EP1009764A1 - VACCINE COMPOSITIONS COMPRISING THE $i(HELICOBACTER PYLORI) FlgE POLYPEPTIDE - Google Patents

VACCINE COMPOSITIONS COMPRISING THE $i(HELICOBACTER PYLORI) FlgE POLYPEPTIDE

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Publication number
EP1009764A1
EP1009764A1 EP98928772A EP98928772A EP1009764A1 EP 1009764 A1 EP1009764 A1 EP 1009764A1 EP 98928772 A EP98928772 A EP 98928772A EP 98928772 A EP98928772 A EP 98928772A EP 1009764 A1 EP1009764 A1 EP 1009764A1
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EP
European Patent Office
Prior art keywords
helicobacter pylori
gly
ala
asn
ser
Prior art date
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EP98928772A
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German (de)
French (fr)
Inventor
Thomas Berglindh
Björn MELLG RD
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AstraZeneca AB
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AstraZeneca AB
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Publication of EP1009764A1 publication Critical patent/EP1009764A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)

Definitions

  • the present invention relates to polypeptides and vaccine compositions for inducing a protective immune response to Helicobacter pylori infection.
  • the invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment or prophylaxis of Helicobacter pylori infection.
  • H. pylori The gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen, involved in several gastroduodenal diseases. Colonization of gastric epithelium by the bacterium leads to active inflammation and progressive chronic gastritis, with a greatly enhanced risk of progression to peptic ulcer disease. A lifelong inflammation of the gastric mucosa is very closely correlated with a significantly enhanced risk for gastric cancer.
  • H. pylori In order to colonize the gastric mucosa, H. pylori uses a number of virulence factors. Such virulence factors comprise several adhesins, with which the bacterium associates with the mucus and/or binds to epithelial cells; urease which helps to neutralize the acid environment; and proteolytic enzymes which makes the mucus more fluid.
  • H. pylori is highly motile, swimming in the mucus and down into the crypts. Motility has been shown to be an essential virulence factor, since non motile H. pylori has failed to infect the mucosa in experimental models Eaton et al. (Infection & Immunity 64(7), 2445-2448, 1996). There are many possible reasons for this, the most obvious being an inability to swim down and attach to mucosal cells and the inability to avoid noxious agents in the stomach.
  • H. pylori Despite a strong apparent host immune response to H. pylori, with production of both local (mucosal) as well as systemic antibodies, the pathogen persists in the gastric mucosa, normally for the life of the host. The reason for this is probably that the spontaneously induced immune-responses are inadequate or directed towards the wrong epitopes of the antigens. Alternatively the immune response could be of the wrong kind, since the immune system might treat H. pylori as a commensal (as indicated from the life-time host/bacteria relationship).
  • the H. pylori cell transforms from a bacillary to a coccoid form.
  • the H. pylori cell is much less sensitive to antibiotics and other anti-bacterial agents.
  • Circumstantial evidence indicate the H. pylori might be transmitted between individuals in this form, possibly via water or direct contact (oral-oral; feacal-oral).
  • An efficient vaccine composition should therefore elicit an immune response towards both the coccoid and the bacillary form of H. pylori. Since systemic immunity probably only plays a limited role in protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach.
  • Flagellar hooks from H. pylori has been shown to be composed of FlgE subunits of 78 kDa (O'Toole et al. Molecular Microbiology, 14(4), 691-703, 1994).
  • the role of the flagellar hook is to connect the flagella with the submembraneous flagellar motor.
  • the part of the hook extruding outside the membrane is short, approximately 60 nanometers (compared to approximately 10 micrometers for the flagella).
  • the hook is probably covered with a sheet (Geis et al. (1993) J. Med. Microbiol. 38(5), 371-377).
  • the amino acid sequence of the FlgE polypeptide has significant resemblance with that of other known hook proteins, including limited homology to other
  • C FlgE specific. Specific FlgE IgG increased in animals given FlgE + CT, but could not be detected in control animals.
  • the purpose of this invention is to provide an antigenic H. pylori polypeptide which can be useful for eliciting a protective immune response against, and for diagnosis of, H. pylori infection.
  • This purpose has been achieved by the recombinant cloning of an H. pylori gene which encodes a well conserved essential polypeptide.
  • the nucleic acid sequence of this gene is similar to the sequence of the flgE gene as published by O'Toole et al, Molecular Microbiology, 14(4), 691- 703, 1994. Being an essential protein for motility, UxeflgE gene is expressed by all H. pylori strains.
  • the H. pylori FlgE polypeptide in spite of the facts that only a small part of the hook protein is existing outside bacteria and that it is probably covered by a sheet, can serve as a therapeutic antigen in an H. pylori infected mouse model, when given together with the adjuvant cholera toxin.
  • the experimental data below thus indicates that the H. pylori FlgE polypeptide, when used as an oral immunogen, acts as a stimulator of an immune response leading to a significant reduction of colonization of H. pylori in mice which were infected with H. pylori one month prior to immunization.
  • the H. pylori FlgE polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections.
  • the FlgE polypeptide will be useful both for the detection of H. pylori infections as well as for the manufacture of vaccine compositions, which when given in an appropriate pharmaceutical formulation will elicit a protective or therapeutic immune response against such infections.
  • the present invention provides a Helicobacter pylori FlgE polypeptide for use in inducing a protective immune response to Helicobacter pylori infection.
  • the term "Helicobacter pylori FlgE polypeptide” is intended to mean the polypeptide which is disclosed by O'Toole et al. in Molecular Microbiology, 14(4), 691-703, 1994, and which is encoded by the gene whose nucleotide sequence is set forth as SEQ ID NO: 1, or can be obtained from the National Center for Biotechnology Information (Accession number U09549), or a substantially similar modified form of the said polypeptide retaining functionally equivalent antigenicity.
  • protective immune response is to be understood as an immune response which makes the composition suitable for therapeutic and /or prophylactic purposes.
  • the term "functionally equivalent antigenicity” is to be understood as the ability to induce a systemic and mucosal immune response while decreasing the number of H. pylori cells associated with the gastric mucosa.
  • the skilled person will be able to identify modified forms of the FlgE polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies.
  • the Helicobacter pylori FlgE polypeptide for use in inducing a protective immune response to Helicobacter pylori infection, has substantially the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing, or is a modified form thereof retaining functionally equivalent antigenicity.
  • the definition of the Helicobacter pylori FlgE polypeptide is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of the Helicobacter pylori FlgE polypeptide and is retaining functionally equivalent antigenicity.
  • Helicobacter pylori FlgE polypeptide includes amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing.
  • the invention provides a vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori FlgE polypeptide as defined above, optionally together with a pharmaceutically acceptable carrier or diluent.
  • an immunologically effective amount is to be understood as an amount which elicits a significant protective Helicobacter pylori response, which will eradicate a H. pylori infection in an infected mammal or prevent the infection in a susceptible mammal.
  • an immunologically effective amount will comprise approximately 1 ⁇ g to 1000 mg, preferably approximately 10 ⁇ g to 100 mg, of H. pylori antigen for oral administration, or approximately less than 100 ⁇ g for parenteral administration.
  • the vaccine composition comprises optionally in addition to a pharmaceutically acceptable carrier or diluent one or more other immunologically active antigens for prophylactic or therapeutic use.
  • Physiologically acceptable carriers and diluents are well known to those skilled in the art and include e.g. phosphate buffered saline (PBS), or, in the case of oral vaccines, HCO3" based formulations or enterically coated powder formulations.
  • the vaccine composition can optionally include or be administered together with acid secretion inhibitors, preferably proton pump inhibitors (PPIs), e.g. omeprazole.
  • PPIs proton pump inhibitors
  • the vaccine can be formulated in known delivery systems such as liposomes, ISCOMs, cochleates, etc. (see e.g. Rabinovich et al. (1994) Science 265, 1401-1404) or be attached to or incorporated into polymer microspheres of degradable or non-degradable nature.
  • the antigens could be associated with live attenuated bacteria, viruses or phages or with killed vectors of the same kind.
  • the antigens can be chemically or genetically coupled to carrier proteins of inert or adjuvantic types (i.e Cholera B subunit).
  • the invention provides in a further aspect a vaccine composition according to above, in addition comprising an adjuvant, such as a cholera toxin.
  • an adjuvant such as a cholera toxin.
  • cholera toxin are known in the art, e.g. from Rappuoli et al. (1995) Int. Arch. Allergy & Immunol. 108(4), 327-333; and Dickinson et al. (1995) Infection and Immunity 63(5), 1617-1623.
  • a vaccine composition according to the invention can be used for both therapeutic and prophylactic purposes. Consequently, the invention includes a vaccine composition according as defined above, for use as a therapeutic or a prophylactic vaccine in a mammal, including man, which is infected by Helicobacter pylori.
  • the term "prophylactic purpose” means to induce an immune response which will protect against future infection by Helicobacter pylori
  • therapeutic purpose means to induce an immune response which can eradicate an existing Helicobacter pylori infections.
  • the vaccine composition according to the invention is preferably administered to any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the gastric, the intestinal (small and large intestine), the rectal and the vaginal mucosa.
  • the mucosal vaccines can be given together with for the purpose appropriate adjuvants.
  • the vaccine can also be given orally, or parenterally, by the subcutaneous, intracutaneous or intramuscular route, optionally together with the appropriate adjuvant.
  • the vaccine composition can optionally be given together with antimicrobial therapeutic agents.
  • the invention proivides the use of a Helicobacter pylori FlgE polypeptide, as defined above, in the manufacture of
  • compositions for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection (i) a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection;
  • the invention provides a method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori FlgE polypeptide as defined above, optionally labelled or coupled to a solid support, is used.
  • the said method could e.g. comprise the steps (a) contacting a said Helicobacter pylori FlgE polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and (b) detecting antibodies from the said body fluid binding to the said FlgE polypeptide.
  • Preferred methods of detecting antibodies are ELISA (Enzyme linked immunoabsorbent assay) methods which are well known in the art.
  • the invention provides a diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method of in vitro diagnosis as described above to be carried out.
  • the said diagnostic kit could e.g. comprise: (a) a Helicobacter pylori FlgE polypeptide; and (b) reagents for detecting antibodies binding to the said FlgE polypeptide.
  • the said reagents for detecting antibodies could e.g. be an enzyme- labelled anti-immunoglobulin and a chromogenic substrate for the said enzyme.
  • the invention provides a method of eliciting in a mammal, including humans, a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a Helicobacter pylori FlgE polypeptide as defined above, or alternatively administering to the said mammal an immunologically effective amount of a vaccine composition as employeed above.
  • Standard protocols and “standard procedures”, when used in the context of molecular cloning techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al, John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T, Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989. Preparation of recombinant Helicobacter pylori FlgE polypeptide
  • Sequence information for the gene encoding for the FlgE polypeptide was obtained from the National Center for Biotechnology Information (Accession number U09549; SEQ ID NO: 1).
  • Sequences were cloned from the J99 strain of H. pylori by amplification cloning using the polymerase chain reaction (PCR).
  • Synthetic oligonucleotide primers (see below) specific for the 5'- and 3'-ends of open reading frames of genes were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA).
  • Forward primers (specific for the 5'-end of the sequence) for FlgE were designed to include an Ncol cloning site at the extreme 5'-terminus, while reverse primers included a EcoRI site at the extreme 5'-terminus to permit cloning of each H. pylori sequence into the reading frame of the pET28b vector.
  • Inserts cloned into the Ncol- EcoRI sites of the pET-28b vector are fused to a vector DNA sequence encoding an additional 20 carboxy-terminal amino including six histidine residues (at the extreme C-terminus).
  • Reverse primer (SEQ ID NO: 4):
  • Genomic DNA prepared from the J99 strain of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994).
  • genomic DNA 50 ng was introduced into a reaction vial containing 2 mM MgCl2, 1 ⁇ M synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H.
  • pylori ORF 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP, and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 ⁇ l.
  • thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Per kin Elmer Cetus/ Gene Amp PCR System 9600 thermal cycler: Denaturation at +94°C for 2 min; 2 cycles at +94°C for 15 sec, +30°C for 15 sec and +72°C for 1.5 min; 23 cycles at +94°C for 15 sec, +58°C for 15 sec and +72°C for 1.5 min; Reactions were concluded at +72°C for 6 minutes.
  • each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). Amplified DNA samples were subjected to digestion with the restriction endonucleases Ndel and EcoRI according to standard procedures. DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave UN irradiation. D ⁇ A contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA). Cloning ofH. pylori DNA sequences into the pET-28b prokaryotic expression vector.
  • the pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI according to standard procedures. Following digestion, DNA inserts were cloned according to standard procedures into the previously digested pET-28b expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli as described below.
  • Competent bacteria E. coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods. Briefly, 1 ⁇ l of ligation reaction was mixed with 50 ⁇ l of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgSO 4 and 20, mM glucose) at +37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 ⁇ g/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below.
  • the pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5 ⁇ , etc. for the purpose of cloning or plasmid preparation.
  • Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase.
  • T7 RNA polymerase is induced by addition of isopropyl- ⁇ -D- thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest.
  • Strains used in our laboratory include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Methods Enzymol. 185, 60-89).
  • H. pylori sequences 50 ng of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 ⁇ g/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 ⁇ g/ml) to an optical density at 600 nm of 0.5 to 1.0 O.D. units, at which point, 1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
  • the concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.H., Rosebrough,N., Farr, A.L. & Randall, R.J. (1951) , using bovine serum albumin as a standard.
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gels (12% or 4 to 25 % gradient acrylamide) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie Brilliant Blue. Molecular mass markers included rabbit skeletal muscle myosin (200 kDa), E.
  • the pellets were first washed with lysis buffer containing 10% glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1% ⁇ -mercaptoethanol, then with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1% ⁇ -mercaptoethanol.
  • the resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials.
  • Inclusion bodies were dissolved in 20 ml 8 M urea in lysis buffer with 1 mM PMSF and 0.1% ⁇ - mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation (100,000 x g for 30 min) . The clear supernatant was filtered and loaded onto a Ni 2+ -NTA agarose column equilibrated in 8 M urea in lysis buffer.
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1 mM PMSF and 0.1% ⁇ - mercaptoethanol, and developed with sequential steps of lysis buffer containing 8 M urea, 1 mM PMSF, 0.1% ⁇ -mercaptoethanol and 20, 100, 200, and 500 mM imidazole.
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding centre (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
  • H. pylori strain 244, originally isolated from an ulcer patient. This strain has earlier proven to be a good colonizer of the mouse stomach. Bacteria from a stock kept at -70°C were grown overnight in Brucella broth supplemented with 10% fetal calf serum, at +37°C in a microaerophilic atmosphere (10% CO 2 , 5% O 2 ). The animals were given an oral dose of omeprazole (400 ⁇ mol/kg) and after 3-5 h an oral inoculation of H. pylori (approximately 10 7 -10 8 CFU/ animal). Infection was checked in control animals 2-3 weeks after the inoculation. 1.3. Immunizations
  • mice One month after infection, two groups of mice (10 mice/group) were immunized 4 times over a 34 day period (day 1, 15, 25 and 35). Purified recombinant FlgE dissolved in PBS plus 0.5% Deoxycholate (DOC) was given at a dose of 100 microgram / mouse.
  • DOC Deoxycholate
  • the animals in both the control as well as the FlgE group were also given 10 ⁇ g/mouse of cholera toxin (CT) with each immunization.
  • Omeprazole 400 ⁇ mol/kg was given orally to all animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Animals were sacrificed 1-2 weeks after final immunization.
  • Group 1 300 ⁇ l PBS with 0.5% DOC containing 10 ⁇ g CT
  • Group 2 300 ⁇ l PBS with 0.5% DOC containing 100 ⁇ g FlgE and 10 ⁇ g CT.
  • mice were sacrificed by CO2 and cervical dislocation. The abdomen and chest cavity was opened and blood sampled by heart puncture. Subsequently the stomach was removed. After cutting the stomach along the greater curvature, it was rinsed in saline and subsequently cut into two identical pieces. An area of 25 mm 2 of the mucosa from the antrum and corpus was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth, diluted and plated onto Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining. 1.5. Antibody measurements
  • Serum antibodies were collected from blood. Prior to centrifugation, the blood was diluted with equal amount of PBS. The serum was kept at -20°C until analysis. Serum antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with FlgE followed by addition of different dilutions of serum. The ELISA was developed with alkaline phosphatase-labelled anti-mouse-Ig-antibodies. The anti-Ig antibodies were of an anti-heavy /anti-light chain type, which should detect all types of antibodies.
  • mice in this study were infected with H. pylori strain 244 one month prior to immunizations. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant FlgE polypeptide. Four weeks after the final immunization, the animals were sacrificed and CFU was determined (Fig. 1). The animals treated with CT alone, were highly infected both in corpus and antrum.
  • CT cholera toxin
  • ORGANISM Helicobacter pylori
  • CTTTAAATTA AAGGATAACC ATG CTT AGG TCT TTA TGG TCT GGT GTC AAT 350
  • AGC TTA GGC ACT AAA CAC GCC ATG CGC ATT GAG TTT TAT AGG AGT GGG 1838 Ser Leu Gly Thr Lys His Ala Met Arg He Glu Phe Tyr Arg Ser Gly 495 500 505 GGA GCG GAT TGG AAT TTT AGA GTG ATC GTG CCT GAG CCT GGG GAA TTA 1886 Gly Ala Asp Trp Asn Phe Arg Val He Val Pro Glu Pro Gly Glu Leu 510 515 520
  • CAA TTT GAC CCT AAA AAT GGT GCT GAT GCC CCC CAA CGC ATC AAT TTA 2030 Gin Phe Asp Pro Lys Asn Gly Ala Asp Ala Pro Gin Arg He Asn Leu 555 560 565 570

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Abstract

The present invention relates to polypeptides and vaccine compositions for inducing a protective immune response to Helicobacter pylori infection. The invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment or prophylaxis of Helicobacter pylori infection.

Description

VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI FlgE POLYPEPTIDE
TECHNICAL HELD
The present invention relates to polypeptides and vaccine compositions for inducing a protective immune response to Helicobacter pylori infection. The invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment or prophylaxis of Helicobacter pylori infection.
BACKGROUND ART
Helicobacter pylori
The gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen, involved in several gastroduodenal diseases. Colonization of gastric epithelium by the bacterium leads to active inflammation and progressive chronic gastritis, with a greatly enhanced risk of progression to peptic ulcer disease. A lifelong inflammation of the gastric mucosa is very closely correlated with a significantly enhanced risk for gastric cancer.
In order to colonize the gastric mucosa, H. pylori uses a number of virulence factors. Such virulence factors comprise several adhesins, with which the bacterium associates with the mucus and/or binds to epithelial cells; urease which helps to neutralize the acid environment; and proteolytic enzymes which makes the mucus more fluid. In addition H. pylori is highly motile, swimming in the mucus and down into the crypts. Motility has been shown to be an essential virulence factor, since non motile H. pylori has failed to infect the mucosa in experimental models Eaton et al. (Infection & Immunity 64(7), 2445-2448, 1996). There are many possible reasons for this, the most obvious being an inability to swim down and attach to mucosal cells and the inability to avoid noxious agents in the stomach.
Despite a strong apparent host immune response to H. pylori, with production of both local (mucosal) as well as systemic antibodies, the pathogen persists in the gastric mucosa, normally for the life of the host. The reason for this is probably that the spontaneously induced immune-responses are inadequate or directed towards the wrong epitopes of the antigens. Alternatively the immune response could be of the wrong kind, since the immune system might treat H. pylori as a commensal (as indicated from the life-time host/bacteria relationship).
In order to understand the pathogenesis and immunology of H. pylori infections, it is of great importance to define the antigenic structure of this bacterium. In particular, there is a need for characterization of surface-exposed, surface associated as well as secreted proteins which, in many bacterial pathogens, have been shown to constitute the main virulence factors, and which can be useful for the diagnosis of H. pylori and in the manufacture of vaccine compositions. If such proteins in addition to being surface associated also are essential for survival and /or colonization their usefulness as a target for vaccine mediated immunotherapy targets increase.
Whenever stressed or threatened, the H. pylori cell transforms from a bacillary to a coccoid form. In the coccoid form, the H. pylori cell is much less sensitive to antibiotics and other anti-bacterial agents. Circumstantial evidence indicate the H. pylori might be transmitted between individuals in this form, possibly via water or direct contact (oral-oral; feacal-oral). An efficient vaccine composition should therefore elicit an immune response towards both the coccoid and the bacillary form of H. pylori. Since systemic immunity probably only plays a limited role in protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach.
Flagellar Hook protein
Flagellar hooks from H. pylori has been shown to be composed of FlgE subunits of 78 kDa (O'Toole et al. Molecular Microbiology, 14(4), 691-703, 1994). The role of the flagellar hook is to connect the flagella with the submembraneous flagellar motor. The part of the hook extruding outside the membrane is short, approximately 60 nanometers (compared to approximately 10 micrometers for the flagella). Like the fagellum of H. pylori the hook is probably covered with a sheet (Geis et al. (1993) J. Med. Microbiol. 38(5), 371-377).
The amino acid sequence of the FlgE polypeptide has significant resemblance with that of other known hook proteins, including limited homology to other
Helicobacter species like mustelae (O'Toole et al., supra). Polyclonal antibodies raised against the FlgE polypeptide showed cross-reactivity against flagellar proteins A and B, possibly indicating the existence of shared epitopes. Production of FlgE knockout H. pylori, resulted in an aflagellar, non-motile bacteria, where FlgE polypeptide still was produced but could only be recovered in the cytoplasm.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1:
Effect of therapeutic immunization of H. pylori infected mice (n=9-10/group) with FlgE polypeptide. Results are given as mean±SEM of number of H. pylori associated with antrum (=A), corpus (=B) or totally (A+C) (=C). Abbreviations: CFU, colony forming units (number of bacteria); unshaded bars=DOC + CT, Phosphate buffered saline with 0.5% deoxycholate given together with cholera toxin 10 μg/mouse; shaded bars=FlgE + CT, mice given 100 μg FlgE and 10 μg cholera toxin. The decrease in cfu was significant in the antrum and as calculated for the whole stomach. ** p<0.01; * p<0.05 (Wilcoxon-Mann-Whittney sign rank test).
Fig. 2:
Serum IgG from mice measured by ELISA technique: response to infection and to immunisation with FlgE. The values are expressed as mean titers ± SEM. n=9- 10/group. ELISA coated with H. pylori strain 244: As a sign of infection H. pylori specific antibodies can be found in serum in animals treated with DOC + CT (=A. Control/244). Following immunization with FlgE + cholera toxin (=B. FlgE/244) this reactivity increased 4 fold (** p<0.01; Wilcoxon-Mann-Whittney sign rank test). C=FlgE specific. Specific FlgE IgG increased in animals given FlgE + CT, but could not be detected in control animals.
DISCLOSURE OF TFIE INVENTION
The purpose of this invention is to provide an antigenic H. pylori polypeptide which can be useful for eliciting a protective immune response against, and for diagnosis of, H. pylori infection. This purpose has been achieved by the recombinant cloning of an H. pylori gene which encodes a well conserved essential polypeptide. The nucleic acid sequence of this gene is similar to the sequence of the flgE gene as published by O'Toole et al, Molecular Microbiology, 14(4), 691- 703, 1994. Being an essential protein for motility, UxeflgE gene is expressed by all H. pylori strains.
It has surprisingly been found that the H. pylori FlgE polypeptide, in spite of the facts that only a small part of the hook protein is existing outside bacteria and that it is probably covered by a sheet, can serve as a therapeutic antigen in an H. pylori infected mouse model, when given together with the adjuvant cholera toxin. The experimental data below thus indicates that the H. pylori FlgE polypeptide, when used as an oral immunogen, acts as a stimulator of an immune response leading to a significant reduction of colonization of H. pylori in mice which were infected with H. pylori one month prior to immunization.
These results strongly support the use of the H. pylori FlgE polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections. As such, the FlgE polypeptide will be useful both for the detection of H. pylori infections as well as for the manufacture of vaccine compositions, which when given in an appropriate pharmaceutical formulation will elicit a protective or therapeutic immune response against such infections.
Consequently, in one aspect the present invention provides a Helicobacter pylori FlgE polypeptide for use in inducing a protective immune response to Helicobacter pylori infection. The term "Helicobacter pylori FlgE polypeptide" is intended to mean the polypeptide which is disclosed by O'Toole et al. in Molecular Microbiology, 14(4), 691-703, 1994, and which is encoded by the gene whose nucleotide sequence is set forth as SEQ ID NO: 1, or can be obtained from the National Center for Biotechnology Information (Accession number U09549), or a substantially similar modified form of the said polypeptide retaining functionally equivalent antigenicity.
The term "protective immune response" is to be understood as an immune response which makes the composition suitable for therapeutic and /or prophylactic purposes.
The term "functionally equivalent antigenicity" is to be understood as the ability to induce a systemic and mucosal immune response while decreasing the number of H. pylori cells associated with the gastric mucosa. The skilled person will be able to identify modified forms of the FlgE polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies.
In a preferred form of the invention, the Helicobacter pylori FlgE polypeptide, for use in inducing a protective immune response to Helicobacter pylori infection, has substantially the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing, or is a modified form thereof retaining functionally equivalent antigenicity.
It is thus to be understood that the definition of the Helicobacter pylori FlgE polypeptide is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of the Helicobacter pylori FlgE polypeptide and is retaining functionally equivalent antigenicity. Included in the definition of the Helicobacter pylori FlgE polypeptide are consequently polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing.
In another aspect, the invention provides a vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori FlgE polypeptide as defined above, optionally together with a pharmaceutically acceptable carrier or diluent.
In the present context the term "immunologically effective amount" is to be understood as an amount which elicits a significant protective Helicobacter pylori response, which will eradicate a H. pylori infection in an infected mammal or prevent the infection in a susceptible mammal. Typically an immunologically effective amount will comprise approximately 1 μg to 1000 mg, preferably approximately 10 μg to 100 mg, of H. pylori antigen for oral administration, or approximately less than 100 μg for parenteral administration.
The vaccine composition comprises optionally in addition to a pharmaceutically acceptable carrier or diluent one or more other immunologically active antigens for prophylactic or therapeutic use. Physiologically acceptable carriers and diluents are well known to those skilled in the art and include e.g. phosphate buffered saline (PBS), or, in the case of oral vaccines, HCO3" based formulations or enterically coated powder formulations.
The vaccine composition can optionally include or be administered together with acid secretion inhibitors, preferably proton pump inhibitors (PPIs), e.g. omeprazole. The vaccine can be formulated in known delivery systems such as liposomes, ISCOMs, cochleates, etc. (see e.g. Rabinovich et al. (1994) Science 265, 1401-1404) or be attached to or incorporated into polymer microspheres of degradable or non-degradable nature. The antigens could be associated with live attenuated bacteria, viruses or phages or with killed vectors of the same kind. The antigens can be chemically or genetically coupled to carrier proteins of inert or adjuvantic types (i.e Cholera B subunit). Consequently, the invention provides in a further aspect a vaccine composition according to above, in addition comprising an adjuvant, such as a cholera toxin. Such pharmaceutically acceptable forms of cholera toxin are known in the art, e.g. from Rappuoli et al. (1995) Int. Arch. Allergy & Immunol. 108(4), 327-333; and Dickinson et al. (1995) Infection and Immunity 63(5), 1617-1623.
A vaccine composition according to the invention can be used for both therapeutic and prophylactic purposes. Consequently, the invention includes a vaccine composition according as defined above, for use as a therapeutic or a prophylactic vaccine in a mammal, including man, which is infected by Helicobacter pylori. In this context the term "prophylactic purpose" means to induce an immune response which will protect against future infection by Helicobacter pylori, while the term "therapeutic purpose" means to induce an immune response which can eradicate an existing Helicobacter pylori infections.
The vaccine composition according to the invention is preferably administered to any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the gastric, the intestinal (small and large intestine), the rectal and the vaginal mucosa. The mucosal vaccines can be given together with for the purpose appropriate adjuvants. The vaccine can also be given orally, or parenterally, by the subcutaneous, intracutaneous or intramuscular route, optionally together with the appropriate adjuvant. The vaccine composition can optionally be given together with antimicrobial therapeutic agents.
In a further aspect, the invention proivides the use of a Helicobacter pylori FlgE polypeptide, as defined above, in the manufacture of
(i) a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection;
(ii) a vaccine for use in eliciting a protective immune response against Helicobacter pylori; and
(iii) a diagnostic kit for diagnosis of Helicobacter pylori infection.
In yet a further aspect, the invention provides a method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori FlgE polypeptide as defined above, optionally labelled or coupled to a solid support, is used. The said method could e.g. comprise the steps (a) contacting a said Helicobacter pylori FlgE polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and (b) detecting antibodies from the said body fluid binding to the said FlgE polypeptide. Preferred methods of detecting antibodies are ELISA (Enzyme linked immunoabsorbent assay) methods which are well known in the art.
In another aspect the invention provides a diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method of in vitro diagnosis as described above to be carried out. The said diagnostic kit could e.g. comprise: (a) a Helicobacter pylori FlgE polypeptide; and (b) reagents for detecting antibodies binding to the said FlgE polypeptide. The said reagents for detecting antibodies could e.g. be an enzyme- labelled anti-immunoglobulin and a chromogenic substrate for the said enzyme.
In yet a further aspect, the invention provides a method of eliciting in a mammal, including humans, a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a Helicobacter pylori FlgE polypeptide as defined above, or alternatively administering to the said mammal an immunologically effective amount of a vaccine composition as denned above.
EXPERIMENTAL METHODS
Throughout this description the terms "standard protocols" and "standard procedures", when used in the context of molecular cloning techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al, John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T, Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989. Preparation of recombinant Helicobacter pylori FlgE polypeptide
DNA sequence Information
Sequence information for the gene encoding for the FlgE polypeptide was obtained from the National Center for Biotechnology Information (Accession number U09549; SEQ ID NO: 1).
PCR Amplification and cloning of DNA sequences containing ORF'sfor membrane and secreted proteins from the J99 Strain of Helicobacter pylori.
Sequences were cloned from the J99 strain of H. pylori by amplification cloning using the polymerase chain reaction (PCR). Synthetic oligonucleotide primers (see below) specific for the 5'- and 3'-ends of open reading frames of genes were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). Forward primers (specific for the 5'-end of the sequence) for FlgE were designed to include an Ncol cloning site at the extreme 5'-terminus, while reverse primers included a EcoRI site at the extreme 5'-terminus to permit cloning of each H. pylori sequence into the reading frame of the pET28b vector. Inserts cloned into the Ncol- EcoRI sites of the pET-28b vector are fused to a vector DNA sequence encoding an additional 20 carboxy-terminal amino including six histidine residues (at the extreme C-terminus).
Forward primer (SEQ ID NO: 3):
5'-TATACC ATG GTG CTT AGGTCTTTAT-3'
Reverse primer (SEQ ID NO: 4):
5'-GCG AAT TCA ATT GCT TAA GAT TCA A-3' Genomic DNA prepared from the J99 strain of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 ng) was introduced into a reaction vial containing 2 mM MgCl2, 1 μM synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP, and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 μl. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Per kin Elmer Cetus/ Gene Amp PCR System 9600 thermal cycler: Denaturation at +94°C for 2 min; 2 cycles at +94°C for 15 sec, +30°C for 15 sec and +72°C for 1.5 min; 23 cycles at +94°C for 15 sec, +58°C for 15 sec and +72°C for 1.5 min; Reactions were concluded at +72°C for 6 minutes.
Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). Amplified DNA samples were subjected to digestion with the restriction endonucleases Ndel and EcoRI according to standard procedures. DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave UN irradiation. DΝA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA). Cloning ofH. pylori DNA sequences into the pET-28b prokaryotic expression vector.
The pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI according to standard procedures. Following digestion, DNA inserts were cloned according to standard procedures into the previously digested pET-28b expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E. coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods. Briefly, 1 μl of ligation reaction was mixed with 50 μl of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at +37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 μg/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below.
Identification of recombinant pET expression plasmids carrying H. pylori sequences
Individual BL21 clones transformed with recombinant pET-28b H. pylori genes were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the expression vector according to standard procedures. Isolation and Preparation ofplasmid DNA from BL21 transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H. pylori ORFs were picked and incubated in 5 ml of LB broth plus 25 μg/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA).
Expression of recombinant H. pylori sequences in E. coli
The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5α, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-β-D- thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest. Strains used in our laboratory include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Methods Enzymol. 185, 60-89).
To express recombinant H. pylori sequences, 50 ng of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 μg/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 μg/ml) to an optical density at 600 nm of 0.5 to 1.0 O.D. units, at which point, 1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 ml cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at +4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification.
Analytical Methods
The concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.H., Rosebrough,N., Farr, A.L. & Randall, R.J. (1951) , using bovine serum albumin as a standard.
Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels (12% or 4 to 25 % gradient acrylamide) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie Brilliant Blue. Molecular mass markers included rabbit skeletal muscle myosin (200 kDa), E. coli β-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa). Purification of FlgE from inclusion bodies
The following steps were carried out at +4°C. Cell pellets were resuspended in lysis buffer with 10% glycerol 200 μg/ml lysozyme, 5 mM EDTA, 1 mM PMSF and 0.1% β-mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was made 0.2% DOC, stirred 10 minutes, then centrifuged (10,000 g x 30 min). The pellets were first washed with lysis buffer containing 10% glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1% β-mercaptoethanol, then with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1% β-mercaptoethanol. The resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials.
The following steps were carried out at room temperature. Inclusion bodies were dissolved in 20 ml 8 M urea in lysis buffer with 1 mM PMSF and 0.1% β- mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation (100,000 x g for 30 min) . The clear supernatant was filtered and loaded onto a Ni2+-NTA agarose column equilibrated in 8 M urea in lysis buffer. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1 mM PMSF and 0.1% β- mercaptoethanol, and developed with sequential steps of lysis buffer containing 8 M urea, 1 mM PMSF, 0.1% β-mercaptoethanol and 20, 100, 200, and 500 mM imidazole. Fractions were monitored by absorbance at OD280 τu:n-' and peak fractions were analyzed by SDS-PAGE. Two bands were visualized by Coomassie Brilliant Blue staining, a major band Mr = 78 kDa and a minor band Mr = 60 kDa. Purity of recombinant FlgE (78 kDa) was assessed at greater than 90%. As with the purification of the soluble proteins, fractions containing the recombinant protein eluted at 100 mM imidazole.
Urea was slowly removed from the FlgE polypeptide by dialysis against TBS containing 0.5% DOC with sequential reduction in urea as follows; 6M, 4M, 3M, 2M, 1M, 0.5 M then 0 M. Each dialysis step was carried for a minimum of 4 hours at room temperature,
After dialysis, samples were concentrated by pressure filtration using Amicon stirred cells. Protein concentrations were then measured by the methods of Perkins, Bradford and Lowry.
EXAMPLES OF THE INVENTION
EXAMPLE 1: THERAPEUTIC IMMUNIZATION
1. Materials & Methods
1.1 Animals
Female SPF BALB/c mice were purchased from Bomholt Breeding centre (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
1.2. Infection
After a minimum of one week of acclimatization, the animals were infected with a type 2 strain of H. pylori (strain 244, originally isolated from an ulcer patient). This strain has earlier proven to be a good colonizer of the mouse stomach. Bacteria from a stock kept at -70°C were grown overnight in Brucella broth supplemented with 10% fetal calf serum, at +37°C in a microaerophilic atmosphere (10% CO2, 5% O2). The animals were given an oral dose of omeprazole (400 μmol/kg) and after 3-5 h an oral inoculation of H. pylori (approximately 107-108 CFU/ animal). Infection was checked in control animals 2-3 weeks after the inoculation. 1.3. Immunizations
One month after infection, two groups of mice (10 mice/group) were immunized 4 times over a 34 day period (day 1, 15, 25 and 35). Purified recombinant FlgE dissolved in PBS plus 0.5% Deoxycholate (DOC) was given at a dose of 100 microgram / mouse.
As an adjuvant, the animals in both the control as well as the FlgE group were also given 10 μg/mouse of cholera toxin (CT) with each immunization. Omeprazole (400 μmol/kg) was given orally to all animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Animals were sacrificed 1-2 weeks after final immunization.
Group 1: 300 μl PBS with 0.5% DOC containing 10 μg CT Group 2: 300 μl PBS with 0.5% DOC containing 100 μg FlgE and 10 μg CT.
1.4. Analysis of infection
The mice were sacrificed by CO2 and cervical dislocation. The abdomen and chest cavity was opened and blood sampled by heart puncture. Subsequently the stomach was removed. After cutting the stomach along the greater curvature, it was rinsed in saline and subsequently cut into two identical pieces. An area of 25 mm2 of the mucosa from the antrum and corpus was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth, diluted and plated onto Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining. 1.5. Antibody measurements
Serum antibodies were collected from blood. Prior to centrifugation, the blood was diluted with equal amount of PBS. The serum was kept at -20°C until analysis. Serum antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with FlgE followed by addition of different dilutions of serum. The ELISA was developed with alkaline phosphatase-labelled anti-mouse-Ig-antibodies. The anti-Ig antibodies were of an anti-heavy /anti-light chain type, which should detect all types of antibodies.
2. Results
2.1. Therapeutic immunization: effects on CFU
The animals in this study were infected with H. pylori strain 244 one month prior to immunizations. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant FlgE polypeptide. Four weeks after the final immunization, the animals were sacrificed and CFU was determined (Fig. 1). The animals treated with CT alone, were highly infected both in corpus and antrum. Animals actively immunized with recombinant FlgE polypeptide and CT had significantly decreased CFU values in the antrum and in the stomach as a whole compared with the CT treated animals (p<0.01 and p<0.05, respectively; Wilcoxon-Mann-Whittney sign rank test).
2.2. Therapeutic immunization: effects on antibody formation and secretion
As a sign of infection H. pylori specific antibodies can be found in serum (Control/ 244). In animals given FlgE + CT the titer against strain 244 (as membrane proteins) increased 4-fold (p<0.01). Only in animals given FlgE + CT could a specific serum IgG titer against FlgE be measured (Fig. 2). FlgE specific IgG increased in animals given FlgE + CT, but could not be detected in control animals.
The results presented show that the recombinant FlgE H. pylori polypeptide is highly immunogenic when given orally, together with cholera toxin as an adjuvant, measured as an increase in systemic FlgE specific Ig antibodies. The immunization with FlgE also resulted in a significant increase in the Ig titers against a p articulate fraction of H. pylori. In addition, a dramatic decrease in number of colonizing H. pylori in the gastric mucosa of the infected mice was found following immunization with FlgE toghether with cholera toxin.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Astra AB
(B) STREET: Vastra Malarehamnen 9
(C) CITY: Sδdertalje
(E) COUNTRY: Sweden
(F) POSTAL CODE (ZIP) : S-151 85
(G) TELEPHONE: +46 8 553 260 00 (H) TELEFAX: +46 8 553 288 20
(ii) TITLE OF INVENTION: Vaccine Compositions V
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2550 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 321..2477
(D) OTHER INFORMATION: /product= "FlgE flagellar hook protein"
(x) PUBLICATION INFORMATION:
(A) AUTHORS: O'Toole, Paul W.
Kostrzynska, Magdalena Trust, Trevor J.
(B) TITLE: Non-motile mutants of Helicobacter pylori and
Helicobacter mustelae defective in flagellar hook production
(C) JOURNAL: Mol . Microbiol .
(D) VOLUME: 14
(E) ISSUE: 4
(F) PAGES: 691-703
(G) DATE: 1994
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1:
AACAAAGCGA TAACTCCTTT GTCTTATTAG CGACACAATT TAACCCATTG ACTTTAAATC 60
GCGCTTCAGC CGAAGAGATT CAAGATCATG AATGCGCGAT TTTGCACTAA AGCGAGTTAG 120
ATTCTTAAAT TTGAGCGATA ACCTTTAAAA AGCGTAATTA AGGGGTGGTG TTACAAAACC 180 CCCTATCCCC TTATGAATTT GACCGATCTT TTTGATTAAC AAAACTTTAA AATCCGCAAT 240
CAATCATTCT AAAAAGCTAT TTAGGAACAA CTTTTGCTTT ATTTTGCATA GATTGAATTT 300
CTTTAAATTA AAGGATAACC ATG CTT AGG TCT TTA TGG TCT GGT GTC AAT 350
Met Leu Arg Ser Leu Trp Ser Gly Val Asn 1 5 10
GGG ATG CAA GCC CAC CAA ATC GCT TTG GAT ATT GAG AGT AAC AAT ATT 398 Gly Met Gin Ala His Gin lie Ala Leu Asp He Glu Ser Asn Asn He 15 20 25
GCG AAC GTG AAT ACC ACT GGT TTT AAG TAT TCT AGG GCT TCT TTT GTG 446 Ala Asn Val Asn Thr Thr Gly Phe Lys Tyr Ser Arg Ala Ser Phe Val 30 35 40
GAT ATG CTT TCT CAA GTC AAA CTC ATC GCT ACC GCA CCC TAT AAA AAC 494 Asp Met Leu Ser Gin Val Lys Leu He Ala Thr Ala Pro Tyr Lys Asn 45 50 55
GGG TTA GCA GGG CAG AAT GAT TTT TCT GTG GGG CTT GGG GTA GGC GTG 542 Gly Leu Ala Gly Gin Asn Asp Phe Ser Val Gly Leu Gly Val Gly Val 60 65 70
GAT GCG ACG ACT AAA ATC TTT TCA CAA GGC AAT ATC CAA AAC ACA GAT 590 Asp Ala Thr Thr Lys He Phe Ser Gin Gly Asn He Gin Asn Thr Asp 75 80 85 90
GTC AAA ACC GAT CTA GCG ATT CAA GGC GAT GGC TTT TTT ATC ATT AAC 638 Val Lys Thr Asp Leu Ala He Gin Gly Asp Gly Phe Phe He He Asn 95 100 105
CCT GAT AGG GGG ATC ACG CGC AAT TTC ACT AGA GAT GGG GAG TTC CTT 686 Pro Asp Arg Gly He Thr Arg Asn Phe Thr Arg Asp Gly Glu Phe Leu 110 115 120
TTT GAC TCG CAA GGG AGT TTG GTT ACC ACC GGC GGG CTT GTG GTG CAA 734 Phe Asp Ser Gin Gly Ser Leu Val Thr Thr Gly Gly Leu Val Val Gin 125 130 135
GGG TGG GTG AGA AAT GGG AGC GAT ACC GGC AAT AAA GGG AGC GAT ACA 782 Gly Trp Val Arg Asn Gly Ser Asp Thr Gly Asn Lys Gly Ser Asp Thr 140 145 150
GAC GCT TTA AAA GTG GAT AAC ACC GGT CCT TTA GAA AAC ATT AGG ATT 830 Asp Ala Leu Lys Val Asp Asn Thr Gly Pro Leu Glu Asn He Arg He 155 160 165 170
GAT CCT GGA ATG GTG ATG CCA GCC AGA GCG AGT AAC CGC ATT TCT ATG 878 Asp Pro Gly Met Val Met Pro Ala Arg Ala Ser Asn Arg He Ser Met 175 180 185
AGG GCG AAT TTA AAC GCT GGA AGG CAT GCC GAT CAA ACA GCG GCG ATA 926 Arg Ala Asn Leu Asn Ala Gly Arg His Ala Asp Gin Thr Ala Ala He 190 195 200
TTC GCT TTG GAT TCT TCA GCC AAA ACC CCT TCA GAT GGC ATT AAT CCG 974 Phe Ala Leu Asp Ser Ser Ala Lys Thr Pro Ser Asp Gly He Asn Pro 205 210 215
GTG TAT GAT TCA GGC ACG AAT CTT GCT CAA GTC GCC GAA GAC ATG GGA 1022 Val Tyr Asp Ser Gly Thr Asn Leu Ala Gin Val Ala Glu Asp Met Gly 220 225 230
TCT TTA TAC AAT GAA GAT GGC GAC GCT CTT TTG TTG AAT GAA AAT CAA 1070 Ser Leu Tyr Asn Glu Asp Gly Asp Ala Leu Leu Leu Asn Glu Asn Gin 235 240 245 250
GGG ATT TGG GTG AGC TAT AAG AGT CCA AAA ATG GTC AAA GAC ATC CTC 1118 Gly He Trp Val Ser Tyr Lys Ser Pro Lys Met Val Lys Asp He Leu 255 260 265
CCT TCT GCA GAA AAC AGC ACG CTT GAA TTG AAT GGC GTT AAG ATT TCT 1166 Pro Ser Ala Glu Asn Ser Thr Leu Glu Leu Asn Gly Val Lys He Ser 270 275 280
TTC ACA AAC GAT TCA GCG GTG AGC CGG ACT TCA AGC TTA GTG GCG GCT 1214 Phe Thr Asn Asp Ser Ala Val Ser Arg Thr Ser Ser Leu Val Ala Ala 285 290 295
AAA AAT GCG ATC AAT GCA GTC AAA AGC CAA ACA GGC ATT GAA GCT TAT 1262 Lys Asn Ala He Asn Ala Val Lys Ser Gin Thr Gly He Glu Ala Tyr 300 305 310
TTA GAC GGC AAG CAA TTG CGT TTG GAA AAC ACC AAT GAA TTA GAC GGC 1310 Leu Asp Gly Lys Gin Leu Arg Leu Glu Asn Thr Asn Glu Leu Asp Gly 315 320 325 330
GAT GAA AAG CTT AAA AAC ATT GTA GTT ACT CAA GCC GGA ACC GGA GCG 1358 Asp Glu Lys Leu Lys Asn He Val Val Thr Gin Ala Gly Thr Gly Ala 335 340 345
TTC GCT AAC TTT TTA GAC GGC GAT AAA GAT GTA ACG GCT TTC AAA TAC 1406 Phe Ala Asn Phe Leu Asp Gly Asp Lys Asp Val Thr Ala Phe Lys Tyr 350 355 360
AGC TAC ACG CAT TCT ATT AGC CCT AAC GCC AAT AGC GGG CAG TTT AGG 1454 Ser Tyr Thr His Ser He Ser Pro Asn Ala Asn Ser Gly Gin Phe Arg 365 370 375
ACC ACT GAA GAC TTG CGC GCC TTA ATC CAG CAT GAC GCT AAT ATC GTT 1502 Thr Thr Glu Asp Leu Arg Ala Leu He Gin His Asp Ala Asn He Val 380 385 390
AAA GAT CCT AGC CTA GCG GAC AAT TAC CAA GAC TCA GCC GCT TCT ATA 1550 Lys Asp Pro Ser Leu Ala Asp Asn Tyr Gin Asp Ser Ala Ala Ser He 395 400 405 410
GGA GTT ACA ATC AAC CAA TAC GGC ATG TTT GAA ATC AAC AAT AAA GAC 1598 Gly Val Thr He Asn Gin Tyr Gly Met Phe Glu He Asn Asn Lys Asp 415 420 425
AAT AAA AAT GTC ATT AAA GAA AAT CTT AAT ATC TTT GTG AGC GGG TAT 1646 Asn Lys Asn Val He Lys Glu Asn Leu Asn He Phe Val Ser Gly Tyr 430 435 440
TCT TCA GAC AGC GTA ACG AAC AAT GTT TTG TTT AAA AAT GCG ATG AAA 1694 Ser Ser Asp Ser Val Thr Asn Asn Val Leu Phe Lys Asn Ala Met Lys 445 450 455
GGG CTT AAT ACC GCT TCT TTA ATT GAA GGG GGA GCG TCA GCG AGC AGT 1742 Gly Leu Asn Thr Ala Ser Leu He Glu Gly Gly Ala Ser Ala Ser Ser 460 465 470
TCT AAA TTC ACC CAC GCT ACG CAT GCG ACA AGC ATT GAT GTG ATA GAC 1790 Ser Lys Phe Thr His Ala Thr His Ala Thr Ser He Asp Val He Asp 475 480 485 490
AGC TTA GGC ACT AAA CAC GCC ATG CGC ATT GAG TTT TAT AGG AGT GGG 1838 Ser Leu Gly Thr Lys His Ala Met Arg He Glu Phe Tyr Arg Ser Gly 495 500 505 GGA GCG GAT TGG AAT TTT AGA GTG ATC GTG CCT GAG CCT GGG GAA TTA 1886 Gly Ala Asp Trp Asn Phe Arg Val He Val Pro Glu Pro Gly Glu Leu 510 515 520
GTA GGG GGG TCA GCG GCT AGG CCT AAT GTG TTT GAA GGA GGC CGT TTG 1934 Val Gly Gly Ser Ala Ala Arg Pro Asn Val Phe Glu Gly Gly Arg Leu 525 530 535
CAC TTC AAT AAT GAC GGA TCG CTT GCA GGC ATG AAC CCG CCT CTT TTG 1982 His Phe Asn Asn Asp Gly Ser Leu Ala Gly Met Asn Pro Pro Leu Leu 540 545 550
CAA TTT GAC CCT AAA AAT GGT GCT GAT GCC CCC CAA CGC ATC AAT TTA 2030 Gin Phe Asp Pro Lys Asn Gly Ala Asp Ala Pro Gin Arg He Asn Leu 555 560 565 570
GCT TTT GGT TCC TCA GGG AGT TTT GAC GGG CTA ACG AGC GTG GAT AAG 2078 Ala Phe Gly Ser Ser Gly Ser Phe Asp Gly Leu Thr Ser Val Asp Lys 575 580 585
ATT TCT GAA ACT TAT GCG ATT GAG CAA AAC GGC TAT CAA GCG GGC GAT 2126 He Ser Glu Thr Tyr Ala He Glu Gin Asn Gly Tyr Gin Ala Gly Asp 590 595 600
TTG ATG GAT GTC CGC TTT GAT TCA GAT GGG GTG CTT TTA GGA GCG TTC 2174 Leu Met Asp Val Arg Phe Asp Ser Asp Gly Val Leu Leu Gly Ala Phe 605 610 615
AGT AAT GGC AGG ACT TTA GCG CTC GCT CAA GTG GCT TTA GCG AAT TTC 2222 Ser Asn Gly Arg Thr Leu Ala Leu Ala Gin Val Ala Leu Ala Asn Phe 620 625 630
GCT AAC GAT GCG GGC TTG CAG GCT TTA GGC GGG AAT GTC TTT TCT CAA 2270 Ala Asn Asp Ala Gly Leu Gin Ala Leu Gly Gly Asn Val Phe Ser Gin 635 640 645 650
ACC GGA AAC TCA GGG CAA GCC TTA ATC GGT GCG GCT AAT ACG GGG CGT 2318 Thr Gly Asn Ser Gly Gin Ala Leu He Gly Ala Ala Asn Thr Gly Arg 655 660 665
AGG GGT TCA ATT TCA GGA TCT AAA CTG GAG TCT AGT AAT GTG GAT TTG 2366 Arg Gly Ser He Ser Gly Ser Lys Leu Glu Ser Ser Asn Val Asp Leu 670 675 680
AGC CGG AGT TTA ACG AAT TTG ATT GTG GTT CAA AGG GGC TTT CAA GCA 2414 Ser Arg Ser Leu Thr Asn Leu He Val Val Gin Arg Gly Phe Gin Ala 685 690 695
AAC TCT AAA GCG GTA ACC ACA TCC GAT CAA ATC CTT AAT ACC CTA TTG 2462 Asn Ser Lys Ala Val Thr Thr Ser Asp Gin He Leu Asn Thr Leu Leu 700 705 710
AAT CTT AAG CAA TAA ACTAAAGGAT TACTCTAATA CAATATAATA GGGGCTAATT 2517
Asn Leu Lys Gin *
715
TAAAGATTAA GGTTTAGTAT GCATGAATAC TCG 2550
( 2 ) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 719 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Arg Ser Leu Trp Ser Gly Val Asn Gly Met Gin Ala His Gin 1 5 10 15
He Ala Leu Asp He Glu Ser Asn Asn He Ala Asn Val Asn Thr Thr 20 25 30
Gly Phe Lys Tyr Ser Arg Ala Ser Phe Val Asp Met Leu Ser Gin Val 35 40 45
Lys Leu He Ala Thr Ala Pro Tyr Lys Asn Gly Leu Ala Gly Gin Asn 50 55 60
Asp Phe Ser Val Gly Leu Gly Val Gly Val Asp Ala Thr Thr Lys He 65 70 75 80
Phe Ser Gin Gly Asn He Gin Asn Thr Asp Val Lys Thr Asp Leu Ala 85 90 95
He Gin Gly Asp Gly Phe Phe He He Asn Pro Asp Arg Gly He Thr 100 105 110
Arg Asn Phe Thr Arg Asp Gly Glu Phe Leu Phe Asp Ser Gin Gly Ser 115 120 125
Leu Val Thr Thr Gly Gly Leu Val Val Gin Gly Trp Val Arg Asn Gly 130 135 140
Ser Asp Thr Gly Asn Lys Gly Ser Asp Thr Asp Ala Leu Lys Val Asp 145 150 155 160
Asn Thr Gly Pro Leu Glu Asn He Arg He Asp Pro Gly Met Val Met 165 170 175
Pro Ala Arg Ala Ser Asn Arg He Ser Met Arg Ala Asn Leu Asn Ala 180 185 190
Gly Arg His Ala Asp Gin Thr Ala Ala He Phe Ala Leu Asp Ser Ser 195 200 205
Ala Lys Thr Pro Ser Asp Gly He Asn Pro Val Tyr Asp Ser Gly Thr 210 215 220
Asn Leu Ala Gin Val Ala Glu Asp Met Gly Ser Leu Tyr Asn Glu Asp 225 230 235 240
Gly Asp Ala Leu Leu Leu Asn Glu Asn Gin Gly He Trp Val Ser Tyr 245 250 255
Lys Ser Pro Lys Met Val Lys Asp He Leu Pro Ser Ala Glu Asn Ser 260 265 270
Thr Leu Glu Leu Asn Gly Val Lys He Ser Phe Thr Asn Asp Ser Ala 275 280 285
Val Ser Arg Thr Ser Ser Leu Val Ala Ala Lys Asn Ala He Asn Ala 290 295 300
Val Lys Ser Gin Thr Gly He Glu Ala Tyr Leu Asp Gly Lys Gin Leu 305 310 315 320
Arg Leu Glu Asn Thr Asn Glu Leu Asp Gly Asp Glu Lys Leu Lys Asn 325 330 335
He Val Val Thr Gin Ala Gly Thr Gly Ala Phe Ala Asn Phe Leu Asp 340 345 350
Gly Asp Lys Asp Val Thr Ala Phe Lys Tyr Ser Tyr Thr His Ser He 355 360 365
Ser Pro Asn Ala Asn Ser Gly Gin Phe Arg Thr Thr Glu Asp Leu Arg 370 375 380
Ala Leu He Gin His Asp Ala Asn He Val Lys Asp Pro Ser Leu Ala 385 390 395 400
Asp Asn Tyr Gin Asp Ser Ala Ala Ser He Gly Val Thr He Asn Gin 405 410 415
Tyr Gly Met Phe Glu He Asn Asn Lys Asp Asn Lys Asn Val He Lys 420 425 430
Glu Asn Leu Asn He Phe Val Ser Gly Tyr Ser Ser Asp Ser Val Thr 435 440 445
Asn Asn Val Leu Phe Lys Asn Ala Met Lys Gly Leu Asn Thr Ala Ser 450 455 460
Leu He Glu Gly Gly Ala Ser Ala Ser Ser Ser Lys Phe Thr His Ala 465 470 475 480
Thr His Ala Thr Ser He Asp Val He Asp Ser Leu Gly Thr Lys His 485 490 495
Ala Met Arg He Glu Phe Tyr Arg Ser Gly Gly Ala Asp Trp Asn Phe 500 505 510
Arg Val He Val Pro Glu Pro Gly Glu Leu Val Gly Gly Ser Ala Ala 515 520 525
Arg Pro Asn Val Phe Glu Gly Gly Arg Leu His Phe Asn Asn Asp Gly 530 535 540
Ser Leu Ala Gly Met Asn Pro Pro Leu Leu Gin Phe Asp Pro Lys Asn 545 550 555 560
Gly Ala Asp Ala Pro Gin Arg He Asn Leu Ala Phe Gly Ser Ser Gly 565 570 575
Ser Phe Asp Gly Leu Thr Ser Val Asp Lys He Ser Glu Thr Tyr Ala 580 585 590
He Glu Gin Asn Gly Tyr Gin Ala Gly Asp Leu Met Asp Val Arg Phe 595 600 605
Asp Ser Asp Gly Val Leu Leu Gly Ala Phe Ser Asn Gly Arg Thr Leu 610 615 620
Ala Leu Ala Gin Val Ala Leu Ala Asn Phe Ala Asn Asp Ala Gly Leu 625 630 635 640
Gin Ala Leu Gly Gly Asn Val Phe Ser Gin Thr Gly Asn Ser Gly Gin 645 650 655
Ala Leu He Gly Ala Ala Asn Thr Gly Arg Arg Gly Ser He Ser Gly 660 665 670
Ser Lys Leu Glu Ser Ser Asn Val Asp Leu Ser Arg Ser Leu Thr Asn 675 680 685
Leu He Val Val Gin Arg Gly Phe Gin Ala Asn Ser Lys Ala Val Thr 690 695 700
Thr Ser Asp Gin He Leu Asn Thr Leu Leu Asn Leu Lys Gin * 705 710 715
(2) INFORMATION FOR SEQ ID NO : 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TATACCATGG TGCTTAGGTC TTTAT 25
(2) INFORMATION FOR SEQ ID NO : 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4: GCGAATTCAA TTGCTTAAGA TTCAA 25

Claims

1. A Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, for use in inducing a protective immune response to Helicobacter pylori infection.
2. A Helicobacter pylori FlgE polypeptide according to claim 1 which has substantially the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing, for use in inducing a protective immune response to Helicobacter pylori infection.
3. A vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2, optionally together with a pharmaceutically acceptable carrier or diluent.
4. A vaccine composition according to claim 3 in addition comprising an adjuvant.
5. A vaccine composition according to claim 4 wherein the adjuvant is a pharmaceutically acceptable form of cholera toxin.
6. A vaccine composition according to any one of claims 3 to 5 for use as a therapeutic vaccine in a mammal, including man, which is infected by Helicobacter pylori.
7. A vaccine composition according to any one of claims 3 to 5 for use as a prophylactic vaccine to protect a mammal, including man, from infection by Helicobacter pylori.
8. Use of a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2 in the manufacture of a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection.
9. Use of a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2 in the manufacture of a vaccine for use in eliciting a protective immune response against Helicobacter pylori.
10. Use of a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2 in the manufacture of a diagnostic kit for diagnosis of Helicobacter pylori infection.
11. A method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2, optionally labelled or coupled to a solid support, is used.
12. A method according to claim 11 comprising the steps
(a) contacting a said Helicobacter pylori FlgE polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and
(b) detecting antibodies from the said body fluid binding to the said FlgE polypeptide.
13. A diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method according to claim 11 or 12 to be carried out.
14. A diagnostic kit according to claim 13, comprising:
(a) a Helicobacter pylori FlgE polypeptide; and
(b) reagents for detecting antibodies binding to the said FlgE polypeptide.
15. A method of eliciting in a mammal a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically ef ective amount of a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2.
16. A method of eliciting in a mammal a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a vaccine composition according to any one of claims 3 to 7.
17. A method according to claim 15 or 16 wherein the said mammal is a human.
EP98928772A 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE $i(HELICOBACTER PYLORI) FlgE POLYPEPTIDE Withdrawn EP1009764A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9702242 1997-06-12
SE9702242A SE9702242D0 (en) 1997-06-12 1997-06-12 Vaccine compositions V
PCT/SE1998/001093 WO1998056816A1 (en) 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI FlgE POLYPEPTIDE

Publications (1)

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EP1009764A1 true EP1009764A1 (en) 2000-06-21

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EP98928772A Withdrawn EP1009764A1 (en) 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE $i(HELICOBACTER PYLORI) FlgE POLYPEPTIDE

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EP (1) EP1009764A1 (en)
JP (1) JP2002507118A (en)
KR (1) KR20010013699A (en)
CN (1) CN1259960A (en)
AR (1) AR012896A1 (en)
AU (1) AU8048798A (en)
BR (1) BR9810026A (en)
CA (1) CA2293293A1 (en)
EE (1) EE9900566A (en)
HU (1) HUP0003164A3 (en)
ID (1) ID23052A (en)
IL (1) IL133144A0 (en)
IS (1) IS5288A (en)
NO (1) NO996132L (en)
NZ (1) NZ501427A (en)
PL (1) PL337503A1 (en)
SE (1) SE9702242D0 (en)
SK (1) SK173099A3 (en)
TR (1) TR199903060T2 (en)
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CN104784687A (en) * 2015-04-27 2015-07-22 苏州大学附属第一医院 Application of flagellar hook protein FlgE of reorganized pseudomonas aeruginosa
CN113425717B (en) * 2021-04-22 2023-06-16 成都欧林生物科技股份有限公司 Medicament for improving efficacy of oral helicobacter pylori vaccine and application thereof
CN116535472B (en) * 2023-05-31 2024-04-30 四川大学华西医院 Helicobacter pylori recombinant protein antigen FlgK and preparation method and application thereof
CN118105473B (en) * 2024-04-30 2024-08-16 成都欧林生物科技股份有限公司 Oral immunogenic composition for preventing or treating Hp infection and application thereof

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US5459041A (en) * 1988-02-18 1995-10-17 Enteric Research Laboratories, Inc. Campylobacter pylori antigens and uses thereof for detection of Campylobacter pylori infection
AR003125A1 (en) * 1995-06-01 1998-07-08 Astra Ab BACTERIAL ANTIGENS FOR THE DIAGNOSIS OF INFECTIONS WITH HELICOBACTER PYLORI, A DNA MOLECLE THAT CODES IT, A VECTOR, A HOST CELL, A PROCEDURE FOR PRODUCING THE POLIPEPTIDE, USE OF ELEPIPETICO, AND PROAPILY USE

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Title
See references of WO9856816A1 *

Also Published As

Publication number Publication date
HUP0003164A2 (en) 2000-12-28
IL133144A0 (en) 2001-03-19
SE9702242D0 (en) 1997-06-12
AU8048798A (en) 1998-12-30
BR9810026A (en) 2000-09-19
TR199903060T2 (en) 2000-09-21
NO996132D0 (en) 1999-12-10
KR20010013699A (en) 2001-02-26
HUP0003164A3 (en) 2001-10-29
PL337503A1 (en) 2000-08-28
CA2293293A1 (en) 1998-12-17
JP2002507118A (en) 2002-03-05
ZA984696B (en) 1999-01-04
ID23052A (en) 2000-01-20
EE9900566A (en) 2000-06-15
SK173099A3 (en) 2000-06-12
WO1998056816A1 (en) 1998-12-17
CN1259960A (en) 2000-07-12
NZ501427A (en) 2000-09-29
AR012896A1 (en) 2000-11-22
NO996132L (en) 2000-01-28
IS5288A (en) 1999-12-08

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