WO2000022135A1

WO2000022135A1 - Helicobacter pylori vaccine

Info

Publication number: WO2000022135A1
Application number: PCT/EP1999/007754
Authority: WO
Inventors: Bernhard Knapp; Klaus-Dieter Diehl; Erika Hundt
Original assignee: Chiron Behring Gmbh & Co.
Priority date: 1998-10-15
Filing date: 1999-10-14
Publication date: 2000-04-20
Also published as: DE19847628C2; DE19847628A1

Abstract

The invention relates to a novel outer membrane protein of Helicobacter pylori with amino acid sequence SEQ ID NO:1 and to variants of said membrane protein and the DNA sequences coding for said proteins. The invention also relates to antibodies directed against said proteins or fragments thereof and to vaccines or medicaments containing said proteins or the antibodies directed against said proteins which can be used for active or passive immunization. The proteins or antibodies disclosed can also be used in a diagnostic method for detecting Helicobacter pylori infection.

Description

Helicobacter py / o / 7 vaccine

The present invention relates to an outer membrane protein of Helicobacter pylori with the amino acid sequence of SEQ ID NO: 1 and the variants of this protein described below as well as DNA sequences coding for these proteins. The present invention further relates to antibodies or fragments thereof directed against this protein, as well as vaccines or medicaments which contain this membrane protein or the antibodies directed against it and which can be used for active or passive immunization. Finally, the present invention relates to a diagnostic method for the detection of a Helicobacter py / oπ infection using the membrane protein according to the invention or the antibody according to the invention and a kit for carrying out this method.

Helicobacter pylori is a gram-negative, microaerophilic and spiral bacterium that colonizes the mucosa of the human intestine. This bacterium causes chronic active gastritis and peptic ulcer, especially duodenal ulcer, and also plays a role in the development of gastric cancer. Helicobacter pylori is therefore an important human pathogen. The shape of the screw and the mobility of four to six flagella allows the bacterium to migrate through the gastric mucus in order to then reach the almost pH-neutral boundary layer between mucus and mucosa. Ammonium ions, which are generated during the enzymatic breakdown of urea by bacterial urease, protect the pathogen from the aggressive stomach acid. The bacterium adheres to the endothelial cells of the stomach using specific adhesins. A consequence of chronic mucous membrane colonization with Helicobacter pylori can be inflammatory granulocytic, later monocytic infiltration of the epithelium, which in turn contributes to tissue destruction via inflammation mediators. The infection stimulates both a local and a systemic humoral immune response without being able to effectively eliminate the pathogen.

Vaccination is the classic way to prevent infectious diseases. The development of a vaccine involves the identification of factors that are decisive for virulence, or of structures that are important for humans Immune system to eliminate a pathogen. Such antigens are usually found in the outer membrane of the pathogen. Thus, in the outer membrane of Helicobacter pylori adhesins of 19.6 kD (Doig et al. (1992)) and 20 kD (Evans et al. (1993)) isolated, fo the possible _candidates. are an experimental vaccine with the aim of inducing antibodies that prevent bacterial adhesion to the mucosal surface. In addition, the outer membrane of Helicobacter pylori has porins with molecular weights of 30 kD (Tufano et al. (1994)), 48 kD, 49 kD, 50 kD, 67 kD (Exner et al. (1995)) and 31 kD (Doig et al. (1995)), and via iron-regulated outer membrane proteins with the molecular weights 48 kD, 50 kD and 77 kD (Worst et al. (1995)), erythrocyte-binding antigens with the molecular weights 25 kD and 59 kD (Huang et al. (1992)) and binding proteins for laminin, collagen I and IV, fibronectin and vitronectin (Kondo et al. (1993)). Proteins with molecular weights of 19 kD (Drouet et al. (1991)), 50 kD (Exner et al., (1995)) and 30 kD (Bölin et al. (1995)) as well as a lipoprotein with 20 kD (Kostrzynska et al. (1994)) and strain-specific, surface-localized antigens of 51 kD, 60 kD and 80 kD (Doig and Trust (1994)). The genes for the proteins with the molecular weights of 20 kD (HpaA; Evans et al. (1993)) and 20 kD (Ipp20; Kostrzynska et al. (1994)) have now been isolated. The N-terminal protein sequences for the adhesin with the molecular weights of 19.6 kD (Doig et al. (1992)), for the porins with the molecular weights of 48 kD, 49 kD, 50 kD, 67 kD (Exner et al. ( 1995)), 30 kD (Tufano (1994)) and 31 kD (Doig et al. (1993)) and for the 50 kD protein (Exner et al. (1995)) have now been determined. The porins with the molecular weights of 48kD, 49kD, 50kD, 67kD (Exner et al., (1995)) and 31 kD (Doig et al., (1995)) became aisHop A, Hop B, Hop C, Hop D and Hop E denotes. These belong to a family of 32 highly homologous outer membrane proteins (Tomb et al. (1997)). Another member of this family was developed by llver et al. (1998) identified as a blood group antigen binding antigen, Bab A. However, it was not possible to develop a satisfactorily effective vaccine with the proteins isolated and characterized to date from Helicobacter pylori.

The technical problem underlying the present invention is therefore to provide an effective vaccine against Helicobacter pylori.

This technical problem is solved by providing the embodiments characterized in the claims. The antigen according to the invention enables the development of a vaccine that induces an immune response against Helicobacter pylori in a host vaccinated with this vaccine. It was possible to characterize an outer (probably iron-regulated) membrane protein with a molecular weight of 97 kD originating from Helicobacter pylori, which surprisingly turned out to provide excellent vaccination protection.

The present invention thus relates to a DNA sequence which encodes an outer membrane protein of Helicobacter pylori with the amino acid sequence of SEQ ID NO: 1 or comprises the nucleic acid sequence of SEQ ID NO: 1.

The present invention also relates to a DNA sequence encoding a protein having the immunogenic properties of the outer membrane protein of Helicobacter pylori, the DNA sequence differing from the DNA sequence of SEQ ID NO: 1 (1) in the codon sequence due to the Degeneration of the genetic code distinguishes (2) hybridizes with the DNA sequence of SEQ ID NO: 1 or (1), or (3) a fragment, an allelic variant or another variant of the DNA sequence of SEQ ID NO: 1 is.

The term rotein used in the present invention with the immunogenic properties of Helicobacter pylori 'outer membrane protein refers to any protein, polypeptide or peptide that (1) can serve as an antigen for antibodies that specifically bind to Helicobacter pyloris or (2) when administered as a vaccine, has a protective effect against infection with Helicobacter pylori. The person skilled in the art can use conventional methods to determine proteins, peptides or polypeptides which have such properties, for example using the methods disclosed in the examples below. For example, these proteins, polypeptides or peptides have 50%, 60%, 70% or 80%, preferably 90%, more preferably 95% and most preferably 98% homology to the membrane protein with the amino acid sequence of SEQ ID NO: 1, where these Homology can be determined, for example, using the "Smith-Waterman" homology search algorithm, for example using the "MPSRCH" program (Oxford Molecular), using an "affinity gap search" with the following parameters "gap open penalty 12, gap extension penalty 1 ".

The term "hybridize" used in the present invention refers to conventional hybridization conditions, preferably to hybridization conditions, in which 5xSSPE, 1% SDS, IxDenhardts solution are used as the solution and the hybridization temperatures are between 35 ° C. and 70 ° C., preferably 65 ° C. After hybridization, washing is preferably carried out first with 2xSSC, 1% SDS and then with 0.2xSSC at temperatures between 35 ° C and 70 ° C, preferably at _J35 ° C (for the definition of SSPE, SSC and Denhardts solution see Sambrook et al ., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Stringent hybridization conditions, as described for example in Sambrook et al., Supra, are particularly preferred.

The terms "variants" or "fragment" used in the present invention encompass DNA molecules that differ from the sequence given in SEQ ID NO: 1 by deletion (s), insertion (s), exchange (s) and / or others Distinguish modifications known in the prior art or comprise a fragment of the original nucleic acid molecule, the protein encoded by these DNA molecules still having the properties mentioned above. This also includes allele variants. Methods for generating the above changes in the nucleic acid sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al., Supra. The person skilled in the art is also able to determine whether a protein encoded by a nucleic acid sequence modified in this way still has the properties mentioned above, for example the methods described in the examples.

In a particularly preferred embodiment, the present invention also relates to DNA sequences which comprise the outer membrane protein of Helicobacter pylori from amino acid no. 17 to 863 (eel 7-863) of SEQ ID NO: 1 (ie the protein without a signal sequence) or encode from amino acid number 254 to 323 (aa254-323) of SEQ ID NO: 1.

The DNA sequences according to the invention can also be inserted into a vector. Thus, the present invention also includes vectors containing these DNA sequences. The term "vector" refers to a plasmid (eg pUC18, pBR322, pBlueScript), a virus or another suitable vehicle. In a preferred embodiment, the DNA molecule according to the invention is functionally linked in the vector to regulatory elements which allow its expression in prokaryotic or eukaryotic host cells. In addition to the regulatory elements, such vectors contain, for example a promoter, typically an origin of replication, and specific genes that allow phenotypic selection of a transformed host cell. The regulatory elements for the expression in prokaryotes, for example E. coli, include the lac-trp promoter or T7 promoter, and for the expression in eukaryon or the AOX1 or GAL1 promoter in yeast, and the CMV, SV40, RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells. Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter. Suitable vectors include, for example, T7-based expression vectors for expression in bacteria (Rosenberg et al. (1987)), pMSXND for expression in mammalian cells (Lee and Nathans (1988)), and baculovirus-derived vectors for expression in insect cells.

General methods known in the art can be used to construct expression vectors containing the DNA sequences of the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods, and in vivo recombination methods, as are described, for example, in Sambrook et al., Supra.

The present invention also relates to host cells containing the vectors described above. These host cells include bacteria, yeast, insect and animal cells, preferably mammalian cells. Preferred mammalian cells are CHO, VERO, BHK, HeLa, COS, MDCK, 293 and WI38 cells. Methods for transforming these host cells, for phenotypically selecting transformants and for expressing the DNA molecules of the invention using the vectors described above are known in the art.

The present invention further relates to a method for producing an outer membrane protein of Helicobacter pylori encoded by the DNA sequences according to the invention or a fragment or a protein with its immunogenic properties, which comprises culturing a host cell described above under conditions which allow expression of the protein (preferably stable expression), and the recovery of the protein from the culture or from the host cells. Suitable methods for the recombinant production of the protein are generally known (see for example Holmgren (1985); LaVallie et al. (1993); Wong (1995); Romanos (1995); Williams (1995); Davies (1995)). Suitable cleaning processes (e.g. preparative Chromatography, affinity chromatography, for example

Immunoaffinity chromatography, HPLC etc.) are generally known.

In a further embodiment, the present invention also relates to an outer membrane protein of Helicobacter pylori encoded by the above-described DNA sequences or obtained by the above method, a fragment thereof or a protein with its immunogenic properties. The proteins of the invention can also be modified according to conventional methods known in the art. These modifications include exchanges, insertions, or deletions of amino acids that modify the structure of the protein, while essentially preserving its immunological properties. The exchanges preferably include "conservative" exchanges of amino acid residues, i.e. Exchanges for biologically similar residues, e.g. the substitution of a hydrophobic residue (e.g. isoleucine, valine, leucine, methionine) for another hydrophobic residue, or the substitution of a polar residue for another polar residue (e.g. arginine for lysine, glutamic acid for aspartic acid etc.). Deletions can lead to the generation of molecules that are significantly smaller in size, i.e. that lack amino acids at the N or C terminus, for example.

The present invention also relates to an immunogenic composition, preferably a vaccine, which contains the outer membrane protein of Helicobacter pylori described above, a fragment thereof or protein with its immunogenic properties. The vaccine according to the invention optionally additionally contains a pharmaceutically acceptable carrier. Suitable carriers and the formulation of such vaccines are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline, water, emulsions, for example oil / water emissions, wetting agents, sterile solutions, etc. The vaccine can be administered orally or parenterally, for example intradermally, subcutaneously or intramuscularly. The appropriate dosage is determined by the attending physician and depends on various factors, for example the type of administration, the age and weight of the recipient, etc. It can be, for example, in the range from 10 μg to 40 μg per patient. In children the dose is reduced to 5 μg and in hemodialysis patients the dose is increased to 40 μg. The present invention thus also relates to the use of the outer membrane protein of Helicobacter pylori described above, a fragment thereof or a protein with its immunogenic properties for producing a vaccine for inducing an immune response against Helicobacter pylori in a recipient vaccinated with the vaccine.

Another preferred embodiment of the present invention relates to antibodies against the outer membrane protein of Helicobacter pylori described above, a fragment thereof or protein with its immunogenic properties. These antibodies can be monocional, polyclonal or synthetic antibodies or fragments thereof, for example Fab, Fv or scFv fragments. These are preferably monocional antibodies. The antibodies according to the invention can be produced according to standard methods, the protein encoded by the DNA sequences according to the invention or a synthetic fragment thereof serving as an immunogen. Methods for obtaining monoclonaier antibodies are known to the person skilled in the art and comprise, for example, as a first step the preparation of polyclonal antibodies using the outer membrane protein according to the invention or fragments thereof (for example synthetic peptides) with suitable ligand sequences, for example the peptides or fragments thereof described in the examples Immunogen for the immunization of suitable animals and the production of B-lymphocytes sensitized to the defined antigen. Then, for example, cell hybrids are produced from antibody-producing cells and bone marrow tumor cells and cloned. A clone is then selected which produces an antibody which is specific for the antigen used. This antibody is then made. Examples of cells that produce antibodies are spleen cells, lymph node lines, B-lymphocytes, etc. Examples of animals that can be immunized for this purpose are mice, rats, horses, goats and rabbits. The myeloma cells can be obtained from mice, rats, humans or other sources. The cell fusion can be carried out, for example, by the well-known Köhler and Milstein method. The hybridomas obtained by cell fusion are screened with the antigen by the enzyme-antibody method or by a similar method. For example, clones are obtained using the limit dilution method. The clones obtained are implanted, for example, intraperitoneally in BALB / c mice, the ascites are removed from the mouse after 10 to 14 days, and the monocional antibody is obtained by known methods (for example ammonium sulfate fractionation, PEG fractionation, ion exchange chromatography, gel chromatography or affinity chromatography).

The antibody obtained can be used directly or a fragment thereof can be used. In this context, the term "fragment" means all parts of the monoclonal antibody (e.g. Fab, Fv or "single chain Fv" fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art.

In a particularly preferred embodiment, the monocional antibody mentioned is an antibody derived from an animal (for example a mouse), a humanized antibody or a chimeric antibody or a fragment thereof. Chimeric, human antibody-like or humanized antibodies have a reduced potential antigenicity, but their affinity for the target is not reduced. The production of chimeras and humanized antibodies or of antibodies similar to human antibodies has been described in detail (see for example Queen et al., 1989; Verhoeyan et al., 1988). Humanized immunoglobulins have variable scaffold areas, which essentially come from a human immunoglobulin (called acceptor immunoglobulin) and the complementarity of the determining areas, which essentially come from a non-human immunoglobulin (e.g. from the mouse) (called Donor immunoglobulin). The constant region (s), if any, also originate essentially from a human immunoglobulin. When administered to human patients, humanized (as well as human) antibodies offer a number of advantages over antibodies from mice or other species: (a) the human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore should Antibody response against such an injected antibody is lower than against a completely foreign mouse antibody or a partially foreign chimeric antibody; (b) since the effector area of the humanized antibody is human, it should interact better with other parts of the human immune system, and (c) injected humanized antibodies have a half-life that is essentially equivalent to that of naturally occurring human antibodies, which it is allows smaller and less frequent doses to be administered compared to antibodies from other species. The antibodies or fragments thereof described above can be used, for example, for immunoprecipitation of the proteins discussed above or for the isolation of related proteins from cDNA expression banks. The antibodies can be bound, for example, in liquid phase immunoassays or to a solid support. The antibodies can be labeled in different ways. Suitable markers and labeling methods are known in the art. Examples of immunoassays are ELISA and RIA. The antibodies according to the invention described above can also be used for passive immunization, ie for combating an already existing infection with Helicobacter pylori, with regard to the preparation of the medicament containing this antibody or the fragments described above, etc., the mode of administration and In principle, the dosage is based on the usual criteria for how these are met for other passive vaccines, for example for Tetagam® or Berigiobin®.

The present invention thus also relates to a medicament containing the above-described antibodies according to the invention or a fragment thereof, or the use thereof for producing a medicament for passive immunization.

The present invention also relates to a hybridoma that produces the monoclonal antibody described above.

Furthermore, the present invention also relates to a diagnostic method for the detection of an acute, chronic or previous infection with Helicobacter pylori, in which antibodies directed against Helicobacter pylori are obtained from a sample with the outer membrane protein of Helicobacter pylori according to the invention, a fragment thereof or protein with its immunogenic properties and then determines whether they are bound to the antigen. The detection takes place in that the antibody against Helicobacter pylori generated by the host is detected in the sample (by means of the antigen according to the invention).

In this diagnostic method, a blood sample is taken, the serum is obtained and brought into contact with the antigen according to the invention, and it is then determined whether antibodies from the serum are bound to the antigen. The diagnostic method according to the invention can be designed as an ELISA, RIA or another common detection method, in which, for example, the antibody bound from the serum is included With the help of a second antibody. In a preferred embodiment of the diagnostic method according to the invention, the antigen or a fragment thereof is immobilized, ie adsorbed, for example, on the wall of a plastic shell in such a way that the specific binding specificity is retained. ,,.

Finally, the present invention relates to a diagnostic kit for carrying out the diagnostic method described above, which contains the outer membrane protein of Helicobacter pylori according to the invention, a fragment thereof or protein with its immunogenic properties or the antibody according to the invention described above or the fragment thereof. Depending on the configuration of the diagnostic kit, the outer membrane protein of Helicobacter pylori described above, the fragment thereof or protein with the same immunogenic properties or the antibody described above or the fragment thereof can be immobilized.

The following examples illustrate the invention.

Example 1: Preparation of the membrane fraction and production of sera in rabbits Helicobacter pylori was cultivated and the membrane fraction was isolated as described in Examples 1 and 2 of WO 98/04702. Three rabbits were immunized subcutaneously with 0.2 mg of the membrane fraction in complete Freund ^'s adjuvant (CFA) at 8 different sites near lymph nodes. The rabbits were then boosted after two weeks with 0.4 mg membrane fraction in CFA as described above and after a further two weeks with 0.1 mg membrane fraction in Aerosil (silicon dioxide) a total of ten times daily. After another week, the rabbits were bled and the sera obtained. The antibodies directed against components of Escherichia coli were removed from the sera by the method of Gruber and Zingales (1995).

Example 2: Preparation of a Helicobacter pylori expression gene bank in the vector λTriplEx

Genomic DNA of the Helicobacter pylori strain ATCC 43504 was obtained using the

DNAzol method prepared by GIBCO / BRL (Catalog No. 10503). This was with the

Restriction enzymes Alul and Haelll according to the Sambrook et al. (1989) partially digested. DNA which was larger than 200 bp was cut out after separation of the partially digested DNA by means of LMP gel electrophoresis, the agarose was digested by gelase (BlOzym Diagnostik GmbH, Hameln) and the remaining DNA was extracted with ethanol after phenol-choroform extraction. The ends of the partially digested DNA fragments were then an Eco RI / Not I adapter (Pharmacia, Cat No. 27-7791) according to the procedure of Sambrook et al at _^. (1989) ligated. The ligation batches were cleaned with a Tip5 column (Qiagen), the ends of the DNA fragments were phosphorylated with polynucleotide kinase and the excess adapter molecules were separated off using 1% LMP agarose gel electrophoresis. DNA larger than 200 bp was excised from the gel, the agarose digested by gelase and the DNA finally precipitated with ethanol.

The Alul or Hae Ill fragments of the genomic DNA provided with the EcoRI adapter were, according to the manufacturer's instructions (Clontech Laboratories Inc., Palo Alto, USA, λTripl Ex ™ library, instructions for use), in dephosphorylated λ TriplEx digested with EcoRI Arms (Cat. No. # 6161-1) ligated and then packed into phage heads using the "Gigapack II Gold" packaging extract (Stratagene GmbH, Heidelberg, Germany; Cat. No. # 200216). λTriplEx is a λ vector that has two translation starting points in two different reading frames and has a thymidine row that allows the ribosomes to be shifted in the reading frame at the start of translation, so that DNA inserted into the vector can be read in all three reading frames and thus the probability of detection via antibodies is drastically increased in an immune screening. The titer of the gene banks could be determined to be 5.0x10 ⁵ pfu / ml (Alul gene bank; 6% not recombinant) and 4.5x10 ⁵ pfu / ml (Hae Ill gene bank; 11% not recombinant). The size of the inserted DNA was determined from eight phage plaques according to a PCR protocol (λ TriplEx ™ library, instructions for use) from 0.5 kb to 5.0 kb.

Example 3: Screening of the Alul library

24000 pfu of the Alul gene bank were plated on two 150 mm LB agar plates with the E. coli strain XL1-Blue MRF 1 (Stratagene GmbH, Heidelberg), the phage plaques were transferred to nitrocellulose, the non-specific binding sites were saturated, the filters with a 1: 500 diluted rabbit sera pool, five times saturated with E. coli, from three immunizations (see Example 1) were incubated and the antibodies bound to the plaques were detected with a secondary antibody conjugated to alkaline phosphatase (Clontech, λTriplEx ™ package leaflet). From both agar plates the positively reacting phage plaques were picked and subjected to another screening in order to separate the PIaques. The second screening resulted in a total of 60 positive clones.

The 60 positive phage plaques were spotted on 2 LB plates and the plaques were transferred to several nitrocellulose filters. Hybridization was then carried out with various gene fragments of the Helicobacter pylori genes ureA, ureB and cat, which were labeled with digoxigenin using the "D1G DNÄ" labeling and detection kit (Boehringer Mannheim). In each case, 14 PIaques could be identified, which were identified using gene probes of ureA, ureB or cat hybridized. When the 60 PIaques were screened repeatedly with the anti-membrane serum, 19 PIaques could no longer be clearly identified, so that only 13 clones were used for further analysis.

Example 4: Characterization of the inserted DNA fragments

Plasmid DNA was obtained from the 13 phage plaques using Clontech's excision protocol (λTriplEx ™ library, patient information leaflet). This is achieved by connecting a plasmid DNA in the λTriplEx vector to the λ-specific DNA via the lox P sites, which allow conversion via site-specific recombination. The inserted DNA of all 13 plasmids was sequenced with the primers from Clontech flanking the multiple cloning site of pTriplEx ("λTriplEx 5 ^' LD-Insert Screening Amplimer", "λTriplEx 3 ^' LD-Insert Screening Amplimer"). Eight of the 13 inserted DNA fragments showed sequences that code for the heat shock protein hsp60 from Helicobacter pylori. Of the remaining five inserted DNA fragments, two proved to be identical, so that the remaining four sequences were subjected to further investigations.

Since the genomic sequence of the Helicobacter py / otv strain 26695 is now known (Tomb et al., 1997), the sequences of the inserted DNA fragments of the four clones could be determined using the program "FastA" (Wisconsin Package, Unix, Version 8, Genetics Computer Group) assigned to certain genes and their amino acid sequences are derived. The deduced amino acid sequences of the four genes were compared to a non-redundant protein database using the program "BlastP" using the website http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-blast (contains " Gen Bank Translations "," PDB "," Swiss Prot "," SPupdate "," PIR "). This made it possible to assign homologous sequences to these four amino acid sequences. An amino acid sequence was found to be homologous to that "(3R) -Hydroxymyristoyl- [acyi carrier proteinjdehydratase" from Yersinia enterolitica (P 32205) was found, which was already described in WO 98/04702 as a 17 kD protein. The second amino acid sequence was found to be homologous to the ATP-dependent DNA helicase RecG from Escherichia coli (P 24230), the third amino acid sequence to be homologous to the biotin carboxyiase of /.nabaena species (Q 06862) and the fourth sequence was found to be homologous the iron-regulated outer membrane protein FrpB from Neisseria meningitidis (U 55377).

The phage clones which code for the four amino acid sequences described were used to isolate from the serum saturated with E. coli against the membrane fraction according to the method of Ozaki et al. (1986) to isolate monospecific antibodies. Western blot analysis against a whole germ lysate from Helicobacter pylori showed that the monospecific antibodies against the clone which codes for the helicase-homologous protein recognize an approximately 60 kD protein and that the monospecific antibodies against the clone which encoded homologous protein for the iron-regulated outer membrane protein, recognizing an approximately 97 kD protein. A monospecific antiserum obtained with a phage clone containing a ureA gene was used as a positive control. This recognizes a clear protein band at approx. 30 kD in the Western blot, which can be assigned to the urease A subunit. The monospecific antibodies were detected using the "Vectastain ABC" kit (Vector Laboratories, Burlingame, USA).

Example 5: Cloning and expression of the gene which codes for the 97 kD protein Since, among the genes found, only the one which codes for the 97 kD protein codes for an outer membrane protein and thus represents a potential candidate for obtaining a vaccine characterized this in more detail. SEQ ID N0: 1 shows the DNA sequence and the deduced amino acid sequence of the gene which codes for the 97 kD antigen. The 16 N-terminal residues of the derived amino acid sequence obviously correspond to a signal sequence (Heijne (1985)). The mature protein of 847 amino acids has a calculated molecular weight of 94.1 kD and an isoelectric point of 9.90. Thus, the molecular weight is quite close to the molecular weight of 97 kD, which was determined by SDS gel electrophoresis. With the help of genomic DNA from the Helicobacter py / otv ^' strain ATCC 43504, the gene fragment which codes for the mature protein of the 97 kD antigen was amplified by PCR and converted into the vector pQE30 (Qiagen, Hilden, according to known methods (Ausubel et al.) , Germany) inserted. Expression of the 97 kD antigen in the £. co // ^' strain "XL1 Blue "(Stratagene GmbH, Heidelberg) was shown by the detection of a protein band at 97 kD after SDS polyacrylamide gel electrophoresis and Coomassie blue staining. According to a Western blot analysis, this protein band reacted intensively with the antiserum which acts against the membrane fraction of H. pylori directed is.t

Example 6: Expression of gene fragments

Since the expression rate of the complete 97 kD antigen was low and many degradation products appeared after expression of the protein in E. coli, subgene regions should be brought to expression which were predicted by the GCG program "Peptide Structure" (Wisconsin Package, Unix, Version 8 ) have a high probability of hydrophilicity and surface localization. Three regions were identified: aa17 - aa69, aa148 - aa224 and aa254 - aa323. These regions were amplified according to known methods and expressed in the vector pQE30 in E. coli "XL1 Blue" cells. In the Coomassie blue-stained SDS-polyacrylamide gel, prominent expression bands could be detected, which were shown with the anti-membrane fraction serum in the Western Blot analysis responded.

Example 7: Purification of the expression products

The expression products were purified by binding the N-terminally fused histidine residues to a nickel chelate column under denatured conditions according to a modified protocol from Qiagen ("The QIA expressionist, a handbook for high level expression and purification of 6xHis-tagged proteins", March 1997). After unlocking the £. co / Z bacteria using a glass ball mill (10 min at 4000 rpm; 0.1-0.2 mm glass balls), the cell pellet in 0.1 M NaH ₂ P0 ₄ , 8 M urea, 0.5 M NaCI, 0.02 M imidazole, pH 8.0 solubilized, the supernatant passed over a nickel chelate column and after washing the bound protein finally with 0.1 M NaH ₂ PO ₄ , 8 M urea, 0.5 M NaCl, 0.5 M imidazole, pH 8.0 eluted. The eluate was gradually dialyzed against various buffers containing 0.02 M Tris / HCl, urea, 0.8 M L-arginine, 0.15 M NaCl, pH 8.0 with decreasing urea concentration. Attempts were also made to keep the proteins soluble in the neutral pH range and without the addition of arginine. The proteins were still soluble in the following buffers:

97 kD antigen 20 imM Tris / HCl, 1 M urea, 0.8 M arginine 0.15 M NaCl, pH 7.5 aa17-69 20 mM Tris / HCl, 0.8 M arginine, 0.15 M NaCI, pH 7.0 aa 148-224 20 mM Tris / HCl, 3 M urea, 0.8 M arginine, 0.15 M NaCl, pH 7.0 aa254-323: 20 mM Tris / HCl, 0.15 M NaCl, pH 7.0

The concentration of the purified proteins was calculated after measuring the extinction using the formula E = ε x c x d (E = extinction; ε = molar extinction coefficient; d = layer thickness of the cuvette).

Example 8: Localization of the 97 kD antigen

Rabbits were immunized with the 97 kD expression product to produce antibodies as described in Example 1. After saturation of the antisera with £. co // ^{'-Bestandteiien} were the antisera for a Western blot with whole cell lysate Anaiyse used pylori H.. The antibodies recognize a specific protein band of 97 kD. This protein band is also recognized by antisera, which are directed against the three subregions aa17-69, aal 48-224 and aa254-323.

Cultured - /. py / or / ^' cells washed with PBS, 1% BSA and 2 × 10 ⁸ cells with the respective antiserum (1: 480) in 100 μl volume incubated overnight at 4 ° C. After washing the cells three times, they were incubated with 20 μg / ml of a goat anti-rabbit Ig (H + L) -FITC-labeled antibody (Southern Biotechnology Associates, Birmingham, USA) for one hour at room temperature in the dark. It was washed again three times and the pellet was taken up in 50 μl PBS, pH 7.2. 10 ul was dropped on a slide and allowed to dry slightly. After "Mounting Medium" (Sigma, Heidelberg, Germany) had been dripped on and coverslipped, the mixture was dried overnight, sealed with nail polish and the bacteria were observed under phase contrast and fluorescence microscopy. The antisera, which are directed against the mature 97 kD antigen, showed a very intense fluorescence of the living bacteria compared to the corresponding null sera, which was not only the localization of the 97 kD protein in the outer membrane of Helicobacter pylori shows, but also makes it clear that certain epitopes of the protein are present on the surface of the bacterium and allow access of the bacterium with antibodies. The antisera, which are directed against the three expressed protein regions of the 97 kD antigen, showed only a weak immunofluorescence compared to the corresponding null sera. Example 9: Preservation of the 97kD antigen

Since surface-localized antigens are often very variable in bacteria and are then not suitable for the development of a split vaccine (vaccine that only contains individual structures of a pathogen), the conservation of the 97 KB antigen was investigated. For this purpose, two bacterial isolates from patients (pat01, pat02) and H. py / or / ^' bacteria from the ATCC strains 51110, 60190, 25392 and 43504 were brought into culture and their DNA was analyzed using the "DNAzol" method (GIBCO / BRL; Catalog No. 10503) prepared. Starting from these DNA samples, as described in Ausubel et al. (1987), amplified various subfragments and made them directly accessible for sequencing. The sequences were evaluated with the aid of "GCG" programs and the deduced amino acid sequences of the 97 kD antigen from all examined H. pylori strains were compared directly with one another (Table 1). The Tomb et al. (1997) published sequence HP 1512 of the H. pylori strain 26695. It was found that only a few positions of the 97 kD antigen have amino acid exchanges which are largely conserved. The 97 kD antigen thus appears to be a highly conserved antigen and is therefore suitable for the development of a vaccine.

Table 1: Comparison of the amino acid sequences of the 97 kD antigen between the Helicobacter py / oπ strains 26695, pat 01, pat 02, ATCC 51110, ATCC 60190, ATCC 25392, and ATCC 43504.

The consensus sequence is given. Deviations are marked for the individual strains in the respective positions. The predicted signal sequence is underlined and the subregions that were also expressed are printed in bold.

50

CONSENSUS MLRNQFRIVF VSCIVASN Q AQE-THTLGK VTTKGERTFE YNNKMΪIDRK

26695 T.

PAT01 K T N

PAT02 N

ATCC 51110 K T

60190 N

25392 S K

ATCC 43504 A S N

51 100

CONSENSUS ELQQRQSNQI RDIFRTRADV NVASGGLMAQ KIYVRGIESR LLRVTIDGVA

26695

PAT01

PAT02

ATCC 51110

60190

25392

ATCC 43504

101 150

CONSENSUS QNGNIFHHDA NTVIDPNMIK EVEVIKGAAN ASAGPGAVAG KLSFTTIDAN

26695

PAT01

PAT02

ATCC 51110

60190

25392

ATCC 43504

151 200

CONSENSUS DFLRKNQTYG AKAEAAFYTN FGYRMNATAA YRGKNWDILA YYNHQNIFYY

26695

PAT01

PAT02

ATCC 51110

60190

25392

ATCC 43504 V

201 250

CONSENSUS RDGNNAFRNL FHPNYDLQDP SNSDMSVGTP SEVNSVLAKI NGYINETDSI

26695

PAT01 p IG G V

PAT02 F IG G V

ATCC 51110

60190 E

25392 E.

ATCC 43504 F IG G V

251 300

CONSENSUS SVSYNLTRDN STRLLRPNTT SALSKANDPG SQPAPFVIDF GKELAHTINF

26695

PAT01 M E

PAT02 M

ATCC 51110

60190

25392

ATCC 43504 301 350

CONSENSUS NHNLSLKYKH EGGPNFNQPR VESTAFLGVR GGNYNPVVNP FAYNSNEPAN 26695 PAT01 PAT02

ATCC 51110 60190 25392

ATCC 43504 D TS D

351 400

CONSENSUS PDYIPEVKE CNNPDNISQC TQGAIRPSNG GYQIGYGAPN SINWQGDΞDS 26695 T T PAT01 H D PAT02 R

ATCC 51110 60190 R 25392 R

ATCC 43504 Q G

401 450

CONSENSUS SGGAQAGYGQ LNATMLSTSA NVYHGLVPKN PDYDMTPPNA QNPSAND TL 26695 PAT01 V PAT02 A

ATCC 51110 60190 A 25392 A

ATCC 43504 SAI GS I

451 500

CONSENSUS GNADAEGTLA RRIFLINSGV NFKVTHPISE DYGNVFEYGM lYQNLSVFSG 26695 PAT01 PAT02

ATCC 51110 V 60190 V 25392

ATCC 43504

501 550

CONSENSUS LDKGKNGYYK NNIDPNDPNG FGLPYRHYYT DQSSQYPQNL NTPNPLYRNM 26695 PAT01 PAT02 A

ATCC 51110 60190 25392

ATCC 43504 A

551 600

CONSENSUS PQNSHAIGNI IGGFMQANYN ILSNVIVGAG TRYDIYTLLD KNGRTHVTSG 26695 PAT 01 PAT 02

ATCC 51110 60190 25392

ATCC 43504 LKL 601 650

CONSENSUS FSPSATVLYN PIESIGLKVS YAYVTKGALP GDGVLMRDPT VIYQRNLRPS 26695 A PAT01 PAT02

ATCC 51110 60190 25392 H A

ATCC 43504

651 700

CONSENSUS IGQNVEFNVD YNSKYFNVRG AAFYQVINNF INSYGQDTSK NGGGNATAKN 26695 v PAT01 PAT02

ATCC 51110 A 60190 F 25392 F

ATCC 43504

701 750

CONSENSUS MSGNLPETIN lYGYEVSGNV RYKNFLGTFS VARSWPTARG HLLADTYALA 26695 PAT01 Q V PAT02 Q V

ATCC 51110 60190 25392

ATCC 43504 Q V

751 800

CONSENSUS ATTGNVFILK ADYDIRRWGL TLTWLSRFVT NMYYEGYSIY YPQYGLIKIH 26695 V PAT01 N H F MR PAT02 N F M

ATCC 51110 60190 V 25392

ATCC 43504 N HK M

801 850

CONSENSUS KPGYGVHNVF INWTPPSKKW QGLRISAVFN NILNKQYVDQ TSVFQASADA 26695 PAT01 S A W PAT02 T R S A W

ATCC 51110 TR 60190 25392

ATCC 43504 S A W

851 879

CONSENSUS PASDMIPKGK RMALPAPGFN ARFEVSYQF 26695 PAT01 D PAT02 N

ATCC 51110 60190 25392

ATCC 43504 Example 10: Detection of the protective properties of the 97 kP protein

Six groups of 10 mice each (CP1; male) were treated on days 1, 8 and 15 with the purified proteins 97 kP (1), 97 kP / aa17-69 (2), 97 kD / aa148-224 (3), 97 kD / aa254-323 (4), a mixture of the three subfragments (5) and - immunized with physiological saline (6). The vaccines for groups 1 to 4 contained 0.2 mg protein / dose in 0.2 ml and were mixed with 10 μg cholera toxin (CT) per dose and administered orally. Group 5 received a mixture of subfragments aa17-69 and aa254-323 (0.1 mg of each fragment and 10 μl CT) and after 10 min 0.1 mg subfragment eel 48-224 together with 5 μg CT. About 20 min before the immunization, 0.2 ml of 0.2 N NaHC0 _{2 were} administered orally. A stress infection with 10 ⁹ cfu of the H. pylori strain 326 was given on days 27, 29 and 31. On day 47, the animals were sacrificed, the stomachs were removed, opened, cleaned with tweezers and then shaken with 5 ml of physiological saline. 0.1 ml of this suspension was plated onto 57 cm ² "Columbia" agar plates containing 5% horse blood and cultured for three days at 37 ° C under microaerophilic conditions ("BBL Jar / Camping Pak Plus", Beiton & Dickinson) . The colonies were counted to 4 cm ² . The results are summarized in Figure 1. The animals which were immunized with the complete 97 kD antigen or with the subfragment 97 kD / aa254-323 showed a protective effect against an exposure to H. pylori in comparison with the other animals.

Figure 1: Protection experiment in mice

H. pylori infection in mice after oral vaccination with the 97 kD protein (1), the subfragments 97kD / aa 17-69 (2), 97kD / aa 148-224 (3), 97kD / aa 254-323 (4) and a mixture of the subfragments (5) and the 97kD protein (5). 6: Infection control.

literature

1. Ausubel et al., (Ed.), Current protocois in Molecular Biology (1987)

2. Bölin et al., J. Clinical Microbiol. 33: 381 (1995)

3. Davies, Curr.Opin. Biotech. 6, 1995, 543

4. Doig et al., J. Bacteriol. 174: 2539 (1992)

5. Doig et al., J. Bacteriol. 177: 5447 (1995)

6. Doig and Trust, Infection and Immunity 62 (1994), 4526

7. Drouet et al., J. Clinical Microbiol. 29: 1620 (1991)

8. Evans et al., J. Bacteriol. 175: 674 (1993)

9. Exner et al., 1995, Infection and Immunity 63, 1567

10. Galfre, Meth. Enzymol. 73 (1981), 3

11. Gruber and Zingales, Biotechniques 19 (1995), 28

12. Holmgren, Annu.Rev.Biochem. 54: 237 (1985)

13. Huang et al., J. Gen. Microbiol. 138: 1503, 1992

14. Ilver et al., Science 279 (1998), 373

15. Koehler and Milstein, Nature 256 (1975), 495

16. Kondo et al., European J. Gastroenterol. Hepatol. 5 (1993), 63

17. Kostrzynska et al., J. Bacteriol. 176: 5938 (1994)

18. LaVallie et al., Bio / Technology 11 (1993), 527

19. Lee and Nathans, J.Biol.Chem. 1987, 263: 3521

20. Ozaki et al., J. of Immunol. Meth. 89: 213 (1986)

21. Romanos, Curr.Opin. Biotech. 6, 1995, 527

22. Rosenberg et al., Gene 56 (1987), 125

23. Sambrook et al., (1989) Molecular Cloning. A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

24. Tomb et al., Nature 388 (1997), 539 25. Tufano et al., Infection and Immunity 62 (1994), 1392

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Claims

claims

1. DNA sequence encoding an outer membrane protein of Helicobacter pylori with the amino acid sequence of SEQ ID NO: 1.

2. DNA sequence according to claim 1, which contains the nucleic acid sequence of SEQ ID NO: 1.

3. DNA sequence encoding a protein with the immunogenic properties of the outer membrane protein of Helicobacter pylori,

(a) which differs from the DNA sequence of claim 2 in the codon sequence due to the degeneration of the genetic code,

(b) which hybridizes with the DNA sequence of claim 2 or 3 (a), or

(c) which is a fragment, an allelic variant or another variant of the DNA sequence of claim 2.

4. DNA sequence according to one of claims 1 to 3, the outer membrane protein of Helicobacter pylori of amino acid No. 17 to 863 (eel 7-863) or a fragment thereof of amino acid No. 254 to 323 (aa254-323 ) coded.

5. Vector which contains the DNA sequence according to one of claims 1 to 4 in functional linkage with an expression sequence.

6. Cell transformed with a vector according to claim 5.

7. The cell of claim 6 which is a mammalian cell, bacterial cell, insect cell or yeast cell.

8. Method for producing an outer membrane protein of Helicobacter pylori. a fragment thereof or a protein with its immunogenic properties, which is characterized by the following steps:

(a) growing a cell according to claim 6 or 7 or a cell transformed with the DNA sequence according to any one of claims 1 to 4 or the vector according to claim 5, and

(b) Obtaining the membrane protein from the medium or from the cell.

9. Outer membrane protein of Helicobacter pylori, a fragment thereof or protein with its immunogenic properties, which is encoded by the DNA sequence according to one of claims 1 to 4 or is obtainable by the method of claim 8.

10. Immunogenic composition containing an outer membrane protein of Helicobacter pylori, a fragment thereof or protein with its immunogenic properties according to claim 9, optionally in combination with a pharmaceutically acceptable carrier.

11. The immunogenic composition of claim 10 which is a vaccine.

12. Use of the outer membrane protein of Helicobacter pylori, a fragment thereof or a protein with its immunogenic properties according to claim 9 for the production of a vaccine for inducing an immune response against Helicobacter pylori in a recipient vaccinated with the vaccine.

13. Antibodies against the outer membrane protein of Helicobacter pylori, a fragment thereof or protein with its immunogenic properties according to claim 9 or a fragment thereof.

14. The antibody of claim 13 which is a monoclonal antibody.

15. The antibody of claim 14, wherein the monocional antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody, or a fragment thereof. - * ^•

16. Hybridoma that produces the antibody of claim 14.

17. Medicament containing an antibody according to any one of claims 13 to 15 or a fragment thereof, optionally in combination with a pharmaceutically acceptable carrier.

18. Use of the antibody according to any one of claims 13 to 15 or a fragment thereof for the manufacture of a medicament for passive immunization against Helicobacter pylori.

19. Diagnostic method for the detection of an acute, chronic or previous infection with Helicobacter pylori, in which a sample containing Helicobacter pylori or antibodies directed against it with an outer membrane protein of Helicobacter pylori, a fragment thereof or protein with its immunogenic properties according to claim 9 or Antibody or the fragment thereof according to any one of claims 13 to 15 and then determines whether these are bound to Helicobacter pylori or antibodies directed against it.

20. Diagnostic kit for carrying out the method according to claim 19, which contains an outer membrane protein of Helicobacter pylori, a fragment thereof or protein with its immunogenic properties according to claim 9 or the antibody according to one of claims 13 to 15 or the fragment thereof.