted C 1-6 alkoxy, -NR a R b , or -OH;
  • each R a and R b is independently H, CV 6 alkyl, substituted CV 6 alkyl, -C(O)R d ,
  • each Rc is independently H, phosphate, diphosphate, triphosphate, C 1-6 alkyl, or substituted C 1-6 alkyl;
  • each Ra is independently H, halo, C 1-6 alkyl, substituted C 1-6 alkyl, C 1-6 20 alkoxy, substituted C 1-6 alkoxy, -NH 2 , -NH(C 1-6 alkyl), -NH(substituted
  • Ci_6 alkyl -N(C 1-6 alkyl) 2 , -N(substituted Ci_ 6 alkyl) 2 , C 6-10 aryl, or heterocyclyl;
  • each Re is independently H, C 1-6 alkyl, substituted C 1-6 alkyl, C 6-10 aryl, substituted C 6-10 aryl, heterocyclyl, or substituted heterocyclyl;
  • each R f is independently H, C 1-6 alkyl, substituted C 1-6 alkyl, -C(O)Ra, phosphate, diphosphate, or triphosphate;
  • each n is independently O, 1, 2, or 3;
  • each p is independently O, 1, or 2; or or (g) a pharmaceutically acceptable salt of any of (a) to (f), a tautomer of any of (a) to (f), or a pharmaceutically acceptable salt of the tautomer.
  • Aminobenzimidazole quinolinone (ABIQ) compounds (101, 102), Hydrapthalamide compounds, Benzophenone compounds, Isoxazole compounds, Sterol compounds, Quinazilinone compounds, Pyrrole compounds (103), Anthraquinone compounds, Quinoxaline compounds, Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole compounds (104).
  • An aminoalkyl glucosaminide phosphate derivative such as RC-529 (106, 107).
  • a phosphazene such as poly[di(carboxylatophenoxy)phosphazene] ("PCPP") as described, for example, in references 108 and 109.
  • PCPP poly[di(carboxylatophenoxy)phosphazene]
  • SIPs Small molecule immunopotentiators
  • the cytokine-inducing agents for use in the present invention may be modulators and/or agonists of Toll-Like Receptors (TLR).
  • TLR Toll-Like Receptors
  • they may be agonists of one or more of the human TLRl, TLR2, TLR3, TLR4, TLR7, TLR8, and/or TLR9 proteins.
  • Preferred agents are agonists of TLR4 (e.g., modified natural lipid As derived from enterobacterial lipopolysaccharides, phospholipid compounds, such as the synthetic phospholipid dimer, E6020), TLR7 (e.g., benzonaphthyridines,
  • the cytokine-inducing agent can be added to the composition at various stages during its production. For example, it may be within an antigen composition, and this mixture can then be added to an oil-in-water emulsion. As an alternative, it may be within an oil-in-water emulsion, in which case the agent can either be added to the emulsion components before emulsification, or it can be added to the emulsion after emulsification. Similarly, the agent may be coacervated within the emulsion droplets.
  • the location and distribution of the cytokine-inducing agent within the final composition will depend on its hydrophilic/lipophilic properties, e.g., the agent can be located in the aqueous phase, in the oil phase, and/or at the oil-water interface.
  • the cytokine-inducing agent can be conjugated to a separate agent, such as an antigen (e.g., CRM197).
  • a separate agent such as an antigen (e.g., CRM197).
  • an antigen e.g., CRM197
  • the adjuvants may be non- covalently associated with additional agents, such as by way of hydrophobic or ionic interactions.
  • cytokine-inducing agents are (a) benzonapthridine compounds; (b) immunostimulatory oligonucleotides and (c) 3dMPL.
  • Immunostimulatory oligonucleotides can include nucleotide
  • references 111, 112, and 113 disclose possible analog substitutions, e.g., replacement of guanosine with 2'-deoxy-7- deazaguanosine.
  • the adjuvant effect of CpG oligonucleotides is further discussed in refs. 114 to 119.
  • a CpG sequence may be directed to TLR9, such as the motif
  • the CpG sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • CpG-A and CpG-B ODNs are discussed in refs. 121-123.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers".
  • CpG7909 also known as PROMUNE (TM) (Coley Pharmaceutical Group, Inc.).
  • PROMUNE TM
  • TpG sequences can be used (127). These oligonucleotides may be free from unmethylated CpG motifs.
  • the immunostimulatory oligonucleotide may be pyrimidine-rich.
  • it may comprise more than one consecutive thymidine nucleotide (e.g., TTTT, as disclosed in ref. 127), and/or it may have a nucleotide composition with >25% thymidine (e.g., >35%, >40%, >50%, >60%, >80%, etc.).
  • it may comprise more than one consecutive cytosine nucleotide (e.g., CCCC, as disclosed in ref.
  • nucleotide composition with >25% cytosine (e.g., >35%, >40%, >50%, >60%, >80%, etc.).
  • oligonucleotides may be free from
  • Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides.
  • 3dMPL also known as 3 de-O-acylated monophosphoryl lipid A or
  • 3-O-desacyl-4'-monophosphoryl lipid A) is an adjuvant in which position 3 of the reducing end glucosamine in monophosphoryl lipid A has been de-acylated.
  • 3dMPL has been prepared from a heptoseless mutant of Salmonella Minnesota, and is chemically similar to lipid A but lacks an acid-labile phosphoryl group and a base-labile acyl group. It activates cells of the monocyte/macrophage lineage and stimulates release of several cytokines, including IL-I, IL- 12, TNF- ⁇ and GM-CSF (see also ref. 128). Preparation of 3dMPL was originally described in reference 129.
  • 3dMPL can take the form of a mixture of related molecules, varying by their acylation (e.g., having 3, 4, 5 or 6 acyl chains, which may be of different lengths).
  • the two glucosamine (also known as 2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their 2-position carbons (i.e., at positions 2 and T), and there is also O-acylation at the 3' position.
  • the group attached to carbon 2 has formula
  • Groups R 1 , R 2 and R 3 are each independently -(CH 2 ) n -CH 3 .
  • the value of n is preferably between 8 and 16, more preferably between 9 and 12, and is most preferably 10.
  • Groups R 1 , R 2 and R 3 can each independently be: (a) -H; (b) -OH; or (c) -
  • R 4 is either -H or -(CH 2 ) m -CH3, wherein the value of m is preferably between 8 and 16, and is more preferably 10, 12 or 14.
  • m is preferably 14.
  • m is preferably 10.
  • m is preferably 12.
  • Groups R 1 ' , R 2 and R 3 are thus preferably -O-acyl groups from dodecanoic acid, tetradecanoic acid or hexadecanoic acid.
  • the 3dMPL has only 3 acyl chains (one on each of positions 2, 2' and 3').
  • the 3dMPL can have 4 acyl chains.
  • the 3dMPL can have 5 acyl chains.
  • the 3dMPL can have 6 acyl chains.
  • the 3dMPL adjuvant used according to the invention can be a mixture of these forms, with from 3 to 6 acyl chains, but it is preferred to include 3dMPL with 6 acyl chains in the mixture, and in particular to ensure that the hexaacyl chain form makes up at least 10% by weight of the total 3dMPL e.g., >20%, >30%, >40%, >50% or more. 3dMPL with 6 acyl chains has been found to be the most adjuvant-active form.
  • 3dMPL is used in the form of a mixture
  • references to amounts or concentrations of 3dMPL in compositions of the invention refer to the combined 3dMPL species in the mixture.
  • 3dMPL can form micellar aggregates or particles with different sizes e.g., with a diameter ⁇ 150nm or >500nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g., small enough to give a clear aqueous suspension of 3dMPL) are preferred for use according to the invention because of their superior activity (130).
  • Preferred particles have a mean diameter less than 220nm, more preferably less than 200nm or less than 150nm or less than 120nm, and can even have a mean diameter less than lOOnm. In most cases, however, the mean diameter will not be lower than 50nm. These particles are small enough to be suitable for filter sterilization. Particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals a mean particle diameter. Where a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this mean, but at least 50% by number (e.g., >60%, >70%, >80%, >90%, or more) of the particles will have a diameter within the range x+25%.
  • 3dMPL can advantageously be used in combination with an oil-in-water emulsion. Substantially all of the 3dMPL may be located in the aqueous phase of the emulsion.
  • the 3dMPL can be used on its own, or in combination with one or more further compounds.
  • 3dMPL in combination with the QS21 saponin (131) (including in an oil-in-water emulsion (132)), with an
  • immunostimulatory oligonucleotide with both QS21 and an immunostimulatory oligonucleotide, with aluminum phosphate (133), with aluminum hydroxide (134), or with both aluminum phosphate and aluminum hydroxide.
  • Fatty adjuvants that can be used with the invention include the oil-in-water emulsions described above, and also include, for example:
  • ER804057 is also called E6020.
  • a phospholipid compound of formula I, II or III, or a salt thereof can be used on its own, or in combination with one or more further compounds.
  • a compound of formula I, II or III can be used in combination with an oil-in-water emulsion or a mineral-containing composition.
  • E6020 is used in combination with an oil-in-water emulsion (e.g. a squalene-water emulsion, such as MF59) or a mineral-containing composition (e.g., a mineral said such as an aluminum salt or a calcium salt).
  • a formulation of a cationic lipid and a (usually neutral) co-lipid such as aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide- diphytanoylphosphatidyl-ethanolamine ("VAXFECTIN (TM)") or
  • Lipopeptides i.e., compounds comprising one or more fatty acid residues and two or more amino acid residues
  • lipopeptides based on glycerylcysteine Specific examples of such peptides include compounds of the following formula
  • each of R 1 and R 2 represents a saturated or unsaturated, aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical having from 8 to 30, preferably 11 to 21, carbon atoms that is optionally also substituted by oxygen functions
  • R 3 represents hydrogen or the radical Ri-CO-O-CH 2 - in which R 1 has the same meaning as above
  • X represents an amino acid bonded by a peptide linkage and having a free, esterified or amidated carboxy group, or an amino acid sequence of from 2 to 10 amino acids of which the terminal carboxy group is in free, esterified or amidated form.
  • the amino acid sequence comprises a D-amino acid, for example, D-glutamic acid (D-GIu) or D-gamma-carboxy-glutamic acid (D-GIa).
  • TLR2 Bacterial lipopeptides generally recognize TLR2, without requiring TLR6 to participate.
  • TLRs operate cooperatively to provide specific recognition of various triggers, and TLR2 plus TLR6 together recognize peptidoglycans, while TLR2 recognizes lipopeptides without TLR6.
  • TLR2 recognizes lipopeptides without TLR6.
  • Synthetic lipopeptides tend to behave similarly, and are primarily recognized by TLR2.
  • Lipopeptides suitable for use as adjuvants include compounds have the formula:
  • each R la and R 1 is independently an aliphatic or cycloaliphatic-aliphatic hydrocarbon group having 7-21 carbon atoms, optionally substituted by oxygen functions, or one of
  • R la and R lb are H;
  • R 2 is an aliphatic or cycloaliphatic hydrocarbon group having 1-21 carbon atoms and optionally substituted by oxygen functions;
  • n 0 or 1 ;
  • Z 1 and Z 2 each independently represent -OH or the N-terminal radical of a D- or L- alpha amino acid of an amino-(lower alkane)-sulfonic acid or of a peptide having up to 6 amino acids selected from the D- and L-alpha
  • aminocarboxylic acids and amino-lower alkyl-sulfonic acids
  • Z 3 is H or -CO-Z 4 , wher Z 4 is -OH or the N-terminal radical of a D- or L-alpha
  • Suitable amides include -NH 2 and NH(lower alkyl), and suitable esters include C1-C4 alkyl esters, (lower alkyl or lower alkane, as used herein, refers to C 1 -Ce straight chain or branched alky Is).
  • the lipopeptide has the formula:
  • LP40 lipopeptide species
  • TLR2 Another example of a lipopeptide species is called LP40, and is an agonist of TLR2.
  • murein lipoproteins are related to a known class of lipopeptides from E. coli, referred to as murein lipoproteins.
  • murein lipopetides Certain partial degradation products of those proteins called murein lipopetides are described in Hantke, et al., Eur. J. Biochem., 34: 284-296 (1973). These comprise a peptide linked to N-acetyl muramic acid and are thus related to Muramyl peptides, which are described in Baschang, et al., Tetrahedron, 45(20): 6331-6360 (1989).
  • Aluminum salt adjuvants are described in Baschang, et al., Tetrahedron, 45(20): 6331-6360 (1989).
  • adjuvants known as "aluminum hydroxide” and "aluminum phosphate” may be used. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present (e.g., see chapter 9 of reference 74).
  • the invention can use any of the "hydroxide” or "phosphate” adjuvants that are in general use as adjuvants.
  • the adjuvants known as "aluminum hydroxide” are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline.
  • Aluminum oxyhydroxide which can be represented by the formula AlO(OH)
  • AlO(OH) can be distinguished from other aluminum compounds, such as aluminum hydroxide Al(0H) 3 , by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm “1 and a strong shoulder at 3090-3100CnT 1 (chapter 9 of ref. 74).
  • IR infrared
  • the degree of crystallinity of an aluminum hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes.
  • the surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption.
  • a fibrous morphology e.g., as seen in transmission electron micrographs
  • the pi of aluminum hydroxide adjuvants is typically about 11, i.e., the adjuvant itself has a positive surface charge at physiological pH.
  • Adsorptive capacities of between 1.8-2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum hydroxide adjuvants.
  • the adjuvants known as "aluminum phosphate” are typically aluminum hydroxyphosphates, often also containing a small amount of sulfate (i.e., aluminum hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO 4 /AI molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPO 4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm "1 (e.g., when heated to 200 0 C) indicates the presence of structural hydroxyls (ch.9 of ref.
  • the PO 4 /AI + molar ratio of an aluminum phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95+0.1.
  • the aluminum phosphate will generally be amorphous, particularly for
  • a typical adjuvant is amorphous aluminum hydroxyphosphate with PO 4 /AI molar ratio between 0.84 and 0.92, included at 0.6mg Al 3 VmI.
  • the aluminum phosphate will generally be particulate (e.g., plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20 ⁇ m (e.g., about 5-10 ⁇ m) after any antigen adsorption.
  • Adsorptive capacities of between 0.7-1.5 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum phosphate adjuvants.
  • Aluminum phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5, e.g., about 5.7.
  • Suspensions of aluminum salts used to prepare compositions of the invention may contain a buffer (e.g., a phosphate or a histidine or a Tris buffer), but this is not always necessary.
  • the suspensions are preferably sterile and pyrogen-free.
  • a suspension may include free aqueous phosphate ions e.g., present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.
  • the suspensions may also comprise sodium chloride.
  • the invention can use a mixture of both an aluminum hydroxide and an aluminum phosphate.
  • there may be more aluminum phosphate than hydroxide e.g., a weight ratio of at least 2: 1 e.g., >5: 1, >6: 1, >7: 1, >8: 1, >9: 1, etc.
  • the concentration of Al +++ in a composition for administration to a patient is preferably less than 10mg/ml e.g., ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
  • a preferred range is between 0.3 and 1 mg/ml.
  • the adjuvant component may include one or more further adjuvant or immunostimulating agents.
  • additional components include, but are not limited to: a benzonaphthyridine compound, a 3-0-deacylated monophosphoryl lipid A adjuvant ('3d-MPL'); and/or an oil-in- water emulsion.
  • 3d-MPL has also been referred to as 3 de-O-acylated monophosphoryl lipid A or as 3-O-desacyl-4'-monophosphoryl lipid A. The name indicates that position 3 of the reducing end glucosamine in monophosphoryl lipid A is de-acylated.
  • an aluminum hydroxide and/or aluminum phosphate adjuvant is useful, particularly in children, and antigens are generally adsorbed to these salts. Squalene-in-water emulsions are also preferred, particularly in the elderly.
  • Useful adjuvant combinations include combinations of ThI and Th2 adjuvants such as CpG and alum, or resiquimod and alum.
  • ThI and Th2 adjuvants such as CpG and alum, or resiquimod and alum.
  • a combination of aluminum phosphate and 3dMPL may be used. Other combinations that may be used include: alum and a
  • benzonapthridine compound or a SMIP a squalene-in-water emulsion (such as MF59) and a benzonapthridine compound or a SMIP
  • E6020 and a squalene-in-water emulsion, such as MF59) or alum a squalene-in-water emulsion, such as MF59 or alum.
  • compositions of the invention may elicit both a cell mediated immune response as well as a humoral immune response.
  • CD8 T cells Two types of T cells, CD4 and CD8 cells, are generally thought necessary to initiate and/or enhance cell mediated immunity and humoral immunity.
  • CD8 T cells can express a CD8 co-receptor and are commonly referred to as Cytotoxic T lymphocytes (CTLs).
  • CTLs Cytotoxic T lymphocytes
  • CD8 T cells are able to recognized or interact with antigens displayed on MHC Class I molecules.
  • CD4 T cells can express a CD4 co-receptor and are commonly referred to as T helper cells.
  • CD4 T cells are able to recognize antigenic peptides bound to MHC class II molecules.
  • the CD4 cells Upon interaction with a MHC class II molecule, the CD4 cells can secrete factors such as cytokines. These secreted cytokines can activate B cells, cytotoxic T cells, macrophages, and other cells that participate in an immune response.
  • Helper T cells or CD4+ cells can be further divided into two functionally distinct subsets: THl phenotype and TH2 phenotypes which differ in their cytokine and effector function.
  • Activated THl cells enhance cellular immunity (including an increase in antigen-specific CTL production) and are therefore of particular value in responding to intracellular infections.
  • Activated THl cells may secrete one or more of IL-2, IFN- ⁇ , and TNF- ⁇ .
  • a THl immune response may result in local inflammatory reactions by activating macrophages, NK (natural killer) cells, and CD8 cytotoxic T cells (CTLs).
  • a THl immune response may also act to expand the immune response by stimulating growth of B and T cells with IL- 12.
  • THl stimulated B cells may secrete IgG2a.
  • Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections.
  • Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10.
  • a TH2 immune response may result in the production of IgGl, IgE, IgA and memory B cells for future protection.
  • An enhanced immune response may include one or more of an enhanced THl immune response and a TH2 immune response.
  • a THl immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a THl immune response (such as IL-2, IFN- ⁇ , and TNF- ⁇ ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a.
  • the enhanced THl immune response will include an increase in IgG2a production.
  • a THl immune response may be elicited using a THl adjuvant.
  • a THl adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant.
  • THl adjuvants suitable for use in the invention may include for example saponin formulations, virosomes and virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides.
  • LPS enterobacterial lipopolysaccharide
  • Immunostimulatory oligonucleotides such as oligonucleotides containing a CpG motif, are preferred THl adjuvants for use in the invention.
  • a TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgGl, IgE, IgA and memory B cells.
  • cytokines associated with a TH2 immune response such as IL-4, IL-5, IL-6 and IL-10
  • an increase in the production of IgGl, IgE, IgA and memory B cells such as IL-4, IL-5, IL-6 and IL-10
  • the enhanced TH2 immune response will include an increase in IgGl production.
  • a TH2 immune response may be elicited using a TH2 adjuvant.
  • a TH2 adjuvant will generally elicit increased levels of IgGl production relative to
  • TH2 adjuvants suitable for use in the invention include, for example, mineral containing compositions, oil-emulsions, and ADP-ribosylating toxins and detoxified derivatives thereof.
  • Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use in the invention.
  • a composition may include a combination of a THl adjuvant and a TH2 adjuvant.
  • a composition elicits an enhanced THl and an enhanced TH2 response, i.e., an increase in the production of both IgGl and IgG2a production relative to immunization without an adjuvant.
  • the composition comprising a combination of a THl and a TH2 adjuvant elicits an increased THl and/or an increased TH2 immune response relative to immunization with a single adjuvant (i.e., relative to immunization with a THl adjuvant alone or immunization with a TH2 adjuvant alone).
  • the immune response may be one or both of a THl immune response and a TH2 response.
  • immune response provides for one or both of an enhanced THl response and an enhanced TH2 response.
  • the enhanced immune response may be one or both of a systemic and a mucosal immune response.
  • the immune response provides for one or both of an enhanced systemic and an enhanced mucosal immune response.
  • the mucosal immune response is a TH2 immune response.
  • the mucosal immune response includes an increase in the production of IgA.
  • compositions of the invention are suitable for administration to mammals, and the invention provides a method of inducing an immune response in a mammal, comprising the step of administering a composition (e.g., an immunogenic
  • compositions of the invention to the mammal.
  • the compositions can be used to produce a vaccine formulation for immunizing a mammal.
  • the mammal is typically a human, and the RSV F protein ecto-domain is typically a human RSV F protein ecto-domain.
  • the mammal can be any other mammal that is susceptible to infection with RSV, such as a cow that can be infected with bovine RSV.
  • the immune response may be raised following administration of a purified RSV F protein, an alphavirus particle, or self-replicating RNA.
  • the invention also provides a composition of the invention for use as a medicament, e.g., for use in immunizing a patient against RSV infection.
  • the invention also provides the use of a polypeptide as described above in the manufacture of a medicament for raising an immune response in a patient.
  • the immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response.
  • Methods for assessing antibody responses after RSV vaccination are well known in the art.
  • compositions of the invention can be administered in a number of suitable ways, such as intramuscular injection (e.g., into the arm or leg), subcutaneous injection, intranasal administration, oral administration, intradermal administration,
  • transcutaneous administration transdermal administration, and the like.
  • the appropriate route of administration will be dependent upon the age, health and other characteristics of the mammal. A clinician will be able to determine an appropriate route of administration based on these and other factors.
  • Immunogenic compositions, and vaccine formulations may be used to treat both children and adults, including pregnant women.
  • a subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old.
  • Preferred subjects for receiving the vaccines are the elderly (e.g., >50 years old, >60 years old, and preferably >65 years), the young (e.g., ⁇ 6 years old, such as 4 - 6 years old, ⁇ 5 years old), and pregnant women.
  • the vaccines are not suitable solely for these groups, however, and may be used more generally in a population.
  • Treatment can be by a single dose schedule or a multiple dose schedule.
  • Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule.
  • the various doses may be given by the same or different routes, e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
  • Administration of more than one dose is particularly useful in immunologically na ⁇ ve patients.
  • Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, and the like.).
  • Vaccine formulations produced using a composition of the invention may be administered to patients at substantially the same time as (e.g., during the same medical consultation or visit to a healthcare professional or vaccination centre) other vaccines.
  • the invention also provides a polypeptide (e.g., recombinant polypeptide) comprising a first domain and a second domain, wherein (i) the first domain comprises a RSV F glycoprotein ectodomain, in whole or part, and (ii) the second domain comprises a heterologous oligomerization domain. Further details are provided above. If the oligomerization domain comprises a heptad sequence (e.g., the sequence from GCN described above) then it is preferably in heptad repeat phase with the HR2 sequence (if present) of the ectodomain.
  • a polypeptide e.g., recombinant polypeptide
  • the first domain comprises a RSV F glycoprotein ectodomain, in whole or part
  • the second domain comprises a heterologous oligomerization domain.
  • the oligomerization domain comprises a heptad sequence (e.g., the sequence from GCN described above) then it is preferably in hept
  • the invention also provides nucleic acid (e.g., DNA) encoding this polypeptide. It also provides vectors including such nucleic acids, and host cells including such vectors. The vectors may be used for, e.g., recombinant expression purposes, nucleic acid immunization, etc.
  • the invention also provides a composition comprising molecules comprising RSV F glycoprotein ectodomains, wherein the ectodomains of at least 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%) of the molecules are present in a pre-fusion conformation.
  • the invention may be used with other members of the Pneumoviridae and Paramyxoviridae, including, but not limited to, bovine respiratory syncytial virus, parainfluenzavirus 1, parainflueznavirus 2, parainfluenzavirus 3, and parainfluenzavirus 5.
  • the invention provides an immunogenic composition comprising a F glycoprotein from a Pneumoviridae or Paramyxoviridae, wherein the F glycoprotein is in pre-fusion conformation.
  • the invention also provides an immunogenic composition comprising a polypeptide that displays an epitope present in a pre-fusion conformation of the F glycoprotein of a Pneumoviridae or Paramyxoviridae, but absent the glycoprotein's post fusion conformation.
  • the invention also provides a polypeptide comprising a first domain and a second domain, wherein (i) the first domain comprises an ectodomain of the F glycoprotein of a Pneumoviridae or Paramyxoviridae, in whole or part, and (ii) the second domain comprises a heterologous oligomerization domain.
  • the invention also provides these polypeptides and compositions for use in immunization, etc.
  • the invention also provides a composition comprising molecules comprising RSV F glycoprotein ectodomains, wherein the ectodomains of at least 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%) of the molecules are present in a pre-fusion conformation or an intermediate conformation.
  • Particular RSV F protein ecto-domain polypeptides are used or included in some embodiments of the invention. Some of the particular RSV F protein ectodomain polypeptides contain altered amino acid sequences from about position 100 to about position 161. The amino acid sequences from position 100 to position 150 for several particular RSV F protein ecto-domain polypeptides are shown in FIG. 1C. Amino acid sequences of several particular RSV F protein ecto-domain polypeptides are presented herein, e.g., in Example 1.
  • composition comprising
  • X may consist exclusively of X or may include something additional e.g., X + Y.
  • the word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • x means, for example, x ⁇ 10%.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform
  • BSE encephalopathy
  • a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.
  • a cell substrate is used for reassortment or reverse genetics procedures, it is preferably one that has been approved for use in human vaccine production e.g., as in Ph Eur general chapter 5.2.3.
  • DPPG 1 ,2-Dipalmitoyl-sn-Glycero-3 [Phosphatidyl-rac-( 1 -glycerol%)
  • LYSOPC MYRISTIC 1 Myristoyl-sn-Glycero-3 -phosphatidylcholine
  • LYSOPC STEARIC 1 Steparoyl-sn-Glycero-3 -phosphatidylcholine
  • polypeptides e.g., that contain signal sequences
  • nucleic acid sequences that may be used to express RSV F polypeptides of the present invention.
  • the presented amino acid sequences include the signal peptide and contain an optional C-terminal linker and His tag (GGSAGSGHHHHHH (SEQ ID NO:90)).
  • GGSAGSGHHHHHH SEQ ID NO:90
  • the polypeptide will usually be processed by the cell to remove the signal peptide and, as described herein, some of the polypeptides will be cleaved, for example at unmodified furin cleavage sites.
  • compositions that contain, all forms of the particular RSV F protein ecto- domain polypeptides disclosed herein, including mature forms, which lack the signal peptide, forms that may be cleaved into subunits that comprise Fi and F 2 , and forms that lack the optional C-terminal His tag.
  • RSV F protein ecto- domain polypeptides disclosed herein, including mature forms, which lack the signal peptide, forms that may be cleaved into subunits that comprise Fi and F 2 , and forms that lack the optional C-terminal His tag.
  • polypeptides that can be produced as monomers include: RSV F Furx (SEQ ID NO:45); RSV F old furx Truncated HIS (SEQ ID NO:88); RSV F Furx Rl 13Q K123N K124N Truncated HIS (SEQ ID NO:89); RSV F delp21 furx Truncated HIS (SEQ ID NO:47); and RSV F delP23 furx Truncated HIS (SEQ ID NO:48).
  • polypeptides that can be produced as trimers include: RSV F N- term Furin Truncated HIS (SEQ ID NO:85); RSV F Fusion Deletion Truncated HIS (SEQ ID NO:67); and RSV F Fusion Deletion 2 Truncated HIS (SEQ ID NO:68).
  • the following polypeptide is a full-length RSV F polypeptide.
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence.
  • CTGTGCACCA CCAACACCAA AGAGGGCAGC AACATCTGCC TGACCCGGAC CGACCGGGGC 1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG
  • the following polypeptide includes the full-length RSV F polypeptide followed by a hexa-histidine tag.
  • the following polypeptide includes the full-length RSV F polypeptide with the trimerization domain of GCN4 (underlined) attached at the C-terminus of the RSV F polypeptide followed by a hexa-histidine tag.
  • GCN4 trimerization domain of GCN4
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence.
  • the following polypeptide includes the full-length RSV F polypeptide with the trimerization domain of GCN4 (underlined) attached at the C-terminus of the RSV F polypeptide followed by a hexa-histidine tag.
  • GCN4 trimerization domain of GCN4
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence.
  • CTGTGCACCA CCAACACCAA AGAGGGCAGC AACATCTGCC TGACCCGGAC CGACCGGGGC 1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG
  • the following polypeptide includes the ecto domain of the RSV F polypeptide followed by a hexa-histidine tag.
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence.
  • CTGTGCACCA CCAACACCAA AGAGGGCAGC AACATCTGCC TGACCCGGAC CGACCGGGGC 1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG
  • the following polypeptide includes the ecto domain of the RSV F polypeptide with the trimerization domain of GCN4 (underlined) inserted into the RSV F polypeptide up stream of where the TM domain of the RSV protein would have been (beginning at a. a. 517) followed by a hexa-histidine tag.
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence. 1 ATGGAACTGC TGATCCTGAA GGCCAACGCC ATCACCACCA TCCTGACCGC CGTGACCTTC
  • the following polypeptide includes the full-length RSV F polypeptide with the post- fusion trimerization domain of the influenza hemagglutinin polypeptide (underlined) attached at the C-terminus of the RSV F polypeptide followed by a hexa-histidine tag.
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence. 1 ATGGAACTGC TGATCCTGAA GGCCAACGCC ATCACCACCA TCCTGACCGC CGTGACCTTC
  • the following polypeptide includes the ecto domain of the RSV F polypeptide with the post- fusion trimerization domain of the influenza hemagglutinin polypeptide (underlined) inserted into the RSV F polypeptide up stream of where the TM domain of the RSV protein would have been (beginning at a. a. 517) followed by a hexa-histidine tag.

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