US20120058146A1 - Immunogens from uropathogenic escherichia coli - Google Patents

Immunogens from uropathogenic escherichia coli Download PDF

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US20120058146A1
US20120058146A1 US13/293,005 US201113293005A US2012058146A1 US 20120058146 A1 US20120058146 A1 US 20120058146A1 US 201113293005 A US201113293005 A US 201113293005A US 2012058146 A1 US2012058146 A1 US 2012058146A1
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seq
amino acid
acid sequence
polypeptides
sequence
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Danilo Gomes Moriel
Francesco Berlanda Scorza
Mariagrazia Pizza
Laura Serino
Maria Rita Fontana
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GSK Vaccines SRL
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Novartis Vaccines and Diagnostics SRL
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is in the field of Escherichia coli biology, and in particular relates to immunogens for use in immunising against extraintestinal pathogenic E. coli (ExPEC) strains.
  • E. coli can also be a deadly pathogen.
  • E. coli strains have traditionally been classified as either commensal or pathogenic, and pathogenic strains are then sub-classified as intestinal or extraintestinal strains. More recent taxonomic techniques such as multilocus enzyme electrophoresis (MLEE) classify E. coli into five phylogenetic groups (A, B1, B2, D & E), and these groupings do not match the traditional ones. For instance, MLEE group B1 includes both commensal and pathogenic strains, and group D includes both intestinal and extraintestinal strains.
  • MLEE group B1 includes both commensal and pathogenic strains
  • group D includes both intestinal and extraintestinal strains.
  • E. coli The extraintestinal pathogenic strains (or ‘ExPEC’ strains [1]) of E. coli fall into MLEE groups B2 and D, and include both uropathogenic (UPEC) strains and meningitis/sepsis-associated (MNEC) strains.
  • UPEC strains cause urinary tract infections (UTIs), and are the most common form of cystitis. They also cause pyelonephritis (and its complications such as sepsis) and catheter-associated infections.
  • UTIs urinary tract infections
  • MNEC strains cause neonatal meningitis (0.1 cases per 1000 live births) with case fatality rates ranging from 25 to 40%, and are also responsible for around 1 ⁇ 6 of sepsis cases.
  • ExPEC vaccines including a need to move away from crude cell lysates and towards better-defined molecules, and a need to identify further antigens that are suitable for inclusion in vaccines, particularly antigens that are prevalent among clinical ExPEC strains without also being found in commensal strains.
  • Reference 10 used the genome sequence of UPEC (O6:K2:H1) strain CFT073 [11,12] to identify sequences not present in non-pathogenic E. coli strains.
  • Reference 13 discloses a comparison of the genome sequence of E. coli human pyelonephritis isolate 536 (O6:K15:H31), an UPEC, with sequence data for strains CFT073 (UPEC), EDL933 (enterohemorrhagic) and MG1655 (non-pathogenic laboratory strain). Genome sequences of pathogenic strains are available in the databases under accession numbers AE005174, BA000007 and NC-004431. A sequence from a non-pathogenic strain is available under accession number U00096.
  • the inventors have identified various genes that can be included in immunogenic compositions specific for pathogenic E. coli strains.
  • the genes are from uropathogenic strains (UPEC) but are absent from non-pathogenic strains, and their encoded proteins have cellular locations which render them accessible to the immune system.
  • UPEC uropathogenic strains
  • the invention relates to a polypeptide comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
  • the invention relates to a polypeptide comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a).
  • polypeptides of this aspect of the invention comprise a fragment which comprises at least one B-cell epitope of (a).
  • polypeptides of the invention can be used in medicine and in the manufacture of a medicament for raising an immune response in a patient.
  • the present invention also relates to a pharmaceutical composition comprising a polypeptide of the invention in admixture with a pharmaceutically acceptable carrier.
  • the invention further relates to a pharmaceutical composition comprising two or more polypeptides of the invention in admixture with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the invention further comprise a vaccine adjuvant.
  • the present invention further relates to immunogenic compositions comprising one or more outer membrane vesicles (OMVs) expressing or overexpressing one or more polypeptides comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a).
  • the immunogenic composition of this aspect of the invention comprises one or more polypeptides comprising a fragment which comprises at least one B-cell epitope of (a).
  • the present invention also relates to methods for raising an immune response in a patient, comprising the step of administering to the patient a pharmaceutical composition or immunogenic composition of the invention.
  • the immune response is protective against ExPEC infection.
  • FIG. 1 shows the % survival of mice after challenge with CFT073 following immunization with heat-inactivated CFT073.
  • the inventors have identified various genes that can be included in immunogenic compositions specific for pathogenic E. coli strains.
  • the genes are from UPEC strains but are absent from non-pathogenic strains, and their encoded proteins have cellular locations which render them accessible to the immune system.
  • the invention provides polypeptides comprising the amino acid sequences disclosed in the examples. These amino acid sequences are given in the sequence listing as SEQ ID NOs 1 to 596 and 597 to 599. Preferred subsets of SEQ ID NOs 1 to 596 are given in Tables 2, 3 and 5.
  • polypeptide may, compared to the sequences of the examples, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid replacements i.e. replacements of one amino acid with another which has a related side chain.
  • conservative amino acid replacements i.e. replacements of one amino acid with another which has a related side chain.
  • Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e.
  • the polypeptides may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to a reference sequence.
  • the polypeptides may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to a reference sequence.
  • Preferred polypeptides include polypeptides that are lipidated, that are located in the outer membrane, that are located in the inner membrane, or that are located in the periplasm. Particularly preferred polypeptides are those that fall into more than one of these categories e.g. lipidated polypeptides that are located in the outer membrane. Lipoproteins may have a N-terminal cysteine to which lipid is covalently attached, following post-translational processing of the signal peptide.
  • Polypeptides that may be lipidated include SEQ ID NOS: 1, 2, 7, 12, 13, 14, 15, 16, 17, 18, 22, 26, 28, 29, 33, 34, 38, 40, 45, 50, 51, 59, 67, 68, 69, 71, 77, 80, 82, 83, 84, 92, 98, 103, 104, 105, 120, 121, 124, 125, 126, 127, 130, 131, 133, 134, 138, 142, 147, 159, 160, 176, 184, 185, 186, 187, 192, 206, 210, 215, 222, 223, 225, 226, 228, 233, 234, 246, 251, 252, 268, 272, 273, 275, 284, 287, 293, 295, 297, 298, 299, 300, 302, 303, 304, 305, 310, 311, 312, 314, 315, 320, 326, 327, 330, 331, 336, 340, 359
  • Preferred polypeptides are those listed in Tables 2, 3 and 5, particularly those listed in Table 3.
  • the invention further provides polypeptides comprising fragments of the amino acid sequences disclosed in the examples.
  • the fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more).
  • the fragment may comprise at least one T-cell or, preferably, a B-cell epitope of the sequence.
  • T- and B-cell epitopes can be identified empirically (e.g. using PEPSCAN [14,15] or similar methods), or they can be predicted (e.g.
  • fragments are those that begin with an amino acid encoded by a potential start codon (ATG, GTG, TTG). Fragments starting at the methionine encoded by a start codon downstream of the indicated start codon are polypeptides of the invention.
  • Polypeptides of the invention can be prepared in many ways e.g. by chemical synthesis (in whole or in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself (e.g. after bacterial culture, or direct from patients), etc.
  • a preferred method for production of peptides ⁇ 40 amino acids long involves in vitro chemical synthesis [27,28].
  • Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [29] chemistry.
  • Enzymatic synthesis [30] may also be used in part or in full.
  • biological synthesis may be used e.g.
  • polypeptides may be produced by translation. This may be carried out in vitro or in vivo. Biological methods are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e.g. of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) [31]. Where D-amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides of the invention may have covalent modifications at the C-terminus and/or N-terminus.
  • Polypeptides of the invention can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, etc.).
  • Polypeptides of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other polypeptides (e.g. free from naturally-occurring polypeptides), particularly from other ExPEC or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i.e. less than about 50%, and more preferably less than about 10% (e.g. 5% or less) of a composition is made up of other expressed polypeptides.
  • Polypeptides of the invention are preferably ExPEC polypeptides.
  • Polypeptides of the invention may be attached to a solid support.
  • Polypeptides of the invention may comprise a detectable label (e.g. a radioactive or fluorescent label, or a biotin label).
  • polypeptide refers to amino acid polymers of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • Polypeptides can occur as single chains or associated chains.
  • Polypeptides of the invention can be naturally or non-naturally glycosylated (i.e. the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring polypeptide).
  • Polypeptides of the invention may be at least 40 amino acids long (e.g. at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500 or more). Polypeptides of the invention may be shorter than 500 amino acids (e.g. no longer than 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400 or 450 amino acids).
  • the invention provides polypeptides comprising a sequence —X—Y— or —Y—X—, wherein: —X— is an amino acid sequence as defined above and —Y— is not a sequence as defined above i.e. the invention provides fusion proteins.
  • —X— is an amino acid sequence as defined above
  • —Y— is not a sequence as defined above i.e. the invention provides fusion proteins.
  • the invention provides a process for producing polypeptides of the invention, comprising the step of culturing a host cell of to the invention under conditions which induce polypeptide expression.
  • the invention provides a process for producing a polypeptide of the invention, wherein the polypeptide is synthesised in part or in whole using chemical means.
  • the invention provides a composition comprising two or more polypeptides of the invention.
  • the invention also provides a hybrid polypeptide represented by the formula NH 2 -A-[-X-L-] n -B—COOH, wherein X is a polypeptide of the invention as defined above, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1.
  • the value of n is between 2 and x, and the value of x is typically 3, 4, 5, 6, 7, 8, 9 or 10.
  • —X— may be the same or different.
  • linker amino acid sequence -L- may be present or absent.
  • the hybrid may be NH 2 —X 1 -L 1 -X 2 -L 2 -COOH, NH 2 —X 1 —X 2 —COOH, NH 2 —X 1 -L 1 -X 2 —COOH, NH 2 —X 1 —X 2 -L 2 -COOH, etc.
  • Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
  • Other suitable linker amino acid sequences will be apparent to those skilled in the art.
  • -A- and —B— are optional sequences which will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
  • Other suitable N-terminal and C-terminal amino acid sequences will be apparent to those skilled in the art.
  • polypeptides of the invention can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the polypeptide and patient serum indicates that the patient has previously mounted an immune response to the protein in question i.e. the protein is an immunogen. This method can also be used to identify immunodominant proteins.
  • the invention provides antibodies that bind to polypeptides of the invention. These may be polyclonal or monoclonal and may be produced by any suitable means (e.g. by recombinant expression). To increase compatibility with the human immune system, the antibodies may be chimeric or humanised [e.g. refs. 32 & 33], or fully human antibodies may be used. The antibodies may include a detectable label (e.g. for diagnostic assays). Antibodies of the invention may be attached to a solid support. Antibodies of the invention are preferably neutralising antibodies.
  • Monoclonal antibodies are particularly useful in identification and purification of the individual polypeptides against which they are directed.
  • Monoclonal antibodies of the invention may also be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA), etc.
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • the monoclonal antibodies produced by the above method may also be used for the molecular identification and characterization (epitope mapping) of polypeptides of the invention.
  • Antibodies of the invention are preferably specific to ExPEC strains of E. coli, i.e. they bind preferentially to ExPEC E. coli relative to other bacteria (e.g. relative to non-ExPEC E. coli and relative to non- E.coli bacteria). More preferably, the antibodies are specific to UPEC strains i.e. they bind preferentially to UPEC bacteria relative to other bacteria, including other ExPEC E. coli.
  • Antibodies of the invention are preferably provided in purified or substantially purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides e.g. where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
  • Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgM i.e. an ⁇ , ⁇ or ⁇ heavy chain), but will generally be IgG. Within the IgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass. Antibodies of the invention may have a ⁇ or a ⁇ light chain.
  • IgA IgG
  • IgM i.e. an ⁇ , ⁇ or ⁇ heavy chain
  • Antibodies of the invention can take various forms, including whole antibodies, antibody fragments such as F(ab′) 2 and F(ab) fragments, Fv fragments (non-covalent heterodimers), single-chain antibodies such as single chain Fv molecules (scFv), minibodies, oligobodies, etc.
  • antibody does not imply any particular origin, and includes antibodies obtained through non-conventional processes, such as phage display.
  • the invention provides a process for detecting polypeptides of the invention, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
  • the invention provides a process for detecting antibodies of the invention, comprising the steps of (a) contacting a polypeptide of the invention with a biological sample (e.g. a blood or serum sample) under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
  • a biological sample e.g. a blood or serum sample
  • Preferred antibodies bind to a polypeptide of the invention with substantially greater affinity than antibodies known in the art.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold, 10 6 -fold etc. stronger than antibodies known in the art.
  • the invention also provides nucleic acid comprising a nucleotide sequence encoding the polypeptides of the invention.
  • the invention also provides nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences. Identity between sequences is preferably determined by the Smith-Waterman homology search algorithm as described above.
  • the invention also provides nucleic acid which can hybridize to these nucleic acids.
  • Hybridization reactions can be performed under conditions of different “stringency”. Conditions that increase stringency of a hybridization reaction of widely known and published in the art [e.g. page 7.52 of reference 34]. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C., 55° C.
  • Hybridization techniques and their optimization are well known in the art [e.g. see refs 34-37, etc.].
  • nucleic acid of the invention hybridizes to a target under low stringency conditions; in other embodiments it hybridizes under intermediate stringency conditions; in preferred embodiments, it hybridizes under high stringency conditions.
  • An exemplary set of low stringency hybridization conditions is 50° C. and 10 ⁇ SSC.
  • An exemplary set of intermediate stringency hybridization conditions is 55° C. and 1 ⁇ SSC.
  • An exemplary set of high stringency hybridization conditions is 68° C. and 0.1 ⁇ SSC.
  • Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the sequences and, depending on the particular sequence, n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferred fragments are those that are common to a nucleic acid sequence of the invention and to a nucleic acid sequence identified in any of references 5, 6, 8, 10 and 11.
  • the invention provides nucleic acid of formula 5′—X—Y—Z-3′, wherein: —X— is a nucleotide sequence consisting of x nucleotides; —Z— is a nucleotide sequence consisting of z nucleotides; —Y— is a nucleotide sequence consisting of either (a) a fragment of a nucleic acid sequence encoding one of SEQ ID NOS: 1 to 596, (b) a fragment of a nucleic acid sequence encoding one of SEQ ID NOS: 597 to 599, or (c) the complement of (a) or (b); and said nucleic acid 5′—X—Y—Z-3′ is neither (i) a fragment of either a nucleic acid sequence encoding one of SEQ ID NOS: 1 to 596 or encoding one of SEQ ID NOS: 597 to 599 nor (ii) the complement of (i).
  • the invention includes nucleic acid comprising sequences complementary to these sequences (e.g. for antisense or probing, or for use as primers).
  • Nucleic acids of the invention can be used in hybridisation reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or ‘gene chips’) and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) and other nucleic acid techniques.
  • hybridisation reactions e.g. Northern or Southern blots, or in nucleic acid microarrays or ‘gene chips’
  • amplification reactions e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.
  • Nucleic acid according to the invention can take various forms (e.g. single-stranded, double-stranded, vectors, primers, probes, labelled etc.). Nucleic acids of the invention may be circular or branched, but will generally be linear. Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary single-stranded forms which make up the double-stranded form. Primers and probes are generally single-stranded, as are antisense nucleic acids.
  • Nucleic acids of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other nucleic acids (e.g. free from naturally-occurring nucleic acids), particularly from other ExPEC or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90% pure. Nucleic acids of the invention are preferably ExPEC nucleic acids.
  • Nucleic acids of the invention may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.
  • nucleases e.g. restriction enzymes
  • ligases or polymerases e.g. using ligases or polymerases
  • Nucleic acid of the invention may be attached to a solid support (e.g. a bead, plate, filter, film, slide, microarray support, resin, etc.). Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to be used in detection techniques e.g. where the nucleic acid is a primer or as a probe.
  • a solid support e.g. a bead, plate, filter, film, slide, microarray support, resin, etc.
  • Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to be used in detection techniques e.g. where the nucleic acid is a primer or as a probe.
  • nucleic acid includes in general means a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. It includes DNA, RNA, DNA/RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases.
  • PNAs peptide nucleic acids
  • the invention includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc. Where nucleic acid of the invention takes the form of RNA, it may or may not have a 5′ cap.
  • Nucleic acids of the invention comprise sequences, but they may also comprise non-ExPEC sequences (e.g. in nucleic acids of formula 5′-X—Y—Z-3′, as defined above). This is particularly useful for primers, which may thus comprise a first sequence complementary to a nucleic acid target and a second sequence which is not complementary to the nucleic acid target. Any such non-complementary sequences in the primer are preferably 5′ to the complementary sequences. Typical non-complementary sequences comprise restriction sites or promoter sequences.
  • Nucleic acids of the invention can be prepared in many ways e.g. by chemical synthesis (at least in part), by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.
  • nucleases e.g. restriction enzymes
  • ligases or polymerases e.g. using ligases or polymerases
  • Nucleic acids of the invention may be part of a vector i.e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types.
  • Vectors may be, for example, “cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, “expression vectors” which are designed for expression of a nucleotide sequence in a host cell, “viral vectors” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors”, which comprise the attributes of more than one type of vector.
  • Preferred vectors are plasmids.
  • a “host cell” includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention.
  • nucleic acid is DNA
  • U in a RNA sequence
  • T in the DNA
  • RNA RNA
  • T in a DNA sequence
  • complement or “complementary” when used in relation to nucleic acids refers to Watson-Crick base pairing.
  • the complement of C is G
  • the complement of G is C
  • the complement of A is T (or U)
  • the complement of T is A.
  • bases such as I (the purine inosine) e.g. to complement pyrimidines (C or T).
  • the terms also imply a direction—the complement of 5′-ACAGT-3′ is 5′-ACTGT-3′ rather than 5′-TGTCA-3′.
  • Nucleic acids of the invention can be used, for example: to produce polypeptides; as hybridization probes for the detection of nucleic acid in biological samples; to generate additional copies of the nucleic acids; to generate ribozymes or antisense oligonucleotides; as single-stranded DNA primers or probes; or as triple-strand forming oligonucleotides.
  • the invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesised in part or in whole using chemical means.
  • the invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.
  • nucleotide sequences of the invention e.g. cloning or expression vectors
  • the invention also provides a kit comprising primers (e.g. PCR primers) for amplifying a template sequence contained within an ExPEC nucleic acid sequence, the kit comprising a first primer and a second primer, wherein the first primer is substantially complementary to said template sequence and the second primer is substantially complementary to a complement of said template sequence, wherein the parts of said primers which have substantial complementarity define the termini of the template sequence to be amplified.
  • the first primer and/or the second primer may include a detectable label (e.g. a fluorescent label).
  • the invention also provides a kit comprising first and second single-stranded oligonucleotides which allow amplification of a ExPEC template nucleic acid sequence contained in a single- or double-stranded nucleic acid (or mixture thereof), wherein: (a) the first oligonucleotide comprises a primer sequence which is substantially complementary to said template nucleic acid sequence; (b) the second oligonucleotide comprises a primer sequence which is substantially complementary to the complement of said template nucleic acid sequence; (c) the first oligonucleotide and/or the second oligonucleotide comprise(s) sequence which is not complementary to said template nucleic acid; and (d) said primer sequences define the termini of the template sequence to be amplified.
  • the non-complementary sequence(s) of feature (c) are preferably upstream of (i.e. 5′ to) the primer sequences.
  • One or both of these (c) sequences may comprise a restriction site [e.g. ref. 38] or a promoter sequence [e.g. 39].
  • the first oligonucleotide and/or the second oligonucleotide may include a detectable label (e.g. a fluorescent label).
  • the invention provides a process for detecting nucleic acid of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.
  • the invention provides a process for detecting in a biological sample (e.g. blood), comprising the step of contacting nucleic acid according to the invention with the biological sample under hybridising conditions.
  • the process may involve nucleic acid amplification (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) or hybridisation (e.g. microarrays, blots, hybridisation with a probe in solution etc.).
  • PCR detection of ExPEC in clinical samples has been reported [e.g. see ref. 40].
  • Clinical assays based on nucleic acid are described in general in ref. 41.
  • the invention provides a process for preparing a fragment of a target sequence, wherein the fragment is prepared by extension of a nucleic acid primer.
  • the target sequence and/or the primer are nucleic acids of the invention.
  • the primer extension reaction may involve nucleic acid amplification (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.).
  • Nucleic acid amplification according to the invention may be quantitative and/or real-time.
  • nucleic acids are preferably at least 7 nucleotides in length (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300 nucleotides or longer).
  • nucleic acids are preferably at most 500 nucleotides in length (e.g. 450, 400, 350, 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 nucleotides or shorter).
  • Primers and probes of the invention, and other nucleic acids used for hybridization are preferably between 10 and 30 nucleotides in length (e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
  • Reference 42 describes the preparation of vesicles from a uropathogenic (UPEC) strain by the knockout of mltA (a murein lytic transglycosylase) or one or more of the components of the E.coli Tol-Pal complex [43], such as tolA, tolQ, tolB, pal and/or tolR.
  • mltA murein lytic transglycosylase
  • Tol-Pal complex such as tolA, tolQ, tolB, pal and/or tolR.
  • These vesicles can be improved by making one or more further genetic changes to the chromosome of the bacterium or through insertion of episomal elements (e.g. expression vectors) in order to increase the amount of and/or immunoaccessibility of protective antigens on the surface the vesicles.
  • One way of obtaining such improvements is to up-regulate the expression of the polypeptides of the invention.
  • Many different genetic strategies for increasing the expression of a target protein are well-known in the art and can be distinguished into two broad categories: one relying on modifications of the chromosome (e.g. replacement of the wild-type promoter with a stronger promoter, inactivation of natural repressor genes, etc.) to increase expression of an endogenous gene, and the other based on recombinant expression by episomal elements (e.g. high-copy number plasmids, vectors harboring an engineered target gene, etc.) or integration of a exogenous gene in the chromosome. Practical examples for each of these approaches can be found in references 44 to 50.
  • Another way of increasing vesicle immunogenicity and selectivity is to down-regulate the expression of immunodominant non-protective antigens or to down-regulate proteins that are homologous to proteins found in commensal strains. Further improvements can be achieved by detoxification of the Lipid A moiety of LPS. Similar changes have been previously described to produce improved vesicles from other Gram-negative pathogens (see for example references 51 & 52).
  • the invention provides a pathogenic Escherichia coli bacterium (particularly a UPEC) having a knockout of mltA and/or of a component of its Tol-Pal complex, and one or more of: (i) a chromosomal gene encoding a polypeptide of the invention under the control of a promoter that provides higher expression levels of the polypeptide than the promoter that is naturally associated with the gene encoding the polypeptide; (ii) an autonomously-replicating extrachromosomal element encoding a polypeptide of the invention; and/or (iii) a genetic modification to reduce the toxicity of the Lipid A moiety of E.coli LPS relative to wild-type LPS.
  • the invention also provides vesicles obtainable by culturing such a bacterium, such as the vesicles that, during culture of the bacterium, are released into the culture medium.
  • compositions comprising: (a) polypeptide, antibody, vesicles and/or nucleic acid of the invention; and (b) a pharmaceutically acceptable carrier.
  • compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.
  • the invention provides immunogenic compositions comprising one or more outer membrane vesicles (OMVs) expressing or overexpressing one or more polypeptides of the invention.
  • OMVs outer membrane vesicles
  • the invention provides an immunogenic composition comprising one or more OMVs expressing or overexpressing one or more polypeptides comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a).
  • the immunogenic composition comprises a polypeptide comprising a fragment which comprises at least one B-cell epitope of an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599.
  • a ‘pharmaceutically acceptable carrier’ includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes).
  • Such carriers are well known to those of ordinary skill in the art.
  • the vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate-buffered physiologic saline is a typical carrier. A thorough discussion of pharmaceutically acceptable excipients is available in ref. 296.
  • compositions of the invention may include an antimicrobial, particularly if packaged in a multiple dose format.
  • compositions of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80.
  • Detergents are generally present at low levels e.g. ⁇ 0.01%.
  • compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity.
  • sodium salts e.g. sodium chloride
  • a concentration of 10 ⁇ 2mg/ml NaCl is typical.
  • compositions of the invention will generally include a buffer.
  • a phosphate buffer is typical.
  • compositions of the invention may comprise a sugar alcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml (e.g. 25 mg/ml), particularly if they are to be lyophilised or if they include material which has been reconstituted from lyophilised material.
  • a sugar alcohol e.g. mannitol
  • a disaccharide e.g. sucrose or trehalose
  • the pH of a composition for lyophilisation may be adjusted to around 6.1 prior to lyophilisation.
  • compositions will usually include a vaccine adjuvant.
  • the adjuvant may be selected from one or more of the group consisting of a TH1 adjuvant and TH2 adjuvant, further discussed below.
  • Adjuvants which may be used in compositions of the invention include, but are not limited to:
  • Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts.
  • the invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 53], or mixtures of different mineral compounds (e.g. a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption to the salt(s) being preferred.
  • Mineral containing compositions may also be formulated as a particle of metal salt [54].
  • a typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /Al molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ /ml.
  • Adsorption with a low dose of aluminium phosphate may be used e.g. between 50 and 100 ⁇ g Al 3+ per conjugate per dose.
  • an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution (e.g. by the use of a phosphate buffer).
  • Suspensions of aluminium 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 aluminium hydroxide and an aluminium phosphate.
  • there may be more aluminium 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.
  • Aluminum salts may be included in vaccines of the invention such that the dose of Al 3+ is between 0.2 and 1.0 mg per dose.
  • 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) [Chapter 10 of ref. 53; see also refs. 55-57, chapter 12 of ref. 58].
  • MF59 is used as the adjuvant in the FLUADTM influenza virus trivalent subunit vaccine.
  • the emulsion advantageously includes citrate ions e.g. 10 mM sodium citrate buffer.
  • Particularly preferred adjuvants for use in the compositions are submicron oil-in-water emulsions.
  • Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80 (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85 (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphophoryloxy)-ethylamine (MTP-PE).
  • MTP-PE N-acetylmuramyl-L-alany
  • An emulsion of squalene, a tocopherol, and Tween 80 can be used.
  • the emulsion may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably ⁇ 1 as this provides a more stable emulsion.
  • One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL- ⁇ -tocopherol and 5 ml squalene), then microfluidising the mixture.
  • the resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm.
  • Triton detergent e.g. Triton X-100
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 (“PluronicTM L121”) can be used.
  • the emulsion can be formulated in phosphate buffered saline, pH 7.4.
  • This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the “SAF-1” adjuvant [61] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the “AF” adjuvant [62] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Saponin formulations may also be used as adjuvants in the invention.
  • Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins isolated from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root).
  • Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
  • Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.
  • the saponin is QS21.
  • a method of production of QS21 is disclosed in ref. 63.
  • Saponin formulations may also comprise a sterol, such as cholesterol [64].
  • ISCOMs immunostimulating complexs
  • phospholipid such as phosphatidylethanolamine or phosphatidylcholine.
  • Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA and QHC.
  • ISCOMs are further described in refs. 64-66.
  • the ISCOMS may be devoid of additional detergent(s) [67].
  • Virosomes and virus-like particles can also be used as adjuvants in the invention.
  • These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome.
  • the viral proteins may be recombinantly produced or isolated from whole viruses.
  • viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q ⁇ -phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1).
  • VLPs are discussed further in refs. 70-75.
  • Virosomes are discussed further in, for example, ref. 76
  • Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • LPS enterobacterial lipopolysaccharide
  • Lipid A derivatives Lipid A derivatives
  • immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • a preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 77. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 ⁇ m membrane [77].
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [78,79].
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. O174 is described for example in refs. 80 & 81.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • the CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded.
  • References 82, 83 and 84 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. 85-90.
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [91].
  • the CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, 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. 92-94.
  • 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, refs. 91 & 95-97.
  • immunostimulatory oligonucleotides include a double-stranded RNA, or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention.
  • the protein is derived from E.coli ( E.coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”).
  • LT E.coli heat labile enterotoxin
  • CT cholera
  • PT pertussis
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 98 and as parenteral adjuvants in ref. 99.
  • the toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits.
  • the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192.
  • LT-K63 LT-K63
  • LT-R72 LT-G192.
  • ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 100-107.
  • Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 108.
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [110], etc.) [111], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, tumor necrosis factor and macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta [112].
  • interleukins e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [110], etc.
  • interferons e.g. interferon- ⁇
  • macrophage colony stimulating factor e.g. interferon- ⁇
  • MIP-1alpha macrophage inflammatory protein-1alpha
  • MIP-1beta MIP-1beta
  • Bioadhesives and mucoadhesives may also be used as adjuvants in the invention.
  • Suitable bioadhesives include esterified hyaluronic acid microspheres [113] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [114].
  • Microparticles may also be used as adjuvants in the invention.
  • Microparticles i.e. a particle of ⁇ 100 nm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200 nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500 nm to ⁇ 10 ⁇ m in diameter
  • materials that are biodegradable and non-toxic e.g. a poly( ⁇ -hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
  • a negatively-charged surface e.g. with SDS
  • a positively-charged surface e.g. with a cationic detergent, such as CTAB
  • liposome formulations suitable for use as adjuvants are described in refs. 115-117.
  • Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [118]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [119] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [120].
  • Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • Phosphazene adjuvants include poly[di(carboxylatophenoxy)phosphazene] (“PCPP”) as described, for example, in refs. 121 and 122.
  • PCPP poly[di(carboxylatophenoxy)phosphazene]
  • muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl- L -alanyl- D -isoglutamine (nor-MDP), and N-acetylmuramyl- L -alanyl- D -isoglutaminyl- L -alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
  • Imidazoquinoline adjuvants include Imiquimod (“R-837”) [123,124], Resiquimod (“R-848”) [125], and their analogs; and salts thereof (e.g. the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in references 126 to 130.
  • thiosemicarbazone compounds as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants in the invention include those described in ref. 131.
  • the thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ .
  • tryptanthrin compounds as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants in the invention include those described in ref. 132.
  • the tryptanthrin compounds are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ .
  • nucleoside analogs can be used as adjuvants, such as (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):
  • Adjuvants containing lipids linked to a phosphate-containing acyclic backbone include the TLR4 antagonist E5564 [136,137]:
  • SIPs Small Molecule Immunopotentiators
  • SMIPs include:
  • One adjuvant is an outer membrane protein proteosome preparation prepared from a first Gram-negative bacterium in combination with a liposaccharide preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteosome and liposaccharide preparations form a stable non-covalent adjuvant complex.
  • Such complexes include “IVX-908”, a complex comprised of Neisseria meningitidis outer membrane and lipopolysaccharides. They have been used as adjuvants for influenza vaccines [138].
  • Further useful adjuvant substances include:
  • the invention may also comprise combinations of one or more of the adjuvants identified above.
  • the following combinations may be used as adjuvant compositions in the invention: (1) a saponin and an oil-in-water emulsion [150]; (2) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [151]; (3) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.
  • RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM); (8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dMPL); and (9) one or more mineral salts (such as an aluminum salt)+an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif).
  • RAS RibiTM adjuvant system
  • Ribi Immunochem containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (C
  • compositions of the invention will preferably elicit both a cell mediated immune response as well as a humoral immune response in order to effectively address a uropathogenic infection.
  • This immune response will preferably induce long lasting (e.g. neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to UPEC-associated antigens.
  • 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. Upon interaction with a MHC class II molecule, the CD4 cells can secrete factors such as cytokines.
  • 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 particular 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.
  • An enhanced 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 EL-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.
  • An enhanced 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 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 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 hereof. Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use in the invention.
  • the invention includes a composition comprising a combination of a TH1 adjuvant and a TH2 adjuvant.
  • a composition comprising a combination of a TH1 adjuvant and a TH2 adjuvant.
  • such 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.
  • aluminium hydroxide or aluminium phosphate adjuvant is particularly preferred, and antigens are generally adsorbed to these salts.
  • Calcium phosphate is another preferred adjuvant.
  • compositions of the invention is preferably between 6 and 8, preferably about 7. Stable pH may be maintained by the use of a buffer. Where a composition comprises an aluminium hydroxide salt, it is preferred to use a histidine buffer [154].
  • the composition may be sterile and/or pyrogen-free. Compositions of the invention may be isotonic with respect to humans
  • compositions may be presented in vials, or they may be presented in ready-filled syringes.
  • the syringes may be supplied with or without needles.
  • a syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses.
  • injectable compositions will usually be liquid solutions or suspensions. Alternatively, they may be presented in solid form (e.g. freeze-dried) for solution or suspension in liquid vehicles prior to injection.
  • compositions of the invention may be packaged in unit dose form or in multiple dose form.
  • vials are preferred to pre-filled syringes.
  • Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5 ml.
  • kits may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
  • the invention provides for a kit comprising a first component and a second component, wherein: the first component comprises one or more polypeptide, antibody, vesicle and/or nucleic acid of the invention; and the second component comprises one or more of the following: instructions for administering a composition to a patient, a syringe or other delivery device, an adjuvant, and/or a pharmaceutically acceptable formulating solution.
  • the invention also provides a delivery device (e.g. a syringe) pre-filled with the immunogenic compositions of the invention.
  • a delivery device e.g. a syringe
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials, and a typical quantity of each meningococcal saccharide antigen per dose is between 1 ⁇ g and 10 mg per antigen.
  • the invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition of the invention.
  • the patient may either be at risk from the disease themselves or may be a pregnant woman (‘maternal immunisation’ [155]).
  • the invention provides nucleic acid, polypeptide, vesicle or antibody of the invention for use as medicaments (e.g. as immunogenic compositions or as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, polypeptide, vesicle or antibody of the invention in the manufacture of: (i) a medicament for treating or preventing disease and/or infection caused by an ExPEC bacterium; (ii) a diagnostic reagent for detecting the presence of or of antibodies raised against an ExPEC bacterium; and/or (iii) a reagent which can raise antibodies against an ExPEC bacterium.
  • Said ExPEC bacterium can be of any serotype or strain.
  • the ExPEC bacterium is a UPEC strain.
  • the invention is useful for the prevention and/or treatment of diseases such as bacteremia, meningitis, a urinary tract infection, pyelonephritis and/or cystitis.
  • diseases such as bacteremia, meningitis, a urinary tract infection, pyelonephritis and/or cystitis.
  • the invention is particularly useful for the treatment of urinary tract infections.
  • the patient is preferably a human.
  • the human is preferably an adult (e.g. aged between 20 and 55).
  • a vaccine intended for children or adolescents may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • Female patients are a preferred subset, with sexually-active females aged 20-55 being a particularly preferred patient group.
  • Another groups of patients is females aged 12-20, particularly for prophylactic use.
  • dogs which may be carriers of ExPEC [156,157].
  • One way of checking efficacy of therapeutic treatment involves monitoring infection after administration of the composition of the invention.
  • One way of checking efficacy of prophylactic treatment involves monitoring immune responses against an administered polypeptide after administration.
  • Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age, or animal models e.g. a mouse model) and then determining standard parameters including ELISA titres (GMT) of IgG. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made.
  • Various mouse models of UTI are available [e.g. refs. 158 & 159-160].
  • Administration of polypeptide antigens is a preferred method of treatment for inducing immunity.
  • Administration of antibodies of the invention is another preferred method of treatment. This method of passive immunisation is particularly useful for newborn children or for pregnant women. This method will typically use monoclonal antibodies, which will be humanised or fully human.
  • compositions of the invention will generally be administered directly to a patient.
  • Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal, transcutaneous, intranasal, sublingual, ocular, aural, pulmonary or other mucosal administration.
  • Intramuscular administration to the thigh or the upper arm is preferred.
  • Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
  • a typical intramuscular dose is 0.5 ml.
  • the invention may be used to elicit systemic and/or mucosal immunity.
  • the enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response.
  • the enhanced immune response includes an increase in the production of IgG1 and/or IgG2a and/or IgA.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose 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. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined.
  • a primary course of vaccination may include 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reinforce an immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose or doses after several months.
  • compositions may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised or a spray-freeze dried composition).
  • the composition may be prepared for topical administration e.g. as an ointment, cream or powder.
  • the composition be prepared for oral administration e.g. as a tablet or capsule, as a spray or as a syrup (optionally flavoured).
  • the composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as spray, drops, gel or powder [e.g. refs 161 & 162].
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient.
  • kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
  • compositions 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) other vaccines e.g. at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H.influenzae type b vaccine, a human papillomavirus vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a pneumococcal conjugate vaccine, a meningococcal conjugate vaccine, etc.
  • they may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional) an antibiotic, and in particular an antibiotic compound active against UPEC.
  • the invention also provides a composition comprising a polypeptide or the invention and one or more of the following further antigens:
  • composition may comprise one or more of these further antigens.
  • antigens of the invention are combined with one or more additional, non- E.coli antigens suitable for use in a vaccine designed to protect females against genitourinary and/or sexually transmitted diseases.
  • the antigens may be combined with an antigen derived from the group consisting of Streptococcus agalactiae, Chlamydia trachomatis, Neisseria gonorrhoeae, papillomavirus and herpes simplex virus.
  • human papillomavirus antigens may be from one or more of HPV 16, HPV 18, HPV 6 and/or HPV 11.
  • Preferred Gonococcal antigens include one or more of ngs13 (OmpA), OmpH, ngs576 (peptidyl-prolyl cis/trans isomerase (PPIase) protein), ngs41 and ngs117.
  • ngs13 OmpA
  • OmpH OmpH
  • ngs576 peptidyl-prolyl cis/trans isomerase (PPIase) protein
  • ngs41 and ngs117 include one or more of ngs13 (OmpA), OmpH, ngs576 (peptidyl-prolyl cis/trans isomerase (PPIase) protein), ngs41 and ngs117.
  • PPIase peptidyl-prolyl cis/trans isomerase
  • HPV antigens include one or more of HPV 16, HPV 18, HPV 6 and HPV 11.
  • Chlamydia trachomatis antigens include one or more of: CT045, CT089, CT242, CT316, CT381. CT396, CT398, CT444, CT467, CT547, CT587, CT823, CT761 and specific combinations of these antigens as disclosed in WO 05/002619.
  • Preferred Chlamydia pneumoniae antigens include one or more of: CPn0324, Cpn0301, Cpn0482, Cpn0503, Cpn0525, Cpn0558, Cpn0584, Cpn0800, Cpn0979, Cpn0498, Cpn0300, Cpn0042, Cpn0013, Cpn450, Cpn0661, Cpn0557, Cpn0904, Clpn0795, Cpn0186 and Cpn0604 and specific combinations of these antigens as disclosed in WO 05/084306.
  • Preferred GBS antigens include one or more of GBS80, GBS 104, GBS 59, GBS 67, GBS 322 and GBS 276.
  • the antigen combinations of the invention are combined with one or more additional, non-ExPEC antigens suitable for use in a vaccine designed to protect elderly or immunocompromised individuals.
  • the antigen combinations may be combined with an antigen derived from the group consisting of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitidies, influenza, and Parainfluenza virus (‘PIV’).
  • Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [186]).
  • diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
  • Saccharide antigens are preferably in the form of conjugates.
  • Carrier proteins for the conjugates include bacterial toxins (such as diphtheria toxoid or tetanus toxoid), the N.meningitidis outer membrane protein [222], synthetic peptides [223,224], heat shock proteins [225,226], pertussis proteins [227,228], protein D from H.influenzae [ 229,230], cytokines [231], lymphokines [231], H.influenzae proteins, hormones [231], growth factors [231], toxin A or B from C.difficile [ 232], iron-uptake proteins [233], artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens [234] such as the N19 protein [235], pneumococcal surface protein PspA [236], pneumolysin [237], etc.
  • a preferred carrier protein is CRM197 protein [2
  • Antigens in the composition will typically be present at a concentration of at least 1 ⁇ g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
  • Antigens are preferably adsorbed to an aluminium salt.
  • the immunogenic compositions described above include polypeptide antigens from UPEC.
  • nucleic acid preferably DNA e.g. in the form of a plasmid
  • Nucleic acid immunisation is now a developed field (e.g. see references 239 to 246 etc.), and has been applied to many vaccines.
  • the nucleic acid encoding the immunogen is expressed in vivo after delivery to a patient and the expressed immunogen then stimulates the immune system.
  • the active ingredient will typically take the form of a nucleic acid vector comprising: (i) a promoter; (ii) a sequence encoding the immunogen, operably linked to the promoter; and optionally (iii) a selectable marker.
  • Preferred vectors may further comprise (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii).
  • (i) & (v) will be eukaryotic and (iii) & (iv) will be prokaryotic.
  • Preferred promoters are viral promoters e.g. from cytomegalovirus (CMV).
  • the vector may also include transcriptional regulatory sequences (e.g. enhancers) in addition to the promoter and which interact functionally with the promoter.
  • Preferred vectors include the immediate-early CMV enhancer/promoter, and more preferred vectors also include CMV intron A.
  • the promoter is operably linked to a downstream sequence encoding an immunogen, such that expression of the immunogen-encoding sequence is under the promoter's control.
  • a marker preferably functions in a microbial host (e.g. in a prokaryote, in a bacteria, in a yeast).
  • the marker is preferably a prokaryotic selectable marker (e.g. transcribed under the control of a prokaryotic promoter).
  • prokaryotic selectable marker e.g. transcribed under the control of a prokaryotic promoter.
  • typical markers are antibiotic resistance genes.
  • the vector of the invention is preferably an autonomously replicating episomal or extrachromosomal vector, such as a plasmid.
  • the vector of the invention preferably comprises an origin of replication. It is preferred that the origin of replication is active in prokaryotes but not in eukaryotes.
  • Preferred vectors thus include a prokaryotic marker for selection of the vector, a prokaryotic origin of replication, but a eukaryotic promoter for driving transcription of the immunogen-encoding sequence.
  • the vectors will therefore (a) be amplified and selected in prokaryotic hosts without polypeptide expression, but (b) be expressed in eukaryotic hosts without being amplified. This arrangement is ideal for nucleic acid immunization vectors.
  • the vector of the invention may comprise a eukaryotic transcriptional terminator sequence downstream of the coding sequence. This can enhance transcription levels.
  • the vector of the invention preferably comprises a polyadenylation sequence.
  • a preferred polyadenylation sequence is from bovine growth hormone.
  • the vector of the invention may comprise a multiple cloning site.
  • the vector may comprise a second eukaryotic coding sequence.
  • the vector may also comprise an IRES upstream of said second sequence in order to permit translation of a second eukaryotic polypeptide from the same transcript as the immunogen.
  • the immunogen-coding sequence may be downstream of an IRES.
  • the vector of the invention may comprise unmethylated CpG motifs e.g. unmethylated DNA sequences which have in common a cytosine preceding a guanosine, flanked by two 5′ purines and two 3′ pyrimidines. In their unmethylated form these DNA motifs have been demonstrated to be potent stimulators of several types of immune cell.
  • Vectors may be delivered in a targeted way.
  • Receptor-mediated DNA therapy techniques are described in, for example, references 247 to 252.
  • Therapeutic compositions containing a nucleic acid are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA can also be used during a gene therapy protocol.
  • Factors such as method of action (e.g. for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy.
  • a larger area of tissue, larger amounts of vector or the same amounts re-administered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions may be required to effect a positive therapeutic outcome.
  • routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.
  • Vectors can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non-viral origin (see generally references 253 to 256).
  • Viral-based vectors for delivery of a desired nucleic acid and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (e.g. references 257 to 267), alphavirus-based vectors (e.g. Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532); hybrids or chimeras of these viruses may also be used), poxvirus vectors (e.g.
  • vaccinia fowlpox, canarypox, modified vaccinia Ankara, etc.
  • adenovirus vectors e.g. see refs. 268 to 273
  • AAV adeno-associated virus
  • Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone [e.g. 274], ligand-linked DNA [275], eukaryotic cell delivery vehicles cells [e.g. refs. 276 to 280] and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in refs. 281 and 282. Liposomes (e.g. immunoliposomes) that can act as gene delivery vehicles are described in refs. 283 to 287. Additional approaches are described in references 288 & 289.
  • non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in ref. 289.
  • the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation [e.g. refs. 290 & 291].
  • Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun [292] or use of ionizing radiation for activating transferred genes [290 & 293].
  • Delivery DNA using PLG ⁇ poly(lactide-co-glycolide) ⁇ microparticles is a particularly preferred method e.g. by adsorption to the microparticles, which are optionally treated to have a negatively-charged surface (e.g. treated with SDS) or a positively-charged surface (e.g. treated with a cationic detergent, such as CTAB).
  • a negatively-charged surface e.g. treated with SDS
  • a positively-charged surface e.g. treated with a cationic detergent, such as CTAB
  • composition “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
  • the N-terminus residues in the amino acid sequences in the sequence listing are given as the amino acid encoded by the first codon in the corresponding nucleotide sequence. Where the first codon is not ATG, it will be understood that it will be translated as methionine when the codon is a start codon, but will be translated as the indicated non-Met amino acid when the sequence is at the C-terminus of a fusion partner.
  • the invention specifically discloses and encompasses each of the amino acid sequences of the sequence listing having a N-terminus methionine residue (e.g. a formyl-methionine residue) in place of any indicated non-Met residue. It also specifically discloses and encompasses each of the amino acid sequences of the sequence listing starting at any internal methionine residues in the sequences.
  • nucleic acids and polypeptides of the invention may include sequences that:
  • nucleic acids and polypeptides of the invention may additionally have further sequences to the N-terminus/5′ and/or C-terminus/3′ of these sequences (a) to (d).
  • polypeptide sequences of the invention are already available in public databases, their annotated functions are also available. Some of the 596 polypeptides have no recognised function in the current annotation (e.g. they are annotated in the databases as ‘hypothetical protein’). Although the inventors have not elucidated the basic underlying biological function of these polypeptides, the invention does now provide a credible utility for these polypeptides, namely in the provision of immunogenic compositions as described herein.
  • Table 4 reports the closest matches to SEQ ID NOS: 1-596 in the non-pathogenic K12 sequences for strains MG1655 [307], W3110 and DH10B.
  • Table 5 shows the 414/596 polypeptides with the strongest matches between two different UPEC genomes, with the underlined SEQ ID numbers being those with 100% conservation. The remaining 182 polypeptides are more-weakly conserved between UPEC strains, and so the Table 5 polypeptides are preferred (particularly underlined ones).
  • polypeptides are cloned, expressed and purified.
  • the purified antigens are used to immunise mice, whose sera are analysed by Western blot, ELISA and FACS, and are further tested in both in vitro and in vivo experiments. Suitable in vitro experiments include testing the ability of antibodies to induce complement-mediated bacterial killing and/or opsonophagocytosis activity, to block binding of ExPEC strains (or the purified antigen) to human epithelial cells (e.g. in bladder cells) or other cell lines, and/or to inhibit adhesion/invasion of E.coli bacteria (e.g. K1 strain) to brain microvascular endothelial cells (BMEC).
  • BMEC brain microvascular endothelial cells
  • Suitable in vivo experiments include active and/or passive systemic immunisations and challenge in mouse models of UTI (adult mice), protection by active or passive immunisations against bacteremia and meningitis in 5-day-old rats challenged with E.coli K1 strain, and immunisation and intraperitoneal infection of adult mice with an ExPEC strain.
  • the importance of the proteins to the bacterial life-cycle is tested by creating isogenic knockout mutants.
  • the mutants can also be used to ensure that sera raised by an antigen are specific for that antigen.
  • Microarrays are used to study expression patterns. Conservation and/or variability is assessed by sequencing the genes from multiple different ExPEC strains.
  • the first step consists in amplifying independently the upstream and downstream regions of the target gene (tolR) and the resistance marker cassette.
  • the two PCR products obtained in step 1 are mixed with the amplification producer of the AB cassette at equimolar concentrations and submitted to a second round of PCR (a three way PCR) to generate a resistance marker cassette flanked by upstream and downstream 500 bp (or more) regions homologous to the target gene.
  • large amounts (1 ⁇ g) of the desired linear DNA are electroporated into lamda-red competent cells.
  • LB media was inoculated with bacteria grown on plates and incubated overnight at 37° C. under gentle shaking. The culture was used to inoculate 200 ml of LB at OD600 0.1. Bacteria were grown to OD600 0.4 (or as specified). Culture was centrifuged for 10 minutes at 4000 ⁇ g and the supernatant was filtered through a 0.22 mm filter to remove residual bacteria.
  • Precipitation was performed by adding to the culture supernatant 10% final of a solution at 100% (w/v) TCA, 0.4% (w/v) deoxycholate. The precipitation was allowed to proceed for 30 minutes at 4° C. Precipitate was recovered by 10 minutes centrifugation at 20000 ⁇ g at 4° C. The pellet was washed once with 10% TCA (w/v) and twice with absolute ethanol. The pellet was dried with speed vac, and stored at ⁇ 20° C.
  • the wild type and mutated strains were subjected to SDS polyacrylamide gel electrophoresis from which it could be observed that there were many more bands in the supernatant of the mutated strains than the wildtype strains. Randomly picked bands demonstrated that all the proteins in the supernatant were membrane proteins.
  • Culture supernatant was ultracentrifuged at 200000 ⁇ g for 2 hours at 4° C. The pellet was washed with PBS, resuspended in PBS, and stored at ⁇ 20° C.
  • Vesicles Prior to the guanidinium denaturation, Vesicles were precipitated with ethanol. 10 ⁇ g of OMV in PBS were precipitate by adding cold absolute ethanol to 90% final. Precipitation was allowed to proceed for 20 minutes at ⁇ 20° C. Precipitate was recovered by 10 minutes centrifugation at 13000 ⁇ g. Pellet was resuspended with 50 ml, 6M guanidinium, 15 mM DTT, 200 mM Tris-HCl, pH 8.0. Denaturation was allowed to proceed for 60 minutes at 60° C. Prior to digestion, solution was diluted 1/8 with a solution of 1.5M Tris pH 8.0 and 5 mg of trypsin were added to the diluted solution. Digestion was allowed to proceed overnight at 37° C. Reaction was stopped by adding 0.1% final of formic acid. Peptides were extracted using Oasis extraction cartridges. Peptides were analyzed by LC coupled MS-MS.
  • Proteins were quantified with the Bradford method, using the BSA as standard.
  • Samples were analyzed with a sodium dodecyl sulfate (SDS) 4-12% polyacrylamide gel, using a Mini-Protean II electrophoresis apparatus. Samples were suspended in SDS sample buffer (0.06 M Tris-HCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10 mg/ml bromophenol blue) and heated to 100° C. for 5 min before SDS-polyacrylamide gel electrophoreis. After the run, gels were stained with Coomassie Blue
  • Protein bands or spots were excised from gels, washed with 50 mM ammonium bicarbonate/acetonitrile (50/50, v/v), and dried with a Speed-Vac centrifuge (Savant). The dried spots were digested at 37° C. for 2 h by adding 7 to 1,0 ml of a solution containing 5 mM ammonium bicarbonate, 0.012 mg of sequencing-grade trypsin. After digestion 0.6 ml were loaded on a matrix pre-spotted target and air-dried. Spots were washed with 0.6 ml of a solution of 70% ethanol, 0.1% trifluoracetic acid. Mass spectra were acquired on an ultraflex MALDI TOF mass spectrometer.
  • Spectra were externally calibrated by using a combination of standards pre-spotted on the target. Protein identification was carried out by both automatic and manual comparisons of experimentally generated monoisotopic peaks of peptides in the mass range of 700 to 3,000 Da with computer-generated fingerprints, using the Mascot program.
  • the first dimension was run using a IPGphor Isoelectric Focusing Unit, applying sequentially 150 V for 35 minutes, 500 V for 35 minutes, 1,000 V for 30 minutes, 2,600 V for 10 minutes, 3,500 V for 15 minutes, 4,200 V for 15 minutes, and finally 5,000 V to reach 10 kVh.
  • the strips were equilibrated by two 10 minute -incubations in 4 M urea, 2 M thiourea, 30% glycerol, 2% SDS, 5 mM TBP, 50 Mm Tris HCl pH 8.8, 2.5% acrylamide, Bromo phenol Blue 0.2%: Proteins were then separated on linear 4-12% precasted polyacrylamide gels.
  • Peptides were separated by nano-LC on a CapLC HPLC system connected to a Q-ToF Micro ESI mass spectrometer equipped with a nanospray source. Samples were loaded onto an Atlantis C18 NanoEase column (100 ⁇ m i.d. ⁇ 100 mm), through a C18 trap column (300 ⁇ m i.d. ⁇ 5 mm). Peptides were eluted with a 50-min gradient from 2% to 60% of 95% ACN, in a solution of 0.1% formic acid at a flow rate of 400 nl/minute.
  • the eluted peptides were subjected to an automated data-dependent acquisition program, using the MassLynx software, version 4.0, where a MS survey scan was used to automatically select multi-charged peptides over the m/z range of 400-2,000 for further MS/MS fragmentation. Up to three different components where subjected to MS/MS fragmentation at the same time. After data acquisition, the individual MS/MS spectra were combined, smoothed and centroided by MassLynx. Search and identification of peptides were performed in batch mode with a licensed version of MASCOT.
  • the MASCOT search parameters were: (1) species: ExPEC (2) allowed number of missed cleavages (only for trypsin digestion): 6; (3) variable post-translational modifications: methionine oxidation; (4) peptide tolerance: ⁇ 500 ppm; (5) MS/MS tolerance: ⁇ 0.3 Da and (6): peptide charge: from +1 to +4. As for the previous platform, only significant hits as defined by MASCOT probability analysis were considered.
  • the score thresholds for acceptance of protein identifications from at least one peptide were set by MASCOT as 18 for trypsin digestion and 36 for proteinase K digestion.
  • the experimental model uses 5 week old—CD1 outbreed mice which are challenged with intraperitoneal inoculation of virulent CFT073 E. coli strain.
  • the challenge dose has been experimentally determined as the amount of bacteria able to kill 80% of adult mice within 72 hours and corresponds to 7 ⁇ 10 7 cfu/mouse for the CFT073 strain.
  • mice are immunized three times by subcutaneous injection of 150 ⁇ l of protein solution using freund's adjuvants as shown in the table below:
  • mice Immunized mice: Day 0 75 ⁇ l of saline solution 75 ⁇ l of protein solution 75 ⁇ l of complete freund's (20 ⁇ g) adjuvant 75 ⁇ l of complete freund's adjuvant Day 21 75 ⁇ l of saline solution 75 ⁇ l of protein solution 75 ⁇ l of incomplete freund's (20 ⁇ g) adjuvant 75 ⁇ l of incomplete freund's adjuvant Day 35 75 ⁇ l of saline solution 75 ⁇ l of protein solution 75 ⁇ l of incomplete freund's (20 ⁇ g) adjuvant 75 ⁇ l of incomplete freund's adjuvant
  • Blood samples are colleccted the day before the first immunization (preimmune serum), at day 34 and 48 (day before challenge). Sera from immunized animals are tested by western blot and ELISA to determine the antibodies titer.
  • E.coli CFT073 strain is streaked on LB agar plate from frozen stock and incubated overnight (ON) at 37° C. in incubator.
  • the culture is centrifuged and the pellet resuspended in the same volume with physiological solution and used for challenge undiluted.
  • the culture is plated using a standard plate count method to verify the inoculum.
  • the protection due to vaccination is evaluated by comparison of the survival in the vaccinated group and the survival in control group of mice at 72 hours from the challenge. Percentage of survival relative to controls is calculated using the formula:
  • Antigens are selected for combining to give a composition of the invention.
  • BALB/c mice are divided into nine groups and immunized as follows:
  • Group Immunizing Composition Route of Delivery 1 Mixture of antigens (10-20 ⁇ g Intra-peritoneal or intra-nasal protein/each) + CFA (Complete or subcutaneous Freund's Adjuvant) 2 Mixture of antigens (5 ⁇ g/ Intra-peritoneal or intra-nasal each) + Al-hydroxide (200 ⁇ g) or subcutaneous 3 Mixture of antigens (10-20 ⁇ g Intra-peritoneal or intra-nasal protein/each) + CpG (10 ug) or subcutaneous 4 Mixture of antigens (10-20 ⁇ g Intra-peritoneal or intra-nasal protein/each) + Al-hydroxide or subcutaneous (200 ⁇ g) + CpG (10 ⁇ g) 5 CFA Intra-peritoneal or intra-nasal or subcutaneous 6 Mixture of antigens (10-20 ⁇ g Intra-peritoneal or Intranasal protein/each) + LTK63 (5 ⁇ g) or subcutaneous 7 Al
  • mice are immunized at two-week intervals. Two to three weeks after the last immunization, all mice are challenged with the appropriate UPEC strain. When mucosal immunization (e.g. intranasal) is used, the animal model is also challenged mucosally to test the protective effect of the mucosal immunogen. Immediately prior to challenge, mice are bled to determine antibody titre to the antigens that were administered.
  • mucosal immunization e.g. intranasal
  • mice are bled to determine antibody titre to the antigens that were administered.
  • virulent bacteria will be grown in appropriate media. Bacteria are harvested by centrifugation, re-suspended, and serially diluted for the challenge inoculum. BALB/c mice are challenged and observed daily for 30 days post-exposure.
  • Total IgG and IgG1/IgG2A subtypes can be measured in mouse sera resulting from the different immunization regimens by using an ELISA assay on whole bacteria and on purified recombinant proteins. Furthermore, assessment of antigen-specific CD4+ and CD8+Th-cells in spleen cells and/or PBMC isolated from immunized mice can be carried out by multi-parametric FACS analysis, to evaluate the cytokine expression profiles of antigen-specific T-cells. In particular production of IFN- ⁇ and IL-5 can be measured after in vitro stimulation of T cells with purified antigens.
  • splenocytes and/or PBMC from mice immunized with each antigen/vaccine formulation may be collected 10-12 days after the last immunization dose and stimulated with UPEC bacteria. After 4 hours of stimulation, Brefeldin A is added to the cells for the following 12 hours, to block cytokine secretion. Afterwards cells are fixed and stained with antibodies to detect UPEC-specific T cells expressing IFN- ⁇ and IL-5.
  • T cells can be isolated from peripheral blood lymphocytes (PBLs) by a variety of procedures known to those skilled in the art.
  • PBLs peripheral blood lymphocytes
  • T cell populations can be “enriched” from a population of PBLs through the removal of accessory and B cells.
  • T cell enrichment can be accomplished by the elimination of non-T cells using anti-MHC class II monoclonal antibodies.
  • other antibodies can be used to deplete specific populations of non-T cells.
  • anti-Ig antibody molecules can be used to deplete B cells and anti-MacI antibody molecules can be used to deplete macrophages.
  • T cells can be further fractionated into a number of different subpopulations by techniques known to those skilled in the art. Two major subpopulations can be isolated based on their differential expression of the cell surface markers CD4 and CD8.
  • CD4+ cells can be enriched using antibodies specific for CD4. The antibodies may be coupled to a solid support such as magnetic beads.
  • CD8+ cells can be enriched through the use of antibodies specific for CD4 (to remove CD4+ cells), or can be-isolated by the use of CD8 antibodies coupled to a solid support.
  • CD4 lymphocytes from UPEC-infected patients can be expanded ex vivo, before or after transduction.
  • the purified T cells are pre-stimulated with various cytokines including but not limited to rIL-2, IL-10, IL-12, and IL-15, which promote growth and activation of lymphocytes.
  • cytokines including but not limited to rIL-2, IL-10, IL-12, and IL-15, which promote growth and activation of lymphocytes.
  • UPEC-specific T cells may be activated by the above described immunogenic polypeptides.
  • UPEC-specific T cells can be CD8+ or CD4+.
  • UPEC-specific CD8+ T cells can be cytotoxic T lymphocytes (CTL) which can kill UPEC-infected cells that display any of the above described polypeptides or fragments thereof complexed with an MHC class I molecule.
  • CTL cytotoxic T lymphocytes
  • Chlamydia-specific CD8+ T cells can be detected by, for example, 51 Cr release assays. 51 Cr release assays measure the ability of UPEC-specific CD8+ T cells to lyse target cells displaying one or more of these epitopes.
  • UPEC-specific CD8+ T cells which express antiviral agents, such as IFN ⁇ , are also contemplated herein and can also be detected by immunological methods, preferably by intracellular staining for IFN- ⁇ or alike cytokines after in vitro stimulation with one or more of the above described UPEC polypeptides.
  • UPEC-specific CD4+ T cells can be detected by a lymphoproliferation assay. Lymphoproliferation assays measure the ability of UPEC-specific CD4+ T cells to proliferate in response to one or more of the above described polypeptides.
  • coli 0157:H7-specific protein 949
  • SID selected interacting domain
  • gonorrhoeae amino acid sequence SEQ ID 7826
  • Bovine herpesvirus 1 US3 protein sequence SEQ ID NO: 9
  • WO03/93425 526 1e ⁇ 01 15/48 11 2 323 I 2 Human PECAM-1 protein SEQ ID NO: 73
  • coli 0157:H7-specific protein 1016
  • influenzae Orf25 polypeptide SEQ ID NO: 49

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Abstract

Disclosed herein are various genes that can be included in immunogenic compositions specific for pathogenic E. coli strains. The genes are from uropathogenic strains but are absent from non-pathogenic strains, and their encoded proteins have cellular locations which render them accessible to the immune system.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a Divisional of U.S. patent application Ser. No. 11/884,825 (Now Patent No. 8,062,644), filed Apr. 4, 2008, which is the National Stage of International Patent Application of PCT/US2006/005912, filed Feb. 17, 2006, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/654,632, filed Feb. 18, 2005, each of which is hereby incorporated by reference in its entirety.
    • TECHNICAL FIELD
  • This invention is in the field of Escherichia coli biology, and in particular relates to immunogens for use in immunising against extraintestinal pathogenic E. coli (ExPEC) strains.
    • BACKGROUND ART
  • Few microorganisms are as versatile as E. coli. As well as being an important member of the normal intestinal microflora of mammals, it has been widely exploited as a host in recombinant DNA technology. In addition, however, E. coli can also be a deadly pathogen.
  • E. coli strains have traditionally been classified as either commensal or pathogenic, and pathogenic strains are then sub-classified as intestinal or extraintestinal strains. More recent taxonomic techniques such as multilocus enzyme electrophoresis (MLEE) classify E. coli into five phylogenetic groups (A, B1, B2, D & E), and these groupings do not match the traditional ones. For instance, MLEE group B1 includes both commensal and pathogenic strains, and group D includes both intestinal and extraintestinal strains.
  • The extraintestinal pathogenic strains (or ‘ExPEC’ strains [1]) of E. coli fall into MLEE groups B2 and D, and include both uropathogenic (UPEC) strains and meningitis/sepsis-associated (MNEC) strains. UPEC strains cause urinary tract infections (UTIs), and are the most common form of cystitis. They also cause pyelonephritis (and its complications such as sepsis) and catheter-associated infections. MNEC strains cause neonatal meningitis (0.1 cases per 1000 live births) with case fatality rates ranging from 25 to 40%, and are also responsible for around ⅙ of sepsis cases.
  • Most previous ExPEC vaccines have been based on cell lysates or on cellular structures. SOLCOUROVAC™ includes ten different heat-killed bacteria including six ExPEC strains, and a successful phase II clinical trial was reported in reference 2. URO-VAXOM™ is an oral tablet vaccine containing lyophilised bacterial lysates of 18 selected E. coli strains [3]. Baxter Vaccines developed a UTI vaccine based on pili from 6 to 10 different strains, but this product has been abandoned. MedImmune developed a product called MEDI 516 based on the FimH adhesin complex [4], but phase II clinical trials shows inadequate efficacy. Moreover, there was a risk with this vaccine that it would also affect non-pathogenic FimH+ve strains in the normal intestinal flora, and it was expected that this vaccine would be effective against UPEC strains only, because of its bladder-specific adherence mechanism, leaving other ExPEC strains uncontrolled.
  • There is thus a need for improved ExPEC vaccines, including a need to move away from crude cell lysates and towards better-defined molecules, and a need to identify further antigens that are suitable for inclusion in vaccines, particularly antigens that are prevalent among clinical ExPEC strains without also being found in commensal strains.
  • One way of addressing these needs was reported in reference 5, where the inventors looked for genes present in genomes of MLEE types B2 and D but absent from MLEE types A and B1. Further comparative approaches, based on subtractive hybridisation, were reported in references 6 and 7. Virulence genes in ExPEC strains have also been identified in reference 8. Reference 9 discloses an analysis of four pathogenicity islands in UPEC E. coli strain 536.
  • Reference 10 used the genome sequence of UPEC (O6:K2:H1) strain CFT073 [11,12] to identify sequences not present in non-pathogenic E. coli strains. Reference 13 discloses a comparison of the genome sequence of E. coli human pyelonephritis isolate 536 (O6:K15:H31), an UPEC, with sequence data for strains CFT073 (UPEC), EDL933 (enterohemorrhagic) and MG1655 (non-pathogenic laboratory strain). Genome sequences of pathogenic strains are available in the databases under accession numbers AE005174, BA000007 and NC-004431. A sequence from a non-pathogenic strain is available under accession number U00096.
  • It is an object of the invention to provide further antigens for use in immunisation against pathogenic E. coli strains, particularly ExPEC strains, and more particularly UPEC strains.
    • SUMMARY OF THE INVENTION
  • The inventors have identified various genes that can be included in immunogenic compositions specific for pathogenic E. coli strains. The genes are from uropathogenic strains (UPEC) but are absent from non-pathogenic strains, and their encoded proteins have cellular locations which render them accessible to the immune system.
  • In one aspect, the invention relates to a polypeptide comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a). In a particular embodiment, polypeptides of this aspect of the invention comprise a fragment which comprises at least one B-cell epitope of (a).
  • In another aspect, the invention relates to a polypeptide comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a). In a particular embodiment, polypeptides of this aspect of the invention comprise a fragment which comprises at least one B-cell epitope of (a).
  • The polypeptides of the invention can be used in medicine and in the manufacture of a medicament for raising an immune response in a patient.
  • The present invention also relates to a pharmaceutical composition comprising a polypeptide of the invention in admixture with a pharmaceutically acceptable carrier. The invention further relates to a pharmaceutical composition comprising two or more polypeptides of the invention in admixture with a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical compositions of the invention further comprise a vaccine adjuvant.
  • The present invention further relates to immunogenic compositions comprising one or more outer membrane vesicles (OMVs) expressing or overexpressing one or more polypeptides comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a). In a particular embodiment, the immunogenic composition of this aspect of the invention comprises one or more polypeptides comprising a fragment which comprises at least one B-cell epitope of (a).
  • The present invention also relates to methods for raising an immune response in a patient, comprising the step of administering to the patient a pharmaceutical composition or immunogenic composition of the invention. In a particular embodiment, the immune response is protective against ExPEC infection.
  • Further aspects of the invention are described below.
    • BRIEF DESCRIPTION OF THE DRAWING
  • FIG. 1 shows the % survival of mice after challenge with CFT073 following immunization with heat-inactivated CFT073.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The inventors have identified various genes that can be included in immunogenic compositions specific for pathogenic E. coli strains. The genes are from UPEC strains but are absent from non-pathogenic strains, and their encoded proteins have cellular locations which render them accessible to the immune system.
  • Polypeptides
  • The invention provides polypeptides comprising the amino acid sequences disclosed in the examples. These amino acid sequences are given in the sequence listing as SEQ ID NOs 1 to 596 and 597 to 599. Preferred subsets of SEQ ID NOs 1 to 596 are given in Tables 2, 3 and 5.
  • The invention also provides polypeptides comprising amino acid sequences that have sequence identity to the amino acid sequences disclosed in the examples. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g. 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). These polypeptides include homologs, orthologs, allelic variants and mutants. Typically, 50% identity or more between two polypeptide sequences is considered to be an indication of functional equivalence. Identity between polypeptides is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.
  • These polypeptide may, compared to the sequences of the examples, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid replacements i.e. replacements of one amino acid with another which has a related side chain. Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In general, substitution of single amino acids within these families does not have a major effect on the biological activity. The polypeptides may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to a reference sequence. The polypeptides may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to a reference sequence.
  • Preferred polypeptides include polypeptides that are lipidated, that are located in the outer membrane, that are located in the inner membrane, or that are located in the periplasm. Particularly preferred polypeptides are those that fall into more than one of these categories e.g. lipidated polypeptides that are located in the outer membrane. Lipoproteins may have a N-terminal cysteine to which lipid is covalently attached, following post-translational processing of the signal peptide.
  • Polypeptides that may be lipidated include SEQ ID NOS: 1, 2, 7, 12, 13, 14, 15, 16, 17, 18, 22, 26, 28, 29, 33, 34, 38, 40, 45, 50, 51, 59, 67, 68, 69, 71, 77, 80, 82, 83, 84, 92, 98, 103, 104, 105, 120, 121, 124, 125, 126, 127, 130, 131, 133, 134, 138, 142, 147, 159, 160, 176, 184, 185, 186, 187, 192, 206, 210, 215, 222, 223, 225, 226, 228, 233, 234, 246, 251, 252, 268, 272, 273, 275, 284, 287, 293, 295, 297, 298, 299, 300, 302, 303, 304, 305, 310, 311, 312, 314, 315, 320, 326, 327, 330, 331, 336, 340, 359, 360, 364, 366, 367, 369, 370, 371, 374, 378, 379, 386, 387, 388, 389, 390, 395, 400, 401, 407, 408, 418, 419, 422, 423, 425, 429, 431, 433, 434, 435, 436, 438, 441, 442, 444, 447, 453, 464, 465, 472, 478, 492, 500, 506, 509, 517, 518, 519, 523, 526, 531, 536, 540, 543, 544, 546, 548, 551, 557, 559, 560, 562, 563, 564, 565, 566, 568, 569, 574, 577, 583, 584, 585, 586, 593 and 594.
  • Preferred polypeptides are those listed in Tables 2, 3 and 5, particularly those listed in Table 3.
  • The invention further provides polypeptides comprising fragments of the amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more). The fragment may comprise at least one T-cell or, preferably, a B-cell epitope of the sequence. T- and B-cell epitopes can be identified empirically (e.g. using PEPSCAN [14,15] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [16], matrix-based approaches [17], TEPITOPE [18], neural networks [19], OptiMer & EpiMer [20,21], ADEPT [22], Tsites [23], hydrophilicity [24], antigenic index [25] or the methods disclosed in reference 26, etc.). Other preferred fragments are (a) the N-terminal signal peptides of the polypeptides of the invention, (b) the polypeptides, but without their N-terminal signal peptides, (c) the polypeptides, but without their N-terminal amino acid residue.
  • Other preferred fragments are those that begin with an amino acid encoded by a potential start codon (ATG, GTG, TTG). Fragments starting at the methionine encoded by a start codon downstream of the indicated start codon are polypeptides of the invention.
  • Other preferred fragments are those that are common to a polypeptide of the invention and to a polypeptide identified in any of references 5, 6, 8, 10 and 11.
  • Polypeptides of the invention can be prepared in many ways e.g. by chemical synthesis (in whole or in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself (e.g. after bacterial culture, or direct from patients), etc. A preferred method for production of peptides <40 amino acids long involves in vitro chemical synthesis [27,28]. Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [29] chemistry. Enzymatic synthesis [30] may also be used in part or in full. As an alternative to chemical synthesis, biological synthesis may be used e.g. the polypeptides may be produced by translation. This may be carried out in vitro or in vivo. Biological methods are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e.g. of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) [31]. Where D-amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides of the invention may have covalent modifications at the C-terminus and/or N-terminus.
  • Polypeptides of the invention can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, etc.).
  • Polypeptides of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other polypeptides (e.g. free from naturally-occurring polypeptides), particularly from other ExPEC or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i.e. less than about 50%, and more preferably less than about 10% (e.g. 5% or less) of a composition is made up of other expressed polypeptides. Polypeptides of the invention are preferably ExPEC polypeptides.
  • Polypeptides of the invention may be attached to a solid support. Polypeptides of the invention may comprise a detectable label (e.g. a radioactive or fluorescent label, or a biotin label).
  • The term “polypeptide” refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can occur as single chains or associated chains. Polypeptides of the invention can be naturally or non-naturally glycosylated (i.e. the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring polypeptide).
  • Polypeptides of the invention may be at least 40 amino acids long (e.g. at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500 or more). Polypeptides of the invention may be shorter than 500 amino acids (e.g. no longer than 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400 or 450 amino acids).
  • The invention provides polypeptides comprising a sequence —X—Y— or —Y—X—, wherein: —X— is an amino acid sequence as defined above and —Y— is not a sequence as defined above i.e. the invention provides fusion proteins. Where the N-terminus codon of a polypeptide-coding sequence is not ATG then that codon will be translated as the standard amino acid for that codon rather than as a Met, which occurs when the codon is translated as a start codon.
  • The invention provides a process for producing polypeptides of the invention, comprising the step of culturing a host cell of to the invention under conditions which induce polypeptide expression.
  • The invention provides a process for producing a polypeptide of the invention, wherein the polypeptide is synthesised in part or in whole using chemical means.
  • The invention provides a composition comprising two or more polypeptides of the invention.
  • The invention also provides a hybrid polypeptide represented by the formula NH2-A-[-X-L-]n-B—COOH, wherein X is a polypeptide of the invention as defined above, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1. The value of n is between 2 and x, and the value of x is typically 3, 4, 5, 6, 7, 8, 9 or 10. Preferably n is 2, 3 or 4; it is more preferably 2 or 3; most preferably, n=2. For each n instances, —X— may be the same or different. For each n instances of [-X-L-], linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH2—X1-L1-X2-L2-COOH, NH2—X1—X2—COOH, NH2—X1-L1-X2—COOH, NH2—X1—X2-L2-COOH, etc. Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences which facilitate cloning, poly-glycine linkers (i.e. Glyn where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. Hisn where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. -A- and —B— are optional sequences which will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct polypeptide trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. His where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal and C-terminal amino acid sequences will be apparent to those skilled in the art.
  • Various tests can be used to assess the in vivo immunogenicity of polypeptides of the invention. For example, polypeptides can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the polypeptide and patient serum indicates that the patient has previously mounted an immune response to the protein in question i.e. the protein is an immunogen. This method can also be used to identify immunodominant proteins.
  • Antibodies
  • The invention provides antibodies that bind to polypeptides of the invention. These may be polyclonal or monoclonal and may be produced by any suitable means (e.g. by recombinant expression). To increase compatibility with the human immune system, the antibodies may be chimeric or humanised [e.g. refs. 32 & 33], or fully human antibodies may be used. The antibodies may include a detectable label (e.g. for diagnostic assays). Antibodies of the invention may be attached to a solid support. Antibodies of the invention are preferably neutralising antibodies.
  • Monoclonal antibodies are particularly useful in identification and purification of the individual polypeptides against which they are directed. Monoclonal antibodies of the invention may also be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA), etc. In these applications, the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme. The monoclonal antibodies produced by the above method may also be used for the molecular identification and characterization (epitope mapping) of polypeptides of the invention.
  • Antibodies of the invention are preferably specific to ExPEC strains of E. coli, i.e. they bind preferentially to ExPEC E. coli relative to other bacteria (e.g. relative to non-ExPEC E. coli and relative to non-E.coli bacteria). More preferably, the antibodies are specific to UPEC strains i.e. they bind preferentially to UPEC bacteria relative to other bacteria, including other ExPEC E. coli.
  • Antibodies of the invention are preferably provided in purified or substantially purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides e.g. where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
  • Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgM i.e. an α, γ or μ heavy chain), but will generally be IgG. Within the IgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass. Antibodies of the invention may have a κ or a λ light chain.
  • Antibodies of the invention can take various forms, including whole antibodies, antibody fragments such as F(ab′)2 and F(ab) fragments, Fv fragments (non-covalent heterodimers), single-chain antibodies such as single chain Fv molecules (scFv), minibodies, oligobodies, etc. The term “antibody” does not imply any particular origin, and includes antibodies obtained through non-conventional processes, such as phage display.
  • The invention provides a process for detecting polypeptides of the invention, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
  • The invention provides a process for detecting antibodies of the invention, comprising the steps of (a) contacting a polypeptide of the invention with a biological sample (e.g. a blood or serum sample) under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
  • Preferred antibodies bind to a polypeptide of the invention with substantially greater affinity than antibodies known in the art. Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold etc. stronger than antibodies known in the art.
  • Nucleic Acids
  • The invention also provides nucleic acid comprising a nucleotide sequence encoding the polypeptides of the invention. The invention also provides nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences. Identity between sequences is preferably determined by the Smith-Waterman homology search algorithm as described above.
  • The invention also provides nucleic acid which can hybridize to these nucleic acids. Hybridization reactions can be performed under conditions of different “stringency”. Conditions that increase stringency of a hybridization reaction of widely known and published in the art [e.g. page 7.52 of reference 34]. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C., 55° C. and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or de-ionized water. Hybridization techniques and their optimization are well known in the art [e.g. see refs 34-37, etc.].
  • In some embodiments, nucleic acid of the invention hybridizes to a target under low stringency conditions; in other embodiments it hybridizes under intermediate stringency conditions; in preferred embodiments, it hybridizes under high stringency conditions. An exemplary set of low stringency hybridization conditions is 50° C. and 10×SSC. An exemplary set of intermediate stringency hybridization conditions is 55° C. and 1×SSC. An exemplary set of high stringency hybridization conditions is 68° C. and 0.1×SSC.
  • Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the sequences and, depending on the particular sequence, n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferred fragments are those that are common to a nucleic acid sequence of the invention and to a nucleic acid sequence identified in any of references 5, 6, 8, 10 and 11.
  • The invention provides nucleic acid of formula 5′—X—Y—Z-3′, wherein: —X— is a nucleotide sequence consisting of x nucleotides; —Z— is a nucleotide sequence consisting of z nucleotides; —Y— is a nucleotide sequence consisting of either (a) a fragment of a nucleic acid sequence encoding one of SEQ ID NOS: 1 to 596, (b) a fragment of a nucleic acid sequence encoding one of SEQ ID NOS: 597 to 599, or (c) the complement of (a) or (b); and said nucleic acid 5′—X—Y—Z-3′ is neither (i) a fragment of either a nucleic acid sequence encoding one of SEQ ID NOS: 1 to 596 or encoding one of SEQ ID NOS: 597 to 599 nor (ii) the complement of (i). The —X— and/or —Z— moieties may comprise a promoter sequence (or its complement).
  • The invention includes nucleic acid comprising sequences complementary to these sequences (e.g. for antisense or probing, or for use as primers).
  • Nucleic acids of the invention can be used in hybridisation reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or ‘gene chips’) and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) and other nucleic acid techniques.
  • Nucleic acid according to the invention can take various forms (e.g. single-stranded, double-stranded, vectors, primers, probes, labelled etc.). Nucleic acids of the invention may be circular or branched, but will generally be linear. Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary single-stranded forms which make up the double-stranded form. Primers and probes are generally single-stranded, as are antisense nucleic acids.
  • Nucleic acids of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other nucleic acids (e.g. free from naturally-occurring nucleic acids), particularly from other ExPEC or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90% pure. Nucleic acids of the invention are preferably ExPEC nucleic acids.
  • Nucleic acids of the invention may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.
  • Nucleic acid of the invention may be attached to a solid support (e.g. a bead, plate, filter, film, slide, microarray support, resin, etc.). Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to be used in detection techniques e.g. where the nucleic acid is a primer or as a probe.
  • The term “nucleic acid” includes in general means a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. It includes DNA, RNA, DNA/RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus the invention includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc. Where nucleic acid of the invention takes the form of RNA, it may or may not have a 5′ cap.
  • Nucleic acids of the invention comprise sequences, but they may also comprise non-ExPEC sequences (e.g. in nucleic acids of formula 5′-X—Y—Z-3′, as defined above). This is particularly useful for primers, which may thus comprise a first sequence complementary to a nucleic acid target and a second sequence which is not complementary to the nucleic acid target. Any such non-complementary sequences in the primer are preferably 5′ to the complementary sequences. Typical non-complementary sequences comprise restriction sites or promoter sequences.
  • Nucleic acids of the invention can be prepared in many ways e.g. by chemical synthesis (at least in part), by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.
  • Nucleic acids of the invention may be part of a vector i.e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, “cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, “expression vectors” which are designed for expression of a nucleotide sequence in a host cell, “viral vectors” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors”, which comprise the attributes of more than one type of vector. Preferred vectors are plasmids. A “host cell” includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention.
  • Where a nucleic acid is DNA, it will be appreciated that “U” in a RNA sequence will be replaced by “T” in the DNA. Similarly, where a nucleic acid is RNA, it will be appreciated that “T” in a DNA sequence will be replaced by “U” in the RNA.
  • The term “complement” or “complementary” when used in relation to nucleic acids refers to Watson-Crick base pairing. Thus the complement of C is G, the complement of G is C, the complement of A is T (or U), and the complement of T (or U) is A. It is also possible to use bases such as I (the purine inosine) e.g. to complement pyrimidines (C or T). The terms also imply a direction—the complement of 5′-ACAGT-3′ is 5′-ACTGT-3′ rather than 5′-TGTCA-3′.
  • Nucleic acids of the invention can be used, for example: to produce polypeptides; as hybridization probes for the detection of nucleic acid in biological samples; to generate additional copies of the nucleic acids; to generate ribozymes or antisense oligonucleotides; as single-stranded DNA primers or probes; or as triple-strand forming oligonucleotides.
  • The invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesised in part or in whole using chemical means.
  • The invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.
  • The invention also provides a kit comprising primers (e.g. PCR primers) for amplifying a template sequence contained within an ExPEC nucleic acid sequence, the kit comprising a first primer and a second primer, wherein the first primer is substantially complementary to said template sequence and the second primer is substantially complementary to a complement of said template sequence, wherein the parts of said primers which have substantial complementarity define the termini of the template sequence to be amplified. The first primer and/or the second primer may include a detectable label (e.g. a fluorescent label).
  • The invention also provides a kit comprising first and second single-stranded oligonucleotides which allow amplification of a ExPEC template nucleic acid sequence contained in a single- or double-stranded nucleic acid (or mixture thereof), wherein: (a) the first oligonucleotide comprises a primer sequence which is substantially complementary to said template nucleic acid sequence; (b) the second oligonucleotide comprises a primer sequence which is substantially complementary to the complement of said template nucleic acid sequence; (c) the first oligonucleotide and/or the second oligonucleotide comprise(s) sequence which is not complementary to said template nucleic acid; and (d) said primer sequences define the termini of the template sequence to be amplified. The non-complementary sequence(s) of feature (c) are preferably upstream of (i.e. 5′ to) the primer sequences. One or both of these (c) sequences may comprise a restriction site [e.g. ref. 38] or a promoter sequence [e.g. 39]. The first oligonucleotide and/or the second oligonucleotide may include a detectable label (e.g. a fluorescent label).
  • The invention provides a process for detecting nucleic acid of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.
  • The invention provides a process for detecting in a biological sample (e.g. blood), comprising the step of contacting nucleic acid according to the invention with the biological sample under hybridising conditions. The process may involve nucleic acid amplification (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) or hybridisation (e.g. microarrays, blots, hybridisation with a probe in solution etc.). PCR detection of ExPEC in clinical samples has been reported [e.g. see ref. 40]. Clinical assays based on nucleic acid are described in general in ref. 41.
  • The invention provides a process for preparing a fragment of a target sequence, wherein the fragment is prepared by extension of a nucleic acid primer. The target sequence and/or the primer are nucleic acids of the invention. The primer extension reaction may involve nucleic acid amplification (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.).
  • Nucleic acid amplification according to the invention may be quantitative and/or real-time.
  • For certain embodiments of the invention, nucleic acids are preferably at least 7 nucleotides in length (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300 nucleotides or longer).
  • For certain embodiments of the invention, nucleic acids are preferably at most 500 nucleotides in length (e.g. 450, 400, 350, 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 nucleotides or shorter).
  • Primers and probes of the invention, and other nucleic acids used for hybridization, are preferably between 10 and 30 nucleotides in length (e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
  • Vesicles
  • Reference 42 describes the preparation of vesicles from a uropathogenic (UPEC) strain by the knockout of mltA (a murein lytic transglycosylase) or one or more of the components of the E.coli Tol-Pal complex [43], such as tolA, tolQ, tolB, pal and/or tolR. These vesicles can be improved by making one or more further genetic changes to the chromosome of the bacterium or through insertion of episomal elements (e.g. expression vectors) in order to increase the amount of and/or immunoaccessibility of protective antigens on the surface the vesicles.
  • One way of obtaining such improvements is to up-regulate the expression of the polypeptides of the invention. Many different genetic strategies for increasing the expression of a target protein are well-known in the art and can be distinguished into two broad categories: one relying on modifications of the chromosome (e.g. replacement of the wild-type promoter with a stronger promoter, inactivation of natural repressor genes, etc.) to increase expression of an endogenous gene, and the other based on recombinant expression by episomal elements (e.g. high-copy number plasmids, vectors harboring an engineered target gene, etc.) or integration of a exogenous gene in the chromosome. Practical examples for each of these approaches can be found in references 44 to 50.
  • Another way of increasing vesicle immunogenicity and selectivity is to down-regulate the expression of immunodominant non-protective antigens or to down-regulate proteins that are homologous to proteins found in commensal strains. Further improvements can be achieved by detoxification of the Lipid A moiety of LPS. Similar changes have been previously described to produce improved vesicles from other Gram-negative pathogens (see for example references 51 & 52).
  • All the above strategies can be used either alone or in combination to obtain improved vesicles for use in immunogenic compositions. The invention provides a pathogenic Escherichia coli bacterium (particularly a UPEC) having a knockout of mltA and/or of a component of its Tol-Pal complex, and one or more of: (i) a chromosomal gene encoding a polypeptide of the invention under the control of a promoter that provides higher expression levels of the polypeptide than the promoter that is naturally associated with the gene encoding the polypeptide; (ii) an autonomously-replicating extrachromosomal element encoding a polypeptide of the invention; and/or (iii) a genetic modification to reduce the toxicity of the Lipid A moiety of E.coli LPS relative to wild-type LPS.
  • The invention also provides vesicles obtainable by culturing such a bacterium, such as the vesicles that, during culture of the bacterium, are released into the culture medium.
  • Pharmaceutical Compositions
  • The invention provides compositions comprising: (a) polypeptide, antibody, vesicles and/or nucleic acid of the invention; and (b) a pharmaceutically acceptable carrier. These compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.
  • In a particular aspect, the invention provides immunogenic compositions comprising one or more outer membrane vesicles (OMVs) expressing or overexpressing one or more polypeptides of the invention. In a particular embodiment, the invention provides an immunogenic composition comprising one or more OMVs expressing or overexpressing one or more polypeptides comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599; (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a); or (d) an amino acid sequence having at least 80% sequence identity to an amino acid sequence of (a) and including a fragment of at least 10 consecutive amino acids from an amino acid sequence of (a). In a further embodiment, the immunogenic composition comprises a polypeptide comprising a fragment which comprises at least one B-cell epitope of an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 120, 219, 221, 305, 371, 400, 489, 555, 565, 597, 598 and 599.
  • A ‘pharmaceutically acceptable carrier’ includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate-buffered physiologic saline is a typical carrier. A thorough discussion of pharmaceutically acceptable excipients is available in ref. 296.
  • Compositions of the invention may include an antimicrobial, particularly if packaged in a multiple dose format.
  • Compositions of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. <0.01%.
  • Compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2mg/ml NaCl is typical.
  • Compositions of the invention will generally include a buffer. A phosphate buffer is typical.
  • Compositions of the invention may comprise a sugar alcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml (e.g. 25 mg/ml), particularly if they are to be lyophilised or if they include material which has been reconstituted from lyophilised material. The pH of a composition for lyophilisation may be adjusted to around 6.1 prior to lyophilisation.
  • Polypeptides of the invention may be administered in conjunction with other immunoregulatory agents. In particular, compositions will usually include a vaccine adjuvant. The adjuvant may be selected from one or more of the group consisting of a TH1 adjuvant and TH2 adjuvant, further discussed below. Adjuvants which may be used in compositions of the invention include, but are not limited to:
  • A. Mineral-Containing Compositions
  • Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 53], or mixtures of different mineral compounds (e.g. a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption to the salt(s) being preferred. Mineral containing compositions may also be formulated as a particle of metal salt [54].
  • A typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with PO4/Al molar ratio between 0.84 and 0.92, included at 0.6mg Al3+/ml. Adsorption with a low dose of aluminium phosphate may be used e.g. between 50 and 100 μg Al3+ per conjugate per dose. Where an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution (e.g. by the use of a phosphate buffer).
  • The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate=more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium 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 aluminium 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 aluminium hydroxide and an aluminium phosphate. In this case there may be more aluminium 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.
  • Aluminum salts may be included in vaccines of the invention such that the dose of Al3+ is between 0.2 and 1.0 mg per dose.
  • B. Oil Emulsions
  • 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) [Chapter 10 of ref. 53; see also refs. 55-57, chapter 12 of ref. 58]. MF59 is used as the adjuvant in the FLUADTM influenza virus trivalent subunit vaccine. The emulsion advantageously includes citrate ions e.g. 10 mM sodium citrate buffer.
  • Particularly preferred adjuvants for use in the compositions are submicron oil-in-water emulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80 (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85 (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphophoryloxy)-ethylamine (MTP-PE). Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in references 55 & 59-60.
  • An emulsion of squalene, a tocopherol, and Tween 80 can be used. The emulsion may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably ≦1 as this provides a more stable emulsion. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm.
  • An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100) can be used.
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 (“Pluronic™ L121”) can be used. The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the “SAF-1” adjuvant [61] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the “AF” adjuvant [62] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
  • Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention.
  • C. Saponin Formulations [Chapter 22 of Ref 53]
  • Saponin formulations may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins isolated from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
  • Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in ref. 63. Saponin formulations may also comprise a sterol, such as cholesterol [64].
  • Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexs (ISCOMs) [chapter 23 of ref. 53]. ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA and QHC. ISCOMs are further described in refs. 64-66. Optionally, the ISCOMS may be devoid of additional detergent(s) [67].
  • A review of the development of saponin based adjuvants can be found in refs. 68 & 69.
  • D. Virosomes and Virus-Like Particles
  • Virosomes and virus-like particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1). VLPs are discussed further in refs. 70-75. Virosomes are discussed further in, for example, ref. 76
  • E. Bacterial or Microbial Derivatives
  • Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 77. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 μm membrane [77]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [78,79].
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. O174 is described for example in refs. 80 & 81.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. References 82, 83 and 84 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. 85-90.
  • The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [91]. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, 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. 92-94. Preferably, the CpG is a CpG-A ODN.
  • Preferably, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “immunomers”. See, for example, refs. 91 & 95-97.
  • Other immunostimulatory oligonucleotides include a double-stranded RNA, or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E.coli (E.coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 98 and as parenteral adjuvants in ref. 99. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 100-107. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 108.
  • Compounds of formula I, II or III, or salts thereof, can also be used as adjuvants:
  • Figure US20120058146A1-20120308-C00001
  • as defined in reference 109, such as ‘ER 803058’, ‘ER 803732’, ‘ER 804053’, ER 804058′, ‘ER 804059’, ‘ER 804442’, ‘ER 804680’, ‘ER 804764’, ER 803022 or ‘ER 804057’ e.g.:
  • Figure US20120058146A1-20120308-C00002
  • F. Human Immunomodulators
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [110], etc.) [111], interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor and macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta [112].
  • G. Bioadhesives and Mucoadhesives
  • Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres [113] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [114].
  • H. Microparticles
  • Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).
  • I. Liposomes (Chapters 13 & 14 of ref 53)
  • Examples of liposome formulations suitable for use as adjuvants are described in refs. 115-117.
  • J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations
  • Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [118]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [119] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [120]. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • K. Phosphazenes e.g. PCPP
  • Phosphazene adjuvants include poly[di(carboxylatophenoxy)phosphazene] (“PCPP”) as described, for example, in refs. 121 and 122.
  • L. Muramyl Peptides
  • Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
  • M. Imidazoquinoline Compounds.
  • Imidazoquinoline adjuvants include Imiquimod (“R-837”) [123,124], Resiquimod (“R-848”) [125], and their analogs; and salts thereof (e.g. the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in references 126 to 130.
  • N. Thiosemicarbazone Compounds.
  • Examples of thiosemicarbazone compounds, as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants in the invention include those described in ref. 131. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
  • O. Tryptanthrin Compounds.
  • Examples of tryptanthrin compounds, as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants in the invention include those described in ref. 132. The tryptanthrin compounds are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.
  • P. Nucleoside Analogs
  • Various nucleoside analogs can be used as adjuvants, such as (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):
  • Figure US20120058146A1-20120308-C00003
  • and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds disclosed in references 133 to 135; (f) a compound having the formula:
  • Figure US20120058146A1-20120308-C00004
      • wherein:
        • R1 and R2 are each independently H, halo, —NRaRb, —OH, C1-6 alkoxy, substituted C1-6 alkoxy, heterocyclyl, substituted heterocyclyl, C6-10 aryl, substituted C6-10 aryl, C1-6 alkyl, or substituted C1-6 alkyl;
        • R3 is absent, H, C1-6 alkyl, substituted C1-6 alkyl, C6-10 aryl, substituted C6-10 aryl, heterocyclyl, or substituted heterocyclyl;
        • R4 and R5 are each independently H, halo, heterocyclyl, substituted heterocyclyl, —C(O)—Rd, C1-6 alkyl, substituted C1-6 alkyl, or bound together to form a 5 membered ring as in R4-5:
  • Figure US20120058146A1-20120308-C00005
          • the binding being achieved at the bonds indicated by a
        • X1 and X2 are each independently N, C, O or S;
        • R8 is H, halo, —OH, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, —OH, —NRaRb, —(CH2)n—O—Rc, —O—(C1-6 alkyl), —S(O)pRe, or —C(O)—Rd;
        • R9 is H, C1-6 alkyl, substituted C1-6 alkyl, heterocyclyl, substituted heterocyclyl or R9a, wherein R9a is:
  • Figure US20120058146A1-20120308-C00006
          • the binding being achieved at the bond indicated by a
            Figure US20120058146A1-20120308-P00001
        • R10 and R11 are each independently H, halo, C1-6 alkoxy, substituted C1-6 alkoxy, —NRaRb, or —OH;
        • each Ra and Rb is independently H, C1-6 alkyl, substituted C1-6 alkyl, —C(O)Rd, C6-10 aryl;
        • each Rc is independently H, phosphate, diphosphate, triphosphate, C1-6 alkyl, or substituted C1-6 alkyl;
        • each Rd is independently H, halo, C1-6 alkyl, substituted C1-6 alkyl, C1-6 alkoxy, substituted C1-6 alkoxy, —NH2, —NH(C1-6 alkyl), —NH(substituted C1-6 alkyl), —N(C1-6 alkyl)2, —N(substituted C1-6 alkyl)2, C6-10 aryl, or heterocyclyl;
        • each Re is independently H, C1-6 alkyl, substituted C1-6 alkyl, C6-10 aryl, substituted C6-10 aryl, heterocyclyl, or substituted heterocyclyl;
        • each Rf is independently H, C1-6 alkyl, substituted C1-6 alkyl, —C(O)Rd, phosphate, diphosphate, or triphosphate;
        • each n is independently 0, 1, 2, or 3;
        • each p is independently 0, 1, or 2; or
      • or (g) a pharmaceutically acceptable salt of any of (a) to (f), a tautomer of any of (a) to (f), or a pharmaceutically acceptable salt of the tautomer.
  • Q. Lipids Linked to a Phosphate-Containing Acyclic Backbone
  • Adjuvants containing lipids linked to a phosphate-containing acyclic backbone include the TLR4 antagonist E5564 [136,137]:
  • Figure US20120058146A1-20120308-C00007
  • R. Small Molecule Immunopotentiators (SMIPs)
  • SMIPs include:
      • N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2,N2-dimethyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-ethyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-methyl-1-(2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • 1-(2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-butyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-butyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-methyl-1-(2-methylpropyl)-N2-pentyl-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-methyl-1-(2-methylpropyl)-N2-prop-2-enyl-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • 1-(2-methylpropyl)-2-[(phenylmethyl)thio]-1H-imidazo[4,5-c]quinolin-4-amine;
      • 1-(2-methylpropyl)-2-(propylthio)-1H-imidazo[4,5-c]quinolin-4-amine ;
      • 2-[[4-amino-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl](methyl)amino]ethanol;
      • 2-[[4-amino-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl](methyl)amino]ethyl acetate;
      • 4-amino-1-(2-methylpropyl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one;
      • N2-butyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-butyl-N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • N2,N2-dimethyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine;
      • 1-{4-amino-2-[methyl(propyl)amino]-1H-imidazo[4,5-c]quinolin-1-yl}-2-methylpropan-2-ol;
      • 1-[4-amino-2-(propylamino)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol;
      • N4,N4-dibenzyl-1-(2-methoxy-2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine.
  • S. Proteosomes
  • One adjuvant is an outer membrane protein proteosome preparation prepared from a first Gram-negative bacterium in combination with a liposaccharide preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteosome and liposaccharide preparations form a stable non-covalent adjuvant complex. Such complexes include “IVX-908”, a complex comprised of Neisseria meningitidis outer membrane and lipopolysaccharides. They have been used as adjuvants for influenza vaccines [138].
  • T. Other Adjuvants
  • Other substances that act as immunostimulating agents are disclosed in references 53 and 58.
  • Further useful adjuvant substances include:
      • Methyl inosine 5′-monophosphate (“MIMP”) [139].
      • A polyhydroxlated pyrrolizidine compound [140], such as one having formula:
  • Figure US20120058146A1-20120308-C00008
        • where R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof. Examples include, but are not limited to: casuarine, casuarine-6-α-D-glucopyranose, 3-epi-casuarine, 7-epi-casuarine, 3,7-diepi-casuarine, etc.
      • A gamma inulin [141] or derivative thereof, such as algammulin.
      • Compounds disclosed in reference 142.
      • Compounds disclosed in reference 143, including: Acylpiperazine compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ) compounds, Benzocyclodione compounds, Aminoazavinyl compounds, Aminobenzimidazole quinolinone (ABIQ) compounds [144,145], Hydrapthalamide compounds, Benzophenone compounds, Isoxazole compounds, Sterol compounds, Quinazilinone compounds, Pyrrole compounds [146], Anthraquinone compounds, Quinoxaline compounds, Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole compounds [147].
      • Loxoribine (7-allyl-8-oxoguanosine) [148].
      • A formulation of a cationic lipid and a (usually neutral) co-lipid, such as aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine (“Vaxfectin™”) or aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine (“GAP-DLRIE:DOPE”). Formulations containing (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium salts are preferred [149].
  • The invention may also comprise combinations of one or more of the adjuvants identified above. For example, the following combinations may be used as adjuvant compositions in the invention: (1) a saponin and an oil-in-water emulsion [150]; (2) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [151]; (3) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) [152]; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [153]; (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dMPL); and (9) one or more mineral salts (such as an aluminum salt)+an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif).
  • The compositions of the invention will preferably elicit both a cell mediated immune response as well as a humoral immune response in order to effectively address a uropathogenic infection. This immune response will preferably induce long lasting (e.g. neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to UPEC-associated antigens.
  • 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). 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. 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-γ, and TNF-β. 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 particular 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. An enhanced 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 EL-2, IFN-γ, and TNF-β), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. Preferably, the enhanced TH1 immune response will include an increase in IgG2a production. An enhanced 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. Preferably, the enhanced TH2 immune response will include an increase in IgG1 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. Immunostimulatory oligonucleotides, such as oligonucleotides containing a CpG motif, are preferred TH1 adjuvants for use in the invention.
  • 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 hereof. Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use in the invention.
  • Preferably, the invention includes a composition comprising a combination of a TH1 adjuvant and a TH2 adjuvant. Preferably, such 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. Still more preferably, 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. Preferably, 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. Preferably, the immune response provides for one or both of an enhanced systemic and an enhanced mucosal immune response. Preferably the mucosal immune response is a TH2 immune response. Preferably, the mucosal immune response includes an increase in the production of IgA.
  • The use of an aluminium hydroxide or aluminium phosphate adjuvant is particularly preferred, and antigens are generally adsorbed to these salts. Calcium phosphate is another preferred adjuvant.
  • The pH of compositions of the invention is preferably between 6 and 8, preferably about 7. Stable pH may be maintained by the use of a buffer. Where a composition comprises an aluminium hydroxide salt, it is preferred to use a histidine buffer [154]. The composition may be sterile and/or pyrogen-free. Compositions of the invention may be isotonic with respect to humans
  • Compositions may be presented in vials, or they may be presented in ready-filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses. Injectable compositions will usually be liquid solutions or suspensions. Alternatively, they may be presented in solid form (e.g. freeze-dried) for solution or suspension in liquid vehicles prior to injection.
  • Compositions of the invention may be packaged in unit dose form or in multiple dose form. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5 ml.
  • Where a composition of the invention is to be prepared extemporaneously prior to use (e.g. where a component is presented in lyophilised form) and is presented as a kit, the kit may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
  • Thus the invention provides for a kit comprising a first component and a second component, wherein: the first component comprises one or more polypeptide, antibody, vesicle and/or nucleic acid of the invention; and the second component comprises one or more of the following: instructions for administering a composition to a patient, a syringe or other delivery device, an adjuvant, and/or a pharmaceutically acceptable formulating solution.
  • The invention also provides a delivery device (e.g. a syringe) pre-filled with the immunogenic compositions of the invention.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials, and a typical quantity of each meningococcal saccharide antigen per dose is between 1 μg and 10 mg per antigen.
  • Pharmaceutical Uses
  • The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition of the invention. The patient may either be at risk from the disease themselves or may be a pregnant woman (‘maternal immunisation’ [155]).
  • The invention provides nucleic acid, polypeptide, vesicle or antibody of the invention for use as medicaments (e.g. as immunogenic compositions or as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, polypeptide, vesicle or antibody of the invention in the manufacture of: (i) a medicament for treating or preventing disease and/or infection caused by an ExPEC bacterium; (ii) a diagnostic reagent for detecting the presence of or of antibodies raised against an ExPEC bacterium; and/or (iii) a reagent which can raise antibodies against an ExPEC bacterium. Said ExPEC bacterium can be of any serotype or strain. Preferably the ExPEC bacterium is a UPEC strain.
  • The invention is useful for the prevention and/or treatment of diseases such as bacteremia, meningitis, a urinary tract infection, pyelonephritis and/or cystitis. The invention is particularly useful for the treatment of urinary tract infections.
  • The patient is preferably a human. The human is preferably an adult (e.g. aged between 20 and 55). A vaccine intended for children or adolescents may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. Female patients are a preferred subset, with sexually-active females aged 20-55 being a particularly preferred patient group. Another groups of patients is females aged 12-20, particularly for prophylactic use.
  • Other possible patient animals include dogs, which may be carriers of ExPEC [156,157].
  • One way of checking efficacy of therapeutic treatment involves monitoring infection after administration of the composition of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses against an administered polypeptide after administration. Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age, or animal models e.g. a mouse model) and then determining standard parameters including ELISA titres (GMT) of IgG. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made. Various mouse models of UTI are available [e.g. refs. 158 & 159-160].
  • Administration of polypeptide antigens is a preferred method of treatment for inducing immunity. Administration of antibodies of the invention is another preferred method of treatment. This method of passive immunisation is particularly useful for newborn children or for pregnant women. This method will typically use monoclonal antibodies, which will be humanised or fully human.
  • Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal, transcutaneous, intranasal, sublingual, ocular, aural, pulmonary or other mucosal administration. Intramuscular administration to the thigh or the upper arm is preferred. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 ml.
  • The invention may be used to elicit systemic and/or mucosal immunity. Preferably the enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response. Preferably, the enhanced immune response includes an increase in the production of IgG1 and/or IgG2a and/or IgA.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose 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. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined. For example, a primary course of vaccination may include 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reinforce an immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose or doses after several months.
  • Bacterial infections affect various areas of the body and so compositions may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised or a spray-freeze dried composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, as a spray or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as spray, drops, gel or powder [e.g. refs 161 & 162]. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
  • Compositions 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) other vaccines e.g. at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H.influenzae type b vaccine, a human papillomavirus vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a pneumococcal conjugate vaccine, a meningococcal conjugate vaccine, etc. Similarly, they may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional) an antibiotic, and in particular an antibiotic compound active against UPEC.
  • Further Antigenic Components of Compositions of the Invention
  • The invention also provides a composition comprising a polypeptide or the invention and one or more of the following further antigens:
      • a saccharide antigen from N.meningitidis serogroup A, C, W135 and/or Y (preferably all four), such as the oligosaccharide disclosed in ref. 163 from serogroup C [see also ref. 164] or the oligosaccharides of ref. 165.
      • an antigen from N.meningitidis serogroup B such as those disclosed in refs. 166-174, etc.
      • a saccharide antigen from Streptococcus pneumoniae [e.g. 175, 176, 177].
      • an antigen from hepatitis A virus, such as inactivated virus [e.g. 178, 179].
      • an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 179, 180].
      • an antigen from hepatitis C virus [e.g. 181].
      • an antigen from HIV [182]
      • a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 183] e.g. the CRM197 mutant [e.g. 184].
      • a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 183].
      • an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 185 & 186].
      • a saccharide antigen from Haemophilus influenzae B [e.g. 164].
      • polio antigen(s) [e.g. 187, 188] such as IPV.
      • measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 183].
      • varicella antigens.
      • influenza antigen(s) [e.g. chapter 19 of ref. 183], such as the haemagglutinin and/or neuraminidase surface proteins. Influenza antigens may be derived from interpandemic (annual) flu strains. Influenza antigens may be derived from strains with the potential to cause a pandemic outbreak (i.e., influenza strains with new haemagglutinin compared to the haemagglutinin in currently circulating strains, or influenza strains which are pathogenic in avian subjects and have the potential to be transmitted horizontally in the human population, or influenza strains which are pathogenic to humans). Influenza antigens may be derived from viruses grown in eggs or cell culture.
      • an antigen from Moraxella catarrhalis [e.g. 189].
      • a saccharide antigen from Streptococcus agalactiae (group B streptococcus).
      • an protein antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 190-195]
      • an antigen from N.gonorrhoeae [e.g. 196-199].
      • an antigen from Chlamydia pneumoniae [e.g. refs. 200 to 206] or a combination of antigens from C.pneumoniae [e.g. 207].
      • an antigen from Chlamydia trachomatis, or a combination of antigens from C. trachomatis [e.g. 208].
      • an antigen from Porphyromonas gingivalis [e.g. 209].
      • rabies antigen(s) [e.g. 210] such as lyophilised inactivated virus [e.g. 211, RabAvert™].
      • antigen(s) from a paramyxovirus such as respiratory syncytial virus (RSV [212, 213]) and/or parainfluenza virus (PIV3 [214]).
      • an antigen from Bacillus anthracis [e.g. 215, 216, 217].
      • an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 191,218, 219].
      • an antigen from Staphylococcus aureus [e.g. 220].
      • an antigen from a virus in the flaviviridae family (genus flavivirus), such as from yellow fever virus, Japanese encephalitis virus, four serotypes of Dengue viruses, tick-borne encephalitis virus, West Nile virus.
      • a pestivirus antigen, such as from classical porcine fever virus, bovine viral diarrhoea virus, and/or border disease virus.
      • a parvovirus antigen e.g. from parvovirus B19.
      • a human papilloma virus (HPV) antigen [221]
  • The composition may comprise one or more of these further antigens.
  • In another embodiment, antigens of the invention are combined with one or more additional, non-E.coli antigens suitable for use in a vaccine designed to protect females against genitourinary and/or sexually transmitted diseases. For example, the antigens may be combined with an antigen derived from the group consisting of Streptococcus agalactiae, Chlamydia trachomatis, Neisseria gonorrhoeae, papillomavirus and herpes simplex virus. Where human papillomavirus antigens are used, they may be from one or more of HPV 16, HPV 18, HPV 6 and/or HPV 11.
  • Preferred Gonococcal antigens include one or more of ngs13 (OmpA), OmpH, ngs576 (peptidyl-prolyl cis/trans isomerase (PPIase) protein), ngs41 and ngs117.
  • Preferred HPV antigens include one or more of HPV 16, HPV 18, HPV 6 and HPV 11.
  • Preferred Chlamydia trachomatis antigens include one or more of: CT045, CT089, CT242, CT316, CT381. CT396, CT398, CT444, CT467, CT547, CT587, CT823, CT761 and specific combinations of these antigens as disclosed in WO 05/002619.
  • Preferred Chlamydia pneumoniae antigens include one or more of: CPn0324, Cpn0301, Cpn0482, Cpn0503, Cpn0525, Cpn0558, Cpn0584, Cpn0800, Cpn0979, Cpn0498, Cpn0300, Cpn0042, Cpn0013, Cpn450, Cpn0661, Cpn0557, Cpn0904, Clpn0795, Cpn0186 and Cpn0604 and specific combinations of these antigens as disclosed in WO 05/084306.
  • Preferred GBS antigens include one or more of GBS80, GBS 104, GBS 59, GBS 67, GBS 322 and GBS 276.
  • In another embodiment, the antigen combinations of the invention are combined with one or more additional, non-ExPEC antigens suitable for use in a vaccine designed to protect elderly or immunocompromised individuals. For example, the antigen combinations may be combined with an antigen derived from the group consisting of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitidies, influenza, and Parainfluenza virus (‘PIV’).
  • Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [186]).
  • Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
  • Saccharide antigens are preferably in the form of conjugates. Carrier proteins for the conjugates include bacterial toxins (such as diphtheria toxoid or tetanus toxoid), the N.meningitidis outer membrane protein [222], synthetic peptides [223,224], heat shock proteins [225,226], pertussis proteins [227,228], protein D from H.influenzae [229,230], cytokines [231], lymphokines [231], H.influenzae proteins, hormones [231], growth factors [231], toxin A or B from C.difficile [232], iron-uptake proteins [233], artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens [234] such as the N19 protein [235], pneumococcal surface protein PspA [236], pneumolysin [237], etc. A preferred carrier protein is CRM197 protein [238].
  • Antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
  • Antigens are preferably adsorbed to an aluminium salt.
  • Nucleic Acid Immunisation
  • The immunogenic compositions described above include polypeptide antigens from UPEC. As an alternative to using proteins antigens in the immunogenic compositions of the invention, nucleic acid (preferably DNA e.g. in the form of a plasmid) encoding the antigen may be used, to give compositions, methods and uses based on nucleic acid immunisation. Nucleic acid immunisation is now a developed field (e.g. see references 239 to 246 etc.), and has been applied to many vaccines.
  • The nucleic acid encoding the immunogen is expressed in vivo after delivery to a patient and the expressed immunogen then stimulates the immune system. The active ingredient will typically take the form of a nucleic acid vector comprising: (i) a promoter; (ii) a sequence encoding the immunogen, operably linked to the promoter; and optionally (iii) a selectable marker. Preferred vectors may further comprise (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). In general, (i) & (v) will be eukaryotic and (iii) & (iv) will be prokaryotic.
  • Preferred promoters are viral promoters e.g. from cytomegalovirus (CMV). The vector may also include transcriptional regulatory sequences (e.g. enhancers) in addition to the promoter and which interact functionally with the promoter. Preferred vectors include the immediate-early CMV enhancer/promoter, and more preferred vectors also include CMV intron A. The promoter is operably linked to a downstream sequence encoding an immunogen, such that expression of the immunogen-encoding sequence is under the promoter's control.
  • Where a marker is used, it preferably functions in a microbial host (e.g. in a prokaryote, in a bacteria, in a yeast). The marker is preferably a prokaryotic selectable marker (e.g. transcribed under the control of a prokaryotic promoter). For convenience, typical markers are antibiotic resistance genes.
  • The vector of the invention is preferably an autonomously replicating episomal or extrachromosomal vector, such as a plasmid.
  • The vector of the invention preferably comprises an origin of replication. It is preferred that the origin of replication is active in prokaryotes but not in eukaryotes.
  • Preferred vectors thus include a prokaryotic marker for selection of the vector, a prokaryotic origin of replication, but a eukaryotic promoter for driving transcription of the immunogen-encoding sequence. The vectors will therefore (a) be amplified and selected in prokaryotic hosts without polypeptide expression, but (b) be expressed in eukaryotic hosts without being amplified. This arrangement is ideal for nucleic acid immunization vectors.
  • The vector of the invention may comprise a eukaryotic transcriptional terminator sequence downstream of the coding sequence. This can enhance transcription levels. Where the coding sequence does not have its own, the vector of the invention preferably comprises a polyadenylation sequence. A preferred polyadenylation sequence is from bovine growth hormone.
  • The vector of the invention may comprise a multiple cloning site.
  • In addition to sequences encoding the immunogen and a marker, the vector may comprise a second eukaryotic coding sequence. The vector may also comprise an IRES upstream of said second sequence in order to permit translation of a second eukaryotic polypeptide from the same transcript as the immunogen. Alternatively, the immunogen-coding sequence may be downstream of an IRES.
  • The vector of the invention may comprise unmethylated CpG motifs e.g. unmethylated DNA sequences which have in common a cytosine preceding a guanosine, flanked by two 5′ purines and two 3′ pyrimidines. In their unmethylated form these DNA motifs have been demonstrated to be potent stimulators of several types of immune cell.
  • Vectors may be delivered in a targeted way. Receptor-mediated DNA therapy techniques are described in, for example, references 247 to 252. Therapeutic compositions containing a nucleic acid are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g. for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy. Where greater expression is desired over, a larger area of tissue, larger amounts of vector or the same amounts re-administered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.
  • Vectors can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally references 253 to 256).
  • Viral-based vectors for delivery of a desired nucleic acid and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (e.g. references 257 to 267), alphavirus-based vectors (e.g. Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532); hybrids or chimeras of these viruses may also be used), poxvirus vectors (e.g. vaccinia, fowlpox, canarypox, modified vaccinia Ankara, etc.), adenovirus vectors, and adeno-associated virus (AAV) vectors (e.g. see refs. 268 to 273). Administration of DNA linked to killed adenovirus [274] can also be employed.
  • Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone [e.g. 274], ligand-linked DNA [275], eukaryotic cell delivery vehicles cells [e.g. refs. 276 to 280] and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in refs. 281 and 282. Liposomes (e.g. immunoliposomes) that can act as gene delivery vehicles are described in refs. 283 to 287. Additional approaches are described in references 288 & 289.
  • Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in ref. 289. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation [e.g. refs. 290 & 291]. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun [292] or use of ionizing radiation for activating transferred genes [290 & 293].
  • Delivery DNA using PLG {poly(lactide-co-glycolide)} microparticles is a particularly preferred method e.g. by adsorption to the microparticles, which are optionally treated to have a negatively-charged surface (e.g. treated with SDS) or a positively-charged surface (e.g. treated with a cationic detergent, such as CTAB).
  • General
  • The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
  • The term “about” in relation to a numerical value x means, for example, x±10%.
  • The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • The N-terminus residues in the amino acid sequences in the sequence listing are given as the amino acid encoded by the first codon in the corresponding nucleotide sequence. Where the first codon is not ATG, it will be understood that it will be translated as methionine when the codon is a start codon, but will be translated as the indicated non-Met amino acid when the sequence is at the C-terminus of a fusion partner. The invention specifically discloses and encompasses each of the amino acid sequences of the sequence listing having a N-terminus methionine residue (e.g. a formyl-methionine residue) in place of any indicated non-Met residue. It also specifically discloses and encompasses each of the amino acid sequences of the sequence listing starting at any internal methionine residues in the sequences.
  • As indicated in the above text, nucleic acids and polypeptides of the invention may include sequences that:
      • (a) are identical (i.e. 100% identical) to the sequences disclosed in the sequence listing;
      • (b) share sequence identity with the sequences disclosed in the sequence listing;
      • (c) have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single nucleotide or amino acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b); and
      • (d) when aligned with a particular sequence from the sequence listing using a pairwise alignment algorithm, a moving window of x monomers (amino acids or nucleotides) moving from start (N-terminus or 5′) to end (C-terminus of 3′), such that for an alignment that extends to p monomers (where p>x) there are p-x+1 such windows, each window has at least x·y identical aligned monomers, where: x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and if x·y is not an integer then it is rounded up to the nearest integer. The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [294], using default parameters (e.g. with Gap opening penalty=10.0, and with Gap extension penalty=0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [295].
  • The nucleic acids and polypeptides of the invention may additionally have further sequences to the N-terminus/5′ and/or C-terminus/3′ of these sequences (a) to (d).
  • The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 296-303, etc.
  • Experimental
  • Below are examples of specific embodiments or modes for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
  • Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
  • MODES FOR CARRYING OUT THE INVENTION Computer-based comparative and predictive tools were used to identify 596 polypeptides that are (a) common to at least two UPEC strains but are not found in non-pathogenic strains, and (b) surface- or membrane-associated. These 596 polypeptides are listed in Table 1, and their amino acid sequences are in the sequence listing as SEQ ID NOS: 1 to 596. Table 1 gives the ‘gi’ (GenInfo Identifier) accession numbers for these 596 sequences [11], and this information can be used to retrieve information including (a) the full sequence database record for the polypeptide and (b) the corresponding coding sequence from within the E.coli genome. For example, the NCBI Entrez system [304] can be queried with ‘26111674’ to give a single record [305], and the ‘CDS’ link within that record can be clicked to reveal the corresponding coding sequence [306].
  • As the polypeptide sequences of the invention are already available in public databases, their annotated functions are also available. Some of the 596 polypeptides have no recognised function in the current annotation (e.g. they are annotated in the databases as ‘hypothetical protein’). Although the inventors have not elucidated the basic underlying biological function of these polypeptides, the invention does now provide a credible utility for these polypeptides, namely in the provision of immunogenic compositions as described herein.
  • Table 4 reports the closest matches to SEQ ID NOS: 1-596 in the non-pathogenic K12 sequences for strains MG1655 [307], W3110 and DH10B.
  • Table 5 shows the 414/596 polypeptides with the strongest matches between two different UPEC genomes, with the underlined SEQ ID numbers being those with 100% conservation. The remaining 182 polypeptides are more-weakly conserved between UPEC strains, and so the Table 5 polypeptides are preferred (particularly underlined ones).
  • Of the 596 sequences, 156 were selected in preference (Table 2). Table 6 shows various pieces of information for these 156 sequences.
  • 66 further preferred antigens were selected (Table 3) as being absent from non-pathogenic strains, surface-associated, present in at least two UPEC strains, and not previously identified as antigens. 19 of these were selected for initial work (marked with ‘+’ in Table 3).
  • These polypeptides are cloned, expressed and purified. The purified antigens are used to immunise mice, whose sera are analysed by Western blot, ELISA and FACS, and are further tested in both in vitro and in vivo experiments. Suitable in vitro experiments include testing the ability of antibodies to induce complement-mediated bacterial killing and/or opsonophagocytosis activity, to block binding of ExPEC strains (or the purified antigen) to human epithelial cells (e.g. in bladder cells) or other cell lines, and/or to inhibit adhesion/invasion of E.coli bacteria (e.g. K1 strain) to brain microvascular endothelial cells (BMEC). Suitable in vivo experiments include active and/or passive systemic immunisations and challenge in mouse models of UTI (adult mice), protection by active or passive immunisations against bacteremia and meningitis in 5-day-old rats challenged with E.coli K1 strain, and immunisation and intraperitoneal infection of adult mice with an ExPEC strain.
  • The importance of the proteins to the bacterial life-cycle is tested by creating isogenic knockout mutants. The mutants can also be used to ensure that sera raised by an antigen are specific for that antigen. Microarrays are used to study expression patterns. Conservation and/or variability is assessed by sequencing the genes from multiple different ExPEC strains.
  • Assays were carried out in order to select predicted surface-exposed proteins, which are specific for UPEC strains and absent in non-pathogenic strains (commensal and laboratory strains). Once selected these proteins are expressed and purified and used to immunize mice.
  • It is known from reference 43 that a mutation in any of the tol-pal genes of E. coli results in the formation of vesicles containing native outer membrane proteins. By comparing the proteins present in vesicles of UPEC strains and non-pathogenic strains it is possible to select a small group of proteins that could be used as potential antigens.
  • Lambda Red-Mediated Gene Manipulation in Commensal and Pathogenic E. coli
  • This method is a rapid PCR-based method used to inactivate the tolR gene from the wild-type E. coli strains [308]. Briefly, the first step consists in amplifying independently the upstream and downstream regions of the target gene (tolR) and the resistance marker cassette. The two PCR products obtained in step 1 are mixed with the amplification producer of the AB cassette at equimolar concentrations and submitted to a second round of PCR (a three way PCR) to generate a resistance marker cassette flanked by upstream and downstream 500 bp (or more) regions homologous to the target gene. In the third step, large amounts (1 μg) of the desired linear DNA are electroporated into lamda-red competent cells.
  • Vesicle Preparation
  • 1. Vesicle Preparation by Precipitation with TCA
  • LB media was inoculated with bacteria grown on plates and incubated overnight at 37° C. under gentle shaking. The culture was used to inoculate 200 ml of LB at OD600 0.1. Bacteria were grown to OD600 0.4 (or as specified). Culture was centrifuged for 10 minutes at 4000×g and the supernatant was filtered through a 0.22 mm filter to remove residual bacteria.
  • The same experiments were also performed under iron limiting conditions by adding Dipyridyl (0.25 mM) to the LB media.
  • Precipitation was performed by adding to the culture supernatant 10% final of a solution at 100% (w/v) TCA, 0.4% (w/v) deoxycholate. The precipitation was allowed to proceed for 30 minutes at 4° C. Precipitate was recovered by 10 minutes centrifugation at 20000×g at 4° C. The pellet was washed once with 10% TCA (w/v) and twice with absolute ethanol. The pellet was dried with speed vac, and stored at −20° C.
  • The wild type and mutated strains were subjected to SDS polyacrylamide gel electrophoresis from which it could be observed that there were many more bands in the supernatant of the mutated strains than the wildtype strains. Randomly picked bands demonstrated that all the proteins in the supernatant were membrane proteins.
  • 2. Vesicle Preparation by Ultracentrifugation
  • Culture supernatant was ultracentrifuged at 200000×g for 2 hours at 4° C. The pellet was washed with PBS, resuspended in PBS, and stored at −20° C.
  • 3. Guanidinium Denaturation of the Vesicles
  • Prior to the guanidinium denaturation, Vesicles were precipitated with ethanol. 10 μg of OMV in PBS were precipitate by adding cold absolute ethanol to 90% final. Precipitation was allowed to proceed for 20 minutes at −20° C. Precipitate was recovered by 10 minutes centrifugation at 13000×g. Pellet was resuspended with 50 ml, 6M guanidinium, 15 mM DTT, 200 mM Tris-HCl, pH 8.0. Denaturation was allowed to proceed for 60 minutes at 60° C. Prior to digestion, solution was diluted 1/8 with a solution of 1.5M Tris pH 8.0 and 5 mg of trypsin were added to the diluted solution. Digestion was allowed to proceed overnight at 37° C. Reaction was stopped by adding 0.1% final of formic acid. Peptides were extracted using Oasis extraction cartridges. Peptides were analyzed by LC coupled MS-MS.
  • 4. Surface Digestion
  • 5 mg of trypsin were added to 10 mg of vesicles in PBS and incubated at 37° C. for 3 hours. Reaction was stopped by adding 0.1% final of formic acid. Peptides were recovered by filtration through a 30 Kda cut-off filter and extracted with Oasis extraction cartridge. Peptides were analyzed with LC coupled MSMS.
  • Vesicle Analysis
  • Protein Quantification
  • Proteins were quantified with the Bradford method, using the BSA as standard.
  • SDS-PAGE
  • Samples were analyzed with a sodium dodecyl sulfate (SDS) 4-12% polyacrylamide gel, using a Mini-Protean II electrophoresis apparatus. Samples were suspended in SDS sample buffer (0.06 M Tris-HCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10 mg/ml bromophenol blue) and heated to 100° C. for 5 min before SDS-polyacrylamide gel electrophoreis. After the run, gels were stained with Coomassie Blue
  • MALDI-TOF Mass Spectrometry.
  • Protein bands or spots were excised from gels, washed with 50 mM ammonium bicarbonate/acetonitrile (50/50, v/v), and dried with a Speed-Vac centrifuge (Savant). The dried spots were digested at 37° C. for 2 h by adding 7 to 1,0 ml of a solution containing 5 mM ammonium bicarbonate, 0.012 mg of sequencing-grade trypsin. After digestion 0.6 ml were loaded on a matrix pre-spotted target and air-dried. Spots were washed with 0.6 ml of a solution of 70% ethanol, 0.1% trifluoracetic acid. Mass spectra were acquired on an ultraflex MALDI TOF mass spectrometer. Spectra were externally calibrated by using a combination of standards pre-spotted on the target. Protein identification was carried out by both automatic and manual comparisons of experimentally generated monoisotopic peaks of peptides in the mass range of 700 to 3,000 Da with computer-generated fingerprints, using the Mascot program.
  • Bi-Dimensional Electrophoresis
  • 200 mg of vesicles were resuspended in an Immobiline re-swelling solution (7M urea, 2M thiourea, 2% (w/v) CHAPS (2% w/v) ASB14, 2% (v/v) IPG buffer pH 3-10 NL, 2 mM TBP, 65 mM DTT), and adsorbed overnight on 7 cm Immobiline DryStrips (pH 3-10 NL). Proteins were then separated by 2D electrophoresis. The first dimension was run using a IPGphor Isoelectric Focusing Unit, applying sequentially 150 V for 35 minutes, 500 V for 35 minutes, 1,000 V for 30 minutes, 2,600 V for 10 minutes, 3,500 V for 15 minutes, 4,200 V for 15 minutes, and finally 5,000 V to reach 10 kVh. For the second dimension, the strips were equilibrated by two 10 minute -incubations in 4 M urea, 2 M thiourea, 30% glycerol, 2% SDS, 5 mM TBP, 50 Mm Tris HCl pH 8.8, 2.5% acrylamide, Bromo phenol Blue 0.2%: Proteins were then separated on linear 4-12% precasted polyacrylamide gels.
  • Gels were stained with colloidal Coomassie Blue and scanned with a Personal Densitometer SI. Images were analyzed with Image Master 2D Elite software.
  • Nano-LC/MS/MS
  • Peptides were separated by nano-LC on a CapLC HPLC system connected to a Q-ToF Micro ESI mass spectrometer equipped with a nanospray source. Samples were loaded onto an Atlantis C18 NanoEase column (100 μm i.d.×100 mm), through a C18 trap column (300 μm i.d.×5 mm). Peptides were eluted with a 50-min gradient from 2% to 60% of 95% ACN, in a solution of 0.1% formic acid at a flow rate of 400 nl/minute. The eluted peptides were subjected to an automated data-dependent acquisition program, using the MassLynx software, version 4.0, where a MS survey scan was used to automatically select multi-charged peptides over the m/z range of 400-2,000 for further MS/MS fragmentation. Up to three different components where subjected to MS/MS fragmentation at the same time. After data acquisition, the individual MS/MS spectra were combined, smoothed and centroided by MassLynx. Search and identification of peptides were performed in batch mode with a licensed version of MASCOT. The MASCOT search parameters were: (1) species: ExPEC (2) allowed number of missed cleavages (only for trypsin digestion): 6; (3) variable post-translational modifications: methionine oxidation; (4) peptide tolerance: ±500 ppm; (5) MS/MS tolerance: ±0.3 Da and (6): peptide charge: from +1 to +4. As for the previous platform, only significant hits as defined by MASCOT probability analysis were considered. The score thresholds for acceptance of protein identifications from at least one peptide were set by MASCOT as 18 for trypsin digestion and 36 for proteinase K digestion.
  • Results
  • As a result of the above analyses, 13 preferred antigens were identified from the CFT073 strain. Namely: gi-26110866 (SEQ ID No 489), gi-26109898 (SEQ ID No 597), gi-26107513 (SEQ ID No 120), gi-26108604 (SEQ ID No 598), gi-26109137 (SEQ ID No 305), gi-26106493 (SEQ ID No 22), gi-26108194 (SEQ ID No 221), gi-26108192 (SEQ ID No 219), gi-26109931 (SEQ ID No 400), gi-26111428 (SEQ ID No 565), gi-26109835 (SEQ ID No 371), gi-26109866 (SEQ ID No 599) and gi-26111414 (SEQ ID No 555). These are listed in Table 7.
  • Antigen Analysis
  • Mouse Model of Systemic Infection
  • To screen a large number of antigens selected by comparative genome analysis between pathogenic and non pathogenic E. coli strains, a protection model based on a classical virulence assay has been established. Alternative experimental models that may also be used include those outlined in references 158,159 and 160.
  • The experimental model (immunization and infection) uses 5 week old—CD1 outbreed mice which are challenged with intraperitoneal inoculation of virulent CFT073 E. coli strain. The challenge dose has been experimentally determined as the amount of bacteria able to kill 80% of adult mice within 72 hours and corresponds to 7×107 cfu/mouse for the CFT073 strain.
  • Immunization Protocol
  • Mice are immunized three times by subcutaneous injection of 150 μl of protein solution using freund's adjuvants as shown in the table below:
  • Control mice: Immunized mice:
    Day 0 75 μl of saline solution 75 μl of protein solution
    75 μl of complete freund's (20 μg)
    adjuvant 75 μl of complete freund's
    adjuvant
    Day 21 75 μl of saline solution 75 μl of protein solution
    75 μl of incomplete freund's (20 μg)
    adjuvant 75 μl of incomplete freund's
    adjuvant
    Day 35 75 μl of saline solution 75 μl of protein solution
    75 μl of incomplete freund's (20 μg)
    adjuvant 75 μl of incomplete freund's
    adjuvant
  • Blood samples are colleccted the day before the first immunization (preimmune serum), at day 34 and 48 (day before challenge). Sera from immunized animals are tested by western blot and ELISA to determine the antibodies titer.
  • Challenge
  • At day 48 E.coli CFT073 strain is streaked on LB agar plate from frozen stock and incubated overnight (ON) at 37° C. in incubator. At Day 49 the ON plate-culture is used to inoculate 50 ml of LB medium to have an O.D.600=0.1, and grown for 1.5 hours at 37° C. under agitation until the bacterial culture reaches an O.D.600=0.6 which corresponds to 7×108 cfu/ml for the CFT073 strain. The culture is centrifuged and the pellet resuspended in the same volume with physiological solution and used for challenge undiluted. The culture is plated using a standard plate count method to verify the inoculum. 100 μl of the cell suspension containing 7×107 CFT073 bacteria is injected intraperitoneally, using a 1 ml syringe, to control and immunized mice The number of deaths in each animal group at 24, 48 and 72 hours after infection are recorded.
  • The protection due to vaccination is evaluated by comparison of the survival in the vaccinated group and the survival in control group of mice at 72 hours from the challenge. Percentage of survival relative to controls is calculated using the formula:
  • rate of survival in vaccine group - rate of survival in control group rate of survival in control group
  • Results
  • Immunization was carried out with heat-inactivated CFT073. As can be seen in FIG. 1, the % survival of the mice after challenge with CFT073 is increased following immunization with heat-inactivated CFT073.
  • Immunisation Studies
  • Antigens are selected for combining to give a composition of the invention. BALB/c mice are divided into nine groups and immunized as follows:
  • Group Immunizing Composition Route of Delivery
    1 Mixture of antigens (10-20 μg Intra-peritoneal or intra-nasal
    protein/each) + CFA (Complete or subcutaneous
    Freund's Adjuvant)
    2 Mixture of antigens (5 μg/ Intra-peritoneal or intra-nasal
    each) + Al-hydroxide (200 μg) or subcutaneous
    3 Mixture of antigens (10-20 μg Intra-peritoneal or intra-nasal
    protein/each) + CpG (10 ug) or subcutaneous
    4 Mixture of antigens (10-20 μg Intra-peritoneal or intra-nasal
    protein/each) + Al-hydroxide or subcutaneous
    (200 μg) + CpG (10 μg)
    5 CFA Intra-peritoneal or intra-nasal
    or subcutaneous
    6 Mixture of antigens (10-20 μg Intra-peritoneal or Intranasal
    protein/each) + LTK63 (5 μg) or subcutaneous
    7 Al-hydroxide (200 μg) + Intra-peritoneal or intra-nasal
    CpG (10 μg) or subcutaneous
    8 CpG (10 μg) Intra-peritoneal or intra-nasal
    or subcutaneous
    9 LTK63 (5 μg) Intra-peritoneal or intra-nasal
    or subcutaneous
  • Mice are immunized at two-week intervals. Two to three weeks after the last immunization, all mice are challenged with the appropriate UPEC strain. When mucosal immunization (e.g. intranasal) is used, the animal model is also challenged mucosally to test the protective effect of the mucosal immunogen. Immediately prior to challenge, mice are bled to determine antibody titre to the antigens that were administered.
  • For the mouse challenge, virulent bacteria will be grown in appropriate media. Bacteria are harvested by centrifugation, re-suspended, and serially diluted for the challenge inoculum. BALB/c mice are challenged and observed daily for 30 days post-exposure.
  • Total IgG and IgG1/IgG2A subtypes can be measured in mouse sera resulting from the different immunization regimens by using an ELISA assay on whole bacteria and on purified recombinant proteins. Furthermore, assessment of antigen-specific CD4+ and CD8+Th-cells in spleen cells and/or PBMC isolated from immunized mice can be carried out by multi-parametric FACS analysis, to evaluate the cytokine expression profiles of antigen-specific T-cells. In particular production of IFN-γ and IL-5 can be measured after in vitro stimulation of T cells with purified antigens. In addition, splenocytes and/or PBMC from mice immunized with each antigen/vaccine formulation may be collected 10-12 days after the last immunization dose and stimulated with UPEC bacteria. After 4 hours of stimulation, Brefeldin A is added to the cells for the following 12 hours, to block cytokine secretion. Afterwards cells are fixed and stained with antibodies to detect UPEC-specific T cells expressing IFN-γ and IL-5.
  • T cells can be isolated from peripheral blood lymphocytes (PBLs) by a variety of procedures known to those skilled in the art. For example, T cell populations can be “enriched” from a population of PBLs through the removal of accessory and B cells. In particular, T cell enrichment can be accomplished by the elimination of non-T cells using anti-MHC class II monoclonal antibodies. Similarly, other antibodies can be used to deplete specific populations of non-T cells. For example, anti-Ig antibody molecules can be used to deplete B cells and anti-MacI antibody molecules can be used to deplete macrophages.
  • T cells can be further fractionated into a number of different subpopulations by techniques known to those skilled in the art. Two major subpopulations can be isolated based on their differential expression of the cell surface markers CD4 and CD8. For example, following the enrichment of T cells as described above, CD4+ cells can be enriched using antibodies specific for CD4. The antibodies may be coupled to a solid support such as magnetic beads. Conversely, CD8+ cells can be enriched through the use of antibodies specific for CD4 (to remove CD4+ cells), or can be-isolated by the use of CD8 antibodies coupled to a solid support. CD4 lymphocytes from UPEC-infected patients can be expanded ex vivo, before or after transduction.
  • Following purification of T cells, the purified T cells are pre-stimulated with various cytokines including but not limited to rIL-2, IL-10, IL-12, and IL-15, which promote growth and activation of lymphocytes.
  • UPEC-specific T cells, may be activated by the above described immunogenic polypeptides. UPEC-specific T cells can be CD8+ or CD4+. UPEC-specific CD8+ T cells can be cytotoxic T lymphocytes (CTL) which can kill UPEC-infected cells that display any of the above described polypeptides or fragments thereof complexed with an MHC class I molecule. Chlamydia-specific CD8+ T cells can be detected by, for example, 51Cr release assays. 51Cr release assays measure the ability of UPEC-specific CD8+ T cells to lyse target cells displaying one or more of these epitopes. UPEC-specific CD8+ T cells which express antiviral agents, such as IFN γ, are also contemplated herein and can also be detected by immunological methods, preferably by intracellular staining for IFN-γ or alike cytokines after in vitro stimulation with one or more of the above described UPEC polypeptides. UPEC-specific CD4+ T cells can be detected by a lymphoproliferation assay. Lymphoproliferation assays measure the ability of UPEC-specific CD4+ T cells to proliferate in response to one or more of the above described polypeptides.
  • It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
  • TABLE 1
    599 pathogenic E. coli sequences
    gi-26106321 (SEQ ID NO: 1) gi-26106323 (SEQ ID NO: 2) gi-26106335 (SEQ ID NO: 3)
    gi-26106336 (SEQ ID NO: 4) gi-26106340 (SEQ ID NO: 5) gi-26106348 (SEQ ID NO: 6)
    gi-26106390 (SEQ ID NO: 7) gi-26106403 (SEQ ID NO: 8) gi-26106445 (SEQ ID NO: 9)
    gi-26106446 (SEQ ID NO: 10) gi-26106449 (SEQ ID NO: 11) gi-26106454 (SEQ ID NO: 12)
    gi-26106474 (SEQ ID NO: 13) gi-26106475 (SEQ ID NO: 14) gi-26106476 (SEQ ID NO: 15)
    gi-26106477 (SEQ ID NO: 16) gi-26106478 (SEQ ID NO: 17) gi-26106479 (SEQ ID NO: 18)
    gi-26106480 (SEQ ID NO: 19) gi-26106483 (SEQ ID NO: 20) gi-26106492 (SEQ ID NO: 21)
    gi-26106493 (SEQ ID NO: 22) gi-26106557 (SEQ ID NO: 23) gi-26106569 (SEQ ID NO: 24)
    gi-26106579 (SEQ ID NO: 25) gi-26106582 (SEQ ID NO: 26) gi-26106583 (SEQ ID NO: 27)
    gi-26106584 (SEQ ID NO: 28) gi-26106585 (SEQ ID NO: 29) gi-26106592 (SEQ ID NO: 30)
    gi-26106594 (SEQ ID NO: 31) gi-26106595 (SEQ ID NO: 32) gi-26106596 (SEQ ID NO: 33)
    gi-26106597 (SEQ ID NO: 34) gi-26106598 (SEQ ID NO: 35) gi-26106601 (SEQ ID NO: 36)
    gi-26106624 (SEQ ID NO: 37) gi-26106625 (SEQ ID NO: 38) gi-26106626 (SEQ ID NO: 39)
    gi-26106627 (SEQ ID NO: 40) gi-26106628 (SEQ ID NO: 41) gi-26106629 (SEQ ID NO: 42)
    gi-26106630 (SEQ ID NO: 43) gi-26106633 (SEQ ID NO: 44) gi-26106635 (SEQ ID NO: 45)
    gi-26106637 (SEQ ID NO: 46) gi-26106645 (SEQ ID NO: 47) gi-26106648 (SEQ ID NO: 48)
    gi-26106649 (SEQ ID NO: 49) gi-26106652 (SEQ ID NO: 50) gi-26106656 (SEQ ID NO: 51)
    gi-26106657 (SEQ ID NO: 52) gi-26106658 (SEQ ID NO: 53) gi-26106659 (SEQ ID NO: 54)
    gi-26106662 (SEQ ID NO: 55) gi-26106663 (SEQ ID NO: 56) gi-26106664 (SEQ ID NO: 57)
    gi-26106689 (SEQ ID NO: 58) gi-26106690 (SEQ ID NO: 59) gi-26106694 (SEQ ID NO: 60)
    gi-26106705 (SEQ ID NO: 61) gi-26106706 (SEQ ID NO: 62) gi-26106710 (SEQ ID NO: 63)
    gi-26106722 (SEQ ID NO: 64) gi-26106723 (SEQ ID NO: 65) gi-26106740 (SEQ ID NO: 66)
    gi-26106931 (SEQ ID NO: 67) gi-26107030 (SEQ ID NO: 68) gi-26107046 (SEQ ID NO: 69)
    gi-26107049 (SEQ ID NO: 70) gi-26107050 (SEQ ID NO: 71) gi-26107051 (SEQ ID NO: 72)
    gi-26107053 (SEQ ID NO: 73) gi-26107067 (SEQ ID NO: 74) gi-26107097 (SEQ ID NO: 75)
    gi-26107223 (SEQ ID NO: 76) gi-26107228 (SEQ ID NO: 77) gi-26107235 (SEQ ID NO: 78)
    gi-26107239 (SEQ ID NO: 79) gi-26107241 (SEQ ID NO: 80) gi-26107246 (SEQ ID NO: 81)
    gi-26107247 (SEQ ID NO: 82) gi-26107248 (SEQ ID NO: 83) gi-26107249 (SEQ ID NO: 84)
    gi-26107251 (SEQ ID NO: 85) gi-26107258 (SEQ ID NO: 86) gi-26107260 (SEQ ID NO: 87)
    gi-26107263 (SEQ ID NO: 88) gi-26107265 (SEQ ID NO: 89) gi-26107266 (SEQ ID NO: 90)
    gi-26107267 (SEQ ID NO: 91) gi-26107269 (SEQ ID NO: 92) gi-26107309 (SEQ ID NO: 93)
    gi-26107393 (SEQ ID NO: 94) gi-26107451 (SEQ ID NO: 95) gi-26107452 (SEQ ID NO: 96)
    gi-26107455 (SEQ ID NO: 97) gi-26107459 (SEQ ID NO: 98) gi-26107461 (SEQ ID NO: 99)
    gi-26107465 (SEQ ID NO: 100) gi-26107468 (SEQ ID NO: 101) gi-26107469 (SEQ ID NO: 102)
    gi-26107470 (SEQ ID NO: 103) gi-26107471 (SEQ ID NO: 104) gi-26107473 (SEQ ID NO: 105)
    gi-26107476 (SEQ ID NO: 106) gi-26107477 (SEQ ID NO: 107) gi-26107480 (SEQ ID NO: 108)
    gi-26107483 (SEQ ID NO: 109) gi-26107484 (SEQ ID NO: 110) gi-26107486 (SEQ ID NO: 111)
    gi-26107498 (SEQ ID NO: 112) gi-26107499 (SEQ ID NO: 113) gi-26107502 (SEQ ID NO: 114)
    gi-26107505 (SEQ ID NO: 115) gi-26107506 (SEQ ID NO: 116) gi-26107507 (SEQ ID NO: 117)
    gi-26107508 (SEQ ID NO: 118) gi-26107509 (SEQ ID NO: 119) gi-26107513 (SEQ ID NO: 120)
    gi-26107514 (SEQ ID NO: 121) gi-26107515 (SEQ ID NO: 122) gi-26107516 (SEQ ID NO: 123)
    gi-26107517 (SEQ ID NO: 124) gi-26107518 (SEQ ID NO: 125) gi-26107524 (SEQ ID NO: 126)
    gi-26107525 (SEQ ID NO: 127) gi-26107528 (SEQ ID NO: 128) gi-26107529 (SEQ ID NO: 129)
    gi-26107530 (SEQ ID NO: 130) gi-26107534 (SEQ ID NO: 131) gi-26107537 (SEQ ID NO: 132)
    gi-26107540 (SEQ ID NO: 133) gi-26107541 (SEQ ID NO: 134) gi-26107542 (SEQ ID NO: 135)
    gi-26107544 (SEQ ID NO: 136) gi-26107548 (SEQ ID NO: 137) gi-26107550 (SEQ ID NO: 138)
    gi-26107554 (SEQ ID NO: 139) gi-26107578 (SEQ ID NO: 140) gi-26107639 (SEQ ID NO: 141)
    gi-26107666 (SEQ ID NO: 142) gi-26107673 (SEQ ID NO: 143) gi-26107682 (SEQ ID NO: 144)
    gi-26107688 (SEQ ID NO: 145) gi-26107690 (SEQ ID NO: 146) gi-26107692 (SEQ ID NO: 147)
    gi-26107693 (SEQ ID NO: 148) gi-26107698 (SEQ ID NO: 149) gi-26107699 (SEQ ID NO: 150)
    gi-26107703 (SEQ ID NO: 151) gi-26107707 (SEQ ID NO: 152) gi-26107708 (SEQ ID NO: 153)
    gi-26107714 (SEQ ID NO: 154) gi-26107719 (SEQ ID NO: 155) gi-26107725 (SEQ ID NO: 156)
    gi-26107730 (SEQ ID NO: 157) gi-26107735 (SEQ ID NO: 158) gi-26107739 (SEQ ID NO: 159)
    gi-26107741 (SEQ ID NO: 160) gi-26107751 (SEQ ID NO: 161) gi-26107753 (SEQ ID NO: 162)
    gi-26107755 (SEQ ID NO: 163) gi-26107757 (SEQ ID NO: 164) gi-26107759 (SEQ ID NO: 165)
    gi-26107765 (SEQ ID NO: 166) gi-26107774 (SEQ ID NO: 167) gi-26107788 (SEQ ID NO: 168)
    gi-26107791 (SEQ ID NO: 169) gi-26107794 (SEQ ID NO: 170) gi-26107808 (SEQ ID NO: 171)
    gi-26107811 (SEQ ID NO: 172) gi-26107813 (SEQ ID NO: 173) gi-26107817 (SEQ ID NO: 174)
    gi-26107823 (SEQ ID NO: 175) gi-26107835 (SEQ ID NO: 176) gi-26107839 (SEQ ID NO: 177)
    gi-26107843 (SEQ ID NO: 178) gi-26107851 (SEQ ID NO: 179) gi-26107854 (SEQ ID NO: 180)
    gi-26107855 (SEQ ID NO: 181) gi-26107858 (SEQ ID NO: 182) gi-26107861 (SEQ ID NO: 183)
    gi-26107864 (SEQ ID NO: 184) gi-26107866 (SEQ ID NO: 185) gi-26107867 (SEQ ID NO: 186)
    gi-26107869 (SEQ ID NO: 187) gi-26107890 (SEQ ID NO: 188) gi-26107912 (SEQ ID NO: 189)
    gi-26107916 (SEQ ID NO: 190) gi-26107917 (SEQ ID NO: 191) gi-26107918 (SEQ ID NO: 192)
    gi-26107919 (SEQ ID NO: 193) gi-26107926 (SEQ ID NO: 194) gi-26107942 (SEQ ID NO: 195)
    gi-26107956 (SEQ ID NO: 196) gi-26107959 (SEQ ID NO: 197) gi-26107960 (SEQ ID NO: 198)
    gi-26107961 (SEQ ID NO: 199) gi-26107962 (SEQ ID NO: 200) gi-26108027 (SEQ ID NO: 201)
    gi-26108028 (SEQ ID NO: 202) gi-26108030 (SEQ ID NO: 203) gi-26108031 (SEQ ID NO: 204)
    gi-26108032 (SEQ ID NO: 205) gi-26108047 (SEQ ID NO: 206) gi-26108073 (SEQ ID NO: 207)
    gi-26108075 (SEQ ID NO: 208) gi-26108076 (SEQ ID NO: 209) gi-26108084 (SEQ ID NO: 210)
    gi-26108096 (SEQ ID NO: 211) gi-26108136 (SEQ ID NO: 212) gi-26108146 (SEQ ID NO: 213)
    gi-26108147 (SEQ ID NO: 214) gi-26108151 (SEQ ID NO: 215) gi-26108157 (SEQ ID NO: 216)
    gi-26108164 (SEQ ID NO: 217) gi-26108174 (SEQ ID NO: 218) gi-26108192 (SEQ ID NO: 219)
    gi-26108193 (SEQ ID NO: 220) gi-26108194 (SEQ ID NO: 221) gi-26108196 (SEQ ID NO: 222)
    gi-26108213 (SEQ ID NO: 223) gi-26108215 (SEQ ID NO: 224) gi-26108216 (SEQ ID NO: 225)
    gi-26108299 (SEQ ID NO: 226) gi-26108374 (SEQ ID NO: 227) gi-26108422 (SEQ ID NO: 228)
    gi-26108563 (SEQ ID NO: 229) gi-26108639 (SEQ ID NO: 230) gi-26108640 (SEQ ID NO: 231)
    gi-26108650 (SEQ ID NO: 232) gi-26108651 (SEQ ID NO: 233) gi-26108654 (SEQ ID NO: 234)
    gi-26108656 (SEQ ID NO: 235) gi-26108657 (SEQ ID NO: 236) gi-26108659 (SEQ ID NO: 237)
    gi-26108660 (SEQ ID NO: 238) gi-26108662 (SEQ ID NO: 239) gi-26108663 (SEQ ID NO: 240)
    gi-26108668 (SEQ ID NO: 241) gi-26108676 (SEQ ID NO: 242) gi-26108677 (SEQ ID NO: 243)
    gi-26108678 (SEQ ID NO: 244) gi-26108680 (SEQ ID NO: 245) gi-26108682 (SEQ ID NO: 246)
    gi-26108683 (SEQ ID NO: 247) gi-26108684 (SEQ ID NO: 248) gi-26108686 (SEQ ID NO: 249)
    gi-26108689 (SEQ ID NO: 250) gi-26108692 (SEQ ID NO: 251) gi-26108708 (SEQ ID NO: 252)
    gi-26108711 (SEQ ID NO: 253) gi-26108712 (SEQ ID NO: 254) gi-26108714 (SEQ ID NO: 255)
    gi-26108715 (SEQ ID NO: 256) gi-26108716 (SEQ ID NO: 257) gi-26108717 (SEQ ID NO: 258)
    gi-26108720 (SEQ ID NO: 259) gi-26108721 (SEQ ID NO: 260) gi-26108722 (SEQ ID NO: 261)
    gi-26108723 (SEQ ID NO: 262) gi-26108724 (SEQ ID NO: 263) gi-26108725 (SEQ ID NO: 264)
    gi-26108726 (SEQ ID NO: 265) gi-26108728 (SEQ ID NO: 266) gi-26108739 (SEQ ID NO: 267)
    gi-26108747 (SEQ ID NO: 268) gi-26108756 (SEQ ID NO: 269) gi-26108765 (SEQ ID NO: 270)
    gi-26108768 (SEQ ID NO: 271) gi-26108769 (SEQ ID NO: 272) gi-26108770 (SEQ ID NO: 273)
    gi-26108775 (SEQ ID NO: 274) gi-26108776 (SEQ ID NO: 275) gi-26108778 (SEQ ID NO: 276)
    gi-26108779 (SEQ ID NO: 277) gi-26108810 (SEQ ID NO: 278) gi-26108811 (SEQ ID NO: 279)
    gi-26108813 (SEQ ID NO: 280) gi-26108816 (SEQ ID NO: 281) gi-26108817 (SEQ ID NO: 282)
    gi-26108818 (SEQ ID NO: 283) gi-26108826 (SEQ ID NO: 284) gi-26108860 (SEQ ID NO: 285)
    gi-26108862 (SEQ ID NO: 286) gi-26108877 (SEQ ID NO: 287) gi-26108896 (SEQ ID NO: 288)
    gi-26108898 (SEQ ID NO: 289) gi-26108915 (SEQ ID NO: 290) gi-26108952 (SEQ ID NO: 291)
    gi-26109000 (SEQ ID NO: 292) gi-26109006 (SEQ ID NO: 293) gi-26109008 (SEQ ID NO: 294)
    gi-26109026 (SEQ ID NO: 295) gi-26109062 (SEQ ID NO: 296) gi-26109065 (SEQ ID NO: 297)
    gi-26109126 (SEQ ID NO: 298) gi-26109127 (SEQ ID NO: 299) gi-26109128 (SEQ ID NO: 300)
    gi-26109129 (SEQ ID NO: 301) gi-26109130 (SEQ ID NO: 302) gi-26109131 (SEQ ID NO: 303)
    gi-26109132 (SEQ ID NO: 304) gi-26109137 (SEQ ID NO: 305) gi-26109141 (SEQ ID NO: 306)
    gi-26109143 (SEQ ID NO: 307) gi-26109160 (SEQ ID NO: 308) gi-26109178 (SEQ ID NO: 309)
    gi-26109277 (SEQ ID NO: 310) gi-26109278 (SEQ ID NO: 311) gi-26109279 (SEQ ID NO: 312)
    gi-26109311 (SEQ ID NO: 313) gi-26109396 (SEQ ID NO: 314) gi-26109400 (SEQ ID NO: 315)
    gi-26109404 (SEQ ID NO: 316) gi-26109406 (SEQ ID NO: 317) gi-26109409 (SEQ ID NO: 318)
    gi-26109428 (SEQ ID NO: 319) gi-26109447 (SEQ ID NO: 320) gi-26109452 (SEQ ID NO: 321)
    gi-26109490 (SEQ ID NO: 322) gi-26109512 (SEQ ID NO: 323) gi-26109518 (SEQ ID NO: 324)
    gi-26109519 (SEQ ID NO: 325) gi-26109522 (SEQ ID NO: 326) gi-26109548 (SEQ ID NO: 327)
    gi-26109581 (SEQ ID NO: 328) gi-26109582 (SEQ ID NO: 329) gi-26109583 (SEQ ID NO: 330)
    gi-26109585 (SEQ ID NO: 331) gi-26109587 (SEQ ID NO: 332) gi-26109613 (SEQ ID NO: 333)
    gi-26109629 (SEQ ID NO: 334) gi-26109630 (SEQ ID NO: 335) gi-26109631 (SEQ ID NO: 336)
    gi-26109636 (SEQ ID NO: 337) gi-26109637 (SEQ ID NO: 338) gi-26109639 (SEQ ID NO: 339)
    gi-26109640 (SEQ ID NO: 340) gi-26109641 (SEQ ID NO: 341) gi-26109642 (SEQ ID NO: 342)
    gi-26109643 (SEQ ID NO: 343) gi-26109645 (SEQ ID NO: 344) gi-26109647 (SEQ ID NO: 345)
    gi-26109648 (SEQ ID NO: 346) gi-26109650 (SEQ ID NO: 347) gi-26109730 (SEQ ID NO: 348)
    gi-26109741 (SEQ ID NO: 349) gi-26109750 (SEQ ID NO: 350) gi-26109751 (SEQ ID NO: 351)
    gi-26109753 (SEQ ID NO: 352) gi-26109760 (SEQ ID NO: 353) gi-26109761 (SEQ ID NO: 354)
    gi-26109766 (SEQ ID NO: 355) gi-26109801 (SEQ ID NO: 356) gi-26109807 (SEQ ID NO: 357)
    gi-26109809 (SEQ ID NO: 358) gi-26109811 (SEQ ID NO: 359) gi-26109812 (SEQ ID NO: 360)
    gi-26109813 (SEQ ID NO: 361) gi-26109816 (SEQ ID NO: 362) gi-26109817 (SEQ ID NO: 363)
    gi-26109826 (SEQ ID NO: 364) gi-26109827 (SEQ ID NO: 365) gi-26109828 (SEQ ID NO: 366)
    gi-26109829 (SEQ ID NO: 367) gi-26109831 (SEQ ID NO: 368) gi-26109833 (SEQ ID NO: 369)
    gi-26109834 (SEQ ID NO: 370) gi-26109835 (SEQ ID NO: 371) gi-26109837 (SEQ ID NO: 372)
    gi-26109841 (SEQ ID NO: 373) gi-26109842 (SEQ ID NO: 374) gi-26109844 (SEQ ID NO: 375)
    gi-26109845 (SEQ ID NO: 376) gi-26109849 (SEQ ID NO: 377) gi-26109852 (SEQ ID NO: 378)
    gi-26109853 (SEQ ID NO: 379) gi-26109855 (SEQ ID NO: 380) gi-26109861 (SEQ ID NO: 381)
    gi-26109862 (SEQ ID NO: 382) gi-26109864 (SEQ ID NO: 383) gi-26109867 (SEQ ID NO: 384)
    gi-26109868 (SEQ ID NO: 385) gi-26109870 (SEQ ID NO: 386) gi-26109871 (SEQ ID NO: 387)
    gi-26109873 (SEQ ID NO: 388) gi-26109877 (SEQ ID NO: 389) gi-26109878 (SEQ ID NO: 390)
    gi-26109879 (SEQ ID NO: 391) gi-26109880 (SEQ ID NO: 392) gi-26109881 (SEQ ID NO: 393)
    gi-26109895 (SEQ ID NO: 394) gi-26109908 (SEQ ID NO: 395) gi-26109915 (SEQ ID NO: 396)
    gi-26109927 (SEQ ID NO: 397) gi-26109928 (SEQ ID NO: 398) gi-26109930 (SEQ ID NO: 399)
    gi-26109931 (SEQ ID NO: 400) gi-26109935 (SEQ ID NO: 401) gi-26109936 (SEQ ID NO: 402)
    gi-26109938 (SEQ ID NO: 403) gi-26109941 (SEQ ID NO: 404) gi-26109951 (SEQ ID NO: 405)
    gi-26109955 (SEQ ID NO: 406) gi-26109956 (SEQ ID NO: 407) gi-26109957 (SEQ ID NO: 408)
    gi-26109990 (SEQ ID NO: 409) gi-26109991 (SEQ ID NO: 410) gi-26109993 (SEQ ID NO: 411)
    gi-26109994 (SEQ ID NO: 412) gi-26109995 (SEQ ID NO: 413) gi-26110009 (SEQ ID NO: 414)
    gi-26110010 (SEQ ID NO: 415) gi-26110011 (SEQ ID NO: 416) gi-26110012 (SEQ ID NO: 417)
    gi-26110024 (SEQ ID NO: 418) gi-26110025 (SEQ ID NO: 419) gi-26110026 (SEQ ID NO: 420)
    gi-26110029 (SEQ ID NO: 421) gi-26110030 (SEQ ID NO: 422) gi-26110031 (SEQ ID NO: 423)
    gi-26110032 (SEQ ID NO: 424) gi-26110033 (SEQ ID NO: 425) gi-26110145 (SEQ ID NO: 426)
    gi-26110198 (SEQ ID NO: 427) gi-26110269 (SEQ ID NO: 428) gi-26110271 (SEQ ID NO: 429)
    gi-26110273 (SEQ ID NO: 430) gi-26110457 (SEQ ID NO: 431) gi-26110458 (SEQ ID NO: 432)
    gi-26110460 (SEQ ID NO: 433) gi-26110461 (SEQ ID NO: 434) gi-26110462 (SEQ ID NO: 435)
    gi-26110465 (SEQ ID NO: 436) gi-26110466 (SEQ ID NO: 437) gi-26110467 (SEQ ID NO: 438)
    gi-26110529 (SEQ ID NO: 439) gi-26110530 (SEQ ID NO: 440) gi-26110532 (SEQ ID NO: 441)
    gi-26110559 (SEQ ID NO: 442) gi-26110564 (SEQ ID NO: 443) gi-26110568 (SEQ ID NO: 444)
    gi-26110614 (SEQ ID NO: 445) gi-26110650 (SEQ ID NO: 446) gi-26110651 (SEQ ID NO: 447)
    gi-26110658 (SEQ ID NO: 448) gi-26110671 (SEQ ID NO: 449) gi-26110674 (SEQ ID NO: 450)
    gi-26110675 (SEQ ID NO: 451) gi-26110685 (SEQ ID NO: 452) gi-26110699 (SEQ ID NO: 453)
    gi-26110700 (SEQ ID NO: 454) gi-26110708 (SEQ ID NO: 455) gi-26110734 (SEQ ID NO: 456)
    gi-26110736 (SEQ ID NO: 457) gi-26110737 (SEQ ID NO: 458) gi-26110738 (SEQ ID NO: 459)
    gi-26110745 (SEQ ID NO: 460) gi-26110746 (SEQ ID NO: 461) gi-26110747 (SEQ ID NO: 462)
    gi-26110749 (SEQ ID NO: 463) gi-26110752 (SEQ ID NO: 464) gi-26110754 (SEQ ID NO: 465)
    gi-26110762 (SEQ ID NO: 466) gi-26110764 (SEQ ID NO: 467) gi-26110766 (SEQ ID NO: 468)
    gi-26110768 (SEQ ID NO: 469) gi-26110769 (SEQ ID NO: 470) gi-26110776 (SEQ ID NO: 471)
    gi-26110780 (SEQ ID NO: 472) gi-26110782 (SEQ ID NO: 473) gi-26110783 (SEQ ID NO: 474)
    gi-26110785 (SEQ ID NO: 475) gi-26110786 (SEQ ID NO: 476) gi-26110788 (SEQ ID NO: 477)
    gi-26110789 (SEQ ID NO: 478) gi-26110794 (SEQ ID NO: 479) gi-26110797 (SEQ ID NO: 480)
    gi-26110801 (SEQ ID NO: 481) gi-26110805 (SEQ ID NO: 482) gi-26110811 (SEQ ID NO: 483)
    gi-26110816 (SEQ ID NO: 484) gi-26110818 (SEQ ID NO: 485) gi-26110835 (SEQ ID NO: 486)
    gi-26110844 (SEQ ID NO: 487) gi-26110856 (SEQ ID NO: 488) gi-26110866 (SEQ ID NO: 489)
    gi-26110976 (SEQ ID NO: 490) gi-26110977 (SEQ ID NO: 491) gi-26110984 (SEQ ID NO: 492)
    gi-26110987 (SEQ ID NO: 493) gi-26111004 (SEQ ID NO: 494) gi-26111005 (SEQ ID NO: 495)
    gi-26111007 (SEQ ID NO: 496) gi-26111008 (SEQ ID NO: 497) gi-26111009 (SEQ ID NO: 498)
    gi-26111010 (SEQ ID NO: 499) gi-26111011 (SEQ ID NO: 500) gi-26111013 (SEQ ID NO: 501)
    gi-26111014 (SEQ ID NO: 502) gi-26111016 (SEQ ID NO: 503) gi-26111024 (SEQ ID NO: 504)
    gi-26111025 (SEQ ID NO: 505) gi-26111026 (SEQ ID NO: 506) gi-26111027 (SEQ ID NO: 507)
    gi-26111070 (SEQ ID NO: 508) gi-26111071 (SEQ ID NO: 509) gi-26111072 (SEQ ID NO: 510)
    gi-26111073 (SEQ ID NO: 511) gi-26111074 (SEQ ID NO: 512) gi-26111075 (SEQ ID NO: 513)
    gi-26111076 (SEQ ID NO: 514) gi-26111094 (SEQ ID NO: 515) gi-26111139 (SEQ ID NO: 516)
    gi-26111140 (SEQ ID NO: 517) gi-26111141 (SEQ ID NO: 518) gi-26111143 (SEQ ID NO: 519)
    gi-26111166 (SEQ ID NO: 520) gi-26111169 (SEQ ID NO: 521) gi-26111170 (SEQ ID NO: 522)
    gi-26111221 (SEQ ID NO: 523) gi-26111225 (SEQ ID NO: 524) gi-26111226 (SEQ ID NO: 525)
    gi-26111229 (SEQ ID NO: 526) gi-26111264 (SEQ ID NO: 527) gi-26111265 (SEQ ID NO: 528)
    gi-26111266 (SEQ ID NO: 529) gi-26111270 (SEQ ID NO: 530) gi-26111272 (SEQ ID NO: 531)
    gi-26111278 (SEQ ID NO: 532) gi-26111279 (SEQ ID NO: 533) gi-26111280 (SEQ ID NO: 534)
    gi-26111281 (SEQ ID NO: 535) gi-26111284 (SEQ ID NO: 536) gi-26111287 (SEQ ID NO: 537)
    gi-26111294 (SEQ ID NO: 538) gi-26111301 (SEQ ID NO: 539) gi-26111302 (SEQ ID NO: 540)
    gi-26111303 (SEQ ID NO: 541) gi-26111304 (SEQ ID NO: 542) gi-26111322 (SEQ ID NO: 543)
    gi-26111323 (SEQ ID NO: 544) gi-26111324 (SEQ ID NO: 545) gi-26111325 (SEQ ID NO: 546)
    gi-26111394 (SEQ ID NO: 547) gi-26111395 (SEQ ID NO: 548) gi-26111398 (SEQ ID NO: 549)
    gi-26111404 (SEQ ID NO: 550) gi-26111405 (SEQ ID NO: 551) gi-26111407 (SEQ ID NO: 552)
    gi-26111409 (SEQ ID NO: 553) gi-26111411 (SEQ ID NO: 554) gi-26111414 (SEQ ID NO: 555)
    gi-26111418 (SEQ ID NO: 556) gi-26111419 (SEQ ID NO: 557) gi-26111420 (SEQ ID NO: 558)
    gi-26111421 (SEQ ID NO: 559) gi-26111422 (SEQ ID NO: 560) gi-26111424 (SEQ ID NO: 561)
    gi-26111425 (SEQ ID NO: 562) gi-26111426 (SEQ ID NO: 563) gi-26111427 (SEQ ID NO: 564)
    gi-26111428 (SEQ ID NO: 565) gi-26111431 (SEQ ID NO: 566) gi-26111441 (SEQ ID NO: 567)
    gi-26111442 (SEQ ID NO: 568) gi-26111443 (SEQ ID NO: 569) gi-26111450 (SEQ ID NO: 570)
    gi-26111453 (SEQ ID NO: 571) gi-26111510 (SEQ ID NO: 572) gi-26111525 (SEQ ID NO: 573)
    gi-26111536 (SEQ ID NO: 574) gi-26111537 (SEQ ID NO: 575) gi-26111542 (SEQ ID NO: 576)
    gi-26111560 (SEQ ID NO: 577) gi-26111573 (SEQ ID NO: 578) gi-26111587 (SEQ ID NO: 579)
    gi-26111589 (SEQ ID NO: 580) gi-26111591 (SEQ ID NO: 581) gi-26111596 (SEQ ID NO: 582)
    gi-26111597 (SEQ ID NO: 583) gi-26111612 (SEQ ID NO: 584) gi-26111614 (SEQ ID NO: 585)
    gi-26111620 (SEQ ID NO: 586) gi-26111626 (SEQ ID NO: 587) gi-26111630 (SEQ ID NO: 588)
    gi-26111638 (SEQ ID NO: 589) gi-26111652 (SEQ ID NO: 590) gi-26111659 (SEQ ID NO: 591)
    gi-26111660 (SEQ ID NO: 592) gi-26111670 (SEQ ID NO: 593) gi-26111671 (SEQ ID NO: 594)
    gi-26111673 (SEQ ID NO: 595) gi-26111674 (SEQ ID NO: 596) gi-26109898 (SEQ ID NO: 597)
    gi-26108604 (SEQ ID NO: 598) gi-26109866 (SEQ ID NO: 599)
  • TABLE 2
    156 preferred antigens
    gi-26106323 (SEQ ID NO: 2) gi-26106348 (SEQ ID NO: 6) gi-26106390 (SEQ ID NO: 7)
    gi-26106557 (SEQ ID NO: 23) gi-26106579 (SEQ ID NO: 25) gi-26106582 (SEQ ID NO: 26)
    gi-26106584 (SEQ ID NO: 28) gi-26106585 (SEQ ID NO: 29) gi-26106592 (SEQ ID NO: 30)
    gi-26106594 (SEQ ID NO: 31) gi-26106595 (SEQ ID NO: 32) gi-26106601 (SEQ ID NO: 36)
    gi-26106649 (SEQ ID NO: 49) gi-26106664 (SEQ ID NO: 57) gi-26106690 (SEQ ID NO: 59)
    gi-26106931 (SEQ ID NO: 67) gi-26107030 (SEQ ID NO: 68) gi-26107223 (SEQ ID NO: 76)
    gi-26107228 (SEQ ID NO: 77) gi-26107241 (SEQ ID NO: 80) gi-26107246 (SEQ ID NO: 81)
    gi-26107247 (SEQ ID NO: 82) gi-26107248 (SEQ ID NO: 83) gi-26107249 (SEQ ID NO: 84)
    gi-26107251 (SEQ ID NO: 85) gi-26107260 (SEQ ID NO: 87) gi-26107263 (SEQ ID NO: 88)
    gi-26107265 (SEQ ID NO: 89) gi-26107309 (SEQ ID NO: 93) gi-26107393 (SEQ ID NO: 94)
    gi-26107452 (SEQ ID NO: 96) gi-26107498 (SEQ ID NO: 112) gi-26107502 (SEQ ID NO: 114)
    gi-26107517 (SEQ ID NO: 124) gi-26107518 (SEQ ID NO: 125) gi-26107550 (SEQ ID NO: 138)
    gi-26107554 (SEQ ID NO: 139) gi-26107666 (SEQ ID NO: 142) gi-26107673 (SEQ ID NO: 143)
    gi-26107688 (SEQ ID NO: 145) gi-26107692 (SEQ ID NO: 147) gi-26107698 (SEQ ID NO: 149)
    gi-26107699 (SEQ ID NO: 150) gi-26107708 (SEQ ID NO: 153) gi-26107719 (SEQ ID NO: 155)
    gi-26107739 (SEQ ID NO: 159) gi-26107741 (SEQ ID NO: 160) gi-26107751 (SEQ ID NO: 161)
    gi-26107759 (SEQ ID NO: 165) gi-26107774 (SEQ ID NO: 167) gi-26107794 (SEQ ID NO: 170)
    gi-26107808 (SEQ ID NO: 171) gi-26107835 (SEQ ID NO: 176) gi-26107843 (SEQ ID NO: 178)
    gi-26107851 (SEQ ID NO: 179) gi-26107855 (SEQ ID NO: 181) gi-26107861 (SEQ ID NO: 183)
    gi-26107864 (SEQ ID NO: 184) gi-26107890 (SEQ ID NO: 188) gi-26107926 (SEQ ID NO: 194)
    gi-26107942 (SEQ ID NO: 195) gi-26107959 (SEQ ID NO: 197) gi-26107960 (SEQ ID NO: 198)
    gi-26107961 (SEQ ID NO: 199) gi-26107962 (SEQ ID NO: 200) gi-26108028 (SEQ ID NO: 202)
    gi-26108073 (SEQ ID NO: 207) gi-26108075 (SEQ ID NO: 208) gi-26108076 (SEQ ID NO: 209)
    gi-26108084 (SEQ ID NO: 210) gi-26108096 (SEQ ID NO: 211) gi-26108136 (SEQ ID NO: 212)
    gi-26108216 (SEQ ID NO: 225) gi-26108422 (SEQ ID NO: 228) gi-26108650 (SEQ ID NO: 232)
    gi-26108651 (SEQ ID NO: 233) gi-26108656 (SEQ ID NO: 235) gi-26108657 (SEQ ID NO: 236)
    gi-26108659 (SEQ ID NO: 237) gi-26108660 (SEQ ID NO: 238) gi-26108663 (SEQ ID NO: 240)
    gi-26108668 (SEQ ID NO: 241) gi-26108779 (SEQ ID NO: 277) gi-26108811 (SEQ ID NO: 279)
    gi-26108813 (SEQ ID NO: 280) gi-26108816 (SEQ ID NO: 281) gi-26108818 (SEQ ID NO: 283)
    gi-26108826 (SEQ ID NO: 284) gi-26108898 (SEQ ID NO: 289) gi-26108915 (SEQ ID NO: 290)
    gi-26109141 (SEQ ID NO: 306) gi-26109160 (SEQ ID NO: 308) gi-26109178 (SEQ ID NO: 309)
    gi-26109396 (SEQ ID NO: 314) gi-26109400 (SEQ ID NO: 315) gi-26109406 (SEQ ID NO: 317)
    gi-26109490 (SEQ ID NO: 322) gi-26109512 (SEQ ID NO: 323) gi-26109519 (SEQ ID NO: 325)
    gi-26109522 (SEQ ID NO: 326) gi-26109548 (SEQ ID NO: 327) gi-26109730 (SEQ ID NO: 348)
    gi-26109753 (SEQ ID NO: 352) gi-26109761 (SEQ ID NO: 354) gi-26109807 (SEQ ID NO: 357)
    gi-26109809 (SEQ ID NO: 358) gi-26109811 (SEQ ID NO: 359) gi-26109831 (SEQ ID NO: 368)
    gi-26109842 (SEQ ID NO: 374) gi-26109844 (SEQ ID NO: 375) gi-26109849 (SEQ ID NO: 377)
    gi-26109861 (SEQ ID NO: 381) gi-26109871 (SEQ ID NO: 387) gi-26109877 (SEQ ID NO: 389)
    gi-26109878 (SEQ ID NO: 390) gi-26109879 (SEQ ID NO: 391) gi-26109908 (SEQ ID NO: 395)
    gi-26109915 (SEQ ID NO: 396) gi-26109927 (SEQ ID NO: 397) gi-26109935 (SEQ ID NO: 401)
    gi-26109936 (SEQ ID NO: 402) gi-26109938 (SEQ ID NO: 403) gi-26109941 (SEQ ID NO: 404)
    gi-26109956 (SEQ ID NO: 407) gi-26109994 (SEQ ID NO: 412) gi-26110029 (SEQ ID NO: 421)
    gi-26110198 (SEQ ID NO: 427) gi-26110532 (SEQ ID NO: 441) gi-26110614 (SEQ ID NO: 445)
    gi-26110650 (SEQ ID NO: 446) gi-26110671 (SEQ ID NO: 449) gi-26110746 (SEQ ID NO: 461)
    gi-26110762 (SEQ ID NO: 466) gi-26110764 (SEQ ID NO: 467) gi-26110766 (SEQ ID NO: 468)
    gi-26110782 (SEQ ID NO: 473) gi-26110785 (SEQ ID NO: 475) gi-26110794 (SEQ ID NO: 479)
    gi-26110844 (SEQ ID NO: 487) gi-26110856 (SEQ ID NO: 488) gi-26110976 (SEQ ID NO: 490)
    gi-26110977 (SEQ ID NO: 491) gi-26110987 (SEQ ID NO: 493) gi-26111303 (SEQ ID NO: 541)
    gi-26111394 (SEQ ID NO: 547) gi-26111411 (SEQ ID NO: 554) gi-26111424 (SEQ ID NO: 561)
    gi-26111431 (SEQ ID NO: 566) gi-26111525 (SEQ ID NO: 573) gi-26111536 (SEQ ID NO: 574)
    gi-26111542 (SEQ ID NO: 576) gi-26111573 (SEQ ID NO: 578) gi-26111614 (SEQ ID NO: 585)
    gi-26111620 (SEQ ID NO: 586) gi-26111630 (SEQ ID NO: 588) gi-26111673 (SEQ ID NO: 595)
  • TABLE 3
    66 preferred antigens
    1 gi-26106348 (SEQ ID NO: 6)
    2 gi-26106390 (SEQ ID NO: 7) +
    3 gi-26106664 (SEQ ID NO: 57)
    4 gi-26106931 (SEQ ID NO: 67) +
    5 gi-26107030 (SEQ ID NO: 68)
    6 gi-26107260 (SEQ ID NO: 87)
    7 gi-26107452 (SEQ ID NO: 96)
    8 gi-26107502 (SEQ ID NO: 114)
    9 gi-26107517 (SEQ ID NO: 124) +
    10 gi-26107518 (SEQ ID NO: 125) +
    11 gi-26107666 (SEQ ID NO: 142)
    12 gi-26107741 (SEQ ID NO: 160)
    13 gi-26107835 (SEQ ID NO: 176) +
    14 gi-26107864 (SEQ ID NO: 184)
    15 gi-26107890 (SEQ ID NO: 188)
    16 gi-26108075 (SEQ ID NO: 208) +
    17 gi-26108076 (SEQ ID NO: 209)
    18 gi-26108084 (SEQ ID NO: 210)
    19 gi-26108096 (SEQ ID NO: 211)
    20 gi-26108136 (SEQ ID NO: 212)
    21 gi-26108216 (SEQ ID NO: 225) +
    22 gi-26108650 (SEQ ID NO: 232) +
    23 gi-26108651 (SEQ ID NO: 233) +
    24 gi-26108656 (SEQ ID NO: 235)
    25 gi-26108657 (SEQ ID NO: 236) +
    26 gi-26108659 (SEQ ID NO: 237) +
    27 gi-26108660 (SEQ ID NO: 238)
    28 gi-26108668 (SEQ ID NO: 241)
    29 gi-26108811 (SEQ ID NO: 279) +
    30 gi-26108813 (SEQ ID NO: 280) +
    31 gi-26108816 (SEQ ID NO: 281)
    32 gi-26108818 (SEQ ID NO: 283)
    33 gi-26109160 (SEQ ID NO: 308)
    34 gi-26109396 (SEQ ID NO: 314)
    35 gi-26109519 (SEQ ID NO: 325)
    36 gi-26109522 (SEQ ID NO: 326) +
    37 gi-26109548 (SEQ ID NO: 327) +
    38 gi-26109753 (SEQ ID NO: 352)
    39 gi-26109761 (SEQ ID NO: 354)
    40 gi-26109807 (SEQ ID NO: 357)
    41 gi-26109809 (SEQ ID NO: 358)
    42 gi-26109831 (SEQ ID NO: 368) +
    43 gi-26109842 (SEQ ID NO: 374)
    44 gi-26109844 (SEQ ID NO: 375)
    45 gi-26109871 (SEQ ID NO: 387)
    46 gi-26109877 (SEQ ID NO: 389) +
    47 gi-26109878 (SEQ ID NO: 390) +
    48 gi-26109941 (SEQ ID NO: 404)
    49 gi-26109956 (SEQ ID NO: 407)
    50 gi-26109994 (SEQ ID NO: 412)
    51 gi-26110029 (SEQ ID NO: 421)
    52 gi-26110614 (SEQ ID NO: 445)
    53 gi-26110650 (SEQ ID NO: 446)
    54 gi-26110746 (SEQ ID NO: 461)
    55 gi-26110766 (SEQ ID NO: 468)
    56 gi-26110782 (SEQ ID NO: 473)
    57 gi-26110844 (SEQ ID NO: 487)
    58 gi-26110976 (SEQ ID NO: 490)
    59 gi-26110977 (SEQ ID NO: 491)
    60 gi-26110987 (SEQ ID NO: 493)
    61 gi-26111303 (SEQ ID NO: 541)
    62 gi-26111424 (SEQ ID NO: 561)
    63 gi-26111536 (SEQ ID NO: 574) +
    64 gi-26111542 (SEQ ID NO: 576)
    65 gi-26111630 (SEQ ID NO: 588)
    66 gi-26111673 (SEQ ID NO: 595)
  • TABLE 4
    K12 hits
    SEQ K12 JW3110 DH10B
    ID e value overlap % id e value overlap % id e value overlap % id
    1 1.E−50  87/100 87 4.E−50  86/100 86 7.E−50  86/100 86
    2 10 10 1.E−17 42/63 66
    3 2.E−29  81/263 30 2.E−29  81/263 30 7.E−26  45/123 36
    4 1.E−32 135/531 25 1.E−32 135/531 25 2.E−32 135/531 25
    5 10 10 10
    6 5.E−07  30/112 26 5.E−07  30/112 26 8.E−07  30/112 26
    7 10 10 10
    8 10 10 10
    9 10 10 10
    10 10 10 10
    11 1.E−31  86/286 30 3.E−31  85/284 29 8.E−24  60/184 32
    12 0.E+00 540/625 86 0.E+00 540/625 86 0.E+00 526/628 83
    13   e−147 259/422 61   e−147 259/422 61   e−144 224/385 58
    14 7.E−86 152/198 76 7.E−86 152/198 76 1.E−85 152/198 76
    15 2.E−75 142/202 70 2.E−75 142/202 70 3.E−75 142/202 70
    16 1.E−79 144/189 76 1.E−79 144/189 76 2.E−79 144/189 76
    17 0.E+00 750/859 87 0.E+00 750/859 87 0.E+00 750/859 87
    18   e−124 210/246 85   e−124 210/246 85   e−123 210/246 85
    19 2.E−93 169/196 86 2.E−93 169/196 86 9.E−71 140/209 66
    20 1.E−80 143/159 89 1.E−80 143/159 89 2.E−80 143/159 89
    21 10 10 3.E−14 33/51 64
    22 0.E+00 672/753 89 0.E+00 672/753 89 0.E+00 418/495 84
    23 10 10 10
    24 10 10 10
    25 10 10 4.E−52 104/247 42
    26 10 10 10
    27 10 10 10
    28 10 10 10
    29 6.E−22  51/146 34 6.E−22  51/146 34 2.E−20  53/148 35
    30 10 10 10
    31 10 10 10
    32 10 10 10
    33 6.E−32 167/753 22 6.E−32 167/753 22 5.E−14  91/420 21
    34 2.E−05  42/170 24 2.E−05  42/170 24 10
    35 1.E−39 108/328 32 1.E−39 108/328 32 6.E−40 109/330 33
    36 10 10 10
    37 2.E−15  95/465 20 2.E−15  95/465 20 4.E−15  95/465 20
    38 10 10 10
    39 10 10 10
    40 10 10 10
    41 10 10 10
    42 10 10 10
    43 10 10 10
    44 2.E−43  96/253 37 2.E−43  96/253 37 3.E−43  96/254 37
    45 4.E−72 148/422 35 4.E−72 148/422 35 5.E−67 141/388 36
    46 2.E−52 122/398 30 2.E−52 122/398 30 6.E−48  93/318 29
    47 2.E−18  260/1249 20 2.E−18  260/1249 20 7.E−12 147/708 20
    48 1.E−18 133/596 22 1.E−18 133/596 22 2.E−24 146/605 24
    49 10 10 10
    50 10 10 10
    51 7.E−06  59/250 23 7.E−06  59/250 23 10
    52 2.E−38 137/455 30 2.E−38 137/455 30 1.E−36 131/573 22
    53 1.E−06  58/216 26 1.E−06  58/216 26 4.E−06  73/329 22
    54 2.E−20  306/1435 21 2.E−20  306/1435 21 3.E−19  334/1518 22
    55 10 10 10
    56 10 10 10
    57 10 10 10
    58 2.E−24  220/1026 21 2.E−24  220/1026 21 2.E−20 165/740 22
    59 10 10 10
    60 10 10 10
    61 5.E−23  82/266 30 5.E−23  82/266 30 8.E−23  82/266 30
    62 1.E−59 123/298 41 2.E−59 123/298 41 2.E−59 123/298 41
    63 4.E−49  97/223 43 3.E−36  87/254 34 4.E−54 110/258 42
    64 5.E−49 124/431 28 5.E−49 124/431 28 6.E−45  98/295 33
    65 10 10 10
    66 10 10 10
    67 10 10 10
    68 5.E−06  46/189 24 5.E−06  46/189 24 2.E−05  37/149 24
    69 2.E−12  43/149 28 4.E−12  42/146 28 7.E−12  42/146 28
    70 2.E−79 148/237 62 2.E−79 148/237 62 3.E−79 148/237 62
    71 3.E−30  74/227 32 3.E−30  74/227 32 2.E−27  84/260 32
    72 4.E−38 110/379 29 4.E−38 110/379 29 6.E−38 110/379 29
    73 2.E−50 119/324 36 1.E−50 119/324 36 3.E−50 119/324 36
    74 10 10 1.E−09 26/34 76
    75 10 10 10
    76 10 10 10
    77 10 10 10
    78 10 10 10
    79 10 10 10
    80 10 10 10
    81 10 10 10
    82 3.E−16  57/172 33 3.E−16  57/172 33 5.E−16  57/172 33
    83 10 10 0.002  24/102 23
    84 10 10 10
    85 10 10 10
    86 10 10 10
    87 6.E−10  84/348 24 6.E−10  84/348 24 7.E−09  44/140 31
    88 10 10 10
    89 10 10 10
    90 7.E−05  48/208 23 7.E−05  48/208 23 5.E−05  52/185 28
    91 10 10 10
    92 1.E−18 37/67 55 1.E−18 37/67 55 2.E−18 37/67 55
    93 10 10 1.E−23 55/79 69
    94 10 10 9.E−47 85/96 88
    95 10 10 10
    96 2.E−05 17/43 39 2.E−05 17/43 39 10
    97   e−101 191/462 41   e−101 191/462 41   e−100 191/462 41
    98 10 10 10
    99 8.E−51  92/114 80 8.E−51  92/114 80 1.E−50  92/114 80
    100 7.E−67 160/414 38 7.E−67 160/414 38 1.E−66 160/414 38
    101 2.E−35 102/322 31 2.E−35 102/322 31 4.E−35 102/322 31
    102 10 10 10
    103 10 10 10
    104 10 10 10
    105 10 10 0.001 27/84 32
    106 10 10 0.005 22/63 34
    107 10 10 10
    108 10 10 10
    109 10 10 10
    110 10 10 3.E−04  76/334 22
    111 10 10 10
    112 10 10 10
    113 10 10 10
    114 10 10 10
    115 10 10 10
    116 8.E−50 130/503 25 8.E−50 130/503 25 1.E−49 130/503 25
    117 10 10 10
    118 10 10 10
    119 10 10 10
    120 4.E−58 117/182 64 4.E−58 117/182 64 2.E−36  87/183 47
    121 8.E−51  95/171 55 8.E−51  95/171 55 7.E−17  50/149 33
    122 6.E−82 144/223 64 6.E−82 144/223 64 8.E−48  94/224 41
    123 0.E+00 546/853 64 0.E+00 546/853 64 0.E+00 384/839 45
    124 8.E−30  69/171 40 8.E−30  69/171 40 4.E−18  52/154 33
    125 6.E−27  68/151 45 6.E−27  68/151 45 5.E−23  70/169 41
    126 0.E+00 387/755 51 0.E+00 387/755 51 0.E+00 156/246 63
    127 10 10 10
    128 4.E−65 157/564 27 4.E−65 157/564 27 8.E−65 157/564 27
    129 10 10 10
    130 10 10 10
    131 10 10 10
    132 1.E−31  86/286 30 3.E−31  85/284 29 8.E−24  60/184 32
    133 4.E−17 132/556 23 4.E−17 132/556 23 7.E−17 132/556 23
    134 10 10 10
    135 1.E−07  36/134 26 1.E−07  36/134 26 2.E−07  36/134 26
    136 5.E−53 118/315 37 5.E−53 118/315 37 9.E−53 118/315 37
    137 0.E+00  917/1091 84 0.E+00  897/1039 86 0.E+00 257/288 89
    138 10 10 10
    139 10 10 1.E−55 113/264 42
    140 10 10 2.E−16 38/43 88
    141 10 10 1.E−15 33/43 76
    142   e−133 232/262 88   e−133 232/262 88   e−133 232/262 88
    143 10 10 1.E−93 162/181 89
    144 10 10 0.009 14/20 70
    145 10 10 10
    146 10 10 6.E−04 14/23 60
    147 10 10 3.E−14 33/57 57
    148   e−179 296/348 85   e−179 296/348 85   e−156 146/167 87
    149 10 10 10
    150 6.E−40  66/107 61 6.E−40  66/107 61 1.E−39  66/107 61
    151 8.E−30 57/71 80 8.E−30 57/71 80 1.E−29 57/71 80
    152 3.E−44  96/154 62 1.E−44  97/155 62 2.E−44  97/154 62
    153 10 10 10
    154 2.E−19  48/118 40 2.E−19  48/118 40 1.E−18  51/121 42
    155 10 10 10
    156 10 10 10
    157 1.E−06 23/42 54 1.E−06 23/42 54 2.E−06 23/42 54
    158 10 10 10
    159 10 10 0.008 25/56 44
    160 10 10 10
    161 10 10 10
    162 8.E−09  69/284 24 1.E−05  45/175 25 1.E−08  69/284 24
    163 10 10 10
    164 10 10 10
    165 10 10 10
    166 10 10 10
    167 10 10 10
    168 10 10 10
    169 1.E−31  86/286 30 3.E−31  85/284 29 8.E−24  60/184 32
    170 10 10 10
    171 10 10 10
    172 10 10 10
    173 10 10 10
    174 10 10 10
    175 1.E−31  86/286 30 3.E−31  85/284 29 8.E−24  60/184 32
    176 5.E−13 33/81 40 5.E−13 33/81 40 8.E−13 33/81 40
    177 8.E−31  65/118 55 8.E−31  65/118 55 1.E−30  65/118 55
    178 10 10 10
    179 10 10 10
    180 1.E−07 24/42 57 1.E−07 24/42 57 2.E−07 24/42 57
    181 10 10 10
    182 10 10 0.005 18/32 56
    183 10 10 1.E−05 33/81 40
    184 10 10 10
    185 2.E−19  64/250 25 2.E−19  64/250 25 3.E−19  64/250 25
    186 2.E−23  63/235 26 2.E−23  63/235 26 4.E−23  63/235 26
    187 5.E−13  59/274 21 5.E−13  59/274 21 8.E−13  59/274 21
    188 10 10 0.002 19/31 61
    189 1.E−11  78/262 29 1.E−11  78/262 29 6.E−14 143/685 20
    190 4.E−32  74/238 31 4.E−32  74/238 31 7.E−32  74/238 31
    191 3.E−46 116/278 41 3.E−46 116/278 41 6.E−46 116/278 41
    192 7.E−06  54/229 23 7.E−06  54/229 23 1.E−05  54/229 23
    193 10 10 10
    194 10 10 10
    195 10 1.E−23 51/92 55 2.E−23 51/92 55
    196 2.E−26  85/314 27 2.E−26  85/314 27 3.E−26  85/314 27
    197 10 10 10
    198 9.E−07  46/194 23 1.E−06  38/158 24 2.E−06  38/158 24
    199 10 10 10
    200 10 10 10
    201 3.E−95 186/362 51 3.E−95 186/362 51 4.E−95 191/383 49
    202 10 10 10
    203 0.E+00  569/1007 56 0.E+00  569/1007 56 0.E+00  546/1003 54
    204 4.E−74 166/456 36 4.E−74 166/456 36 3.E−71  90/190 47
    205 9.E−50 115/382 30 6.E−47 108/367 29 1.E−33  67/184 36
    206 10 10 2.E−27 52/65 80
    207 6.E−17 41/54 75 6.E−17 41/54 75 1.E−16 41/54 75
    208 2.E−12 32/86 37 2.E−12 32/86 37 3.E−12 32/86 37
    209 6.E−09  33/124 26 6.E−09  33/124 26 5.E−08 22/56 39
    210 10 10 10
    211 10 10 0.002 13/15 86
    212 2.E−21 50/56 89 2.E−21 50/56 89 4.E−21 50/56 89
    213 10 10 10
    214 10 10 10
    215 10 10 10
    216   e−119 199/226 88   e−119 199/226 88   e−119 199/226 88
    217 10 10   e−121 232/327 70
    218 10 10 1.E−13 35/45 77
    219 0.E+00 497/862 57 0.E+00 497/862 57 0.E+00 231/536 43
    220 1.E−75 133/208 63 1.E−75 133/208 63 1.E−52 100/217 46
    221 4.E−62 121/187 64 4.E−62 121/187 64 6.E−37  84/171 49
    222 2.E−54  97/194 50 2.E−54  97/194 50 3.E−49  77/156 49
    223   e−167 285/541 52   e−167 285/541 52   e−166 285/541 52
    224 1.E−72 163/443 36 1.E−72 163/443 36 2.E−72 163/443 36
    225 2.E−13  42/102 41 2.E−13  42/102 41 3.E−13  42/102 41
    226 10 10 3.E−13 34/40 85
    227 9.E−40 76/86 88 10 2.E−39 76/86 88
    228 10 10 10
    229 10 10 9.E−15 35/42 83
    230   e−108 205/382 53   e−108 205/382 53   e−114 149/247 60
    231 10 10 10
    232 9.E−05  86/394 21 9.E−05  86/394 21 10
    233 10 10 10
    234 10 10 10
    235 10 10 10
    236 10 10 10
    237 10 10 10
    238 10 10 10
    239 10 10 10
    240 10 10 10
    241 10 10 10
    242 1.E−20  99/376 26 1.E−20  99/376 26 3.E−20  99/376 26
    243 3.E−60 172/595 28 3.E−60 172/595 28 6.E−60 172/595 28
    244 2.E−60 169/578 29 2.E−60 169/578 29 3.E−60 169/578 29
    245 1.E−57 238/911 26 1.E−57 238/911 26 2.E−21  47/142 33
    246 10 10 10
    247 7.E−26  83/277 29 7.E−26  83/277 29 1.E−25  83/277 29
    248 10 10 0.002 32/127 25
    249 10 10 10
    250 4.E−80 169/408 41 4.E−80 169/408 41 7.E−76 112/232 48
    251 2.E−15  87/336 25 2.E−15  87/336 25 3.E−15  87/336 25
    252 2.E−22  95/372 25 2.E−22  95/372 25 3.E−18  84/322 26
    253 1.E−63  269/1057 25 1.E−63 269/1057 25 6.E−28  64/193 33
    254 6.E−17  88/436 20 6.E−17  88/436 20 0.007  45/208 21
    255 3.E−63 258/939 27 3.E−63 258/939 27 6.E−36  62/192 32
    256 8.E−86  298/1062 28 8.E−86  298/1062 28 6.E−47  70/191 36
    257 4.E−27 110/434 25 4.E−27 110/434 25 7.E−27 110/434 25
    258   e−102  312/1088 28   e−102 312/1088 28 1.E−25  80/229 34
    259 5.E−23  84/328 25 5.E−23  84/328 25 9.E−23  84/328 25
    260 10 10 10
    261 10 10 10
    262 9.E−41  97/285 34 9.E−41  97/285 34 2.E−40  97/285 34
    263 4.E−24 109/441 24 1.E−23 108/436 24 6.E−24 109/448 24
    264 7.E−22  67/215 31 7.E−22  67/215 31 1.E−21  67/215 31
    265 6.E−64 249/954 26 6.E−64 249/954 26 2.E−50  64/189 33
    266 10 10 10
    267 10 10 10
    268 10 10 10
    269 10 10 10
    270 10 10 10
    271 1.E−39 108/328 32 1.E−39 108/328 32 6.E−40 109/330 33
    272 2.E−05  42/170 24 2.E−05  42/170 24 10
    273 6.E−32 167/753 22 6.E−32 167/753 22 5.E−14  91/420 21
    274 10 10 10
    275 10 10 10
    276 3.E−60 102/153 66 3.E−60 102/153 66 5.E−60 102/153 66
    277 10 10 6.E−38  80/175 45
    278 0.E+00 338/471 71 0.E+00 338/471 71 0.E+00 198/290 68
    279 10 10 10
    280 10 10 10
    281 10 10 10
    282 10 10 10
    283 7.E−25  98/406 24 7.E−25  98/406 24 7.E−23  82/315 26
    284 10 10 4.E−09 24/33 72
    285 10 10 10
    286 10 10 10
    287 10 10 10
    288 0.E+00 520/641 81 0.E+00 408/526 77 0.E+00 776/924 83
    289 10 10 4.E−26 60/83 72
    290 10 10 6.E−19 43/49 87
    291   e−164 277/313 88   e−164 277/313 88   e−172 295/352 83
    292 10 10 8.E−16 36/42 85
    293 10 10 3.E−30 60/72 83
    294 10 10   e−155 177/224 79
    295 0.E+00 1036/1252 82 0.E+00 1036/1252 82 0.E+00 521/682 76
    296 2.E−78 133/153 86 2.E−78 133/153 86 3.E−78 133/153 86
    297 10 10 5.E−14 30/37 81
    298 6.E−75 151/295 51 4.E−73 148/290 51 2.E−74 150/295 50
    299 2.E−67 123/152 80 2.E−67 123/152 80 4.E−67 123/152 80
    300 1.E−51  98/162 60 1.E−51  98/162 60 2.E−51  98/162 60
    301 2.E−57 106/167 63 2.E−57 106/167 63 3.E−57 106/167 63
    302   e−107 186/250 74   e−107 186/250 74   e−107 186/250 74
    303 0.E+00 444/578 76 0.E+00 444/578 76 0.E+00 681/883 77
    304 4.E−72 137/189 72 4.E−72 137/189 72 7.E−67  91/124 73
    305 0.E+00 351/456 76 0.E+00 351/456 76 0.E+00 351/456 76
    306 10 10 10
    307   e−103  390/1363 28   e−103  390/1363 28   e−131 290/918 31
    308 3.E−43 81/91 89 3.E−43 81/91 89 5.E−43 81/91 89
    309 7.E−51  98/114 85 10 4.E−53 102/119 85
    310 5.E−05 104/439 23 5.E−05 104/439 23 5.E−06 207/951 21
    311 10 10 10
    312 7.E−71 159/458 34 9.E−71 159/458 34 5.E−67 139/378 36
    313 10 10 2.E−06 22/37 59
    314 10 10 10
    315 10 10 9.E−09  61/152 40
    316 10 10 0.005 18/32 56
    317 10 10 10
    318 1.E−07 24/42 57 1.E−07 24/42 57 2.E−07 24/42 57
    319 1.E−79 148/165 89 3.E−79 147/165 89 5.E−79 147/165 89
    320 10 10 10
    321 10 10 10
    322 10 10 10
    323 10 10   e−133 258/343 75
    324 10 10 10
    325 5.E−31  62/134 46 5.E−31  62/134 46 8.E−31  62/134 46
    326 10 10 10
    327 10 10 0.004 25/90 27
    328 10 10 7.E−07 23/36 63
    329 10 10 10
    330 2.E−49 115/308 37 2.E−49 115/308 37 2.E−41 100/273 36
    331 1.E−16  46/153 30 1.E−16  46/153 30 7.E−13  36/118 30
    332   e−131 224/291 76   e−131 224/291 76   e−122 209/255 81
    333 10 10 2.E−17 40/45 88
    334 10 10 10
    335 10 10 10
    336 4.E−14  40/104 38 4.E−14  40/104 38 4.E−08  45/127 35
    337 10 10 10
    338 10 10 10
    339 10 10 10
    340 10 10 10
    341 10 10 10
    342 10 10 10
    343 10 10 0.006 24/88 27
    344 10 10 10
    345 6.E−30  77/250 30 6.E−30  77/250 30 1.E−29  77/250 30
    346 4.E−44  97/184 52 4.E−44  97/184 52 1.E−36  76/148 51
    347   e−115 218/523 41   e−115 218/523 41   e−115 218/523 41
    348 10 10 2.E−15 36/49 73
    349   e−165 298/343 86   e−165 298/343 86   e−145 271/343 79
    350 10 10 10
    351 10 10 10
    352 2.E−23  66/215 30 2.E−23  66/215 30 3.E−23  68/209 32
    353 2.E−11  46/158 29 2.E−11  46/158 29 4.E−11  46/158 29
    354 10 7.E−10 31/46 67 2.E−15  49/101 48
    355 10 10 10
    356 10 10 10
    357 2.E−23  74/229 32 2.E−23  74/229 32 4.E−23  74/229 32
    358 10 10 10
    359 10 10 10
    360 10 10 10
    361 10 10 0.003  32/100 32
    362 2.E−73 179/543 32 2.E−73 179/543 32 3.E−73 179/543 32
    363 10 10 10
    364 10 10 10
    365 4.E−05  32/119 26 4.E−05  32/119 26 3.E−04  32/137 23
    366 2.E−05  46/185 24 2.E−05  46/185 24 10
    367 3.E−09  58/177 32 3.E−09  58/177 32 4.E−08  52/157 33
    368 10 10 10
    369   e−162 316/830 38   e−146 306/844 36 0.E+00 359/827 43
    370 3.E−08  42/160 26 3.E−08  42/160 26 6.E−05  38/139 27
    371 2.E−16  63/183 34 2.E−16  63/183 34 2.E−11  38/130 29
    372 3.E−22  55/159 34 3.E−22  55/159 34 6.E−22  55/159 34
    373 10 10 10
    374 6.E−06 25/83 30 6.E−06 25/83 30 1.E−05 25/83 30
    375 2.E−07  45/177 25 2.E−07  45/177 25 3.E−07  45/177 25
    376 10 10 10
    377 10 10 10
    378 10 10 10
    379 7.E−81 214/679 31 7.E−81 214/679 31 1.E−80 214/679 31
    380 5.E−54 108/122 88 2.E−53 107/122 87 10
    381 10 10 10
    382 7.E−21 212/963 22 7.E−21 212/963 22 8.E−18 179/807 22
    383 10 10 10
    384 10 10 10
    385 10 10 10
    386 10 10 10
    387 3.E−09  86/354 24 3.E−09  86/354 24 7.E−08  96/395 24
    388   e−108 176/316 55   e−108 176/316 55   e−107 176/316 55
    389 6.E−99 159/348 45 6.E−99 159/348 45 1.E−98 159/348 45
    390 2.E−87 144/235 61 2.E−87 144/235 61 4.E−87 144/235 61
    391 10 10 10
    392 0.E+00 316/472 66 0.E+00 316/472 66   e−170 273/410 66
    393 4.E−79 152/284 53 4.E−79 152/284 53 7.E−79 152/284 53
    394   e−138 241/456 52   e−138 241/456 52   e−138 241/456 52
    395 10 10 10
    396 10 10 4.E−51  94/107 87
    397 10 10 10
    398 10 10 10
    399 10 10 0.004  43/246 17
    400 2.E−08  54/204 26 2.E−08  54/204 26 10
    401 10 10 10
    402 10 10 10
    403 10 10 10
    404 5.E−06  34/156 21 5.E−06  34/156 21 10
    405 2.E−26 137/573 23 2.E−26 137/573 23 2.E−21  91/374 24
    406 2.E−39 100/356 28 2.E−39 100/356 28 2.E−39 104/376 27
    407 3.E−25  97/340 28 3.E−25  97/340 28 6.E−25  97/340 28
    408 8.E−55 100/184 54 8.E−55 100/184 54 1.E−54 100/184 54
    409   e−122 232/464 50   e−122 232/464 50   e−121 232/475 48
    410   e−108 189/338 55   e−108 189/338 55 1.E−44 108/341 31
    411 5.E−33  89/327 27 5.E−33  89/327 27 9.E−33  89/327 27
    412 1.E−06  32/154 20 1.E−06  32/154 20 10
    413 4.E−77 151/414 36 4.E−77 151/414 36 3.E−79 154/423 36
    414 2.E−10  51/206 24 2.E−10  51/206 24 4.E−10  51/206 24
    415 3.E−08  60/221 27 3.E−08  60/221 27 6.E−05  46/162 28
    416 2.E−42 102/313 32 2.E−42 102/313 32 3.E−42 102/313 32
    417 4.E−53 127/321 39 4.E−53 127/321 39 4.E−53 128/324 39
    418 10 10 10
    419 7.E−14  64/227 28 7.E−14  64/227 28 3.E−13  62/221 28
    420 2.E−09  40/126 31 2.E−09  40/126 31 3.E−09  40/126 31
    421 10 10 2.E−18 40/59 67
    422 1.E−54 110/185 59 1.E−54 110/185 59 3.E−50 103/172 59
    423 0.E+00 451/803 56 0.E+00 348/818 42 0.E+00 453/803 56
    424 2.E−68 117/227 51 2.E−68 117/227 51 1.E−63  99/196 50
    425 1.E−59 134/357 37 1.E−59 134/357 37 7.E−55 119/313 38
    426 2.E−58 113/257 43 2.E−58 113/257 43 3.E−58 113/258 43
    427 10 10 1.E−36  75/107 70
    428 3.E−63 133/312 42 3.E−63 133/312 42 6.E−63 133/312 42
    429 9.E−36  95/292 32 9.E−36  95/292 32 2.E−35  95/292 32
    430 10 10 10
    431 10 10 10
    432 10 10 10
    433 1.E−14  45/163 27 1.E−14  45/163 27 1.E−12  57/223 25
    434 2.E−37  84/227 37 2.E−37  84/227 37 4.E−37  84/227 37
    435 2.E−17  59/183 32 2.E−17  59/183 32 4.E−17  59/183 32
    436 0.E+00 404/834 48 0.E+00 404/834 48 0.E+00 256/536 47
    437 2.E−53 102/224 45 2.E−53 102/224 45 4.E−43  88/218 40
    438 6.E−11  53/180 29 6.E−11  53/180 29 1.E−10  53/180 29
    439 9.E−13 33/93 35 9.E−13  33/93 35 1.E−12 33/93 35
    440   e−100 178/437 40   e−100 178/437 40 2.E−99 178/437 40
    441 10 10 10
    442 1.E−29 172/695 24 1.E−29 172/695 24 2.E−29 172/695 24
    443 2.E−22  67/220 30 2.E−22  67/220 30 4.E−22  67/220 30
    444 2.E−55 126/321 39 2.E−55 126/321 39 4.E−54 117/284 41
    445 10 10 3.E−43  83/100 83
    446 5.E−61 112/149 75 5.E−61 112/149 75 2.E−56 63/74 85
    447 0.E+00 339/390 86 0.E+00 339/390 86 0.E+00 361/425 84
    448 10 10   e−107 182/230 79
    449 10 10 3.E−64 113/136 83
    450 10 10 10
    451 3.E−17  244/1144 21 3.E−17  244/1144 21 1.E−13 157/679 23
    452 10 10 8.E−35 67/87 77
    453 1.E−44 115/390 29 1.E−44 115/390 29 2.E−44 117/394 29
    454 3.E−18  46/139 33 3.E−18  46/139 33 4.E−14 24/72 33
    455   e−143 243/332 73   e−143 243/332 73   e−148 251/344 72
    456 9.E−69 126/305 41 3.E−68 123/296 41 2.E−68 126/305 41
    457 2.E−36  96/280 34 2.E−36  96/280 34 7.E−36  87/232 37
    458 2.E−75 142/328 43 2.E−75 142/328 43 3.E−75 142/328 43
    459 2.E−23 51/99 51 2.E−23 51/99 51 4.E−23 51/99 51
    460 10 10 10
    461 2.E−22  91/347 26 2.E−22  91/347 26 7.E−18  86/330 26
    462   e−103 177/416 42   e−103 177/416 42   e−103 177/416 42
    463 2.E−31 103/382 26 2.E−31 103/382 26 3.E−31 103/382 26
    464 6.E−95 184/480 38 6.E−95 184/480 38 1.E−94 184/480 38
    465 3.E−89 169/470 35 3.E−89 169/470 35 6.E−89 169/470 35
    466 10 10 10
    467 10 10 10
    468 10 10 10
    469 10 10 10
    470 10 10 10
    471 2.E−43  98/237 41 2.E−43  98/237 41 4.E−43  98/237 41
    472   e−115 203/392 51   e−115 203/392 51   e−112 185/345 53
    473 3.E−13 32/47 68 3.E−13 32/47 68 5.E−13 32/47 68
    474 1.E−07  52/206 25 1.E−07  52/206 25 2.E−07  52/206 25
    475 10 10 10
    476 3.E−15  56/188 29 3.E−15  56/188 29 5.E−15  56/188 29
    477 2.E−54 110/268 41 2.E−54 110/268 41 3.E−54 110/268 41
    478 10 10 10
    479 10 10 10
    480   e−143 243/382 63   e−143 243/382 63   e−142 243/382 63
    481   e−128 215/344 62   e−128 215/344 62   e−128 215/344 62
    482 10 10 10
    483 10 10 10
    484 3.E−40  97/226 42 3.E−40  97/226 42 2.E−20 49/89 55
    485 10 10 10
    486 10 5.E−46 87/97 89 4.E−56 105/120 87
    487 10 10 10
    488 10 10 3.E−18 41/52 78
    489 1.E−38  73/118 61 1.E−38  73/118 61 9.E−49  92/143 64
    490 10 10 5.E−07 27/36 75
    491 10 10 1.E−15 34/47 72
    492 10 10 10
    493 10 10 10
    494 2.E−27 55/82 67 2.E−27 55/82 67 4.E−27 55/82 67
    495 10 10 10
    496   e−105 213/487 43   e−105 213/487 43   e−105 213/487 43
    497 0.E+00 452/658 68 0.E+00 452/659 68 0.E+00 434/663 65
    498 3.E−17  64/268 23 3.E−17  64/268 23 5.E−15  62/273 22
    499 4.E−49 101/294 34 4.E−49 101/294 34 5.E−46  97/282 34
    500 3.E−31 106/400 26 3.E−31 106/400 26 10
    501 2.E−75 155/319 48 2.E−75 155/319 48 8.E−69 123/232 53
    502   e−119 216/466 46   e−119 216/466 46   e−115 214/461 46
    503   e−100 214/511 41   e−100 214/511 41 2.E−94 134/277 48
    504 10 10 10
    505 10 10 10
    506 10 10 10
    507 5.E−31  83/202 41 5.E−31  83/202 41 3.E−44  91/195 46
    508   e−103 186/260 71   e−103 186/260 71   e−103 186/260 71
    509   e−112 199/297 67   e−112 199/297 67   e−107 106/154 68
    510   e−118 211/292 72   e−118 211/292 72   e−118 130/181 71
    511   e−108 194/288 67   e−108 194/288 67   e−102 171/258 66
    512 2.E−34  99/316 31 2.E−34  99/316 31 3.E−34  99/316 31
    513 9.E−26 112/428 26 9.E−26 112/428 26 6.E−22  91/377 24
    514 9.E−97 175/424 41 9.E−97 175/424 41 2.E−96 175/424 41
    515 10 10 10
    516   e−106 169/262 64   e−106 169/262 64   e−106 169/262 64
    517 2.E−19 140/630 22 2.E−19 140/630 22 3.E−19 140/630 22
    518 9.E−30 132/525 25 9.E−30 132/525 25 2.E−18 120/566 21
    519 8.E−25 121/497 24 8.E−25 121/497 24 5.E−21 119/542 21
    520   e−137 239/427 55   e−137 239/427 55 7.E−58 74/198 37
    521 1.E−34 121/426 28 1.E−34 121/426 28 3.E−34 121/426 28
    522 1.E−56 138/416 33 1.E−56 138/416 33 9.E−52 128/428 29
    523 10 10 10
    524 1.E−96 170/272 62 1.E−96 170/272 62 5.E−91 137/205 66
    525 6.E−92 160/252 63 6.E−92 160/252 63 5.E−78 115/172 66
    526 3.E−30  90/271 33 3.E−30  90/271 33 3.E−29  88/271 32
    527 2.E−07 107/560 19 2.E−07 107/560 19 10
    528 1.E−79 156/420 37 1.E−79 156/420 37 7.E−64 144/420 34
    529 2.E−35  88/254 34 2.E−35  88/254 34 4.E−35  88/254 34
    530 10 10 10
    531 2.E−15  47/204 23 2.E−15  47/204 23 3.E−15  47/204 23
    532 1.E−77 176/458 38 1.E−77 176/458 38 1.E−61 112/289 38
    533   e−115 216/386 55   e−115 216/386 55   e−114 216/386 55
    534   e−116 196/290 67   e−116 196/290 67   e−116 196/290 67
    535 2.E−39 134/486 27 2.E−39 134/486 27 1.E−38 126/430 29
    536 1.E−20  62/231 26 1.E−20  62/231 26 4.E−13  97/410 23
    537 10 10 6.E−08 23/35 65
    538 10 10 9.E−18 37/44 84
    539 5.E−27  79/275 28 5.E−27  79/275 28 5.E−26  73/241 30
    540 10 10 10
    541 2.E−12  36/109 33 2.E−12  36/109 33 3.E−12  36/109 33
    542 7.E−33  77/224 34 7.E−33  77/224 34 1.E−32  77/224 34
    543 2.E−47 109/277 39 2.E−47 109/277 39 3.E−46 110/263 41
    544 4.E−66 121/266 45 3.E−66 121/266 45 6.E−66 121/266 45
    545 5.E−71 145/332 43 5.E−71 145/332 43 1.E−70 145/332 43
    546 2.E−42 146/504 28 2.E−42 146/504 28 3.E−41 144/502 28
    547 10 10 1.E−31  63/129 48
    548 2.E−59 100/137 72 2.E−59 100/137 72 3.E−59 100/137 72
    549 10 10 10
    550 10 10 10
    551 10 10 10
    552 7.E−22  67/217 30 2.E−21  66/215 30 1.E−17  42/118 35
    553 10 10 10
    554 10 10 0.006 15/43 34
    555   e−103 254/698 36   e−103 254/698 36   e−103 254/698 36
    556 5.E−31  84/286 29 2.E−30  83/284 29 8.E−24  60/184 32
    557 10 10 10
    558 4.E−05  32/119 26 4.E−05  32/119 26 3.E−04  32/137 23
    559 2.E−05  46/185 24 2.E−05  46/185 24 10
    560 3.E−09  58/177 32 3.E−09  58/177 32 4.E−08  52/157 33
    561 10 10 10
    562 3.E−51  99/243 40 3.E−51  99/243 40 5.E−51  99/243 40
    563   e−162 316/830 38   e−146 306/844 36 0.E+00 359/827 43
    564 3.E−08  42/160 26 3.E−08  42/160 26 6.E−05  38/139 27
    565 9.E−15  60/182 32 9.E−15  60/182 32 7.E−12  50/184 27
    566 10 10 10
    567 3.E−85 169/437 38 3.E−85 169/437 38 5.E−85 169/437 38
    568 10 10 10
    569 2.E−18  58/215 26 2.E−18  58/215 26 2.E−13  50/195 25
    570 10 10 10
    571 7.E−31  85/286 29 2.E−30  84/284 29 6.E−24  65/202 32
    572 10 10 3.E−22 43/49 87
    573 10 10   e−114 196/221 88
    574 10 10 10
    575   e−122 200/424 47   e−122 200/424 47   e−122 200/424 47
    576 7.E−24  75/255 29 7.E−24  75/255 29 1.E−23  75/255 29
    577 3.E−47 107/304 35 3.E−47 107/304 35 5.E−47 107/304 35
    578 10 10 9.E−40  91/126 72
    579 3.E−63 145/495 29 3.E−63 145/495 29 7.E−35  86/326 26
    580 1.E−84 164/298 55 1.E−84 164/298 55 1.E−82 161/295 54
    581 10 10 10
    582 2.E−35  71/132 53 2.E−35  71/132 53 3.E−34  70/131 53
    583 9.E−43  85/227 37 9.E−43  85/227 37 2.E−42  85/227 37
    584 10 10 10
    585 10 10 10
    586 10 10 0.006 17/50 34
    587 10 10 10
    588 10 10 1.E−48 103/138 74
    589 10 10 7.E−04 22/42 52
    590 10 10 10
    591   e−105 185/294 62   e−105 185/294 62   e−105 185/294 62
    592 10 10 10
    593 2.E−66 130/414 31 2.E−66 130/414 31 1.E−67 136/419 32
    594 5.E−08 27/95 28 5.E−08 27/95 28 5.E−06 22/74 29
    595 10 10 10
    596   e−179 321/549 58   e−179 321/549 58 2.E−96 181/334 54
    597 0  927/1041 89 0  891/1040 89 0 267/288 92
    598  1.E−175 295/329 89 1.00e0175 295/329 89  1.E−174 395/329 89
    599 3.00E−12  146/711 20 3.00e−12  146/711 20 5.00E−12  146/711 20
    A blank box indicates no hit above 10% identity in that strain
  • TABLE 5
    SEQ ID NOs with strongest inter-UPEC hits
     1  3  4  6  7  11  12  13  14  15  16  17  18  19  20  22
     24  33  34  35  37  41  42  44  45  46  47  48  50  51  52  53
     54  55  56  57  58  60  61  62  63  64  65  67  68  69  70  71
     72  73  75  82  87  96  97  99 100 101 106 110 111 114 115 116
    117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
    133 134 135 136 137 140 142 151 152 154 156 157 158 160 162 169
    175 176 177 180 182 184 185 186 187 188 189 190 191 192 194 195
    196 201 203 204 205 208 209 210 211 212 214 215 216 219 220 221
    222 223 224 225 227 230 232 233 234 235 236 237 238 239 241 242
    243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
    259 261 262 263 264 265 266 268 269 270 271 272 273 274 275 276
    278 279 280 281 283 285 286 288 291 295 296 298 299 300 301 302
    303 304 305 307 308 310 311 312 314 316 318 325 326 327 329 330
    331 332 334 335 336 340 341 342 343 344 345 346 347 349 350 351
    352 353 354 355 357 358 360 361 362 363 364 365 366 367 368 369
    370 371 372 374 375 379 380 382 387 388 389 390 392 393 394 399
    400 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418
    419 420 421 422 423 424 425 426 428 429 431 432 433 434 435 436
    437 438 439 440 442 443 444 445 446 447 450 451 453 454 455 456
    457 458 459 461 462 463 464 465 468 471 472 473 474 476 477 480
    481 482 484 485 486 487 489 490 491 492 493 494 496 497 498 499
    500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515
    516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531
    532 533 534 535 536 539 541 542 543 544 545 546 548 549 552 553
    555 556 557 558 559 560 561 562 563 564 565 567 568 569 571 574
    575 576 577 579 580 582 583 588 591 592 593 594 595 596
  • TABLE 6
    %
    SEQ Iden- % Tar-
    ID PSORT LP TMDs Top hit HitLen e tities Query get
    2 I * 1 Propionibacterium acnes transporter-related polypeptide|WO03/33515 221 3e−01 21/69 24 9
    6 I 3 Propionibacterium acnes immunogenic polypeptide|WO03/33515 318 2e−10  49/171 27 15
    7 O * 0 AmEPV late transcription factor-2 (AMV047)|WO200212526 259 0.011  33/114 23 12
    23 I 1 Enterohaemorragic E. coli 0157:H7-specific protein SEQ ID NO: 1295| 470 0.059 16/57 14 3
    JP2002355074
    25 I 1 Human XB51|JP2003164298 273 3e−01 14/28 4 5
    26 I * 1 Protein encoded by Prokaryotic essential gene|WO200277183 324 9e−02 15/38 9 4
    28 P * 0 Protein sequence SEQ ID 2281|EP1258494 503 2e−02 21/81 9 4
    29 O * 0 Novel human diagnostic protein|WO200175067 1395 6e−17  42/120 25 3
    30 I 1 Leishmania extended antigen LmgSP10|US2002169285 320 1e−02  36/157 9 11
    31 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 249 6e−02 27/95 11 10
    32 I 1 Acinetobacter baumannii protein|U.S. Pat. No. 6,562,958 284 1e−06  53/215 19 18
    36 I 2 C. glutamicum homeostasis and adaptation protein SEQ ID 32| 513 4e−02 15/33 10 2
    WO03/40290
    49 I 1 Prostate cancer marker protein|WO03/09814 338 4e−01 16/52 30 4
    57 I 1 Photorhabdus luminescens protein sequence|WO200294867 149 4e−02 15/41 36 10
    59 O * 0 Protein encoded by Prokaryotic essential gene|WO200277183 512 4e−02 13/40 20 2
    67 I * 1 Mycobacterium tuberculosis nutrient starvation-inducible protein| 429 4e−02 17/43 15 3
    WO03/04520
    68 I * 5 Acinetobacter baumannii protein|U.S. Pat. No. 6,562,958 273 6e−23  68/204 10 24
    76 I 2 Plasmodium falciparum chromosome 2 related protein 980 2e−05  42/172 13 4
    SEQ ID NO: 152|WO200025728
    77 P * 0 Mycoplasma genitalium outlier protein|US2003039963 1139 9e−02 13/37 19 1
    80 O * 0 Novel human diagnostic protein|WO200175067 812 2e−01  33/111 7 4
    81 I 2 Histophilus somni holin fragment|WO03/59392 74 3e−02 15/52 21 20
    82 P * 0 Heamin-binding protein from ORF5 of plasmid pRAP501|WO9321323 178 2e−20  61/163 35 34
    83 I * 3 Hepatitis GB virus protein sequence SEQ ID NO: 53|U.S. 177 0.033 23/75 16 12
    Pat. No. 6,051,374
    84 P * 0 Novel human secreted and transmembrane protein|WO03/72035 591 0.004 26/80 18 4
    85 I 1 Mycobacterium species protein sequence 46C|WO9909186 119 5e−02 28/84 15 23
    87 I 8 Enzyme EPS11 involved in exopolysaccharide 504 3e−23  86/320 25 17
    biosynthesis|WO9962316
    88 I 1 Herbicidally active polypeptide SEQ ID NO 2856|WO200210210 638 3e−01  35/139 18 5
    89 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 734 8e−02 12/24 7 1
    93 I 1 Human GPCR protein SEQ ID NO: 1506|EP1270724 156 1e−01 20/66 15 12
    94 I 0 Plasmodium falciparum chromosome 2 related protein|WO200025728 471 3e−02 16/74 16 3
    96 I 0 Protein encoded by Prokaryotic essential gene|WO200277183 87 6e−09 28/47 51 32
    112 I 0 Staphylococcus aureus protein|WO200294868 190 1e−01 12/36 15 6
    114 I 1 Novel human secreted and transmembrane protein|US2003008352 1894 2e−04 23/61 23 1
    124 O * 0 E. coli proliferation associated protein SEQ ID 365|WO200044906 176 6e−28  69/171 39 39
    125 O * 0 E. coli proliferation associated protein SEQ ID 366|WO200044906 167 4e−25  68/151 40 40
    138 O * 0 Protein encoded by Prokaryotic essential gene|WO200277183 312 4e−02  26/101 17 8
    139 I 1 Human parathyroid cell calcium receptor 5.2 1088 7e−02  32/118 10 2
    (HuPCaR 5.2)|U.S. Pat. No. 5,688,938
    142 I * 2 Protein encoded by Prokaryotic essential gene|WO200277183 285 2e−02  32/158 12 11
    143 I 2 Human Down syndrome-cell adhesion molecule DS-CAM2|WO9817795 1571 3e−01  27/104 11 1
    145 I 2 Protein encoded by Prokaryotic essential gene|WO200277183 679 3e−01 17/55 23 2
    147 P * 0 Human NS protein sequence SEQ ID NO: 159|WO200206315 354 1e−01 14/41 23 3
    149 I 1 Enterohaemorragic E. coli 0157:H7-specific protein|JP2002355074 55 0.003 18/29 41 32
    150 I 0 Human p53 modifying protein, SEQ ID 214|WO200299122 409 4e−02 13/42 11 3
    153 I 1 Enterohaemorragic E. coli 0157:H7-specific protein: 949|JP2002355074 50 1e−19 46/50 92 92
    155 I 0 Plasmodium falciparum chromosome 2 related protein|WO200025728 1700 5e−02 14/34 11 0
    159 P * 0 Novel human diagnostic protein|WO200175067 260 4e−02 25/56 46 9
    160 I * 1 Mouse PG1 protein sequence|WO9932644 354 4e−02 14/37 14 3
    161 I 2 Alloiococcus otitis antigenic protein SEQ ID NO: 1640|WO03/48304 225 9e−02 16/46 15 7
    165 I 0 A. niger metalloprotease polypeptide|WO200268623 791 3e−01  29/110 24 3
    167 I 0 Pinus radiata cell signalling involved protein SEQ ID: 128|WO200042171 120 2e−01 19/54 35 15
    170 I 1 Enterohaemorragic E. coli 0157:H7-specific protein: 881|JP2002355074 53 2e−13 38/42 15 71
    171 I 0 Enterohaemorragic E. coli 0157:H7-specific protein: 615|JP2002355074 50 1e−21 48/50 96 96
    176 P * 0 Protein encoded by Prokaryotic essential gene|WO200277183 239 3e−11 33/81 26 13
    178 I 0 Human colon cancer antigen protein SEQ ID NO: 7716|WO200122920 134 4e−02 11/20 8 8
    179 I 3 Human GPCR protein SEQ ID NO: 1630|EP1270724 523 0.014  23/115 16 4
    181 I 0 Bovine herpesvirus 1 US3 protein sequence SEQ ID NO: 9|WO03/12049 468 2e−02 17/56 7 3
    183 I 1 Novel human diagnostic protein|WO200175067 260 0.007 32/75 38 12
    184 I * 1 N. gonorrhoeae amino acid sequence SEQ ID 7826|WO200279243 508 4e−02 28/90 28 5
    188 I 1 Lactococcus lactis protein ycdH|FR2807446 486 3e−01 18/61 24 3
    194 I 1 MeCP2-Tat dMT fusion protein|WO200285948 561 0.029 11/22 15 2
    195 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 80 1e−17 44/80 47 55
    197 I 1 S. cinnamonensis CapK homologue|WO200168867 427 3e−18  91/350 21 21
    198 I 4 Protein encoded by Prokaryotic essential gene|WO200277183 219 4e−11  49/178 24 22
    199 I 8 Protein of drug metabolising enzyme - 2804794CD1|WO200266654 445 2e−28  78/231 20 17
    200 I 4 Synechocystis delta-6-desaturase polypeptide|US2002108147 359 2e−19  84/347 23 23
    202 I 0 Protein encoded by Prokaryotic essential gene|WO200277183 615 4e−01 14/41 29 2
    207 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 230 1e−14 41/54 65 17
    208 I 1 Acinetobacter baumannii protein|U.S. Pat. No. 6,562,958 358 2e−11 29/86 28 8
    209 I 1 Acinetobacter baumannii protein|U.S. Pat. No. 6,562,958 515 1e−06  29/124 21 5
    210 I * 2 Bacterial polypeptide|U.S. Pat. No. 6,605,709 320 3e−27  55/105 45 17
    211 I 1 S cerevisiae apoptosis associated protein YOR010C|WO200102550 730 8e−02 15/53 20 2
    212 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 240 4e−19 50/56 64 20
    225 P * 0 Listeria monocytogenes protein|WO200177335 100 2e−26 57/97 55 56
    228 P * 0 Drosophila melanogaster polypeptide SEQ ID NO 19890|WO200171042 460 5e−02  37/129 13 8
    232 I 1 Photorhabdus luminescens protein sequence|WO200294867 393 5e−13  93/377 19 23
    233 O * 0 Photorhabdus luminescens protein sequence|WO200294867 205 2e−11  50/183 27 24
    235 I 2 Photorhabdus luminescens protein sequence|WO200294867 513 9e−02 22/58 7 4
    236 O * 0 Alloiococcus otitis antigenic protein SEQ ID NO: 1664|WO03/48304 309 1e−02 15/30 7 4
    237 I 1 38 kd regression associated antigen|U.S. Pat. No. 5,242,823 403 6e−02  33/129 20 8
    238 I 1 Enterohaemorragic E. coli 0157:H7-specific protein: 1084|JP2002355074 131 1e−08 26/45 37 19
    240 I 0 Human polypeptide SEQ ID NO 21835|WO200164835 81 2e−01 11/40 24 14
    241 I 1 Aspergillus fumigatus essential gene protein|WO200286090 367 0.005  32/116 25 8
    277 I 1 Bacterial polypeptide|U.S. Pat. No. 6,605,709 338 3e−01 20/64 11 5
    279 I 0 Escherichia coli polypeptide SEQ ID NO 1320|WO200166572 367 0.017  45/217 12 12
    280 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 266 2e−02  28/105 7 10
    281 I 9 Streptococcus pneumoniae polypeptide SEQ ID NO 329|WO200283855 477 7e−07  34/118 7 7
    283 I 11 Protein encoded by Prokaryotic essential gene|WO200277183 431 6e−25 105/421 25 24
    284 P * 0 Novel human diagnostic protein|WO200175067 285 7e−05 23/29 44 8
    289 I 1 H. pylori selected interacting domain (SID) protein|WO200266501 268 2e−01 17/65 20 6
    290 I 0 Novel human diagnostic protein|WO200175067 750 2e−01 16/23 32 2
    306 I 1 S. pneumoniae antigenic protein SP092|U.S. Pat. No. 6,573,082 641 0.020  40/139 22 6
    308 O * 0 Listeria monocytogenes protein|WO200177335 439 9e−02 16/53 17 3
    309 I 3 Novel human diagnostic protein|WO200175067 285 0.001 17/23 14 5
    314 I * 1 N. gonorrhoeae amino acid sequence SEQ ID 7826|WO200279243 508 8e−02 28/90 28 5
    315 I * 3 Novel human diagnostic protein|WO200175067 261 2e−04  35/100 23 13
    317 I 0 Bovine herpesvirus 1 US3 protein sequence SEQ ID NO: 9|WO03/12049 468 5e−02 18/56 6 3
    322 I 1 M. truncatula squalene epoxidase|WO03/93425 526 1e−01 15/48 11 2
    323 I 2 Human PECAM-1 protein SEQ ID NO: 73|WO200078808 738 4e−02 18/55 5 2
    325 I 1 E. coli proliferation associated protein sequence: 300|WO200044906 138 2e−29  62/134 44 44
    326 P * 0 Cucumber raffinose synthase|JP11123080 784 1e−01 21/68 8 2
    327 P * 0 Predicted partial sequence of clone CXC-AMN20|WO9634112 262 1e−05  70/286 20 26
    348 I 2 Human polypeptide SEQ ID NO 21123|WO200164835 73 5e−02 15/47 30 20
    352 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 279 4e−29  72/225 30 25
    354 I 2 Novel human enzyme polypeptide|WO200155301 190 3e−13 40/61 26 21
    357 I 2 Protein encoded by Prokaryotic essential gene|WO200277183 898 7e−26  82/252 16 9
    358 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 206 5e−28  57/124 43 27
    359 P * 0 Drosophila melanogaster polypeptide SEQ ID NO 5868|WO200171042 1176 3e−01 19/75 23 1
    368 I 1 S. pneumoniae transport protein|WO9826072 293 4e−02 23/82 11 7
    374 P * 0 Novel human diagnostic protein|WO200175067 701 0.012 24/82 27 3
    375 I 4 Bacterial polypeptide|U.S. Pat. No. 6,605,709 184 0.001  36/168 23 19
    377 I 0 Rat Protein P26039, SEQ ID NO 11184|WO03/16475 2541 4e−02 22/67 20 0
    381 I 1 Photorhabdus luminescens protein sequence|WO200294867 323 2e−01 30/88 12 9
    387 I * 9 C glutamicum protein fragment SEQ ID NO: 4758|EP1108790 405 5e−08  79/354 19 19
    389 P * 0 Enterohaemorragic E. coli 0157:H7-specific protein: 1016|JP2002355074 108 3e−20 42/80 11 38
    390 O * 0 Protein encoded by Prokaryotic essential gene|WO200277183 230 6e−15  58/207 23 25
    391 I 1 Cephalosporin C acetylesterase|JP04144688 231 2e−18  67/218 24 29
    395 O * 0 Protein encoded by Prokaryotic essential gene|WO200277183 312 4e−02  26/101 17 8
    396 I 1 C glutamicum protein fragment SEQ ID NO: 5209|EP1108790 261 8e−02 15/30 6 5
    397 I 0 Acinetobacter sp. recombinase|US2003087403 324 9e−03 20/50 39 6
    401 O * 0 Streptococcus sobrinus glucosyltransferase-U|WO03/75845 1554 9e−02  30/139 9 1
    402 I 1 Lactococcus lactis protein yjeD|FR2807446 543 3e−15  77/331 22 14
    403 I 1 Fukuyama-type congenital muscular dystrophy-causing prtn|JP11313682 461 9e−06  42/167 10 9
    404 I 6 Protein encoded by Prokaryotic essential gene|WO200277183 264 1e−16  60/237 23 22
    407 I * 5 Photorhabdus luminescens protein sequence|WO200294867 367 2e−23  86/345 24 23
    412 I 4 Photorhabdus luminescens protein sequence|WO200294867 139 3e−24  50/121 31 35
    421 I 1 Lactococcus lactis protein yqbF|FR2807446 320 0.093 27/78 22 8
    427 I 1 Streptococcus polypeptide SEQ ID NO 7746|WO200234771 139 2e−02  30/110 12 21
    441 O * 0 Human ORFX protein sequence SEQ ID NO: 18748|WO200192523 130 8e−02 14/42 10 10
    445 I 1 Novel human diagnostic protein|WO200175067 717 3e−04 24/33 14 3
    446 I 4 Haemophilus influenzae essential gene|WO200218601 165 7e−11  31/126 20 18
    449 I 1 Human polypeptide SEQ ID NO 21123|WO200164835 73 6e−02 21/65 15 28
    461 I 1 Bacterial polypeptide|U.S. Pat. No. 6,605,709 376 1e−20  87/358 23 23
    466 I 3 Arabidopsis thaliana protein fragment SEQ ID NO: 49986|EP1033405 382 2e−02 18/54 9 4
    467 I 1 Listeria monocytogenes protein|WO200177335 400 6e−19  90/309 20 22
    468 I 1 H. influenzae Orf25 polypeptide SEQ ID NO: 49|WO03/55905 664 2e−08  62/287 10 9
    473 I 1 Listeria monocytogenes protein|WO200177335 95 4e−17 44/48 53 46
    475 I 1 Human Protein P57723, SEQ ID NO 11321|WO03/16475 403 9e−02 18/36 18 4
    479 I 1 Novel human secreted/transmembrane prtn PRO1110|US2003215911 322 3e−01 13/42 20 4
    487 I 2 Human polypeptide SEQ ID NO 16040|WO200164835 94 2e−02 21/89 7 22
    488 I 0 Human polypeptide SEQ ID NO 18650|WO200164835 115 4e−01 15/32 26 13
    490 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 277 9e−02 20/78 4 7
    491 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 622 3e−01 14/39 2 2
    493 I 1 Bacterial polypeptide|U.S. Pat. No. 6,605,709 168 3e−06  36/118 21 21
    541 I 4 Acinetobacter baumannii protein|U.S. Pat. No. 6,562,958 146 2e−14  43/130 32 29
    547 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 1765 8e−02 22/64 14 1
    554 I 1 Human GPCR protein SEQ ID NO: 1864|EP1270724 294 0.058 30/72 34 10
    561 I 1 S. pneumoniae transport protein|WO9826072 293 4e−02 23/82 11 7
    566 O * 0 Enterohaemorragic E. coli 0157:H7-specific protein; 202|JP2002355074 48 4e−18 39/48 26 81
    573 I 1 Propionibacterium acnes predicted ORF-encoded polypeptide| 215 4e−10  52/191 23 24
    WO03/33515
    574 O * 0 Photorhabdus luminescens protein sequence|WO200294867 157 2e−10  46/134 22 29
    576 I 0 Alloiococcus otitis antigenic protein SEQ ID NO: 5934|WO03/48304 262 9e−26  90/273 34 34
    578 I 1 Streptococcus pneumoniae polypeptide SEQ ID NO 382|WO200283855 456 3e−02 17/35 12 3
    585 O * 0 Bifidobacterium longum NCC2705 ORF amino acid sequence SEQ ID 180 2e−01 14/42 12 7
    NO: 298|EP1227152
    586 O * 0 Human secreted polypeptide|US2003100051 266 1e−01 11/33 11 4
    588 I 1 Novel human diagnostic protein|WO200175067 1387 1e−01 22/83 16 1
    595 I 1 Protein encoded by Prokaryotic essential gene|WO200277183 345 3e−01 15/32 27 4
    Columns:
    PSORT: predicted location by the PSORT algorithm. I = inner membrane; O = outer membrane; P = periplasm
    LP: a star indicates a lipoprotein
    TMDs: number of transmembrane domains
    Top hit: closest match in patent database
    HitLen: length of the closest match
    e: ‘expected’ value in BLAST analysis
    Identities: number of identical residues over length of alignment, and length of the alignment
    % Query/% Target: % sequence identity from perspective of ExPEC or database sequence
  • TABLE 7
    Protein gi# SEQ ID Annotation Psort TMD
    upec-4519 26110866 489 Putative conserved I 1
    protein
    upec-3573 26109898 597 Antigen 43 precursor I 1
    upec-1196 26107513 120 F1C major fimbrial O 0
    subunit
    upec-2283 26108604 598 Outer membrane porin p C 0
    upec-2814 26109137 305 Long-chain fatty acid O 0
    upec-0179 26106493 22 Ferrichrome-iron O 0
    receptor precursor
    upec-1875 26108194 221 Type-1 fimbrial protein I 1
    upec-1873 26108192 219 Outer membrane usher I 1
    upec-3606 26109931 400 KpsD protein P 0
    upec-5079 26111428 565 PapA protein O 0
    upec-3510 26109835 371 PapA protein O 0
    upec-3541 26109866 599 IutA protein C 0
    upec-5065 26111414 555 Putative iron-regulated I 1
    protein
    Columns:
    PSORT: predicted location by the PSORT algorithm. I = inner membrane; O = outer membrane; P = periplasm; C = cytoplasm
    TMD: number of transmembrane domains
    % ID: percentage identitical residues over length of alignment
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Claims (12)

We claim:
1. A polypeptide composition comprising: an isolated polypeptide comprising (a) an amino acid sequence that is the full-length amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598 and 599; (b) an amino acid sequence that is at least 80% identical to the full-length amino acid sequence of (a); (c) an amino acid sequence which is a fragment of at least 10 consecutive amino acids from the full-length amino acid sequence of (a); or (d) an amino acid sequence that is at least 80% identical to the full-length amino acid sequence of (a) and includes a fragment of at least 10 consecutive amino acids from the full-length amino acid sequence of (a).
2. The polypeptide composition of claim 1, wherein the isolated polypeptide comprises (i) an amino acid sequence that is the full-length amino acid sequence of (a); (ii) an amino acid sequence that is at least 90% identical to the full-length amino acid sequence of (a); (iii) an amino acid sequence which is a fragment of at least 16 consecutive amino acids from the full-length amino acid sequence of (a); or (d) an amino acid sequence that is at least 90% identical to the full-length amino acid sequence of (a) and includes a fragment of at least 16 consecutive amino acids from the full-length amino acid sequence of (a).
3. The polypeptide composition of claim 1, wherein said fragment comprises at least one B-cell epitope of the full-length amino acid sequence of (a).
4. The polypeptide composition of claim 2, wherein said fragment comprises at least one B-cell epitope of the full-length amino acid sequence of (a).
5. A pharmaceutical composition comprising the polypeptide composition of any one of claims 1 to 4, in admixture with a pharmaceutically acceptable carrier.
6. A pharmaceutical composition comprising the polypeptide composition of any one of claims 1 to 4 with two or more polypeptides of any one of claims 1 to 4, in admixture with a pharmaceutically acceptable carrier.
7. An immunogenic composition comprising one or more outer membrane vesicles (OMVs) expressing or overexpressing one or more isolated polypeptides of claim 1.
8. The immunogenic composition of claim 7, wherein said fragment comprises at least one B-cell epitope of the full-length amino acid sequence of (a).
9. A method for raising an immune response in a subject, comprising the step of administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier in admixture with the polypeptide composition of any one of claims 1 to 4 or the immunogenic composition of claim 7 or claim 8.
10. The method of claim 9, wherein the pharmaceutical composition further comprises an adjuvant.
11. The method of claim 9, wherein the immune response is a protective immune response.
12. The method of claim 10, wherein the immune response is a protective immune response.
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