HELICOBACTER PYLORI WITH MODIFIED LIPOPOLYSACCHARIDE BIOSYNTHESIS

Abstract: <i>Helicobacter pylori</i> can be manipulated at a genetic level to prevent the synthesis of auto-reactive LPS, without destroying the physical integrity or function of the bacterial outer membrane. The bacterium is particularly useful for preparing cellular vaccines which do not induce antibodies which cross-react with self epitopes. Cellular <i>H.pylori</i> vaccines are provided which comprise mutant <i>H.pylori</i> which either does not express LPS or, preferably, expresses a mutant form of LPS.

Description: HEL1COBACTER PYLORI WITH MODIFIED LIPOPOLYSACCHARIDE BIOSYNTHESIS All documents cited herein are incorporated by reference in their entirety. TECHNICAL FIELD This invention is in the field of vaccines against Helicobacter pylori. BACKGROUND ART Helicobacter pylori is a Gram-negative bacterium that inhabits the gastric mucosa. It is responsible for gastritis and peptic ulcers, and has been linked with gastric cancer. Vaccines against H. pylori have been proposed, including sub-unit vaccines and whole-cell vaccines. Lipopolysaccharides (LPS), also termed endotoxins, are found in the cell envelopes of Gram- negative bacteria. They are generally the main surface antigens of Gram-negative bacteria and are essential for the physical integrity and function of the bacterial outer membrane. They are typically removed from pharmaceutical preparations {e.g. vaccines) because of their pyrogenicity. H. pylori LPS has three major components: lipid A, which anchors LPS to the outer membrane; a core oligosaccharide unit; and the O-antigen, a polysaccharide consisting of repeating units of 3- 6 sugars. H. pylori lipid A has unusually long 3-hydroxy fatty acids and has as its backbone a D-glucosamine disaccharide which carries a phosphate group at position 1 but which is not phosphorylated at position 4' [Muotiala et al. (1992) Infect, lmmun. 60: 1714-16]. H. pylori LPS possesses unique biological properties [Moran (1996) Aliment Pharmacol Ther 10(suppl.l):39-50]. It has low immunogenicity, low toxicity, low pyrogenicity, and low mitogenicity. These properties are partly due to the absence of a 4' phosphate on the lipid A moiety. There is thus no pyrogenic requirement to remove LPS from H. pylori compositions. The O-specific chain of the LPS mimics Lewis-X and Lewis-Y blood group antigens in structure. As these antigens are present in the gastric mucosa, the LPS may camouflage H.pyloή and aid its survival. LPS may also be relevant in the development of autoimmunity in H.pylori- associated disease, as auto-antibodies against gastric mucosa have been observed in H./?y/øπ ' -positive patients [Appelmelk et al. (1996) Infect, lmmun. 64:2031-40]. Thus any need to remove LPS from H.pylori compositions (e.g. whole-cell vaccines) arises from the need to avoid the induction of autoantibodies, rather than pyrogenicity. There have been several reports of mutations in genes involved in H.pylori LPS biosynthesis [e.g. Kwon et al. (1998) Curr. Microbiol. 37: 144-48; McGowan et al. (1998) Mol. Microbiol. 30: 19-31; Wang et al. (1999) Mol. Microbiol. 31: 1265-74; Appelmelk et al. (1999) Infect, lmmun. 67:5361-66]. The role of LPS or of its auto-antigen moieties in gastric colonisation is, however, unknown. Furthermore, mutations in the lipid A biosynthetic pathway are typically lethal [Raetz (1993) 7. Bacteriol. 175:5745-5753]. It is an object of the invention to provide an improved way of providing H.pylori compositions that lack auto-reactive LPS, and to provide vaccines based thereon. DISCLOSURE OF THE INVENTION The invention is based on the finding that H.pylori can be manipulated at a genetic level to prevent the synthesis of auto-reactive LPS, without destroying the physical integrity or function of the bacterial outer membrane. This gives a bacterium which is particularly useful for cellular vaccines which avoid problems of vaccine-induced autoimmunity In a first aspect, the invention provides a cellular H.pylori vaccine comprising mutant H.pylori which either does not express LPS or, preferably, expresses a mutant form of LPS. When administered to a patient, the vaccine does not induce antibodies which cross-react with human self epitopes. The cellular vaccine of the invention will typically be inactivated, either by chemical means (e.g. treatment with glutaraldehyde, formalin, B-propiolactone, or ethylenimines) or physical means (e.g. by heating or irradiation). Further details on bacterial inactivation can be found in Manclark & Cowell (1984), pp. 69-106 of Bacterial Vaccines (ed. Germanier, Academic Press, Orlando). The vaccine may take the form of whole cells, or may be a cellular lysate. The vaccine may be for prophylactic or therapeutic use. Where the mutant H.pylori expresses a mutant form of LPS, the LPS preferably does not contain Lewis-X and/or Lewis-Y antigens. The invention also provides a method for treating or preventing H.pylori infection, comprising administering an effective amount of a vaccine of the invention. In a second aspect, the invention provides mutant bacteria for use in the vaccine. The mutant bacteria preferably are not capable of gastric colonisation. The invention provides a Helicobacter pylori bacterium which has a mutation that affects LPS biosynthesis, wherein either LPS is not expressed, or LPS which is expressed does not induce antibodies which cross-react with human self epitopes. Preferably, the LPS does not contain Lewis-X and/or Lewis-Y antigens. The invention also provides a Helicobacter pylori bacterium which has a mutation that affects LPS biosynthesis upstream of the attachment of the O-antigen. Preferably the mutation reduces the enzymatic activity of a β-l,4-galactosyltransferase. More particularly, the invention provides a Helicobacter pylori bacterium which expresses a mutant form of LPS, characterised in that the bacterium has a mutation in gene HP 1031 and/or gene HP0563. Exemplary HP1031 and HP0563 sequences from fully-sequenced strains J99 and 26695 are shown in Figure 1, and it will be appreciated that the equivalent genes in other strains of H.pylori can readily be determined using this information. The mutation may be a point mutation affecting a single nucleotide e.g. a mutation which changes an amino acid responsible for the activity of the gene, or a mutation inside or outside the coding region which attenuates its transcription or translation. Several point mutations may be present in the gene. As an alternative, the mutation may be more substantial e.g. a deletion or insertion of several amino acids. Preferably, the mutation is a knock-out mutation i.e. the gene is removed or completely de-activated from the bacterium such that its protein product is not expressed. Knock-out HP1031 mutants are viable bacteria but, advantageously, are incapable of gastric colonisation, thus being particularly suitable for vaccine preparation. Knock-out mutants of the invention are preferably isogenic knock-out mutants. The invention also provides the mutant bacteria of the invention for use in vaccination. The invention also provides the use of mutant bacteria of the invention in the manufacture of a vaccine for the treatment or prophylaxis of H.pylori infection. The invention also provides a method for treating or preventing H.pylori infection, comprising administering an effective amount of a mutant bacterium of the invention. The invention also provides a method for producing a bacterium of the invention, comprising the step of mutating a gene which is involved in LPS biosynthesis, such that the mutation gives a bacterium which either does not express LPS, or expresses LPS which does not contain Lewis-X and/or Lewis-Y antigens. The mutation may be in a gene which encodes an enzyme involved in LPS biosynthesis upstream of the attachment of the O-antigen. Preferably, it is in a gene that encodes a β-l ,4-galactosyltransferase. The mutation may be in gene HP1031 and/or gene HP0563. Vaccines of the invention may comprise one or more adjuvants. Preferred adjuvants include: (1) aluminium salts {e.g. hydroxide, phosphate, sulphate); (2) oil-in-water emulsions (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59™, containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE - WO90/14837) formulated into submicron particles (b) SAF (10% squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr- MDP) either microfluidized or vortexed, and (c) Ribi™ adjuvant system (2% squalene, 0.2% Tween 80, and one or more of bacterial cell wall components monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS); (3) saponin adjuvants, such as Stimulon™, optionally in the form of ISCOMs; (4) Freund's complete and incomplete adjuvants; (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. IFNγ), M-CSF, TNF, etc.; (6) mucosal adjuvants, such as LT or CT mutants, as described in WO95/17211 e.g. LT-K63, LT-R72 etc. The vaccines will typically be formulated with a pharmaceutically acceptable carriers, which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, poly- meric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as adjuvants. The vaccine will also typically be formulated with diluents (e.g. water, saline, glycerol, ethanol, etc.) Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Generally, the vaccine will be administered parenterally e.g. by injection, either subcutaneously or intramuscularly. They may also be administered to mucosal surfaces (e.g. oral or intranasal), or in the form of pulmonary formulations, suppositories, or transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents. The vaccine will comprise an immunologically effective amount of antigen i.e. an amount which, when administered to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount may vary depending upon the health and physical condition of the individual to be treated, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, a physician's of the medical situation, and other relevant factors. The amount will fall in a rel- atively broad range which can be readily determined through routine trials. BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows sequences for HP0563 and HP1031 from strain J99 [Aim et al. (1999) Nature 397: 176-180] and strain 26695 [Tomb et al. (1997) Nature 388: 539-547]. Figure 2 shows the results of PCR amplification of HP1031 after mutagenesis. Figure 3 shows immunoblots of LPS preparations after proteinase K digestion. Figure 4 shows the structures of Lewis-related antigens. Abbreviations: Gal=D-galactose; Fuc=L-fucose; GlcNac=N-acetyl-D-glucosamine. MODES FOR CARRYING OUT THE INVENTION Bacterial strains and growth Three H.pylori strains were used: strain SSI [Lee et al. (1997) Gastroenterology 112: 1386- 1397], clinical isolate G27 (Hospital of Grosseto, Italy [Xiang et al. (1995) Infect. lmmun. 63: 94-98]) and clinical isolate IRIS01 (Hospital of Siena, Italy). These were stored at -80°C in Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, UK) containing 20% glycerol, 10% foetal calf serum and 0.2% cyclodextrin (Sigma, Poole, UK). Strains were grown on Helicobacter selective agar consisting of Columbia agar supplemented with 10% horse defribinated blood plus vancomicin (10 mg/ml), cefsulodin (5 mg/ml), polymixin B (2.5 U/ml), cyclohexamide (50 mg/ml), trimethoprim (5 mg/ml), amphotericin B (8 mg/ml) and β-cyclodextrin 0.2%. Bacteria were grown at 37°C using the Anaereojar system with the Campygen atmosphere generating system (Oxoid, Basingstoke, UK). The identity of H.pylori was confirmed by characteristic colony morphology, carbofucsin stain and urease activity test. Escherichia coli strains were routinely grown in Luria-Bertani (LB) broth or on LB agar. DH10B electrocompetent cells were used for all transformations and were prepared for electroporation as follows: 5ml of an overnight 20ml starter bacterial culture was inoculated into 450ml fresh LB broth and incubated at 37°C under shaking until OD 55 o=0.7. The culture was then spun down at 2600g (JA10=3800rpm) for 15min at 4°C and the pellet was then washed three times with cold 10% glycerol (450ml, 225ml and 22.5ml respectively). The pellet was resuspended in glycerol, aliquoted, and immediately frozen in liquid nitrogen (stored at -80°C). One aliquot was used for each transformation: cells were kept on ice and, when defrosted, added to a 2ml cold ligation, gently mixed and transferred to a 1 mm electroporation cuvette, where a pulse of 2.0V was applied, giving a time constant of 4.0-4.7. 975ml SOC medium was then added to the electroporated cells and the mix was gently transferred to a tube and incubated at 37°C for 30min. The 1ml suspension was then plated on LB selective plates and incubated at 37°C for 16- 24 hours. Antibiotics used for selection purposes were ampicillin (100 mg/ml) and kanamycin (20 mg/ml for H. pylori and 50 mg/ml for E. coli). Generation ofHP563 and HP1031 mutant constructs HP563 and HP 1031 are both annotated as LPS biosynthesis enzymes. Oligonucleotide primers, each containing a 5' terminal restriction site (Kpnl, Pstl, Pstl and Notl respectively) were designed for each gene to amplify by PCR two regions of the genes, J563I and J563II, and J 103 II and J 103 III, respectively. These were synthesised on an automatic synthesiser (Applied Biosystems Inc) by the phosphoramidite coupling method: Template DΝA was prepared from bacteria grown for 48h on Helicobacter-selective agar. Bacteria were harvested and washed in PBS. The pellet was resuspended in 350ml STE, to which 2 ml lysozyme (100 mg/ml in STE, freshly prepared) and 4ml RNAase A (lOmg/ml) were added. After gently mixing, the suspensions were incubated at 37°C for 30min and then at 65°C for 15min, following the addition of 35 ml SDS 10%. A final 1.5 h incubation at 50°C after the addition of 1ml Proteinase K (100 mg/ml in STE, freshly prepared) was performed, then DNA was purified by phenol/chloroform extractions. PCR amplifications were performed on a GeneA p PCR system 9700 (Perkin Elmer). Annealing temperatures were determined according to primer length and GC content. Extension times were determined according to the length of the PCR product (30s for 1 kb). PCR mixtures contained 50mM KCl, 20mM Tris-HCl pH 8.6, 2.5mM MgCl 2 , 0.2mM dNTPs, lOmg/ml BSA, 30pmol of each primer, 2.5U Taq Polymerase, and lmg/ml template DNA. PCR products were digested with the appropriate restriction enzymes (KpnMPstl for J563I and J1031I, and PstUNotl for J563I and J1031II) and cloned into pSK (KpnVNotl digested), to create pJ563 and pJ 1031 plasmids. Purified plasmid DNA (isolated from E.coli chromosomal DNA using the Boehringer Mannheim High Pure™ kit according to the manufacturers 's instructions) was then electroporated into E.coli DH10B competent cells and the transformants were screened by PCR using either vector or insert specific primers. The km cassette was cloned into the unique Pstl site of pJ563 and p J 1031 , the ends of which were previously dephosphorylated to avoid self-ligation, to create pJ563::km and pJ 1031::km. The construct was electroporated into E.coli DH10B cells and the resultant kanamycin resistant clones were analysed by PCR using gene specific primers. All clones analysed were found to contain the correct insert size of 2.4 kb (insert 1.0 kb + kanamycin cassette 1.4 kb) for J 1031, and of 2.6 kb (insert 1.2 kb) for J563, which confirmed the insertion of the kanamycin cassette. Mutagenesis ofHP563 and HP1031 in H.pylori HP563 and HP1031 genes in H.pylori strains G27, SSI and IRIS01 were mutated. To inactivate the genes, the constructs pJ563::km and pJ1031::km (which are unable to replicate in H.pylori) were introduced directly into each H.pylori strain by natural transformation as follows: wild-type H.pylori were grown on blood agar plates for 24h. A light loopfull of these fresh cultures was then re-streaked on fresh non-selective plate in a small area, evenly distributed and incubated for 6h, in order to have logarithmic growth. 1-10 mg (10-20 ml in H 2 O) of DNA obtained from minipreps of the E.coli strain bearing the plasmid of interest was then added to the growing bacteria and spread evenly to fully mix DNA and cells. Plates were incubated for further 16h in a controlled micro-aerophilic atmosphere of 8% O 2 and 10% CO and then the transformation mix was streaked on one full selective kanamycin plate. Single colonies appeared in 3-6 days and were then expanded and analysed. Transformation with 2.2μg of pCM12:km DNA yielded ~103 kanamycin-resistant transformants for G27, whilst transformation with l Oμg of pCM12:km DNA yielded -100 kanamycin-resistant transformants for SSI and for IR1S01 strains. Transformation with 5μg of pJ1031 :km DNA produced -103 kanamycin-resistant transformants for G27, whilst -100 kanamycin-resistant transformants were obtained for SSI and for IRIS01 strains. Putative mutants selected on kanamycin blood agar plates were screened by PCR to confirm that the clones resulted from allelic replacement rather than plasmid integration. Genomic DNA isolated from the transformants and from the parental strains was analysed by PCR using gene specific primers. These primers amplified a 2.4 kb product for the mutant strains of gene HP1031 and a 2.6 kb product for HP563, consistent with the insertion of a 1.4 kb kanamycin cassette into the wild-type gene. As expected, a 1.3 kb and a 1.0 kb products respectively were amplified when gene specific primers were used with the parental strains as templates (Fig. 2). Sequencing of the above mentioned PCR products was performed using kanU (kanamycin upstream) and kanD (kanamycin downstream) as sequencing primers. 100% sequence identity with HP563 and HP1031 nucleotide sequences from strain 26695 confirmed that the insertion of the kanamycin cassette took place in the target gene. SDS-PAGE of LPS Bacteria grown on solid medium were harvested with a sterile swab and washed in PBS. The pellets were weighed and solubilised in sample buffer containing glycerol (20%), Tris-HCl pH 6.8 (0.125M), SDS (4.6%), and 2-mercaptoethanol (5%), at a concentration of approximately 250 mg/ml sample buffer. Samples were boiled at 100°C for 10-15 min, protein content was determined by the Bradford method, and dilutions with lysing buffer were performed to reach a protein concentration of about 25mg/ml for all samples. 5ml of proteinase K (PK) (Preston & Penner, 1987) (100 mg/ml in lysing buffer) were added to each boiled lysate and incubated at 60°C for 2h. Protein content was determined to assess the percentage of undigested, contaminating protein. Finally, bromophenol blue was added to final concentration of 0.004%. PK-treated samples were applied to gel and electrophoresed in a modified Laemmli gel system. The LPS preparations were boiled for 10 min before loading. No SDS was incorporated into the separating gel (15% acrylamide) or stacking gel (4% acryl) and a Tris-glycine buffer was used as running buffer. Electrophoresis was carried out in a slab-gel apparatus (Biorad) with a constant current of 50 mA/slab gel for about 2 1 /2h until the bromophenol blue migrates 1 cm from the end of the gel. After electrophoresis, gels were either fixed and resolved by silver stain, or transferred to a nitrocellulose membrane [Kittelberger & Hilbink (1993) J. Biochem. Biophys. Methods 26: 81-86; Tsai & Frasch (l 982) Anal. Biochem. 119: 115-119]. To fix gels, SDS-PAGE gels were incubated overnight in 40% ethanol, 5% acetic acid, and then gently agitated in 0.7% periodic acid (in fixative) for 10 min. After 3 x 15 min. washes with deionised water the gels were stained for 10 min in 0.5% silver nitrate. Following 3 x 10 min. washes with deionised water, the stain was developed with 0.05% formaldehyde in 0.005% citric acid. Stain was stopped (5% acetic acid) when required intensity was reached. LPS from wild-type H.pylori strains showed several ladder bands in the high MW range (resembling those of the smooth-type LPS of enterobacteria) and one or two bands in the low MW range (characteristic of rough-type LPS [Moran et al. (1992) J. Bacteriol. 174: 1370- 1377]). As expected, the LPS banding of the wild-type strains exhibited different patterns. When the LPS from the mutant strains were subjected to the same analyses, no major differences were noticed with those from the respective wild-type counterparts. Western blots of LPS Based on the SDS-PAGE results, LPS preparations from wild-type and from mutant strains were subjected to western blotting. Immunoblots of LPS preparations were performed using polyclonal mouse anύ-H.pylori G27 hyperimmune serum. Aliquots of LPS preparations were layered onto the surface of a 12.5% polyacrylamide gel in a Mini-PR OTEAN II slab cell (Biorad Laboratories). Following electrophoresis at 15 mA/mini gel, LPS was transferred to nitrocellulose membranes by electroblotting for 2.5h at 70V. After non-specific binding was blocked with a 3% solution of skim milk in 0.1% Triton-PBS, the membrane was incubated overnight at room temperature with 1:500 dilutions of serum samples. HRP-conjugated goat anti-mouse IgG diluted 1:2000 in blocking solution was used, and incubated for 3h at room temperature. Reactive bands were visualized using hydrogen peroxide as the HRP substrate and p-chloro-naphtol as the colour development reagent. The antiserum revealed several bands on the wild-type strains G27, SSI and IRIS1. As expected by the intrinsic variability of LPS in H.pylori strains, the patterns of bands differed in the three wild-type strains (Fig. 3). The HP563 knock-out mutants showed bands which did not differ significantly from those of the respective wild-type strains. LPS preparations from mutant strains G27Δ1031 , SS1Δ1031 and IRIS1 Δ1031 only showed a single major band of apparent MW -25 kDa. The G27Δ1031 mutant in particular expressed a significantly truncated LPS structure. Thus mutants not expressing HP1031 lack an important portion of their LPS. Lewis antigen expression Based on the western blot data, LPS of mutant strains was examined for Lewis epitopes (Appelmelk et al. 1996). Lysed cells of wild-type strain G27 and the HP563 and HP1031 mutants were tested by ELISA using the following monoclonal antibodies: anti-Lewis X, anti- Lewis Y, anti-H type 1, anti-Lewis A, anti-Lewis B, and anti-i antigen: NB: - means OD <0.3; + means 0.3<OD<1.3; ++ means 1.3<OD<2.3; +++ means OD>2.3. The wild-type parental strain (G27) was strongly positive for Lewis X, Lewis Y, H-type 1 and i- antigen expression. The HP563 mutant strain was positive for all Lewis determinants except H-type 1. Based on known biosynthetic pathways known, this suggests that HP563 codes for a β-1-3- galactosyltransferase (Fig. 4), responsible for attaching galactose to 3-N-acetyl-D-glucosamine to form H-type 1. As Lewis Y and H-type 2 antigens are still present in the mutant G27Δ563 strain, the absence of H-type 1 cannot be attributed to the lack of α-1-2 fucosyltransferase activity, as this would also have resulted in the disappearance of Lewis Y and H-type 2. The HP1031 mutant was completely negative for the expression of all known Lewis antigens. This suggests that this isogenic mutant bears a heavily truncated LPS, possibly missing all O-side chains. HP1031 appears to be involved in LPS biosynthesis upstream of the O side chain attachment step e.g. a β-l,4-galactosyltransferase. Self-reactivity Crude extracts or formaldehyde-treated whole-cell bacteria [Marchetti et al. (1995) Science 267:1655-58; Marchetti et al. (1998) Vaccine 16:33-7] were used to immunise BALB/c mice intraperitoneally with alum adjuvant. Ten days after the third immunisation, serum samples were taken from each mouse and tested by immunofluorescence on human gastric section [Negrini et al. (1991) Gastroenterology 101 :437-445; (1996) Gastroenterology 111:655-665]. In some experiments, serum samples from mice immunised with the wild type G27 strain were first incubated with a lysate of this same strain or with the G27Δ563 or G27Δ1031 mutant. Immunofluorescence measurements were then made as described above: The results show that the G27Δ1031 mutant does not contain the cross-reacting epitope(s) since pre-asdorption of anti-G27 antisera with the mutant strain does not abrogate cross-reactivity with human gastric cells. Gastric colonisation The HP 1031 knockout strains were tested for their ability to colonise the stomach of mice. Infection and read-out for colonisation were carried out using the methods described in Marchetti et al. (1995). Results were as follows These results show convincingly that removal of HP1031 activity destroys the ability of H.pylori to colonise the stomach. It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Claims: CLAIMS 1. A cellular Helicobacter pylori vaccine comprising a mutant H.pylori which either does not express LPS, or expresses a mutant form of LPS. 2. The vaccine of claim 1, wherein the vaccine does not induce antibodies which cross-react with human self epitopes. 3. The vaccine of claim 1 or claim 2, in the form of whole cells or a cellular lysate. 4. The vaccine of any preceding claim, further comprising an adjuvant. 5. A Helicobacter pylori bacterium which has a mutation that affects LPS biosynthesis, wherein either LPS is not expressed, or LPS which is expressed does not induce antibodies which cross-react with human self epitopes. 6. The bacterium of claim 5, wherein LPS which is expressed does not contain Lewis-X and/or Lewis-Y antigen. 7. The bacterium of claim 5, having a mutation that affects LPS biosynthesis upstream of the attachment of the O-antigen. 8. The bacterium of claim 5, having a mutation that reduces the enzymatic activity of a β-1,4- galactosyltransferase. 9. The bacterium of claim 5, having a mutation in gene HP 1031 and/or gene HP0563. 10. The bacterium of any one of claims 5 to 9 for use in vaccination. 1 1. The use the bacterium of any one of claims 5 to 9 in the manufacture of a vaccine for the treatment or prophylaxis of H.pylori infection. 12. A method for treating or preventing H.pylori infection, comprising administering an effective amount of the vaccine of any one of claims 1 to 4.

Inventors: DEL GIUDICE GUISEPPE, RAPPUOLI RINO, DEL GIUDICE, GUISEPPE, RAPPUOLI, RINO
Applicants: CHIRON SPA, DEL GIUDICE GUISEPPE, RAPPUOLI RINO, CHIRON S.P.A., DEL GIUDICE, GUISEPPE, RAPPUOLI, RINO
Application year: 2001
Main classification: A61K39
IPC codes: A61P31, C07K14, C12N9, C12N15