Classifications

• • 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
  • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/04—Drugs for disorders of the respiratory system for throat disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • 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
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• This invention is in the field of Haemophilus influenzae immunology and vaccinology. BACKGROUND ART
• Haemophilus influenzae is a small, non-motile, Gram-negative coccobacillus. It is a respiratory pathogen that causes a wide spectrum of human infections, including: asymptomatic colonization of the upper respiratory tract (i.e. carriage); infections that extend from colonized mucosal surfaces to cause otitis media (inflammation of the middle ear), bronchitis, conjunctivitis, sinusitis, urinary tract infections and pneumonia; and invasive infections, such as bacteremia, septic arthritis, epiglottitis, pneumonia, empyema, pericarditis, cellulitis, osteomyelitis and meningitis. H.influen ⁇ ae was the first bacterium for which a complete genome sequence was published [1].
• H. influenzae strains are either capsulated (typeable) or non-capsulated (non-typeable), and there are six major serological types of capsulated strains (a to f). 95% of Hinfluenzae-causnd invasive diseases are caused by H.influenz ⁇ e type b ('Hib') strains. The most serious manifestation of Hib disease is meningitis, but the introduction in the 1980s of vaccines based on conjugated Hib capsular saccharides has hugely reduced incidence of this disease.
• H.influenz ⁇ e non-typeable H.influenz ⁇ e
• NHi non-typeable H.influenz ⁇ e
• OM otitis media
• Hearing loss is the most common complication of OM, with behavioral, educational and language development delays being additional consequences of early onset OM with effusion.
• Acute OM is the most common bacterial infection in children in the USA.
• the non-typeable H. influenzae biogroup aegyptius causes epidemic conjunctivitis and Brazilian purpuric fever (BPF) [2], with BPF having a mortality of up to 70%.
• BPF Brazilian purpuric fever
• polypeptides for use in the development of vaccines for preventing and/or treating infections caused by non-typeable H.influenzae strains.
• polypeptides for use in improved vaccines for preventing and/or treating otitis media.
• the polypeptides may also be useful for diagnostic purposes, and as targets for antibiotics.
• the invention provides polypeptides comprising the H.influenzae amino acid sequences disclosed in the examples. These amino acid sequences are the even SEQ ID NOs between 2 and 5080. There are thus 2540 amino acid sequences, and these are referred to as NTHr ⁇ r ⁇ ?, where nnnn is a number between 0001 and 2832 (there are 292 ⁇ Y ⁇ nnnn numbers that have no sequence; see Table I). Further NTHi sequences of the invention are given as SEQ ID NOS: 5088 onwards.
• polypeptide may, compared to the NTHi sequences of the examples, include one or more (e.g.
• 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;
• non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• uncharged polar i.e. glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine.
• the polypeptides may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to the NTHi sequences of the examples.
• 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
• polypeptides of the invention are listed in Table II, including polypeptides that are lipidated, that are located in the outer membrane, that are located in the inner membrane, that are located in the periplasm, or that are not found in non-pathogenic H.influenzae strains. Particularly preferred polypeptides are those that fall into more than one of these categories e.g. polypeptides that are located in the outer membrane and are also not found in non-pathogenic H.influenzae strains.
• a particularly preferred polypeptide is NTH0867.
• NTH0861, NTH0863 and NTH0865 are also preferred.
• these four proteins are embodied by SEQ ID NOS: 1566, 5095, 1570, 5094, 1574, 5903, 1578 and 5092. Within these eight SEQ ID NOS, 1566-1578 are preferred.
• the invention further provides polypeptides comprising fragments of the H.influenzae 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 [4,5] or similar methods), or they can be predicted (e.g. using the Jameson- Wolf antigenic index [6], matrix-based approaches [7], TEPITOPE [8], neural networks [9], OptiMer & EpiMer [10, 11], ADEPT [12], Tsites [13], hydrophilicity [14], antigenic index [15] or the methods disclosed in reference 16 etc.).
• NTHi polypeptides of the invention are (a) the N-terminal signal peptides of the NTHi polypeptides of the invention, (b) the NTHi polypeptides, but without their N-terminal signal peptides, (c) the NTHi polypeptides, but without their N-terminal amino acid residue.
• 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 [17,18].
• Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [19] chemistry.
• Enzymatic synthesis [20] may also be used in part or in full.
• 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.) [21]. 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 Haemophilus or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i.e.
• Polypeptides of the invention are preferably H.influenzae polypeptides. Polypeptides of the invention preferably have the function indicated in Table III for the relevant sequence. 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).
• 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).
• 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-] generous-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 ⁇ -L ⁇ -X 2 -L 2 -COOH, NH 2 -X ⁇ -X 2 -COOH, NH 2 -X ⁇ -L,-X 2 -COOH, NH 2 -X ⁇ -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. 22 & 23], 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 (RLA) 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 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 IgGl, IgG2, IgG3 or IgG4 subclass. Antibodies of the invention may have a K 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.
• 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.
• a biological sample e.g. a blood or serum sample
• the invention provides nucleic acid comprising the H. influenzae nucleotide sequences disclosed in the examples. These nucleic acid sequences are the odd SEQ ID NOs between 1 and 5080.
• the invention also provides nucleic acid comprising nucleotide sequences having sequence identity to the H.influenzae nucleotide sequences disclosed in the examples. 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 the H.influenzae nucleic acid disclosed in the examples. 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 24].
• 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 x SSC, 6 x SSC, 1 x SSC, 0.1 x 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 x SSC, 1 x SSC, 0.1 x SSC, or de-ionized water.
• nucleic acid of the invention hybridizes to a target of the invention 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 x SSC.
• An exemplary set of intermediate stringency hybridization conditions is 55°C and 1 x SSC.
• An exemplary set of high stringency hybridization conditions is 68°C and 0.1 x SSC.
• Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the H.influenzae 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).
• 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 one of the odd-numbered SEQ ID NOS: 1 to 5079, or (b) the complement of (a); and said nucleic acid 5'-X-Y-Z-3' is neither (i) a fragment of one of the odd-numbered SEQ ID NOS: 1 to 5079 nor (ii) the complement of (i).
• the -X- and/or -Z- moieties may comprise a promoter sequence (or its complement).
• the invention also provides nucleic acid encoding the polypeptides and polypeptide fragments of the invention.
• the invention includes nucleic acid comprising sequences complementary to the sequences disclosed in the sequence listing (e.g. for antisense or probing, or for use as primers), as well as the sequences in the orientation actually shown.
• 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 Haemophilus 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 H.influenzae 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 NTHi sequences, but they may also comprise non-NTHi 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 PCAV 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.
• the invention also provides a kit comprising primers (e.g. PCR primers) for amplifying a template sequence contained within a Haemophilus bacterium (e.g. H.influenzae) 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 Haemophilus 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 compementary 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.28] or a promoter sequence [e.g. 29].
• the first oligonucleotide and/or the second oligonucleotide may include a detectable label (e.g. a fluorescent label).
• the template sequence may be any part of a genome sequence.
• 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 H.influenzae 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 H.influenzae in clinical samples has been reported [e.g. see refs. 30 & 31].
• Clinical assays based on nucleic acid are described in general in ref. 32.
• 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).
• compositions comprising: (a) polypeptide, antibody, 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.
• a 'pharmaceutically acceptable carriers' 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. 138.
• 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 15-30mg/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.
• 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. 33], or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred.
• the mineral containing compositions may also be formulated as a particle of metal salt [34].
• Aluminium phosphates are particularly preferred, particularly in compositions which include a H.influenzae saccharide antigen, and a typical adjuvant is amorphous aluminium hydroxyphosphate with P0 /A1 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 lOO ⁇ g Al 3+ per conjugate per dose. Where there is more than one conjugate in a composition, not all conjugates need to be adsorbed.
• Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 33; see also ref. 35] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A) may also be used.
• CFA Complete Freund's adjuvant
• IF A 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. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), 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. QS21 is marketed as StimulonTM.
• 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. 36.
• Saponin formulations may also comprise a sterol, such as cholesterol [37].
• Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexs (ISCOMs) [chapter 23 of ref. 33], ISCOMs typically also include a phospholipid such as phosphatidylethanolamkie or phosphatidylcholine.
• the ISCOM includes one or more of QuilA, QHA & QHC. ISCOMs are further described in refs. 37-39.
• the ISCOMS may be devoid of additional detergent [40].
• 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 pi).
• influenza virus such as HA or NA
• Hepatitis B virus such as core or capsid proteins
• Hepatitis E virus measles virus
• Sindbis virus Rotavirus
• Foot-and-Mouth Disease virus Retrovirus
• Norwalk virus Norwalk virus
• human Papilloma virus HIV
• RNA-phages Q ⁇ -phage (such as coat proteins)
• 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. 50. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 ⁇ m membrane [50].
• Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [51,52].
• Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174.
• OM-174 is described for example in refs. 53 & 54.
• 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 55, 56 and 57 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. 58-63.
• the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [64].
• the CpG sequence may be specific for inducing a Thl 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. 65-67.
• 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. 64 & 68-70.
• 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”).
• E.coli E.coli heat labile enterotoxin
• CT cholera
• PT pertussis
• 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.
• the use of ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 73- 80. 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. 81, specifically incorporated herein by reference in its entirety.
• 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 [82], etc.) [83], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
• cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [82], etc.) [83], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
• interleukins e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [82], etc.
• interferons e.g. interferon- ⁇
• macrophage colony stimulating factor e.g.
• Bioadhesives and mucoadhesives may also be used as adjuvants in the invention.
• Suitable bioadhesives include esterified hyaluronic acid microspheres [84] 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 [85].
• Microparticles may also be used as adjuvants in the invention.
• Microparticles i.e. a particle of -lOOnm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500nm 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. 86-88. J. Polyoxyethylene ether and polyoxyethylene ester formulations
• Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [89]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [90] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [91].
• 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.
• PCPP Polyphosphazene
• PCPP formulations are described, for example, in refs. 92 and 93.
• 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-s7i-glycero-3- hydroxyphosphoryloxy)-ethylamine MTP-PE) .
• thr-MDP N-acetyl- muramyl-L-threonyl-D-isoglutamine
• nor- MDP N-acetyl-normuramyl-L-alanyl-D-isoglutamine
• NTP-PE N-acetylmuramyl-L
• imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues (e,g. "Resiquimod 3M"), described further in refs. 94 and 95.
• the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
• the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [96]; (2) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL) [97]; (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); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).
• MPL monophosphorylipid A
• TDM trehalose dimycolate
• CWS cell wall skeleton
• LPS cell wall skeleton
• 3dMPL non-toxic derivative of LPS
• Other substances that act as immunostimulating agents are disclosed in chapter 7 of ref. 33.
• 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.
• 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 [100].
• 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 0.5ml.
• 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.
• 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 l ⁇ g and lOmg 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').
• the invention provides nucleic acid, polypeptide, 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, or antibody of the invention in the manufacture of: (i) a medicament for treating or preventing disease and/or infection caused by H.influenzae; (ii) a diagnostic reagent for detecting the presence of H.influenzae or of antibodies raised against H.influenzae; and/or (iii) a reagent which can raise antibodies against H.influenzae. Said H.influenzae serotype or strain, but is preferably a non-typeable H. influenzae.
• Said disease may be, for instance, otitis media (including acute otitis media), bronchitis, conjunctivitis, sinusitis, a urinary tract infection, pneumonia, bacteremia, septic arthritis, epiglottitis, pneumonia, empyema, pericarditis, cellulitis, osteomyelitis, lower respiratory tract infection or meningitis.
• otitis media including acute otitis media
• bronchitis conjunctivitis
• sinusitis a urinary tract infection
• pneumonia bacteremia
• septic arthritis epiglottitis
• pneumonia empyema
• pericarditis cellulitis
• osteomyelitis lower respiratory tract infection or meningitis.
• the invention is particularly useful for preventing inflammation of the middle ear, by eliciting an immune response that prevents bacteria from moving from the throat to the middle ear via the eustachian tube, where the middle ear is then colonised.
• the patient is preferably a human.
• the human is preferably a child (e.g. a toddler or infant); where the vaccine is for therapeutic use, the human is preferably an adult.
• a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
• One way of checking efficacy of therapeutic treatment involves monitoring NT ⁇ i infection after administration of the composition of the invention.
• a chinchilla model [146]) and then determining standard parameters including ELISA titres (GMT) of IgG.
• GTT ELISA titres
• 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.
• 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, vaginal, topical, transdermal, intranasal, 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.
• 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. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined.
• Bacterial infections affect various areas of the body and so 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 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, 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 101 & 102].
• NTH0861, NTH0863, NTH0865 and NTH0867 are particularly preferred for use with the invention (particularly in vaccines), and these four proteins can be used in combinations.
• the invention provides a composition comprising: (a) a NTH0861 protein; and (b) at least one further NTHi protein.
• the invention also provides a composition comprising: (a) a NTH0863 protein; and (b) at least one further NTHi protein.
• the invention also provides a composition comprising: (a) a NTH0865 protein; and (b) at least one further NTHi protein.
• the invention also provides a composition comprising: (a) a NTH0867 protein; and (b) at least one further NTHi protein.
• the further NTHi protein can be selected from proteins of the invention as described above.
• compositions comprising two or more (i.e. 2, 3 or 4) of NTH0861, NTH0863, NTH0865 and/or NTH0867.
• Preferred compositions comprise: (1) NTH0861 and NTH0863; (2) NTH0861 and NTH0865; (3) NTH0861 and NTH0867; (4) NTH0863 and NTH0865; (5) NTH0863 and NTH0867; (6) NTH0865 and NTH0867; (7) NTH0861, NTH0863 and NTH0865; (8) NTH0861, NTH0863 and NTH0867; (9) NTH0863, NTH0865 and NTH0867; (10) NTH0861, NTH0865 and NTH0867; (11) NTH0861, NTH0863 , NTH0865 and NTH0867.
• the NTH0861 protein preferably comprises SEQ ID NO: 1566, SEQ ID NO: 5095, or an amino acid sequence having sequence identity (as described above) to SEQ ID NO: 1566 and/or to SEQ ID NO: 5095.
• the NTH0863 protein preferably comprises SEQ ID NO: 1570, SEQ ID NO: 5094, or an amino acid sequence having sequence identity (as described above) to SEQ ID NO: 1570 and/or to SEQ ID NO: 5094.
• the NTH0865 protein preferably comprises SEQ ID NO: 1574, SEQ ID NO: 5093, or an amino acid sequence having sequence identity (as described above) to SEQ ID NO: 1574 and/or to SEQ ID NO: 5093.
• the NTH0867 protein preferably comprises SEQ ID NO: 1578, SEQ ID NO: 5092, or an amino acid sequence having sequence identity (as described above) to SEQ ID NO: 1578 and/or to SEQ ID NO: 5092.
• compositions of the invention are 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. 103 from serogroup C [see also ref. 104] or the oligosaccharides of ref. 105.
• a saccharide antigen from Streptococcus pneumoniae e.g. 106, 107, 108].
• an antigen from hepatitis A virus such as inactivated virus [e.g. 109, 110].
• an antigen from hepatitis B virus such as the surface and/or core antigens [e.g. 110, 111].
• diphtheria antigen such as a diphtheria toxoid [e.g. chapter 3 of ref. 112] e.g. the CRM 1 97 mutant [e.g. 113].
• tetanus antigen such as a tetanus toxoid [e.g. chapter 4 of ref. 112].
• 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. 114 & 115].
• - polio antigen(s) e.g. 116, 117
• IPV. - measles, mumps and/or rubella antigens e.g. chapters 9, 10 & 11 of ref. 112].
• influenza antigen(s) e.g. chapter 19 of ref. 112
• haemagglutinin and/or neuraminidase surface proteins such as the haemagglutinin and/or neuraminidase surface proteins.
• Streptococcus pyogenes group A streptococcus [e.g. 120, 121, 122].
• composition may comprise one or more of these further antigens.
• Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [115]).
• 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 diphtheria toxin, tetanus toxin, the N.meningitidis outer membrane protein [124], synthetic peptides [125,126], heat shock proteins [127,128], pertussis proteins [129,130], protein D from H.influenzae [131], cytokines [132], lymphokines [132], streptococcal proteins, hormones [132], growth factors [132], toxin A or B from C.diffic ⁇ le [133], iron-uptake proteins [134], etc.
• a preferred carrier protein is the CRM197 diphtheria toxoid [135].
• 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.
• nucleic acid preferably DNA e.g. in the form of a plasmid
• encoding the antigen may be used.
• Antigens are preferably adsorbed to an aluminium salt. Screening methods
• the invention provides a process for determining whether a test compound binds to a polypeptide of the invention. If a test compound binds to a polypeptide of the invention and this binding inhibits the life cycle of the H. influenzae bacterium, then the test compound can be used as an antibiotic or as a lead compound for the design of antibiotics.
• the process will typically comprise the steps of contacting a test compound with a polypeptide of the invention, and determining whether the test compound binds to said polypeptide.
• Preferred polypeptides of the invention for use in these processes are enzymes (e.g. tRNA synthetases), membrane transporters and ribosomal polypeptides.
• test compounds include polypeptides, polypeptides, carbohydrates, lipids, nucleic acids (e.g. DNA, RNA, and modified forms thereof), as well as small organic compounds (e.g. MW between 200 and 2000 Da).
• the test compounds may be provided individually, but will typically be part of a library (e.g. a combinatorial library).
• Methods for detecting a binding interaction include NMR, filter-binding assays, gel-retardation assays, displacement assays, surface plasmon resonance, reverse two-hybrid etc.
• a compound which binds to a polypeptide of the invention can be tested for antibiotic activity by contacting the compound with GBS bacteria and then monitoring for inhibition of growth.
• the invention also provides a compound identified using these methods.
• the process comprises the steps of: (a) contacting a polypeptide of the invention with one or more candidate compounds to give a mixture; (b) incubating the mixture to allow polypeptide and the candidate compound(s) to interact; and (c) assessing whether the candidate compound binds to the polypeptide or modulates its activity.
• a candidate compound has been identified in vitro as a compound that binds to a polypeptide of the invention then it may be desirable to perform further experiments to confirm the in vivo function of the compound in inhibiting bacterial growth and/or survival.
• the method comprise the further step of contacting the compound with a NT ⁇ i bacterium and assessing its effect.
• the polypeptide used in the screening process may be free in solution, affixed to a solid support, located on a cell surface or located intracellularly.
• the binding of a candidate compound to the polypeptide is detected by means of a label directly or indirectly associated with the candidate compound.
• the label may be a fluorophore, radioisotope, or other detectable label.
• the invention provides a computer-readable medium (e.g. a floppy disk, a hard disk, a CD-ROM, a DVD etc.) and/or a computer memory and/or a computer database containing one or more of the sequences in the sequence listing.
• a computer-readable medium e.g. a floppy disk, a hard disk, a CD-ROM, a DVD etc.
• a computer memory and/or a computer database containing one or more of the sequences in the sequence listing e.g. a floppy disk, a hard disk, a CD-ROM, a DVD etc.
• 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.
• Alternative start codons can be used in biology.
• amino acid sequences in the sequence listing are based on particular start codons, but downstream start codons may alternatively be used.
• the invention specifically discloses and encompasses each of the amino acid sequences of the sequence listing, starting at any methionine residue from the sequence that is downstream of the N-terminal residue shown in the sequence listing (e.g. SEQ ID NOS: 5088, 5089 & 5090).
• nucleic acids and polypeptides of the invention may include sequences that: (a) are identical (i.e.
• 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 138-145, etc.
• Genome sequencing has been carried out on a low-passage clinical NTHi isolate (strain 86-028NP [146]). A total of 2540 coding sequences were identified in the genome, and these are given in the sequence listing together with their inferred translation products. Annotation of 1489 of the polypeptide sequences is given in Table III. From the sequenced material, polypeptide-coding sequences of particular interest were selected for further work, with particular attention to immunogenic proteins for vaccine development. Lipoproteins
• NTH0094 NTH0163, NTH0167, NTH0255, NTH0289, NTH0838, NTH0909, NTH0997, NTH1000, NTH1016, NTHI 174, NTH1298, NTH1313, NTH1413, NTH1416, NTH1552, NTH1623, NTH1675, NTH1680, NTH1739, NTH1873, NTH1922, NTH1942, NTH1974, NTH2142, NTH2169, TH2251, NTH2349, NTH2356, NTH2358, NTH2524, NTH2588, NTH2595, NTH2641, NTH2673, NTH2715, NTH2754, NTH2758, and NTH2769.
• Lipoproteins are surface-exposed and, as such, they represent accessible immunological targets e.g. for diagnostic and for immunisation purposes. Moreover, it has been found in B.burgdorferi [147] that OspA protein is immunogenic in a lipidated form but is non-immunogenic in a non-lipidated form, and the authors concluded that post-translational lipid attachment is a critical determinant of OspA immunogenicity.
• H.influenzae is a Gram-negative bacterium, its cell wall includes an outer membrane.
• the following 48 were identified as being located in this outer membrane: NTH0017, NTH0193, NTH0227, NTH0241, NTH0252, NTH0270, NTH0283, NTH0432, NTH0498, NTH0502, NTH0504, NTH0512, NTH0539, NTH0638, NTH0647, NTH0648, NTH0788, NTH0839, NTH0867, NTH0914, NTH1054, NTH1075, NTH1082, NTH1200, NTH1203, NTH1214, NTH1290, NTH1390, NTH1582, NTH1652, NTH1666, NTH1744, NTH1819, NTH1845, NTH1900, NTH1953, NTH1956, NTH1958, NTH1962, NTH2039, NTH2232, NTH2234, NTH2235, N
• OMPs Outer membrane proteins
• NTHI 845 is an Aida-like autotransporter ('Lav') and is a preferred protein of the invention. It is conserved between NTHi strains, including one known to cause meningitis, but there is no corresponding gene in the Rd sequence or in strain R2846.. It lies between the tmk and holb genes.
• a preferred form of NTH1845 starts at Met-22 (i.e. SEQ ID NO: 5090).
• Periplasm As H.influenzae is a Gram-negative bacterium, it has a periplasm between its cell cytoplasmic membrane and its outer membrane. Of the 2540 coding sequences, the following 105 were identified as being located in the periplasm: NTH0014, NTH0049, NTH0057, NTH0059, NTH0106, NTH0119, NTH0120,
• Inner membrane As H.influenzae is a Gram-negative bacterium, it has an inner membrane. Of the 2540 coding sequences, the following 740 were identified as being located in the inner membrane: NTH0002, NTH0015, NTH0019, NTH0020, NTH0021, NTH0025, NTH0032, NTH0034, NTH0035, NTH0041, NTH0043, NTH0045, NTH0048, NTH0050, NTH0052, NTH0060, NTH0061, NTH0067, NTH0073, NTH0076, NTH0085, NTH0089, NTH0091, NTH0097, NTH0107, NTH0110, NTH0116, NTH0118, NTH0121, NTH0128, NTH0134, NTH0135, NTH0151, NTH0155, NTH0157, NTH0158, NTH0180, NTH0184, NTH0185, NTH0186, NTH0189, N
• NTH1201 NTH1205 NTH1216, NTH1220, NTH1221, NTH1224, NTH1225, NTH1226, NTH1229, NTH1230, NTH1231, NTH1233 NTH1234, NTH1236, NTH1238, NTH1240, NTH1241, NTH1250, NTH1252, NTH1254, NTH1255, NTH1256, NTH1262 NTH1273, NTH1275, NTH1279, NTH1280, NTH1281, NTH1282, NTH1283, NTH1286, NTH1287, NTH1293, NTH1295 NTH1297, NTH1300, NTH1302, NTH1305, NTH1306, NTH1307, NTH1308,
• NTH1069 in particular has been identified as a virulence-associated protein.
• a preferred form of this protein starts at Met-27 (i.e. SEQ ID NO: 5088).
• Another preferred form starts at Met-19 (SEQ ID NO: 5089).
• Adhesin An adhesin having amino acid sequence SEQ ID NO: 5091 has been identified in a NTHi strain isolated from a patient with meningitis. It is homologous to the Hia adhesin from N.meningitidis.
• the genome sequence of the serotype d strain KW20 [1,3] was published in 1995. As serotype d strains are generally not pathogens, but the sequenced NTHi strain is from a clinical infection, expressed NTHi sequences that are not seen in serotype d are likely to be the proteins that are involved in pathogenic mechanisms. Blocking these proteins, either by antibiotic treatment or by antibody binding, thus has therapeutic potential.
• Protein NMB0419 from Neisseria meningitidis has been found to participate in the meningococcal invasion mechanism [2]. The protein was shown to modulate bacterial interaction with monolayers of human repiratory epithelial cells, promoting invasion. A homologous protein BPF001 is seen in H.influenzae biogroup aegyptius, but study of this protein was not possible.
• the NTHi genome includes a region (SEQ ID NO: 5081) encoding a string of four polypeptides (NTH0861, NTH0863, NTH0865 and NTH0867) with strong similarity to NMB0419. This region and the four polypeptides are shown below: GCCAGCTTGCTGCCTGCGGTCGACTCTANGAGACCCCTACAACAAAACATAATGCTAAAC
• AAAAAATGTTTGGTTGTTTCAATTGTTGATTAATAAATGGACGATTTCTATGCGATTC ⁇ C (SEQ ID NO: 1578) NTH0867 M R F T AAAAACACTTTTTACCACCGCACTTTTAGGTGCTTCTATCTTTTCTTTTCAATCCACCGC K T L F T T A L L G A S I F S F Q S T A TTGGGCGGATACGCTGGAACAGCAATTCCAACAAGGTTCTGAAGCCACTACGAGAGGCGA W A D T L E Q Q F Q Q G S E A T T K G D TTATCAAACCACCTTTAAATTTTTGTTACCTCTGGCGGAGCAGGGAAATGCAGAGGCTCA Y Q T T F K F L P A E Q G N A E A Q ATTGATGTTGGGCGTGATGTATGCAAGAGGAATAGGCGTCAAACAAGATGair2 , 2?TGAAGC L M L G V M Y A R G I G V K Q D D F E A GGTGAAGTGGT
• ATCCATTCATTCAATTTAATAGGAAAACAAAATGAAACTCACAAAAACACTTCTTACCAC (SEQ ID NO: 1570) NTH0863 M K T K T L L T T CGCACTTTTCGGTGCTTCTGTATTTTCTTTTCAATCCACCGCTTGGGCGGATACGCTGGA A L F G A S V F S F Q S T A A D T L E ACAGCAATTCCAACAAGGTTTAACCGCTTATGAGCAAAGCAACTATCAAACCGCCTTTAA Q Q F Q Q Q G L T A Y E Q S N Y Q T A F K ACTTTGGTTACCTATGGCAGAGCAGGGATATGCAAAGGCTCAATTTAATTTGGGCGTGAT L W L P M A E Q G Y A K A Q F N L G V M GTATGCTAAGGGGCAAGGCGTCAAACAAGATG ⁇ TGAAGCGGTGAAGTGGTTTCGCAA Y A K G Q G V K Q D D F E A V K W F R K AGCGG
• NTH0861 AEQG NTH0863 AEQG NTH0865 AEQGDADAQLNLGAMYAIGRGVKQDGVEAVKWFRKAAEQGNAKAQNGLGMMYDGGLGIKQ NTH0867 AEQG BPF001 AEQGEEFGLLFLG ETYEDGEGVEK NMB0419 AAQGVVQAQYNLG VIYAEGRGVRQ
• NTH0861, NTH0863, NTH0865 and NTH0867 are of particular interest for immunisation purposes.
• NTH0867 in particular is an outer-membrane protein that is not seen in the Rd genome, and is of special interest.
• NTHi chronic otitis media
• Moraxella catarrhalis and Streptococcus pneumoniae a protein that has homologs in the Mxatarrhalis genome.
• proteins have been identified that in the NTHi genome that have homologs in the Mxatarrhalis genome, namely NTH0861 (SEQ ID NO: 1566), NTH0863 (SEQ ID NO: 1570), NTH0865 (SEQ ID NO: 1574) and NTH0867 (SEQ ID NO: 1578).
• These proteins can thus be used as antigens for a general AOM vaccine.
• the corresponding M.catarrhalis antigens can also be used, either on their own or in combination with the NTHi 5 antigens.
• SEQ ID NO: 1566 is given as SEQ ID NO: 5095: 10 20 30 40 50 60 70 80 I I 1 I I I I 1566 MKLTKTLLTTA LGASVLSFQSTAWADTLEQQFQQGLTAYEQSNYQTAFK WLP AEQGDANVQFN GVMYAEGQGVKQD
• SEQ ID NO: 1570 is given as SEQ ID NO: 5094: 10 20 30 40 50 60 70 80 1 I I I I I 1 1570 MKLTKTL TTA FGASVFSFQSTAADTLEQQFQQGLTAYEQSNYQTAFKLWLPMAEQGYAKAQFNLGVMYAKGQGVKQD 5094 MKLTKTLLTTA GASVFSFQSTA ADTPEQQFQQGSTAYEQSDYQTAFK WLPMAEQGDANVQFNLGV YAKGQGVKQD
• SEQ ID NO: 1574 is given as SEQ ID NO: 5093: 10 20 30 40 50 60 70 80
• SEQ ID NO: 1578 is given as SEQ ID NO: 5092: 10 20 30 40 50 60 70 80 I I I I I I I I
• Preferred NTH0861 proteins have identity to both of SEQ ID NOS: 1566 and 5095.
• Preferred NTH0863 proteins have identity to both of SEQ ID NOS: 1570 and 5094.
• Preferred NTH0865 proteins have identity to both of SEQ ID NOS: 1574 and 5093.
• Preferred NTH0867 proteins have identity to both of SEQ ID NOS: 1578 and 5092.
• NTH1575 NTH1580 NT ⁇ 1581 NTH1582 NTH1584 NTH1586 NTH1587 NTH1588
• NTH1658 NTH1662 NTH1664 NTH1666 NTH1668 NTH1671 NTHI 675 NTH1676 NTH1677 NTH1680 NTH1686 NTH1687 NTH1688 NTH1689 NTH1690 NTH1692
• NTH1820 NTH1821 NTH1822 NTH1824 NTH1828 NTH1829 NTH1832 NTH1833 NTH1835 NTH1836 NTH1837 NTH1842 NTH1845 NTH1849 NTH1850 NTH1854
• NTH2501 NTH2505 NTH2508 NTH2510 NTH2524 NTH2525 NTH2527 NTH2528 NTH2531 NTH2532 NTH2533 NTH2534 NTH2536 NTH2537 NTH2541 NTH2542
• EP-B-0509612. [29; EP-B-0505012. [3o; Yadav et al. (2003) Lett Appl Microbiol 37(3):190-5.

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