CA2293293A1 - 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 THE HELICOBACTER PYLORI FIgE POLYPEPTIDE
TECHNICAL FIELD
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.
to BACKGROUND ART
Helicobacter pylori The gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen, involved in several gastroduodenal diseases. Colonization of gastric epithelium by the bacterium leads to active inflammation and progressive chronic gastritis, with a greatly enhanced risk of progression to peptic ulcer disease. A
lifelong inflammation of the gastric mucosa is very closely correlated with a significantly enhanced risk for gastric cancer.
In order to colonize the gastric mucosa, H, pylori uses a number of virulence factors. Such virulence factors comprise several adhesins, with which the as bacterium associates with the mucus and/or binds to epithelial cells;
unease 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 3o experimental models Eaton et al. (Infection & Immunity 64(7}, 2445-2448,1996).

There are many possible reasons for this, the most obvious being an inability to swim down and attach to mucosal cells and the inability to avoid noxious agents in the stomach.
s 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 ~o 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 is 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 zo 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 is antibiotics and other anti-bacterial agents. Circumstantial evidence indicate the H.
pylori might be transmitted between indi~ iuals in ~.zis form, possibly via water or direct contact (oral-oral; feacal-oral). An efricient vaccine composition should therefore elicit an immune response towards both the coccoid and the bacillary form of H. pylori. Since systemic immunity probably only plays a limited role in protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach.
Flagellar Hook protein Flagellar hooks from H. pylori has been shown to be composed of FIgE 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 fiagellar motor. The part of the hook extruding outside the membrane is short, io 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 FIgE polypeptide has significant resemblance with is 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 FIgE polypeptide showed cross-reactivity against flagellar proteins A
and B, possibly indicating the existence of shared epitopes. Production of FIgE
knockout H. pylori, resulted in an aflagellar, non-motile bacteria, where FlgE
2o polypeptide still was produced but could only be recovered in the cytoplasm.
BRIEF DESCRIPTION OF THE DRAWINGS
2s Fig. 1:
Effect of therapeutic immunization of H. pylori infected mice (n=9-10/group) with FIgE polypeptide. Results are given as mean~SEM of number of H. pylori associated with antnzln (=A), corpus (=B) or totally (A+C) (=C).
Abbreviations:
CFU, colony forming units (number of bacteria); unshaded bars=DOC + CT, so Phosphate buffered saline with 0.5% deoxycholate given together with cholera toxin 10 ~g/mouse; shaded bars=FIgE + CT, mice given 100 ~,g FIgE and 10 ~.g 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 FIgE. 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 to specific antibodies can be found in serum in animals treated with DOC + CT
(=A.
Control/244). Following immunization with FIgE + cholera toxin (=B. FIgE/244) this reactivity increased 4 fold (** p<0.01; Wilcoxon-Mann-Whittney sign rank test). C=FIgE specific. Specific FIgE IgG increased in animals given FIgE +
CT, but could not be detected in control animals.
IS
DISCLOSURE OF THE INVENTION
The purpose of this invention is to provide an antigenic H. pylori polypeptide zo which can be useful for eliciting a protective immune response against, and for diagnosis of, H. pylori infection. This purpose has been achieved by the recombinant cloning of an H. pylori gene which encodes a well conserved essential polypeptide. The nucleic acid sequence of this gene is similar to the sequence of the f 1gE gene as published by O'Toole et al., Molecular Microbiology,14(4), 25 703,1994. Be"- ; an essen!-ial protein for motility, the flgE gene is expressed by all T ?. pylori stra~ . ~.
It has surprisingly been found that the H. pylori FIgE polypeptide, in spite of the facts that only a small part of the hook protein is existing outside bacteria and that 3o it is probably covered by a sheet, can serve as a therapeutic antigen in an H. pylori infected mouse model, when given together with the adjuvant cholera toxin. The experimental data below thus indicates that the H. pylori FlgE poiypeptide, when used as an oral immunogen, acts as a stimulator of an immune response leading to a significant reduction of colonization of H. pylori in mice which were infected with H. pylori one month prior to immunization.
These results strongly support the use of the H. pylori FlgE polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections.
As such, the FlgE polypeptide will be useful both for the detection of H.
pylori ~o 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 cs FIgE polypeptide for use in inducing a protective immune response to Helicobacter pylori infection. The term "HeIicobacter pylori FIgE 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 2o 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 ~s 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 to identify modified forms of the FIgE polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies.
In a preferred form of the invention, the Helicobacter pylori FIgE
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.
~o It is thus to be understood that the definition of the Helicobacter pylori FIgE
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, is small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of the Helicobacter pylori FIgE
polypeptide and is retaining functionally equivalent antigenicity. Included in the definition of the Helicobacter pylori FIgE polypeptide are consequently polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95%
2o 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 2s immunogenically effective amount of a Helicobacter pylori FIgE polypeptide as defined above, optionally together with a pharmaceutically acceptable carrier nr diluent.
In the present context the term "immunologically effective amount" is to be 3o understood as an amount which elicits a significant protective Helicobacter pylori WO 98I568~6 PCT/SE98I01093 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 ~,g to 1000 mg, preferably approximately 10 ~.g to 100 mg, of H. pylori antigen for oral administration, or approximately less than 100 ~.g for parenteral administration.
The vaccine composition comprises optionally in addition to a pharmaceutically acceptable carrier or diluent one or more other immunologically active antigens for prophylactic or therapeutic use. Physiologically acceptable carriers and ~o 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, HC03- based formulations or enterically coated powder formulations.
The vaccine composition can optionally include or be administered together with is 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 zo 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 ~s 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 3o and prophylactic purposes. Consequently, the invention includes a vaccine composition according as defined above, for use as a therapeutic or a prophylactic vaccine in a mammal, including man, which is infected by Helicobacter pylori.
In this context the term "prophylactic purpose" means to induce an immune response which will protect against future infection by Helicobacter pylori, while the term "therapeutic purpose" means to induce an immune response which can eradicate an existing Helicobacter pylori infections.
The vaccine composition according to the invention is preferably administered to any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the ~o 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 ~s with antimicrobial therapeutic agents.
In a further aspect, the invention proivides the use of a Helicobacter pylori FIgE
polypeptide, as defined above, in the manufacture of (i) a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori 2o infection;
(ii) a vaccine for use in eliciting a protective immune response against HeIicobacter pylori; and (iii) a diagnostic kit for diagnosis of Helicobacter pylori infection.
2s In yet a : ,: r ther aspect, the invention provides a method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori FIgE 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 FIgE polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and (b) detecting antibodies from the said body fluid binding to the said FlgE polypeptide. Preferred methods of detecting antibodies are ELISA (Enzyme linked immunoabsorbent assay) methods which are well known in the art.
In another aspect the invention provides a diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method of in vitro diagnosis as described above to be carried out.
The said diagnostic kit could e.g. comprise: (a) a Helicobacter pylori FIgE
io polypeptide; and (b) reagents for detecting antibodies binding to the said FIgE
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, ~s 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 FIgE polypeptide as defined above, or alternatively administering to the said mammal an immunologically effective amount of a vaccine composition as defined above.
EXPERIMENTAL METHODS
Throughout this description the terms "standard protocols" and "standard is 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, so Cold Spring Harbor, NY 1989.

Preparation of recombinant Helicobacter pylori FIgE polypeptide DNA sequence Information Sequence information for the gene encoding for the FIgE polypeptide was obtained from the National Center for Biotechnology Information (Accession number U09549; SEQ ID NO: 1).
~o PCR Amplification and cloning of DNA sequences containing ORF's for membrane and secreted proteins from the j99 Strain of HeIicobacter pylori.
Sequences were cloned from the J99 strain of H. pylori by amplification cloning using the polymerase chain reaction (PCR). Synthetic oligonucleotide primers (see is 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 2o 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).
zs Forward primer (SEQ ID NO: 3):

5'-TAT ACC ATG GTG CTT AGG TCT TTA T-3' Reverse primer (SEQ ID I'~lU: 4):
5'-GCG AAT TCA ATT GCT TAA GAT TCA A-3' WO 98/5681b PCT/SE98/01093 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 s introduced into a reaction vial containing 2 mM MgCl2,1 ~.M synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP, and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume io of 100 ~.1. 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 +94°C for 2 min;
2 cycles at +94°C for 15 sec, +30°C for 15 sec and +72°C
for 1.5 min;
~s 23 cycles at +94°C for 15 sec, +58°C for 15 sec and +72°C for 1.5 min;
Reactions were concluded at +72°C for 6 minutes.
Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, 2o 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
2s contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).

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 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 ~o Competent bacteria, E. coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H.
pylori sequences according to standard methods. Briefly,1 ~.l of ligation reaction was mixed with 50 ~.1 of electrocompetent cells and subjected to a high voltage is pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5%
yeast extract, 2.0% tryptone,10 mM NaCI, 2.5 mM KC1,10 mM MgCl2,10 mM MgS04 and 20, mM glucose) at +37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 ~.g/ml kanamycin sulfate for growth overnight.
Transformed colonies of BL21 were then picked and analyzed to evaluate cloned 2o 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 2s were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific fo~ ~ ..~h H. pylori sequence, tl. ' were used in ~. _ original PCR amplification cloning reactions. Successful amp~ification verified the integration of the H. pylori sequences in the expression vector according to standard procedures.

Isolation and Preparation of plasmid DNA from BL22 transformants Individual clones of recombinant pET-28b vectors carrying properly cloned H.
pylori ORFs were picked and incubated in 5 ml of LB broth plus 25 ~.g/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA).
io 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, DHSa, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for is T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lacl gene, the lacUV5 promoter and the gene for T7 RNA
polymerase. T7 RNA polymerase is induced by addition of isopropyl-~i-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 is described above (provided by Novagen as part of the pET expression system kit).
Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 ~,g/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 ~.g/ml) to an optical density at 600 nm of 0.5 to 1.0 O.D.

units, at which point,1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C.
Pellets were resuspended in 50 ml cold 10 mM Tris-HCI, pH 8.0, 0.1 M NaCI and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at +4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification.
~o Analytical Methods The concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid is 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.
2o 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 ~3-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 2s kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).

Purification of FIgE from inclusion bodies The following steps were carried out at +4°C. Cell pellets were resuspended in lysis buffer with 10% glycerol 200 ~.g/ml lysozyme, 5 mM EDTA,1 mM PMSF and s 0.1% (3-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% (3-mercaptoethanol, then with lysis buffer containing 1 M urea, l mM PMSF and 0.1% ~i-mercaptoethanol. The ~o 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% ~3-is mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation (100,000 x g for 30 min) . The clear supernatant was filtered and loaded onto a Ni2+-NTA agarose column equilibrated in 8 M urea in lysis buffer. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, l mM PMSF and 0.1% ~i-2o mercaptoethanol, and developed with sequential steps of lysis buffer containing 8 M urea, 1 mM PMSF, 0.1% (3-mercaptoethanol and 20,100, 200, and 500 mM
imidazole. Fractions were monitored by absorbance at OD28p 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.
2s Purity of recombinant FIgE (7$ 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 FIgE polypeptide by dialysis against TBS
3o containing 0.5% DOC with sequential reduction in urea as follows; 6M, 4M, 3M, 2M,1M, 0.5 M then 0 M. Each dialysis step was carried for a minimum of 4 hours at room temperature, After dialysis, samples were concentrated by pressure filtration using Amicon s stirred cells. Protein concentrations were then measured by the methods of Perkins, Bradford and Lowry.
EXAMPLES OF THE INVENTION
EXAMPLE 1: THERAPEUTIC IMMUNIZATION
1. Materials f~ Methods is 1.1 Animals Female SPF BALB/c mice were purchased from Bomholt Breeding centre (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
1.2. Infection After a minimum of one week of acclimatization, the animals were infected with a type 2 strain of H. pylori (strain 244, originally isolated from an ulcer patient). This 2s strain has earlier proven to be a good colonizer of the mouse stomach.
Bacteria from a stock kept at -70°C were gro«~n overnight in Brucella broth supplemented with 10% fetal calf serum, at +37°C L~ a microaerophilic atmosphere (10% CO2, 5%
02). The animals were given an oral dose of omeprazole (400 ~,mol/kg) and after 3-5 h an oral inoculation of H. pylori (approximately 10~-10$ CFU/animal).
3o Infection was checked in control animals 2-3 weeks after the inoculation.

1.3. Immunizations One month after infection, two groups of mice (10 mice/group) were immunized 4 times over a 34 day period (day 1,15, 25 and 35). Purified recombinant FIgE
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 IO ~.g/mouse of cholera toxin (CT) with each immunization. Omeprazole io (400 ~mol/kg) was given orally to all animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Animals were sacrificed weeks after final immunization.
Group I: 300 ~1 PBS with 0.5% DOC containing 10 ~.g CT
Group 2: 300 ~,l PBS with 0.5% DOC containing 100 ~.g FIgE and 10 ~.g CT.
is 1.4. Analysis of infection The mice were sacrificed by C02 and cervical dislocation. The abdomen and chest cavity was opened and blood sampled by heart puncture. Subsequently the 2o stomach was removed. After cutting the stomach along the greater curvature, it was rinsed in saline and subsequently cut into two identical pieces. An area of 25 mm2 of the mucosa from the antrum and corpus was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth, diluted and plated onto Blood Skirrow plates. The plates were incubated under 2s microaerophilic conditions for 3-5 days and the number of colonies was counted.
The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.

1.5. Antibody measurements Serum antibodies were collected from blood. Prior to centrifugation, the blood was diluted with equal amount of PBS. The serum was kept at -20°C until analysis.
Serum antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with FIgE 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.
io 2. Results 2.1. Therapeutic immunization: effects on CFU
is 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 FIgE 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 2o and antrum. Animals actively immunized with recombinant FIgE polypeptide and CT had significantly decreased CFU values in the antrum and in the stomach as a whole compared with the CT treated animals (p<0.01 and p<0.05, respectively;
Wilcoxon-Mann-Whittney sign rank test).
is 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 FIgE + CT the titer against strain 244 (as membrane proteins) increased 4-fold (p<0.01). Only in animals given FIgE + CT
so could a specific serum IgG titer against FIgE be measured (Fig. 2).

FIgE specific IgG increased in animals given FIgE + CT, but could not be detected in control animals.
The results presented show that the recombinant FIgE H. pylori polypeptide is highly immunogenic when given orally, together with cholera toxin as an adjuvant, measured as an increase in systemic FIgE specific Ig antibodies. The immunization with FIgE also resulted in a significant increase in the Ig titers against a particulate fraction of H. pylori. In addition, a dramatic decrease in io number of colonizing H. pylori in the gastric mucosa of the infected mice was found following immunization with FIgE toghether with cholera toxin.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Astra AB
(B) STREET: Vastra Malarehamnen 9 (C) CITY: Sodertalje (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:

ATG CTT AGG
TCT TTA TGG
TCT GGT GTC
AAT

Met sn Leu Arg Ser Leu Trp Ser Gly Val A

Giy MetGln AlaHisGln IleAlaLeuAsp IleGluSer AsnAsnIle Ala AsnVal AsnThrThr GlyPheLysTyr SerArgAla SerPheVal Asp MetLeu SerGlnVal LysLeuIleAla ThrAlaPro TyrLysAsn Gly LeuAla GlyGlnAsn AspPheSerVal GlyLeuGly ValGlyVal Asp AlaThr ThrLysIle PheSerGlnGly AsnIleGln AsnThrAsp Val LysThr AspLeuAla IleGlnGlyAsp GlyPhePhe IleIleAsn Pro AspArg GlyIleThr ArgAsnPheThr ArgAspGly GluPheLeu Phe AspSer GlnGlySer LeuValThrThr GlyGlyLeu ValValGln Gly TrpVal ArgAsnGly SerAspThrGly AsnLysGly SerAspThr Asp AlaLeu LysValAsp AsnThrGlyPro LeuGluAsn IleArgIle Asp ProGly MetValMet ProAlaArgAla SerAsnArg IleSerMet Arg AlaAsn LeuAsnAla GlyArgHisAla AspGlnThr AlaAlaIle Phe AlaLeu AspSerSer AlaLysThrPro SerAspGly IleAsnPro Val TyrAsp SerGlyThr AsnLeuAlaGln ValAlaGlu AspMetGly SerLeuTyr AsnGluAsp GlyAspAlaLeu LeuLeuAsn GluAsnGln GlyIleTrp ValSerTyr LysSerProLys MetValLys AspIleLeu ProSerAla GluAsnSer ThrLeuGluLeu AsnGlyVal LysIleSer PheThrAsn AspSerAla ValSerArgThr SerSerLeu ValAlaAla LysAsnAla IleAsnAla ValLysSerGln ThrGlyIle GluAlaTyr LeuAspGly LysGlnLeu ArgLeuGluAsn ThrAsnGlu LeuAspGly AspGluLys LeuLysAsn IleValValThr GlnAlaGly ThrGlyAla PheAlaAsn PheLeuAsp GlyAspLysAsp ValThrAla PheLysTyr SerTyrThr HisSerIle SerProAsnAla AsnSerGly GlnPheArg ThrThrGlu AspLeuArg AlaLeuIleGln HisAspAla AsnIleVal LysAspPro SerLeuAla AspAsnTyrGln AspSerAla AlaSerIle GlyValThr IleAsnGln TyrGlyMetPhe GluIleAsn AsnLysAsp AsnLysAsn ValIleLys GluAsnLeuAsn IlePheVal SerGlyTyr SerSerAsp SerValThr AsnAsnValLeu PheLysAsn AlaMetLys GGGCTTAAT ACCGCTTCT TTAATTGAAG-'9GGAGCGTCA GCGAGCAGT 1742 GlyLeuAsn ThrAlaSer LeuIleGlur GlyA.aSer AlaSerSer ,~

SerLysPhe ThrHisAla ThrHisAlaThr SerIleAsp ValIleAsp SerLeuGly ThrLysHis AlaMetArgIle GluPheTyr ArgSerGly Gly Ala Asp Trp Asn Phe Arg Val Ile Val Pro Glu Pro Gly Glu Leu Val Gly Gly Ser Ala Ala Arg Pro Asn Val Phe Glu Gly Gly Arg Leu AAC

His PheAsnAsnAsp GlySerLeuAla GlyMetAsn ProProLeuLeu Gln PheAspProLys AsnGlyAlaAsp AlaProGln ArgIleAsnLeu Ala PheGlySerSer GlySerPheAsp GlyLeuThr SerValAspLys Ile SerGluThrTyr AlaIleGluGln AsnGlyTyr GlnAlaGlyAsp Leu MetAspValArg PheAspSerAsp GlyValLeu LeuGlyAlaPhe Ser AsnGlyArgThr LeuAlaLeuAla GlnValAla LeuAlaAsnPhe Ala AsnAspAlaGly LeuGlnAlaLeu GlyGlyAsn ValPheSerGln Thr GlyAsnSerGly GlnAlaLeuIle GlyAlaAla AsnThrGlyArg Arg GlySerIleSer GlySerLysLeu GluSerSer AsnValAspLeu Ser ArgSerLeuThr AsnLeuIleVal ValGlnArg GlyPheGlnAla Asn SerLysAlaVal ThrThrSerAsp GlnIleLeu AsnThrLeuLeu CAATATAATA
GGGGCTAATT

Asn LeuLysGln . (2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 719 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear WO 98/5681b PCT/SE98/01093 (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 Ile Ala Leu Asp Ile Glu Ser Asn Asn Ile Ala Asn Val Asn Thr Thr Gly Phe Lys Tyr Ser Arg Ala Ser Phe Val Asp Met Leu Ser Gln Val Lys Leu Ile Ala Thr Ala Pro Tyr Lys Asn Gly Leu Ala Gly Gln Asn Asp Phe Ser Val Gly Leu Gly Val Gly Val Asp Ala Thr Thr Lys Ile Phe Ser Gln Gly Asn Ile Gln Asn Thr Asp Val Lys Thr Asp Leu Ala Ile Gln Gly Asp Gly Phe Phe Ile Ile Asn Pro Asp Arg Gly Ile Thr Arg Asn Phe Thr Arg Asp Gly Glu Phe Leu Phe Asp Ser Gln Gly Ser Leu Val Thr Thr Gly Gly Leu Val Val Gln Gly Trp Val Arg Asn Gly Ser Asp Thr Gly Asn Lys Gly Ser Asp Thr Asp Ala Leu Lys Val Asp Asn Thr Gly Pro Leu Glu Asn Ile Arg Ile Asp Pro Gly Met Val Met Pro Ala Arg Ala Ser Asn Arg Ile Ser Met Arg Ala Asn Leu Asn Ala Gly Arg His Ala Asp Gln Thr Ala Ala Ile Phe Ala Leu Asp Ser Ser Ala Lys Thr Pro Ser Asp GIy Ile Asn Pro Val Tyr Asp Ser Gly Thr Asn Leu Ala Gln Val Ala Glu Asp Met Gly Ser Leu Tyr Asn Glu Asp Gly Asp Ala Leu Leu Leu Asn Glu Asn Gln Gly Ile Trp Val Ser Tyr Lys Ser Pro Lys Met Val Lys Asp Ile Leu Pro Ser Ala Glu Asn Ser Thr Leu Glu L~PU Asn Gly Val Lys Ile Ser Phe Thr Asn Asp Ser Ala Val Ser Arg Thr Ser Ser Leu Val Ala Ala Lys Asn Ala Ile Asn AIa Val Lys Ser Gln Thr Gly Ile Glu Ala Tyr Leu Asp Gly Lys Gln Leu Arg Leu Glu Asn Thr Asn Glu Leu Asp Gly Asp Glu Lys Leu Lys Asn Ile Val Val Thr Gln Ala Gly Thr Gly Ala Phe Ala Asn Phe Leu Asp Gly Asp Lys Asp Val Thr Ala Phe Lys Tyr Ser Tyr Thr His Ser Ile Ser Pro Asn Ala Asn Ser Gly Gln Phe Arg Thr Thr Glu Asp Leu Arg Ala Leu Ile Gln His Asp Ala Asn Ile Val Lys Asp Pro Ser Leu Ala Asp Asn Tyr Gln Asp Ser Ala Ala Ser Ile Gly Val Thr Ile Asn Gln Tyr Gly Met Phe Glu Ile Asn Asn Lys Asp Asn Lys Asn Val Ile Lys Glu Asn Leu Asn Ile Phe Val Ser Gly Tyr Ser Ser Asp Ser Val Thr Asn Asn Val Leu Phe Lys Asn Ala Met Lys Gly Leu Asn Thr Ala Ser Leu Ile Glu Gly Gly Ala Ser Ala Ser Ser Ser Lys Phe Thr His Ala Thr His Ala Thr Ser Ile Asp Val Ile Asp Ser Leu Gly Thr Lys His Ala Met Arg Ile Glu Phe Tyr Arg Ser Gly Gly Ala Asp Trp Asn Phe Arg Val Ile Val Pro Glu Pro Gly Glu Leu Val Gly Gly Ser Ala Ala Arg Pro Asn Val Phe Glu Gly Gly Arg Leu His Phe Asn Asn Asp Gly Ser Leu Ala Gly Met Asn Pro Pro Leu Leu Gln Phe Asp Pro Lys Asn Gly Ala Asp Ala Pro Gln Arg Ile Asn Leu Ala Phe Gly Ser Ser Gly Ser Phe Asp Gly Leu Thr Ser Val Asp Lys Ile Ser Glu Thr Tyr Ala Ile Glu Gln Asn Gly Tyr Gln Ala Gly Asp Leu Met Asp Val Arg Phe Asp Ser Asp Gly Val Leu Leu Gly Ala Phe Ser Asn Gly Arg Thr Leu Ala Leu Ala Gln Val Ala Leu Ala Asn Phe Ala Asn Asp Ala Gly Leu Gln Ala Leu Gly Gly Asn Val Phe Ser Gln Thr Gly Asn Ser Gly Gln Ala Leu Ile Gly Ala Ala Asn Thr Gly Arg Arg Gly Ser Ile Ser Gly Ser Lys Leu Glu Ser Ser Asn Val Asp Leu Ser Arg Ser Leu Thr Asn Leu Ile Val Val Gln Arg GIy Phe Gln Ala Asn Ser Lys Ala Val Thr Thr Ser Asp Gln Ile Leu Asn Thr Leu Leu Asn Leu Lys Gln (2) INFORMATION FOR SEQ ID N0: 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:

(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:

Claims (29)

1. A vaccine composition comprising a Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, for inducing in a mammal a protective immune response to Helicobacter pylori infection.

2. 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.

3. 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.

4. 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.

5. The vaccine composition of any preceding claim, including a pharmaceutically acceptable carrier or diluent.

6. The vaccine composition according to any preceding claim, comprising an adjuvant.

7. The vaccine composition according to claim 6, wherein the adjuvant is a pharmaceutically acceptable form of cholera toxin.

8. The vaccine composition of any preceding claim, including a substance selected from liposomes, ISCOMs, cochleates and polymer microspheres.

9. The vaccine composition of any one of claims 1 to 7, comprising a vector selected from live attenuated bacteria, viruses and phages.

10. Use of a Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, in the manufacture of a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection in a mammal.

11. Use of a Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, in the manufacture of a vaccine composition for use in eliciting a protective immune response in a mammal against Helicobacter pylori.

12. The use according to claim 10 or 11, wherein the FlgE polypeptide is as defined in any one of claims 2 to 4.

13. The use according to any one of claims 10 to 12, wherein the mammal is a human.

14. The use according to any one of claims 10 to 13, wherein the composition includes a pharmaceutically acceptable carrier or diluent.

15. The use according to any one of claims 10 to 14, wherein the composition comprises an adjuvant.

16. The use according to claim 15, wherein the adjuvant is a pharmaceutically acceptable form of cholera toxin.

17. The use according to any one of claims 10 to 16, wherein the composition includes a substance selected from liposomes, ISCOMs, cochleates and polymer microspheres.

18. The use according to any one of claims 10 to 16, comprising a vector selected from live attenuated bacteria, viruses and phages.

19. A method of in vitro diagnosis of Helicobacter pylori infection in a mammal, the method comprising the steps of (a) contacting a Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, with a body fluid taken from the mammal; and (b) detecting antibodies from the said body fluid binding to the said FlgE
polypeptide.

20. The method of claim 19, wherein the FlgE polypeptide is as defined in any one of claims 2 to 4.

21. The method of claim 19 or 20, wherein the FlgE polypeptide is bound to a solid support.

22. The method of any one of claims 19 to 21, wherein the mammal is a human.

23. A diagnostic kit for use in the method of claim 19, wherein the kit comprises a Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity.

24. The kit of claim 23, wherein the FlgE polypeptide is as defined in any one of claims 2 to 4.

25. A method of eliciting in a mammal a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the mammal a Helicobacter pylori FlgE polypeptide, or a modified form thereof retaining functionally equivalent antigenicity.

26. The method of claim 25, wherein the FlgE polypeptide is as defined in any one of claims 2 to 4.

27. The method of claim 25 or 26, wherein the mammal is a human.

28. The method of any one of claims 25 to 27, wherein the method is a prophylactic method.

29. The method of any one of claims 25 to 27, wherein the method is a therapeutic method.