EP0980204A1 - POLYPEPTIDES D'HELICOBACTER DE 76 kDa, 32 kDa ET 50 kDa ET MOLECULES DE POLYNUCLEOTIDES CORRESPONDANTES - Google Patents

POLYPEPTIDES D'HELICOBACTER DE 76 kDa, 32 kDa ET 50 kDa ET MOLECULES DE POLYNUCLEOTIDES CORRESPONDANTES

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Publication number
EP0980204A1
EP0980204A1 EP98914395A EP98914395A EP0980204A1 EP 0980204 A1 EP0980204 A1 EP 0980204A1 EP 98914395 A EP98914395 A EP 98914395A EP 98914395 A EP98914395 A EP 98914395A EP 0980204 A1 EP0980204 A1 EP 0980204A1
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EP
European Patent Office
Prior art keywords
asn
ala
leu
ser
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98914395A
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German (de)
English (en)
Other versions
EP0980204A4 (fr
Inventor
Harold Kleanthous
Ling Lissolo
Jean-François TOMB
Charles Miller
Amal Al-Garawi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merieux Oravax
Human Genome Sciences Inc
Original Assignee
Merieux Oravax
Human Genome Sciences Inc
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Publication date
Priority claimed from US08/834,666 external-priority patent/US20020044949A1/en
Priority claimed from US08/831,310 external-priority patent/US20020026035A1/en
Application filed by Merieux Oravax, Human Genome Sciences Inc filed Critical Merieux Oravax
Publication of EP0980204A1 publication Critical patent/EP0980204A1/fr
Publication of EP0980204A4 publication Critical patent/EP0980204A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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

  • Polypeptide derivatives that are encoded by polynucleotides of the invention include, e.g., fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins.
  • Polypeptide fragments of the invention can be derived from a polypeptide having a sequence homologous to the sequences of any of SEQ ID NOs:2-22 (even numbers), 66, and 68, to the extent that the fragments retain the substantial antigenicity of the parent polypeptide (specific antigenicity).
  • Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial part of the parent polypeptide, while retaining specific antigenicity. Generally, polypeptide derivatives should be about at least 12 amino acids in length to maintain antigenicity.
  • polypeptide fragments and polypeptides having large internal deletions can be constructed using standard methods (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), for example, by PCR, including inverse PCR, by restriction enzyme treatment of the cloned DNA molecules, or by the method of Kunkel et al (Proc. Natl. Acad. Sci. USA 82:448, 1985; biological material available at Stratagene).
  • a polypeptide derivative can also be produced as a fusion polypeptide that contains a polypeptide or a polypeptide derivative of the invention fused, e.g., at the - or C-terminal end, to any other polypeptide (hereinafter referred to as a peptide tail).
  • a product can be easily obtained by translation of a genetic fusion, i.e., a hybrid gene.
  • Vectors for expressing fusion polypeptides are commercially available, and include the pMal-c2 or pMal-p2 systems of New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention.
  • a recombinant expression system can be selected from procaryotic and eucaryotic hosts.
  • Eucaryotic hosts include, for example, yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells.
  • yeast cells e.g., Saccharomyces cerevisiae or Pichia pastoris
  • mammalian cells e.g., COS1, NIH3T3, or JEG3 cells
  • arthropods cells e.g., Spodoptera frugiperda (SF9) cells
  • plant cells e.g., a procaryotic host such as E. coli is used.
  • Bacterial and eucaryotic cells are available from a number of different sources that are known to those skilled in the art,
  • Promoters and signal peptide-encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (U.S. Patent No. 5,028,530; Cagnon et al, Protein Engineering 4(7): 843, 1991); the promoter of the bacteriophage T7 RNA polymerase gene, which is functional in a number of E. coli strains expressing T7 polymerase (U.S. Patent No. 4,952,496); the OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al, J. Bact. 169:5692, 1987).
  • Antibodies useful for immunoaffinity purification of the polypeptides of the invention can be obtained using methods described below.
  • Polynucleotides of the invention can also be used in DNA vaccination methods, using either a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid.
  • Therapeutic or prophylactic efficacy of a polynucleotide of the invention can be evaluated as is described below.
  • a vaccine vector such as a poxvirus, containing a polynucleotide molecule of the invention placed under the control of elements required for expression; (ii) a composition of matter containing a vaccine vector of the invention, together with a diluent or carrier; (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Helicobacter in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Helicobacter infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit an immune response, e.g., a protective or therapeutic immune response to Helicobacter; and (v) a method for preventing and/or treating a Helicobacter (
  • Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively also see, e.g., Tartaglia et al, Virology 188:217, 1992, for a description of a vaccinia virus vector, and Taylor et al, Vaccine 13:539, 1995, for a description of a canary poxvirus vector.
  • Poxvirus vectors capable of expressing a polynucleotide of the invention can be obtained by homologous recombination, as described in Kieny et al (Nature 312: 163, 1984) so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells.
  • Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens, and their use as oral vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and in WO 92/11361.
  • Preferred routes of administration for these vectors include all mucosal routes. Most preferably, the vectors are administered intranasally or orally. Others bacterial strains useful as vaccine vectors are described by
  • the desmin promoter (Li et al, Gene 78:243, 1989; Li et al, J. Biol. Chem. 266:6562, 1991 ; Li et al, J. Biol. Chem. 268: 10403, 1993) is tissue-specific and drives expression in muscle cells. More generally, useful promoters and vectors are described, e.g., in WO 94/21797 and by Hartikka et al. (Human Gene Therapy 7: 1205, 1996).
  • a polynucleotide encoding a cytokine such as interleukin-2 (IL-2) or interleukin- 12 (IL-12), can also be added to the composition so that the immune response is enhanced.
  • additional polynucleotides are placed under appropriate control for expression.
  • DNA molecules of the invention and/or additional DNA molecules to be included in the same composition are earned in the same plasmid.
  • a polynucleotide can be associated with agents that assist in cellular uptake. It can be, e.g., (i) complemented with a chemical agent that modifies cellular permeability, such as bupivacaine (see, e.g.,
  • Liposomes A Practical Approach, RPC New Ed, IRL Press, 1990, for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.
  • Cationic lipids can also be used for gene delivery.
  • Such lipids include, for example, LipofectinTM, which is also known as DOTMA (N-[l-DOTMA) (N-[l-DOTMA) (N-[l-DOTMA) (N-[l-DOTMA)
  • probe refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to polynucleotide molecules having sequences homologous to those shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or to a complementary or anti-sense sequence of any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • probes are significantly shorter than the full- length sequences shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67.
  • they can contain from about 5 to about 100, preferably from about 10 to about 80 nucleotides.
  • probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of a sequence as shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67, or a sequence complementary to such sequences.
  • Probes can contain modified bases, such as inosine, methyl-5- deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2, 6- purine.
  • Sugar or phosphate residues can also be modified or substituted.
  • a deoxyribose residue can be replaced by a poly amide (Nielsen et al, Science 254: 1497, 1991) and phosphate residues can be replaced by ester groups such as diphosphate, alkyl, arylphosphonate, and phosphorothioate esters.
  • the 2'-hydroxyl group on ribonucleotides can be modified by addition of, e.g., alkyl groups.
  • Probes of the invention can be used in any conventional hybridization method, such as in dot blot methods (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982), Southern blot methods (Southern, J. Mol. Biol. 98:503, 1975), northern blot methods (identical to Southern blot to the exception that RNA is used as a target), or a sandwich method (Dunn et al, Cell 12:23, 1977). As is known in the art, the latter technique involves the use of a specific capture probe and a specific detection probe that have nucleotide sequences that are at least partially different from each other.
  • Primers used in the invention usually contain about 10 to 40 nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), an elongation process, or a reverse transcription method. In a diagnostic method involving PCR, the primers can be labeled.
  • polypeptides that can be produced by expression of the polynucleotides of the invention can be used as vaccine antigens.
  • a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.
  • Specific antigenicity can be determined using a number of methods, including Western blot (Towbin et al, Proc. Natl. Acad. Sci. USA 76:4350, 1979), dot blot, and ELISA methods, as described below.
  • the product to be screened is fractionated by SDS-PAGE, as described, for example, by Laemmli (Nature 227:680, 1970).
  • a filter such as a nitrocellulose membrane
  • the material is incubated with the monospecific hyperimmune antiserum, which is diluted in a range of dilutions from about 1 :50 to about 1 :5000, preferably from about 1 : 100 to about 1 :500.
  • Specific antigenicity is shown once a band co ⁇ esponding to the product exhibits reactivity at any of the dilutions in the range.
  • the product to be screened can be used as the coating antigen.
  • a purified preparation is preferred, but a whole cell extract can also be used. Briefly, about 100 ⁇ l of a preparation of about 10 ⁇ g protein/ml is distributed into wells of a 96-well ELISA plate. The plate is incubated for about 2 hours at 37°C, then overnight at 4°C. The plate is washed with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer) and the wells are saturated with 250 ⁇ l PBS containing 1% bovine serum albumin (BSA), to prevent non-specific antibody binding.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • a composition of matter containing a polypeptide of the invention together with a diluent or carrier containing a therapeutically or prophylactically effective amount of a polypeptide of the invention
  • a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention containing a therapeutically or prophylactically effective amount of a polypeptide of the invention
  • a method for inducing an immune response against Helicobacter in a mammal by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, e.g., a protective immune response to Helicobacter
  • a method for preventing and/or treating a Helicobacter e.g., H. pylori, H. felis, H.
  • a polypeptide or polypeptide derivative can be formulated into or with liposomes, such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs), to facilitate delivery and/or enhance the immune response.
  • liposomes such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs)
  • VLPs virus-like particles
  • Adjuvants other than liposomes can also be used in the invention and are well known in the art (see, for example, the list provided below).
  • Administration can be achieved in a single dose or repeated as necessary at intervals that can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly or monthly intervals.
  • An appropriate dose depends on various parameters, including the nature of the recipient (e.g., whether the recipient is an adult or an infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), and can be readily determined by one skilled in the art.
  • a vaccine antigen of the invention can be administered mucosally in an amount ranging from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
  • Polypeptides and polypeptide derivatives of the invention can also be used as diagnostic reagents for detecting the presence of anti-Helicobacter antibodies, e.g., in blood samples.
  • Such polypeptides can be about 5 to about 80, preferably, about 10 to about 50 amino acids in length and can be labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • a polypeptide or polypeptide derivative is produced and can be purified using known methods.
  • the polypeptide or polypeptide derivative can be produced as a fusion protein containing a fused tail that facilitates purification.
  • the fusion product can be used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise monospecific antibodies against the polypeptide or polypeptide derivative.
  • the eighth aspect of the invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
  • the antibodies of the invention can be of any isotype, e.g., IgG or IgA, and polyclonal antibodies can be of a single isotype or can contain a mixture of isotypes.
  • the antibodies of the invention which can be raised to a polypeptide or polypeptide derivative of the invention, can be produced and identified using standard immunological assays, e.g., Western blot assays, dot blot assays, or ELISA (see, e.g., Coligan et al, Current Protocols in Immunology, John Wiley & Sons, Inc., New York, NY, 1994).
  • the antibodies can be used in diagnostic methods to detect the presence of Helicobacter antigens in a sample, such as a biological sample.
  • the antibodies can also be used in affinity chromatography methods for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, the antibodies can also be used in prophylactic and therapeutic passive immunization methods.
  • a polypeptide reagent can be used for detecting the presence of anti-Helicobacter antibodies in a sample, e.g., a blood sample, while an antibody of the invention can be used for screening a sample, such as a gastric extract or biopsy sample, for the presence of Helicobacter polypeptides.
  • the reagent e.g., the antibody, polypeptide, or polypeptide derivative of the invention
  • the reagent can be in a free ⁇ tate or can be immobilized on a solid support, such as, for example, on the interior surface of a tube or on the surface, or within pores, of a bead. Immobilization can be achieved using direct or indirect means. Direct means include passive adso ⁇ tion (i.e., non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with the reagent is first attached to the solid support.
  • an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in recognition of antibodies in biological samples.
  • Indirect means can also employ a ligand-receptor system, for example, a molecule, such as a vitamin, can be grafted onto the polypeptide reagent and the corresponding receptor can be immobilized on the solid phase. This concept is illustrated by the well known biotin-streptavidin system.
  • the antibody can be polyclonal or monospecific, and preferably is of the IgG type.
  • Purified IgGs can be prepared from an antiserum using standard methods (see, e.g., Coligan et al, supra). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described, for example, by Harlow et al (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).
  • Therapeutic or prophylactic efficacy can be evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the H. felis mouse model and the procedures described by Lee et al. (Eur. J. Gastroenterology & Hepatology 7:303, 1995) or Lee et al. (J. Infect. Dis. 172: 161, 1995).
  • the H. felis strain of the model can be replaced with another Helicobacter strain.
  • the efficacy of polynucleotide molecules and polypeptides from H. pylori is, preferably, evaluated in a mouse model using an H.
  • an antibody of the invention can be administered to the gastric mucosa of mice previously challenged with an H. pylori strain, as described, e.g., by Lee et al. (supra). Then, after an appropriate period of time, the bacterial load of the mucosa can be estimated by assessing urease activity, as compared to a control. Reduced urease activity indicates that the antibody is therapeutically effective.
  • Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).
  • Additional LT mutants that can be used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E.
  • MPLA bacterial monophosphoryl lipid A
  • coli Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri
  • saponins and polylactide glycolide (PLGA) microspheres
  • PLGA polylactide glycolide
  • Adjuvants useful for both mucosal and parenteral administrations such as polyphosphazene (WO 95/2415), can also be used.
  • Any pharmaceutical composition of the invention, containing a polynucleotide, polypeptide, polypeptide derivative, or antibody of the invention, can be manufactured using standard methods.
  • the invention also includes methods in which gastroduodenal infections, such as Helicobacter infection, are treated by oral administration of a Helicobacter polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antisecretory agent, a bismuth salt, an antacid, sucralfate, or a combination thereof.
  • antibiotics including, e.g., macrolides, tetracyclines, ⁇ -lactams, aminoglycosides, quinolones, penicillins, and derivatives thereof
  • antibiotics include, e.g., amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin, cefuroxime, and erythromycin
  • antisecretory agents including, e.g., H 2 - receptor antagonists (e.g., cimetidine, ranitidine, famotidine, nizatidine and roxatidine), proton pump inhibitors (e.g., omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs (e.g., misoprostil and enprostil), and anticholinergic agents (e.g., pirenzepine
  • H 2 - receptor antagonists e.g., cimetidine, ranitidine, fa
  • compositions for carrying out these methods i.e., compositions containing a Helicobacter antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
  • Amounts of the above-listed compounds used in the methods and compositions of the invention can readily be determined by one skilled in the art.
  • one skilled in the art can readily design treatment/immunization schedules.
  • the non- vaccine components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.
  • a 76 kDa protein band containing GHPO 386, GHPO 789, and GHPO 1516 (hereinafter the "purified 76 kDa proteins"), GHPO 1360,- and GHPO 750 were purified from Helicobacter pylori strain ATCC number 43579 (American Type Culture Collection, Rockville, Maryland) by immunoaffinity- based chromatography using the methods described below in Example 1, and were shown to be effective vaccine antigens as follows.
  • the pellet (Cl) is suspended in 1% N-octyl-D-glucopyranoside (NOG; 30 ml/L; Sigma). The bacterial suspension is incubated for 1 hour at room temperature while stirring, spun in a centrifuge at 17,600 x g for 30 minutes at 4°C, and the pellet (C2) is recovered.
  • NOG N-octyl-D-glucopyranoside
  • Pefabloc (Buffer A), and is homogenized with an ultra-turrax (3821, Janke and Kungel). Lysozyme and EDTA are added at 0.1 mg/ml and 1 mM, respectively.
  • Each pellet is resuspended in 2 ml of 10 mM NaP0 4 (pH 7.0) containing 1 M NaCl, 0.1% Sarkosyl, 100 ⁇ M PMSF, and 6 M urea (buffer D).
  • the solubilized sample is dialyzed, in order, against 100 ml buffer D containing 4 M urea, 100 ml buffer D containing 2 M urea and 0.5% Sarkosyl, and twice against 100 ml buffer D that does not contain urea or Sarkosyl.
  • the dialyses are earned out for 1 hour each while stirring at room temperature.
  • the last dialysate is incubated for 30 minutes in an ice bath, and then is spun in a centrifuge at low speed for 10 minutes at 4°C.
  • the supernatant is recovered, filtered through a Millipore filter (0.45 ⁇ m), and stored at -20°C.
  • the hyperimmune serum prepared as described above is applied to a Protein A Sepharose Fast Flow column (Pharmacia) that is equilibrated with
  • the 76 kDa protein is adsorbed and eluted as follows.
  • the membrane fraction Cs2d is suspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 ⁇ m membrane.
  • the supernatant is applied to the column, which is equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour.
  • the column is washed with 20 column volumes of 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and then with 2 to 6 volumes 10 mM phosphate buffer (pH 6.8).
  • the supernatant is dialyzed against 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 ⁇ m membrane.
  • the filtered supernatant is applied to the column, which is equilibrated with 50 mM Tris- HCL (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour.
  • the column is washed with 20 column volumes of 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then with 2 to 6 volumes of 10 mM phosphate buffer (pH 6.8).
  • the antigen is eluted with 100 mM glycine buffer (pH 2.5).
  • the eluate is collected in 3 ml fractions, to each of which is added 150 ⁇ l 1 M phosphate buffer (pH 8.0).
  • the optical density of each fraction is measured at 280 nm, and fractions containing the 50 or 32 kDa protein are pooled and stored at -70°C.
  • One hundred and forty mg of protein from the membrane fraction Cs2d resuspended in buffer B are applied to the column.
  • the column is washed with 0.1 M NaCl in buffer B, and then a 0.1-0.5 M NaCl gradient is applied to the column.
  • the fraction eluted between 0.35 and 0.45 M NaCl is further purified on a 10 ml S-Sepharose column (diameter: 1.5 cm; height: 5 cm; up to 10 mg protein/ml of gel), which is prepared according to the manufacturer's instructions (Pharmacia).
  • the fraction obtained is dialyzed against 50 mM acetate (pH 5.0) containing 100 ⁇ M Pefabloc and 0.1% Zwittergent 3-14, and then is applied to the column, which is equilibrated with the acetate buffer.
  • Membrane fraction C4 is solubilized in 50 mM NaC0 3 buffer (pH 9.5) at room temperature for 30 minutes under stirring. The suspension is then centrifuged at 200,000 x g for 30 minutes at 4°C. This allows the 32 and 35 kDa proteins to be separated, since the 35 kDa protein is insoluble in the NaC0 3 buffer. The supernatant is dialyzed against 50 mM NaP0 4 buffer (pH 7.0), and then is applied to an SP-Sepharose column, which is equilibrated with the NaP0 4 buffer. The column is washed with the NaP0 4 buffer, and then an 0-0.5 M
  • the 50 kDa protein can also be purified as follows.
  • a 40 ml Q-Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia), washed, and equilibrated with buffer B (pH 9.5) (50 mM NaC0 3 , 100 ⁇ M Pefabloc, and
  • the chromatography is monitored by UV detection at 280 nm at the column exit.
  • One hundred and forty mg of protein solubilized as is described above are applied to the column, which is then washed with buffer B until the absorbance at 280 nm is stabilized.
  • the proteins are eluted with a 0.1-
  • Fraction 9 which conesponds to the beginning of the washing at 1 M NaCl and contains acidic proteins, is further purified as follows.
  • a 10 ml DEAE Sepharose column (diameter: 1.5 cm, height: 5 cm) is prepared according to the manufacturer's instructions (Pharmacia) (up to 10 mg protein/ml of gel). The column is washed and equilibrated with buffer B. Chromatography is monitored as is described above. Fraction 9 is dialyzed against buffer B and contains about 10 mg protein.
  • Fraction 9 is applied to the DEAE-Sepharose column. The column is washed with buffer B until the absorbance at 280 nm is stabilized. The proteins are eluted with a 0-0.5 M NaCl gradient in buffer B (10 fold V ⁇ ), followed by washing in buffer B, containing 1 M NaCl (2 fold V ⁇ ). Fractions are recovered and analyzed by SDS-PAGE. The 50 kDa protein is found in the fractions eluted at 0.3-0.4 M NaCl.
  • EXAMPLE 2 Identification of genes in the H. pylori genome, such as genes encoding the 76 kDa proteins, the 32 kDa protein (GHPO 1360), and the 50 kDa protein (GHPO 750) identification of signal sequences, and primer design for amplification of genes lacking signal sequences
  • the H. pylori genome was provided as a text file containing a single contiguous string of nucleotides that had been determined to be 1.76 Megabases in length.
  • the complete genome was split into 17 separate files using the program SPLIT (Creativity in Action), giving rise to 16 contigs, each containing 100,000 nucleotides, and a 17 th contig containing the remaining 76,000 nucleotides.
  • a header was added to each of the 17 files using the format: >hpg0.txt (representing contig 1), .hpgl .txt (representing contig 2), etc.
  • ORFs open reading frames
  • FASTA Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988.
  • FASTA was used for searching either a DNA sequence against either of the gene databases (" ⁇ " and/or "N"), or a peptide sequence against the ORF library ("O").
  • TFASTX was used to search a peptide sequence against all possible reading frames of a DNA database (" ⁇ " and/or "N” libraries).
  • ⁇ " and/or "N” libraries - Potential frameshifts also being resolved, FASTX was used for searching the translated reading frames of a DNA sequence against either a DNA database, or a peptide sequence against the protein database.
  • the deduced protein encoded by a target gene sequence is analyzed using the PROTEAN software package (DNAStar, Inc.). This analysis predicts those areas of the protein that are hydrophobic by using the Kyte-Doolittle algorithm, and identifies any potential polar residues preceding the hydrophobic core region, which is typical for many signal sequences. For confirmation, the target protein is then searched against a PROSITE database (DNAStar, Inc.) consisting of motifs and signatures. Characteristic of many signal sequences and hydrophobic regions in general, is the identification of predicted prokaryotic lipid attachment sites. Where confirmation between the two approaches is apparent at the N-terminus of any protein, putative cleavage sites are sought.
  • this includes the presence of either an Alanine (A), Serine (S), or Glycine (G) residue immediately after the core hydrophobic region.
  • A Alanine
  • S Serine
  • G Glycine
  • C Cysteine
  • Helicobacter pylori strain ORV2001 stored in LB medium containing 50%) glycerol at -70°C, is grown on Colombia agar containing 7% sheep blood for 48 hours under microaerophilic conditions (8-10% C0 2 , 5-7% 0 2 , and 85- 87% N 2 ). Cells are harvested, washed with PBS (pH 7.2), and DNA is then extracted from the cells using the Rapid Prep Genomic DNA Isolation kit (Pharmacia Biotech). 3.B. PCR amplification
  • N-terminal primer 5'-CTGAATTCGATTTCAAGGAGAAAACATGAAA-3' (SEQ ID NO:59); and C-terminal primer:
  • N-terminal primer 5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID NO:61);
  • C-terminal primer 5'-CCGCTCGAGTTAAGTAAGCGAACACATATTCAA-3' (SEQ ID NO:57).
  • GHPO 896 N-terminal primer:
  • GHPO 1360 5'-CCGCTCGAGTTAGTAAGCAAACACATAATTGTG-3' (SEQ ID NO: 64).
  • N-terminal primer 5'-CGCGGATCCGAATGAAAAAAAATATCTTAAAT-3' (SEQ ID NO:69);
  • GHPO 750 5*-CCGCTCGAGTTACTTGTTGATAACAATTTT-3' (SEQ ID NO:70).
  • C-terminal primer 5'-CCGCTCGAGTTATTCAATAATATTGCTCAC-3* (SEQ ID NO:72).
  • GHPO 711 N-terminal primer:
  • C-terminal primer 5'-CCCCTCGAGTTAATAGGCAAACAC-3' (SEQ ID NO:84).
  • the N-terminal and C-terminal primers for each clone both include a 5' clamp and a restriction enzyme recognition sequence for cloning pu ⁇ oses (BamHl (GGATCC) andXhol (CTCGAG) recognition sequences).
  • Amplification of gene-specific DNA is carried out using a heat-stable DNA Polymerase (e.g., Thermalase DNA Polymerase (Amresco)) according to the manufacturer's instructions.
  • the reaction mixture which is brought to a final volume of 100 ⁇ l with distilled water, is as follows: dNTPs mix 200 ⁇ M
  • amplification reaction conditions can readily be determined by one skilled in the art. In the present case, the following conditions were used.
  • GHPO 386 and GHPO 789 in a reaction containing Taq DNA polymerase (Appligene), a denaturing step was canied out at 95 °C for 30 seconds, followed by an annealing step at 50 °C for one minute, and an extension step at 72 °C for 2 minutes and 30 seconds. Twenty five cycles were canied out.
  • GHPO 896 in a reaction containing Taq DNA polymerase, a denaturing step was carried out at 97 °C for 30 seconds, followed by an annealing step at 50°C for one minute, and an extension step at 72°C for 2 minutes and 30 seconds.
  • GHPO 1516 was clone GHPO 1516, instead of Taq DNA polymerase, and the annealing temperature was 55 °C.
  • Vent DNA polymerase was used for clone GHPO 1516, instead of Taq DNA polymerase, and the annealing temperature was 55 °C.
  • GHPO 1360 and GHPO 750 Thermalase DNA polymerase was used for GHPO 1360 and GHPO 750.
  • a denaturing step was canied out at 95 °C for 30 seconds, followed by an annealing step at 55 °C for one minute, and an extension step at 72 °C for 2 minutes. Thirty cycles were carried out.
  • GHPO 711 Vent DNA polymerase was used for GHPO 711
  • the ligation reaction (20 ⁇ l) is canied out at 14 °C overnight and then is used to transform 100 ⁇ l fresh E. coli XL 1 -blue competent cells (Novagen).
  • the cells are incubated on ice for 2 hours, then heat-shocked at 42 °C for 30 seconds, and returned to ice for 90 seconds.
  • the samples are then added to 1 ml LB broth in the absence of selection and grown at 37 °C for 2 hours.
  • the cells are then plated out on LB agar containing kanamycin (50 ⁇ g/ml) at a lOx and neat dilution and incubated overnight at 37 °C. The following day, 50 colonies are picked onto secondary plates and incubated at 37 °C overnight.
  • PCR is performed with the gene-specific primers under the conditions stated above and transformant DNA is confirmed to contain the desired insert. If PCR-positive, one of the five plasmid DNA samples (500 ng) extracted from the E. coli XL 1 -blue cells is used to transform competent BL21 ( ⁇ DE3) E. coli competent cells (Novagen; as described previously). Transformants (10) are picked onto selective kanamycin (50 ⁇ g/ml) containing LB agar plates and stored as a research stock in LB containing 50% glycerol.
  • One ml of frozen glycerol stock prepared as described in 3. C. is used to inoculate 50 ml of LB medium containing 25 ⁇ g/ml of kanamycin in a 250 ml Erlenmeyer flask.
  • the flask is incubated at 37°C for 2 hours or until the absorbance at 600 nm (OD 600 ) reaches 0.4-1.0.
  • the culture is stopped from growing by placing the flask at 4°C overnight.
  • 10 ml of the overnight culture are used to inoculate 240 ml LB medium containing kanamycin (25 ⁇ g/ml), with the initial OD 600 about 0.02-0.04.
  • Four flasks are inoculated for each ORF.
  • the cells are grown to an OD 600 of 1.0 (about 2 hours at 37°C), a 1 ml sample is harvested by centrifugation, and the sample is analyzed by SDS- PAGE to detect any leaky expression.
  • the remaining culture is induced with 1 mM IPTG and the induced cultures are grown for an additional 2 hours at 37°C.
  • the final OD 600 is taken and the cells are harvested by centrifugation at 5,000 x g for 15 minutes at 4°C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Two hundred and fifty ml of buffer are used for a 1 L culture and the cells are recovered by centrifugation at 12,000 x g for 20 minutes. The supernatant is discarded and the pellets are stored at -45°C.
  • Pellets obtained from 3.D. are thawed and resuspended in 95 ml of 50 mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to final concentrations of 100 ⁇ M and 100 ⁇ g/ml, respectively.
  • the mixture is homogenized with magnetic stining at 5°C for 30 minutes.
  • Benzonase (Merck) is added at a 1 U/ml final concentration, in the presence of 10 mM MgCl2, to ensure total digestion of the DNA.
  • the suspension is sonicated (Branson Sonifier 450) for 3 cycles of 2 minutes each at maximum output.
  • the target protein is produced in a soluble form (i.e., in the supernatant obtained in 3.E.)
  • NaCl and imidazole are added to the supernatant to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 10 mM imidazole (buffer A).
  • the mixture is filtered through a 0.45 ⁇ m membrane and loaded onto an IMAC column (Pharmacia HiTrap chelating Sepharose; 1 ml) that has been charged with nickel ions according to the manufacturer's recommendations.
  • the column is washed with 50 column volumes of buffer A and the recombinant target protein is eluted with 5 ml of buffer B (50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM imidazole).
  • buffer B 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM imidazole.
  • the elution profile is monitored by measuring the absorbance of the fractions at 280 nm. Fractions conesponding to the protein peak are pooled, dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 ⁇ m membrane, and stored at -45°C. 3.E.2. Insoluble fraction If the target protein is expressed in the insoluble fraction (pellets obtained from 3.E.), purification is conducted under denaturing conditions.
  • NaCl, imidazole, and urea are added to the resuspended pellet to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10 mM imidazole, and 6 M urea (buffer C). After complete solubilization, the mixture is filtered through a 0.45 ⁇ m membrane and loaded onto an IMAC column.
  • the purification procedures on the IMAC column are the same as described in 3.E.I ., except that 6 M urea is included in all buffers used and 10 column volumes of buffer C are used to wash the column after protein loading, instead of 50 column volumes.
  • the protein fractions eluted from the IMAC column with buffer D (buffer C containing 500 mM imidazole) are pooled.
  • Arginine is added to the solution to final concentration of 0.5 M and the mixture is dialyzed against PBS containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1 M, and 0.5 M) to progressively decrease the concentration of urea.
  • the final dialysate is filtered through a 0.22 ⁇ m membrane and stored at -45°C.
  • a first alternative involves the use of a mild denaturant, N-octyl glucoside (NOG). Briefly, a pellet obtained in 3.E. is homogenized in 5 mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCl (pH 7.9) by microfluidization at a pressure of 15,000 psi and is clarified by centrifugation at 4,000-5,000 x g. The pellet is recovered, resuspended in 50 mM NaP0 4 (pH 7.5) containing 1-2% weight
  • NOG N-octyl glucoside
  • the NOG-soluble impurities are removed by centrifugation.
  • the pellet is extracted once more by repeating the preceding extraction step.
  • the pellet is dissolved in 8 M urea, 50 mM Tris (pH 8.0).
  • the urea-solubilized protein is diluted with an equal volume of 2 M arginine, 50 mM Tris (pH 8.0), and is dialyzed against 1 M arginine for 24-48 hours to remove the urea.
  • the final dialysate is filtered through a 0.22 ⁇ m membrane and stored at -45°C.
  • a second alternative involves the use of a strong denaturant, such as guanidine hydrochloride. Briefly, a pellet obtained in 3.E.
  • a protection test is described above that was canied out for testing the protective activity of the purified, native proteins. This test can also be used for testing the protective efficacy of recombinant proteins. Alternatively, the following test can be used.
  • New Zealand rabbits are injected both subcutaneously and intramuscularly with 100 ⁇ g of a purified fusion polypeptide, as obtained in 3.E.I . or 3.E.2., in the presence of Freund's complete adjuvant and in a total volume of approximately 2 ml. Twenty one and 42 days after the initial injection, booster doses, which are identical to priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Fifteen days after the last injection, animal serum is recovered, decomplemented, and filtered through a 0.45 ⁇ m membrane. 3.G.2. Mouse hyperimmune ascites fluid
  • mice are also injected intraperitoneally with sarcoma 180/TG cells CM26684 (Lennette et al, Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections , 5th Ed., Washington DC, American Public Health Association, 1979). Ascites fluid is collected 10-13 days after the last injection.
  • EXAMPLE 4 Methods for producing transcriptional fusions lacking His- tags Methods for amplification and cloning of DNA encoding the polypeptides of the invention as transcriptional fusions lacking His-tags are described as follows. Two PCR primers for each clone are designed based upon the sequences of the polynucleotides that encode them (SEQ ID NOs: 1-21 (odd numbers), 65, and 67). These primers can be used to amplify DNA encoding the polypeptides of the invention from any Helicobacter pylori strain, including, for example, ORV2001 and the H. pylori strain deposited with the American Type Culture Collection (ATCC, Rockville, Maryland) as ATCC number 43579, as well as from other Helicobacter species.
  • ATCC American Type Culture Collection
  • the N-terminal primers are designed to include the ribosome binding site of the target gene, the ATG start site, the signal sequence (if any), and the cleavage site.
  • the N-terminal primers can include a 5' clamp and restriction endonuclease recognition site, such as that for BamHl (GGATCC), which facilitates subsequent cloning.
  • the C-terminal primers can include a restriction endonuclease recognition site, such as that fo ⁇ Xhol (CTCGAG), which can be used in subsequent cloning, and a TAA stop codon. Specific primers that can be used are listed above.
  • Amplification of a genes encoding the polypeptides of the invention can be canied out using Vent DNA polymerase (New England Biolabs) or Taq DNA polymerase (Appligene) under the conditions described above in Example 3.
  • Vent DNA polymerase New England Biolabs
  • Taq DNA polymerase Appligene
  • Thermalase DNA polymerase or Pwo DNA polymerase can be used, according to instructions provided by the manufacturers.
  • a single PCR product for each clone is amplified and can be cloned into BamHl-XhoI cleaved pET24, resulting in construction of transcriptional fusions that permit expression of the proteins without ⁇ is-tags.
  • the expressed products can be purified as denatured proteins that are refolded by dialysis into 1 M arginine.
  • Cloning into pET 24 allows transcription of genes from the T7 promoter, which is supplied by the vector, but relies upon binding of the RNA-specific DNA polymerase to the intrinsic ribosome binding site of the genes, and thereby expression of the complete ORF.
  • the amplification, digestion, and cloning protocols are as described above for constructing translational fusions.
  • An immune serum as prepared as is described in section 3.G., is applied to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100 mM Tris-HCl (pH 8.0). The resin is washed by applying 10 column volumes of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH 8.0) to the column. IgG antibodies are eluted with 0.1 M glycine buffer (pH 3.0) and are collected in 5 ml fractions to each of which is added 0.25 ml 1 M Tris-HCl (pH 8.0).
  • the optical density of the eluate is measured at 280 nm and the fractions containing the IgG antibodies are pooled, dialyzed against 50 mM Tris-HCl (pH 8.0), and, if necessary, stored frozen at -70°C.
  • CNBr-activated Sepharose 4B gel (1 g of dried gel provides for approximately 3.5 ml of hydrated gel; gel capacity is from 5 to 10 mg coupled IgG/ml of gel) manufactured by Pharmacia (17-0430- 01) is suspended in 1 mM HC1 buffer and washed using a buchner by adding small quantities of 1 mM HC1 buffer. The total volume of buffer is 200 ml per gram of gel.
  • Purified IgG antibodies are dialyzed for 4 hours at 20+5 °C against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The antibodies are then diluted in 500 mM phosphate buffer (pH 7.5) to a final concentration of 3 mg/ml.
  • IgG antibodies are mixed with the gel overnight at 5+3 °C.
  • the gel is packed into a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5), and 1 column volume of 50 mM sodium phosphate buffer, containing 500 mM NaCl (pH 7.5).
  • the gel is then transfened to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at room temperature, and washed twice with 2 column volumes of PBS.
  • the gel is then stored in 1/10,000 PBS/merthiolate.
  • the amount of IgG antibodies coupled to the gel is determined by measuring the optical density (OD) at 280 nm of the IgG solution and the direct eluate, plus washings.
  • OD optical density
  • the adsorbed gel is washed with 2 to 6 volumes of 10 mM sodium phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine buffer (pH 2.5).
  • the eluate is recovered in 3 ml fractions, to each of which is added 150 ⁇ l of 1 M sodium phosphate buffer (pH 8.0). Abso ⁇ tion is measured at 280 nm for each fraction; those fractions containing the antigen are pooled and stored at -20°C.
  • EXAMPLE 6 The GHPO 1360 polypeptide is useful as a serodiagnostic tool for H. pylori infection
  • H. pylori proteins The reactivity of patient sera against H. pylori proteins was analyzed by immunoblot technique. Briefly, total lysate of H. pylori strain ORV2001 was subjected to SDS-PAGE electrophoresis (BioRad protean II system) on a 12.5% gel. Proteins were electrotransfened onto a nitrocellulose paper for immunoblot assay. After blocking, the nitrocellulose paper was incubated with patient sera (1 :500 diluted in blocking buffer) for one hour at room temperature, washed, and further incubated with peroxidase-conjugated goat anti-human IgG. The positive bands were revealed by incubation with the appropriate substrates. The results showed that the H. y/or.
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • AAAC ATG AAA AAA CAC ATC CTT TCA TTA GCT TTA GGC TCG CTT TTA GTT 409
  • AGC ACG CAA ACA ACC GCG ACA ACC ACG CAA GAC GGC GTA ACG ATC ACC 1129 Ser Thr Gin Thr Thr Ala Thr Thr Thr Gin Asp Gly Val Thr He Thr 220 225 230 235
  • CTTTTTTA AACCCTCTTT TTTTAAGGGG TTTCTTTTTA AAGCTTTTTTTT GAAGTCTTTT 2859
  • MOLECULE TYPE Genomic DNA
  • FEATURE

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Abstract

L'invention porte sur des polypeptides d'Hélicobacter de 76 kDa, 32 kDa et 50 kDa pouvant être utilisés dans des méthodes de vaccination pour prévenir ou traiter les infections par l'Hélicobacter, et sur les polynucléotides codant pour eux. L'invention porte également sur des méthodes diagnostiques utilisant lesdits polypeptides.
EP98914395A 1997-04-01 1998-03-31 POLYPEPTIDES D'HELICOBACTER DE 76 kDa, 32 kDa ET 50 kDa ET MOLECULES DE POLYNUCLEOTIDES CORRESPONDANTES Withdrawn EP0980204A4 (fr)

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Application Number Priority Date Filing Date Title
US08/834,666 US20020044949A1 (en) 1997-04-01 1997-04-01 76 kda helicobacter polypeptides and corresponding polynucleotide molecules
US831310 1997-04-01
US834666 1997-04-01
US08/831,310 US20020026035A1 (en) 1997-04-01 1997-04-01 Helicobacter ghpo 1360 and ghpo 750 polypeptides and corresponding polynucleotide molecules
PCT/US1998/006421 WO1998043479A1 (fr) 1997-04-01 1998-03-31 POLYPEPTIDES D'HELICOBACTER DE 76 kDa, 32 kDa ET 50 kDa ET MOLECULES DE POLYNUCLEOTIDES CORRESPONDANTES

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DATABASE EMBL [Online] 25 August 1997 (1997-08-25), "Helicobacter pylori 26695 section 16 of 134 of the complete genome." XP002321192 retrieved from EBI accession no. EM_PRO:AE000538 Database accession no. AE000538 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE EMBL [Online] 25 August 1997 (1997-08-25), "Helicobacter pylori 26695 section 20 of 134 of the complete genome." XP002321189 retrieved from EBI accession no. EM_PRO:AE000542 Database accession no. AE000542 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE EMBL [Online] 25 August 1997 (1997-08-25), "Helicobacter pylori 26695 section 21 of 134 of the complete genome." XP002321188 retrieved from EBI accession no. EM_PRO:AE000543 Database accession no. AE000543 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE EMBL [Online] 25 August 1997 (1997-08-25), "Helicobacter pylori 26695 section 27 of 134 of the complete genome." XP002321179 retrieved from EBI accession no. EM_PRO:AE000549 Database accession no. AE000549 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE EMBL [Online] 25 August 1997 (1997-08-25), "Helicobacter pylori 26695 section 3 of 134 of the complete genome." XP002321181 retrieved from EBI accession no. EM_PRO:AE000525 Database accession no. AE000525 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE EMBL [Online] 25 August 1997 (1997-08-25), "Helicobacter pylori 26695 section 63 of 134 of the complete genome." XP002321187 retrieved from EBI accession no. EM_PRO:AE000585 Database accession no. AE000585 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE EMBL Entry AE000599 25 August 1997 (1997-08-25), TOMB, J.-F. ET AL.: "Helicobacter pylori 26695 section 77 of 134 of the complete genome" XP002305071 Database accession no. AE000599 & TOMB, J.-F. ET AL.: "The complete genome sequence of the gastric pathogen Helicobacter pylori" NATURE, vol. 388, 1997, pages 539-547, *
DATABASE UniProt [Online] 1 January 1998 (1998-01-01), "Outer membrane protein (Omp2)." XP002321180 retrieved from EBI accession no. UNIPROT:O24870_HELPY Database accession no. O24870 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt [Online] 1 January 1998 (1998-01-01), "Outer membrane protein (Omp27)." XP002321185 retrieved from EBI accession no. UNIPROT:O25791_HELPY Database accession no. O25791 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt [Online] 1 January 1998 (1998-01-01), "Outer membrane protein (Omp29) (Omp5)." XP002321184 retrieved from EBI accession no. UNIPROT:O34523_HELPY Database accession no. O34523 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt [Online] 1 January 1998 (1998-01-01), "Outer membrane protein (Omp6)." XP002321190 retrieved from EBI accession no. UNIPROT:O25015_HELPY Database accession no. O25015 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt [Online] 1 January 1998 (1998-01-01), "Outer membrane protein (Omp9)." XP002321178 retrieved from EBI accession no. UNIPROT:O25086_HELPY Database accession no. O25086 & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt [Online] 1 November 1995 (1995-11-01), "Elongation factor Tu (EF-Tu)." XP002321195 retrieved from EBI accession no. UNIPROT:EFTU_WOLSU Database accession no. EFTU_WOLSU & LUDWIG W ET AL: "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes" ANTONIE VAN LEEUWENHOEK, vol. 64, no. 3-4, 1993, pages 285-305, ISSN: 0003-6072 *
DATABASE UniProt [Online] 1 November 1997 (1997-11-01), "Elongation factor Tu (EF-Tu)." XP002321194 retrieved from EBI accession no. UNIPROT:EFTU_HELPY Database accession no. EFTU_HELPY & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt [Online] 1 November 1997 (1997-11-01), "Hypothetical protein HP0175 precursor." XP002321191 retrieved from EBI accession no. UNIPROT:Y175_HELPY Database accession no. Y175_HELPY & TOMB J-F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTIC PATHOGEN HELICOBACTER PYLORI" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 388, 7 August 1997 (1997-08-07), pages 539-547, XP002914020 ISSN: 0028-0836 *
DATABASE UniProt Entry O25556 1 January 1998 (1998-01-01), TOMB, J.-F. ET AL.: "Outer membrane protein (Omp19)" XP002305070 Database accession no. O25556 & TOMB, J.-F. ET AL.: "The complete genome sequence of the gastric pathogen Helicobacter pylori" NATURE, vol. 388, 1997, pages 539-547, *
DATABASE UniProt Entry O25840_HELPY 1 January 1998 (1998-01-01), TOMB, J.-F. ET AL.: "Outer membrane protein (Omp28)" XP002321176 Database accession no. O25840 & TOMB, J.-F. ET AL.: "The complete genome sequence of the gastric pathogen Helicobacter pylori" NATURE, vol. 388, 1997, pages 539-547, *
SCHMITT W ET AL: "GENETIC ANALYSIS OF THE HELICOBACTER PYLORI VACUOLATING CYTOTOXIN: STRUCTURAL SIMILARITIES WITH THE IGA PROTEASE TYPE OF EXPORTED PROTEIN" MOLECULAR MICROBIOLOGY, BLACKWELL SCIENTIFIC, OXFORD, GB, vol. 12, no. 2, 1 April 1994 (1994-04-01), pages 307-319, XP000605827 ISSN: 0950-382X *
See also references of WO9843479A1 *

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WO1998043479A1 (fr) 1998-10-08
CA2286893A1 (fr) 1998-10-08

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