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WO2002000851A2 - Helicobacter dd-heptosyltransferase - Google Patents

Helicobacter dd-heptosyltransferase Download PDF

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Publication number
WO2002000851A2
WO2002000851A2 PCT/CA2001/000969 CA0100969W WO0200851A2 WO 2002000851 A2 WO2002000851 A2 WO 2002000851A2 CA 0100969 W CA0100969 W CA 0100969W WO 0200851 A2 WO0200851 A2 WO 0200851A2
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WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
pylori
sequence
seq
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PCT/CA2001/000969
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English (en)
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WO2002000851A3 (fr
Inventor
Koji Hiratsuka
Eleonora Altman
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National Research Council Of Canada
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Publication date
Priority claimed from PCT/CA2000/000777 external-priority patent/WO2001000796A2/fr
Application filed by National Research Council Of Canada filed Critical National Research Council Of Canada
Priority to EP01947090A priority Critical patent/EP1299527A2/fr
Priority to US10/451,685 priority patent/US20040110261A1/en
Priority to CA002417692A priority patent/CA2417692A1/fr
Priority to AU2001268885A priority patent/AU2001268885A1/en
Publication of WO2002000851A2 publication Critical patent/WO2002000851A2/fr
Publication of WO2002000851A3 publication Critical patent/WO2002000851A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to novel heptosyltransferase polynucleotides and polypeptides.
  • Helicobacter pylori is a spiral Gram negative bacterium which colonizes the human stomach. It is estimated that up to 50% of the human population is infected with K. pylori (Dunn et al, 1997). Thus, H. pylori remains one of the most prevalent bacterial pathogens worldwide.
  • H. pylori Infection by H. pylori is associated with chronic superficial and active gastritis (B laser, 1990), which may eventually develop into peptic ulcers (Blaser, 1995; Graham, 1991). Furthermore, prolonged infection by H. pylori can lead to the development of gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (Dunn, et al, 1997; Parsonnet, et al, 1994). H. pylori has been declared a human carcinogen by the International Agency for Cancer Research. Many research initiatives worldwide are aimed at determining the reasons why H. pylori produces such a variety of pathogenic outcomes.
  • Genomic mapping of several H. pylori strains showed a variation in the arrangement of several genetic markers (Jiang et al, 1996). This variability was also observed in gene organization, gene content and nucleotide sequence between the genome sequences of two H. pylori strains that have been determined and annotated (Tombs, et al, 1997; Aim, et al, 1999).
  • H. pylori may play several roles in pathogenesis.
  • H. pylori may play several roles in pathogenesis.
  • H. pylori LPS has been implicated in causing abnormal acid secretion and in inducing apoptosis of epithelial cells and gastritis in mice (Piotrowski, et al, 1997a; Sakagami, et al, 1997; Kidd, et al, 1997; Piotrowski, et al, 1997b; Ootsubo, et al, 1997; Okumura, et al, 1998).
  • H. pylori LPS may also be involved in triggering inflammatory response. Additionally, some strains of H.
  • O-antigen polysaccharide chains which mimic Lewis blood group antigens (Aspinall, et al, 1997; Monteiro et al, 1998b) which are naturally expressed in the human gastric mucosa.
  • Such antigenic mimicry may play a role in evasion of the host immune system.
  • this mimicry may give rise to pathogenic autoimmune antibodies by the host (Appelmelk, et al, 1997).
  • the exposure of LPS at the bacterial cell surface would make it an obvious putative colonization factor.
  • Edwards et al. (2000) showed that the O-chain polysaccharide found in the LPS of many strains of H. pylori may be involved in the adhesion of the pathogen to gastric epithelial cells.
  • Logan et al. (2000) have shown O-antigen to be an important H. pylori factor for the colonization of the murine stomach.
  • Applicants have identified, cloned and characterized a gene involved in assembly of the core polysaccharide of the LPS molecule.
  • the gene encodes a DD-heptosyltransferase (DDHepT) obtainable from Helicobacter that is responsible for adding DDHepII to the core LPS structure.
  • DHepT DD-heptosyltransferase
  • a truncated LPS with no O-antigen resulted.
  • mutations were introduced into the mouse-colonizing strain, H. pylori SSI, the mutant strain was unable to colonize the murine stomach.
  • a polypeptide of the invention is referred to herein as "DDHepT” or “DDHepT polypeptide” and a polynucleotide encoding a polypeptide of the invention is referred to herein as "DdhepT gene” or “DdhepV
  • the present invention contemplates an isolated polynucleotide encoding a DD-HepT polypeptide of the invention, including mRNAs, DNAs, cDNAs, genomic DNAs, PNAs, as well as antisense analogs and biologically, diagnostically, prophylactically, clinically or therapeutically useful variants or fragments thereof, and compositions comprising same.
  • the present invention contemplates an isolated polynucleotide comprising a sequence that comprises at least 18 nucleotides and hybridizes under stringent conditions to the complementary nucleic acid sequence of SEQ. ID. NO. 1, 3, or 5or a degenerate form thereof.
  • the polynucleotide comprises a region encoding DD-HepT polypeptides comprising a sequence set out in SEQ ID NO: 1, 3, or 5 which includes a full length polynucleotide or a variant thereof.
  • the polynucleotide encodes a polynucleotide designated herein as HP0479.
  • the polynucleotides of the invention permit identification of untranslated nucleic acid sequences or regulatory sequences which specifically promote expression of genes operatively linked to the promoter regions.
  • the invention therefore contemplates a polynucleotide encoding a regulatory sequence of a polynucleotide of the invention such as a promoter sequence, preferably a regulatory sequence of a DDhepT gene.
  • the polynucleotides encoding a mature polypeptide of the invention may include only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequences (e.g.
  • polynucleotides of the invention may be inserted into an appropriate expression vector, and the vector may contain the necessary elements for the transcription and translation of an inserted coding sequence. Accordingly, recombinant expression vectors may be constructed which comprise a polynucleotide of the invention, and where appropriate one or more transcription and translation elements linked to the polynucleotide.
  • Vectors are contemplated within the scope of the invention which comprise regulatory sequences of the invention, as well as chimeric gene constructs wherein a regulatory sequence of the invention is operably linked to a polynucleotide sequence encoding a heterologous protein (i.e. a protein not naturally expressed in the host cell), and a transcription termination signal.
  • a vector can be used to transform host cells to express a polypeptide of the invention, or a heterologous protein. Therefore, the invention further provides host cells containing a vector of the invention.
  • the invention also contemplates an isolated DD-HepT polypeptide encoded by a polynucleotide of the invention.
  • the invention provides a DD-HepT from Helicobacter comprising the amino acid sequence of SEQ ID NO:2, 4, or 6 or a variant thereof.
  • Further embodiments of the invention provide biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof and compositions comprising a polypeptide of the invention.
  • variants of a polypeptide of the invention encoded by naturally occurring alleles of a DDhepT gene.
  • Polypeptides of the invention may be obtained as an isolate from natural cell sources, but they are preferably produced by recombinant procedures.
  • the invention provides a method for preparing a polypeptide of the invention utilizing an isolated polynucleotide of the invention.
  • a method for preparing a DDHepT polypeptide comprising: (a) transferring a recombinant expression vector of the invention having a polynucleotide sequence encoding a DD-HepT, into a host cell;
  • the invention further broadly contemplates a recombinant DD-HepT obtained using a method of the invention.
  • a polypeptide of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins or chimeric proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
  • the invention further contemplates antibodies having specificity against an epitope of a polypeptide of the invention.
  • Antibodies may be labeled with a detectable substance and used to detect polypeptides of the invention in biological samples, tissues, and cells.
  • the invention also permits the construction of nucleotide probes which are unique to the polynucleotides of the invention or to polypeptides of the invention. Therefore, the invention also relates to a probe comprising a sequence encoding a polypeptide of the invention, or a part thereof.
  • the probe may be labeled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a polynucleotide of the invention including polynucleotides encoding a polypeptide which displays one or more of the properties of a polypeptide of the invention.
  • the invention provides a method for evaluating a test compound or agent for its ability to modulate the activity of a polypeptide or polynucleotide of the invention. For example a substance which inhibits or enhances the catalytic activity of a polypeptide of the invention may be evaluated.
  • “Modulate” refers to a change or an alteration in the biological activity of a polypeptide of the invention. Modulation may be an increase or a decrease in activity, a change in characteristics, or any other change in the biological, functional, or immunological properties of the polypeptide.
  • the invention provides methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: (a) contacting a polypeptide or polynucleotide of the invention with a test compound under conditions to permit binding to or other interaction between the test compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the test compound, wherein the binding or interaction is associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the test compound; and
  • test compound binds to or interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the test compound with the polypeptide or polynucleotide.
  • Compounds which modulate the biological activity of a polypeptide of the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of a polynucleotide or polypeptide of the invention in cells and organisms, in the presence, and in the absence of the compounds.
  • Methods are also contemplated that identify compounds or substances (e.g. polypeptides) which bind to regulatory sequences (e.g. promoter sequences, enhancer sequences, negative modulator sequences).
  • regulatory sequences e.g. promoter sequences, enhancer sequences, negative modulator sequences.
  • Still another aspect of the invention provides a method of conducting a drug discovery business comprising:
  • step (a) providing one or more systems or methods for identifying modulators of a polypeptide or polynucleotide of the invention; (b) conducting therapeutic profiling of modulators identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and
  • step (c) formulating a pharmaceutical composition including one or more modulators identified in step (b) as having an acceptable therapeutic profile.
  • the subject method may also include a step of establishing a distribution system for distributing the pharmaceutical composition for sale, and may optionally include establishing a sales group for marketing the pharmaceutical composition.
  • a method of conducting a target discovery business comprising: (a) providing one or more systems or methods for identifying modulators of a polypeptide or polynucleotide of the invention;
  • step (b) optionally conducting therapeutic profiling of modulators identified in (a) for efficacy and toxicity in animals; and (c) licensing to a third party the rights for further drug development and/or sales for modulators identified in step (a), or analogs thereof.
  • substances and compounds identified using the methods of the invention, antibodies, and antisense polynucleotides may be used to modulate the biological activity of a polypeptide or polynucleotide of the invention, and they may be used in the prevention and treatment of disease.
  • the substances and compounds are inhibitors of polypeptides of the invention that are useful as antibacterial agents.
  • agonists and antagonists of a DDHepT preferably bacteriostatic or bacteriocidal agonists or antagonists.
  • the polynucleotides and polypeptides of the invention may be formulated into compositions for administration to a cell or to a multicellular organism. Therefore, the present invention also relates to a composition comprising one or more of a polynucleotide or polypeptide of the invention, antibody or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, excipient or diluent.
  • a method for treating or preventing a disease comprising administering to a patient in need thereof, a composition of the invention.
  • products, compositions and methods for assessing DDHepT expression, treating disease, assaying genetic variation, and administering a polypeptide or polynucleotide of the invention to an organism to raise an immunological response against a bacteria Having provided novel DDHepT polypeptides, and polynucleotides encoding same, the invention accordingly further provides methods for preparing oligosaccharides e.g. two or more saccharides.
  • the invention relates to a method for preparing an oligosaccharide comprising contacting a reaction mixture comprising an activated D-glycero- ⁇ -D-manno-heptose ("DDHepIP), and an acceptor in the presence of a polypeptide of the invention.
  • DDHepIP activated D-glycero- ⁇ -D-manno-heptose
  • processes for utilizing polypeptides or polynucleotides of the invention for in vitro purposes related to scientific research, synthesis of DNA, and manufacture of vectors.
  • a computer readable medium having stored thereon a member selected from the group consisting of: (a) a polynucleotide comprising the sequence of SEQ ID NO. 1, 3, or 5; (b) a polypeptide comprising the sequence of SEQ ID NO. 2, 4, or 6; (c) a data set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO. 1, 3, or 5; (d) a data set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO. 2, 4, or 6; (e) a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, or 5; and (f) a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO. 2, 4, or 6.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, or 5 in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a polypeptide sequence comprising the sequence of SEQ ID NO. 2, 4, or 6 in a computer readable medium; and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: (a) providing a first polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, or 5 in a computer readable medium; and (b) screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: (a) providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, or 5 in a computer readable medium; and (b) comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: (a) providing a polypeptide sequence comprising the sequence of SEQ ID NO. 2, 4, or 6 in a computer readable medium; and (b) comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: (a) providing a first polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, or 5 in a computer readable medium; and (b) screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • Figure 1 shows a PCR amplification of H. pylori DNA.
  • Panel A PCR amplification using primers HP0479-F1 and HP0479-R1; Lane 1 - 250 bp ladder DNA size marker, Lane 2 - HP0479 product from 26695, Lane 3 -HP0479 product from strain 0:3 Lane 4 - HP0479 product from Sydney strain, Lane 5 - HP0479 product from Type strain (ATC43504), Lane 6 -HP0479 product from strain PJ1. Expected size of the HP0479 PCR product using these primers was 1242 bp.
  • Panel B PCR amplification using primers HP0479-GF1 and HP0479-GR1.
  • Lanes 1,8, and 17 - 250 bp ladder DNA size marker Lane 2 - H. pylori 26695, Lane 3 - H. pylori 26695 HP0479 mutant 1, Lane 4 - H. pylori 26695 HP0479 mutant 2, Lane 5 - H. pylori Sydney strain, Lane 6 - H. pylori Sydney strain HP0479 mutant 1, Lane 7 - H. pylori Sydney strain HP0479 mutant 2, Lane 9 - H. pylori strain PJ1, Lane 10 - H. pylori strain PJ1 HP0479 mutant 1, Lane 11 - H pylori strain 0:3, Lane 12 - H.
  • FIG. 2 shows ClustalW multiple sequence alignment of HP0479 homologs from various H. pylori strains.
  • HP0479S HP0479 homolog in Sydney strain.
  • JHP0431 HP0479 homolog in J99.
  • HP0479P HP0479 homolog in PJ1.
  • HP04793 HP0479 homolog in 0:3.
  • HP0479G HP0479 from 26695.
  • Figure 3 shows a blot of SDS-PAGE analysis of H. pylori whole cell LPS samples.
  • Lane 1 - H. pylori 26695 LPS Lane 2 - H. pylori 0479GM1 LPS, Lane 3 - H. pylori Sydney strain LPS, Lane 4 - H. pylori 0479SM1 LPS, Lane 5 - H. pylori strain PJ1 LPS, Lane 6 - H. pylori 0479PM1 LPS, Lane 1 - H. pylori strain 0:3 LPS, Lane 8 - H. pylori HP04793M1 LPS, Lane 9 - H. pylori ATCC 43504 LPS, Lane 10 - H. pylori HP0479TM1 LPS.
  • Figure 4 shows blots of SDS-PAGE analysis of whole cell LPS samples from H. pylori mutant and parental strains.
  • Panel A show LPS silver stained.
  • Panel B shows LPS immunoblotted with mouse monoclonal antibody raised against Lewis X.
  • Panel C shows LPS immunoblotted with mouse monoclonal antibody raised against Lewis Y.
  • Panel D shows LPS immunoblotted with rabbit polyclonal antibody raised against PJ1.
  • the arrow in panel B shows the position of a faint band in lane 2.
  • Figure 5 shows a blot of a Silver stain of an SDS-PAGE gel of H. pylori Sydney strain and H. pylori 0480M1 whole cell LPS samples. Lane 1 - H. pylori Sydney strain LPS, Lane 2 - H pylori 0480SM1 LPS.
  • FIG. 6 CZE-ES-MS and CZE-MS-MS (+ion mode) analysis of 0.1M sodium acetate buffer treated LPS from H. pylori 0479 mutants. Separation conditions: lOmM ammonium acetate containing 5% methanol, pH 9.0, +25kV.
  • A Extracted mass spectra at 14.6 min;
  • B Tandem mass spectrum of precursor ions at m/z 1612;
  • C Tandem mass spectrum of precursor ions at m/z 1392;
  • D Tandem mass spectrum of precursor ions at m/z 1271;
  • E Tandem mass spectrum of precursor ions at m/z 1246. Separation conditions as in (A) except nitrogen collision gas; E ⁇ at ,: 60eV (laboratory frame of reference).
  • Figure 7 shows the structure of H. pylori HP0479 mutant and parent LPS from strains 26695 and
  • Figure 8 shows a flow cytometric analysis of the adhesion of H.pylori SSI strain and its mutants to Hutu 80 cells.
  • Figure 9 shows the molecular structure of Helicobacter pylori LPS. DETAILED DESCRIPTION OF THE INVENTION
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • Complementarity between two single-stranded molecules may be "partial”, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules.
  • the term "consisting essentially of or “consisting of a polynucleotide sequence” refers to the disclosed polynucleotide sequence, and also encompasses polynucleotide sequences which are identical except for a base change or substitution therein. As known to those skilled in the art, a limited number of base changes or substitutions may be made in a short oligonucleotide sequence resulting in a sequence maintaining substantial function (ranging from approximately 50% to greater than 100% of the activity) of the original unmodified sequence.
  • Disease(s) means a condition or disease caused by or related to infection by a bacteria that comprises a polypeptide or polynucleotide of the invention.
  • “Host cell” is a cell which has been transformed or transfected, or is capable of being transformed or transfected by an exogenous polynucleotide sequence.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. “Identity” also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the match between strings of such sequences. “Identity” may be calculated by conventional 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.
  • BLASTP BLASTN
  • FASTA Altschul, S. F. et al., J. Molec. Biol 215: 403-410 (1990).
  • the BLAST X program 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 Smith Waterman algorithm known in the art may also be used to determine identity.
  • Parameters for comparison of polypeptide sequences include the following: (1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970); (2) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); (3) Gap Penalty: 12; and (4) Gap Length Penalty: 4.
  • a useful publicly available program with these parameters is the "gap" program from Genetics Computer Group, Madison Wis.
  • the above-mentioned comparison parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
  • Parameters for comparison of polynucleotide sequences include the following: (1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970); (2) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:109
  • Polynucleotide embodiments may include an isolated polynucleotide comprising a polynucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the sequence of SEQ ID NO:l, 3, or 5, where the polynucleotide sequence may be identical to the sequence of SEQ ID NO: 1, 3, or 5 or may include up to a certain integer number of nucleotide alterations as compared to the sequence of SEQ ID NO: 1, 3, or 5.
  • the alterations may be selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • the alterations may occur at the 5' or 3' terminal positions of the sequence of SEQ ID NO: 1, 3, or 5 or anywhere between those terminal positions, interspersed either individually among the nucleotides in the sequence of SEQ ID NO: 1, 3, or 5 or in one or more contiguous groups within this sequence.
  • the number of nucleotide alterations can be determined by multiplying the total number of nucleotides in SEQ ID NO:l, 3, or 5 by the integer defining the percent identity divided by 100 and then subtracting that product from the total number of nucleotides in SEQ ID NO:l, 3, or 5.
  • Polypeptide embodiments may 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 sequence of SEQ ID NO:2, 4, or 6 where the polypeptide sequence may be identical to the sequence of SEQ ID NO: 2, 4, or 6 or may include up to a certain integer number of amino acid alterations as compared to the sequence.
  • the alterations may be selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and where the 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 is determined by multiplying the total number of amino acids in SEQ ID NO: 2, 4, or 6 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, 4, or 6.
  • isolated refers to a polynucleotide or polypeptide changed and/or removed from its natural environment, purified or separated, or substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation, or any other recombinant method is "isolated” even if it is still present in an organism, which may be living or non-living.
  • an isolated polynucleotide or polypeptide is at least 60% free, more preferably at least 75% free, and most preferably at least 90%) free from other components with which they are naturally associated.
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, including mRNAs, DNAs, cDNAs and genomic DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • the term also includes triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such triple-stranded regions may be from the same molecule or from different molecules.
  • the regions may include all or one or more of the molecules, but typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described herein that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are within the meaning of the term “polynucleotide(s)”.
  • Polynucleotide(s) also includes DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples.
  • DNA and RNA that serve many useful purposes known to those of skill in the art and the term " ⁇ olynucleotide(s)” embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)” also includes short polynucleotides often referred to as oligonucleotide(s). The term “polynucleotides” and in particular DNA or RNA, refers only to the primary and secondary structure and it does not limit it to any particular tertiary forms.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacteria polypeptide and more particularly a polypeptide of Helicobacter pylori having an amino acid sequence set out in SEQ ID NO: 2, 4, or 6.
  • the term also contemplates polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (e.g. interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. The term includes both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) as used herein includes those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and research literature, and they are well known to those of skill in the art.
  • Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • modifications include, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoyl
  • Polypeptides may be branched or cyclic, with or without branching. These polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods.
  • Variant(s) refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of an encoded polypeptide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring variant such as an allelic variant, or it may be a variant that is not known to occur naturally. Mutagenesis techniques, direct synthesis, and other recombinant methods known to skilled artisans may be used to produce non-naturally occurring variants of polynucleotides and polypeptides.
  • a "ligand" refers to a compound or entity that associates with a polypeptide of the invention or part thereof, including acceptor molecules or analogues or parts thereof, and donor molecules or analogues or parts thereof.
  • a "donor molecule” refers to a molecule capable of donating a sugar to an acceptor molecule, via the action of a DDHepT polypeptide.
  • the donor molecule may be di- or poly-saccharides, sugar 1- phosphates, or, most commonly, nucleotide diphosphosugars (ADP-sugars), or nucleotide phosphosugars.
  • the donor molecule is ADP- mannoheptose.
  • An acceptor molecule is capable of accepting a sugar from a donor molecule, via the action of a DDHepT polypeptide. It may, for example, comprise a terminal sugar residue for transfer purposes.
  • the acceptor molecule or aglycone can be, for example, a lipid, a protein, a heterocyclic compound, an antibiotic, a peptide, an amino acid, an aromatic or aliphatic alcohol or thiol or another carbohydrate residue.
  • the acceptor molecule is or comprises a terminal D- ⁇ -D-heptose. (See Figure 9 for the LPS structure).
  • An analogue of a donor or acceptor molecule is one which mimics the donor or acceptor molecule binding to a DDHepT polypeptide but which is incapable (or has a significantly reduced capacity) to take part in the transfer reaction.
  • the invention provides isolated polynucleotides, (including a full length DDhepT gene) that encode DDHepT polypeptides, or fragments, variants, homologs thereof, and polynucleotides having substantial identity thereto, and variants thereof.
  • the polynucleotides encode polypeptides that retain substantially the same biological function or activity of a mature DDHepT.
  • an isolated polynucleotide is contemplated which comprises: (i) a polynucleotide encoding a polypeptide having substantial sequence identity, preferably at least 50%, more preferably at least 70% sequence identity, with an amino acid sequence of SEQ. ID. NO. 2, 4, or 6; (ii) polynucleotides complementary to (i);
  • polynucleotides differing from any of the polynucleotides of (i) or (ii) in codon sequences due to the degeneracy of the genetic code;
  • a polynucleotide comprising at least 10, 15, or 18, preferably at least 20 nucleotides and capable of hybridizing under stringent conditions to a polynucleotide of SEQ. ID. NO. 1, 3, or 5 or to a degenerate form thereof;
  • the isolated polynucleotide comprises:
  • polynucleotide having substantial sequence identity preferably at least 50%, more preferably at least 70% sequence identity with a sequence of SEQ. ID. NO. 1, 3, or 5;
  • polynucleotides differing from any of the nucleic acids of (i) to (ii) in codon sequences due to the degeneracy of the genetic code; or (iv) a fragment, or allelic or species variation of (i), (ii) or (iii).
  • the isolated nucleic acid comprises a polynucleotide encoded by an amino acid sequence of SEQ. ID. NO. 2, 4, or 6 or comprises or consists essentially of a polynucleotide of SEQ. ID. NO. 1, 3, or 5 wherein T can also be U.
  • the DNA sequence set out in SEQ ID NO: 1, 3, or 5 contains an open reading frame encoding a polypeptide comprising the amino acid residues set forth in SEQ ID NO: 2, 4, or 6, respectively, with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art.
  • a polynucleotide of the present invention has substantial sequence identity using the preferred computer programs cited herein, for example at least 50%, 60%, 70%, 75%, 80%, 90%), more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence of SEQ. ID. NO. 1, 3, or 5.
  • Isolated nucleic acid molecules encoding a polypeptide of the invention and having a sequence which differs from a polynucleotide of SEQ. ID. NO. 1, 3, or 5 due to degeneracy in the genetic code are also within the scope of the invention.
  • DNA sequence variations within DDhepT may result in silent mutations which do not affect the amino acid sequence.
  • Variations in one or more nucleotides may exist among organisms within a genus due to natural allelic variation. Any and all such nucleic acid variations are within the scope of the invention.
  • DNA sequence variations may also occur which lead to changes in the amino acid sequence of a polypeptide of the invention. These amino acid variations are also within the scope of the present invention.
  • species variations i.e. variations in nucleotide sequence naturally occurring among different species are within the scope of the invention.
  • the invention contemplates the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequences, including those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence.
  • a polynucleotide of the invention may also contain non-coding sequences, including, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids.
  • the additional sequences may be a marker sequence that facilitates purification of the fused polypeptide, the sequences may play a role in processing of a polypeptide 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. Additional sequences may be at the amino or carboxyl-terminal end or interior to the mature polypeptide.
  • Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression. Also included in the invention are polynucleotides of the formula:
  • Ri and R 2 is independently any nucleic acid residue
  • m is an integer between 1 and 3000 or zero, preferably between 1 and 1000
  • n is an integer between 1 and 3000 or zero, preferably between 1 and 1000
  • Z is a polynucleotide sequence of the invention, particularly a sequence selected from SEQ ID NO: 1, 3, or 5.
  • Any stretch of nucleotide residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • X and Y together define a covalent bond and the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • Fragments of a polynucleotide of the invention include fragments that are a stretch of at least about 10, 15, 18, 20, 40, 50, 100, or 150 nucleotides, more typically at least 50 to 100 nucleotides but less than 2 kb. It will further be appreciated that variant forms of the polynucleotides of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention. Polynucleotides that encode for variants of polypeptides of the invention are particularly contemplated that have an amino acid sequence of SEQ ID NO: 2, 4, or 6, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1, or no amino acid residues are substituted.
  • Another aspect of the invention provides a polynucleotide which hybridizes under selective conditions, e.g. high stringency conditions, to a polynucleotide which comprises a sequence which encodes a polypeptide of the invention.
  • the sequence encodes an amino acid sequence of SEQ. ID. NO. 2, 4, or 6 or part thereof and comprises at least 18 nucleotides. Selectivity of hybridization occurs with a certain degree of specificity rather than being random.
  • hybridization may occur at 30°C in 750 mM NaCl, 75mM trisodium citrate, and 1% SDS, preferably 37°C in 500mM NaCl, 500 mM trisodium citrate, 1% SDS, 35% formamide, and lOO ⁇ g/ml denatured salmon sperm DNA (ssDNA), and more preferably 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • the stringency may be selected based on the conditions used in the wash step.
  • Wash step stringency conditions may be defined by salt concentration and by temperature. Generally, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • a stringent salt concentration for the wash step is preferably less than about 30 mM NaCl and 3mM trisodium citrate, and more preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions will generally include temperatures of a least about 25°C, more preferably at least about 68°C.
  • the wash steps will be carried out at 42°C in 15 mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS.
  • the wash steps are carried out at 68°C in 15 mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. Variations on these conditions will be readily apparent to those skilled in the art.
  • polynucleotides of the inventions are preferably derived from Helicobacter pylori, however, they may be obtained from organisms of the same taxonomic genus. They may also be obtained from organisms of the same taxonomic family or order.
  • An isolated polynucleotide of the invention which comprises DNA can be isolated by preparing a labeled nucleic acid probe based on all or part of a nucleic acid sequence of SEQ. ID. NO. 1, 3, or 5.
  • the labeled nucleic acid probe is used to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library).
  • a cDNA library can be used to isolate a cDNA encoding a polypeptide of the invention by screening the library with the labeled probe using standard techniques.
  • a genomic DNA library can be similarly screened to isolate a genomic clone encompassing a DDhepT gene.
  • Polynucleotides isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.
  • An isolated polynucleotide of the invention which is DNA can also be isolated by selectively amplifying a polynucleotide of the invention.
  • “Amplifying” or “amplification” refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).
  • PCR polymerase chain reaction
  • a nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al, Biochemistry, 18, 5294-5299 (1979).
  • cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Louis, FL).
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL.
  • RNA can be isolated by cloning a cDNA encoding a polypeptide of the invention into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes the polypeptide.
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques.
  • a bacteriophage promoter e.g. a T7 promoter
  • a polynucleotide of the invention may be engineered using methods known in the art to alter the DDhepT encoding sequence for a variety of purposes including modification of the cloning, processing, and/or expression of the gene product. Procedures such as DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleic acid molecules. Mutations may be introduced by oligonucleotide-mediated site-directed mutagenesis to create for example new restriction sites, change codon preference, or produce variants.
  • Polynucleotides of the invention may be chemically synthesized using standard techniques. Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid- phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
  • Determination of whether a particular polynucleotide is a DDhepT gene or encodes a polypeptide of the invention can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the expressed protein in the methods described herein.
  • a cDNA encoding a polypeptide of the invention can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.
  • the polynucleotides of the invention may be extended using a partial nucleotide sequence and various PCR-based methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR which uses universal and nested primers to amplify unknown sequences from genomic DNA within a cloning vector may be employed (See Sarkar, G, PCR Methods Applic. 2:318-322, 1993).
  • Inverse PCR which uses primers that extend in divergent directions to amplify unknown sequences from a circularized template may also be used.
  • the template in inverse PCR is derived from restriction fragments adjacent to known sequences in human and yeast artificial chromosome DNA (See e.g.
  • oligo d(T) library does not yield a full-length cDNA
  • random-primed libraries which often include sequences containing the 5' regions of genes. Genomic libraries may be useful for extending the sequence into 5 'non-translated regulatory regions.
  • capillary electrophoresis systems may be employed to analyse the size or confirm the sequence of PCR or sequencing products.
  • the system may use flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Commercially available software e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer
  • the polynucleotides isolated using the methods described herein are mutant DDhepT gene alleles.
  • the mutant alleles may be isolated from organisms either known or proposed to contribute to a disease.
  • Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein.
  • a cDNA of a mutant DDhepT gene may be isolated using PCR as described herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutation(s) responsible for the loss or alteration of function of the mutant gene product.
  • a genomic library can also be constructed using DNA from an organism suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from organisms known to express the mutant allele.
  • a polynucleotide encoding a normal DDhepT gene or any suitable fragment thereof, may then be labeled and used as a probe to identify the corresponding mutant allele in such libraries.
  • Clones containing mutant sequences can be purified and subjected to sequence analysis.
  • an expression library can be constructed using cDNA from RNA isolated from organisms known or suspected to express a mutant DDhepT allele. Gene products from putatively mutant organisms may be expressed and screened, for example using antibodies specific for a polypeptide as described herein. Library clones identified using the antibodies can be purified and subjected to sequence analysis.
  • Antisense molecules and ribozymes are contemplated within the scope of the invention.
  • Antisense refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • Ribozymes are enzymatic RNA molecules that can be used to catalyze the specific cleavage of RNA.
  • Antisense molecules and ribozymes may be prepared by any method known in the art for the synthesis of polynucleotides. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding a polypeptide of the invention.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O- methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • a polypeptide of the invention includes a polypeptide of SEQ.ID. NO: 2, 4, or 6, particularly those which have the biological activity of a DDHepT.
  • polypeptides comprising an amino acid sequence of SEQ.ID. NO. 2, 4, or 6 include truncations or fragments, and variants, and homologs.
  • Truncated polypeptides may comprise peptides of between 3 and 70 amino acid residues, ranging in size from a tripeptide to a 50 mer polypeptide, preferably 30 to 50 amino acids.
  • fragments of a polypeptide of the invention are provided having an amino acid sequence of at least five consecutive amino acids of SEQ.ID. NO. 2, 4, or 6 where no amino acid sequence of five or more, six or more, seven or more, or eight or more, consecutive amino acids present in the fragment is present in a polypeptide other than a DDHepT of the invention.
  • the fragment is a stretch of amino acid residues of at least 12 to 20 contiguous amino acids from particular sequences such as the sequences of SEQ.ID. NO.
  • the fragments may be immunogenic and preferably are not immunoreactive with antibodies that are immunoreactive to polypeptides other than a DDHepT of the invention. Particularly preferred are fragments that are antigenic or immunogenic in an animal, especially in a human.
  • a fragment may be characterized by structural or functional attributes such as fragments that comprise 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 regions, and high antigenic index regions.
  • the invention provides biologically active fragments which are those fragments that mediate activities of a DDHepT, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
  • fragments comprising domains of enzymes that confer a function essential for viability of Helicobacter species or the ability to initiate, maintain, or cause disease in an individual, particularly a human.
  • Truncated polypeptides may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end.
  • the truncated polypeptides may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.
  • a truncated polypeptide or fragment 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, of a single larger polypeptide.
  • polypeptides of the invention may also include variants of a DDHepT of the invention, and/or truncations thereof as described herein, which may include, but are not limited to a polypeptide of the invention containing one or more amino acid substitutions, insertions, and/or deletions.
  • Amino acid substitutions may be of a conserved or non-conserved nature. conserveed amino acid substitutions involve replacing one or more amino acids of a DDHepT amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog is preferably functionally equivalent to a DDHepT of the invention.
  • Non-conserved substitutions involve replacing one or more amino acids of the DDHepT amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
  • amino acid insertions may be introduced into a polypeptide of the invention.
  • Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length.
  • Deletions may consist of the removal of one or more amino acids, or discrete portions from the DDHepT amino acid sequence.
  • the deleted amino acids may or may not be contiguous.
  • the lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 50 amino acids.
  • Allelic variants of a DDHepT at the protein level differ from one another by only one, or at most, a few amino acid substitutions.
  • a species variation of a DDHepT polypeptide is a variation which is naturally occurring among different species of an organism.
  • the polypeptides of the invention include homologs of a DDHepT and/or truncations thereof as described herein.
  • DDHepT homologs include proteins whose amino acid sequences are comprised of the amino acid sequences of DDHepT regions from other species that hybridize under selective hybridization conditions (see discussion of selective and in particular stringent hybridization conditions herein) with a probe used to obtain a polypeptide. These homologs will generally have the same regions which are characteristic of a DDHepT polypeptide.
  • a protein comprising an amino acid sequence which has at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% identity, more preferably 95%, 96%, 97%, 98%>, or 99% identity with an amino acid sequence of SEQ. ID. NO. 2, 4, or 6 will be a homolog of a polypeptide of the invention.
  • a percent amino acid sequence homology or identity is calculated using the methods described herein, preferably the computer programs described herein.
  • the invention also contemplates isoforms of polypeptides of the invention.
  • An isoform contains the same number and kinds of amino acids as a polypeptide of the invention, but the isoform has a different molecular structure.
  • the isoforms contemplated by the present invention preferably have the same properties as a polypeptide of the invention as described herein.
  • the present invention also provides a polypeptide of the invention conjugated with a selected protein, or a marker or other glycosyltransferase, to produce fusion proteins or chimeric proteins. Also included in the invention are polypeptides of the formula:
  • each occurrence of Ri and R 2 is independently any amino acid residue
  • m is an integer between 1 and 1000 or zero, preferably between 1 and 1000
  • n is an integer between 1 and 3000 or zero, preferably between 1 and 1000
  • Z is a polypeptide of the invention, particularly a sequence selected from SEQ ID NO: 2, 4, or 6. Any stretch of amino acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • X and Y together define a covalent bond the polypeptide of the above formula is a closed, circular polypeptide.
  • a polypeptide of the invention may be prepared using recombinant DNA methods. Accordingly, polynucleotides of the present invention having a sequence which encodes a polypeptide of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the polypeptide. Possible expression vectors include but are not limited to chromosomal, episomal and virus-derived vectors, so long as the vector is compatible with the host cell used.
  • vectors are 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 and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids
  • the invention therefore contemplates a recombinant expression vector comprising a polynucleotide of the invention, and the necessary regulatory sequences for the transcription and translation of the inserted sequence.
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art.
  • the necessary regulatory sequences may be supplied by the native polypeptide and/or its flanking regions.
  • the invention further provides a recombinant expression vector comprising a polynucleotide of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a polynucleotide sequence of SEQ. ID. NO. 1, 3, or 5. Regulatory sequences linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance a viral promoter and/or enhancer, or regulatory sequences can be chosen which direct tissue or cell type specific expression of antisense RNA.
  • the recombinant expression vectors of the invention may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • marker genes are genes encoding a protein such as G418, dhfr, npt, als, pat and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, trpB, hisD, herpes simplex virus thymidine kinase, adenine phosphoribosyl transferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
  • Visible markers such as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants, and also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. et al. (1995)Mol. Biol. 55:121-131).
  • the markers can be introduced on a separate vector from the nucleic acid of interest.
  • the recombinant expression vectors may also contain genes that encode a fusion moiety which provides increased expression of the recombinant polypeptide; increased solubility of the recombinant polypeptide; and aid in the purification of the target recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site may be added to the target recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion protein.
  • Typical fusion expression vectors include pGEX (Amrad Corp, Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
  • the vectors may be introduced into host cells to produce a transformed or transfected host cell.
  • the terms "transfected " and “transfection” encompass the introduction of nucleic acid (e.g. a vector) into a cell by one of many standard techniques. A cell is "transformed" by a nucleic acid when the transfected nucleic acid effects a phenotypic change.
  • Prokaryotic cells can be transfected or transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation.
  • Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection.
  • Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, or mammalian cells.
  • Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).
  • host cells examples include bacterial cells, such as Streptococci, Staphylococci, Enterococci, E. coli, Helicobacter, Streptomyces, and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
  • bacterial cells such as Streptococci, Staphylococci, Enterococci, E. coli, Helicobacter, Streptomyces, and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • a host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e.g. glycosylation) and processes (e.g. cleaves) the polypeptide in a desired fashion.
  • Host systems or cell lines may be selected which have specific and characteristic mechanisms for post- translational processing and modification of proteins. For long-term high-yield stable expression of the polypeptide, cell lines and host systems which stably express the gene product may be engineered.
  • Host cells and in particular cell lines produced using the methods described herein may be particularly useful in screening and evaluating compounds that modulate the activity of a polypeptide of the invention.
  • Polypeptides of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). Protein synthesis may be performed using manual procedures or by automation. Automated synthesis may be carried out, for example, using an Applied Biosystems 431 A peptide synthesizer (Perkin Elmer). Various fragments of the polypeptides of the invention may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • N-terminal or C-terminal fusion polypeptides or chimeric polypeptides comprising a polypeptide of the invention conjugated with other molecules, (e.g. markers) may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of a polypeptide of the invention, and the sequence of a selected molecule with a desired biological function (e.g.marker protein).
  • the resultant fusion proteins contain a polypeptide of the invention fused to the selected molecule as described herein.
  • molecules which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), protein A, hemagglutinin (HA), and truncated myc.
  • Antibodies include immunoglobulins, glutathione-S-transferase (GST), protein A, hemagglutinin (HA), and truncated myc.
  • Antibodies as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunoglobulin expression library.
  • oligopeptides, peptides, or fragments used to induce antibodies to a polypeptide of the invention have an amino acid sequence consisting of at least 5 amino acids and more preferably at least 10 amino acids.
  • the oligopeptides, etc. can be identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule.
  • Antibodies having specificity for a polypeptide of the invention may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, etc. may be prepared using methods known to those skilled in the art.
  • Antibodies against polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols.
  • Monoclonal antibodies may be obtained by any technique known in the art that provides antibodies produced by continuous cell line cultures. (See for example, Kohler, G. and Milstein, C, Nature 256. 495-497 (1975); Kozbor et al, Imnmunology Today 4: 72 (1983); Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
  • Single chain antibodies to polypeptides of this invention can be prepared using methods known in the art (e.g. U.S. Pat. No. 4,946,778).
  • Transgenic mice, or other organisms such as other mammals may be used to express humanized antibodies.
  • Phage display technology may also be utilized to select antibody genes 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-DDHepT or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • Chain shuffling can also be used to improve the affinity of these antibodies (Clackson, T. et al, (1991) Nature 352, 624-628).
  • the polynucleotides, polypeptides, and antibodies of the invention may be used in the prognostic and diagnostic evaluation of disease. (See below).
  • Methods for detecting polynucleotides and polypeptides of the invention can be used to monitor disease in eukaryotes particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising a DDhepT gene or polypeptide of the invention, by detecting and localizing the polynucleotides and polypeptides.
  • the applications of the present invention also include methods for the identification of agents (eg. compounds) which modulate the biological activity of a polypeptide of the invention (See below).
  • the compounds, antibodies, etc. may be used for the treatment of disease. (See below). Diagnostic and Prognostic Methods
  • polynucleotides of the invention may be used, for example, for: (1) the detection of the presence of DDhepT gene mutations, or the detection of either over- or under-expression of DDHepT mRNA relative to a non-disorder state; and (2) the detection of either an over- or an under-abundance of a polypeptide of the invention relative to a non-disorder state or the presence of a modified (e.g., less than full length) polypeptide of the invention.
  • DDhepT gene mutations or the detection of either over- or under-expression of DDHepT mRNA relative to a non-disorder state
  • a modified polypeptide of the invention e.g., less than full length
  • the methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising at least one specific polynucleotide or antibody described herein, which may be conveniently used, e.g., in clinical settings, to screen and diagnose individuals and to screen and identify or monitor disease in individuals.
  • Nucleic acid-based detection techniques and peptide detection techniques are described below.
  • the samples that may be analyzed using the methods of the invention include those which are known or suspected to contain a polynucleotide or polypeptide of the invention.
  • the methods may be performed on biological samples including but not limited to cells, lysates of cells which have been incubated in cell culture, genomic DNA (in solutions or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), an extract from cells or a tissue (e.g. bone, muscle, cartilage, skin), and biological fluids such as serum, urine, blood, and CSF.
  • the samples may be derived from a patient or a culture.
  • the invention provides a process for diagnosing disease, preferably bacterial infections, more preferably infections by Helicobacter pylori, comprising determining from a sample derived from an individual an increased level of expression of a polynucleotide of the invention.
  • Increased or decreased expression of a polynucleotide of the inventon can be measured using any of the methods well known in the art.
  • a polynucleotide of the invention may be used in southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dipstick, pin, ELISA assays or microarrays utilizing fluids or tissues from patients to detect altered expression. Such qualitative or quantitative methods are well known in the art and some methods are described below.
  • the polynucleotides of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of polynucleotides of the invention in biological materials.
  • Suitable probes include polynucleotides based on nucleic acid sequences encoding at least 5 sequential amino acids from regions of a polynucleotide of the invention (see SEQ. ID. No. 1, 3, or 5), preferably they comprise 15 to 30 nucleotides.
  • a nucleotide probe may be labeled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as ⁇ F, ⁇ H, ⁇ C or the like.
  • detectable substances which may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization.
  • Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleic acid probes may be used to detect hepT genes, preferably in human cells.
  • the nucleotide probes may also be useful for example in the diagnosis or prognosis of disease, and in monitoring the progression of a disease condition, or monitoring a therapeutic treatment.
  • the probe may be used in hybridization techniques to detect DDhepT genes. The technique generally involves contacting and incubating a sample from a patient or other cellular source with a probe of the present invention under conditions favourable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.
  • the detection of polynucleotides of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art.
  • Suitable primers can be routinely designed by one of skill in the art. (See SEQ ID NO 7 through 14).
  • primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Madison Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50%) or more, and to anneal to the template at temperatures of about 60°C to 72° C.
  • Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities in cells involving DDHepT structure, including point mutations, insertions, and deletions. For example, direct sequencing, single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and oligonucleotide hybridization may be utilized. Mutations in the DNA sequence of a DDhepT gene may be used to diagnose infection and to serotype and/or classify the infectious agent. Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to the mutations in a DDhepT gene. The polymorphisms may be used to identify species of organisms that are likely to cause disease.
  • RT-PCR may be used to detect mutations in the RNA.
  • RT-PCR may be used in conjunction with automated detection systems such as for example GeneScan.
  • the primers and probes may be used in the above described methods in situ i.e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.
  • Oligonucleotides derived from any of the polynucleotides of the invention may be used as targets in microarrays.
  • “Microarray” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon, or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • the microarrays can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image) and to identify genetic variants, mutations, and polymorphisms. This information can be useful in determining gene function, diagnosing disease, and in developing and monitoring the activity of therapeutic agents (Heller, R. et al. (1997) Proc. Natl. Acad, Sci. 94:2150-55).
  • the polynucleotides of the present invention are useful for chromosome identification.
  • the sequences can be specifically targeted to, and can hybridize with a particular location on an individual microbial chromosome, particularly & Helicobacter pylori chromosome.
  • the mapping of relevant sequences to a chromosome is an important step in correlating those sequences with genes associated with microbial pathogenicity and disease, or to precise chromosomal regions critical to the growth, survival, and/or ecological niche of an organism.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data to define a genetic relationship between the gene and another gene or phenotype by, for example, linkage analysis.
  • RNA or genomic sequence between microbes of different phenotypes may also be determined.
  • a mutation or sequence observed in some or all of the organisms of a certain phenotype but not in organisms lacking that phenotype, will likely be the causative agent for the phenotype.
  • chromosomal regions may be identified that confer pathogenicity, growth characteristics, survival characteristics, and/or ecological niche.
  • the polynucleotides of the invention may be used in differential screening and differential display methods known in the art. (e.g. see Chuang et al J. Bacteriol. 175: 2026, 1993). Genes are identified which are expressed in an organism by identifying mRNA present using randomly primed RT-PCR.
  • Antibodies specifically reactive with a polypeptide of the invention or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used to detect the polypeptides in various samples. They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of a polypeptide of the invention, or abnormalities in the structure of the polypeptides. Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on a disease. In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies.
  • the antibodies of the invention may also be used in vitro to determine the level of DDHepT expression in cells genetically engineered to produce a DDHepT.
  • the invention provides a diagnostic method for detecting over-expression of a polypeptide of the invention compared to normal control tissue samples. The method may be used to detect the presence of an infection.
  • the antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a polypeptide of the invention, and the antibodies.
  • assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests.
  • the antibodies may be used to detect and quantify polypeptides of the invention in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
  • Antigenic polypeptides of the invention or fragments thereof may be used in immunoassays to detect antibody levels and correlations can be made with diseases such as gastroduodenal disease and with duodenal ulcer in particular. Immunoassays based on well defined recombinant antigens can be developed. Antibodies to Helicobacter pylori HepT polypeptides within biological samples such as blood or serum samples may be detected.
  • the antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a polypeptide of the invention, to localise it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a polypeptide of the invention.
  • an antibody of the invention may be labeled and a polypeptide may be localised in tissues and cells based upon detection of the label.
  • Various methods of labeling polypeptides are known in the art and may be used to label antibodies and polypeptides of the invention.
  • detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3 H, l4 C, 35 S, 125 1, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached via spacer arms of various lengths to
  • an antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies etc.
  • the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite.
  • the support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip).
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against a polypeptide of the invention.
  • the antibody having specificity against a polypeptide of the invention is a rabbit IgG antibody
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • a polypeptide of the invention may be localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • a polypeptide of the invention may also be detected by assaying for DDHepT activity as described herein. For example, a sample may be reacted with an acceptor molecule and a donor molecule under conditions where a DDHepT is capable of transferring the donor molecule to the acceptor molecule to produce a donor-acceptor complex.
  • Methods for Identifying or Evaluating Substances/Compounds The invention provides methods for identifying substances that modulate the biological activity of a polypeptide of the invention including substances that interfere with, or enhance the activity of the polypeptide.
  • the substances and compounds identified using the methods of the invention include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries including libraries made of D- and/or L- configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, antisense, oligosaccharides, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)2, and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules.
  • peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries including libraries made of D- and/or L- configuration amino acids, phospho
  • the substance or compound may be an endogenous physiological compound or it may be a natural or synthetic compound.
  • a substance of the invention may be a natural substrate or ligand (e.g. an acceptor or donor molecule) or a structural or functional mimetic.
  • the substance may be a small molecule ligand in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • Substances which modulate a polypeptide of the invention can be identified based on their ability to associate with (or bind to) a polypeptide of the invention. Therefore, the invention also provides methods for identifying substances which associate with a polypeptide of the invention. Substances identified using the methods of the invention may be isolated, cloned and sequenced using conventional techniques.
  • a substance that associates with a polypeptide of the invention may be an agonist or antagonist of the biological or immunological activity of the polypeptide.
  • antagonist refers to a molecule which decreases the biological or immunological activity of the polypeptide.
  • Agonists and antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that associate with a polypeptide of the invention (including ligands or mimetics thereof).
  • Substances which can associate with a polypeptide of the invention may be identified by reacting the polypeptide with a test substance which potentially associates with the polypeptide, under conditions which permit the association, and removing and/or detecting polypeptide associated with the test substance.
  • Substance-polypeptide complexes, free substance, or non-complexed polypeptide may be assayed, or the activity of the polypeptide may be assayed.
  • Conditions which permit the formation of substance- polypeptide complexes may be selected having regard to factors such as the nature and amounts of the substance and the polypeptide.
  • the substance-polypeptide complex, free substance or non-complexed polypeptide may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against a polypeptide of the invention or the substance, or labeled polypeptide, or a labeled substance may be utilized.
  • the antibodies, polypeptide, or substances may be labeled with a detectable substance as described above.
  • a polypeptide of the invention, or the substance used in the method of the invention may be insolubilized.
  • a polypeptide, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubilized polypeptide or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • the invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a polypeptide of the invention, by assaying for an agonist or antagonist (i.e. enhancer or inhibitor) of the association of the polypeptide with a substance which associates with the polypeptide.
  • the basic method for evaluating if a compound is an agonist or antagonist of the association of a polypeptide of the invention and a substance that associates with the polypeptide is to prepare a reaction mixture containing the polypeptide and the substance under conditions which permit the formation of substance- polypeptide complexes, in the presence of a test compound.
  • the test compound may be initially added to the mixture, or may be added subsequent to the addition of the polypeptide and substance.
  • Control reaction mixtures without the test compound or with a placebo are also prepared.
  • the formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of the polypeptide and substance.
  • the reactions may be carried out in the liquid phase or the polypeptide, substance, or test compound may be immobilized as described herein.
  • the substance is a natural substrate or ligand of a polypeptide of the invention, or a structural or functional mimetic thereof. It will be understood that the agonists and antagonists i.e.
  • inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the polypeptide or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites.
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of a polypeptide of the invention with a substance which is capable of associating with or binding to the polypeptide.
  • the invention may be used to assay for a compound that competes for the same binding site of a polypeptide of the invention.
  • the invention provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of polypeptides or polynucleotides of the invention, particularly those compounds that are bacteriostatic and/or bacteriocidal.
  • the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising a polypeptide of the invention and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a test compound that may be an agonist or antagonist.
  • test compound to agonize or antagonize the polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i.e., without inducing the effects of a polypeptide of the invention are most likely to be good antagonists.
  • Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to calorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in polynucleotide or polypeptide activity, and binding assays known in the art.
  • an assay for antagonists is a competitive assay that combines a polypeptide of the invention and a potential antagonist with molecules that bind a polypeptide of the invention, a recombinant binding molecule, natural substrate or ligand, or substrate or ligand mimetic, under appropriate conditions for a competitive inhibition assay.
  • the polypeptide can be labeled, such as by radioactivity or a colorimetric compound, such that the number of polypeptides bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
  • Agents that modulate a polypeptide of the invention can be identified based on their ability to interfere with or enhance the activity of a polypeptide of the invention.
  • the invention provides a method for evaluating a compound for its ability to modulate the activity of a polypeptide of the invention comprising (a) reacting an acceptor molecule and a donor molecule for a polypeptide of the invention in the presence of a test compound; (b) measuring transfer of a sugar of the donor molecule to the acceptor molecule, and (c) carrying out steps (a) and (b) in the absence of the test compound to determine if the compound interferes with or enhances transfer of the sugar of the donor molecule to the acceptor molecule by the polypeptide.
  • the acceptor molecule or donor molecule may be labeled with a detectable substance as described herein, and the interaction of the polypeptide of the invention with the acceptor molecule and donor molecule will give rise to a detectable change.
  • the detectable change may be colorimetric, photometric, radiometric, potentiometric, etc.
  • the activity of a polypeptide of the invention may also be determined using methods based on HPLC (Koenderman et al, FEBS Lett. 222:42, 1987) or methods employing synthetic oligosaccharide acceptors attached to hydrophobic aglycones (Palcic et al Glycoconjugate 5:49, 1988; and Pierce et al, Biochem. Biophys. Res. Comm. 146: 679, 1987).
  • a polypeptide of the invention is reacted with the acceptor and donor molecules at a pH and temperature and in the presence of a metal cofactor, usually a divalent cation, effective for the polypeptide to transfer the sugar of the donor molecule to the acceptor molecule, and where one of the components is labeled, to produce a detectable change.
  • a metal cofactor usually a divalent cation
  • the buffer, acceptor and donor molecules may be used as an assay composition. Other compounds such as EDTA and detergents may be added to the assay composition.
  • the polypeptide may be obtained from natural sources or produced using recombinant methods as described herein.
  • the reagents suitable for applying the methods of the invention to evaluate compounds that modulate a polypeptide of the invention may be packaged into convenient kits providing the necessary materials packaged into suitable containers.
  • the kits may also include suitable supports useful in performing the methods of the invention.
  • a substance that inhibits a polypeptide may be identified by treating a cell which expresses the polypeptide with a test substance, and analyzing the lipopolysaccharide structures on the cell. Lipopolysaccharide can be analyzed using the methods described herein. Cells that have not been treated with the substance or which do not express the polypeptide may be employed as controls.
  • Substances which inhibit transcription or translation of a DDhepT gene may be identified by transfecting a cell with an expression vector comprising a recombinant molecule of the invention, including a reporter gene, in the presence of a test substance and comparing the level of expression of a DDhepT, or the expression of the protein encoded by the reporter gene with a control cell transfected with the nucleic acid molecule in the absence of the substance.
  • the method can be used to identify transcription and translation inhibitors of a DDhepT gene.
  • polynucleotides and polypeptides of the invention and substances or compounds identified by the methods described herein, antibodies, and antisense nucleic acid molecules of the invention may be used to treat diseases.
  • diseases that may be treated include diseases associated with organisms that contain a polypeptide or polynucleotide of the present invention.
  • the organsims are from the Helicobacter family, and are particularly Helicobacter pylori species.
  • H. pylori infects the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France; http://www.uicc.ch/ecp/ecp2904.htm).
  • H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen.
  • Preferred agonists of the invention found using screens provided by the invention will be useful in the treatment of H pylori infection, and they should decrease the advent of H. py/o ⁇ ' -induced cancers, such as gastrointestinal carcinoma.
  • the agonists should also be useful in the treatment of gastric ulcers and gastritis.
  • the proteins, substances, antibodies, and compounds etc. may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • the substances may be administered to living organisms including humans, and animals.
  • Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage periods may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration.
  • the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the compound.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA 1985).
  • the compositions include, albeit not exclusively, solutions of the substances or compounds in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • the labeling would include amount, frequency, and method of administration.
  • the compositions, substances, compounds etc. may be indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment (e.g. chemotherapy or radiotherapy).
  • they can be used in combination with anti-proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-inf ⁇ ammatories.
  • anti-bacterial agents In particular, they can be used in combination with anti-bacterial agents. They can be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.
  • Polynucleotides of the invention or any fragment thereof, or antisense sequences may be used for therapeutic purposes.
  • Antisense to a polynucleotide encoding a polypeptide of the invention may be used in situations to block the synthesis of the polypeptide.
  • cells may be transformed with sequences complementary to polynucleotides of the invention.
  • antisense sequences may be used to modulate activity of a polypeptide of the invention, or to achieve regulation of gene function.
  • Sense or antisense oligomers or larger fragments can be designed from various locations along the coding or regulatory regions of sequences encoding a polypeptide of the invention.
  • Expression vectors may be derived from retroviruses, adenoviruses, herpes or vaccinia viruses or from various bacterial plasmids for delivery of nucleic acid sequences to the target organ, tissue, or cells.
  • Vectors that express antisense nucleic acid sequences of OOhepT can be constructed using techniques well known to those skilled in the art (see for example, Sambrook et al. (supra)).
  • Genes encoding a DDHepT can be turned off by transforming a cell or tissue with expression vectors that express high levels of a polynucleotide of the invention. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even if they do not integrate into the DNA, the vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases.
  • Modification of gene expression may be achieved by designing antisense molecules, DNA, RNA, or Peptide nucleic acid (PNA), to the control regions of a DDHepT gene i.e. the promoters, enhancers, and introns.
  • the antisense molecules are oligonucleotides derived from the transcription initiation site
  • Inhibition can also be achieved by using triple-helix base-pairing techniques.
  • Triple helix pairing causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules (see Gee J.E. et al (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co, Mt.
  • An antisense molecule may also be designed to block translation of mRNA by inhibiting binding of the transcript to the ribosomes.
  • Ribozymes may be used to catalyze the specific cleavage of RNA. Ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • hammerhead motif ribozyme molecules may be engineered that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a polypeptide of the invention.
  • Specific ribosome cleavage sites within any RNA target may be initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • Short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the cleavage site of the target gene may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED 50 /LD 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. Mutant Organisms
  • the invention provides novel mutants of Helicobacter bacteria, in particular mutants of H. pylori, having mutated (deactivated) DDhepT genes.
  • mutated refers to a sudden heritable change in the phenotype of an organism which can be spontaneous or induced by known mutagenic agents, including radiation and various chemicals.
  • TnlO transposon
  • Methods are known in the art that can be used to generate mutations to produce the mutant bacteria of the present invention.
  • the transposon, TnlO can be used to produce chromosomal deletions in a wide variety of bacteria (Kleckner et al, J. Mol. Biol. 116:125-159, 1977; EPO Pub. No. 315,682; U.S. Pat. No. 5,387,744.
  • methods may be used that involve introducing specific deletions in a DDhepT gene in an organism.
  • a specific deletion in the selected gene can be generated by either of two general methods.
  • the first method generates a mutation in a gene isolated from a population of clones contained in a genomic DNA library using restriction enzymes and the second method generates the mutation in a gene of known sequence using PCR.
  • the position of the gene on a vector is identified using transposon tagging and a restriction map of the recombinant DNA in the vector is generated.
  • Information derived from the transposon tagging allows all or a portion of a gene to be excised from the vector using the known restriction enzyme sites.
  • the second method is based upon PCR. Divergent PCR primers are used to amplify the upstream and downstream regions flanking a specified segment of the DDhepT DNA to be deleted from the gene, generating a PCR product consisting of the cloning vector and upstream and downstream flanking nucleotide sequences (Innes et al. Eds, PCR Protocols, 1990, Academic Press, New York). In a variation of this method, PCR products are produced representing portions of the gene or flanking sequence, which are then joined together in a cloning vector.
  • Mutagenesis of a cloned DDhepT gene may also be carried out by insertion of a marker into an insertion site in the gene.
  • a marker for example, a kanamycin resistance marker may be ligated into an insertion site created in a DDhepT gene by reverse PCR (See Example 1).
  • the DNA containing the mutant gene can be introduced into the bacterial host by transformation using chemical means or electroporation, by recombinant phage infection, or by conjugation.
  • the mutant gene is introduced into the chromosomes of the bacteria which can be accomplished using any of a number of methods well known in the art such as, for example, methods using temperature-sensitive replicons (Hamilton et al, J. Bacteriol. 171:4617-4622, 1989), linear transformation of recBC mutants (Jasin et al, J. Bacteriol. 159:783-786, 1984), or host restricted replicons known as suicide vectors (Miller et al, J. Bacteriol. 170:2575-2583, 1988).
  • the particular method used is coupled with an appropriate counter selection method such as, for example, by using PCR, nucleic acid hybridization, or an immunological method.
  • Mutant bacteria of the invention include H. plyori 0479GM1; H. pylori 0479M1, H. pylori
  • the invention also provides modified LPS molecules from mutants of the invention.
  • the modified LPS molecules from mutants of the invention.
  • LPS may be isolated from the mutant bacteria and at least partially purified using techniques well known to those skilled in the art. Preparations of at least 70%), particularly 80%, more particularly 90%, most particularly 95 % pure LPS are preferred. The purity of an LPS preparation is expressed as the weight percentage of the total Helicobacter antigens present in the preparation. The purified LPS can be used as antigen either directly or after being conjugated to a suitable carrier protein. The structures of LPS of mutant bacteria are shown in Figure 7. Methods for Preparing Oligosaccharides
  • the invention relates to a method for preparing an oligosaccharide comprising contacting a reaction mixture comprising an activated donor molecule and an acceptor molecule in the presence of a polypeptide of the invention.
  • the oligosaccharides are prepared on a carrier that is non-toxic to a mammal, in particular a lipid isoprenoid or polyisoprenoid alcohol.
  • a carrier is dolichol phosphate.
  • the oligosaccharide may be attached to a carrier via a labile bond allowing for chemical removal of the oligosaccharide from the lipid carrier.
  • the oligosaccharide transferase may be used to transfer the oligosaccharide from a lipid carrier to a protein.
  • mutant bacteria expressing the truncated LPS and the modified LPS isolated from such mutants are useful sources of antigens in vaccination against Helicobacter bacteria, in particular against H. pylori.
  • Such vaccines are normally prepared from dead bacterial cells, using methods well known to those skilled in the art, and usually contain various auxiliary components, such as an appropriate adjuvant and a delivery system.
  • a delivery system aiming at mucosal delivery is preferred.
  • the antigenic preparation is administered orally to the host, but parenteral administration is also possible.
  • Live vaccines based on H. pylori mutants may also be prepared, but would normally require an appropriate vector for mucosal delivery.
  • Vaccines of the present invention are useful in preventing and reducing the number of
  • Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with an antigen (e.g. modified
  • LPS LPS
  • T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Helicobacter pylori infection. Also provided are methods whereby such immunological response slows bacterial replication.
  • a further aspect of the invention relates to an immunological composition which, when introduced into an individual capable of having induced within it an immunological response, induces an immunological response in such individual to Helicobacter wherein the composition comprises a modified
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+T cells.
  • a modified LPS may be fused with a molecule which may not by itself produce antibodies, but is capable of stabilizing the modified LPS and producing an antigen which will have immunogenic and protective properties.
  • a molecule which may not by itself produce antibodies, but is capable of stabilizing the modified LPS and producing an antigen which will have immunogenic and protective properties.
  • examples of such molecules are Hpoprotein D from Hemophilus influenzae, glutathione-S-transferase (GST) or beta-galactosidase.
  • the molecule may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
  • the invention provides methods using the modified LPS in immunization experiments in animal models of infection with Helicobacter to identify 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 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 Helicobacter pylori infection, in mammals, particularly humans.
  • the modified LPS may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by preventing colonization.
  • the invention also includes a vaccine formulation which comprises a modified LPS of the invention together with a suitable carrier.
  • the formulation is preferably 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, bacteriostats and solutes which render the formulation insotonic 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 ampules 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 may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • H. pylori cultures were grown either on solid Columbia Blood agar (Difco) supplemented with Horse Blood (5%), Vancomycin (10 mg/L), Nalidixic acid (1.1 mg/L;), Bacitracin (20 mg/L), Polymyxin B (0.33 mg/L), and Amphotericin A (5 mg/L) or in Brucella broth supplemented with Fetal Bovine Calf Serum (5-10%), Vancomycin (10 mg/L), Nalidixic acid (1.1 mg/L;), Bacitracin (20 mg/L), Polymyxin B (0.33 mg/L), and Amphotericin A (5 mg/L). All H.
  • pylori cultures were incubated at 37°C in a tri-gas incubator with a gas mixture of 85%> N 2 , 10% C0 2 and 5% 0 2 until the desired amount of growth was obtained, normally 2-3 days.
  • gas mixture of 85%> N 2 , 10% C0 2 and 5% 0 2 until the desired amount of growth was obtained, normally 2-3 days.
  • liquid media cultures were also shaken at 100 rpm.
  • E. coli DH5 ⁇ was used (Table 1). E. coli cultures were grown in Luria broth supplemented, when needed, with Ampicillin (100 mg/L; SIGMA), X-gal (20 mg/L; Gibco/BRL), IPTG (lOOmM; Gibco/BRL), or Kanamycin (20 mg/L; SIGMA).
  • Plasmids The plasmid pUC19 was used to clone PCR-amplified H. pylori genes as well as a suicide vector to transform H. pylori strains.
  • a 1398 bp Smal fragment from the plasmid pIP1433 (Labigne-Roussel, et al., 1988) was used which contained a Campylobacter coli Kanamycin marker.
  • PCR Polymerase Chain Reaction
  • H. pylori HP0479 Amplification of the H. pylori HP0479 gene for cloning was carried out using the heat stable DNA polymerase Pwol (Roche Biochemicals). This enzyme also carries a 3'-5' proofreading activity which increases the fidelity of replication and produces a blunt-ended product.
  • the primers used for this purpose, HP0479-F1 (SEQ ID NO: 7) and HP0479-R1 (SEQ ID NO: 8), are listed in Table 2. An annealing temperature of 60°C was used for amplification. PCR amplification was carried out on the cloned HP0479 H pylori genes to generate blunt ends for insertion of a C.
  • HP0479-mutFl SEQ ID NO: 9
  • HP0479-mutRl SEQ ID NO: 10.
  • PCR using Taq DNA polymerase was used to ascertain if the C. jejuni Kanamycin marker had been inserted into the appropriate genes.
  • the primers [HP0479-GF1 (SEQ ID NO: 11) and HP0479-GR1 (SEQ ID NO: 12)] and annealing temperatures used for amplification are listed in Table 2. Cloning of the HP0479 gene The HP0479 gene ofH.
  • pylori 26695 was cloned using the cloning vector pUC19.
  • Polymerase chain reaction (PCR) was used to amplify the gene from genomic DNA of H. pylori 26695, as well as the HP0479 homologs from the genomes of the type strain (ATCC 43504), 0:3, PJ1 and Sydney strain.
  • the amplified PCR fragments were analyzed by agarose gel electrophoresis to insure that a product of the expected size was obtained. Subsequently, the PCR fragments were cloned into the Smal site of the pUC19 vector using T4 DNA ligase with protocols described by the manufacturer (Gibco/BRL). The ligation mixture was used to transform E.
  • coli DH5 ⁇ using methods described by Chung and Miller (1988). Selection of clones was carried out using standard white/blue selection on LB agar supplemented with X-gal, IPTG and Ampicillin. Plasmid DNA was isolated and purified using a plasmid isolation kit (Qiagen). Insertion of the gene was confirmed and orientation of insertion was determined by restriction endonuclease analysis using the enzyme Hindlll. One clone, p0479-l, carrying the HP0479 gene from H. pylori 26695, was used for mutagenesis. The cloned HP0479 gene and the cloned HP0479 homologs from the other strains were sequenced for comparisons at the DNA and amino acid levels.
  • Mutagenesis of the cloned HP0479 gene Mutagenesis of the cloned HP0479 gene was carried out by the insertion of a Kanamycin resistance marker from Campylobacter coli, carried on a 1348 bp Smal fragment from the plasmid pIP1433 (Labigne-Roussel, et al., 1988). A blunt-ended insertion site was created in the cloned HP0479 gene by reverse PCR using primers HP0479-mutFl and HP0479-mutRl. The Kanamycin cassette was ligated into the insertion site using T4 ligase.
  • the ligation mix was used to transform DH5 ⁇ , as above (Chung and Miller, 1988), and selection was carried out on LB-agar supplemented with Ampicillin and Kanamycin. Insertion of the Kanamycin cassette and orientation of insertion was confirmed by restriction endonuclease analysis using the enzyme Ec ⁇ Rl.
  • One plasmid, p0479- Kl was used for transformation of H. pylori strains. Transformation of H. pylori with p0479-Kl Natural transformation of H pylori was carried out following a protocol modified from those of Haas et al., 1993. Briefly, H. pylori cultures were grown on Columbia Blood agar as described above, for 2-3 days.
  • Bacterial growth was suspended in 3 ml of Brucella broth (see media above) and adjusted to an optical density (OD 60 o) of 0.1-0.2 with Brucella broth.
  • One ml of diluted culture was mixed with 10-50 ⁇ l (5-25 ⁇ g) of plasmid DNA in a sterile 24-well cell-culture dish. The cells were incubated at 37°C for 4-6 hours in the tri-gas incubator. Aliquots of the transformed cells were then plated onto Columbia Blood agar with Kanamycin and the plates were incubated in the tri-gas incubator for 3-7 days until colonies were visible.
  • HP0480-F 1 GATAACCTCATCACGCTTAG
  • HP0480-R1 GATAACCTCATCACGCTTAG
  • TTCAATCCATTCTAACGC (SEQ ID NO: 14) with Pwol as described above with an annealing temperature of 60°C.
  • the gene was cloned into pUC19 and mutated by insertion of the C. coli Chloramphenicol cassette from the plasmid pRY109 (Yao et al., 1993) into a unique Smal restriction site.
  • the mutated gene was transformed into 26695 and Sydney strain as described above and the LPS of whole cells was analyzed by SDS-PAGE.
  • Membrane fraction analysis Membrane fractions were prepared from overnight (18 h) liquid cultures of HP0479 mutant strains and parental strains using the protocols described by Logan et al. (2000). The fractions were analyzed by SDS-PAGE and stained using Coomasie blue.
  • LPS H. pylori strains were cultivated as previously described (Logan et al. 2000). The wet cell mass obtained by centrifugation of the bacterial growth was washed successively, once with ethanol, twice with acetone, and twice with light petroleum ether and air-dried. LPS was extracted from the air-dried cellular material by the hot phenol-water extraction procedure of Westphal and Jann (1965). LPS was obtained from the aqueous phase after extensive dialysis and lyophilization. H. pylori LPS from parental strains was further purified by ultracentrifugation and the pellet suspended in distilled water and lyophilized.
  • Glycoses were determined by GLC as their alditol acetate derivatives. Samples (0.2-0.5 mg) were hydrolyzed with 2M trifluoroacetic acid (TFA) for 16 h at 100°C and evaporated to dryness under a stream of nitrogen. The liberated glycoses were reduced with sodium borohydride (NaBH 4 ) and acetylated (Ac 2 0) as previously described (York et al. 1985). The configuration of peracetylated heptitol derivatives was determined to be L-glycero-D-manno or D-glycero-D-manno by comparison of their GLC retention times with that of an authentic standard.
  • TFA trifluoroacetic acid
  • Hexoses were determined to have the D-configuration by GLC analysis of their acetylated (R)-2-octyl glycoside derivatives (Gerwig et al. 1979). Methylation analysis was performed on lipopolysaccharide samples (1-3 mg) with iodomethane in dimethylsulfoxide containing an excess of sodium hydroxide (Ciucanu and Kerek, 1984) and permethylated alditol acetates were characterized by GLC-MS in the El mode.
  • FAB-MS analysis in a positive mode was performed on permethylated LPS samples using a JEOL AX505H double focusing sector mass spectrometer. 6kV Xenon atom was used to ionize the sample.
  • the sample was typically mixed with a solution of 1 : 1 thioglycerol/glycerol, although thioglycerol and glycerol alone was also used for some samples.
  • Electrospray mass spectrometry Samples were analyzed on a crystal Model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex, Concord, Canada) via a microlonspray interface.
  • a sheath solution (isopropanol-methanol, 2:1) was delivered at a flow rate of 1 ⁇ L/min to a low dead volume tee (250 ⁇ m i.d, Chromatographic Specialities, Brockville, Canada). All aqueous solutions were filtered through a 0.45- ⁇ m filter (Millipore, Bedford, MA, USA) before use.
  • An electrospray stainless steel needle (27 gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column.
  • the separation were obtained on about 90 cm length bare fused-silica capillary using 10 mM ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5% methanol.
  • a voltage of 25 kV was typically applied at the injection.
  • the outlet of the capillary was tapered to ca. 15 ⁇ m i.d. using a laser puller (Sutter Instruments, Novato, CA, USA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full- mass-scan mode.
  • mice were inoculated by gavage twice 5 days apart with bacterial suspensions of approximately 10 9 organisms and 10 6 organisms respectively. In the second experiment the mice were given three inocula spread over 5 days with bacterial suspensions of approximately 10 9 organisms per dose.
  • Cell culture HuTu-80 (ATCC HTB-40), derived from a human duodenal adenocarcinoma, was obtained from the American Type Culture Collection. It was maintained in tissue culture flasks as adherent monolayers in Minimal Essential Media (MEM) (Gibco) supplemented with 10% (vol/vol) FBS without antibiotics.
  • MEM Minimal Essential Media
  • FBS fetal bovine serum
  • adherence assays cells were trypsinized with 0.25% trypsin (Sigma) (10 min, 37 °C), centrifuged at 200 x g for 5 min, washed once with PBS and resuspended in PBS, pH 7.4 at a final concentration of 1x10*.
  • Adherence assay The fluorescently labeled bacteria were added to mammalian cells at a ratio of
  • H. pylori DD-heptosyl transferases Computer database searches of the genome of H. pylori 26695 were conducted using the BLAST search engine (Altschul, et al, 1997) for genes which showed structural homology to heptosyl-transferases. Several different heptosyl-transferases were used as the query sequences for these searches including the WaaC genes from E. coli, S. typhimurium, Campylobacter coli, Campylobacter jejuni, Campylobacter hyoilei, and H. pylori 26695 (HP0279). One gene, HP0479, was identified through this process.
  • HP0479 gene from H. pylori strains The primers used for PCR amplification, HP0479-F1 and HP0479-R1 (Table 2), were chosen from regions flanking the gene from the total genome sequence of H. pylori 26695. Primer HP0479-F1 starts 140 bp from the start of the HP0479 gene while primer HP0479-R1 ends 49 bp downstream of the gene. With these primers a PCR product of 1242 bp was expected. Amplification was carried out as described in Materials and Methods. A PCR product of approximately the correct size was amplified from DNA from H. pylori strains 26695, Sydney, ATCC 43504, PJl, and strain 0:3 ( Figure 1, Panel A).
  • the fragments were cloned into the Smal site of pUC19 and transformed into E. coli DH5 ⁇ . Standard blue/white selection was carried out and several clones were isolated. The orientation of the cloned fragments was determined by restriction endonuclease analyses.
  • the clones were designated p0479G (from 26695 genome strain), p0479S (from Sydney strain), p04793 (from strain 0:3), p0479T (from the type strain ATCC43504) and p0479P (from PJl).
  • the DNA sequence of the HP0479 genes from p0479G, p0479S, p04793, and p0479P was determined. Amino acid sequences were predicted from the DNA sequence and these were aligned with the sequence of the HP0479 homolog from J99, JHP0431 ( Figure 2).
  • Mutagenesis of the cloned HP0479 gene PCR was used to generate a blunt-ended insertion site within the HP0479 gene of p0479G for insertion of the Kanamycin cassette.
  • the primers used to generate the blunt-ended insertion site, HP0479-mutFl and HP0479-mutRl, are shown on Table 2.
  • the thermostable polymerase Pwol was used to amplify the fragment for insertion mutagenesis.
  • H. pylori with the mutated HP0479 gene Since the pUC19 plasmid was not compatible with H. pylori, it acted as a suitable suicide vector for the transfer of the mutated HP0479 gene into H. pylori strains. Selection for Kanamycin resistance was used to isolate H. pylori that had incorporated the mutated HP0479 gene. The HP0479 mutant gene was introduced into several strains of H. pylori using this method. The H. pylori strains mutagenized were strain 26695, Sydney strain, strain 0:3, Type strain (ATCC 43504), and strain PJl. Kanamycin resistant transformants were obtained for all strains.
  • the mutant H. pylori strains were designated H. pylori 0479GM1 (26695 mutant), H. pylori 0479SM1 (Sydney strain mutant), H. pylori 04793M1 (0:3 mutant), H. pylori 0479TM1 (ATCC 43504 type strain mutant) and H. pylori 0479PM1 (PJl mutant).
  • HP0480 gene was amplified as described in Materials and Methods, cloned into pUC19 and mutagenized by the insertion of a C. coli Chloramphenicol cassette into a unique Smal site. This mutant plasmid was then transformed into H. pylori 26695 and Sydney strain. Chloramphenicol resistant strains were isolated and whole cell LPS samples were run on SDS-PAGE. Figure 5 shows that the LPS profiles on SDS-PAGE of the parental and the 0480SM1 H. pylori Sydney strains are identical.
  • FAB-MS analysis in the positive mode of the permethylated LPS from each strain indicated the presence of primary glycosyl oxonium ions at m/z 260 [GlcNAc] + and m/z 434 [Fuc,GlcNAc] + and secondary glycosyl oxonium ions at m/z 228 (260-32) [GlcNAc and m/z 402 (434-32) [Fuc,GlcNAc] + .
  • H. pylori Sydney strain This strain has been shown to consistently colonize mice and has been universally used as a mouse colonization model for H. pylori (Lee et al., 1997; Ferrero et al., 1998; Conlan, et al., 1999; Logan et al, 2000). In this study two separate trials were carried out. In each trial 10 mice were given H. pylori Sydney strain wild type cultures and 10 were given H. pylori 479SM1.
  • H. pylori was administered twice orogastrically 5 days apart with approximately 10 6 to 10 8 bacteria as determined by plate counts of the inoculum. One week and 12 weeks later, 5 mice from each group were sacrificed, their stomachs homogenized, and plate counts were performed to determine the extent of colonization.
  • H. pylori was administered three times over a 5 day period with approximately 1 x 10 9 doses of either organism. In this trial the mice were sacrificed two weeks and four weeks after bacterial inoculation. In both trials the parental strain was able to establish colonization which persisted at significant levels even after 12 weeks (Tables 5 and 6). In contrast, the mutant strain was never detected in the stomachs of any of the mice challenged with it (Tables 5 and 6).
  • PCR was carried out on the stomach homogenates using the HP0479-GF1 and HP0479- GR1 primers.
  • the expected PCR products were obtained from the mice inoculated with the H. pylori SSI, but not from the stomach homogenates of mice inoculated with mutant strain (data not shown).
  • Lipopolysaccharides are the main surface antigens of Gram-negative bacteria, and are essential for the physical integrity and function of the bacterial outer membrane. Despite the importance of LPS in bacterial pathogenesis, H. pylori LPS has received limited attention and its' role in the pathogenic process has not been clearly established. Similar to LPS from other species, the basic structure of H. pylori LPS consists of three distinct regions: O-chain polysaccharide composed of repeating units, covalentiy linked to a core oligosaccharide, which in turn is attached to a hydrophobic lipid A moiety (Raetz et al., 1990). The structure of H.
  • pylori LPS is unique among Gram-negative bacteria having four consecutive heptosyl residues in the core region (two O-glycero-D-manno-heptose (DD-heptose) and two L-glycero-D-manno- heptose (LD-heptose) residues).
  • the core region also includes a trisaccharide moiety branching from DDHepI ( Figure 7)(Aspinall and Monteiro, 1996; Aspinall et al., 1996; Monteiro et al., 1998a; Monteiro et al., 2000b). This core structure is conserved among H. pylori species and no structural variability in this region has been reported so far.
  • This heptan region may vary from strain to strain (Aspinall et al., 1997; Monteiro, et al., 2000b) and in some strains O-chain was found to be directly linked to the 0-2 position of DDHepII.
  • the length of the O-chain polysaccharide varies considerably from strain to strain and phase variation in the population of a given strain has been reported (Monteiro, et al, 2000a; Monteiro, et al., 2000b;, Appelmelk et al., 1998; Aspinall et al, 1997). This ability is thought to contribute to pathogenicity by allowing the organism to survive and adapt to various environments (Appelmelk, et al., 1998).
  • the core of if. pylori LPS is a unique combination of both DD and LD forms of heptose.
  • some strains further extend this polyheptose region by the addition of DD-heptose between the core and the O-antigen ( Figure 7)(Aspinall et al., 1996; Aspinall and Monteiro 1996; Aspinall et al, 1997).
  • Figure 7 (Aspinall et al., 1996; Aspinall and Monteiro 1996; Aspinall et al, 1997).
  • none of the transferases involved in the biosynthesis of the core or the addition of DD- heptose to the heptan-linking region have been functionally identified. From the standpoint of developing therapeutic intervention strategies against H. pylori, this region is of particular interest as it provides a fairly constant target for H pylori without the variation that is seen in the O-antigen.
  • L-glycero-a-D-manno- heptose L-glycero-a-D-manno- heptose
  • DD-heptose D-glycero-a-D-manno- eptose
  • LD-heptose and DD-heptose are structurally related, differing stereochemically at the glycero moiety of the molecule, some homology may exist between LD-and DD- heptosyl transferases.
  • computer searches were used to identify proteins in H. pylori that could be potential DD-heptosyl transferases.
  • the HP0479 gene of H. pylori 26695 was identified as a putative heptosyl-transferase.
  • HP0479 had significant homology in conserved regions with the other transferases. According to the classification of glycosyl transferase families as described by Campbell et al. (1997) HP0479 belongs to family 9, which includes heptosyl-transferases.
  • HP0479 Mutational analysis of HP0479 showed that H. pylori strains carrying mutations of this gene had truncated LPS. Western blots of whole cell LPS samples immunoblotted against Lewis antigens also showed a loss of O-antigen. The LPS profiles of all of the mutant strains of H. pylori tested were the same. This showed that HP0479 did not encode a heptosyl transferase involved in the synthesis of the heptan linker region that is present in the 26695 and 0:3 strains, but not present in Sydney strain. Polar mutational effects were ruled out as mutations to the gene immediately downstream of HP0479, HP0480, produced wild type LPS.
  • HP0479 is the first DD-heptosyltransferase from H. pylori that has been functionally identified.
  • Structural data from several other strains of H. pylori show that there may potentially be up to 7 or more heptosyl-transferase genes involved in the assembly ofH. pylori LPS.
  • Average mass units were used for calculation of molecular mass values based on proposed composition as follows: Hex, 162.15; HexNAc, 203.20; Fuc, 146.14; Hep, 192.17; KDO, 220.18; PEA, 123.05; H 2 0, 18.02; b observed fragment ion corresponds to anhydro-KDO-containing glycoform; c the most abundant glycoform (based on the fragment ion intensity).

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Abstract

Cette invention se rapporte à des polynucléotides et polypeptides nouvellement identifiés, et à leur production et à leurs utilisations, ainsi qu'à leurs variants, agonistes et antagonistes, et à leurs utilisations. Cette invention concerne en particulier de nouveaux polynucléotides et polypeptides d'heptosyltransférase.
PCT/CA2001/000969 2000-06-28 2001-06-28 Helicobacter dd-heptosyltransferase WO2002000851A2 (fr)

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WO2011094489A1 (fr) 2010-01-29 2011-08-04 Vertex Pharmaceuticals Incorporated Thérapies pour traiter une infection par le virus de l'hépatite c
EP2399575A2 (fr) 2006-08-11 2011-12-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés, utilisations et compositions pour le traitement d'une infection par un virus de la famille de Flaviviridae par l'inhibition de récepteur farnésoïde X (FXR)
WO2012107589A1 (fr) 2011-02-11 2012-08-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques pour le traitement et la prévention des infections à vhc
WO2017175239A1 (fr) 2016-04-05 2017-10-12 Council Of Scientific & Industrial Research Protéine de type glycosyltransférase recombinante multifonctionnelle dépendant des nucléotides et son procédé de glycosylation

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SK165197A3 (en) * 1995-06-07 1999-01-11 Astra Ab Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics
CA2377427A1 (fr) * 1999-06-28 2001-01-04 National Research Council Of Canada Glycosyltransferases de helicobacter pylori utilisees comme nouvelle cible dans la prevention et le traitement des infections par h. pylori

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EP2399575A2 (fr) 2006-08-11 2011-12-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés, utilisations et compositions pour le traitement d'une infection par un virus de la famille de Flaviviridae par l'inhibition de récepteur farnésoïde X (FXR)
EP2399988A2 (fr) 2006-08-11 2011-12-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Système de culture cellulaire pour la réplication du virus de l'hépatite C par l'activation ou l'inhibition de récepteur farnésoïde X (FXR) et méthode de diagnostic pour l'infection avec VHC
WO2011094489A1 (fr) 2010-01-29 2011-08-04 Vertex Pharmaceuticals Incorporated Thérapies pour traiter une infection par le virus de l'hépatite c
WO2012107589A1 (fr) 2011-02-11 2012-08-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques pour le traitement et la prévention des infections à vhc
WO2017175239A1 (fr) 2016-04-05 2017-10-12 Council Of Scientific & Industrial Research Protéine de type glycosyltransférase recombinante multifonctionnelle dépendant des nucléotides et son procédé de glycosylation
US11485991B2 (en) 2016-04-05 2022-11-01 Council Of Scientific & Industrial Research Multifunctional recombinant nucleotide dependent glycosyltransferase protein and its method of glycosylation thereof

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EP1299527A2 (fr) 2003-04-09
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