SK173099A3 - Vaccine compositions comprising the helicobacter pylori flge polypeptide - Google Patents

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 a FlgE polypeptide of Helicobacter pylori

Technical field

The present invention relates to polypeptides and vaccine compositions for inducing a protective immune response against Helicobacter pylon infection. The invention further relates to the use of Helicobacter pylori polypeptides for the preparation of compositions for the treatment or prophylaxis of Helicobacter pylori infection.

BACKGROUND OF THE INVENTION

Helicobacter pylori

The Gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen that is responsible for some gastroduodenal diseases. Colonization of the stomach epithelium by bacteria leads to active inflammation and progressive chronic gastritis, with a significantly increased risk of progression to peptic ulcer disease. Long-term gastric mucosal inflammation is in a very close correlation with a significantly increased risk of gastric cancer.

A number of virulent factors are involved in the colonization of the gastric mucosa of H. pylori. Such virulent factors include adhesins (adhesions) by which the bacterium attaches to the mucosa and / or binds to epithelial cells; ureas that help neutralize the acidic environment; and proteolytic enzymes that make the mucosa more fluid. In addition, H. pylori is very mobile, running down the mucosa and down into the interior. Mobility has been shown to be an essential virulence factor because H. pylori has failed to infect the mucosa in experimental models performed by Eaton et al. (Infection & Immunity 64 (7), 2455-2448 1996).

There are many possible causes for this, the most understandable of which is the inability to flow down and reach the mucosal cells and the inability to avoid harmful agents in the stomach.

Despite a strong apparent host immune response to H. pylori, producing both local (mucosal) and systemic antibodies, the pathogen persists in the gastric mucosa normally during the life of the patient. The reason for this is probably that spontaneously induced immune responses are inadequate or directed against

- incorrect epitope of antigens. Alternatively, the immune response may be of the wrong species since the immune system may consider H. pylori to be symbiotic (as shown by the host / bacterial life ratio).

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 the exposed surface, the bonded surface as well as the secreted proteins, which have been shown to be major virulent factors in many bacterial pathogens and which may be useful in diagnosing H. pylori and in preparing vaccine compositions. When such proteins, in addition to being surface-linked, are also essential for survival and / or colonization, their utility as a target for vaccine-mediated immunotherapeutic targets increases.

In stress or at risk, a H. pylori cell is transformed from a bacillary form to a cocoid form. In a cocoid form, the H. pylori cell is less sensitive to antibiotics and other antibacterial agents. Detailed research suggests that H. pylori may be transmitted between individuals in this form, probably by water or direct contact (oral-oral, faecal-oral). Thus, an effective vaccine composition should elicit an immune response against both forms of H. pylori, cocoid and bacillary. Since systemic immunity is likely to play only a limited role in protecting against mucosal infections, it is also important that the vaccine composition enhances the protective immune mechanism locally in the stomach.

Flagellam hook protein

H. pylori flagellar hooks have been shown to consist of the 78kDa FlgE subunits (O'Toole et al., Molecular Microbiology, 14 (4), 691-703, 1994). The task of Flagelame hook is to connect Flagela with submembrane Flagellar hook movement. The portion of the extruded hook is short, about 60 nanometers (compared to about 10 micrometers for Flagela). Like Flagela H. pylori, the hook is probably covered with a coating (Geis et al., (1993), J. Med. Microbiol. 38 (5), 371-377).

The -3 amino acid sequence of the FlgE polypeptide has significant similarity to other known hook proteins, including limited homology to other Helicobacter species such as mustelae (O'Toole et al., Supra). Polyclonal antibodies raised against FlgE polypetide showed cross-reactivity to Flagela proteins A and B, probably indicating the existence of common epitopes. FlgE production paralyzes H. pylon, resulting in an aflagelama immobilized bacterium where FlgE polypeptide was produced but could be recaptured in the cytoplasm.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1

Effect of therapeutic immunization of H. pylori-infected mice (n = 9-10 / group) with FlgE polypeptide. Results are presented as mean ± SEM of the number of H. pylori associated with anthra (= A), body (= B) or total (A + B) (= C). Abbreviations: CFU, colony forming units (number of bacteria); unhatched bars = DOC + CT, phosphate buffered saline with 0.5% deoxycholate was co-administered with cholera toxin 10 µg / mouse; shaded bars = FlgE + CT, mice received 100 µg FlgE and 10 µg cholera toxin. The decrease in CFU was a significant vantrume and was calculated for the entire stomach.

** p <0.01; * p <0.05 (Wilcoxon-Mann-Whittney character classification test)

Figure 2

IgG serum from mice measured by ELISA methodology: response to infection and immunization with FlgE. Values were expressed as mean titers ± SEM, n = 9-10 / group. ELISA coated with H. pylori 244; specific antibodies could be detected as a symptom of H. pylori infection in the sera of animals treated with DOC + CT (= A, control / 244). Subsequent immunization with FlgE + cholera toxin (= B, FlgE / 244) increased this reactivity fourfold (** p <0.01; (Wicoxon-Mann-Whittney character test). C = FlgE specific. Specific FlgE increased in animals that received FlgE + CT but could not be determined for control animals.

4. Summary of the Invention

It is an object of the present invention to provide an antigenic H. pylori polypeptide that can be used to elicit a protective immune response against H. pylori and to diagnose H. pylori infection. This goal was achieved by recombinant cloning of the H. pylori gene, which encodes a well conserved parent polypeptide. The nucleic acid sequence of this gene is similar to that of the FlgE gene as reported by O'Toole et al., Molecular Microbiology, 14 (4), 691-703, 1994. As a basic motility protein, the FlgE gene is expressed by all H. pylori strains.

Surprisingly, it has been found that FlgE H. pylori polypeptide, despite the fact that only a small portion of the hook protein exists outside the bacteria and that it is likely to be coated, may serve as a therapeutic antigen in a model of H. pylori-infected mice when co-administered with an adjuvant. cholera toxin. Thus, the experimental data below indicates that FlgE polypeptide of H. pylori, when used as an oral immunogen, acts as an immune response stimulator leading to a significant reduction in H. pylori colonization in mice infected with H. pylori one month prior to immunization. .

These results strongly support the use of the H. pylori FlgE polypeptide in oral vaccine formulations for use in treating or preventing H. pylori infections in humans. FlgE protein itself will be useful in both H. pylori infections as well as in the manufacture of vaccine compositions which, when administered in suitable pharmaceutical formulations, will produce a protective or therapeutic immune response to such infections.

Therefore, the invention further provides a Helgobacter pylori FlgE polypeptide for use in eliciting a protective immune response against infections. The term "FlgE polypeptide Helicobacter pylori" is intended to be the polypeptide described by O'Toole et al. in Molecular Microbiology, 14 (4), 691-703, 1994, and which is encoded by a gene whose nucleotide sequence is shown as SEQ ID NO. 1, or may be obtained from the National Center for Biotechnology Information (Accession Number U09549) or a substantially similar modified form of said polypeptide that retains functional equivalent antigenicity.

-5 "Protective immune response" means an immune response that makes the composition suitable for therapeutic and / or prophylactic purposes.

By "functional equivalent antigenicity" is meant the ability to elicit a systemic and mucosal immune response, reducing the number of H. pylori cells associated with the gastric mucosa. Those of skill in the art will be able to identify modified forms of FlgE polypeptide that retain functional equivalent antigenicity, using known procedures, such as imaging epitopes with antibodies elicited in vivo.

In a preferred embodiment of the invention, the Heiicobacter pylori FlgE polypeptide for use in eliciting a protective immune response against Heiicobacter pylori infection, has substantially the amino acid sequence shown as SEQ ID NO. 2 of the Sequence Listing, or a modified form thereof that retains functional equivalent antigenicity.

Therefore, it is to be understood that the definition of the FlgE polypeptide of Heiicobacter pylori is not strictly limited to a polypeptide having an amino acid sequence that is identical to SEQ ID NO. 2 in the sequence list. Rather, the invention encompasses polypeptides that carry modifications, such as substitutions, small knockouts, insertions, or inversions, but which nevertheless have substantially the biological activity of the Heiicobacter pylori FlgE polypeptide and retain functional equivalent antigenicity. Therefore, polypeptides having an amino acid sequence of at least 90% homology, preferably at least 95% homology, with the amino acid sequence shown as SEQ ID No 2 in the Sequence Listing are included in the definition of Hegicobacter pylori FlgE polypeptide.

The invention further provides a vaccine composition for eliciting a protective immune response against a Heiicobacter pylori infection comprising an immunogenically effective amount of a Heiicobacter 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 understood to be an amount that elicits a significant protective response of Heiicobacter pylori that eradicates H. pylori infection in infected mammals or prevents infection in susceptible mammals. Typically, the immunologically effective amount will comprise

About 1 gg to 1000 mg, preferably about 10 µg to 100 mg, of H. pylori antigen for oral administration or about less than 100 µg for parenteral administration.

The vaccine composition optionally comprises, in addition to a pharmaceutically acceptable carrier or diluent, one or more other pharmaceutically effective antigens for prophylactic or therapeutic use. Physiologically acceptable carriers and diluents are well known to those skilled in the art and include, for example, phosphate buffered saline (PBS) or, in the case of oral vaccines, HCO3- or enteric-coated powder formulations.

The vaccine composition may optionally contain or be administered together with secretion inhibitors, preferably proton pump inhibitors (PPIs), for example omeprazole. The vaccine may then be formulated in known delivery systems such as liposomes, ISCOM, cochley, and the like (see, for example, Rabinovich et al., (1994), Science 265, 1401-1404) or attached or incorporated into the microspheres of a degradable or polymer. non-degradable nature. Antigens may be associated with live attenuated bacteria, viruses or phages, or with killed vectors of the same species. The antigens may be chemically or genetically linked to carrier or inert type carrier proteins (e.g., cholera B subunit). Furthermore, the invention relates to a vaccine composition according to the above, which additionally comprises an adjuvant such as cholera toxin. Such pharmaceutically acceptable forms of cholera toxin are known in the art, for example, 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.

The vaccine composition of the invention can be used for both therapeutic and prophylactic purposes. Thus, the invention encompasses a vaccine composition as defined above for use as a medicament or prophylactic vaccine for a mammal, including a human, who is infected with Helicobacter pylori. In this context, the term "prophylactic purpose" means inducing an immune response that will prevent future infection with Helicobacter pylori, while the term "therapeutic purpose" means inducing an immune response that may eradicate existing Helicobacter pylori infections.

The vaccine composition of the invention is preferably administered through the mammalian mucosa, for example, through the buccal, nasal, tonsillar, stomach or intestinal (small and large intestine), rectal and vaginal mucosa. The mucosal vaccine may be administered together with excipients suitable for this purpose. The vaccine can also be administered orally or parenterally, subcutaneously, intracutaneously or intramuscularly, optionally together with suitable excipients. The vaccine composition may optionally be administered together with a suitable excipient. Optionally, the active ingredient composition may be co-administered with an antimicrobial therapeutic agent.

The invention further relates to the use of a Helgobacter pylori FlgE polypeptide as defined above for the preparation of (i) a composition for the treatment, prophylaxis or diagnosis of a Helicobacter pylon infection;

(ii) a vaccine for use in eliciting a protective immune response against Helicobacter pylon; and (iii) a diagnostic kit for diagnosing Helicobacter pylori infection.

In addition, the invention still provides a method for in vitro diagnosing Helicobacter pylori infection, which comprises at least one step, wherein the Helicobacter pylori FlgE polypeptide as defined above is optionally used labeled or associated with a solid support. For example, said method could comprise the steps of (a) contacting said Helicobacter pylori FlgE polypeptide, optionally bound to a solid support, with a body fluid removed from the mammal; and (b) detecting antibodies from said body fluid by binding to said FlgE polypeptide. Preferred methods for detecting antibodies are ELISA (Enzyme-linked immunoabsorbed assay) methods, which are well known in the art.

The present invention further provides a diagnostic kit for detecting a Helicobacter pylori infection in a mammal, including a human, comprising components that enable the method of in vitro diagnosis as described above to be performed. Said diagnostic kit may comprise, for example: (a) a Helicobacter pylori FlgE polypeptide;

(B) reagents for detecting antibodies binding to said FlgE polypeptide. Said antibody detection reagents could be, for example, an enzyme-labeled anti-immunoglobulin and a chromogenic substrate for said enzyme.

Still further, the invention provides a method of inducing in a mammal, including a human, a protective immune response against Helicobacter pylon infection, said method comprising the step of administering to said mammal an immunologically effective amount of a Helicobacter pylori FlgE polypeptide as defined above or alternatively administering to said mammal an immunologically effective amount. the vaccine composition as defined above.

Experimental methods

Throughout this specification, the terms "standard protocol" and "standard procedure" used in the context of genetic engineering methods are to be understood as protocols and procedures found in a standard laboratory manual such as: Current Protocols in Molecular Biology, Ed. F. Ausubel et al., John Woley and Sons, Inc. 1994, or Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A laboratory manual, 2 ^nd 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 encoded by 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 ORFs for membranes and secreted proteins from the J99 strain Helicobacter pylori.

The sequences were cloned from the J99 strain of H. pylori by amplification cloning using a polymerase chain reaction (PCR). Synthetic oligonucleotide primers (see below) specific for the 5 'and 3' ends of the open reading frames (GibcoBRL Life Technologies, Gaithersburg, MD) were designed and obtained. The original primers (specific for the 5'-ends of the sequence) for FlgE were designed to include

The 99 NcoI cloned site at the extreme 5'-end, wherein the reverse primers included an EcoRI site at the extreme 5'-end to allow cloning of each H. pylori sequence into the reading frame of the pET-28b vector. The inserts cloned into the NcoEcoR1 sites of the pET-28b vector were fused to a DNA sequence vector encoding additional 20 carboxy-terminal amino acids, including six histidine residues (at the extreme C-terminus).

Original 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'

Genomic DNA prepared from Helicobacter pylori J99 strain was used as a source of DNA matrix for PCR amplification reactions (Current Protocols in Molecular Biology, Ed. F. Ausubel et al., John Wiley and Sons, Inc. 1994). To amplify the DNA sequence containing the H. pylori ORF, genomic DNA (50 ng) was introduced into a reaction vial containing 2 mM MgCl2, 1 μΜ of synthetic oligonucleotide primers (parent and reverse primers) complementary to the defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP and 2.5 units of heat stabilized DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 μΙ. The following cyclic conditions were used to obtain amplified DNA products for each ORF used, using a Perkin Elmer Cetus / GeneAmp 9600 thermal cycle PCR system:

Denaturation at +94 ° C for 2 min;

cycles at +94 ° C for 15 sec., +30 ° C for 15 sec. and +72 ^e C for 1.5 min;

cycles at +94 ° C for 15 sec., +58 ° C for 15 sec. and + 72 ° C for 1.5 min; Reactions were concluded at + 72 ^e C for 6 minutes.

After thermal cycling reactions were completed, each amplified DNA sample was washed and purified using a Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). The amplified DNA samples were digested

-10 with Nde I and Eco RI restriction endonucleases using standard procedures. DNA samples were then subjected to agarose gel electrophoresis on a 1.0% NuSeive (FMC BioProducts, Rockland, ME, USA). DNA was visualized by exposure to ethidium bromide and UV long wave irradiation. DNA contained in aliquots of isolated zagarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).

Cloning of H. pylori DNA sequences into pET-28b prokaryotic expression vector pET-28b vector was prepared for cloning by digestion with s / col and EcoRI according to standard procedures. After digestion, the DNA inserts were cloned according to standard procedures into the originally digested pET-28b expression vector. The ligation reaction products were then used to transform the BL21 strain of E. coli as described below.

Transformation of a suitable bacterium with recombinant plasmids

Appropriate E. coli strain BL21 or E. coli strain BL21 (DE3) was transformed with recombinant pET expression plasmids carrying cloned H. pylori sequences according to standard procedures. Briefly, 1 μΙ of the ligation reaction mixture was mixed with 50 μΙ of electrocompetent cells and subjected to a high voltage pulse, after which the samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl). , 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO _4, and 20 mM glucose) at + 37 ° C with shaking for one hour. Samples were then plated on LB agar plates containing 25 µg / ml kanamynsulfate on and allowed to grow overnight. Transformed BL21 colonies were then harvested and analyzed for evaluation of the rounded inserts described below.

Identification of recombinant pET expression plasmids carrying H. pylori sequences

Individual BL21 clones transformed with recombinant H. pylori pET-28b genes were analyzed by PCR amplification of the cloned inserts using the original and reverse primers, specifically for each H. pylori sequence that

Were used in the original PCR amplification cloning reactions. Subsequent amplification verified the integration of H. pylon sequences in the expression vector according to standard procedures.

Isolation and preparation of plasmid DNA from BL21 transformants

Individual clones of recombinant pET-28b vectors carrying appropriately cloned H. pylori ORFs were harvested and incubated overnight in 5 ml LB broth plus 25 µg / ml kanamycin sulfate. The following day, plasmid DNA was isolated and purified using a Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA).

Expression of the recombinant H. pylon sequence in E. coli

The pET vector can be propagated in any K-12 strain of E. coli, for example HMS174, HB101, JM109, DH5α, and the like, for cloning or plasmid preparation purposes. Expression hosts include E. coli strains containing a chromosomal copy of the T7 RNA polymerase gene. These hosts are the bacteriophage DE3 lysogens, a lambda derivative that carries the lacI gene, the lacUV5 promoter, and the T7 RNA polymerase gene. T7 RNA polymerase is induced by the addition of isopropyl-β-Dtiogalactoside (IPTG) and T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its significant gene. 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).

For the expression of recombinant H. pylori sequences, 50 ng of plasmid DNA, isolated as described above, was used to transform the respective BL21 (DE3) bacterium as described above (provided by Novagen as part of the pET expression system kit). The transformed cells were cultured in SOC medium for one hour and the cultures were then plated on LB plates containing 25 µg / ml kanamycin sulfate. The following day, the 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 the expression of H. pylori recombinant DNA constructs.

After inducing expression of the sIPTG gene, the bacteria were granulated by centrifugation in a Sorval RC-3B centrifuge at 3500 x g for 15 minutes at 4 ° C. The granules were resuspended in 50 ml cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). The cells were then centrifuged at 2000 x g for 20 minutes at + 4 ° C. The wet granules were weighed and frozen at -80 ° C until ready for protein purification.

Analytical methods

Concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from the amino acid content (Perkins, S.J., 1986 Eur. J. Biochem, 157, 169-180). Protein concentrations were also measured using the method described by 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 dodecylsulfate-polyacrylamide (SDS-PAGE) gels (acrylamide gradient of 12% or 4 to 25%) were obtained from BioRad (Hercules, CA, USA) and stained using Coomasie Brilliant Blue. Molecular weight markers containing 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 carbon 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 implied bodies

The following steps were performed at +4 ° C. The cell granules were resuspended in lysis buffer with 10% glycerol, 200 µg / ml lysosome, 5 mM EDTA, mM PMSF and 0.1% β-mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was treated with 0.2% DOC, stirred for 10 minutes, then centrifuged (10,000 x g for 30 minutes). The granules 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 granules consisted primarily

-13 from implied bodies that did not contain disrupted cells and membranous materials.

The following steps were performed at room temperature, the implied bodies were dissolved in 20 ml of 8 M urea in lysis solution with 1 mM PMSF and 0.1% β-mercaptoethanol and incubated at room temperature for one hour. Substances that did not dissolve were removed by centrifugation (100.00 xg for 30 min). The clear supernatant was filtered off and loaded onto a Ni ²⁺ -NTA agarose column which was equilibrated with 8 M urea in lysis buffer. The column was washed with 250 ml (50 bed volumes) lysis buffer containing 8 M urea, 1 mM PMSF and 0.1% β-mercaptoethanol and developed with stepwise lysis solution containing 8 mM urea, 1 mM PMSF, 0.1% β-mercaptoethanol and 20, 100, 200 and 500 mM imidazole. Fractions were monitored by absorbance at OD 280 nm and fractions peaks were analyzed by SDS-PAGE. Two bands were visualized by staining using Coomassie Brilliant Blue, the main band Mr = 78 kDa and the minor band Mr = 60 kDa. The purity of recombinant FlgE (78 kDa) was evaluated to be greater than 90%. As with the purification of soluble proteins, the fractions containing the recombinant protein were eluted at 100 mM midazole.

Urea was slowly removed from FlgE polypeptide by dialysis versus TBS containing 0.5% DOC with the following gradual decrease in urea: 6 M, 4 M, 3 M, 2 M, 1 M, 0.5 M and then 0 M. Each dialysis step was performed for at least 4 hours at room temperature.

After dialysis, samples were concentrated by pressure filtration using Amicon stirred cells. Protein concentrations were then determined using Perkins, Bradford and Lowry procedures.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Therapeutic immunization

1. Material and methods

- ΜΙ. 1. Animals

Female SPF BALB / c mice were obtained from the Bomholt Breeding Center (Denmark).

They were kept in conventional macrocolumn cages with free water and food. At delivery, the animals were 4-6 weeks of age.

1.2. infection

After at least one week of acclimatization, the animals were infected with type 2 strain H. pylori (strain 244, originally isolated from an ulcerative patient). This strain has previously been shown to be a good colonizer in the stomach of mice. Bacteria from the strain maintained at -70 ° C were grown overnight in Brucel 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 pmol / kg) and after 3-5 hours an oral inoculation of H. pylori (approximately 10 ⁷ to 10 ⁸ CFU / animal). Infection was examined in control animals 2 to 3 weeks after inoculation.

1.3 Immunizations

One month after infection, two groups of mice (10 mice / group) were immunized four times over a 34-day period (days 1, 15, 25 and 35). Purified recombinant FlgE dissolved in PBS plus 0.5% deoxycholate (DOC) was administered at a dose of 100 micrograms / mouse.

As an adjuvant, animals in both control groups and FlgE group were given 10 µg / mouse cholera toxin (CT) with each immunization. Omeprazole (400 μηιοΙ / kg) was administered orally to all animals 3 to 5 hours prior to immunization as a way to protect antigens from acid degradation. Animals were sacrificed 1-2 weeks after the last immunization.

Group 1: 300 μΙ PBS with 0.5% DOC containing 10 gg CT

Group 2: 300 μΙ PBS with 0.5% DOC containing 100 μg FlgE and 10 μg CT.

1.4. Analysis of infection

-15Mice were sacrificed using CO ₂ and neck breakage. The abdomen and thoracic cavity were opened and blood was collected by puncture from the heart. Subsequently, the stomach was removed. After cutting the stomach along the greatest curvature, the stomach was washed with saline and then cut into two equal parts. The 25 mm ^{2 area of the} mucosa of the zantrum and the corpus were scraped separately using a surgical scalpel. The scraped mucosa was suspended in Brucel broth, diluted and plated on Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies counted. The identity of H. pylon was evaluated by urea and a catalase and direct microscopy or Gram stain assay.

1.5. Measurement of antibodies

Serum antibodies were collected from blood. Prior to centrifugation, blood was diluted with an equal amount of PBS. The serum was maintained at -20 ° C until analysis. Serum antibodies were measured using ELISA, wherein the plates were coated with either fragmented H. pylon strain or FlgE, and various serum dilutions were then added. ELISA was developed with alkaline phosphatase labeled mouse anti-Ig antibodies. Anti-Ig antibodies were of the anti-heavy / anti-light chain type, which should detect all types of antibodies.

2. Results

2.1. Therapeutic immunization: effect on CFU

Animals in this study were infected with strain H. H. pylori one month before immunization. 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 last immunization, animals were sacrificed and CFU was determined (Figure 1). Animals treated with CT alone were highly infected in both the corpus and the antrum. The animals actively immunized with the recombinant FlgE polypeptide and CT showed a significant decrease in CFU values in the antrum and stomach compared to animals immunized with CT (p <0.01 and p <0.05; Wilcoxon-Mann-Whittney character classification test).

2.2 Therapeutic immunization: effect on antibody production and secretion

As a symptom of H. pylori infection, specific antibodies can be found in the serum (control / 244). In animals receiving FlgE + CT, titer to strain 244 (as membrane proteins) increased fourfold (p <0.01). Only in the animals they received

FlgE + CT could be measured for specific IgG titer to FlgE (Figure 2).

FlgE specific IgG was increased in animals receiving FlgE + CT but could not be detected in control animals.

The results show that the recombinant H. pylon FlgE polypeptide is highly immunogenic when administered orally, together with cholera toxin as an adjuvant, measured as an increase in systemic FlgE specific Ig antibodies. Immunization with FlgE also resulted in a significant increase in ig titers relative to the fragmented H. pylon fraction. In addition, a dramatic decrease in the number of H. pylori colonizing the mucosa of the stomach of infected mice was found after immunization with FlgE together with cholera toxin.

-17 Sequence Listing (1) General (i) Applicant (A) Name: Astra AB (B) Street: Västra Mälarehamnen 9 (C) City: Södertälje (E) Country: Sweden (F) Post Code (ZIP): S- 151 85 (G) Telephone: +46 8 553 260 00 (H) Fax: +46 8 553 288 20 (i) Title of the invention: Vaccine compositions containing the Helicobacter pylori FlgE polypeptide (ii) Number of sequences: 4 (iii) Computer readable form:

(A) Media Type: Floppy Disk (B) Computer: IBM PC Compatible (C) Operating System: PC-DOS / MS-DOS (D) Software: Patentln Reease # 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) String: double (D) Topology: linear (ii) Molecule type: cDNA (vi) Original source:

(A) Organism: Helicobacter pylori (ix) Characteristics:

(A) Name / Key: CDS (B) Localization: 321..2477 (D) Other Information: / Product = “FlgE Flagellam Hook Protein (x) Editorial Information:

-18 (A) Authors: O'Toole, Paul W., Kostrzynska, Magdalena, Trust, Trevor J.

(B) Title: Non-motile mutants of Helicobacter pylon and Helicobacter mustelae defective in flagellar hook production (C) Magazine: Mol. Microbiology (D) Volume: 14 (E) Number: 4 (F) Pages: 691 - 703 (G) Date: 1994 (xi) Description of Sequence: SEQ ID NO: 1:

AACAAAGCGA TAACTCCTTT GTCTTATTAG CGACACAATT TAACCCATTG ACTTTAAATC GCGCTTCAGC cgaagagatt caagatcatg aatgcgcgat tttgcactaa AGCGAGTTÄG attcttaäat ttgagcgata

19CCCTATCCCC TTATGAATTT

CÄATCATTCT AAAAAGCTAT

CTTTAAATTA AAGGATAACC

GACCGATCTT TTTGATTAAC AAAACTTTAA AATCCGCAAT

TTAGGAACAA CTTTTGCTTT ATTTTGCATA GATTGAATTT

ATG CTT AGG TCT TTA TGG TCT GGT GTC AAT Met Leu Arg Ser Leu Tro Ser Gly Val Asn

10

GGG ATG CAA GCC CAC CAA ATC GCT TTG GAT ATT GAG AGT AAC AAT ATT Gly Met gin Ala His 15 gin Ile Ala Leu Aso 20 Ile Glu Ser same time same time 25 Ile GCG AAC GTG AAT ACC ACT GGT TTT AAG TAT TCT AGG GCT TCT TTT GTG Ala same time wall same time 30 Thr Thr Gly Phe Lys 35 Tyr Ser Arg Ala Ser 40 Phe wall GAT ATG CTT TCT CAA GTC AAA CTC ATC GCT ACC GCA CCC TAT AAA AAC Asp Met Leu 45 Ser gin wall Lys Leu 50 Ile Ala Thr ! · Ala for 55 Tyr Lys same time GGG TTA GCA GGG CAG AAT GAT TTT TCT GTG GGG CTT GGG GTA GC-C GTG Gly Leu 60 Ala Gly gin same time Aso 65 Phe Ser wall Gly Leu 70 Gly wall Gly wall GAT GCG ACG ACT AAA ATC TTT TCA CAA GGC AAT ATC CAA AAC ACA GAT Aso 75 Ala Thr Thr Lys Ile 30 Phe Ser gin Gly same time 85 Ile gin same time Thr Aso 90 GTC AAA ACC GAT CTA GCG ATT CAA GGC GAT GGC TTT TTT ATC ATT AAC wall Lys Thr Asp Leu 95 Ala Ile gin Gly Aso 10Ô Gly Phe Phe Ile Ile 105 same time CCT GAT AGG GGG ATC ACG CGC AAT TTC ACT AGA GAT GGG GAG TTC CTT for Asp Arg Gly Ile Thr Arg same time Phe 115 Thr Arg Asp Gly Glu 120 Phe Leu fflipip GAC TCG CAA GGG AGT TTG GTT ACC ACC GGC GGG CTT GTG GTG CAA Phe Asp Ser 125 gin Gly Ser Leu wall 130 Thr Thr Gly Gly Leu 135 wall wall gin GGG TGG GTG AGA AAT GGG AGC GAT ACC GGC AAT AAA GGG AGC GAT ACA Gly Trp 140 wall Arg same time Gly Ser 145 Asp Thr Gly same time Lys 150 Gly Ser Asp Thr GAC GCT TTA AAA GTG GAT AAC ACC GGT CCT TTA GAA AAC ATT AGG ATT Asp 155 Ala Leu Lys wall Asp 160 same time Thr Gly for Leu 165 Glu same time Ile Arg Ile 170 GAT CCT GGA ATG GTG ATG CCA GCC AGA GCG AGT AAC CGC ATT TCT ATG Asp for Gly Met wall 175 Met for Ala Arg Ala 180 Ser same time Arg Ile Ser 185 Met AGG GCG AAT TTA AAC GCT GGA AGG CAT GCC GAT CAA ACA GCG GCG ATA Arg Ala same time Leu 190 same time Ala Gly Arg His 195 Ala Asp gin Thr Ala 200 Ala Ile TTC GCT TTG GAT TCT TCA GCC AAA ACC CCT TCA GAT GGC ATT AAT CCG Phe Ala Leu 205 Asp Ser Ser Ala Lys 210 Thr for Ser Asp Gly 215 ill same time for GTG TAT GAT TCA GGC ACG AAT CTT GCT CAA GTC GCC GAA GAC ATG GGA wall Tyr Asp Ser Gly Thr same time Leu Ala gin wall Ala Glu Asp Met Gly

220 225 230 'CT TTA TAC AAT GAA GAT GG GAT GCT CTT TTG TTG AAT GAA AAT CAA

Ser Leu Tyr same time Glu Asp Gly Asp Ala . Leu Leu Leu , Asn . Glu . same time i Gin 235 240 245 250th GGG ATT TGG GTG AGC TAT AAG AGT CCA AAA ATG GTC : AAA GAC ATC CTC Gly Ile Trp wall Ser Tyr Lys Ser for Lys Met wall Lys Asp Ile Leu 255 260 265 CCT TCT GCA GAA AAC AGC ACG CTT GAA TTG AAT GGC GTT AAG ATT TCT for Ser Ala Glu same time Ser Thr Leu Glu Leu same time Gly wall Lys Ile Ser 270 275 280 TTC ACA AAC GAT TCA GCG GTG AGC CGG ACT TCA AGC TTA GTG GCG GCT Phe Thr same time Asp Ser Ala wall Ser Arg Thr Ser Ser Leu wall Ala Ala 285 290 295 AAA AAT GCG ATC AAT GCA .GTC .aaa AGC CAA ACA GGC ATT GAA GCT TAT Lys 'Asn Ala Ile same time Ala wall Lys Ser gin 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 Leu Asp Gly Lys gin Leu Arg Leu Glu same time Thr same time Glu Leu Asp Gly 315 320 325 330 GAT GAA AAG CTT AAA AAC ATT GTA GTT ACT CAA GCC GGA ACC GGA GCG Asp Glu Lys Leu Lys same time Ile wall wall Thr gin Ala Gly Thr Gly Ala 335 340 345 TTC GCT AAC TTT TTA GAC GC-C GAT AAA GAT GTA ACG GCT TTC AAA TAC Phe Ala same time Phe Leu Asp Gly Asp Lys Aso wall Thr Ala Phe Lys Tyr 350 355 360 AGC TAC ACG CAT TCT ATT AGC CCT AAC GCC AAT AGC GGG CAG TTT AGG Ser Tyr Thr His Ser Ile Ser for same time Ala same time Ser Gly gin Phe Arg 365 370 375 ACC ACT GAA GAC TTG CGC GCC TTA ATC CAG CAT GAC GCT AAT ATC GTT Thr Thr Glu Asp Leu Arg Ala Leu Ile gin His Asp Ala same time Ile wall 380 385 390 AAA GAT CCT AGC CTA GCG GAC AAT TAC CAA GAC TCA GCC GCT TCT ATA Lys Asp for Ser Leu Ala Asp same time Tyr gin asd 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 Gly wall Thr Ile same time gin Tyr Gly Met Phe Glu Ile same time same time Lys Asp 415 420 425 AAT AAA AAT GTC ATT AAA GAA AAT CTT AAT ATC TTT GTG AGC GGG TAT same time Lys same time wall Ile Lys Glu same time Leu same time Ile Phe wall Ser Gly Tyzr 430 435 440 TCT TCA GAC AGC GTA ACG AAC AAT GTT TTG TTT AAA AAT GCG ATG AAA Ser Ser Aso Ser wall Thr same time A.sn wall Leu Phe Lys same time Ala Met Lys 445 450 455 GGG CTT AAT ACC GCT ψΓ · ΠΊ TTA ATT GAA GGG GGA LG TCA G77 AGC AGT Gly Leu same time Tr.r Ala apply.` Leu Ile Glu Gly Gly . and Ser A_S. Ser Ser 460 465 470 AAA TTC ACC CAC GCT ACG CAT GCG ACA AGC ATT GAT GTG ATA GAC Ser Lys Phe Thr His Ala Thr His Ala Thr Ser Ile Asp wall Ile Asp 475 480 485 490 AGC TTA GGC ACT AAA CAC GCC ATG CGC ATT GAG TTT TAT AGG AGT GGG Ser Leu Gly Thr Lys His Ala Met Arg ill Glu Phe Tyr Arg Ser Gly

495 500 505

-21GGA GAT GAT TGG AAT TTT AGA GTG ATC GTG CCT GAG CCT GGG GAA TTA Gly Ala Asp

(+420) 510 515 520

GTA GGG GGG TCA GCG GCT AGG CCT AAT GTG TTT GAA GGA GGC CGT TTG Val Gly Gly Ser Ala Ala

525 530 535

CAC TTC AAT AAT GAC TCG CTT GCA GGC ATG AAC CCG CCT CTT TTG His Phe Asn Asp Gly Ser Leu Ala Gly Met same time 550 For Leu Leu 540 545 CAA TTT GAC CCT AAA AAT GGT GCT GAT GCC CCC CAA CGC ATC AAT TTA gin Phe Asp for Lys same time Gly Ala Asp Ala for gin Arg Ile same time Leu 555 560 565 570 GCT TTT GGT TCC TCA GGG AGT TTT GAC GGG CTA ACG AGC GTG GAT AAG Ala Phe Gly Ser Ser Gly Ser Phe Asp Gly Leu Thr Ser wall Aso Lys 575 580 585 ATT TCT GAA ACT TAT GCG ATT GAG CAA AAC GGC TAT CAA GCG GGC GAT Ile Ser Glu Thr Tyr Ala Ile Glu gin same time 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 Leu Met Aso wall Arg Phe Asp Ser Asp Gly wall Leu Leu Gly Ala Phe 605 610 615 AGT AAT GGC AGG ACT TTA GCG CTC GCT CAA GTG GCT TTA GCG AAT TTC Ser same time Gly Arg Thr Leu Ala Leu Ala gin wall Ala Leu Ala same time Phe 620 625 630 GCT AAC GAT GCG GGC TTG CAG GCT TTA GGC GGG AAT GTC TTT TCT CAA Ala same time Asp Ala Gly Leu gin Ala Leu Gly Gly same time wall Phe Ser gin 635 640 645 650 ACC GGA AAC TCA GGG CAA GCC TTA ATC GGT GCG GCT AAT ACG GGG CGT Thr Gly same time Ser Gly gin Ala Leu Ile Gly Ala Ala same time Thr Gly Arg 655 660 665 AGG GGT TCA ATT TCA GGA TCT AAA CTG GAG TCT AGT AAT GTG GAT TTG Arg Gly Ser Ile Ser Gly Ser Lys Leu Glu Ser Ser same time wall Asp Leu 670 675 680 AGC CGG AGT TTA ACG AAT TTG ATT GTG GTT CAA AGG GGC TTT CAA GCA Ser Arg Ser Leu Thr same time Leu Ile wall wall 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 same time Ser Lys Ala wall Thr Thr Ser Asp gin Ile Leu same time Thr Leu Leu 700 705 710

AAT CTT AAG CAA TAA ACTAAAGGAT TACTCTAATA CAATATAATA GGGGCTAATT Asn Leu Lys Gin

715

TAAAGATTAA GGTTTAGTAT GCATGAATAC TCG

(2) Information for SEQ ID NO: 2:

(i) Sequence characteristics:

(A) Length: 719 amino acids (B) Type: amino acid (C) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 2:

Met Leu Arg Ser Leu Trp Ser Gly wall same time Gly  Met gin Ala His gin 1 5 10 15 Ile Ala Leu Aso Ile Glu Ser same time same time Ile Ala same time wall same time Thr Thr 20 25 30 Gly Phe Lys Tyr Ser Arg Ala Ser Phe wall Asp Met Leu Ser gin wall 35 40 45 Lys Leu Ile Ala Thr Ala for Tyr Lys same time Gly Leu Ala Gly gin same time 50 55 60 Aso Phe Ser wall Gly Leu Gly wall Gly wall Aso Ala Thr Thr Lys Ile 65 70 75 80 Phe Ser gin Gly same time Ile gin same time Thr Asp wall Lys Thr Asp Leu Ala 85 90 95 Ile gin Gly Aso Gly Phe Phe Ile Ile same time ? ro Asp Arg Gly Ile Thr 10O 105 110 Arg same time Phe Thr Arg Asp Gly Glu Phe Leu Phe Asp Ser gin Gly Ser 115 120 125 Leu wall Thr Thr Gly Gly Leu wall wall gin Gly Trp wall Arg same time Gly 130 135 140 Ser Asp Thr Gly same time Lys Gly Ser Asp Thr Aso Ala Leu Lys wall Asp 145 150 155 160 same time Thr Gly for Leu Glu same time Ile Arg Ile Asp for Gly Met wall Met 165 170 175 for Ala Arg Ala Ser same time Arg Ile Ser Met Arg Ala same time Leu same time Ala 180 185 190 Gly Arg His Ala Asp gin Thr Ala Ala Ile Phe Ala Leu Asp Ser Ser 195 200 205 Ala Lys Thr for Ser Asp Gly Ile same time for wall Tyr Asp Ser Gly Thr 210 215 220 same time Leu Ala gin wall Ala Glu Asp Met Gly Ser Leu Tyr same time Glu Asp 225 230 235 240 Gly Asp Ala Leu Leu Leu same time Glu same time gin Gly Ile Trp wall Ser Tyr 245 250 255 Lys Ser for Lys Met wall Lys Asp Ile Leu for Ser Ala Glu same time Ser 260 265 270 Thr Leu Glu Leu same time Gly wall Lys Ile Ser Phe Thr A.- • -sp WITH-:: Ala 275 280 2SL wall Ser Arg Thr Ser Ser Leu wall Ala Ala Lys same time Ala Ile same time Ala 290 295 300 wall Lys Ser gin Thr Gly Ile Glu Ala Tyr Leu Asp Gly Lys gin Leu 305 310 315 320 Arg Leu Glu same time Thr same time Glu Leu Asp Gly Asp Glu Lys Leu Lys A.sn

(+420) 325 330 335

Ile wall wall Thr gin Ala Gly Thr Gly Ala Phe Ala same time Phe Leu Asp 340 345 350 Gly Asp Lys Asp wall Thr Ala Phe Lys Tyr Ser Tyr Thr His Ser Ile 355 360 3 65 Ser for same time Ala same time Ser Gly gin Phe Arg Thr Thr Glu Asp Leu Arg 370 375 380 Ala Leu Ile gin His Asp Ala same time Ile wall Lys ASD for Ser Leu Ala 385 390 395 400 Asp same time Tyr gin Aso Ser Ala Ala Ser Ile Gly wall Thr Ile same time gin 405 410 415 Tyr Gly Met Phe Glu Ile same time same time Lys Asp same time Lys same time wall Ile Lys 420 425 430 Glu same time Leu same time .Zle Phe wall Ser Gly Tyr Ser Ser Aso Ser wall Thr 435 440 445 same time same time wall Leu Phe Lys same time Ala Met Lys Gly Leu same time 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 Th.2ľ Ser ill Asp wall Ile Aso Ser Leu Gly Thr LVS His 485 490 495 Ala Met Arg ill Glu Phe Tyr Arg Ser Gly Gly Ala Asp Trp A.sn Phe 500 505 510 Arg wall Ile wall for Glu for Gly Glu Leu wall Gly Gly Ser Ala Ala 515 520 525 Arg for same time wall Phe Glu Gly Gly Arg Leu His Phe same time same time Asp Gly 530 535 540 Ser Leu Ala. Gly Het same time for for Leu Leu gin Phe Asp for Lys same time 545 550 555 560 Gly Ala Asp Ala for gin Arg Ile same time Leu Ala Phe Gly Ser Ser Gly 565 570 575 Ser Phe Asp Gly Leu Thr Ser wall Asp Lys Ile Ser Glu Thr Tyr Ala 580 585 590 Ile Glu gin same time Gly Tyr gin Ala Gly Asp Leu Met Aso wall Arg Phe 595 600 605 Asp Ser Asp Gly wall Leu Leu Gly Ala Phe Ser same time Gly Arg tn ' Leu 610 615 620 Ala Leu Ala gin wall Ala Leu Ala same time Phe Ala same time Asp Ala Gly Leu 625 630 635 640 gin Ala Leu Gly Gly same time wall Phe Ser gin Thr Gly same time Ser Gly gin 645 650 655 Ala Leu Ile Gly Ala Ala same time Thr Gly Arg Arg Gly Ser Ile Ser Gly 660 665 670

Ser Lys Leu Glu Ser Ser Asn Val Asp Ser Le Ser Ser Ser Le Thr Asn

-25675 680 685

Leu il Val Val Gin Arg Gly? He Gin Ala Asn Ser Lys Ala Val Th '· 690 695 700

Thr Ser Asp Gin White 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) String: single (D) Topology: linear (ii) Molecule type: Other nucleic acids (A) Description: / dese = PRC primer ”(xi) Sequence description: SEQ ID NO: 3:

TATACCATGG TGCTTAGGTC TTTAT (2) Information for SEQ ID NO: 4:

(i) Sequence characteristics:

(A) Length: 25 base pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: Other nucleic acids (A) Description: / dese = “PCR primer” (xi ) Sequence Description: SEQ ID NO: 4:

GCGAATTCAA TTGCTTAAGA TTCAA

Claims (28)

PATENT CLAIMS A vaccine composition comprising a FlgE polypeptide of Helicobacter pylori or a modified form thereof that retains functional equivalent antigenicity, to elicit a protective immune response against Helicobacter pylori infection in a mammal. The vaccine composition of claim 1, wherein the FlgE polypeptide comprises substantially the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing. The vaccine composition of claim 2, wherein the polypeptide comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 2. The vaccine composition of claim 2, wherein the polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO: 2. The vaccine composition according to any one of the preceding claims, characterized in that it comprises a pharmaceutically acceptable carrier or diluent. Vaccine composition according to any one of the preceding claims, characterized in that it contains an adjuvant. The vaccine composition of claim 6, wherein the adjuvant is a pharmaceutically acceptable form of cholera toxin. Vaccine composition according to any one of the preceding claims, characterized in that it comprises a substance selected from liposomes, ISCOMs, cochlea and polymeric microspheres. The vaccine composition according to any one of claims 1 to 7, characterized in that it comprises a vector selected from live attenuated bacteria, viruses and phages. -2710. The use of a FlgE polypeptide of Helicobacter pylori, or a modified form thereof that retains functional equivalent antigenicity, for the manufacture of a composition for the treatment, prophylaxis or diagnosis of a Helicobacter pylori infection in a mammal. Use of a FlgE polypeptide of Helicobacter pylori, or a modified form thereof, which retains functional equivalent antigenicity, for the manufacture of a vaccine composition for use in eliciting a protective immune response against Helicobacter pylori in a mammal. The use of claim 10 or 11, wherein the FlgE polypeptide is a polypeptide as defined in any one of claims 2 to 4. The use of any one of claims 10 to 12, wherein the mammal is a human. Use according to any one of claims 10 to 13, wherein the composition comprises a pharmaceutically acceptable carrier or diluent. The use of any one of claims 10 to 14, wherein the composition comprises an excipient. The use of claim 15, wherein the excipient is a pharmaceutically acceptable form of cholera toxin. The use according to any one of claims 10 to 16, wherein the composition comprises a substance selected from selected from liposomes, ISCOMs, cochlea and polymeric microspheres. The use of any one of claims 10 to 16, comprising a vector selected from live attenuated bacteria, viruses and phages. 19. A method of diagnosing a Helicobacter pylori infection in a mammal, comprising the steps of (a) contacting a FlgE polypeptide of Helicobacter pylori or a modified form thereof that retains functional equivalent antigenicity with a body fluid collected from the mammal; and (B) detecting antibodies from said body fluid by binding to said FlgE polypeptide. The method of claim 19, wherein the FlgE polypeptide is a polypeptide as defined in any one of claims 2 to 4. The method of claim 19 or 20, wherein the FlgE polypeptide is bound to a solid support. The method of any one of claims 19 to 21, wherein the mammal is a human. The diagnostic kit for use in the method of claim 19, wherein the kit comprises a FlgE polypeptide of Helicobacter pylori or a modified form thereof that retains functional equivalent antigenicity. The diagnostic kit of claim 23, wherein the FlgE polypeptide is a polypeptide as defined in any one of claims 2 to 4. 25. A method of inducing a protective immune response against Helicobacter pylori infection in a mammal, said method comprising the step of administering to the mammal a FlgE polypeptide of Helicobacter pylori or a modified form thereof that retains functional equivalent antigenicity. The method of claim 25, wherein the FlgE polypeptide is a polypeptide as defined in any one of claims 2 to 4. The method of claim 25 or 26, wherein the mammal is a human. The method of any one of claims 25 to 27, wherein the method is a prophylactic method. The method of any one of claims 25 to 27, wherein the method is a therapeutic method.