AU8048798A - Vaccine compositions comprising the (helicobacter pylori) flge polypeptide - Google Patents

Description

WO 98/56816 PCT/SE98/01093 1 VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI FigE POLYPEPTIDE TECHNICAL FIELD 5 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. 10 BACKGROUND ART Helicobacter pylori 15 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 20 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 25 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 30 experimental models Eaton et al. (Infection & Immunity 64(7), 2445-2448, 1996).

WO 98/56816 PCT/SE98/01093 2 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. 5 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 10 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 15 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 20 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 25 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 WO 98/56816 PCT/SE98/01093 3 protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach. Flagellar Hook protein 5 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, 10 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 15 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 20 polypeptide still was produced but could only be recovered in the cytoplasm. BRIEF DESCRIPTION OF THE DRAWINGS 25 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, 30 Phosphate buffered saline with 0.5% deoxycholate given together with cholera WO 98/56816 PCT/SE98/01093 4 toxin 10 gg/mouse; shaded bars=FlgE + CT, mice given 100 gg FlgE and 10 gg 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). 5 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 10 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. 15 DISCLOSURE OF THE INVENTION The purpose of this invention is to provide an antigenic H. pylori polypeptide 20 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 theflgE gene as published by O'Toole et al., Molecular Microbiology, 14(4), 691 25 703, 1994. Being an essential protein for motility, the figE 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 30 it is probably covered by a sheet, can serve as a therapeutic antigen in an H. pylori WO 98/56816 PCT/SE98/01093 5 infected mouse model, when given together with the adjuvant cholera toxin. The experimental data below thus indicates that the H. pylori FigE 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 5 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 10 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 15is 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 20 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 25 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 30 of H. pylori cells associated with the gastric mucosa. The skilled person will be able WO 98/56816 PCT/SE98/01093 6 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. 5 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. 10 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, 15 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% 20 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 25 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 30 understood as an amount which elicits a significant protective Helicobacter pylori WO 98/56816 PCT/SE98/01093 7 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 gg to 1000 mg, preferably approximately 10 Rg to 100 mg, of H. pylori antigen for oral administration, or 5 approximately less than 100 gg 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 10 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, HCO 3- based formulations or enterically coated powder formulations. The vaccine composition can optionally include or be administered together with 15is 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 20 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 25 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 30 and prophylactic purposes. Consequently, the invention includes a vaccine WO 98/56816 PCT/SE98/01093 8 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 s 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 0to 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 15 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 20 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. 25 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 WO 98/56816 PCT/SE98/01093 9 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. 5 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 10 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, 15 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 defined above. 20 EXPERIMENTAL METHODS Throughout this description the terms "standard protocols" and "standard 25 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, 30 Cold Spring Harbor, NY 1989.

WO 98/56816 PCT/SE98/01093 10 Preparation of recombinant Helicobacter pylori FIgE polypeptide DNA sequence Information 5 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). o10 PCR Amplification and cloning of DNA sequences containing ORF's for 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 15is 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 NcoI 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 20 sequence into the reading frame of the pET28b vector. Inserts cloned into the NcoI 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). 25 Forward primer (SEQ ID NO: 3): 5'-TAT ACC ATG GTG CTT AGG TCT TTA T-3' Reverse primer (SEQ ID NO: 4): 5'-GCG AAT TCA ATT GCT TAA GAT TCA A-3' WO 98/56816 PCT/SE98/01093 11 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 5 introduced into a reaction vial containing 2 mM MgC1 2 , 1 jtM 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 10 of 100 jtl. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler: Denaturation at +94oC for 2 min; 2 cycles at +94oC for 15 sec, +30'C for 15 sec and +72oC for 1.5 min; 15is 23 cycles at +94oC for 15 sec, +58oC for 15 sec and +72oC for 1.5 min; Reactions were concluded at +72oC 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, 20 Gaithersburg, MD, USA). Amplified DNA samples were subjected to digestion with the restriction endonucleases NdeI 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 UV irradiation. DNA 25 contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).

WO 98/56816 PCT/SE98/01093 12 Cloning of H. pylori DNA sequences into the pET-28b prokaryotic expression vector. The pET-28b vector was prepared for cloning by digestion with NcoI and EcoRI according to standard procedures. Following digestion, DNA inserts were cloned 5 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 10 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 gl of ligation reaction was mixed with 50 gl of electrocompetent cells and subjected to a high voltage 15is pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaC1, 2.5 mM KC1, 10 mM MgC1 2 , 10 mM MgSO 4 and 20, mM glucose) at +37C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 gg/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned 20 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 25 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.

WO 98/56816 PCT/SE98/01093 13 Isolation and Preparation of plasmid DNA from BL21 transformants Individual clones of recombinant pET-28b vectors carrying properly cloned H. 5 pylori ORFs were picked and incubated in 5 ml of LB broth plus 25 tg/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA). o10 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, DH5a., etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for 15 T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lacI gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-3-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 20 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 25 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 gg/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 gg/ml) to an optical density at 600 nm of 0.5 to 1.0 O.D.

WO 98/56816 PCT/SE98/01093 14 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 5 centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4oC. Pellets were resuspended in 50 ml cold 10 mM Tris-HC1, pH 8.0, 0.1 M NaC1 and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at +4oC. Wet pellets were weighed and frozen at -80oC until ready for protein purification. 10 Analytical Methods The concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid 15is 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. 20 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 -galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 25 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).

WO 98/56816 PCT/SE98/01093 15 Purification of FlgE from inclusion bodies The following steps were carried out at +4oC. Cell pellets were resuspended in lysis buffer with 10% glycerol 200 gg/ml lysozyme, 5 mM EDTA, 1 mM PMSF and 5 0.1% -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% P-mercaptoethanol, then with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1% P-mercaptoethanol. The o10 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% 03 15 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% P3 20 mercaptoethanol, and developed with sequential steps of lysis buffer containing 8 M urea, 1 mM PMSF, 0.1% P-mercaptoethanol and 20, 100, 200, and 500 mM imidazole. Fractions were monitored by absorbance at OD 280 nm, 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. 25 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 30 containing 0.5% DOC with sequential reduction in urea as follows; 6M, 4M, 3M, WO 98/56816 PCT/SE98/01093 16 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 5 stirred cells. Protein concentrations were then measured by the methods of Perkins, Bradford and Lowry. EXAMPLES OF THE INVENTION 10 EXAMPLE 1: THERAPEUTIC IMMUNIZATION 1. Materials & Methods 15 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. 20 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 25 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 0 C in a microaerophilic atmosphere (10% CO 2 , 5% 02). The animals were given an oral dose of omeprazole (400 imol/kg) and after 3-5 h an oral inoculation of H. pylori (approximately 107-108 CFU/animal). 30 Infection was checked in control animals 2-3 weeks after the inoculation.

WO 98/56816 PCT/SE98/01093 17 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 5 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 gg/mouse of cholera toxin (CT) with each immunization. Omeprazole 10 (400 gmol/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 gl PBS with 0.5% DOC containing 10 gg CT Group 2: 300 gl PBS with 0.5% DOC containing 100 gg FlgE and 10 gg CT. 15 1.4. Analysis of infection The mice were sacrificed by CO 2 and cervical dislocation. The abdomen and chest cavity was opened and blood sampled by heart puncture. Subsequently the 20 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 25 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.

WO 98/56816 PCT/SE98/01093 18 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. 5 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. 10 2. Results 2.1. Therapeutic immunization: effects on CFU 15is 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 20 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<0.0 5 , respectively; Wilcoxon-Mann-Whittney sign rank test). 25 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 30 could a specific serum IgG titer against FlgE be measured (Fig. 2).

WO 98/56816 PCT/SE98/01093 19 FlgE specific IgG increased in animals given FlgE + CT, but could not be detected in control animals. 5 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 particulate fraction of H. pylori. In addition, a dramatic decrease in 10 number of colonizing H. pylori in the gastric mucosa of the infected mice was found following immunization with FlgE toghether with cholera toxin.

WO 98/56816 PCT/SE98/01093 20 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Astra AB (B) STREET: Vdstra Malarehamnen 9 (C) CITY: S6dertalje (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: PatentIn 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 WO 98/56816 PCT/SE98/01093 21 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 Gln Ala His Gln Ile Ala Leu Asp Ile Glu Ser Asn Asn Ile 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 Gln Val Lys Leu Ile 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 Gln 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 Ile Phe Ser Gln Gly Asn Ile Gln 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 Ile Gln Gly Asp Gly Phe Phe Ile Ile 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 Ile 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 Gln Gly Ser Leu Val Thr Thr Gly Gly Leu Val Val Gln 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 Ile Arg Ile 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 Ile 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 Gln Thr Ala Ala Ile 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 Ile 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 Gln 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 WO 98/56816 PCT/SE98/01093 22 Ser Leu Tyr Asn Glu Asp Gly Asp Ala Leu Leu Leu Asn Glu Asn Gln 235 240 245 250 GGG ATT TGG GTG AGC TAT AAG AGT CCA AAA ATG GTC AAA GAC ATC CTC 1118 Gly Ile Trp Val Ser Tyr Lys Ser Pro Lys Met Val Lys Asp Ile 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 Ile 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 Ile Asn Ala Val Lys Ser Gln Thr Gly Ile 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 Gln 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 Ile Val Val Thr Gln 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 Ile Ser Pro Asn Ala Asn Ser Gly Gln 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 Ile Gln His Asp Ala Asn Ile 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 Gln Asp Ser Ala Ala Ser Ile 395 400 405 410 GGA GTT ACA ATC AAC CAA TAC GGC ATG TTT GAA ATC AAC AAT AAA GAC 1598 Gly Val Thr Ile Asn Gln Tyr Gly Met Phe Glu Ile 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 Ile Lys Glu Asn Leu Asn Ile 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 Ile 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 Ile Asp Val Ile 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 Ile Glu Phe Tyr Arg Ser Gly 495 500 505 WO 98/56816 PCT/SE98/01093 23 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 Ile 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 Gln Phe Asp Pro Lys Asn Gly Ala Asp Ala Pro Gln Arg Ile 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 Ile Ser Glu Thr Tyr Ala Ile Glu Gln Asn Gly Tyr Gln 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 Gln 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 Gln Ala Leu Gly Gly Asn Val Phe Ser Gln 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 Gln Ala Leu Ile 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 Ile 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 Ile Val Val Gln Arg Gly Phe Gln 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 Gln Ile Leu Asn Thr Leu Leu 700 705 710 AAT CTT AAG CAA TAA ACTAAAGGAT TACTCTAATA CAATATAATA GGGGCTAATT 2517 Asn Leu Lys Gln * 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 WO 98/56816 PCT/SE98/01093 24 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Leu Arg Ser Leu Trp Ser Gly Val Asn Gly Met Gln Ala His Gln 1 5 10 15 Ile Ala Leu Asp Ile Glu Ser Asn Asn Ile Ala Asn Val Asn Thr Thr 20 25 30 Gly Phe Lys Tyr Ser Arg Ala Ser Phe Val Asp Met Leu Ser Gln Val 35 40 45 Lys Leu Ile Ala Thr Ala Pro Tyr Lys Asn Gly Leu Ala Gly Gln Asn 50 55 60 Asp Phe Ser Val Gly Leu Gly Val Gly Val Asp Ala Thr Thr Lys Ile 65 70 75 80 Phe Ser Gln Gly Asn Ile Gln Asn Thr Asp Val Lys Thr Asp Leu Ala 85 90 95 Ile Gln Gly Asp Gly Phe Phe Ile Ile Asn Pro Asp Arg Gly Ile Thr 100 105 110 Arg Asn Phe Thr Arg Asp Gly Glu Phe Leu Phe Asp Ser Gln Gly Ser 115 120 125 Leu Val Thr Thr Gly Gly Leu Val Val Gln 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 Ile Arg Ile Asp Pro Gly Met Val Met 165 170 175 Pro Ala Arg Ala Ser Asn Arg Ile Ser Met Arg Ala Asn Leu Asn Ala 180 185 190 Gly Arg His Ala Asp Gln Thr Ala Ala Ile Phe Ala Leu Asp Ser Ser 195 200 205 Ala Lys Thr Pro Ser Asp Gly Ile Asn Pro Val Tyr Asp Ser Gly Thr 210 215 220 Asn Leu Ala Gln 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 Gln Gly Ile Trp Val Ser Tyr 245 250 255 Lys Ser Pro Lys Met Val Lys Asp Ile Leu Pro Ser Ala Glu Asn Ser 260 265 270 Thr Leu Glu Leu Asn Gly Val Lys Ile Ser Phe Thr Asn Asp Ser Ala 275 280 285 Val Ser Arg Thr Ser Ser Leu Val Ala Ala Lys Asn Ala Ile Asn Ala 290 295 300 Val Lys Ser Gln Thr Gly Ile Glu Ala Tyr Leu Asp Gly Lys Gln Leu 305 310 315 320 Arg Leu Glu Asn Thr Asn Glu Leu Asp Gly Asp Glu Lys Leu Lys Asn WO 98/56816 PCT/SE98/01093 25 325 330 335 Ile Val Val Thr Gln 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 Ile 355 360 365 Ser Pro Asn Ala Asn Ser Gly Gln Phe Arg Thr Thr Glu Asp Leu Arg 370 375 380 Ala Leu Ile Gln His Asp Ala Asn Ile Val Lys Asp Pro Ser Leu Ala 385 390 395 400 Asp Asn Tyr Gln Asp Ser Ala Ala Ser Ile Gly Val Thr Ile Asn Gln 405 410 415 Tyr Gly Met Phe Glu Ile Asn Asn Lys Asp Asn Lys Asn Val Ile Lys 420 425 430 Glu Asn Leu Asn Ile 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 Ile Glu Gly Gly Ala Ser Ala Ser Ser Ser Lys Phe Thr His Ala 465 470 475 480 Thr His Ala Thr Ser Ile Asp Val Ile Asp Ser Leu Gly Thr Lys His 485 490 495 Ala Met Arg Ile Glu Phe Tyr Arg Ser Gly Gly Ala Asp Trp Asn Phe 500 505 510 Arg Val Ile 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 Gln Phe Asp Pro Lys Asn 545 550 555 560 Gly Ala Asp Ala Pro Gln Arg Ile Asn Leu Ala Phe Gly Ser Ser Gly 565 570 575 Ser Phe Asp Gly Leu Thr Ser Val Asp Lys Ile Ser Glu Thr Tyr Ala 580 585 590 Ile Glu Gln Asn Gly Tyr Gln 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 Gln Val Ala Leu Ala Asn Phe Ala Asn Asp Ala Gly Leu 625 630 635 640 Gln Ala Leu Gly Gly Asn Val Phe Ser Gln Thr Gly Asn Ser Gly Gln 645 650 655 Ala Leu Ile Gly Ala Ala Asn Thr Gly Arg Arg Gly Ser Ile Ser Gly 660 665 670 Ser Lys Leu Glu Ser Ser Asn Val Asp Leu Ser Arg Ser Leu Thr Asn WO 98/56816 PCT/SE98/01093 26 675 680 685 Leu Ile Val Val Gln Arg Gly Phe Gln Ala Asn Ser Lys Ala Val Thr 690 695 700 Thr Ser Asp Gln Ile Leu Asn Thr Leu Leu Asn Leu Lys Gln * 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 (17)

1. A Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, for use in inducing a protective immune 5 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 10 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 15is together with a pharmaceutically acceptable carrier or diluent.

4. A vaccine composition according to claim 3 in addition comprising an adjuvant. 20

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 25 Helicobacter pylori. WO 98/56816 PCT/SE98/01093 28

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. 5

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 10 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. 15

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. 20

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. 25

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. WO 98/56816 PCT/SE98/01093 29

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. 5

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 effective amount of a Helicobacter pylori FlgE polypeptide as defined in claim 1 or 2. 10

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. 15

17. A method according to claim 15 or 16 wherein the said mammal is a human.