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
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
EP98928772A
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German (de)
English (en)
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 date
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Publication of EP1009764A1 publication Critical patent/EP1009764A1/en
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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
    • C07K14/195—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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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
SE9702242A SE9702242D0 (sv) 1997-06-12 1997-06-12 Vaccine compositions V
SE9702242 1997-06-12
PCT/SE1998/001093 WO1998056816A1 (en) 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI FlgE POLYPEPTIDE

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CN113425717B (zh) * 2021-04-22 2023-06-16 成都欧林生物科技股份有限公司 一种提高口服幽门螺杆菌疫苗效力的药剂及其应用
CN116535472B (zh) * 2023-05-31 2024-04-30 四川大学华西医院 一种幽门螺杆菌重组蛋白抗原FlgK及其制备方法与应用
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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
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NO996132L (no) 2000-01-28
CN1259960A (zh) 2000-07-12
CA2293293A1 (en) 1998-12-17
WO1998056816A1 (en) 1998-12-17
PL337503A1 (en) 2000-08-28
NZ501427A (en) 2000-09-29
SK173099A3 (en) 2000-06-12
AR012896A1 (es) 2000-11-22
EE9900566A (xx) 2000-06-15
HUP0003164A2 (hu) 2000-12-28
TR199903060T2 (xx) 2000-09-21
SE9702242D0 (sv) 1997-06-12
ZA984696B (en) 1999-01-04
JP2002507118A (ja) 2002-03-05
NO996132D0 (no) 1999-12-10
IS5288A (is) 1999-12-08
KR20010013699A (ko) 2001-02-26
AU8048798A (en) 1998-12-30
ID23052A (id) 2000-01-20
IL133144A0 (en) 2001-03-19
HUP0003164A3 (en) 2001-10-29
BR9810026A (pt) 2000-09-19

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