WO2000050599A1 - Haemophilus antigen - Google Patents

Haemophilus antigen Download PDF

Info

Publication number
WO2000050599A1
WO2000050599A1 PCT/EP2000/001423 EP0001423W WO0050599A1 WO 2000050599 A1 WO2000050599 A1 WO 2000050599A1 EP 0001423 W EP0001423 W EP 0001423W WO 0050599 A1 WO0050599 A1 WO 0050599A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
amino acid
sequence
Prior art date
Application number
PCT/EP2000/001423
Other languages
French (fr)
Inventor
Jean-Louis Ruelle
Joelle Thonnard
Original Assignee
Smithkline Beecham Biologicals S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Biologicals S.A. filed Critical Smithkline Beecham Biologicals S.A.
Priority to EP00909227A priority Critical patent/EP1157113A1/en
Priority to AU31582/00A priority patent/AU3158200A/en
Priority to CA002363829A priority patent/CA2363829A1/en
Priority to JP2000601163A priority patent/JP2002536998A/en
Publication of WO2000050599A1 publication Critical patent/WO2000050599A1/en

Links

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
    • 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
    • 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 relates to methods for the production of polynucleotides, (herein referred to as "BASB070” polynucleotide(s)”), polypeptides encoded by them (referred to herein as “BASB070” or “BASB070” polypeptide(s)”), and recombinant materials.
  • the invention relates to methods for using such polypeptides and polynucleotides, including vaccines against bacterial infections.
  • the invention relates to diagnostic assays for detecting infection of certain pathogens.
  • Haemophilus influenza is a non-motile Gram negative bacterium. Man is its only natural host. H. influenzae isolates are usually classified according to their polysaccharide capsule. Six different capsular types designated a through f have been identified. Isolates that fail to agglutinate with antisera raised against one of these six serotypes are classified as nontypeable, and do not express a capsule.
  • H. influenzae type b is clearly different from the other types in that it is a major cause of bacterial meningitis and systemic diseases.
  • Nontypeable H. influenzae (NTHi) are only occasionally isolated from the blood of patients with systemic disease.
  • NTHi is a common cause of pneumonia, exacerbation of chronic bronchitis, sinusitis and otitis media.
  • Otitis media is an important childhood disease both by the number of cases and its potential sequelae. More than 3.5 millions cases are recorded every year in the United States, and it is estimated that 80 % of children have experienced at least one episode of otitis before reaching the age of 3 (1). Left untreated, or becoming chronic, this disease may lead to hearing loss that can be temporary (in the case of fluid accumulation in the middle ear) or permanent (if the auditive nerve is damaged). In infants, such hearing losses may be responsible for delayed speech learning.
  • Streptococcus pneumoniae Three bacterial species are primarily isolated from the middle ear of children with otitis media: Streptococcus pneumoniae, NTHi and M. catarrhalis. These are present in 60 to 90 % of cases. A review of recent studies shows that S. pneumoniae and NTHi together represent about 30 %, and -M catarrhalis about 15 % of otitis media cases (2). Other bacteria can be isolated from the middle ear (H. influenza type B, S. pyogenes, ...) but at a much lower frequency (2 % of the cases or less).
  • Epidemiological data indicate that, for the pathogens found in the middle ear, the colonization of the upper respiratory tract is an absolute prerequisite for the development of an otitis; other factors are however also required to lead to the disease (3-9). These are important to trigger the migration of the bacteria into the middle ear via the Eustachian tubes, followed by the initiation of an inflammatory process. These other factors are unknown todate. It has been postulated that a transient anomaly of the immune system following a viral infection, for example, could cause an inability to control the colonization of the respiratory tract (5). An alternative explanation is that the exposure to environmental factors allows a more important colonization of some children, who subsequently become susceptible to the development of otitis media because of the sustained presence of middle ear pathogens (2).
  • H. influenzae Various proteins of H. influenzae have been shown to be involved in pathogenesis or have been shown to confer protection upon vaccination in animal models.
  • the NTHi 115 k-Da Hia protein (17) is highly similar to the Hsf adhesin expressed by H. influenzae type b strains (18).
  • Another protein, the Hap protein shows similarity to IgAl serine proteases and has been shown to be involved in both adhesion and cell entry (19).
  • OMP outer membrane proteins
  • P6 is a conserved peptidoglycan-associated lipoprotein making up 1-5 % of the outer membrane (25). Later a lipoprotein of about the same mol. wt. was recognized, called PCP (P6 crossreactive protein) (26). A mixture of the conserved lipoproteins P4, P6 and PCP did not reveal protection as measured in a chinchilla otitis-media model (27). P6 alone appears to induce protection in the chinchilla model (28).
  • NTHi expresses a dual human transferrin receptor composed of TbpA and TbpB when grown under iron limitation. Anti-TbpB protected infant rats. (32). Hemoglobin / haptoglobin receptors have also been described for NTHi (33). A receptor for Haem: Hemopexin has also been identified (34). A lactoferrin receptor is also present in NTHi, but is not yet characterized (35). A protein resembling neisserial FrpB-protein has not been described in NTHi.
  • a 80kDa OMP the D15 surface antigen, provides protection against NTHi in a mouse challenge model. (36).
  • a 42kDa outer membrane lipoprotein, LPD is conserved amongst Haemophilus influenzae and induces bactericidal antibodies (37).
  • a minor 98kDa OMP (38) was found to be a protective antigen, this OMP may very well be one of the Fe- limitation inducible OMPs or high molecular weight adhesins that have been characterized thereafter.
  • H. Influenzae produces IgA 1 -protease activity (39).
  • IgAl- proteases of NTHi reveals a high degree of antigenic variability (40).
  • NTHi, OMP26, a 26-kDa protein has been shown to enhance pulmonary clearance in a rat model (41 ).
  • the NTHi HtrA protein has also been shown to be a protective antigen. Indeed, this protein protected Chinchilla against otitis media and protected infant rats against H. influenzae type b bacteremia (42)
  • the frequency of NTHi infections has risen dramatically in the past few decades. This phenomenon has created an unmet medical need for new anti-microbial agents, vaccines, drug screening methods and diagnostic tests for this organism.
  • the present invention aims to meet that need. In particular the present invention aims to meet the need for a vaccine effective against NTHi.
  • the present invention relates to recombinant materials and methods for the production of B ASB070, in particular BASB070 polypeptides and BASB070 polynucleotides, for use especially in therapeutic or prophylactic vaccines.
  • the invention relates to methods for using such polypeptides and polynucleotides, including prevention and treatment of microbial diseases, amongst others.
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting expression or activity of BASB070 polynucleotides or polypeptides.
  • BASB070 encodes a polypeptide that has the features of a surface-exposed molecule recognisable by the immune system.
  • the polypeptide encoded by BASB070 contains a signal peptide, indicating that it is exported at least to the periplasm between the inner and outer membranes of the bacterium.
  • the polypeptide has similarities to other known surface-exposed proteins and potential similarity to other known immunogenic and immunoprotective peptides.
  • BASB070 is 26% identical to the HasR protein of Serratia marcescens in an 817 amino acid overlap.
  • S. marcescens HasR is a receptor for the HasA hemophore protein. It is a TonB dependent protein. It has the characteristics of an integral outer membrane protein with a ⁇ -barrel 3D structure.
  • the ⁇ -barrels formed by the integral outer membrane proteins are composed of anti-parallel, amphipathic ⁇ -strands. Their external loops contain frequently immunodominant B-cell epitopes.
  • BASB070 is sufficiently closely related to the HasR protein of Serratia marcescens to say that BASB070 is also an integral outer membrane protein with a ⁇ -barrel conformation. BASB070 or fragments of it therefore provide potential vaccine antigens.
  • the invention relates to the use of BASB070 polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to the use of polypeptides and polynucleotides of a BASB070 of Haemophilus influenzae, which is related by amino acid sequence homology to Serratia marcescens HasR hemophore receptor polypeptide.
  • the invention relates especially to the use of BASB070 having the nucleotide and amino acid sequences set out in SEQ ID NO:l or 3 and SEQ ID NO:2 or 4 respectively.
  • the invention further relates to uses of polynucleotides and polypeptides which have at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% or exact identity to the sequences identified in SEQ ID NO: 1 or 3 and SEQ ID NO:2 or 4.
  • the invention also relates to novel NTHi polynucleotide and polypeptide sequences disclosed herein.
  • BASB070 polypeptides of Haemophilus influenzae referred to herein as "BASB070”and “ “BASB070 polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • the present invention further provides uses for:
  • polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence which has at least 85% identity, more preferably at least 90%) identity, yet more preferably at least 95% identity, even more preferably at least 91-99% or exact identity to SEQ ID NO:l or 3 over the entire length of SEQ ID NO:l or 3 respectively; or (c) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide which has at least 85% identity, more preferably at least 90%) identity, yet more preferably at least 95% identity, even more preferably at least 97- 99%> or exact identity, to the amino acid sequence of SEQ ID NO:2 or 4.
  • the BASB070 polypeptides provided in SEQ ID NO:2 or 4 are the BASB070 polypeptides from Haemophilus influenzae strains Rd KW20 and ntHi 3224.
  • the invention also provides uses for immunogenic fragments of a BASB070 polypeptide, that is, a contiguous portion of the BASB070 polypeptide which has the same or substantially the same immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 4. That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises the BASB070 polypeptide.
  • an immunogenic fragment may include, for example, the BASB070 polypeptide lacking an N-terminal leader sequence, and/or a transmembrane domain and.or a C-terminal anchor domain.
  • the immunogenic fragment of BASB070 comprises substantially all of the extracellular domain of a polypeptide which has at least 85%) identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 91-99% identity, more preferably at least more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 or 4 over the entire length of SEQ ID NO:2 or 4.
  • a fragment is a polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing", or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of SEQ ID NO:2 or 4 or of a variant thereof, such as a continuous series of residues that includes an amino- and/or carboxyl -terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, are also preferred.
  • fragments characterized by structural or functional attributes such as fragments that comprise beta-barrels, alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2 or 4, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2 or 4.
  • variants in which several, 5-10, 1-5, 1-3 , 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
  • polypeptides, or immunogenic fragments, for use in the invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
  • the invention relates to the use of genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • the proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non-fused protein.
  • the fusion partner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein.
  • the fusion partner will be both an immunological fusion partner and expression enhancing partner.
  • Fusion partners include protein D from Haemophilus influenzas and the non-structural protein from influenza virus, NS1 (hemagglutinin).
  • Another fusion partner is the protein known as LytA.
  • LytA Preferably the C terminal portion of the molecule is used.
  • Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase LytA, (coded by the lytA gene ⁇ Gene, 43 (1986) page 265-272 ⁇ ) an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
  • the C-terminal domain of the LytA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE.
  • the present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • Polypeptides for use in the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • a polypeptide for use in the invention is derived from Haemophilus influenzae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide for use in the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • polynucleotide comprises a region encoding BASB070 polypeptides comprising a sequence set out in SEQ ID NO.T or 3 which includes a full length gene, or a variant thereof.
  • the BASB070 polynucleotides provided in SEQ ID NO: 1 or 3 are the BASB070 polynucleotides from Haemophilus influenzae strains Rd KW20 and ntHi 3224.
  • a polynucleotide of the invention encoding BASB070 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria, followed by obtaining a full length clone.
  • a polynucleotide sequence for use in the invention such as a polynucleotide sequence given in SEQ ID NO:l or 3
  • a library of clones of chromosomal DNA of Haemophilus influenzae in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions.
  • sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence.
  • DNA sequence set out in SEQ ID NO:l or 3 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in SEQ ID NO:2 or 4 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
  • the polynucleotide of SEQ ID NO:l between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 2740 of SEQ ID NO:l, encodes the polypeptide of SEQ ID NO:2.
  • the present invention provides uses for an isolated polynucleotide comprising or consisting of:
  • a polynucleotide encoding a polypeptide for use in the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45 - 65° C and an SDS concentration from 0.1 - 1%) with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO:l or 3 or a fragment thereof; and isolating a full- length gene and/or genomic clones containing said polynucleotide sequence.
  • the invention provides uses for a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in SEQ ID NO: 1 or 3. Also provided by the invention are uses for a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence.
  • the polynucleotide may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals.
  • the polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequence fused to them.
  • Polynucleotides for use with the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
  • the nucleotide sequence encoding BASB070 polypeptide of SEQ ID NO:2 or 4 may be identical to the polypeptide encoding sequence contained in nucleotides 1 to 2739 of SEQ ID NO:l, or the polypeptide encoding sequence contained in nucleotides 1 to 2754 of SEQ ID NO:3, respectively. Alternatively it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2 or 4.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Haemophilus influenzae BASB070 having an amino acid sequence set out in SEQ ID NO:2 or 4.
  • polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
  • the invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of SEQ ID NO:2 or 4.
  • Fragments of polynucleotides of the invention may be used, for example, to synthesize ull- length polynucleotides of the invention.
  • BASB070 variants that have the amino acid sequence of BASB070 polypeptide of SEQ ID NO:2 or 4 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
  • silent substitutions, additions and deletions that do not alter the properties and activities of BASB070 polypeptide.
  • polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding BASB070 polypeptide having an amino acid sequence set out in SEQ ID NO:2 or 4, and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 90% identical over its entire length to a polynucleotide encoding BASB070 polypeptide and polynucleotides complementary thereto.
  • polynucleotides at least 95% identical over their entire length to the same are particularly preferred.
  • those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99%> being the more preferred.
  • Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of SEQ ID NO: l or 3.
  • polynucleotides that hybridize, particularly under stringent conditions, to BASB070 polynucleotide sequences, such as the polynucleotides in SEQ ID NO:l or 3.
  • the invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein.
  • the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein.
  • stringent conditions and “stringent hybridization conditions” mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences.
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
  • a coding region of a B ASB070 gene may be isolated by screening using a DNA sequence provided in SEQ ID NO:l or 3 to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • PCR Nucleic acid amplification
  • PCR Nucleic acid amplification
  • the PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence).
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • the invention also provides uses for polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
  • inactive precursors When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al, Hum Mol Genet (1992) 1: 363, Manthorpe et al, Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (Wu et al, J Biol Chem.
  • the invention relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • Recombinant polypeptides for use in the present invention may be prepared using genetically engineered host cells comprising expression systems by processes well known in the art. Accordingly, in a -further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, ( 1986) and Sambrook, et al. ,
  • MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of 5 streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Neisseria, Moraxella and Haemophilus influenzae
  • fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus insect cells such as cells o ⁇ Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CN-1 and Bowes melanoma cells
  • plant cells such l o as cells of a gymnosperm or angiosperm.
  • vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from
  • transposons from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • 20 expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate D ⁇ A sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in
  • Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well-known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • the expression system may also be a recombinant live microorganism, such as a virus or bacterium.
  • the gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses.
  • Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Dialoguelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella, Shigella, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
  • poxviruses e.g; vaccinia, fowlpox, canarypox
  • alphaviruses Semliki Forest Virus, Kunststoffuelian Equine Encephalitis Virus
  • adenoviruses adeno-associated virus
  • picornaviruses
  • This invention is also related to the use of BASB070 polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of BASB070 polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the BASB070 gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials.
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis.
  • RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways.
  • amplification, characterization of the species and strain of infectious or resident organism present in an individual may be made by an analysis of the genotype of a selected polynucleotide of the organism.
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species.
  • Point mutations can be identified by hybridizing amplified DNA to labeled BASB070 polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics.
  • Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al, Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
  • an array of oligonucleotides probes comprising BASB070 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
  • the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1 or 3, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or 4 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2 or 4.
  • kits may comprise a substantial component.
  • Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents.
  • Detection of a mutated form of a polynucleotide of the invention, preferably SEQ ID NO:l or 3, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide.
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide or polypeptide level by a variety of techniques, such as those described elsewhere herein.
  • the nucleotide sequences of the present invention are also valuable for organism chromosome identification.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an organism's chromosome, particularly to a Haemophilus influenzae chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention may be an important step in correlating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data may be found on-line in a sequence database.
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.
  • the differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
  • Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example.
  • RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
  • RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
  • PCR primers complementary to a polynucleotide encoding BASB070 polypeptide can be used to identify and analyze mutations.
  • the invention further provides primers for, among other things, amplifying BASB070 DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material.
  • the primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or give a prognosis for the infection or its stage or course, or to serotype and/or classify the infectious agent.
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Haemophilus influenzae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1 or 3].
  • Increased or decreased expression of a BASB070 polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over- or under- expression of BASB070 polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example.
  • Assay techniques that can be used to determine levels of a BASB070 polypeptide, in a sample derived from a host, such as a bodily material, are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
  • the polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high-density arrays or grids. These high-density arrays are particularly useful for diagnostic and prognostic purposes.
  • a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention may be used for probing, such as using hybridization or nucleic acid amplification, using probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual.
  • Such a presence may indicate the presence of a pathogen, particularly Haemophilus influenzae, and may be useful in diagnosing and/or giving a prognosis for disease or a course of disease.
  • a grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO: 1 is preferred. Also preferred is a gridcomprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2.
  • polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.
  • antibodies against BASB070 polypeptides or polynucleotides there are provided antibodies against BASB070 polypeptides or polynucleotides.
  • Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols.
  • any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al. , Immunology Today 4: 72 (1983); Cole et al. , pg.
  • phage display technology may be utilized to select genes for antibodies with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti- BASB070 or from naive libraries (McCafferty, et al, (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et ⁇ /., (1991) Nature 352: 628).
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
  • antibodies against BASB070-polypeptide or B ASB070-polynucleotide may be employed to treat infections, particularly bacterial infections.
  • Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
  • the antibody or variant thereof is modified to make it less immunogenic in the individual.
  • the antibody may most preferably be "humanized," where the complementarity determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
  • a mimotope is generally a peptide sequence, sufficiently similar to the native peptide (sequentially or structurally), which is capable of binding to the binding site of the native peptide.
  • a mimotope is capable of being recognised by antibodies which recognise the native peptide; or is capable of raising antibodies which recognise the native peptide, optionally when coupled to a suitable carrier.
  • T cell recognition a mimotope is capable of being recognised by the same T cells that recognise the native peptide; or is capable of generating a T cell response which recognises the native peptide.
  • Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids.
  • the peptides may be modified for the purposes of ease of conjugation to a protein carrier.
  • the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail.
  • the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide and/or to increase the affinity of the peptide for a particular ligand.
  • Mimotopes may also be retro sequences of the natural peptide sequences, in that the sequence orientation is reversed; or alternatively the sequences may be entirely or at least in part comprised of D-stereoisomer amino acids (inverso sequences). Also, the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are of the D-stereoisomer form. Retro, inverso and retro- inverso peptides are described in WO95/24916 and WO94/05311.
  • peptide mimotopes may be identified using antibodies which are capable themselves of binding to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267 Bl). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native polypeptide.
  • One particularly important aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, preferably humans, which comprises inoculating the individual with a BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or variant thereof, adequate to produce antibody and/or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Haemophilus influenzae infection. Also provided are methods whereby such immunological response slows bacterial replication.
  • Yet another aspect of the invention relates to a method of inducing an immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or a variant thereof, for expressing BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not.
  • an immunological response such as, to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that
  • nucleic acid vectors may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.
  • a further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a BASB070 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant BASB070 polynucleotide and/or polypeptide encoded therefrom, or a fragment, or a mimotope, or a variant thereof, and/or comprises DNA and/or RNA which encodes and expresses an antigen of said B ASB070 polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention, such as a fragment or a mimotope or a variant.
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.
  • a BASB070 polypeptide or a fragment thereof may be fused with a co-protein or chemical moiety which may or may not by itself produce antibodies or induce a T cell response, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties.
  • fused recombinant protein preferably further comprises an antigenic co-protein, such as lipoprotein D from Haemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co- protein which solubilizes the protein and facilitates production and purification thereof.
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein.
  • the co-protein may be attached to either the amino- or carboxy-terminus of the first protein.
  • a BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or a variant thereof may be present in or encoded by a vector, such as the live recombinant vectors described above for example live bacterial vectors.
  • a vector such as the live recombinant vectors described above for example live bacterial vectors.
  • non-live vectors for the BASB070 polypeptide for example bacterial outer-membrane vesicles or "blebs”.
  • OM blebs are derived from the outer membrane of the two-layer membrane of Gram-negative bacteria and have been documented in many Gram-negative bacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett. 163 :223-228) including C.
  • a non-exhaustive list of bacterial pathogens reported to produce blebs also includes: Bordetell pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Esherichia coli, Haemophilus influenza, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa and Yersinia enterocolitica.
  • Blebs have the advantage of providing outer-membrane proteins in their native conformation and are thus particularly useful for vaccines. Blebs can also be improved for vaccine use by engineering the bacterium so as to modify the expression of one or more molecules at the outer membrane.
  • a desired immunogenic protein at the outer membrane such as the BASB070 polypeptide
  • the expression of outer-membrane molecules which are either not relevant (e.g. unprotective antigens or immunodominant but variable proteins) or detrimental (e.g. toxic molecules such as LPS, or potential inducers of an autoimmune response) can be downregulated.
  • the non-coding flanking regions of the BASB070 gene contain regulatory elements important in the expression of the gene. This regulation takes place both at the transcriptional and translational level.
  • the sequence of these regions can be obtained by DNA sequencing. This sequence information allows the determination of potential regulatory motifs such as the different promoter elements, terminator sequences, inducible sequence elements, repressors, elements responsible for phase variation, the shine-dalgarno sequence, regions with potential secondary structure involved in regulation, as well as other types of regulatory motifs or sequences.
  • This sequence information allows the modulation of the natural expression of the BASB070 gene.
  • the upregulation of the gene expression may be accomplished by altering the promoter, the shine-dalgarno sequence, potential repressor or operator elements, or any other elements involved.
  • downregulation of expression can be achieved by similar types of modification.
  • the expression of the gene can be put under phase variation control, or it may be uncoupled from this regulation.
  • the expression of the gene can be put under the control of one or more inducible elements allowing regulated expression. Examples of such regulation include, but are not limited to, induction by temperature shift, addition of inductor substrates like selected carbohydrates or their derivatives, trace elements, vitamins, co-factors, metal ions, etc.
  • modifications as described above can be introduced by several different means.
  • the modification of sequences involved in gene expression can be carried out in vivo by random mutagenesis followed by selection for the desired phenotype.
  • Another approach consists in isolating the region of interest and modifying it by random mutagenesis, or site-directed replacement, insertion or deletion mutagenesis.
  • the modified region can then be reintroduced into the bacterial genome by homologous recombination, and the effect on gene expression can be assessed.
  • the sequence knowledge of the region of interest can be used to replace or delete all or part of the natural regulatory sequences.
  • the regulatory region targeted is isolated and modified so as to contain the regulatory elements from another gene, a combination of regulatory elements from different genes, a synthetic regulatory region, or any other regulatory region, or to delete selected parts of the wild-type regulatory sequences. These modified sequences can then be reintroduced into the bacterium via homologous recombination into the genome.
  • a non-exhaustive list of preferred promoters that could be used for up-regulation of gene expression includes the promoters porA, porB, lbpB, tbpB, pi 10, 1st, hpuAB from N. meningitidis or N.
  • gonorrohea ompCD, copB, lbpB, ompE, UspAl, UspA2, TbpB from M. Catarrhalis; pi, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from H. influenzae.
  • the expression of the gene can be modulated by exchanging its promoter with a stronger promoter (through isolating the upstream sequence of the gene, in vitro modification of this sequence, and reintroduction into the genome by homologous recombination).
  • Upregulated expression can be obtained in both the bacterium as well as in the outer membrane vesicles shed (or made) from the bacterium.
  • the described approaches can be used to generate recombinant bacterial strains with improved characteristics for vaccine applications. These can be, but are not limited to, attenuated strains, strains with increased expression of selected antigens, strains with knock-outs (or decreased expression) of genes interfering with the immune response, strains with modulated expression of immunodominant proteins, strains with modulated shedding of outer-membrane vesicles.
  • a modified upstream region of the BASB070 gene which modified upstream region contains a heterologous regulatory element which alters the expression level of the BASB070 protein located at the outer membrane.
  • the upstream region according to this aspect of the invention includes the sequence upstream of the BASB070 gene.
  • the upstream region starts immediately upstream of the BASB070 gene and continues usually to a position no more than about 1000 bp upstream of the gene from the ATG start codon.
  • the upstream region can start immediately preceding the gene of interest, or preceding the first gene in the operon.
  • a modified upstream region according to this aspect of the invention contains a heterologous promotor at a position between 500 and 700 bp upstream of the ATG.
  • the invention provides the BASB070 polypeptide, in a modified bacterial Bleb.
  • the invention further provides modified host cells capable of producing the non-live membrane- based bleb vectors.
  • the invention further provides vectors comprising the BASB070 gene having a modified upstream region containing a heterologous regulatory element.
  • Vaccine antigens may be provided in a variety of other forms known in the art, depending on the properties of the protein.
  • Lipoproteins for example, because of the hydrophobicity of the lipids added to their N-terminus, are able to aggregate and to form micelles. The particulate nature of these structures can enhance the immunogenicity of the lipoprotein, as compared to the unlipidated version of the protein.
  • the size of the micelles may also have an impact on the immunogneicity of the lipoprotein and this can be modified for example by adjusting the extraction procedure.
  • compositions particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
  • polynucleotide or particular fragments thereof which have been shown to encode non- variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Haemophilus influenzae.
  • Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Haemophilus influenzae infection, in mammals, particularly humans.
  • the invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as any pharmaceutically acceptable carrier.
  • a suitable carrier such as any pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each could be administered via a mucosal surface such as intranasally, or administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation of the invention may also include adjuvant systems for enhancing the immunogenicity of the formulation.
  • An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed THl -type responses (cell-mediated response), and TH2-type immune responses (humoral response).
  • Extreme THl -type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses.
  • mice THl -type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies.
  • TH2- type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
  • cytokines the driving force behind the development of these two types of immune responses.
  • High levels of THl -type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
  • THl and TH2-type immune responses are not absolute. In reality an individual will support an immune response which is described as being predominantly THl or predominantly TH2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989) THl and TH2 cells: different patterns qflymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, pl45-173). Traditionally, THl-type responses are associated with the production of the IFN- ⁇ and IL-2 cytokines by T-lymphocytes.
  • cytokines often directly associated with the induction of THl-type immune responses are not produced by T-cells, such as IL-12.
  • TH2- type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.
  • the best indicators of the THl :TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of THl or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or (in the murine system) the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses.
  • a THl-type adjuvant is one which preferentially stimulates isolated T-cell populations to produce a high ratio of THl-type cytokines when re-stimulated with antigen in vitro, and promotes development of both CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulin responses associated with THl-type isotype.
  • Adjuvants which are capable of preferential stimulation of the THl cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.
  • 3 De-O-acylated monophosphoryl lipid A is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 Bl (SmithKline Beecham Biologicals SA).
  • the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (European Patent number 0 689 454).
  • 3D-MPL will be present in the range of lO ⁇ g - lOO ⁇ g preferably 25-50 ⁇ g per dose wherein the antigen will typically be present in a range 2-50 ⁇ g per dose.
  • Another preferred adjuvant comprises QS21, an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina. Optionally this may be admixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with a carrier.
  • QS21 an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina.
  • this may be admixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with a carrier.
  • 3D-MPL 3 De-O-acylated monophosphoryl lipid A
  • Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THl stimulating adjuvants when formulated together with an antigen.
  • Further adjuvants which are preferential stimulators of THl cell responses include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
  • THl stimulating adjuvants such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of THl cell response.
  • QS21 can be formulated together with 3D-MPL.
  • the ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1 ; preferably 1 :5 to 5 : 1 and often substantially 1 : 1.
  • the preferred range for optimal synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
  • a carrier which enhances immunogenicity is also present in the vaccine composition according to the invention.
  • a carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.
  • a preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tween 80.
  • a metabolisible oil such as squalene, alpha tocopherol and Tween 80.
  • the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion.
  • the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
  • QS21 and 3D-MPL will be present in a vaccine in the range of l ⁇ g - 200 ⁇ g, such as 10-100 ⁇ g, preferably lO ⁇ g - 50 ⁇ g per dose.
  • the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80.
  • the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion.
  • Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
  • Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier.
  • a non-toxic oil e.g. squalane or squalene
  • an emulsifier e.g. Tween 80
  • the aqueous carrier may be, for example, phosphate buffered saline.
  • the present invention also provides a polyvalent vaccine composition
  • a polyvalent vaccine composition comprising a vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating other bacterial or viral diseases, cancers, autoimmune diseases and related conditions.
  • a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.
  • compositions for purposes of compositions, kits and administration
  • compositions comprising a BASB070 polynucleotide and/or BASB070 polypeptide for administration to a cell or to a multicellular organism.
  • the invention also relates to compositions comprising a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists.
  • the polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual.
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal intradermalor transdermal routes among others.
  • the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route.
  • Preferred forms of systemic administration include injection, typically by intramuscular or subcutaneous injection. Other injection routes, such as intradermal, intraperitoneal, or intravenous can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, solutions, powders and the like.
  • the dosage level of the active agent will be from 0.01 ⁇ g/kg to 10 ⁇ g/kg, typically around 1 ⁇ g/kg.
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • Suitable dosages are in the range of 0.1-100 ⁇ g/kg of subject.
  • a vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.
  • a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
  • Sequence Databases Sequences in a Tangible Medium, and Algorithms
  • Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3-dimensional structures as well as to identify further sequences of similar homology.
  • These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as the GCG program package.
  • sequence analysis includes, for example, methods of sequence homology analysis, such as identity and similarity analysis, DNA, RNA and protein structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, codon usage analysis, nucleic acid base trimming, and sequencing chromatogram peak analysis.
  • a computer based method for performing homology identification. This method comprises the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
  • a computer based method for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Computer program methods to determine identity between two sequences include, but are not limited to, the GAP program in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN (Altschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988).
  • the BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • Parameters for polypeptide sequence comparison include the following:
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100%) identity to the reference sequence of SEQ ID NO:l or 3, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: l or 3 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO:l or 3
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non- integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 or 4 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l or 3, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100%) identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO: 1 or 3 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO:l or 3, or:
  • n n is the number of nucleic acid alterations
  • x n is the total number of nucleic acids in SEQ ID NO:l or 3
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2 or 4, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or 4 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2 or 4
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2 or 4, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2 or 4, or:
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2 or 4
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA including single and double-stranded regions.
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Disease(s) means any disease caused by or related to infection by bacteria, including, for example, otitis media, acute otitis media, recurrent otitis media, otitis media with effusion, sinusitis, conjuctivitis, rhinopharyngitis, laryngitis, obstructive laryngitis, alveolitis, bronchitis, chronic bronchitis, enhancement of chronic obstructive pulmonary disease, complications of cystic fibrosis, pericarditis, endocarditis, osteomyelitis, arthritis, genitouinary tract colonization and neonatal infection, bacteremia, septicemia, meningitis.
  • Example 1 BASB070 gene from Haemophilus influenzae strain Rd KW20 and non-typeable Haemophilus influenzae (NTHi) strain 3224.
  • the BASB070 gene of SEQ ID NO: 1 comes from Heamophilus influenzae strain Rd KW20.
  • the translation of the BASB070 polynucleotide sequence is shown in SEQ ID NO:2.
  • B BASB070 in NTHi strain 3224.
  • the sequence of the BASB070 gene comes from the sequencing of NTHi strain 3224.
  • the Ncol and Asp 718 restriction sites CC ATG G and GG TAC C were engineered into specifically designed forward and reverse amplification primers, respectively, permitting directional cloning of a BASB070 PCR product into the commercially available E. coli expression plasmid pBADg ⁇ II(A) (Invitrogen, USA, ampicillin resistant).
  • This plasmid provides the signal peptide from the bacteriophage fd pill protein such that a mature BASB070 protein can be targeted to the periplasm of E. coli.
  • the BASB070 PCR product was purified from the amplification reaction using Wizard PCR prepTM (Promega) according to the manufacturers instructions.
  • coli Top 10 cells according to methods well known in the art. Following a -2-3 hour outgrowth period at 37°C in -1.0 ml of LB broth, transformed cells were plated on LB agar plates containing Ampicillin (50 ⁇ g/ml). Individual ampicillin-resistant colonies were selecteded and analyzed by whole cell-based PCR to verify that transformants contained the BASB070 D ⁇ A insert. Transformants that produced the expected PCR product were identified as strains containing a BASB070 expression construct. Expression plasmid containing strains were then analyzed for the inducible expression of recombinant BASB070.
  • the following strains of Haemophilus influenzae are provided as a useful reference for the present invention.
  • the BASB070 gene utilised in accordance with the invention is not limited with regard to the strain, but it may correspond to theBASB070 gene as found in any of the strains listed below or any related strain. This information is provided merely for convenience to those of skill in the art and is not an admission that any provision of a deposit is required for enablement.
  • strain 3219C (ET7) strain 3241A (ET30) strain 840645 (ET51) strain 901905U (ET60) strain A840177 (ET40) strain A840177 (ET69)
  • HiRd strain An example of a HiRd strain is described in R.D. Fleischmann et al., Science . Vol 269: 496-512 (1995) and K. W. Wilcox et al., J. Bact. Vol 122: 443 (1975) with the strain name KW20. This strain was deposited by the authors with the American Type Culture Collection under deposit number ATCC 51907.
  • SEQ ID NO:4 Haemophilus influenzae BASB070 polypeptide sequence deduced from the polynucleotide of SeQ ID NO:3

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pulmonology (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides vaccine compositions comprising BASB070 polypeptides and polynucleotides encoding BASB070 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing BASB070 polypeptides to screen for antibacterial compounds.

Description

HAEMOPHILUS ANTIGEN
FIELD OF THE INVENTION
This invention relates to methods for the production of polynucleotides, (herein referred to as "BASB070" polynucleotide(s)"), polypeptides encoded by them (referred to herein as "BASB070" or "BASB070" polypeptide(s)"), and recombinant materials. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including vaccines against bacterial infections. In a further aspect, the invention relates to diagnostic assays for detecting infection of certain pathogens.
BACKGROUND OF THE INVENTION
Haemophilus influenza is a non-motile Gram negative bacterium. Man is its only natural host. H. influenzae isolates are usually classified according to their polysaccharide capsule. Six different capsular types designated a through f have been identified. Isolates that fail to agglutinate with antisera raised against one of these six serotypes are classified as nontypeable, and do not express a capsule.
The H. influenzae type b is clearly different from the other types in that it is a major cause of bacterial meningitis and systemic diseases. Nontypeable H. influenzae (NTHi) are only occasionally isolated from the blood of patients with systemic disease.
NTHi is a common cause of pneumonia, exacerbation of chronic bronchitis, sinusitis and otitis media.
Otitis media is an important childhood disease both by the number of cases and its potential sequelae. More than 3.5 millions cases are recorded every year in the United States, and it is estimated that 80 % of children have experienced at least one episode of otitis before reaching the age of 3 (1). Left untreated, or becoming chronic, this disease may lead to hearing loss that can be temporary (in the case of fluid accumulation in the middle ear) or permanent (if the auditive nerve is damaged). In infants, such hearing losses may be responsible for delayed speech learning.
Three bacterial species are primarily isolated from the middle ear of children with otitis media: Streptococcus pneumoniae, NTHi and M. catarrhalis. These are present in 60 to 90 % of cases. A review of recent studies shows that S. pneumoniae and NTHi together represent about 30 %, and -M catarrhalis about 15 % of otitis media cases (2). Other bacteria can be isolated from the middle ear (H. influenza type B, S. pyogenes, ...) but at a much lower frequency (2 % of the cases or less).
Epidemiological data indicate that, for the pathogens found in the middle ear, the colonization of the upper respiratory tract is an absolute prerequisite for the development of an otitis; other factors are however also required to lead to the disease (3-9). These are important to trigger the migration of the bacteria into the middle ear via the Eustachian tubes, followed by the initiation of an inflammatory process. These other factors are unknown todate. It has been postulated that a transient anomaly of the immune system following a viral infection, for example, could cause an inability to control the colonization of the respiratory tract (5). An alternative explanation is that the exposure to environmental factors allows a more important colonization of some children, who subsequently become susceptible to the development of otitis media because of the sustained presence of middle ear pathogens (2).
Various proteins of H. influenzae have been shown to be involved in pathogenesis or have been shown to confer protection upon vaccination in animal models. Adherence of NTHi to human nasopharygeal epithelial cells has been reported (10). Apart from fimbriae and pili (11-15), many adhesins have been identified in NTHi. Among them, two surface exposed high-molecular- weight proteins designated HMW1 and HMW2 have been shown to mediate adhesion of NTHi to epithelial cells (16). Another family of high molecular weight proteins has been identified in NTHi strains that lack proteins belonging to HMW1/HMW2 family. The NTHi 115 k-Da Hia protein (17) is highly similar to the Hsf adhesin expressed by H. influenzae type b strains (18). Another protein, the Hap protein shows similarity to IgAl serine proteases and has been shown to be involved in both adhesion and cell entry (19).
Five major outer membrane proteins (OMP) have been identified and numerically numbered.
Original studies using H. influenzae type b strains showed that antibodies specific for PI and P2 protected infant rats from subsequent challenge (20-21). P2 was found to be able to induce bactericidal and opsonic antibodies, which are directed against the variable regions present within surface exposed loop structures of this integral OMP (22-23). The lipoprotein P4 also could induce bactericidal antibodies (24).
P6 is a conserved peptidoglycan-associated lipoprotein making up 1-5 % of the outer membrane (25). Later a lipoprotein of about the same mol. wt. was recognized, called PCP (P6 crossreactive protein) (26). A mixture of the conserved lipoproteins P4, P6 and PCP did not reveal protection as measured in a chinchilla otitis-media model (27). P6 alone appears to induce protection in the chinchilla model (28).
Another fimbrin is described with homology to P5, which in itself has sequence homology to the integral Escherichia coli OmpA (29-30). This paradox needs further investigation to clarify the nature and role of pilin, pilin-associated proteins, pilin- excreting proteins and P5. It is however shown that NTHi adhere to mucus by way of fimbriae. (29). P5 appears to undergo antigenic drift during persistent infections with NTHi (31).
In line with the observations made with gonococci and meningococci, NTHi expresses a dual human transferrin receptor composed of TbpA and TbpB when grown under iron limitation. Anti-TbpB protected infant rats. (32). Hemoglobin / haptoglobin receptors have also been described for NTHi (33). A receptor for Haem: Hemopexin has also been identified (34). A lactoferrin receptor is also present in NTHi, but is not yet characterized (35). A protein resembling neisserial FrpB-protein has not been described in NTHi.
A 80kDa OMP, the D15 surface antigen, provides protection against NTHi in a mouse challenge model. (36). A 42kDa outer membrane lipoprotein, LPD is conserved amongst Haemophilus influenzae and induces bactericidal antibodies (37). A minor 98kDa OMP (38), was found to be a protective antigen, this OMP may very well be one of the Fe- limitation inducible OMPs or high molecular weight adhesins that have been characterized thereafter. H. Influenzae produces IgA 1 -protease activity (39). IgAl- proteases of NTHi reveals a high degree of antigenic variability (40). Another OMP of NTHi, OMP26, a 26-kDa protein has been shown to enhance pulmonary clearance in a rat model (41 ). The NTHi HtrA protein has also been shown to be a protective antigen. Indeed, this protein protected Chinchilla against otitis media and protected infant rats against H. influenzae type b bacteremia (42)
Background References 1. Klein, JO (1994) Clin.Inf.Dis 19:823
2. Murphy, TF (1996) Microbiol.Rev. 60:267 3. Dickinson, DP et al. (1988) J. Infect.Dis. 158:205
4. Faden, HL et al. (1991) Ann.Otorhinol.Laryngol. 100:612
5. Faden, HL et al (1994) J. Infect.Dis. 169:1312
6. Leach, AJ et al. (1994) Pediatr.Infect.Dis.J. 13:983 7. Prellner, KP et al. (1984) Acta Otolaryngol. 98:343
8. Stenfors, L-E and Raisanen, S. (1992) J.Infect.Dis. 165:1148
9. Stenfors, L-E and Raisanen, S. (1994) Acta Otolaryngol. 113:191
10. Read, RC. et al. (1991) J. Infect. Dis. 163:549
11. Brinton, CC. et al. (1989) Pediatr. Infect. Dis. J. 8:S54 12. Kar, S. et al. (1990) Infect. Immun. 58:903
13. Gildorf, JR. et al. (1992) Infect. Immun. 60:374
14. St. Geme, JW et al. (1991) Infect. Immun. 59:3366
15. St. Geme, JW et al. (1993) Infect. Immun. 61 : 2233
16. St. Geme, JW. et al. (1993) Proc. Natl. Acad. Sci. USA 90:2875 17. Barenkamp, SJ. et JW St Geme (1996) Mol. Microbiol. (In press)
18. St. Geme, JW. et al. (1996) J. Bact. 178:6281
19. St. Geme, JW. et al. (1994) Mol. Microbiol. 14:217
20. Loeb, MR. et al. (1987) Infect. Immun. 55:2612
21. Musson, RS. Jr. et al. (1983) J. Clin. Invest. 72:677 22. Haase, EM. et al. (1994) Infect. Immun. 62:3712
23. Troelstra, A. et al. (1994) Infect. Immun. 62:779
24. Green, BA. et al. (1991) Infect.Immun.59:3191
25. Nelson, MB. et al. (1991) Infect. Immun. 59:2658
26. Deich, RM. et al. (1990) Infect. Immun. 58:3388 27. Green, BA. et al. (1993) Infect.immun. 61 : 1950
28. Demaria, TF. et al. (1996) Infect. Immun. 64:5187
29. Miyamoto, N., Bakaletz, LO (1996) Microb. Pathog. 21 :343
30. Munson, RS.j.r. et al. (1993) Infect. Immun. 61 :1017 31. Duim, B. et al. (1997) Infect. Immun. 65:1351
32. Loosmore, SM. et al(1996) Mol.Microbiol. 19:575
33. Maciver, I. et al. (1996) Infect. Immun. 64:3703
34. Cope, LD. et al. (1994) Mol.Microbiol. 13:868 35. Schryvers, AB. et al. (1989) J. Med. Microbiol. 29:121
36. Flack, FS. et al. (1995) Gene 156:97
37. Akkoyunlu, M. et al. (1996) Infect. Immun. 64:4586
38. Kimura, A. et al. (1985) Infect. Immun. 47:253
39. Mulks, MH. et Shoberg, RJ (1994) Meth. Enzymol. 235:543 40. Lomholt, H. Alphen, Lv, Kilian, M. (1993) Infect. Immun. 61 :4575
41. Kyd, J.M. and Cripps, A.W. (1998) Infect. Immun. 66:2272
42. Loosmore, S.M. et al. (1998) Infect. Immun. 66:899
The frequency of NTHi infections has risen dramatically in the past few decades. This phenomenon has created an unmet medical need for new anti-microbial agents, vaccines, drug screening methods and diagnostic tests for this organism. The present invention aims to meet that need. In particular the present invention aims to meet the need for a vaccine effective against NTHi.
SUMMARY OF THE INVENTION
The present invention relates to recombinant materials and methods for the production of B ASB070, in particular BASB070 polypeptides and BASB070 polynucleotides, for use especially in therapeutic or prophylactic vaccines. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including prevention and treatment of microbial diseases, amongst others. In a further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting expression or activity of BASB070 polynucleotides or polypeptides.
It has been discovered that BASB070 encodes a polypeptide that has the features of a surface-exposed molecule recognisable by the immune system. For example, the polypeptide encoded by BASB070 contains a signal peptide, indicating that it is exported at least to the periplasm between the inner and outer membranes of the bacterium. Furthermore the polypeptide has similarities to other known surface-exposed proteins and potential similarity to other known immunogenic and immunoprotective peptides.
BASB070 is 26% identical to the HasR protein of Serratia marcescens in an 817 amino acid overlap. S. marcescens HasR is a receptor for the HasA hemophore protein. It is a TonB dependent protein. It has the characteristics of an integral outer membrane protein with a β-barrel 3D structure. The β-barrels formed by the integral outer membrane proteins are composed of anti-parallel, amphipathic β-strands. Their external loops contain frequently immunodominant B-cell epitopes. BASB070 is sufficiently closely related to the HasR protein of Serratia marcescens to say that BASB070 is also an integral outer membrane protein with a β-barrel conformation. BASB070 or fragments of it therefore provide potential vaccine antigens.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION The invention relates to the use of BASB070 polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to the use of polypeptides and polynucleotides of a BASB070 of Haemophilus influenzae, which is related by amino acid sequence homology to Serratia marcescens HasR hemophore receptor polypeptide. The invention relates especially to the use of BASB070 having the nucleotide and amino acid sequences set out in SEQ ID NO:l or 3 and SEQ ID NO:2 or 4 respectively.
The invention further relates to uses of polynucleotides and polypeptides which have at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% or exact identity to the sequences identified in SEQ ID NO: 1 or 3 and SEQ ID NO:2 or 4.
The invention also relates to novel NTHi polynucleotide and polypeptide sequences disclosed herein.
Polypeptides
In one aspect of the invention there are provided uses for polypeptides of Haemophilus influenzae referred to herein as "BASB070"and " "BASB070 polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
The present invention further provides uses for:
(a) an isolated polypeptide which comprises an amino acid sequence which has at least 85%o identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO:2 or 4;
(b) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence which has at least 85% identity, more preferably at least 90%) identity, yet more preferably at least 95% identity, even more preferably at least 91-99% or exact identity to SEQ ID NO:l or 3 over the entire length of SEQ ID NO:l or 3 respectively; or (c) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide which has at least 85% identity, more preferably at least 90%) identity, yet more preferably at least 95% identity, even more preferably at least 97- 99%> or exact identity, to the amino acid sequence of SEQ ID NO:2 or 4.
The BASB070 polypeptides provided in SEQ ID NO:2 or 4 are the BASB070 polypeptides from Haemophilus influenzae strains Rd KW20 and ntHi 3224.
The invention also provides uses for immunogenic fragments of a BASB070 polypeptide, that is, a contiguous portion of the BASB070 polypeptide which has the same or substantially the same immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 4. That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises the BASB070 polypeptide. Such an immunogenic fragment may include, for example, the BASB070 polypeptide lacking an N-terminal leader sequence, and/or a transmembrane domain and.or a C-terminal anchor domain. In a' preferred aspect the immunogenic fragment of BASB070 according to the invention comprises substantially all of the extracellular domain of a polypeptide which has at least 85%) identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 91-99% identity, more preferably at least more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 or 4 over the entire length of SEQ ID NO:2 or 4.
A fragment is a polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with BASB070 polypeptides, fragments may be "free-standing", or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of SEQ ID NO:2 or 4 or of a variant thereof, such as a continuous series of residues that includes an amino- and/or carboxyl -terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise beta-barrels, alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2 or 4, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2 or 4.
Particularly preferred are variants in which several, 5-10, 1-5, 1-3 , 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
The polypeptides, or immunogenic fragments, for use in the invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
In one aspect, the invention relates to the use of genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non-fused protein. The fusion partner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein. Preferably the fusion partner will be both an immunological fusion partner and expression enhancing partner.
Fusion partners include protein D from Haemophilus influenzas and the non-structural protein from influenza virus, NS1 (hemagglutinin). Another fusion partner is the protein known as LytA. Preferably the C terminal portion of the molecule is used. Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page 265-272}) an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LytA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.coli C- LytA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C- LytA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798}. It is possible to use the repeat portion of the LytA molecule found in the C terminal end starting at residue 178, for example residues 188 - 305.
The present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
Polypeptides for use in the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
It is most preferred that a polypeptide for use in the invention is derived from Haemophilus influenzae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide for use in the invention may also be obtained, for example, from organisms of the same taxonomic family or order. Polynucleotides
It is an object of the invention to provide uses for polynucleotides that encode BASB070 polypeptides, particularly polynucleotides that encode the polypeptide herein designated BASB070, for use in or in preparation of the vaccine compositions described herein.
In a particularly preferred embodiment the polynucleotide comprises a region encoding BASB070 polypeptides comprising a sequence set out in SEQ ID NO.T or 3 which includes a full length gene, or a variant thereof.
The BASB070 polynucleotides provided in SEQ ID NO: 1 or 3 are the BASB070 polynucleotides from Haemophilus influenzae strains Rd KW20 and ntHi 3224.
Using the information provided herein, such as a polynucleotide sequence set out in SEQ ID NO: 1 or 3, a polynucleotide of the invention encoding BASB070 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence for use in the invention, such as a polynucleotide sequence given in SEQ ID NO:l or 3, typically a library of clones of chromosomal DNA of Haemophilus influenzae in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence.
Moreover, the DNA sequence set out in SEQ ID NO:l or 3 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in SEQ ID NO:2 or 4 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID NO:l, between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 2740 of SEQ ID NO:l, encodes the polypeptide of SEQ ID NO:2. The polynucleotide of SEQ ID NO:3, between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 2755 of SEQ ID NO:3, encodes the polypeptide of SEQ ID NO:4.
In a further aspect, the present invention provides uses for an isolated polynucleotide comprising or consisting of:
(a) a polynucleotide sequence which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:l or 3 over the entire length of SEQ ID NO:l or 3 respectively; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO:2 or 4 over the entire length of SEQ ID NO:2 or 4 respectively. A polynucleotide encoding a polypeptide for use in the present invention, including homologues and orthologs from species other than Haemophilus influenzae, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45 - 65° C and an SDS concentration from 0.1 - 1%) with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO:l or 3 or a fragment thereof; and isolating a full- length gene and/or genomic clones containing said polynucleotide sequence.
The invention provides uses for a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in SEQ ID NO: 1 or 3. Also provided by the invention are uses for a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides for use with the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression. The nucleotide sequence encoding BASB070 polypeptide of SEQ ID NO:2 or 4 may be identical to the polypeptide encoding sequence contained in nucleotides 1 to 2739 of SEQ ID NO:l, or the polypeptide encoding sequence contained in nucleotides 1 to 2754 of SEQ ID NO:3, respectively. Alternatively it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2 or 4.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Haemophilus influenzae BASB070 having an amino acid sequence set out in SEQ ID NO:2 or 4. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of SEQ ID NO:2 or 4.
Fragments of polynucleotides of the invention may be used, for example, to synthesize ull- length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding BASB070 variants, that have the amino acid sequence of BASB070 polypeptide of SEQ ID NO:2 or 4 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of BASB070 polypeptide.
Further preferred for use in the invention are polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding BASB070 polypeptide having an amino acid sequence set out in SEQ ID NO:2 or 4, and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 90% identical over its entire length to a polynucleotide encoding BASB070 polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 95% identical over their entire length to the same are particularly preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99%> being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of SEQ ID NO: l or 3.
In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to BASB070 polynucleotide sequences, such as the polynucleotides in SEQ ID NO:l or 3.
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
A coding region of a B ASB070 gene may be isolated by screening using a DNA sequence provided in SEQ ID NO:l or 3 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al, PNAS USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
The invention also provides uses for polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
In accordance with one particular aspect of the invention, there is provided the use of a polynucleotide as described herein for therapeutic or prophylactic purposes, in particular genetic immunization. This is described in more detail later on in the section headed "Vaccines".
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al, Hum Mol Genet (1992) 1: 363, Manthorpe et al, Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (Wu et al, J Biol Chem. (1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al, Science (1989) 243: 375), particle bombardment (Tang et al, Nature (1992) 356:152, Eisenbraun et al, DNA Cell Biol (1993) 12: 791) and in vivo infection using cloned retroviral vectors (Seeger et al, PNAS USA (1984) 81 : 5849).
Vectors, Host Cells, Expression Systems
The invention relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
Recombinant polypeptides for use in the present invention may be prepared using genetically engineered host cells comprising expression systems by processes well known in the art. Accordingly, in a -further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, ( 1986) and Sambrook, et al. ,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of 5 streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Neisseria, Moraxella and Haemophilus influenzae; fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus insect cells such as cells oϊDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CN-1 and Bowes melanoma cells; and plant cells, such l o as cells of a gymnosperm or angiosperm.
A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from
15 transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The
20 expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DΝA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in
25 Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well-known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
The expression system may also be a recombinant live microorganism, such as a virus or bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses. Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella, Shigella, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of BASB070 polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of BASB070 polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the BASB070 gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein. Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled BASB070 polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al, Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASB070 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1 or 3, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or 4 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2 or 4.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferably SEQ ID NO:l or 3, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide or polypeptide level by a variety of techniques, such as those described elsewhere herein.
The nucleotide sequences of the present invention are also valuable for organism chromosome identification. The sequence is specifically targeted to, and can hybridize with, a particular location on an organism's chromosome, particularly to a Haemophilus influenzae chromosome. The mapping of relevant sequences to chromosomes according to the present invention may be an important step in correlating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data may be found on-line in a sequence database. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.
The differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers complementary to a polynucleotide encoding BASB070 polypeptide can be used to identify and analyze mutations.
The invention further provides primers for, among other things, amplifying BASB070 DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or give a prognosis for the infection or its stage or course, or to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Haemophilus influenzae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1 or 3]. Increased or decreased expression of a BASB070 polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over- or under- expression of BASB070 polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a BASB070 polypeptide, in a sample derived from a host, such as a bodily material, are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
The polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high-density arrays or grids. These high-density arrays are particularly useful for diagnostic and prognostic purposes. For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as using hybridization or nucleic acid amplification, using probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual. Such a presence may indicate the presence of a pathogen, particularly Haemophilus influenzae, and may be useful in diagnosing and/or giving a prognosis for disease or a course of disease. A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO: 1 is preferred. Also preferred is a gridcomprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2.
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.
In certain preferred embodiments of the invention there are provided antibodies against BASB070 polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al. , Immunology Today 4: 72 (1983); Cole et al. , pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985). Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.
Alternatively, phage display technology may be utilized to select genes for antibodies with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti- BASB070 or from naive libraries (McCafferty, et al, (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et α/., (1991) Nature 352: 628).
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against BASB070-polypeptide or B ASB070-polynucleotide may be employed to treat infections, particularly bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized," where the complementarity determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
Mimotopes
In a further aspect, the present invention relates to mimotopes of the polypeptide of the invention. A mimotope is generally a peptide sequence, sufficiently similar to the native peptide (sequentially or structurally), which is capable of binding to the binding site of the native peptide. Thus where an antibody-binding peptide is concerned, a mimotope is capable of being recognised by antibodies which recognise the native peptide; or is capable of raising antibodies which recognise the native peptide, optionally when coupled to a suitable carrier. In the case of T cell recognition, a mimotope is capable of being recognised by the same T cells that recognise the native peptide; or is capable of generating a T cell response which recognises the native peptide. Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides may be modified for the purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine. In addition it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic terminus distal from the conjugated terminus of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier protein. Thereby presenting the peptide in a conformation which most closely resembles that of the peptide as found in the context of the whole native molecule. For example, the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Alternatively, the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide and/or to increase the affinity of the peptide for a particular ligand.
Mimotopes may also be retro sequences of the natural peptide sequences, in that the sequence orientation is reversed; or alternatively the sequences may be entirely or at least in part comprised of D-stereoisomer amino acids (inverso sequences). Also, the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are of the D-stereoisomer form. Retro, inverso and retro- inverso peptides are described in WO95/24916 and WO94/05311.
Alternatively, peptide mimotopes may be identified using antibodies which are capable themselves of binding to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267 Bl). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native polypeptide.
Vaccines
One particularly important aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, preferably humans, which comprises inoculating the individual with a BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or variant thereof, adequate to produce antibody and/or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Haemophilus influenzae infection. Also provided are methods whereby such immunological response slows bacterial replication.
Yet another aspect of the invention relates to a method of inducing an immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or a variant thereof, for expressing BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not. One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vectors may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex. A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a BASB070 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant BASB070 polynucleotide and/or polypeptide encoded therefrom, or a fragment, or a mimotope, or a variant thereof, and/or comprises DNA and/or RNA which encodes and expresses an antigen of said B ASB070 polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention, such as a fragment or a mimotope or a variant. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.
A BASB070 polypeptide or a fragment thereof may be fused with a co-protein or chemical moiety which may or may not by itself produce antibodies or induce a T cell response, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Haemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co- protein which solubilizes the protein and facilitates production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein. The co-protein may be attached to either the amino- or carboxy-terminus of the first protein.
In a vaccine composition according to the invention, a BASB070 polynucleotide and/or polypeptide, or a fragment, or a mimotope, or a variant thereof may be present in or encoded by a vector, such as the live recombinant vectors described above for example live bacterial vectors. Also suitable are non-live vectors for the BASB070 polypeptide, for example bacterial outer-membrane vesicles or "blebs". OM blebs are derived from the outer membrane of the two-layer membrane of Gram-negative bacteria and have been documented in many Gram-negative bacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett. 163 :223-228) including C. trachomatis and C. psittaci. A non-exhaustive list of bacterial pathogens reported to produce blebs also includes: Bordetell pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Esherichia coli, Haemophilus influenza, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa and Yersinia enterocolitica.
Blebs have the advantage of providing outer-membrane proteins in their native conformation and are thus particularly useful for vaccines. Blebs can also be improved for vaccine use by engineering the bacterium so as to modify the expression of one or more molecules at the outer membrane. Thus for example the expression of a desired immunogenic protein at the outer membrane, such as the BASB070 polypeptide, can be introduced or upregulated (e.g. by altering the promoter). Instead or in addition, the expression of outer-membrane molecules which are either not relevant (e.g. unprotective antigens or immunodominant but variable proteins) or detrimental (e.g. toxic molecules such as LPS, or potential inducers of an autoimmune response) can be downregulated. These approaches are discussed in more detail below.
The non-coding flanking regions of the BASB070 gene contain regulatory elements important in the expression of the gene. This regulation takes place both at the transcriptional and translational level. The sequence of these regions, either upstream or downstream of the open reading frame of the gene, can be obtained by DNA sequencing. This sequence information allows the determination of potential regulatory motifs such as the different promoter elements, terminator sequences, inducible sequence elements, repressors, elements responsible for phase variation, the shine-dalgarno sequence, regions with potential secondary structure involved in regulation, as well as other types of regulatory motifs or sequences.
This sequence information allows the modulation of the natural expression of the BASB070 gene. The upregulation of the gene expression may be accomplished by altering the promoter, the shine-dalgarno sequence, potential repressor or operator elements, or any other elements involved. Likewise, downregulation of expression can be achieved by similar types of modification. Alternatively, by changing phase variation sequences, the expression of the gene can be put under phase variation control, or it may be uncoupled from this regulation. In another approach, the expression of the gene can be put under the control of one or more inducible elements allowing regulated expression. Examples of such regulation include, but are not limited to, induction by temperature shift, addition of inductor substrates like selected carbohydrates or their derivatives, trace elements, vitamins, co-factors, metal ions, etc.
Such modifications as described above can be introduced by several different means. The modification of sequences involved in gene expression can be carried out in vivo by random mutagenesis followed by selection for the desired phenotype. Another approach consists in isolating the region of interest and modifying it by random mutagenesis, or site-directed replacement, insertion or deletion mutagenesis. The modified region can then be reintroduced into the bacterial genome by homologous recombination, and the effect on gene expression can be assessed. In another approach, the sequence knowledge of the region of interest can be used to replace or delete all or part of the natural regulatory sequences. In this case, the regulatory region targeted is isolated and modified so as to contain the regulatory elements from another gene, a combination of regulatory elements from different genes, a synthetic regulatory region, or any other regulatory region, or to delete selected parts of the wild-type regulatory sequences. These modified sequences can then be reintroduced into the bacterium via homologous recombination into the genome. A non-exhaustive list of preferred promoters that could be used for up-regulation of gene expression includes the promoters porA, porB, lbpB, tbpB, pi 10, 1st, hpuAB from N. meningitidis or N. gonorrohea;, ompCD, copB, lbpB, ompE, UspAl, UspA2, TbpB from M. Catarrhalis; pi, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from H. influenzae.
In one example, the expression of the gene can be modulated by exchanging its promoter with a stronger promoter (through isolating the upstream sequence of the gene, in vitro modification of this sequence, and reintroduction into the genome by homologous recombination). Upregulated expression can be obtained in both the bacterium as well as in the outer membrane vesicles shed (or made) from the bacterium.
In other examples, the described approaches can be used to generate recombinant bacterial strains with improved characteristics for vaccine applications. These can be, but are not limited to, attenuated strains, strains with increased expression of selected antigens, strains with knock-outs (or decreased expression) of genes interfering with the immune response, strains with modulated expression of immunodominant proteins, strains with modulated shedding of outer-membrane vesicles.
Thus, also provided by the invention is a modified upstream region of the BASB070 gene, which modified upstream region contains a heterologous regulatory element which alters the expression level of the BASB070 protein located at the outer membrane. The upstream region according to this aspect of the invention includes the sequence upstream of the BASB070 gene. The upstream region starts immediately upstream of the BASB070 gene and continues usually to a position no more than about 1000 bp upstream of the gene from the ATG start codon. In the case of a gene located in a polycistronic sequence (operon) the upstream region can start immediately preceding the gene of interest, or preceding the first gene in the operon. Preferably, a modified upstream region according to this aspect of the invention contains a heterologous promotor at a position between 500 and 700 bp upstream of the ATG.
Thus, the invention provides the BASB070 polypeptide, in a modified bacterial Bleb. The invention further provides modified host cells capable of producing the non-live membrane- based bleb vectors. The invention further provides vectors comprising the BASB070 gene having a modified upstream region containing a heterologous regulatory element.
Further provided by the invention are processes to prepare the host cells and bacterial blebs according to the invention.
Vaccine antigens may be provided in a variety of other forms known in the art, depending on the properties of the protein. Lipoproteins for example, because of the hydrophobicity of the lipids added to their N-terminus, are able to aggregate and to form micelles. The particulate nature of these structures can enhance the immunogenicity of the lipoprotein, as compared to the unlipidated version of the protein. The size of the micelles may also have an impact on the immunogneicity of the lipoprotein and this can be modified for example by adjusting the extraction procedure.
Also provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non- variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Haemophilus influenzae. Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Haemophilus influenzae infection, in mammals, particularly humans.
The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as any pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each could be administered via a mucosal surface such as intranasally, or administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation of the invention may also include adjuvant systems for enhancing the immunogenicity of the formulation.
An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed THl -type responses (cell-mediated response), and TH2-type immune responses (humoral response).
Extreme THl -type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice THl -type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. TH2- type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines. High levels of THl -type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
The distinction of THl and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly THl or predominantly TH2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989) THl and TH2 cells: different patterns qflymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, pl45-173). Traditionally, THl-type responses are associated with the production of the IFN-γ and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of THl-type immune responses are not produced by T-cells, such as IL-12. In contrast, TH2- type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.
58 It is known that certain vaccine adjuvants are particularly suited to the stimulation of either THl or TH2 - type cytokine responses. Traditionally the best indicators of the THl :TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of THl or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or (in the murine system) the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses.
Thus, a THl-type adjuvant is one which preferentially stimulates isolated T-cell populations to produce a high ratio of THl-type cytokines when re-stimulated with antigen in vitro, and promotes development of both CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulin responses associated with THl-type isotype. Adjuvants which are capable of preferential stimulation of the THl cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 Bl (SmithKline Beecham Biologicals SA).
Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (European Patent number 0 689 454). 3D-MPL will be present in the range of lOμg - lOOμg preferably 25-50μg per dose wherein the antigen will typically be present in a range 2-50μg per dose.
Another preferred adjuvant comprises QS21, an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina. Optionally this may be admixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with a carrier.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THl stimulating adjuvants when formulated together with an antigen.
Further adjuvants which are preferential stimulators of THl cell responses include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
Combinations of different THl stimulating adjuvants, such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of THl cell response. For example, QS21 can be formulated together with 3D-MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1 ; preferably 1 :5 to 5 : 1 and often substantially 1 : 1. The preferred range for optimal synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
Preferably a carrier which enhances immunogenicity is also present in the vaccine composition according to the invention. Such a carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tween 80. In a particularly preferred aspect the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion. Additionally the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of lμg - 200μg, such as 10-100μg, preferably lOμg - 50μg per dose.
Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210.
The present invention also provides a polyvalent vaccine composition comprising a vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating other bacterial or viral diseases, cancers, autoimmune diseases and related conditions. Such a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.
While the invention has been described with reference to certain BASB070 polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions comprising a BASB070 polynucleotide and/or BASB070 polypeptide for administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal intradermalor transdermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intramuscular or subcutaneous injection. Other injection routes, such as intradermal, intraperitoneal, or intravenous can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, solutions, powders and the like. For administration to mammals, and particularly humans, it is expected that the dosage level of the active agent will be from 0.01 μg/kg to 10 μg/kg, typically around 1 μg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
Wide variations in the required dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Sequence Databases, Sequences in a Tangible Medium, and Algorithms Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3-dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as the GCG program package.
Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded protein sequences. Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, DNA, RNA and protein structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, codon usage analysis, nucleic acid base trimming, and sequencing chromatogram peak analysis.
A computer based method is provided for performing homology identification. This method comprises the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
A computer based method is also provided for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.
DEFINITIONS
"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H, and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GAP program in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN (Altschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.
Parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, j. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 8
Gap Length Penalty: 2
A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison Wl. These are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below. (1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100%) identity to the reference sequence of SEQ ID NO:l or 3, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: l or 3 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:l or 3 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO:l or 3, or:
nn < xn - (xn • y),
wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:l or 3, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non- integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 or 4 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l or 3, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100%) identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO: 1 or 3 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO:l or 3, or:
nn < xn - (xn • y),
wherein nn is the number of nucleic acid alterations, xn is the total number of nucleic acids in SEQ ID NO:l or 3, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2 or 4, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or 4 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2 or 4, or:
»a ≤ xa " (xa • y)>
wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO:2 or 4, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2 or 4, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2 or 4, or:
na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO:2 or 4, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
"Individual(s)," when used herein with reference to an organism, means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living. Similarly, a polynucleotide or polypeptide whose expression is specifically altered by genetic manipulation is "isolated" even though the polynucleotide or polypeptide may be present in the organism in which it is naturally" present. "Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA including single and double-stranded regions.
"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Disease(s)" means any disease caused by or related to infection by bacteria, including, for example, otitis media, acute otitis media, recurrent otitis media, otitis media with effusion, sinusitis, conjuctivitis, rhinopharyngitis, laryngitis, obstructive laryngitis, alveolitis, bronchitis, chronic bronchitis, enhancement of chronic obstructive pulmonary disease, complications of cystic fibrosis, pericarditis, endocarditis, osteomyelitis, arthritis, genitouinary tract colonization and neonatal infection, bacteremia, septicemia, meningitis.
EXAMPLES:
The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.
Example 1: BASB070 gene from Haemophilus influenzae strain Rd KW20 and non-typeable Haemophilus influenzae (NTHi) strain 3224.
A: BASB070 in Hi strain Rd. The BASB070 gene of SEQ ID NO: 1 comes from Heamophilus influenzae strain Rd KW20. The translation of the BASB070 polynucleotide sequence is shown in SEQ ID NO:2.
B: BASB070 in NTHi strain 3224. The sequence of the BASB070 gene comes from the sequencing of NTHi strain 3224.
Using the MegAlign program from the DNASTAR software package, an alignment of the polynucleotide sequences of SEQ ID NO:l and 3 was performed, and is displayed in Figure 1; a pairwise comparison of identities shows that the two BASB070 polynucleotide gene sequences are 90.3 % identical. Using the same MegAlign program, an alignment of the polypeptide sequences of SEQ ID NO:2 and 4 was performed, and is displayed in Figure 2; a pairwise comparison of identities shows that the two BASB070 protein sequences are 90.6 % identical. These data show that the BASB070 gene among the two strains NTHi 3224 and Hi RD are conserved but that there are also variable regions between them.
Example 2: Construction of Plasmid to Express Recombinant BASB070
A: Cloning of BASB070.
The Ncol and Asp 718 restriction sites CC ATG G and GG TAC C were engineered into specifically designed forward and reverse amplification primers, respectively, permitting directional cloning of a BASB070 PCR product into the commercially available E. coli expression plasmid pBADgΙII(A) (Invitrogen, USA, ampicillin resistant). This plasmid provides the signal peptide from the bacteriophage fd pill protein such that a mature BASB070 protein can be targeted to the periplasm of E. coli. The BASB070 PCR product was purified from the amplification reaction using Wizard PCR prepTM (Promega) according to the manufacturers instructions. To produce the required Ncol and Asp 718 termini necessary for cloning, purified PCR product was sequentially digested to completion with Ncol anάAsp 718 restriction enzymes as recommended by the manufacturer (Boehringer Mannheim). Digested BASB070 PCR products and pBAD were gel-purified and ligated together using an approximately 5- fold molar excess of the digested fragment to the vector: A standard -20 μl ligation reaction (~16°C, -16 hours), using methods well known in the art, was performed using T4 DΝA ligase (-2.0 units / reaction, Boehringer Mannheim). An aliquot of the ligation was used to transform electro-competent E. coli Top 10 cells according to methods well known in the art. Following a -2-3 hour outgrowth period at 37°C in -1.0 ml of LB broth, transformed cells were plated on LB agar plates containing Ampicillin (50 μg/ml). Individual ampicillin-resistant colonies were selecteded and analyzed by whole cell-based PCR to verify that transformants contained the BASB070 DΝA insert. Transformants that produced the expected PCR product were identified as strains containing a BASB070 expression construct. Expression plasmid containing strains were then analyzed for the inducible expression of recombinant BASB070.
B: Expression Analysis of PCR-Positive Transformants. For each PCR-positive transformant identified above, -5.0 ml of LB broth containing ampicillin (50 μg/ml) was inoculated with cells from the patch plate and grown overnight at 37 °C with shaking (-250 rpm). An aliquot of the overnight seed culture (-1.0 ml) was inoculated into a 125 ml erlenmeyer flask containing -25 ml of LB AMPICILLINE broth and grown at 37 °C with shaking (-250 rpm) until the culture turbidity reached O.D.600 of -0.5, i.e. mid-log phase (usually about 1.5 - 2.0 hours). At this time approximately half of the culture (-12.5 ml) was transferred to a second 125 ml flask and expression of recombinant BASB070 protein induced by the addition of L- Arabinose to a final concentration of 0.2 % (w/v). Incubation of both the arabinose- induced and non-induced cultures continued for an additional -4 hours at 37 °C with shaking. Samples (-1.0 ml) of both induced and non-induced cultures were removed after the induction period and the cells collected by centrifugation in a microcentrifuge at room temperature for -3 minutes. Individual cell pellets were suspended in ~50μl of sterile water, then mixed with an equal volume of 2X Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in boiling water bath for -3 min to denature protein. Equal volumes (~15μl) of both the crude arabinose-induced and the non- induced cell lysates were loaded onto duplicate 12% Tris/glycine polyacrylamide gel (1 mm thick Mini-gels, Novex). The induced and non-induced lysate samples were electrophoresed together with prestained molecular weight markers under conventional conditions using a standard SDS/Tris/glycine running buffer. Following electrophoresis, one gel was stained with commassie brilliant blue R250 (BioRad) and then destained to visualize novel BASB070 arabinose-inducible protein(s). NTHi Strains
The following strains of Haemophilus influenzae are provided as a useful reference for the present invention. The BASB070 gene utilised in accordance with the invention is not limited with regard to the strain, but it may correspond to theBASB070 gene as found in any of the strains listed below or any related strain. This information is provided merely for convenience to those of skill in the art and is not an admission that any provision of a deposit is required for enablement.
strain 3219C (ET7) strain 3241A (ET30) strain 840645 (ET51) strain 901905U (ET60) strain A840177 (ET40) strain A840177 (ET69)
All of the above strains were described in van Alphen, L., Caugant, D.A, Duim, B.A., O'Rouke, M.,
Bowler, L.D. (1997) Differences in genetic diversity of non-encapsulated H. influenzae from various diseases. Microbiology, 143: 1423-1431.
HiRd Strains
An example of a HiRd strain is described in R.D. Fleischmann et al., Science . Vol 269: 496-512 (1995) and K. W. Wilcox et al., J. Bact. Vol 122: 443 (1975) with the strain name KW20. This strain was deposited by the authors with the American Type Culture Collection under deposit number ATCC 51907.
SEQUENCE INFORMATION
BASB070 Polynucleotide and Polypeptide Sequences
SEQ ID NO: 1 Haemophilus influenzae BASB070 polynucleotide sequence from strain Rd KW20
ATGAAGAAAGCTATAAAATTAAATTTAATTACACTTGGCCTAATTAATACGATCGGTATGACGATTACACAAGCTCAAGC CGAAGAAACATTAGGACAAATTGATGTAGTGGAAAAAGTTATATCAAACGATAAAAAACCTTTCACTGAAGCCAAAGCCA AAAGTACACGTGAAAATGTCTTTAAGGAAACACAAACCATTGACCAAGTGATTCGAAGTATCCCTGGTGCATTTACTCAA CAAGATAAAGGCTCGGGTGTCGTTTCTGTGAATATTCGTGGCGAAAATGGATTAGGTCGTGTCAATACTATGGTTGATGG TGTAACACAGACCTTCTATTCTACAGCCTTAGACTCAGGTCAATCAGGCGGAAGTTCTCAATTTGGTGCGGCAATCGATC CTAATTTTATTGCAGGTGTAGATGTTAATAAAAGCAACTTTTCAGGAGCAAGCGGTATAAATGCGTTAGCAGGCAGTGCT AATTTTAGAACATTAGGCGTTAATGATGTTATTACCGATGACAAACCATTTGGCATTATTCTGAAAGGAATGACAGGGAG TAATGCCACTAAATCCAATTTTATGACAATGGCTGCTGGCAGAAAATGGCTTGATAATGGTGGCTATGTAGGCGTGGTGT ATGGTTATAGCCAACGTGAAGTATCTCAAGATTACCGTATCGGTGGCGGAGAACGATTAGCATCATTAGGGCAGGATATT CTCGCGAAAGAAAAAGAAGCTTATTTTCGTAATGCGGGTTATATTTTAAATCCTGAAGGGCAATGGACACCTGATTTAAG CAAAAAACATTGGTCTTGTAACAAACCAGATTATCAGAAAAATGGTGATTGTAGTTATTATCGTATTGGATCTGCTGCAA AGACTAGAAGAGA-^TTCTAC-^GAATTATTAACA-^ATGGAAAAAAACCTAAGGATATTGAAAAGCTCCAAAAAGGTAAT GATGGAATTGAAGAAACTGACAAATCATTTGAACGTAATAAAGATCAATATAGTGTTGCACCGATTGAGCCGGGTAGTTT GCAATCTCGTTCTCGTAGCCATTTATTAAAATTTGAATATGGCGATGATCACCAAAATTTAGGGGCGCAATTACGCACGT TGGATAATAAAATTGGTTCTCGCAAAATTGAAAACCGTAATTACCAAGTCAATTATAACTTCAATAATAACAGCTATCTT GATCTTAATTTAATGGCTGCACATAACATTGGAAAAACTATTTATCCTAAAGGCGGTTTTTTTGCTGGCTGGCAAGTGGC AGATAAACTTATCACTAAAAATGTCGCAAATATTGTTGATATAAACAACAGCCATACTTTCTTACTGCCAAAAGAAATTG ATTTAAAAACCACATTAGGTTTTAACTATTTTACCAATGAATACAGTAAAAACCGTTTTCCAGAAGAATTAAGTTTGTTT TATAACGATGCTTCACATGATCAAGGCTTATATTCACACAGTAAAAGAGGGCGATATTCTGGCACAAAAAGTTTATTACC ACAACGTTCAGTAATCTTACAACCTTCTGGCAAGCAAAAATTTAAAACCGTGTATTTTGATACCGCACTTTCTAAAGGCA TTTATCATTTAAATTACAGCGTGAATTTTACCCATTATGCCTTTAATGGTGAGTATGTAGGTTACGAAAATACAGCGGGT CAACAAATTAATGAACCTATTTTGCATAAATCAGGGCATAAAAAGGCATTCAATCATTCTGCCACATTAAGTGCAGAACT GAGTGATTATTTTATGCCATTTTTTACTTATTCACGCACTCACAGAATGCCGAATATTCAAGAGATGTTTTTCTCTCAAG TGTCTAATGCAGGGGTAAACACAGCATTAAAACCTGAACAATCTGACACCTATCAACTAGGCTTTAATACTTATAAAAAA GGTCTCTTCACTCAAGACGATGTGCTAGGCGTAAAATTAGTAGGCTATCGTAGCTTTATTAAAAACTATATCCATAATGT TTATGGTGTTTGGTGGCGAGATGGCATGCCTACGTGGGCAGAAAGTAATGGATTTAAATATACTATTGCTCATCAAAATT ATAAGCCTATTGTGAAAAAGAGCGGCGTCGAGTTAGAAATTAACTATGACATGGGACGTTTTTTTGCGAATGTCTCTTAT GCATATCAACGAACAAATCAACCAACCAATTATGCCGATGCCAGCCCGCGTCCGAATAATGCTTCACAAGAAGACATTTT GAAACAAGGTTATGGCTTATCTCGTGTTTCAATGCTACCAAAAGACTACGGCAGATTAGAGCTTGGCACACGTTGGTTTG ATCAAAAATTAACCTTAGGTCTGGCAGCTCGTTATTATGGAAAAAGTAAACGTGCGACAATTGAAGAAGAATATATCAAT GGATCTCGCTTTAAAAAAAATACCTTGCGTCGTGAAAATTACTATGCCGTGAAAAAAACGGAAGATATTAAAAAACAACC GATTATTTTAGATTTACACGTCAGCTATGAACCAATCAAAGATTTGATTATTAAAGCGGAAGTACAAAATCTATTAGATA AACGTTATGTTGATCCGTTAGATGCTGGAAATGACGCGGCTTCGCAACGTTATTATTCAAGTTTAAATAATTCTATAGAA TGTGCGCAAGATTCTTCTGCTTGCGGTGGTTCAGATAAAACCGTGCTTTATAACTTTGCACGTGGAAGAACTTATATTCT GAGTTTAAACTATAAATTCTAA SEQ ID NO:2
Haemophilus influenzae BASB070 polypeptide sequence deduced from the polynucleotide of SeQ ID NO: 1
MKKAIKLNLITLGLINTIGMTITQAQAEETLGQIDVVEKVISNDKKPFTEAKAKSTRENVFKETQTIDQVIRSIPGAFTQ QDKGSGVVSVNIRGENG GRVNTMVDGVTQTFYSTALDSGQSGGSSQFGAAIDPNFIAGVDVNKSNFΞGASGINALAGSA NFRTLGVNDVITDDKPFGIILKGMTGSNATKSNFMTMAAGRKWLDNGGYVG VYGYSQREVSQDYRIGGGERLASLGQDI LAKEKEAYFRNAGYI NPEGQWTPDLSKKHWΞCNKPDYQKNGDCΞYYRIGSAAKTRREI QELLTNGKKPKDIEKLQKGN DGIEETD SFERNKDQYSVAPIEPGΞLQSRSRSH LKFEYGDDHQNLGAQ RTLDNKIGSRKIENRNYQVNYNFNNNSYL DLN MAAHNIGKTIYPKGGFFAG QVADKLITKNVANIVDINNSHTFLLPKEIDLKTTLGFNYFTNEYSKNRFPEE ΞLF YNDAΞHDQGLYSHΞKRGRYSGTKSLLPQRSVILQPSGKQKFKTVYFDTALSKGIYHLNYSVNFTHYAFNGEYVGYENTAG QQINEPILHKSGHKKAFNHSATLSAELΞDYFMPFFTYSRTHRMPNIQEMFFSQVΞNAGVNTALKPEQΞDTYQLGF TYKK GLFTQDDVLGVKLVGYRSFIKNYIHNVYGV RDGMPTWAESNGFKYTIAHQNYKPIVKKSGVE EINYDMGRFFANVΞY AYQRTNQPTNYADAΞPRPNNAΞQEDI KQGYGLSRVSM PKDYGRLELGTR FDQKLTLGLAARYYGKSKRATIEEEYIN GSRFKKNTLRRENYYAVKKTEDIKKQPIILDLHVΞYEPIKDLIIKAEVQNLLDKRYVDPLDAGNDAASQRYYΞSLNNSIE CAQDSSACGGΞ--KTVLYNFARGRTYII-SLNYKF
SEQ ID NO:3 Haemophilus influenzae BASB070 polynucleotide sequence from strain ntHi 3224
ATGAAGAAAGCTATAAAATTAAATTTAATTACACTTAGCCTAATCAATACAATCGGTATGACGATTACACAAGCTCAAGC CGAAGAAACATTAGGGCAAATTGATGTCGTAGAAAAAGTGATATCAAATGACAAAAAACCTTTCACTGAAGCCAAAGCCA AAAGTACGCGTGAAAATGTCTTTAAGGAAACACAAACCATTGACCAAGTCATTCGGAGCATTCCTGGGGCATTTACTCAA CAAGATAAAGGCTCGGGTGTGGTTTCTGTAAATATTCGTGGCGAAAATGGATTAGGTCGTGTCAATACGATGGTTGATGG TGTAACCCAAACCTTCTATTCTACAGCCTTAGACTCTGGTCAATCAGGCGGAAGTTCTCAATTTGGTGCGGCAATCGACC CTAATTTTATTGCAGGTGTAGATGTTAATAAAAGCAACTTTTCGGGAGCAAGCGGTATAAATGCCTTAGCAGGCAGTGCT AATTTTAGAACATTAAGCGTTAATGATGTGATTACCGATGACAAACCATTCGGCATTATTCTGAAAGGAATGACAGGGAG CAATGCCACTAAATCCAATTTTATGACGACAGCTGCAGGCAGAAAATGGCTTGATAATGGTGGCTATGTAGGCGTAGTGT ATGGTTATAGCCAACGTGAAGTTTCACAAGATTATCGTATAGGTGGCGGAGAACGATTAGCATCATTAGGGCAAGATATT CTTGCTAAAGAAAAAGAAAAGATTTTTCGTAATGATGGTTATGTTTTAAATTCTGCTGGACAATGGGCACCTGATTTAAA CAAACCACATTGGTCTTGTAATACCCCGAGTTCTTTAAAAGATAAAAGTATGAGTACATCTTGTAAGCCTTATCGTCTTG GACCTGCTGCAACGACTAGACAAGAAATTCTAAAAGAATTATTAGAAGATGGAAAAGAACCTAAGGATATTGAAAAGCTC CAAAAAAGTAATGATGGAATTGAAGAAACTGAAAAATCATTTGAACGTAATAAAGATCAATATGACGTCGCCCCTATTGA GCCTGGTAGTTTGCAATCTCGTTCACGTAGTCATTTATTAAAATTTGAATATAGCGATGATCACCATACGCTAGGGGCGC AAATACGTACCCTTGATAATAAAATTGGTTCTCGCAAAATTGAAAACCGTAATTACCAAGTCAATTATAACTTCAATAAT AACAGCTATCTTGATCTTAATTTAATGGCTGCACATAACATTGGCAAAACTATTTATCCTAAGGGTGGTTTTTTTGCTGG CTGGCAAGTGGCAGACAAACTTATCACAAAAAATGTGGCAAATATTGTTGATATAAATAACAGCCATACTTTCTTACTGC CAAAAGAAATCGATTTAAAAACCACATTAGGGTTTAACTATTTTACCAATGAATACAGTAAAAACCGTTTTCCAGAAGAA TTAAGTTTGTTTTATGTGAATGAATCACATGATCAAGGCTTATATTCACTCAGTAATAAAGGGCGATATTCTGGCTCAAA AGGTTTATTACCACAACGTTCAGTAATCTTACAACCTTCTGGCAAGCAAAAATTTAAAACAGTGTATTTTGATACCGCAC TTTCTAAAGGTATTTATCATTTAAATTACAGCGTGAATTTTACCCATTATGCCTTTAATGGTGAGTATGTTGGATATAAA AATACAGCAGATAAAATTAATGAACCTATTTTGCATAAATCAGGGCATAAAAAGGCATTCAATCATTCTGCTACATTAAG TGCAGAGCTAAGTGATTATTTTATGCCATTTTTTACTTATTCACGCACACACAGAATGCCGAATATTCAAGAGATGTTTT TCTCTCAAGTGTCTGATGCTGGGGTAAACACCGCATTAAAACCTGAACAATCTGACACCTATCAACTAGGCTTTAATACT TATAAAAAAGGTCTATTCACTCAAGACGATGTATTAGGCATCAAATTAGTGGGCTATCGTAGCTTTATTAAAAACTATAT CCACAATGTGTATGGAGATTGGTCACGAGATGGTGTTATGCCAGAGTGGGCAAGACTCAATGGTTTTCGTCTGACGATTG CTCATCAAAATTATCAACCAATAGTGAAAAAAAGCGGAGCTGAGTTAGAGCTCAATTATGATATGGGGCGTTTTTTTGCA AATCTGTCTTATGCTTATCAACGTACTAATCAGCCAACCAATTATGCCGATGCCAGCTCACGTCCGCGTAATGCTTCAAA AGAAGAGATTTTGAAACAAGGTTATGGTTTATCACGAATCTCTATGTTACCAAAGGACTACGGTAGATTAGAGCTTGGCA CACGCTGGTTTGATCAAAAATTAACTCTTGGTATCGCAGCCCGTTACTATGGAAAAAGTAAACGTGCTACAACTCAAGAA GAATACATCAACGGCTCTCGCTATGAAAAAAATACTACGCGCGACAGAATTTATTATGCTATTAAAAAGACAGAAGAGAT TAAAAAACAACCTATTATTTTAGATTTACACGTCAGCTATGAACCAATCAAAGATTTGATTATTAAAGCGGAAGTACAAA ATCTATTAGATAAACGTTATGTTGATCCGTTAGATGCTGGAAATGATGCGGCTTCGCAACGTTATTATTCAAGTTTAAAT GATTCTTTAGCCTGTAAAATAAATGAATCAACCTGTAATGATGGTTCAGAGAAAACTGTGCTTTATAACTTTGCACGTGG AAGAACTTATATTCTGAGTTTGAACTATAAATTCTAG
SEQ ID NO:4 Haemophilus influenzae BASB070 polypeptide sequence deduced from the polynucleotide of SeQ ID NO:3
MKKAIKLNLITLS INTIGMTITQAQAEETLGQIDVVEKVISNDKKPFTEA AKSTRENVFKETQTIDQVIRSIPGAFTQ QDKGSGVVSVNIRGENG GRVNTMVDGVTQTFYSTALDSGQΞGGΞSQFGAAIDPNFIAGVDVNKΞNFSGASGINALAGSA NFRT SVNDVITDDKPFGII KGMTGSNATKSNFMTTAAGRK LDNGGYVGVVYGYSQREVSQDYRIGGGERLASLGQDI LAKEKEKIFRNDGYVLNSAGQWAPDLNKPHWΞCNTPSSLKDKS ΞTΞCKPYRLGPAATTRQEILKE EDGKEPKDIEK QKSNDGIEETEKSFERNKDQYDVAPIEPGSLQSRSRΞHL KFEYSDDHHTLGAQIRT DNKIGSRKIENRNYQVNYNFNN NSYLDLNLMAAHNIGKTIYPKGGFFAGWQVADKLITKNVANIVDINNSHTFLLPKEID KTTLGFNYFTNEYSKNRFPEE LSLFYVNEΞHDQGLYS SNKGRYSGSKGLLPQRSVI QPSGKQKFKTVYFDTALSKGIYHLNYSVNFTHYAFNGEYVGYK NTADKINEPILHKSGHKKAFNHSAT SAELSDYFMPFFTYSRTHRMPNIQE FFSQVSDAGVNTALKPEQSDTYQLGFNT YKKG FTQDDV GI VGYRSFIKNYIHNVYGD SRDGVMPEWAR NGFRLTIAHQNYQPIVKKSGAELELNYDMGRFFA NLSYAYQRTNQPTNYADASSRPRNASKEEILKQGYGLSRISM PKDYGRLE GTR FDQKLTLGIAARYYGKSKRATTQE EYINGSRYEKNTTRDRIYYAIKKTEEIKKQPIILD HVSYEPIKDLIIKAEVQN LDKRYVDP DAGNDAAΞQRYYΞΞLN DSLACKINESTCNDGSEKTVLYNFARGRTYILSLNYKF

Claims

1. A vaccine composition comprising an effective amount of a polypeptide which polypeptide comprises an amino acid sequence which has at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or 4 or to an immunogenic fragment thereof, or which polypeptide comprises a mimotope of the said amino acid sequence or immunogenic fragment, together with a pharmaceutically acceptable carrier.
2. A vaccine composition according to claim 1 wherein the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO: 2 or 4 or to an immunogenic fragment thereof.
3. A vaccine composition comprising an effective amount of a polynucleotide which polynucleotide comprises a nucleotide sequence which has at least 85% identity to the nucleotide sequence of SEQ ID NO: 1, 3 or to a fragment thereof which encodes an immunogenic polypeptide, together with a pharmaceutically acceptable carrier.
4. The vaccine composition according to any one of claims 1 to 3 wherein said composition comprises at least one other Haemophilus influenzae antigen.
5. An expression vector or a recombinant live microorganism comprising an isolated polynucleotide which polynucleotide comprises a nucleotide sequence which has at least 85% identity to the amino acid sequence of SEQ ID NO: 1 or 3 or to a fragment thereof that encodes an immunogenic polypeptide.
6. A host cell comprising the expression vector of claim 5 or a membrane of said host cell expressing an isolated polypeptide comprising an amino acid sequence which has at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or 4, or to an immunogenic fragment thereof.
7. A process for producing a polypeptide comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or 4 or to an immunogenic fragment, comprising culturing a host cell of claim 6 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.
8. A process for expressing a polynucleotide, which polynucleotide comprises a nucleotide sequence which has at least 85% identity to the nucleotide sequence of SEQ ID NO: 1 or 3 or to a fragment thereof that encodes an immunogenic polypeptide, the process comprising transforming a host cell with an expression vector comprising said polynucleotide and culturing said host cell under conditions sufficient for expression of said polynucleotide.
9. An antibody specific for the polypeptide of SEQ ID NO: 2 or 4 or an immunologically active fragment of the antibody.
10. A method of diagnosing a Haemophilus influenzae infection, comprising identifying a polypeptide which comprises an amino acid sequence which has at least 85% identity to the amino acid sequence of SEQ ID NO: 2or 4 or a fragment thereof, or an antibody that is specific for said polypeptide, present within a biological sample from an animal suspected of having such an infection.
11. Use of a composition comprising an immunologically effective amount of a polypeptide which comprises an amino acid sequence which has at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or 4 or to an immunogenic fragment thereof, or which polypeptide comprises a mimotope of the said amino acid sequence or immunogenic fragment, in the preparation of a medicament for use in generating an immune response in a mammal.
12. Use of a composition comprising an immunologically effective amount of a polynucleotide which comprises a nucleotide sequence which has at least 85% identity to the nucleotide sequence of SEQ ID NO: 1 or 3 or to a fragment thereof that encodes an immunogenic polypeptide, in the preparation of a medicament for use in generating an immune response in a mammal.
13. A therapeutic composition useful in treating humans with Haemophilus influenzae disease comprising at least one antibody directed against the polypeptide of SEQ ID NO: 2 or 4 and a suitable pharmaceutical carrier.
14. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a fragment or a mimotope thereof.
15. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 14.
16. The polynucleotide of claim 15 comprising the nucleotide sequence of SEQ ID NO: 3 a fragment thereof.
PCT/EP2000/001423 1999-02-24 2000-02-22 Haemophilus antigen WO2000050599A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00909227A EP1157113A1 (en) 1999-02-24 2000-02-22 Haemophilus antigen
AU31582/00A AU3158200A (en) 1999-02-24 2000-02-22 Haemophilus antigen
CA002363829A CA2363829A1 (en) 1999-02-24 2000-02-22 Haemophilus antigen
JP2000601163A JP2002536998A (en) 1999-02-24 2000-02-22 Hemophilus antigen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9904183.2 1999-02-24
GBGB9904183.2A GB9904183D0 (en) 1999-02-24 1999-02-24 Novel compounds

Publications (1)

Publication Number Publication Date
WO2000050599A1 true WO2000050599A1 (en) 2000-08-31

Family

ID=10848363

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/001423 WO2000050599A1 (en) 1999-02-24 2000-02-22 Haemophilus antigen

Country Status (6)

Country Link
EP (1) EP1157113A1 (en)
JP (1) JP2002536998A (en)
AU (1) AU3158200A (en)
CA (1) CA2363829A1 (en)
GB (1) GB9904183D0 (en)
WO (1) WO2000050599A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002032944A2 (en) * 2000-10-17 2002-04-25 Glaxosmithkline Biologicals Sa Basb208 nucleotide and amino acid sequences from haemophilus influenzae

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019090A1 (en) * 1992-03-16 1993-09-30 Barenkamp Stephen J High molecular weight surface proteines of non-typeable haemophilus
EP0680765A1 (en) * 1994-05-05 1995-11-08 American Cyanamid Company Vaccine for nontypable haemophilus influenzae strain
WO1996033276A1 (en) * 1995-04-21 1996-10-24 Human Genome Sciences, Inc. NUCLEOTIDE SEQUENCE OF THE HAEMOPHILUS INFLUENZAE Rd GENOME, FRAGMENTS THEREOF, AND USES THEREOF
WO1997001638A1 (en) * 1995-06-27 1997-01-16 Cortecs International Limited Omp26 antigen from haemophilus influenzae

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019090A1 (en) * 1992-03-16 1993-09-30 Barenkamp Stephen J High molecular weight surface proteines of non-typeable haemophilus
EP0680765A1 (en) * 1994-05-05 1995-11-08 American Cyanamid Company Vaccine for nontypable haemophilus influenzae strain
WO1996033276A1 (en) * 1995-04-21 1996-10-24 Human Genome Sciences, Inc. NUCLEOTIDE SEQUENCE OF THE HAEMOPHILUS INFLUENZAE Rd GENOME, FRAGMENTS THEREOF, AND USES THEREOF
WO1997001638A1 (en) * 1995-06-27 1997-01-16 Cortecs International Limited Omp26 antigen from haemophilus influenzae

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FLEISCHMANN R D ET AL: "WHOLE-GENOME RANDOM SEQUENCING AND ASSEMBLY OF HAEMOPHILUS INFLUENZAE RD", SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 269, no. 5223, 28 July 1995 (1995-07-28), pages 496 - 512, XP000517090, ISSN: 0036-8075 *
FLEISCHMANN R. ET AL.: "Haemophilus infuenzae Rd section 116; AC U32801; P45114", EBI DATABASE, 1995, XP002144210 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002032944A2 (en) * 2000-10-17 2002-04-25 Glaxosmithkline Biologicals Sa Basb208 nucleotide and amino acid sequences from haemophilus influenzae
WO2002032944A3 (en) * 2000-10-17 2002-11-14 Smithkline Beecham Biolog Basb208 nucleotide and amino acid sequences from haemophilus influenzae

Also Published As

Publication number Publication date
CA2363829A1 (en) 2000-08-31
AU3158200A (en) 2000-09-14
JP2002536998A (en) 2002-11-05
GB9904183D0 (en) 1999-04-14
EP1157113A1 (en) 2001-11-28

Similar Documents

Publication Publication Date Title
US20110129475A1 (en) Basb 205 polypeptides and polynucleotides from haemophilus influenzae
US20080233123A1 (en) Polypeptide from haemophilus influenzae
US20040241638A1 (en) Novel compounds
WO2002034768A2 (en) Polypeptides and polynucleodides from haemophilus influenzae and their use
EP1337554B1 (en) Novel compounds
EP1326885B1 (en) Non typeable Haemophilus influenzae BASB201 polypeptides and polynucleotides and uses thereof
US20040039169A1 (en) Haemophilus Influenzae basb202 polypeptide, production, vaccine and diagnostic use
EP1157113A1 (en) Haemophilus antigen
WO2000047737A1 (en) Haemophilus influenzae rd outer membrane sequences used as vaccine
EP1409528A2 (en) Novel compounds
US20080226637A1 (en) Haemophilus influenzae BASB206 gene and protein
CA2434822A1 (en) Haemophilus influenza basb212 polynucleotides, polypeptides and use thereof
CA2438012A1 (en) H. influenzae antigen basb213
WO2002034778A2 (en) Baseb209 polypeptides and polynucleotides from haemophilus influenzae
CA2436306A1 (en) Basb221 polypeptides and polynucleotides encoding basb221 polypeptides
CA2425362A1 (en) Basb208 nucleotide and amino acid sequences from haemophilus influenzae
WO2002079237A2 (en) Non-typeable haemophilus influenzae immunogenic polypeptides and use thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2363829

Country of ref document: CA

Ref country code: CA

Ref document number: 2363829

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2000909227

Country of ref document: EP

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 601163

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 2000909227

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2000909227

Country of ref document: EP