US20120164176A1 - Rsv f protein compositions amd methods for making same - Google Patents

Rsv f protein compositions amd methods for making same Download PDF

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US20120164176A1
US20120164176A1 US13/383,897 US201013383897A US2012164176A1 US 20120164176 A1 US20120164176 A1 US 20120164176A1 US 201013383897 A US201013383897 A US 201013383897A US 2012164176 A1 US2012164176 A1 US 2012164176A1
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rsv
protein
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polypeptides
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Kurt Swanson
Philip DORMITZER
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Novartis AG
Novartis Vaccines and Diagnostics Inc
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    • 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
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    • A61K2039/55505—Inorganic adjuvants
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    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • 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)
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
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    • 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
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    • 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.
  • paramyxoviruses such as RSV, like other enveloped viruses such as influenza virus and HIV, require fusion of the viral membrane with a host cell's membrane.
  • RSV conserved fusion protein
  • the conserved fusion protein fuses the viral and cellular membranes by coupling irreversible protein refolding with juxtaposition of the membranes.
  • the RSV F protein initially folds into a metastable “pre-fusion” conformation. During cell entry, the pre-fusion conformation undergoes refolding and conformational changes to its stable “post-fusion” conformation.
  • 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.
  • F 0 is also cleaved at two sites (approximately 109/110 and approximately 136/137) by cellular proteases (in particular furin) in the trans-Golgi.
  • F 1 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.
  • 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.
  • 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.
  • 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, homogeneous, immunogenic preparations are difficult to obtain.
  • 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 NO:48, 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 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.
  • 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 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
  • 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.
  • 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 F 1 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.
  • FIG. 1 shows the schematic of the wild type RSV F ( FIG. 1A ) 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. 1B ).
  • 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
  • FIG. 1A 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. 1B is a general schematic of the RSV F ectodomain construct depicting the shared features with the schematics in FIG. 1A and including an optional HIS 6 -tag (HIS TAG).
  • HIS 6 -tag HIS 6 -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 R113Q, K123N, K124N-SEQ ID NO:5; Furx R113Q, 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 Deletion1-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.
  • the symbol “-” indicates that the amino acid at that position is
  • 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 (Blossum 62 symbol comparison table, gap open penalty: 12, gap extension penalty: A2, F protein of the strain A2 (accession number AF035006) (SEQ ID NO:102); CP52, F protein of the CP52 strain (accession number AF013255) (SEQ ID NO:103); B, F protein of the B strain (accession number AF013254) (SEQ ID NO:104); long, F protein of the long strain (accession number AY911262) strain (SEQ ID NO:105), and 18537strain, F protein of the 18537 strain (accession number Swiss Prot P13843) (SEQ ID NO:106).
  • a consensus of F protein sequences is also shown (SEQ ID NO:107)
  • 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 mls, 65 mls and 77 mls 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 mls.
  • the protein can form rosettes, which now migrate on SEC in the void volume at approximately 47 mls ( FIG. 6B ).
  • the cleaved trimer species of RSV F fusion peptide deletion is purified from the trimer peak at approximately 65 mls retention time ( FIG. 6C ) while the uncleaved Delp21 Furx construct ( ⁇ p21 Furx) is purified from the monomer peak at approximately 77 mls ( FIG. 6D ).
  • 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 conformations ( FIG. 7B ).
  • FIG. 7D Shown in FIG. 7D are three preparations of either ⁇ p21 (Delp21) furx RSV F (labeled Monomer), Fusion peptide 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 (2wp1), 3 weeks after the 1 st vaccination (3wp1), 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 F1/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.
  • 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 characterized by a 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 F 1 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 F 1 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 macromolecular components present in a composition is substantially higher than that present in a crude preparation.
  • 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.
  • 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 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 (HN 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 ).
  • F 1 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 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 F 0 form with or without the signal peptide, or can comprises two separate peptide chains (e.g., an F 1 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 acid 109 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 2) 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 2). 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 conformation.
  • a viral replicon e.g., alphavirus replicon particles
  • 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).
  • mutations in the p27 region (amino acids 110-136 of SEQ ID NOS: 1 or 2), including deletion of the p27 region in whole or in part.
  • furin cleavage mutations can be combined, if desired, with other mutations described herein, such as trypsin cleavage mutations and fusion peptide mutations.
  • 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 (about amino acids 110-136 of SEQ ID NOS: 1 or 2) 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 2). 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 (SEQ ID NO: 14) PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNDKIEEILSKIYHIENEIARIKKLIGE (HA) (SEQ ID NO: 15) PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNEKFHQIEKEFSEVEGRIQDLEK (Idealized helix) (SEQ ID NO: 16) PLVFPSDEFDASISQINEKINQILAFIRKIDELLHNIN (foldon short) (SEQ ID NO: 17) PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNGSGYIPEAPRDGQAYVRKDGEWVLLSTFL; or (foldon long) (SEQ ID NO: 18) PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNNKNDDKGSGYIPEAPRDGQAYVRKDGEWVLLS TFL
  • 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
  • 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
  • Many 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 (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., AGE1.CR and AGE1.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., AGE1.CR and AGE1.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 Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insert cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. Avian cell expression systems are also known to those of skill in the art and described in, e.g., U.S. Pat. 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.
  • bacterial and mammalian cell expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.
  • Recombinant constructs encoding RSV F protein ecto-domains can be prepared in suitable vectors using conventional methods.
  • 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), and/or one or more translation signals; and 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 al., J. Gen. Virol., 85:3677-3687 (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.
  • a “tag” that facilitates purification, such as an epitope tag or a HIS tag.
  • Such 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 underrepresented or at a lower percentage than for isolated native F proteins, or it may be a derivative of a native F protein.
  • 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 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 oligomerization 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., gp160
  • ATCase E. coli aspartate transcarbamoylase
  • this subunit of ATCase may be used with the present invention.
  • 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 (GSGYIPEAPRDGQ AYVRKDGEWVLLSTFL—SEQ ID NO:19) (12). Thus the 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, Ile or Ala and position d is usually Leu or Ala.
  • Residues e and g are often Glu or Gln, with Arg and Lys also prominent at position g.
  • 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).
  • 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 (NC1) 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 NC1 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.
  • the post-fusion 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 Ile 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, I492, V495, I499, S502, I506, 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.
  • the post-fusion 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 conformation such as the HRA fold in the pre-fusion conformation. In addition, the energy barrier of the transitions leading to the post-fusion conformation may be increased. While one of skill in the art will appreciate that stabilizing the starting conformation or destabilizing the end conformation can have the effect of increasing the energy barrier, other modifications that affect the transition state itself may be introduced.
  • the amino acids N-terminal to the HRB domain 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 FIG. 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 several advantages.
  • 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 produced 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 F 1 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 F 1 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,
  • 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
  • Many 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 (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., AGE1.CR and AGELCR.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., AGE1.CR and AGELCR.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 Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insert cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. Avian cell expression systems are also known to those of skill in the art and described in, e.g., U.S. Pat. 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.
  • bacterial and mammalian cell expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al., 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.
  • substitutions R108N, R109N, R108N/R109N, which inhibit cleavage at 109/110, and the substitution K131Q 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. Sci. 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. 85:3677687 (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 F 1 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 F 1 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 F 1 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.
  • trypsin solution trypsin from bovine plasma diluted to a 1 mg/ml concentration in 25 mM Tris pH 7.5, 300 mM NaCl
  • trypsin trypsin from bovine plasma diluted to a 1 mg/ml concentration in 25 mM Tris pH 7.5,
  • 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 al., 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
  • 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 R113Q 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 F 1 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 F 1 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 F 1 -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 position 109/110, but not at the furin cleavage site at about position 136/137, and are secreted into the media as an F 1 subunit that is associated with an F 2 subunit.
  • C-terminal uncleaved RSV F protein ecto-domains can be cleaved further to produce a F 1 subunit, in which the amino terminus is from position 110 to about position 161, that is associated with a F 2 subunit.
  • F 1 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 F 1 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 F 1 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 F 1 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 F 1 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 F 1 subunit that is associated with an F 2 subunit, wherein the amino terminus of the F 1 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 F 1 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.
  • 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.
  • 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 RSV F protein ecto-domain polypeptides.
  • 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 F 1 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 F 1 subunit is from about position 110 to about position 132. More preferably, the amino terminus of the F 1 subunit is about position 110. It is particularly preferred that the amino terminus of the F 1 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.
  • Preferred 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).
  • 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.
  • 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 (ATCC VR-68, ATCC VR-12, ATCC
  • 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), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).
  • 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), DlinDMA (cationic, unsaturated), and/or DMPG (anionic, saturated).
  • DSPC zwitterionic, saturated
  • DlinDMA cationic, unsaturated
  • DMPG anionic, saturated
  • 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, DlinDMA, PEG-DMPG and cholesterol is used in the examples.
  • a separate aspect of the invention is a liposome comprising DSPC, DlinDMA, 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 ⁇ 50 nm, and LUVs have a diameter >50 nm.
  • Liposomes useful with of the invention are ideally LUVs with a diameter in the range of 50-220 nm.
  • compositions comprising a population of LUVs with different diameters: (i) at least 80% by number should have diameters in the range of 20-220 nm, (ii) the average diameter (Zav, by intensity) of the population is ideally in the range of 40-200 nm, and/or (iii) 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 (ii) an aqueous solution of the nucleic acid and (iii) 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.
  • cationic materials e.g. cationic lipids
  • 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 PREPANDRIXTM product.
  • 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.
  • D- ⁇ -tocopherol and 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.
  • Preferred emulsions comprise squalene, a shark liver oil which is a branched, unsaturated terpenoid (C 30 H 50 ; [(CH 3 ) 2 C[ ⁇ CHCH 2 CH 2 C(CH 3 )] 2 ⁇ CHCH 2 —] 2 ; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN 7683-64-9).
  • squalene a shark liver oil which is a branched, unsaturated terpenoid (C 30 H 50 ; [(CH 3 ) 2 C[ ⁇ CHCH 2 CH 2 C(CH 3 )] 2 ⁇ CHCH 2 —] 2 ; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN 7683-64-9).
  • 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 5-20 mM 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: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3′-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol), dimethyldioctadecyl-ammonium (DDA e.g. the bromide), 1,2-Dimyristoyl-3-Trimethyl-AmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP).
  • DOTAP 1,2-dioleoyloxy-3-(trimethylammonio)propane
  • DC Cholesterol 3′-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol
  • DDA dimethyldio
  • 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 CAC
  • N,N′,N′-polyoxyethylene (10)-N-tallow-1,3-diaminopropane dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammonium methoxide, cetyldimethyleth
  • 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 ( ⁇ GluPhCnN), ditetradecyl glutamate ester with pendant amino group (Cl4GIuC
  • the cationic lipid is preferably biodegradable (metabolisable) and biocompatible.
  • an emulsion can include a non-ionic surfactant and/or a zwitterionic surfactant.
  • surfactants include, but are 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 DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such
  • 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-100) 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 50-750 nm.
  • the average droplet size is less than 250 nm e.g. less than 200 nm, less than 150 nm.
  • the average droplet size is usefully in the range of 80-180 nm.
  • 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. These these typically use the techniques of dynamic light scattering and/or single-particle optical sensing e.g. the AccusizerTM and NicompTM series of instruments available from Particle Sizing Systems (Santa Barbara, USA), or the ZetasizerTM instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan).
  • 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 220 nm 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 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 TH1 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.
  • 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 ecto-domain polypeptides, or C-terminal uncleaved RSV F protein ecto-domain polypeptides, and cleaving them to produce F 1 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.
  • the composition is an immunogenic composition, it will elicit an immune response when administered to a mammal, such as a human.
  • the 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 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290-310 mOsm/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 5-20 mM 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 0.5 ml, although a half dose (i.e., about 0.25 ml) 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 TH1 adjuvant and/or a TH2 adjuvant.
  • Adjuvants which may be used in compositions of the invention include, but are not limited to:
  • 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 benzonaphthyridine compounds, etc.
  • 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). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used.
  • 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 Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • oil-in-water emulsions 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 DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol
  • Non-ionic surfactants are preferred.
  • Preferred surfactants for including in the emulsion are TWEEN 80TM (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
  • surfactants can be used e.g., TWEEN 80TM/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (TWEEN 80TM) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) 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 80TM) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, 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 80TM
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100, 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:
  • 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; interleukin-12; TNF- ⁇ ; TNF- ⁇ ; and GM-CSF.
  • Preferred agents elicit the release of cytokines associated with a Th1-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:
  • 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 TLR1, 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, imidazoquinolines) and/or TLR9 (e.g., CpG oligonucleotides). These agents are useful for activating innate immunity pathways.
  • TLR4 e.g., modified natural lipid As derived from enterobacterial lipopolysaccharides, phospholipid compounds, such as the
  • 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 modifications/analogs such as phosphorothioate modifications and can be double-stranded or (except for RNA) single-stranded.
  • 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 GTCGTT or TTCGTT (120).
  • the CpG sequence may be specific for inducing a Th1 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”. See, for example, references 120 & 124-126.
  • a useful CpG adjuvant is CpG7909, also known as PROMUNETM (Coley Pharmaceutical Group, Inc.).
  • 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.).
  • cytosine nucleotide e.g., CCCC, as disclosed in ref 127
  • it may have a nucleotide composition with >25% cytosine (e.g., >35%, >40%, >50%, >60%, >80%, etc.).
  • cytosine nucleotide e.g., >35%, >40%, >50%, >60%, >80%, etc.
  • These oligonucleotides may be free from unmethylated CpG motifs.
  • 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-1, 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 2′), and there is also O-acylation at the 3′ position.
  • the group attached to carbon 2 has formula —NH—CO—CH 2 —CR 1 R 1′ .
  • the group attached to carbon 2′ has formula —NH—CO—CH— 2 —CR 2 R 2′ .
  • the group attached to carbon 3′ has formula —O—CO—CH 2 —CR 3 R 3′ .
  • a representative structure is:
  • 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) —O—CO—R 4 ,where R 4 is either —H or —(CH 2 ) m —CH 3 , wherein the value of m is preferably between 8 and 16, and is more preferably 10, 12 or 14. At the 2 position, m is preferably 14. At the 2′ position, m is preferably 10. At the 3′ position, 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.
  • 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 ⁇ 150 nm or >500 nm. 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 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and can even have a mean diameter less than 100 nm. In most cases, however, the mean diameter will not be lower than 50 nm.
  • 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:
  • 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 R 1 —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-Glu) or D-gamma-carboxy-glutamic acid (D-Gla).
  • 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:
  • 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).
  • the 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)
  • 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 /Al 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 3164 cm ⁇ 1 (e.g., when heated to 200° C.) indicates the presence of structural hydroxyls (ch. 9 of ref. 74)
  • the PO 4 /Al 3+ 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 hydroxyphosphate salts.
  • a typical adjuvant is amorphous aluminum hydroxyphosphate with PO 4 /Al molar ratio between 0.84 and 0.92, included at 0.6 mg Al 3+ /ml.
  • 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 10 mg/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.
  • a maximum of 0.85 mg/dose is preferred.
  • 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-O-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 Th1 and Th2 adjuvants such as CpG and alum, or resiquimod and alum.
  • a combination of aluminum phosphate and 3dMPL may be used.
  • alum and a benzonapthridine compound or a SMIP include: alum and a benzonapthridine compound or a SMIP, a squalene-in-water emulsion (such as MF59) and a benzonapthridine compound or a SMIP, and E6020 and 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: TH1 phenotype and TH2 phenotypes which differ in their cytokine and effector function.
  • Activated TH1 cells enhance cellular immunity (including an increase in antigen-specific CTL production) and are therefore of particular value in responding to intracellular infections.
  • Activated TH1 cells may secrete one or more of IL-2, IFN- ⁇ , and TNF- ⁇ .
  • a TH1 immune response may result in local inflammatory reactions by activating macrophages, NK (natural killer) cells, and CD8 cytotoxic T cells (CTLs).
  • a TH1 immune response may also act to expand the immune response by stimulating growth of B and T cells with IL-12.
  • TH1 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 IgG1, IgE, IgA and memory B cells for future protection.
  • An enhanced immune response may include one or more of an enhanced TH1 immune response and a TH2 immune response.
  • a TH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 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 TH1 immune response will include an increase in IgG2a production.
  • a TH1 immune response may be elicited using a TH1 adjuvant.
  • a TH1 adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant.
  • TH1 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 TH1 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 IgG1, IgE, IgA and memory B cells.
  • the enhanced TH2 immune response will include an increase in IgG1 production.
  • a TH2 immune response may be elicited using a TH2 adjuvant.
  • a TH2 adjuvant will generally elicit increased levels of IgG1 production relative to immunization of the antigen without adjuvant.
  • 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 TH1 adjuvant and a TH2 adjuvant.
  • a composition elicits an enhanced TH1 and an enhanced TH2 response, i.e., an increase in the production of both IgG1 and IgG2a production relative to immunization without an adjuvant.
  • the composition comprising a combination of a TH1 and a TH2 adjuvant elicits an increased TH1 and/or an increased TH2 immune response relative to immunization with a single adjuvant (i.e., relative to immunization with a TH1 adjuvant alone or immunization with a TH2 adjuvant alone).
  • the immune response may be one or both of a TH1 immune response and a TH2 response.
  • immune response provides for one or both of an enhanced TH1 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 composition) of the invention to the mammal.
  • a composition e.g., an immunogenic composition
  • 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.
  • intramuscular injection e.g., into the arm or leg
  • subcutaneous injection intranasal administration
  • oral administration intradermal administration
  • transcutaneous administration transdermal administration
  • transdermal administration e.g., transcutaneous administration
  • transdermal administration e.g., transcutaneous administration
  • 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. In a multiple dose 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 (typically two doses) 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.). P
  • 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, parainfluenzavirus 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° A 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 ecto-domain 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” encompasses “including” as well as “consisting” and “consisting essentially of” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X+Y.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • 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 encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials.
  • TSEs transmissible spongiform encaphalopathies
  • BSE bovine spongiform 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.
  • DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine
  • DLPA 1,2-Dilauroyl-sn-Glycero-3-Phosphate
  • DLPC 1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine
  • DLPE 1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine
  • DLPG 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . .
  • DLPS 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine
  • DMG 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine
  • DMPA 1,2-Dimyristoyl-sn-Glycero-3-Phosphate
  • DMPC 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine
  • DMPE 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine
  • DMPG 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . .
  • DMPS 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine
  • DOPA 1,2-Dioleoyl-sn-Glycero-3-Phosphate
  • DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine
  • DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine
  • DOPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . .
  • DPPS 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine
  • DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine
  • DSPA 1,2-Distearoyl-sn-Glycero-3-Phosphate
  • DSPC 1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine
  • DSPE 1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine
  • DSPG 1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . .
  • PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine
  • SMPC 1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine
  • SOPC 1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine
  • SPPC 1-Stearoyl,2-palmitoyl-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 F 1 and F 2 , and forms that lack the optional C-terminal His tag.
  • the following examples are merely illustrative of the scope of the present invention and therefore are not intended to limit the scope in any way.
  • 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 R113Q 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).
  • RSV F Furx SEQ ID NO:45
  • RSV F old furx Truncated HIS SEQ ID NO:88
  • RSV F Furx R113Q K123N K124N Truncated HIS SEQ ID NO:89
  • RSV F delp21 furx Truncated HIS SEQ ID NO:47
  • 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.
  • 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.
  • 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.
  • the following polypeptide includes the ecto domain of the RSV F polypeptide followed by a hexa-histidine tag.
  • 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.
  • 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.
  • 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.
  • 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 HRB domain deleted followed by a hexa-histidine tag.
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence.
  • the following polypeptide includes just the ecto domain of the RSV F polypeptide.
  • nucleic acid sequence is the optimized coding sequence for the foregoing polypeptide sequence.
  • the RSV F ECTO and truncated constructs lacking the transmembrane domain and cytoplasmic tail region with either wild-type furin cleavage sites or harboring knock-out mutations to the furin cleavage sites and with or without prefusion stabilization mutations, were cloned into a pFastBac baculovirus expression vector (Invitrogen).
  • Several of these constructs contain a C-terminal flexible linker followed by a His6-tag sequence used for chelating purification.
  • the production of high-titer baculovirus stocks were passaged in Sf9 insect cells.
  • Proteins were expressed by infecting either Sf9, Tn5 or High Five insect cells with the required baculovirus and harvesting the media supernatant two or three days post infection, monitored by western blot using an anti-RSV F or anti-6HIS antibody.
  • Large scale expression media was concentrated/purified by one of two general strategies for eliminating the deleterious Effect of the ferritin present in insect cell media from corrupting the chelating resin.
  • the first approach was to concentrate the approximately 10-20 liters of insect expression media down to approximately 300 mls using a GE Healthcare Hollotube fiber concentration column. Copper sulfate was added to this concentrated mixture to a final concentration of 500 ⁇ M and the resulting solution was loaded onto 5 ml HiTrap chelating columns. The bound HIS-tagged protein was then eluted from the column with 25 mM Tris pH 7.5, 300 mM NaCl and a gradient of imidazole.
  • the elution was dialyzed against 25 mM Tris buffer pH 7.5, and the resulting solution was loaded onto a 5 ml Hitrap chelation column charged with NiSO4 and eluted with 25 mM Tris pH 7.5, 300 mM NaCl and a gradient of imidazole.
  • HPLC-SEC was performed using a Biorad SEC column (18 mm) with a 25 mM Tris pH 7.5, 300 mM NaCl mobile phase. Using Biorad HPLC-SEC standards to calibrate the system, we found that the RSV rosettes (representing cleaved-postfusion conformations) elute in the column void volume of the analysis, while RSV monodispersed trimers (presumed trimers from subsequent EM analysis) elute with an apparent molecular weight of approximately 100 kDa.
  • FPLC-SEC was performed on a GE Healtcare FPLC using a 16/60 Superdex 200 column with 25 mM Tris pH 7.5, 300 mM NaCl mobile phase.
  • GE Healthcare High molecular weight standards to calibrate the system, we found that the RSV rosettes elute in the column void volume of the analysis, while RSV monodispersed trimers elute with an apparent molecular weight of approximately 100 kDa.
  • Protein solutions of approximately 50 micrograms per ml RSV F constructs were absorbed onto glow-discharged carbon coated grids and were negatively stained with 2% sodium phosphotungstate (pH 7.0) or 0.75% Urynal-formate (unquantified low pH).
  • the grids were observed on a Technai Spirit or JOEL 1230 transmission electron microscope operating between 80-120 kV with a magnification between 20,000 to 150,000 depending on required resolution.
  • a number of methods are available to determine the conformation of the RSV F protein to assay whether a modification to the RSV F polypeptide or added molecule disfavors the post-fusion conformation. Examples include liposome association, conformation specific monoclonal antibodies (including as used in FACS, ELISA, etc.), electron microscopy, differential protease sensitivity between the conformations, gel filtration chromatography, analytical ultracentrifugation, dynamic light scattering, deuterium exchange NMR experiments, mass spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, tryptophan spectroscopy, and X-ray crystallography.
  • Liposome association may be used to assay the conformation of the RSV F protein. Soluble forms of the RSV F protein in the pre-fusion conformation will not associate with liposomes while the post-fusion conformation will associate with liposomes.
  • Liposomes may be prepared as follows: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol in chloroform (available from Avanti Polar Lipids) are mixed at an 8:2:5 molar ratio. The chloroform is evaporated under argon. A lipid film will form that is dried under vacuum overnight and resuspended in PBS at 40 mM total lipid. After five freeze-thaw cycles, the lipids are vortexed and extruded 21 times through two 100- ⁇ m filters by using a miniextruder (available from Avanti Polar Lipids).
  • the liposome association assay may be performed. For each sample to be tested, 2 ⁇ g of the RSV F polypeptide to test is cleaved with 25 milliunits of trypsin (available from Worthington Biochemical) in 100 mM phosphate buffer (pH 7.1) for 30 min at 25° C. After cleavage, 40 pg of soybean trypsin inhibitor (available from Worthington Biochemical) is added to each sample to end the reaction. The samples are pretreated at 60° C. for 30 min which would induce a conformational shift from the pre-fusion to the post-fusion forms in native isolated RSF F protein.
  • trypsin available from Worthington Biochemical
  • Liposomes (40 ⁇ l per sample) and PBS are added (80 ⁇ l final volume), and the samples are incubated at 60° C. for 30 min. Sucrose is added to a final concentration of 50% (500 ⁇ l final volume). The samples are overlaid with 500 ⁇ l each of 40% sucrose, 25% sucrose, and PBS and are spun in a TLS55 rotor at 49,000 rpm for 3 h at 25° C. Fractions (500 ⁇ l) are collected from the top of the gradients. Proteins are solubilized in 0.5% Triton X-100 and precipitated by using 12.5% vol/vol trichloroacetic acid. Polypeptides are separated by SDS/PAGE and transferred to PVDF membranes. Blots are probed with anti-RSV F monoclonal antibodies.
  • Electron microscopy was used to assay the conformational distribution of RSV F polypeptides.
  • RSV F polypeptides in the pre-fusion form have a “ball and stem” shape with a length of ⁇ 12 nm.
  • RSV F polypeptides in the post-fusion form have a “golf tee” shape with a length of ⁇ 16 nm.
  • the fusion peptides at the narrow end of the “golf tees” aggregate to form a rosette structures.
  • electron microscopy may be used to assay the distribution of conformations in a sample of RSV F polypeptides owing to the readily distinguishable shapes.
  • the RSV F protein ecto-domain constructs encoding polypeptides that lack the transmembrane domain and cytoplasmic tail region with either wild-type furin cleavage sites or harboring knock-out mutations to the furin cleavage sites and/or fusion peptide mutations mutations, were cloned into a pFastBac baculovirus expression vector (Invitrogen).
  • Several of these constructs contain a C-terminal flexible linker followed by a HIS 6 -tag sequence used for chelating purification.
  • the production of high-titer baculovirus stocks were obtained by passage in Sf9 insect cells.
  • Proteins were expressed by infecting either Sf9, Tn5 or High Five insect cells with the required baculovirus and harvesting the conditioned media supernatant two or three days post infection. Protein production was monitored by western blot using an anti-RSV F or anti-HIS 6 antibody.
  • CuCl 2 was added to media supernatant to a final concentration of 500 ⁇ M.
  • approximately four to ten milliliters of chelating resin (Chelating Resin, BioRad) was added, and the slurry was rocked for at least thirty minutes at 4 degrees centigrade, and the resin and media were separated using a gravity column.
  • the resin was washed with approximately ten times the column volume of equilibration buffer (25 mM Tris pH 7.5, 300 mM NaCl), and the F protein ecto-domain was eluted with approximately ten-times the column volume of elution buffer (equilibration buffer with 250 mM imidazole).
  • Elutions from the imidazole gradient in either case were evaluated using anti-HIS 6 western blots and/or Coomassie-stained SDS-PAGE gels. Fractions containing pure constructs were collected, dialyzed against different buffer/saline solutions and were concentrated for subsequent analysis using Millipore Centriprep concentrators and/or Vivaspin concentration units. In some instances, monomers, trimers or rosettes were further purified using size exclusion chromatography.
  • Size exclusion chromatography was used to purify and analyze RSV F protein ecto-domain monomers, trimers and rosettes. This method also allowed uncleaved RSV F protein ecto-domains to be purified away from host cell or media derived lipid and lipoprotein contaminants. Two methods were developed, HPLC-SEC and FPLC-SEC, which may also serve as an efficient purification step.
  • HPLC-SEC was performed using a Biorad SEC column (18 mm) with a 25 mM Tris pH 7.5, 300 mM NaCl mobile phase. Using Biorad HPLC-SEC standards to calibrate the system, we found that the RSV rosettes (representing cleaved, postfusion conformations) elute in the column void volume of the analysis, while RSV F monomers elute with an apparent molecular weight of approximately 75-85 kDa.
  • FPLC-SEC was performed on a GE Healthcare FPLC using a 16/60 Superdex 200 column with 25 mM Tris pH 7.5, 300 mM NaCl as a mobile phase.
  • GE Healthcare High molecular weight standards to calibrate the system, we found that the RSV rosettes elute in the column void volume of the analysis, while RSV monodispersed trimers elute with an apparent molecular weight of approximately 140-160 kDa and RSV F monomers elute with an apparent molecular weight of approximately 75-85 kDa.
  • trypsin digestion of Delp23 Furdel monomers is done with 1:1000 trypsin:RSV F by weight, or 10-15 BAEE units of trypsin for 1 mg of RSV F antigen.
  • trypsin from bovine plasma Sigma Aldrich, T8802: 10,000-15,000 BAEE units/mg trypsin was diluted to a 1 mg/ml concentration in 25 mM Tris pH 7.5, 300 mM NaCl.
  • RSV F protein ecto-domain polypeptide diluted in 25 mM Tris pH 7.5, 300 mM NaCl
  • trypsin solution final mass ratio 0.001:1 trypsin:RSV F or approximately 10-15 BAEE units of trypsin to each milligram of RSV F
  • progress of the cleavage reaction was monitored by SDS-PAGE gel.
  • the cleavage reaction was stopped using a trypsin inhibitor.
  • the cleaved RSV F protein was further purified by size exclusion chromatography.
  • RSV F protein ecto-domain polypeptides (approximately 50 micrograms per ml), were absorbed onto glow-discharged carbon-coated grids and were negatively stained with 2% sodium phosphotungstate (pH 7.0) or 0.75% uranyl-formate (unquantified low pH).
  • the grids were observed on a Technai Spirit or JOEL 1230 transmission electron microscope operating between 80-120 kV with a magnification between 20,000 to 150,000 depending on required resolution.
  • This assay is based on the Wako Pure Chemical Industries, Ltd. assay Phospholipids C choline oxidase—DAOS method (Cat. No 433-36201).
  • the assay protocol is modified only to reduce the amount of material used in the assay, and to decrease the sample dilution in the reaction relative to the general protocol from the vendor.
  • To determine the lipid content of the RSV F sample generate the color reagent by dissolving one bottle of Color Reagent with one bottle of Buffer (color reagent is stable for 1 week at 4° C.). Dilute the 300 mg/dL (3 mg/ml) phospholipid standard to 1.5, 1.0, 0.75, 0.5 and 0.25 mg/ml with distilled water.
  • a water blank and sample reaction For each standard, a water blank and sample reaction, add to a microcentrifuge tube 10 ⁇ l of color reagent and 2 ⁇ l of either standard, distilled water (0 mg/ml standard) or sample. Centrifuge reactions briefly to ensure proper mixing and incubate the tubes for 15 min at 37° C. Record the absorbance at 595 nm for each standard point and generate a standard curve. Record the 595 nm absorbance for each sample and calculate the phospholipids concentration from the prepared calibration curve.
  • Serum anti-RSV F protein IgG and RSV neutralizing antibody titers were measured 2 weeks after the 1 st vaccination (2wp1) and 2 weeks after the 2 nd vaccination (2wp2), or 3 weeks after the 1 st vaccination (3wp1) and 2 weeks after the 2 nd vaccination (2wp2).
  • Anti-RSV F protein IgG FIG. 8A
  • RSV F protein coated plates and horse radish peroxidase-conjugated chicken anti-cotton rat IgG detection antibody. Data are presented as log 10 geometric mean titers (GMT)+standard deviations of individual cotton rats.
  • RSV neutralization titers FIG. 8B ) were measured by plaque reduction neutralization test (PRNT).
  • the soluble RSV F ecto-domain (with un-mutated furin cleavage sites) was expressed, but could not be purified from lipid and lipoprotein impurities derived from the host cells or culture media using size exclusion chromatography. These RSV F ecto-domain polypeptides elute in the SEC column void volume along with the lipid and lipoprotein contaminants.
  • FIG. 1 Several constructs were prepared for making RSV F ecto-domain polypeptides that contain mutations to the furin cleavage sites, including the Furdel constructs. See, FIG. 1 . Polypeptides produced by expression of the Furdel constructs were secreted from the cells as an ⁇ 65 kDa uncleaved species. The Furdel mutation also prevents fusion peptide exposure, which in turn prevents rosette formation. As a result, soluble RSV F Furdel migrated in the included volume of a Superdex 200 preparatory column, resulting in separation from both the lipid debris, which eluted in the void volume, and insect protein impurity.
  • RSV F furdel polypeptides were monomers, trimers or a mixture of monomers and trimers was assessed further using analytical ultracentrifugation.
  • Analytical ultracentrifugation studies were performed using protein purified from the monomer peak from the SEC purification. Sedimentation velocity data of the uncleaved RSV F showed a step pattern suggesting two species in solution. Analysis of the sedimentation velocity experiment showed that the uncleaved RSV F ecto-domain had a high population of monomer and a minor population of apparent trimer in the solution. Equilibrium run data was collected and attempts to fit the data to either an ideal monomer model or a monomer-trimer equilibrium model were performed.
  • FIGS. 6A-6D The principle peaks containing monomers, trimers or rosettes of trimers are indicated by an asterisk in FIGS. 6A-6D , with the retention time of the Superdex P200 16/60 column (GE Healthcare) is indicated in milliliters. On a calibrated column, the approximate retention times of 47 mls, 65 mls and 77 mls correspond to the column void volume, the retention of F trimers and the retention of the monomers, respectively.
  • the uncleaved Delp23 Furdel ( ⁇ p23 Furdel) construct was purified from the monomer peak.
  • RSV F protein ecto-domain polypeptides in uncleaved form or after trypsin cleavage were assessed by EM.
  • the RSV F Furdel and delp23 Furdel constructs have arginine residues remaining in the furin cleavage site. These arginines are susceptible to trypsin cleavage.
  • the uncleaved F 0 species was converted to the F 1 /F 2 species, in which the fusion peptide is exposed.
  • EM analysis confirmed that following trypsin cleavage the uncleaved RSV ecto-domains formed rosettes of trimers by virtue of their fusion peptides, as has been observed for related fusion proteins.
  • RSV F ectodomain subunits including Delp21 Furx, Delp23 Furdel and Fusion Peptide Deletion constructs, were expressed in HiFive insect cells (Invitrogen) using the pFAST Bac baculovirus system.
  • the RSV F subunit was purified from large scale expressions, 10-25 liters, via a two step chelating method that reduced the deleterious effect of the ferritin contaminant present in insect cell media, which can corrupt the chelating resin.
  • CuSO 4 was added to media supernatant to a final concentration of 500 ⁇ M.
  • chelating resin (Chelating Resin, BioRad) was added to each 1 liter of media, the slurry was rocked for at least thirty minutes at 4° C., and the resin and media were separated using a gravity column. The resin was washed with approximately two-times the resin volume of equilibration buffer (25 mM Tris pH 7.5, 300 mM NaCl), and the F protein ecto-domain was eluted with approximately two-times the column volume of elution buffer (equilibration buffer with 250 mM imidazole).
  • equilibration buffer 25 mM Tris pH 7.5, 300 mM NaCl
  • Size exclusion chromatography was used to purify and analyze RSV F protein ectodomain uncleaved monomers and cleaved trimers. This method also allowed uncleaved RSV F protein ectodomains to be purified away from host cell or media derived lipid and lipoprotein contaminants.
  • the uncleaved Delp23 Furdel construct was initially purified as a monomer and subsequently protease treated and re-purified using SEC to purify homogeneous rosettes (see below).
  • Two methods were developed for analysis of RSV F oligamerization, HPLC-SEC and FPLC-SEC, which may also serve as an efficient purification step.
  • HPLC-SEC was performed using a Biorad SEC column (18 mm) with a 25 mM Tris pH 7.5, 300 mM NaCl mobile phase. Using Biorad HPLC-SEC standards to calibrate the system, we found that the RSV rosettes (representing cleaved, postfusion conformations) elute in the column void volume of the analysis while RSV F monomers elute with an apparent molecular weight of approximately 75-85 kDa.
  • FPLC-SEC was performed on a GE Healthcare FPLC using a 16/60 Superdex 200 column with 25 mM Tris pH 7.5, 300 mM NaCl as a mobile phase.
  • GE Healthcare High molecular weight standards to calibrate the system, we found that the RSV rosettes elute in the column void volume of the analysis, while RSV monodispersed trimers elute with an apparent molecular weight of approximately 140-160 kDa and RSV F monomers elute with an apparent molecular weight of approximately 75-85 kDa.
  • Trypsin from bovine plasma (Sigma Aldrich, T8802: 10,000-15,000 BAEE units/mg trypsin) was diluted to a 1 mg/ml concentration in 25 mM Tris pH 7.5, 300 mM NaCl.
  • a 1 mg/ml solution of RSV F protein ecto-domain polypeptide (diluted in 25 mM Tris pH 7.5, 300 mM NaCl) was treated with one microliter of trypsin solution (final mass ratio 0.001:1 trypsin:RSV F or approximately 10-15 BAEE units of trypsin to each milligram of RSV F) for 1 hour at 37° C. Progress of the cleavage reaction was monitored by SDS-PAGE gel.
  • the cleavage reaction was stopped using a trypsin inhibitor (Gibco Soy Bean Trypsin Inhibitor using equal mass of inhibitor to trypsin). It was found that an incubation period was required between the cleavage step and subsequent rosette purification to allow higher efficiency of monomer to rosette conversion. A one to 6 hour incubation period at 37° C. was given to provide higher rosette formation efficiency.
  • the cleaved RSV F protein was further purified from unconverted monomer species using size exclusion chromatography (as described above) where homogeneous rosettes can be collected in the column void volume fractions.
  • RSV F Fusion Peptide Deletion constructs which do not contain a HIS-tag, were purified by cation purification.
  • CHO material containing expressed RSV F trimer antigen was concentrated to approximately one-tenth the original volume on a GE Healthcare hollow fiber cartridge concentration system (MWCO 10,000 kDa).
  • the concentrated solution was then buffer exchanged four times with an equivalent volume of 25 mM Sodium Acetate pH 6.0, 25 mM NaCl.
  • the resulting solution, containing concentrated RSV F trimer in the acetate/saline buffer was loaded onto a pre-charged GE Healthcare HiTrap CM column which had been equilibrated with acetate/saline buffer.
  • the protein was eluted from the column using a step gradient of 25 mM acetate buffer containing either 25, 150, 250, 500 or 1000 mM NaCl (the 250 mM and 500 mM NaCl fractions containing the bulk of the eluted material). This material could be further purified using a SEC purification similar to the protocol above.
  • RSV-F trimer RSV-F-fusion-peptide-deletion-trun
  • rosette RSV-F-delp23-furdel-trunc, cleaved subunits, each formulated with alum or MF59
  • the antigen used for ELISA in this study was RSV-F-fusion-peptide-deletion-trunc (Table 4). Neutralization was against infectious RSV, strain Long (Table 5). All combinations were immunogenic, eliciting high titer RSV-F-specific IgG and RSV neutralizing antibody responses that were boosted by a second vaccination, and afforded protection from nasal RSV challenge.
  • mice Female cotton rats ( Sigmodon hispidis ) were obtained from Harlan Laboratories. Groups of animals were immunized intramuscularly (i.m., 100 ⁇ l) with the indicated vaccines on days 0 and 21. Serum samples were collected 3 weeks after the first immunization (3wp1) and 2 weeks after the second immunization (2wp2). Immunized or unvaccinated control animals were challenged intranasally (i.n.) with 1 ⁇ 10 5 pfu RSV Long 4 weeks after the final immunization. Blood collection and RSV challenge were performed under anesthesia with 3% isoflurane using a precision vaporizer.
  • ELISA enzyme-linked immunosorbent assay
  • Serum samples were tested for the presence of neutralizing antibodies by microneutralization assay. Two-fold serial dilutions of heat inactivated (HI)-serum (in PBS with 5% HI-fetal bovine serum(FBS)) were added to an equal volume of RSV, strain Long previously titered to give approximately 115 PFU/25 ⁇ l. Serum/virus mixtures were incubated for 2 hours at 37° C. and 5% CO2, to allow virus neutralization to occur, and then 25 ⁇ l of this mixture (containing approximately 115 PFU) was inoculated on duplicate wells of HEp-2 cells in 96 well plates.
  • HI heat inactivated
  • FBS 5% HI-fetal bovine serum
  • the cells were overlayed with 0.75% Methyl Cellulose/EMEM 5% HI-FBS and incubated for 42 hours.
  • the number of infectious virus particles was determined by detection of syncytia formation by immunostaining followed by automated counting.
  • the neutralization titer is defined as the reciprocal of the serum dilution producing at least a 60% reduction in number of synctia per well, relative to controls (no serum).
  • Viral load in the lung was determined by plaque assay. Specifically, lungs were harvested 5 days post RSV infection and one right lobe was placed into 2.5 ml Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) with 25% sucrose and disrupted with a tissue homogenizer. Cell-free supernatants from these samples were stored at ⁇ 80° C. To assay for infectious virus, dilutions of clarified lung homogenate (in PBS with 5% HI-FBS) were inoculated on confluent HEp-2 cell monolayers in a volume of 200 ⁇ l/well of a 12-well plate.
  • DMEM Dulbecco's Modified Eagle Medium
  • PFU plaque forming units
  • cDNA synthesis and PCR is performed in a single tube using the SuperScript III Platinum One-Step Quantitative RT-PCR kit (Invitrogen) with 5 ⁇ L of eluted RNA, 10 ⁇ M of each primer, and 50 ⁇ M of the probe (primers and probes from Integrated DNA Technologies).
  • Forward primer TTGGATCTGCAATCGCCA (SEQ ID NO:72).
  • Reverse primer CTTTTGATCTTGTTCACTTCTCCTTCT (SEQ ID NO:73).
  • Probe 5′-carboxyfluorescein (FAM)-TGGCACTGCTGTATCTAAGGTCCTGCACT-tetramethylcarboxyrhodamine(TAMRA)-3′ (SEQ ID NO:74).
  • Amplification and detection is performed with an ABI Prism 7900HT or 7500 (Applied Biosystems).
  • a threshold cycle value (Ct) is defined for each sample as the cycle number at which the fluorescent signal first becomes detectable above a set threshold. PFU equivalents for each sample is then determined based on a standard curve of Ct verses the logarithm of defined copy number of viral RNA.
  • the cotton rat as a model has been used extensively in the study of RSV pathogenesis and immunity because of the many similarities between RSV-induced disease in cotton rats and humans. Two important parallels are the efficacy of neutralizing antibodies, and the enhanced lung histopathology associated with formalin-inactivated RSV vaccination. Cotton rats are also more susceptible to RSV infection than other small animals such as mice.
  • Plasmid DNA encoding alphavirus replicon ( FIG. 4 , SEQ ID NO:77) served as a template for synthesis of RNA in vitro.
  • SEQ ID NO:77 the full length surface fusion glycoprotein of RSV (RSV-F) was used ( FIG. 4 ).
  • RSV-F full length surface fusion glycoprotein of RSV
  • the positive-stranded RNA was translated to produce four non-structural proteins, which together replicated the genomic RNA and transcribed abundant subgenomic mRNAs encoding the heterologous gene product. Due to the lack of expression of the alphavirus structural proteins, replicons are incapable of inducing the generation of infectious particles.
  • a bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA facilitates the synthesis of the replicon RNA in vitro and the hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A)-tail generates the correct 3′-end through its self-cleaving activity.
  • HDV hepatitis delta virus
  • run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage derived DNA-dependent RNA polymerase. Transcriptions were performed for 2 hours at 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion, Austin, Tex.). Following transcription, the template DNA was digested with TURBO DNase (Ambion, Austin, Tex.).
  • RNA was precipitated with LiCl and reconstituted in nuclease-free water.
  • Uncapped RNA was capped post-transcriptionally with Vaccinia Capping Enzyme (VCE) using the ScriptCap m 7 G Capping System (Epicentre Biotechnologies, Madison, Wis.) as outlined in the user manual.
  • Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water.
  • concentration of the RNA samples was determined by measuring the optical density at 260 nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis.
  • 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DlinDMA) was synthesized using a previously published procedure [Heyes, J., Palmer, L., Bremner, K., MacLachlan, I. Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids. Journal of Controlled Release, 107: 276-287 (2005)].
  • 1,2-Diastearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Genzyme. Cholesterol was obtained from Sigma-Aldrich (St. Lois, Mo.).
  • 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) PEG DMG 2000
  • 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) was obtained from Avanti Polar Lipids (Alabaster, Ala.).
  • Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 minutes to form a homogenous mixture. Then, 755 ⁇ L of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipid was used to form LNPs with 250 ⁇ g RNA at a 8:1 N:P (Nitrogen to Phosphate) ratio.
  • the protonatable nitrogen on DlinDMA (the cationic lipid) and phosphates on the RNA are used for this calculation.
  • Each ⁇ g of self-replicating RNA molecule was assumed to contain 3 nmoles of anionic phosphate, each ⁇ g of DlinDMA was assumed to contain 1.6 nmoles of cationic nitrogen.
  • a 2 mL working solution of RNA was also prepared from a stock solution of ⁇ 1 ⁇ g/ ⁇ L in 100 mM citrate buffer (pH 6) (Teknova, Hollister, Calif.)).
  • RNA working solution Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts, San Diego, Calif.) and washed with plenty of MilliQ water before use to decontaminate the vials of RNAses.
  • RNase Away solution Molecular BioProducts, San Diego, Calif.
  • One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described below).
  • the working lipid and RNA solutions were heated at 37° C. for 10 minutes before being loaded into 3 cc luer-lok syringes (BD Medical, Franklin Lakes, N.J.). 2 mL of citrate buffer (pH 6) was loaded in another 3 cc syringe.
  • RNA and the lipids were connected to a T mixer (PEEKTM 500 ⁇ m ID junction, Idex Health Science, Oak Harbor, Wash.) using FEP tubing ([fluorinated ethylene-propylene] 2 mm ID ⁇ 3 mm OD, Idex Health Science, Oak Harbor, Wash.).
  • the outlet from the T mixer was also FEP tubing (2 mm ID ⁇ 3 mm)
  • the third syringe containing the citrate buffer was connected to a separate piece of tubing (2 mm ID ⁇ 3 mm OD). All syringes were then driven at a flow rate of 7 mL/min using a syringe pump (kdScientific, model no.
  • the two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring).
  • the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation, AnnArbor, Mich., USA).
  • a Mustang Q membrane an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation, AnnArbor, Mich., USA.
  • 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) were successively passed through the Mustang membrane. Liposomes were warmed for 10 minutes at 37° C. before passing through the mustang filter.
  • liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1 ⁇ PBS (from Teknova) using the Tangential Flow Filtration (TFF) system before recovering the final product.
  • TFF Tangential Flow Filtration
  • the TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez, Calif.) and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes (part number P/N: X1AB-100-20P) with a 100 kD pore size cutoff and 8 cm 2 surface area were used.
  • formulations were diluted to the required RNA concentration with 1 ⁇ PBS (from Teknova).
  • the primary emulsions were passed three to five times through a Microfluidezer M110S or M110PS homogenizer with an ice bath cooling coil at a homogenization pressure of approximately 15 k-20 k PSI (Microfluidics, Newton, Mass.).
  • the 20 ml batch samples were removed from the unit and stored at 4° C.
  • the table below describes the composition of CNE17.
  • the number of nitrogens in solution were calculated from the cationic lipid concentration, DOTAP for example has 1 nitrogen that can be protonated per molecule.
  • the RNA concentration was used to calculate the amount of phosphate in solution using an estimate of 3 nmols of phosphate per microgram of RNA. By varying the amount of RNA:Lipid the N/P ratio can be modified.
  • RNA was complexed to CNE17 at a nitrogen/phosphate ratios (N/P) of 10:1. Using these values the RNA was diluted to the appropriate concentration in RNase free water and added directly into an equal volume of emulsion while vortexing lightly. The solution was allowed to sit at room temperature for approximately 2 hours. Once complexed the resulting solution was diluted to the required concentration prior to administration.
  • Electroporation was a very effective method for the delivery of pDNA vaccines and this technique was used to deliver self-replicating RNA.
  • Mice were anesthetized under isofluorane, both hind legs were closely shaven to expose the area on the limb to be treated.
  • a dose of 30 ul of vaccine was injected to the calf muscle of the hind limb using a 1 ⁇ 2 cc insulin syringe.
  • the muscle was electroporated using the Elgen® DNA Delivery System (Inovio, San Diego). The instrument parameters are as follows: 60V, 2 pulses each at 60 ms. Another dose was similarly delivered to the second limb, followed by electroporation.
  • VRP Viral Replicon Particles
  • VRPs viral replicon particles
  • VCR alphavirus chimeric replicons
  • VEEV Venezuelan equine encephalitis virus
  • PS Sindbis virus packaging signal
  • VRPs were packaged into VRPs by co-electroporating them into baby hamster kidney (BHK) cells along with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes (see FIG. 2 of Perri et al).
  • BHK baby hamster kidney
  • the VRPs were then harvested and titrated by standard methods and inoculated into animals in culture fluid or other isotonic buffers.
  • Perri S Greer C E, Thudium K, Doe B, Legg H, Liu H, Romero R E, Tang Z, Bin Q, Dubensky T W, Jr. et al (2003)
  • An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and Sindbis viruses is a potent gene-based vaccine delivery vector. J Virol 77: 10394-10403
  • the RSV F trimer is a recombinant protein comprising the ectodomain of RSV F with a deletion of the fusion peptide region preventing association with other trimers.
  • the resulting construct forms a homogeneous trimer, as observed by size exclusion chromatography, and has an expected phenotype consistent with a postfusion F conformation as observed by electron microscopy.
  • the protein was expressed in insect cells and purified by virtue of a HIS-tagged in fusion with the construct's C-terminus followed by size exclusion chromatography using conventional techniques. The resulting protein sample exhibits greater than 95% purity.
  • trimer protein was adsorbed on 2 mg/mL alum using 10 mM Histidine buffer, pH 6.3 and isotonicity adjusted with sodium chloride to 150 mM.
  • F-subunit protein was adsorbed on alum overnight with gentle stirring at 2-8° C.
  • the pH and osmolality of the final vaccine was targeted as 6.5-7.5 and 240-360 mOsm/kg.
  • the vaccine was characterized for protein adsorption by SDS-PAGE (Invitrogen Corporation, USA) and for endotoxin content by LAL assay (Charles River Laboratories, USA). The vaccine was mixed by gentle inversion prior to immunization.
  • mice Groups of 10 female BALB/c mice aged 8-10 weeks and weighing about 20 grams were immunized at day 0 and day 21 with bleeds taken at days 14, 35 and 49. All animals were injected in the quadriceps in the two hind legs each getting an equivalent volume (50 ⁇ l per site) for a total of 100 ⁇ l of vaccine to deliver 10 ⁇ g antigen dose. When measurement of T cell responses was required, spleens were harvested at day 35 or 49.
  • mice Female cotton rats ( Sigmodon hispidis ) were obtained from Harlan Laboratories. All experiments were approved and performed according to Novartis Animal Care and Use Committee. Groups of animals were immunized intramuscularly (i.m., 100 ⁇ l) with the indicated vaccines on days 0 and 21. Serum samples were collected 2 weeks after each immunization. Immunized or unvaccinated control animals were challenged intranasally (i.n.) with 1 ⁇ 10 5 PFU RSV 4 weeks after the final immunization. Blood collection and RSV challenge were performed under anesthesia with 3% isoflurane using a precision vaporizer.
  • Antigen-stimulated cultures contained 1 ⁇ 10 6 splenocytes, RSV F peptide 85-93 (1 ⁇ 10 ⁇ 6 M), RSV F peptide 249-258 (1 ⁇ 10 ⁇ 6 M), RSV F peptide 51-66 (1 ⁇ 10 ⁇ 6 M), anti-CD28 mAb (1 mcg/mL), and Brefeldin A (1:1000). Unstimulated cultures did not contain RSV F peptides, and were otherwise identical to the stimulated cultures.
  • % cytokine-positive cells For each subset in a given sample the % cytokine-positive cells was determined. The % RSV F antigen-specific T cells was calculated as the difference between the % cytokine-positive cells in the antigen-stimulated cultures and the % cytokine-positive cells in the unstimulated cultures. The 95% confidence limits for the % antigen-specific cells were determined using standard methods ( Statistical Methods, 7 th Edition , G. W. Snedecor and W. G. Cochran).
  • the cultures for the secreted cytokines assay were similar to those for the intracellular cytokines immunofluorescence assay except that Brefeldin A was omitted. Culture supernatants were collected after overnight culture at 37° C., and were analyzed for multiple cytokines using mouse Th1/Th2 cytokine kits from Meso Scale Discovery. The amount of each cytokine per culture was determined from standard curves produced using purified, recombinant cytokines supplied by the manufacturer.
  • ELISA enzyme-linked immunosorbent assay
  • Serum samples were tested for the presence of neutralizing antibodies by a plaque reduction neutralization test (PRNT).
  • PRNT plaque reduction neutralization test
  • Two-fold serial dilutions of HI-serum (in PBS with 5% HI-FBS) were added to an equal volume of RSV Long previously titered to give approximately 115 PFU/25 ⁇ l.
  • Serum/virus mixtures were incubated for 2 hours at 37° C. and 5% CO2, to allow virus neutralization to occur, and then 25 ⁇ l of this mixture (containing approximately 115 PFU) was inoculated on duplicate wells of HEp-2 cells in 96 well plates. After 2 hours at 37° C.
  • the cells were overlayed with 0.75% Methyl Cellulose/EMEM 5% HI-FBS and incubated for 42 hours.
  • the number of infectious virus particles was determined by detection of syncytia formation by immunostaining followed by automated counting.
  • the neutralization titer is defined as the reciprocal of the serum dilution producing at least a 60% reduction in number of synctia per well, relative to controls (no serum).
  • Viral load in the lung was determined by plaque assay. Specifically, lungs were harvested 5 days post RSV infection and one right lobe was placed into 2.5 ml Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) with 25% sucrose and disrupted with a tissue homogenizer. Cell-free supernatants from these samples were stored at ⁇ 80° C. To assay for infectious virus, dilutions of clarified lung homogenate (in PBS with 5% heat-inactivated fetal bovine serum, HI-FBS) were inoculated on confluent HEp-2 cell monolayers in a volume of 200 ⁇ l/well of a 12-well plate.
  • DMEM Dulbecco's Modified Eagle Medium
  • Minimal (+) change contained one or a few small foci; mild (++) change was composed of small- to medium-size foci; moderate (+++) change contained frequent and/or moderately-sized foci; and marked (++++) change showed extensive to confluent foci affecting most/all of the tissue.
  • the A317 replicon which expresses the surface fusion glycoprotein of RSV (RSV-F) was used for this experiment.
  • Cotton rats Sigmodon hispidus ), 8 animals per group, were given bilateral intramuscular vaccinations (50 ⁇ L per leg) on days 0 and 21 with naked self-replicating RNA (A317, 1 ⁇ g or 10 ⁇ g), self-replicating RNA formulated in LNP [RV01(01), A317, 0.1 ⁇ g or 1 ⁇ g), VRPs (5 ⁇ 10 6 IU) expressing RSV-F, F-trimer/alum subunit (10 ⁇ g), or formalin inactivated RSV vaccine (5200 FI-pfu).
  • F-specific serum IgG titers of cotton rats ( Sigmodon hispidus ), 8 animals per group, after intramuscular vaccinations on days 0 and 21. Serum was collected for antibody analysis on days 14 (2wp1) and 35 (2wp2), all animals were challenged with 1 ⁇ 10 5 pfu RSV intranasally on day 49. Lungs were collected on day 54 (5dpc) for determination of viral load and lung pathology. Data are represented as geometric mean titers of 8 individual cotton rats per group. If an individual animal had a titer of ⁇ 25 (limit of detection) it was assigned a titer of 5.
  • RSV serum neutralization titers of cotton rats ( Sigmodon hispidus ), 8 animals per group, after intramuscular vaccinations on days 0 and 21. Serum was collected for analysis on days 14 (2wp1) and 35 (2wp2). Data are represented as 60% plaque reduction neutralization titers. Geometric mean titer of 2 pools of 4 cotton rats per group. If an individual animal had a titer of ⁇ 25 (limit of detection) it was assigned a titer of 5.
  • Lung viral titers 5 days post RSV challenge pfu/g lung vaccine dose 5dpc A317 10 ⁇ g 397 A317 1 ⁇ g 659 CNE17 1 ⁇ g 414 CNE17 0.1 ⁇ g 572 RV01(01) 1 ⁇ g 445 RV01(01) 0.1 ⁇ g 716 VRP 5 ⁇ 10 6 IU 359 F-trimer/alum 10 ⁇ g 190 FI-RSV 5200 FI-pfu 5248 none (challenged) 728618 Table 9: Lung viral titers 5 days post RSV challenge of cotton rats ( Sigmodon hispidus ), 8 animals per group, after intramuscular vaccinations on days 0 and 21.
  • Lung alveolitis scores 5 days post RSV challenge # of cotton rats with indicated alveolitis score vaccine dose 0 1 2 3 4 A317l 10 ⁇ g 8 A317l 1 ⁇ g 8 CNE17 1 ⁇ g 8 CNE17 0.1 ⁇ g 7 1 RV01(01) 1 ⁇ g 6 2 RV01(01) 0.1 ⁇ g 8 VRP 5 ⁇ 10 6 IU 3 4 1 F-trimer/alum 10 ⁇ g 7 1 FI-RSV 5200 FI-pfu 1 4 3 none (challenged) 5 3 Table 10.
  • All replicon RNA vaccines provided protection from a nasal RSV challenge, reducing the lung viral load great than 3 order of magnitude when measured 5 days later.
  • the magnitude and protective capacity of the immune response generated by 1 ⁇ g replicon RNA formulated with Liposomes was within 2-fold the response elicited by 5 ⁇ 10 6 VRPs.
  • the alum adjuvanted trimer subunit elicited the highest total anti-F IgG ELISA titers, elicited the highest neutralization titers, and elicited the greatest degree of protection from RSV titers in the lung on challenge of any of the vaccine preparations tested in this study.
  • the A317 replicon that expresses the surface fusion glycoprotein of RSV was used for this experiment.
  • BALB/c mice, 10 animals per group were given bilateral intramuscular vaccinations (50 ⁇ L per leg) on days 0 and 21 with VRP's expressing RSV-F (1 ⁇ 10 6 IU), naked self-replicating RNA (A317, 1 ⁇ g), self-replicating RNA delivered using electroporation (A317, 10 ⁇ g), self-replicating RNA formulated in liposomes [RV01(01), A317, 0.1 ⁇ g or 1 ⁇ g) and self-replicating RNA formulated with CNE17 (A317, 0.1 ⁇ g or 1 ⁇ g). Serum was collected for antibody analysis on days 14 (2wp1), 35 (2wp2) and 49 (4wp2). Spleens were harvested from 5 mice per group at day 49 (4wp2) for T cell analysis.
  • F-specific serum IgG titers of mice 10 animals per group, 14 days after intramuscular vaccination. Data are represented as titers for individual mice and the geometric mean titers of 10 individual mice per group. If an individual animal had a titer of ⁇ 25 (limit of detection) it was assigned a titer of 5.
  • F-specific serum IgG titers of mice 10 animals per group, after intramuscular vaccinations on days 0 and 21. Serum was collected for antibody analysis on day 35 (2wp2). Data are represented as titers for individual mice and the geometric mean titers of 10 individual mice per group. If an individual animal had a titer of ⁇ 25 (limit of detection) it was assigned a titer of 5.
  • F-specific serum IgG titers of mice 10 animals per group, after intramuscular vaccinations on days 0 and 21. Serum was collected for antibody analysis on days 49 (4wp2). Data are represented as titers for individual mice and the geometric mean titers of 10 individual mice per group. If an individual animal had a titer of ⁇ 25 (limit of detection) it was assigned a titer of 5.
  • Serum was collected for analysis on day 35 (2wp2). Data are represented as 60% plaque reduction neutralization titers of individual mice and the geometric mean titer of 10 individual mice per group. If an individual animal had a titer of ⁇ 40 (limit of detection) it was assigned a titer of 20. NA not assayed.
  • Serum was collected for analysis on day 49 (4wp2). Data are represented as 60% plaque reduction neutralization titers of individual mice and the geometric mean titer of 10 individual mice per group. If an individual animal had a titer of ⁇ 40 (limit of detection) it was assigned a titer of 20. NA not assayed.
  • Liposome formulation significantly enhanced immunogenicity, as determined by increased F-specific IgG titers (8-30-fold increase), neutralization titers, and CD4 and CD8 T cell responses, relative to the naked RNA control.
  • F-specific IgG titers and neutralization titers for RV01(01) at both the 0.1 and 1.0 ⁇ g doses were equivalent to the VRP (1 ⁇ 10 6 IU).
  • T cell responses for the LNP formulation were equivalent at the higher dose to the VRP (1 ⁇ 10 6 IU).
  • Formulation of the self-replicating RNA with CNE17 significantly enhanced immunogenicity, as determined by increased F-specific IgG titers (2-5-fold increase), neutralization titers, and CD4 and CD8 T cell responses, relative to the naked RNA control. Electroporation of RNA enhanced immunogenicity relative to the naked RNA control, but was significantly lower than Liposome delivery.
  • the A317 replicon that expresses the surface fusion glycoprotein of RSV was used for this experiment.
  • RV01(01) liposome formulation had a Z average particle diameter of 158 nm with a polydispersity index of 0.14, the encapsulation efficiency was 96%.
  • F-specific serum IgG titers on day 14 and 35 are shown in tables 18 and 19 and T cell responses at day 49 are shown in tables 20 and 21.
  • F-specific serum IgG titers of mice 8 animals per group, after intramuscular vaccinations on days 0 and 21. Serum was collected for antibody analysis on days 14 (2wp1) and 35 (2wp2). Data are represented as individual animals and the geometric mean titers (GMT) of 8 individual cotton rats per group. If an individual animal had a titer of ⁇ 25 (limit of detection) it was assigned a titer of 5.
  • F-specific splenic CD8 + T cell frequencies on day 49 (Expt. 10-1018, 4wp2). Shown are net (antigen-specific) cytokine-positive frequency (%) ⁇ 95% confidence half-interval. Net frequencies shown in bold indicate stimulated responses that were statistically significantly > 0.
  • Liposome formulation significantly enhanced immunogenicity, as determined by increased F-specific IgG titers and T cell frequencies, relative to the naked RNA controls.
  • the F-specific IgG titers and CD8 T cell frequencies for RV01(01) at the 10 ⁇ g RNA dose were enhanced relative to the VRP group (1 ⁇ 10 6 IU).
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