EP1337647A2 - Helicobacter cystein rich protein a (hcpa) et ses utilisations - Google Patents

Helicobacter cystein rich protein a (hcpa) et ses utilisations

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Publication number
EP1337647A2
EP1337647A2 EP01996552A EP01996552A EP1337647A2 EP 1337647 A2 EP1337647 A2 EP 1337647A2 EP 01996552 A EP01996552 A EP 01996552A EP 01996552 A EP01996552 A EP 01996552A EP 1337647 A2 EP1337647 A2 EP 1337647A2
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hcpa
pylori
protein
cells
seq
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Ludwig Deml
Wulf Schneider
Norbert Lehn
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • HcpA Helicobacter cysteine rieh protein A
  • the present invention relates to polynucleotides, comprising a nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7 or 9, or its fragment or derivative, or a polynucleotide which corresponds to the nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7 , 9 or 11 hybridizes, encoding the Helicobacter cysteine rieh protein A (HcpA).
  • the present invention further relates to the polypeptides which are encoded by the nucleic acid sequence or fragment or derivative of the nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7, 9 or 11.
  • the present invention further relates to the use of the polynucleotides and polypeptides.
  • H. pylori Helicobacter pylori
  • H. pylori is a gram-negative, spiral, microaerophilic, polar flagellated bacterium that colonizes the stomach.
  • the role of H. pylori as an etiological agent of gastritis is widely accepted. All patients infected with H. pylori develop chronic gastritis, which is usually asymptomatic. On the basis of chronic gastritis, depending on bacterial and host-dependent factors and the environmental conditions, several diseases can develop:
  • the H. pylori infection can also lead to the formation of MALT lymphoma or ⁇ on-Hodgkin lymphoma of the stomach.
  • H. pylori strains are classified based on the expression of various pathogenicity factors. Despite a large number of studies, however, no markers with clinically relevant prognostic value have been evaluated.
  • Persistent H. pylori infection affects the integrity of the gastric epithelium and leads to chronic inflammation of the surrounding tissue. This is caused by several factors.
  • H. pylori requires mechanisms of acid tolerance for the efficient colonization of the stomach. By expressing large amounts of urease, H. pylori can break down urea present in the stomach to CO2 and NH3 and thus buffer the gastric acid.
  • the polar arranged flagella enable the germ to move from the acidic gastric lumen through the mucus to the neutral milieu on the gastric epithelium.
  • Adhesion of H. pylori to the gastric epithelium is mediated by several factors, such as the BabA adhesive. The vacuolating cytotoxin VacA. About 50% of the H.
  • Cag Pathogenicity Island The Cag PAI codes for 31 open reading frames (ORFs), including for the immunodominant antigen CagA.
  • ORFs open reading frames
  • CagA positive strains are referred to as type I strains and show a strong association with peptic ulcer and gastric adenocarcinoma in infected patients.
  • CagA negative strains type II strains
  • CagA generally do not express functional VacA and only induce mild gastritis in the mouse model of H. pylori infection.
  • the Cag PAI of type I strains codes for essential components of a type IV secretion system, which enables the direct translocation of proteins from the bacterial cytoplasm into the cytoplasm of eukaryotic cells.
  • the secretion system is functionally active and translocates CagA into the target cell.
  • Type I strains induce strong secretion of hiterleukin 8 (IL-8) in vitro after adherence to gastric epithelial cell lines.
  • IL-8 hiterleukin 8
  • H. pylori infection is characterized by massive granulocyte infiltration of the mucosa.
  • the infiltrates are caused by H. pylori activates and contributes to tissue damage by releasing reactive oxygen species and nitrogen oxide radicals as well as proinflammatory cytokines, e.g. IL-8.
  • the binding of neutrophils to the endothelium is facilitated by the neutrophil activating protein from H. pylori reinforced.
  • Specific cell-mediated immune response In addition to massive granulocyte infiltration
  • CD4 and CD8 T lymphocytes and B cells can also be detected in the inflammatory area.
  • the T-eleven response of the CD4 T cells is from
  • Th-1 type The Th-1 response is reflected in the cytokine pattern. Infected in the mucosa
  • Patients have significantly more IL-8, IL-1ß, IL-6, TNF- ⁇ , TGF-ß and IFN- ⁇ secreting mononuclear and polymorphonuclear cells compared to controls.
  • the number of T cells secreting IFN- ⁇ is also significantly increased in infected individuals, while the number of T cells producing IL-4 remains unchanged compared to control subjects.
  • NK cells in the mucosa may also release IFN- ⁇ .
  • living H. pylori isolates induce the release of IL-12 by PBMCs and of IFN- ⁇ by NK cells.
  • Excessive Th-1 response has been demonstrated in patients with peptic ulcer. Isolated local T cells show, after restimulation with H.
  • Th-1 and Th-2 cytokines are secreted equally.
  • About 50% of the Th-1 clones are specific for the immunodominant antigen CagA.
  • IFN- ⁇ seems to play a key role in this.
  • the extent of the inflammation can be reduced by treatment with neutralizing antibodies against IFN- ⁇ .
  • IFN- ⁇ deficient mice are colonized by clinical H. pylori isolates, but the infiltration of the mucosa is completely avoided, as are the lesions associated with the inflammation.
  • the humoral immune response to an H. pylori infection is characterized not only by the IgA response, which is desirable for efficient defense against mucosal infections, but also by an IgG response.
  • IgA response triggered by an H. pylori infection alone is not sufficient to prevent the persistence of the germ.
  • B lymphocyte proliferation and immunoglobulin secretion is not only mediated directly by H. pylori antigens, but also indirectly by H. pylori specific T lymphocytes.
  • the MALT lymphoma associated with H. pylori infection is that of H. pylori specific T cell proliferation mediated hyperproliferative B cells monoclonally, but not H. pylori specifically.
  • antibodies are an important marker in the diagnosis of H. pylori infection.
  • antibodies IgG or IgA
  • individual antigens are detected by ELISA or Western blot analysis.
  • individual antigens correlate more strongly with the development of certain diseases - antibodies against VacA and CagA characterize type I strains, and MALT lymphoma correlates with serum antibodies against FldA - they do not allow prognosis regarding the course of infection.
  • the serology is often supplemented by genotyping additional loci.
  • IFN- ⁇ induces an increased expression of MHC-II proteins on the gastric epithelium.
  • MHC-II proteins also serve as specific receptors for H. pylori.
  • OMP outer membrane proteins from H. pylori can bind directly to MHC-II proteins and induce apoptosis as superantigens.
  • Th-1 cells also contribute to an increased apoptosis rate via the Fas / Fas ligand route. Direct cell damage.
  • IFN- ⁇ also increases the permeability of the gastric epithelium by dysregulating the tight junctions.
  • the granulocytes infiltrating the mucosa release both tissue-damaging reactive oxygen species and nitrogen oxide radicals.
  • pro-inflammatory cytokines induce the release of gastrin or repress secretion of somatostatin and may therefore be involved in gastric hyperchloremia.
  • H. pylori infection When diagnosing an H. pylori infection, a distinction is made between endoscopic and non-endoscopic methods.
  • endoscopy biopsies are taken from several locations on the stomach.
  • H. pylori is detected in the biopsies by culture, histological examination including immunohistological characterization, urease rapid test, the polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH).
  • PCR polymerase chain reaction
  • FISH fluorescent in situ hybridization
  • endoscopically directly visible changes in the stomach that correlate with an H. pylori infection are strong indicators of the presence an infection.
  • H. pylori infection cannot be detected endoscopically by detecting H. pylori-specific antibodies in the blood. The detection of H.
  • the H. pylori infection is detected in the air after application of appropriately labeled urea.
  • the degradation products in the blood or urine can be detected analogously.
  • the diagnosis enables statements regarding the resistance spectrum of the isolate, but markers with a prognostic value have not been evaluated.
  • the therapy regimens have developed from classic bismuth-containing triplet therapy to dual therapy (omeprazole plus amoxicillin) to modified triplet therapy with proton pump inhibitors, clarithromycin and metronidazole or amoxilin. Disadvantages of the therapeutic agents used hitherto lie in the sometimes poor tolerance of the medication and the appearance of multi-resistant H. pylori strains.
  • Vaccines Prevention of infection would be better than treatment.
  • a successful vaccination against H. pylori infection could be found in a variety of animal models e.g. after orogastral administration of H. pylori antigens in combination with mucosal adjuvants such as the cholera toxin and the heat-labile enterotoxin of E. coli.
  • mucosal adjuvants such as the cholera toxin and the heat-labile enterotoxin of E. coli.
  • a large number of different immunogens in test animals provide protection against subsequent exposure to H pylori or even enable the eradication of an existing Helicobacter infection (therapeutic immunization).
  • the most efficient immunogens so far include bacterial sonicates of the purified urease holoenzyme, urease subunits or components thereof, purified VacA cytotoxin and the GroEL (HspB) and GroES (HspA) heat shock protein homologues.
  • HspB purified VacA cytotoxin and the GroEL
  • HspA GroES
  • the object of the present invention is to provide substances for therapy, diagnosis and vaccination of and against diseases with H pylori.
  • the object is achieved by the subject-matter defined in the patent claims.
  • FIG. 1A shows the complete nucleic acid sequence of the hcpA gene from the H pylori strain 60190RE, which was isolated from an ulcer patient (SEQ ID NO: 5 and 6).
  • FIG. 1B shows the nucleic acid and amino acid sequence of the HcpA protein from H. pylori 60190RE expressed in the pET 21 a (+) system. Compared to the wild-type HcpA protein, additional amino acids are highlighted in bold. A histidine (His) tag (6 x His) was fused to the C-terminus of the protein to facilitate purification.
  • His histidine
  • FIG. IC shows the nucleic acid and amino acid sequence of the HcpA protein from Helicobacter pylori 60190RE expressed in the pRBI-PDI system.
  • the OMP-A signal sequence of the protein is underlined in the amino acid sequence shown.
  • the expressed protein contains an OMPA sequence from E. coli as the leader sequence and a C-terminal leucine residue (SEQ ID NO: 9 and 10).
  • FIG. ID shows the nucleic acid and amino acid sequence of the functional C-terminus of the HcpA protein from Helicobacter pylori 60190RE (SEQ ID NO: 11 and 12).
  • FIG. 2 shows the purification of HcpA polypeptides expressed in Escherichia coli (E. coli) by metal affinity chromatography.
  • A Chromatogram: 2 ml of solubilized inclusion bodies were diluted 1:10 in running buffer (6 M urea buffered to pH 8.0) and cleaned at a flow rate of 1 ml / min. The elution was carried out by jumping to pH 4.0 (in 6 M urea). The elution fractions are underlined.
  • B Coomassie stained gel from purified HcpA. The concentration of the protein purified in this way is 2.7 mg / ml.
  • FIG. 3 shows the expression of HcpA in the pRBI-PDI system.
  • A Chromatogram of cation exchange chromatography. Heat-precipitated periplasm was applied to a Porös CM column at a flow rate of 10 ml / min and the HcpA protein in a salt gradient of 0 to 1 M NaCl (buffer B: 50 mM MES pH 6.4, 1 M NaCl) isolated at about 500 mM NaCl in one peak.
  • Buffer B 50 mM MES pH 6.4, 1 M NaCl
  • B Coomassie stained SDS PAGE of an HcpA cleaning. HcpA (24 kDa) and DsbC (20 kDa) are clearly visible in the total lysate-induced bacteria.
  • HcpA is significantly enriched in the periplasm, contaminating cell proteins are largely separated. The heat precipitation does not lead to any significant loss of HcpA, while above all high molecular weight proteins are separated.
  • the purified HcpA is band-pure even when concentrated by a factor of 10. (Lane 1: total lysate of non-induced cells, 2: total lysate of induced cells, 3: periplasma, 4: heat-precipitated periplasma, 5: eluted HcpA, 6: HcpA 10 times concentrated)
  • C UV-visible spectrum of native HcpA.
  • HcpA Natively expressed HcpA was measured in the spectrophotometer in analogy to the refolded protein (100 ⁇ l, layer thickness 1 cm). The concentration is 34.5 ⁇ M (852 ⁇ g / ml).
  • D Cross-linking of HcpA with disuccinimidyl suberate (DSS). 5 ⁇ g HcpA in PBS were cross-linked with DSS and separated on SDS PAGE.
  • the transcription factor MarR (15 kDa as monomer) from E. coli was used as a positive control. While the apparent molecular weight of HcpA is not increased by the crosslinking, it leads to a doubling of the apparent molecular weight in MarR.
  • Figure 4 shows the expression and secretion of HcpA in clinical H. pylori isolates. 5 ⁇ g total lysate from different H. pylori strains was examined in a Western blot for the expression of HcpA. The blot was stripped as a loading control and detected with an ⁇ RecA serum. RecA is a cytoplasmic protein ubiquitous in H. pylori with a molecular weight of approximately 40 kDa. The expression in the total lysate is compared with the secretion in the culture supernatant. 100 ⁇ l each of an overnight culture of H.
  • pylori in BB-FKS-DENT was precipitated with trichloroacetic acid (TCA) / sodium dodecyl sulfate (NaDoc) and HcpA was determined in a Western blot (0: negative control: medium alone).
  • TCA trichloroacetic acid
  • NaDoc sodium dodecyl sulfate
  • HcpA was determined in a Western blot (0: negative control: medium alone).
  • TCA trichloroacetic acid
  • NaDoc sodium dodecyl sulfate
  • FIG. 5 shows the schematic representation of the HcpA ORF from H. pylori 60190RE.
  • the start codons are printed in bold.
  • the protein expressed via the internal translation start is extended by the dipeptide, MA 'in comparison to the processed full-length protein.
  • Mittl et al. have expressed HcpA in this form and demonstrated the processing of start methionine in E. coli by N-terminal sequencing.
  • the HcpA natively expressed in this work has the sequence EPDAKELVN (Edman sequencing) at the N-terminal.
  • FIG. 6 shows the coiled-coil region at the C-terminus of HcpA in comparison with ORF 1098.
  • hydrophobic amino acids form the positions, a 'and, d' (highlighted in gray).
  • ORF 1098 a glutamate residue is replaced by glycine (highlighted in red). This violates the helical structure and no coiled coil can form.
  • FIG. 7 shows the reactivity of HcpA in the ELISA against control sera with a defined Helicobacter status from Mikrogen.
  • the sera were diluted 1:50 in PBS.
  • the reactivity of the sera against H. pylori total lysate was determined in a recomb test by Mikrogen and compared with the culture and histology results of the corresponding patients.
  • "Correctly positive” and “Correctly negative” mean that the results of Mikrogen's test match the gold standard, "False negative” and “False positive” diverging. , + ': Positive control serum.
  • FIG. 8 shows the seroreactivity of a collective of adult patients against HcpA in the ELISA. In each case 68%> of the measured values lie in the area of the bars (68%> confidence).
  • FIG. 9 shows the receiving operating characteristics of the HcpA ELISA in the patient population examined.
  • the c t off assigned to the circled point was subsequently used to evaluate the raw data.
  • cut-off values that, in extreme cases, classify all samples correctly.
  • the proportion of correctly identified samples cannot be increased disproportionately with respect to the incorrectly identified samples by varying the cut off.
  • A Dose dependency of IFN- ⁇ release when incubated with Periplasma from H. pylori 60190RE.
  • the periplasm of H. pylori 60190RE contains HcpA ( Figure 14 C) and shows a bell-shaped dose dependency.
  • the maximum amount of IFN- ⁇ released is about 500 pg / ml.
  • B Dose dependency of IFN- ⁇ release when incubated with recombinant HcpA.
  • HcpA leads to a very strong induction of IFN- ⁇ (75,000 pg / ml).
  • periplasma and HcpA were digested with chymotrypsin. Chymotrypsin alone did not induce IFN- ⁇ .
  • C Neutralization of HcpA with a polyldonal rabbit serum against HcpA. 60 ng HcpA were incubated for 1 h with serum in variable volume ratios (ad 100 ⁇ l PBS) and then the resulting IFN- ⁇ release was determined in spleen cell suspensions. Serum alone did not induce IFN- ⁇ .
  • FIG. 11 shows the cytokine pattern induced by HcpA in murine spleen cells.
  • Spleen cells from Balb / c mice (ages 46, 118, 208 days) were isolated independently of one another and incubated for 36 h with HcpA in variable concentration. The culture supernatant was replaced by a capture ELISA determines the corresponding cytokines. The values given are mean values and standard deviations from double samples from 3 independent experiments.
  • A Concentration dependence of the HcpA effect.
  • B Cytokine pattern induced by 1.3 ⁇ g HcpA / well (PBS, CpG: IFN- ⁇ induction by PBS (buffer control) and CpG-ODNs (positive control)).
  • C Cytokine pattern induced by 1 ⁇ g C-terminal HcpA fragment (mean and standard deviation from triplicates).
  • FIG. 12 shows the development of the antibody titer (total IgG) against HcpA after immunization of sterile Balb / c mice.
  • 5 mice per group were immunized subcutaneously in the tail root by injection of 10 ⁇ g HcpA in 100 ⁇ l PBS and boosted at intervals of 6 weeks.
  • the antibody titer was determined two weeks after the immunization by endpoint dilution in the ELISA.
  • the titer given is the maximum dilution at which the positive serum still showed at least three times the OD492 of the corresponding negative serum.
  • Long-term immunization titers were determined two months after the last immunization.
  • the immunization was carried out either without adjuvation or with adjuvation with aluminum hydroxide (Alum) or CpG oligonucleotides (CpG-ODNs). Naive control animals were only injected with PBS. The mean values of the respective groups are shown as a line graphic.
  • FIG. 13 Ratio of specific antibody isotypes after immunization of Balb / c mice. The mean values and standard deviation of the titer of each experimental group are shown. The isotype ratios IgGl: IgG2a were determined by averaging the isotype ratios of the individual animals and are noted on the bar. For naive animals and animals immunized with HcpA without adjuvant, no isotype ratios were determined after the basic immunization and the first booster immunization due to an undetectable HcpA-specific antibody response.
  • FIG. 14 shows the production of an isogenic HcpA knock out mutant of H. pylori 2802.
  • A The lisertion of the cat cassette was confirmed by PCR of the hcpA gene with the primers Hp211for and Hp211rev. The hcpA gene from the mutated strains is extended by the inserted cat cassette. (0: water control of the PCR)
  • B control of the sertion in the Southern blot. 2 ⁇ g DNA from wild-type and mutant H pylori 2802 were digested with BamHI, electrophoretically (
  • C Detection of the HcpA knock out in the Western blot. Cyto- and periplasma of the corresponding strains were isolated and 5 ⁇ g cytoplasm (C) and the corresponding amount of periplasma (P) were analyzed for the presence of the HcpA protein in a Western blot. The control of the periplasm preparation was carried out by stripping the blot and redetection with an ⁇ -RecA antibody. D: Induction of IFN- ⁇ [pg / ml] in spleen cells from Balb / c mice by incubation with 10 ⁇ g water extract of the respective bacterial strain. The mean ⁇ standard deviation from triple measurements are shown.
  • FIG. 15 Co-cultivation of H. pylori with murine spleen cells.
  • H. pylori 2802 (wild type) or the isogenic HcpA knock out mutant H. pylori 2802hcpA cat (hiock out) growing logarithmically on agar plates were resuspended in inoculation medium, washed three times with PBS and resuspended in RPMI medium. The cell number was determined by measuring the optical density.
  • Murine (C57BL / 6) spleen cell suspensions were cocultivated with increasing multiplicity of infection (MOI) of the H. pylori strains for 36 h and the cytokine release in the supernatant was determined. The mean and standard deviation from triplicates are shown.
  • the isogenic HcpA laiock out mutant induces a significantly lower IFN- ⁇ secretion than the wild-type bacterium.
  • H pylori is a gram-negative bacterium that colonizes the human stomach. The infection necessarily leads to superficial gastritis and is a risk factor for the development of peptic ulcer, MALT lymphoma and adenocarcinoma. H pylori is classified by the WHO as a type I carcinogen. Histological hallmark of H pylori infection is massive inflammatory infiltration of the mucosa. Despite the strong inflammatory reaction, the germ persists for life. The mucosal immune response is of the T helper 1 type with interferon- ⁇ (IFN- ⁇ ) as the leading cytokine. Although about half of the world's population is infected with the bacterium, only a few patients become symptomatic.
  • IFN- ⁇ interferon- ⁇
  • HcpA Helicobacter cysteine rieh protein A
  • Karita and co-workers Karita et al., Infect. Immun. (1997), 65: 4158-64) (Genbank Accession No. AF008565)
  • the HcpA ORF is in the H. pylori strain 60190 together with the GidA and DapE ORF in an operon that contains nucleotides 3703 (start codon) up to and including nucleotide 4455 (Stop codon).
  • the hcpA reading frame corresponds to ORF 211 (Tomb et ah, Nature (1997), 388: 539-47).
  • the HcpA protein belongs to a gene family that is ubiquitous in H pylori and has no significant sequence similarity to other proteins. Processed HcpA contains 12 cysteine residues with a molecular weight of 24 kDa. HcpA functions as a ß-lactamase.
  • the hcpa polynucleotides used in the experiments on which the invention is based come from the H. pylori strain 60190RE, which was isolated from a stomach biopsy of an ulcer patient.
  • the hcpa sequence of the H pylori strain 60190RE has at least 22 nucleotide exchanges in comparison with all hcpa sequences published to date.
  • the internal translation start has a strong Shine-Dalgarno sequence (GAGG) at a suitable distance from the start codon ATG.
  • the protein expressed via the internal translation start has a molecular weight of 24 kDa and differs from the processed form of the HcpA protein already described by the presence of 2 additional N-terminal amino acids. Due to the lack of a signal sequence, this variant of the HcpA protein should be localized in the cytoplasm. The majority of the strains examined express the HcpA protein. In contrast, the secretion of HcpA in the culture supernatant is strain-dependent. Secretion does not take place via the postulated sec-dependent route and not via the Cag apparatus. The release of the protein correlates serologically with the antibody titer. In the investigated collective, HcpA is only strongly secreted in isolates from ulcer patients.
  • the HcpA polypeptide (Fig. 1B) was obtained with yields of 50 mg / 1 culture in the bacterial pET System (Stratagene) expressed in the denatured state. Under defined redox conditions (5 mM N-acetylcysteine) the protein could be folded back into its native state. Alternatively, the protein was expressed natively in the periplasm in a bicistronic vector system with the disulfide isomerase DsbC. The HcpA protein shows great heat stability, is completely disulfide bridged and is present as a monomer. Natively expressed HcpA protein could be crystallized. An X-ray structure with a resolution of 2.6 ⁇ can be determined from this.
  • HcpA is immunogenic in humans and is suitable as a diagnostic marker for H. pylori infection (p ⁇ 0.001).
  • HcpA mediates immunomodulation.
  • Recombinant HcpA induces an extremely strong release of IFN- ⁇ in murine spleen cell cultures. Cytokine induction can be inhibited by chymotrypsin digestion or neutralization with HcpA-specific antiserum.
  • Th-2 cytokines IL-4, IL-5 and IL-10 are not induced, nor was TNF- ⁇ detected in the spleen cell cultures after 36 hours of stimulation.
  • the induced cytokine pattern corresponds to that of the H. pylori mucosa of infected patients. There is also an excessive Th-1 response in patients with ulcers. IFN- ⁇
  • HcpA Induction of inflammation plays a key role.
  • the strong IFN- ⁇ induction by HcpA in the mouse model corresponds clinically with the increased anti-HcpA titer in patients with ulcer.
  • Different clinical isolates do not differ in terms of the expression of HcpA, but in the extent of the secretion of the protein in the culture supernatant. Isolates from patients with ulcers show a very strong HcpA secretion.
  • An isogenic HcpA laiock out mutant was generated by hiseragenic mutagenesis. In comparison to the wild type strain, aqueous extract of the knock out mutant shows a significantly reduced IFN- ⁇ induction in the spleen model.
  • wild type strains of H H.
  • HcpA represents a potential virulence factor both with regard to immunomodulation and with regard to the strong attenuation rank in liquid medium
  • Balb / c mice were immunized with the HcpA protein in the absence and presence of adjuvants. The immunization induced a very high and long-lasting antibody response.
  • Adjuvant-specific led the Immunization to a Th-2 polarization (Alum) or a Th-1 polarization (CpG-ODNs) of the observed immune response.
  • HcpA protein could be neutralized by the generated HcpA-specific sera.
  • the present invention describes the use of the H pylori cysteine protein A (HcpA) and polypeptides derived therefrom, as well as gene sequences coding for HcpA polypeptides, and polynucleotides derived therefrom for use in the diagnosis, therapy and prevention of Helicobacter infections.
  • HcpA H pylori cysteine protein A
  • the present invention includes not only polynucleotides encoding HcpA polypeptides, but also their use for the diagnosis, therapy and prevention of Helicobacter infections.
  • HcpA polypeptides and vectors containing polynucleotides encoding HcpA can be used as a vaccine adjuvant or coadjuvant to increase or modulate the immune response in prophylactic or therapeutic vaccines in which interferons have a positive effect.
  • HcpA polypeptides and vectors containing polynucleotides encoding HcpA can be used in the clinical field as therapeutic agents to stimulate interferon production in mammals and especially in humans.
  • the invention further comprises the production and use of HcpA-negative Helicobacter isolates.
  • hcpA describes a genetic region which is found, for example, in the H pylori strain 60190 together with the GidA and DapE ORF in an operon which comprises the nucleotides 3703 (start codon) to 4455 stop codon) (see Karita et al., Infect. Immun. (1997), 65: 4158-64), Genbank Accession No. AF008565).
  • Cao and co-workers describe HcpA as an immunogenic protein secreted into the culture supernatant Molecular weight of 27 kDa (Cao et ah, Infect. Immun. (1998), 66: 2984-6).
  • the hcpa gene used in the experiments on which the invention is based comes from the H.
  • the hcpa sequence of the H. pylori strain 60190RE has 22 nucleotide changes compared to all hcpa sequences of 60190 published so far.
  • Nucleotide and amino acid sequences of ⁇ cpa polypeptides from H. pylori 60190RE are listed in FIGS. 1A to D (SEQ ID NO: 5 to 10) and SEQ ID NO: 1 to 4.
  • SEQ ID NO: 1 and 2 represent the nucleotide or amino acid sequence of the processed ⁇ cpA, SEQ ID NO: 3 and 4 of the ⁇ cpA expressed via the internal ATG.
  • ⁇ cpA proteins according to the invention also include those polypeptides which have only minor modifications, in particular conservative ones Have amino acid changes to the natural amino acid sequence of the ⁇ cpA protein.
  • Polypeptide molecules which have essentially the same amino acid sequence as the ⁇ cpA protein or have only a few amino acid substitutions or N and / or C-tenninally fused amino acids which do not significantly change the effect of the ⁇ cpA protein according to the invention are in the definition of the invention ⁇ cpA protein included.
  • polypeptide used in this patent specification denotes a polymer of amino acids of any length; thus the term polypeptide also includes the terms epitope, peptide, oligopeptide, protein and polyprotein. Also included in this term are polypeptides that contain post-translational modifications such as e.g. Have glycosylations, acetylations, phosphorylations and similar modifications. Furthermore, this term encompasses, for example, polypeptides which have one or more analogs of amino acids (e.g. unnatural amino acids), polypeptides with substituted linkages, and other modifications which are state of the art, regardless of whether they occur naturally or are not of natural origin.
  • a "recombinant protein or polypeptide” can be defined by a natural occurring genomic polynucleotide, encoded by cDNA, a semi-synthetic polynucleotide or a synthetically produced polynucleotide. It is not limited to polynucleotide sequences that occur completely or in part naturally, it can also be encoded by polynucleotide sequences that are found in naturally occurring Helicobacter isolates or that do not occur in nature. This term thus also includes the situation in which the coding nucleic acid sequence of the HcpA protein has been genetically modified (for example by mutagenesis) or has been synthesized from oligonucleotides with the aim of producing one or more modified polypeptides.
  • the nucleotides to be changed can also be chosen at random if the amino acid sequence of the polypeptide is not impaired by this, or the conservative or non-conservative amino acid exchanges introduced by the change in the nucleic acid sequence result in a protein that has substantially comparable properties to that by the natural gene sequence encoding protein.
  • the nucleic acid sequence coding for the HcpA protein can be changed such that the coded protein has no amino acid exchanges. This is due to the fact that the genetic code is degenerate, which means that many amino acids are encoded by more than one codon. Often, the preferred or less preferred codon usage of a particular host organism can be determined based on available literature (see, e.g., T.
  • polynucleotide used in this patent specification denotes the polymeric form of a nucleotide of any length, preferably deoxyribonucleotides, and is used interchangeably in this patent specification with the terms “oligonucleotide” and “oligomer”. This term refers only to the primary structure of the molecule. The term includes double and single stranded DNA as well as antisense polynucleotides. Furthermore, the term includes all known types of modified polynucleotides, such as common markings, for example methylations, ends, cap structures, and a substitution of natural hit nucleotide linkages by analogous intemucleotide modifications such as, for example, uncharged Links (e.g.
  • methylphosphonates phosphotriesters, phosphoamidates, carbamates, etc.
  • loaded links e.g. phosphosothioates, phosphorodithioates, etc.
  • molecules such as proteins (including toxins, antibodies, signal peptides, poly-L-lysine etc. ) and include alkylators.
  • oligomer also includes primers and probes.
  • An oligomer can be of any size.
  • Typical probes include less than 1000 nucleotides, more often less than 500 nucleotides, most often even less than 250 nucleotides; sometimes they can be less than 100 nucleotides, or even less than 75 nucleotides, and very rarely are they less than 50 nucleotides in length.
  • Typical primers include less than 100 nucleotides, more often less than 50 nucleotides, most often even less than 30 nucleotides; they can sometimes be less than 15 nucleotides.
  • genomic encompasses all or fragments of the genetic material of an organism.
  • parent polynucleotide and “parent strand” refer to single- or double-stranded nucleic acid molecules which contain the desired target sequence. They can be a biological probe.
  • target region denotes a region within a nucleic acid that is to be duplicated or detected.
  • binding partner describes a molecule that is capable of binding to a ligand molecule with high specificity; an example of this is an antigen and an antibody specific for the antigen.
  • Coupled denotes attachment by means of covalent bonding or strong, noncovalent interactions (eg hydrophobic interactions, hydrogen bridge bonds, etc.). Covalent bonds can be, for example, esters, ethers, phosphoesters, amides, peptides, hnides, carbon-sulfur bonds, carbon-phosphate bonds or similar bonds.
  • surface denotes a solid or polymorphic material to which a desired binding partner can be anchored. Suitable surfaces include glass, plastic, metal, polymeric gels, or similar materials, and they can be found in the form of spheres, cups, dipsticks, membranes, and similar structures.
  • label refers to any atom or group that can be used to generate a detectable (preferably quantifiable) signal and that can be coupled to a polynucleotide or polypeptide.
  • a "replicon” is a genetic element, e.g. a plasmid, a chromosome, a virus, a cosmid, which is an autonomous unit for replication of polynucleotides in cells and which controls its own replication.
  • vector or "gene transfer vector” used here denotes naturally occurring or artificially created organisms and constructs for the uptake, multiplication, expression or transfer of nucleic acids in cells.
  • Vectors are, for example, viruses such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, pox viruses, alpha viruses, baculoviruses, rabies viruses or herpes viruses.
  • Vectors are also bacteria, for example, such as listeria, shigella or salmonella.
  • Vectors are also, for example, liposomes or naked DNA, such as bacterial plasmids and MIDGES, virus-derived plasmids, phagemids, cosmids, bacteriophages or artificially produced nucleic acids such as artificial chromosomes. Vectors have the ability to multiply autonomously in a cell.
  • control sequences describes polynucleotide sequences that are required for the expression of coding sequences with which they are linked. The properties of such control sequences differ depending on the host organism. In prokaryotes, such control sequences usually include a promoter, a ribosomal entry site and a transcription termination sequence; in eukaryotes, such control sequences usually include promoters and transcription termination sequences.
  • control sequence also includes at least all components whose presence is required for expression and also includes additional components with a whose presence is conducive to expression, such as leader sequences and the sequences of a fusion partner.
  • immunogenic is defined as the ability of a polypeptide to induce a humoral and / or cellular immune response, either alone or by association with a carrier and / or in the presence or absence of adjuvants.
  • neutralization is defined as an immune response that is capable of either partially or completely preventing infection with an infectious agent.
  • epitope is defined as an antigenic component of a peptide, polypeptide or protein; an epitope can include 3 or more amino acids in a spatial conformation unique to the epitope. Typically, an epitope consists of at least 5 amino acids or, more commonly, at least 8-10 amino acids. Methods for determining the spatial conformation of the amino acids are known and prior art and include, for example, X-ray structure analysis and 2-dimensional nuclear magnetic resonance spectroscopy. Antibodies that recognize the same epitope can be quickly recognized in simple bioassays because each of these antibodies has the ability to prevent the binding of one of the other antibodies to the target antigen.
  • neutralization is also defined as an immune response that is able to suppress the (immune) modulatory effect of a polypeptide either partially or completely.
  • Immunogenic compositions used as vaccines include an immunologically effective concentration of the antigenic polypeptide or polynucleotide and, if necessary, any of the substances already listed.
  • immunologically effective concentration means that the administration of this concentration of active substance to an individual, either in the form of a single dose or repeated administration, is suitable for the treatment or prevention of the disease to be treated.
  • individual refers to an animal susceptible to H. pylori infection and also includes primates, including humans; is however not limited to this.
  • vaccine defines an immunogen, or other substance that is capable of providing partial or complete protection, as well as compositions that are useful for the treatment of an individual. These compositions can include modified bacteria and viruses (e.g., chemically or However, bacterial or viral cleavage products that have either been produced recombinantly or have been purified from cell lysates and contain genetic material administered by means of polynucleotide vaccines are not, however, limited to these.
  • Recombinant host cells describe, for example, microorganisms, insect cells and mammalian cells which can be used as receivers for non-viral, bacterial and viral vectors, or have already been used. These terms also include progenitor cells of the described cells that have been transformed.
  • cell line refers to a population of cells which has the ability to grow and divide continuously in vitro over a long period of time. Often these cell lines represent idonal populations derived from a single progenitor cell. Cells derived from a defined clonal cell line are not always exactly identical to the original cell or culture, and a defined cell line can contain such variants. The term “cell line” also includes immortalized cells.
  • microorganism used here denotes viruses and prokaryotic and eukaryotic microbes such as e.g. Archebacteria, bacteria, unicellular organisms and fungi; the latter group includes, for example, yeast and filamentous fungi.
  • transformation denotes the introduction of an exogenous polynucleotide into bacteria or into eukaryotic cells such as, for example, yeast cells, insect cells, mammalian cells or fungi, regardless of the method used.
  • the exogenous polynucleotide can be episomal, in non-integrated form, for example as a plasmid, or integrated in the genome.
  • purified and isolated mean that a molecule e.g. a polypeptide or a nucleotide sequence is present in the most complete possible absence of comparable biological macromolecules.
  • purified used in the patent means a proportionate weight of the desired product of at least 75%, preferably at least 85%, better at least 95% and optimally at least 98%, of the total weight of the biological macromolecules present (water, buffer and other small molecules, especially molecules with a molecular mass of less than 1000 are not counted among the biological macromolecules).
  • derivatives used here denotes nucleic acids which also encode the HcpA protein, although their nucleic acid sequence differs from that of the comparison sequence. The differences are preferably about 20%, about 10%, about 5% or less than 5%. Derivatives also denote polypeptides with the biological activity of the HcpA protein which have individual amino acid modifications such as substitutions, additions, insertions, deletions, including modifications of approximately 1 to 20 amino acids, approximately 15, 10, 7, 5, 3 or another number of Amino acids ranging from 1 to about 20 amino acids.
  • fragments used here denotes parts of nucleic acid or amino acid sequences, provided that they encode or have at least one functionally important region (domain, sequence or structural motif) of the HcpA protein.
  • fragment in particular denotes the C terminus of the HcpA Proteins according to SEQ ID NO: 10.
  • the present invention relates to polynucleotides which have a nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7 or 9, and to their fragments or derivatives which encode polypeptides which have the biological activity of HcpA proteins.
  • the present invention further relates to polynucleotides that hybridize with polynucleotides that have a nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7 or 9. Polynucleotides which hybridize under stringent hybridization conditions are preferred.
  • Hybridization conditions are: hybridization in 4 x SSC at 65 ° C (alternatively in 50% o formamide and 4 x SSC at 42 ° C), followed by several washing steps in 0.1 x SSC at 65 ° C for a total of about one hour.
  • the present invention further relates to polypeptides encoded by the polynucleotides described above according to SEQ ID NO: 1, 3, 5, 7 or 9 or their fragments or derivatives.
  • the present invention further relates to polypeptides comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12 or 4.
  • the invention preferably relates to the C-terminus of HcpA according to SEQ ID NO: 11 or according to SEQ ID NO: 12.
  • the nucleic acid sequence coding for the H. pylori HcpA can be expressed in a variety of different expression systems in different cells, the following list listing only a few examples, e.g. Bacteria such as Bacillus subtilis, E.
  • yeast cells such as Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Yarrowia lipicaedes, Autograph cells, Autograph cells, Autograph cells, Autograph cells Drosophila melanogaster, Spodoptera frugiperda, or Trichoplusia ni, mammalian cells such as Chinese hamster ovai ⁇ (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), or plant cells.
  • the expression systems used for the various cells can be the vectors or gene transfer vectors known in the prior art or vectors developed in the future.
  • the vectors which are used for cloning and / or expression of the polynucleotides according to the invention are selected according to the criterion as to whether they are suitable for replicating and / or expressing the desired nucleic acids in the host cell in which the nucleic acids are to be replicated and expressed .
  • suitable vectors for gene expression in various types of suitable host cells are listed in the publications by Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et a (1989).
  • the control elements, such as promoters, used in the vectors can be selected so that they have the desired activity in the respective host cell.
  • polypeptide in addition to the direct expression of the polypeptide according to the invention, expression of the polypeptide as a fusion protein is also possible.
  • the DNA sequence of an endogenous bacterial or other stable polypeptide can be fused to the 5 'end of the sequence according to the invention.
  • a fusion protein is formed from this during expression.
  • Accessory proteins - for example chaperones, substrates, disulfide isomerases or prolyl-cis-trans isomerases - can also be co-expressed.
  • the polynucleotide according to the invention can also be used to encode a signal peptide
  • Each sequence e.g. to which the 5 'end of the coding gene sequence must be provided. This allows the production of proteins that reach the surface of the cell or are secreted.
  • amino acid sequence sequence is designated as the signal peptide, all for the
  • secretion of foreign proteins from the host cells required information.
  • a processing sequence between the signal peptide and the foreign protein e.g. a protease cleavage site which enables in vivo or in vitro cleavage of the signal peptide from the desired foreign protein.
  • an expression vector includes a combination of a promoter, a polyadenylation signal, and a transcription termination sequence. Erihancers, introns with functional splice donor and acceptor sites as well as leader sequences can, if necessary, be built into the constructs in a modular manner. Expression constructs are often included in a replicon, such as in extrachromosomal elements (e.g. plasmids).
  • Another aspect of the present invention relates to the use of the polynucleotides according to the invention or their fragments or derivatives, or the polypeptides according to the invention as Medicaments, preferably for the therapy of diseases in which INF- ⁇ is involved, as a vaccine, preferably for diseases associated with Helicobacter, as adjuvants for immunizations, or as a diagnostic agent.
  • HcpA polypeptides can be used alone or in combination with one or more other polypeptides as a vaccine candidate for the prevention or therapeutic treatment of microbial and viral infections, but especially Helicobacter infections.
  • the polypeptides and / or polynucleotides administered together with the HcpA polypeptide (s) can either originate from H. pylori or other pathogens.
  • a preferred immunogenic composition of an H. pylori vaccine includes, for example, in addition to the HcpA polypeptide (s), preferably the Helicobacter VacA cytotoxin, catalase, Lpp20 lipoprotein, L7 / L12 ribosomal proteins, 50/52 kDa antigen, the CagA protein, and / or the urease holoenzyme.
  • HcpA polypeptide preferably the Helicobacter VacA cytotoxin, catalase, Lpp20 lipoprotein, L7 / L12 ribosomal proteins, 50/52 kDa antigen, the CagA protein, and / or the urease holoenzyme.
  • the GroWL (HspB), GroEL, GroES, and GroEE (HspA) heat shock protein homologues can be added to the HspA polypeptide or any composition of the above vaccine candidates.
  • the HcpA polypeptide can be used as a component of Helicobacter lysates or in combination with Helicobacter lysates as a vaccine.
  • the HcpA polyprotein can be used in any combination with any other immunogenic polypeptide from various Helicobacter isolates as a vaccine. Suitable components of Helicobacter that can be combined with the HcpA polypeptide in a vaccine composition are described, for example, in the reviews by Guidice et al. 2001 (Annu. Rev. hnmunol. 2001; 19: 523-63).
  • the HcpA polyprotein can be used in any combination with immunogenic polypeptides of any microorganisms and other organic material as a vaccine.
  • These polypeptides can originate in particular from viruses, bacteria and fungi.
  • these polypeptides can also be of human origin; for example, they can be derived from polypeptides characteristic of T nore.
  • the HcpA polypeptide and said polypeptides can be administered either separately or in combination.
  • polynucleotides encoding HcpA polypeptides can also be used alone, as an integral part of vectors and / or in combination with any of the polypeptide vaccines mentioned above.
  • HcpA polypeptides and for HcpA Polynucleotides encoding polypeptides can be combined alone or as an integral part of vectors, in combination with any non-Helicobacter-derived polypeptides suitable for inducing an immune response against any microorganism (bacteria, viruses, fungi).
  • polynucleotides or vectors coding for HcpA which contain polynucleotides coding for HcpA can be combined with any polynucleotides coding for other Helicobacter polypeptides or vectors which contain polynucleotides coding for other Helicobacter polypeptides.
  • polynucleotides encoding HcpA or vectors which contain polynucleotides encoding HcpA can be combined with any immunologically important coding polynucleotides, or vectors which contain any polynucleotides encoding immunologically important polypeptides.
  • These polynucleotides can in particular originate from viruses, bacteria and fungi. However, these polynucleotides can also code for polypeptides of human origin (for example polypeptides characteristic of tumors).
  • the HcpA polynucleotide and said polynucleotides can be administered either separately or in combination.
  • Suitable vectors for the transfer of the desired polynucleotides include, for example, plasmids, layered DNA, recombinant viruses, such as, for example, recombinant vaccinia viruses (for example MVA, NY-VAC, bird pox viruses), recombinant adenoviruses, recombinant rabies viruses, recombinant adeno-associated alphaviruses, recombinant viruses, recombinant viruses Forest viruses, but also recombinant bacteria such as, but not exclusively, attenuated Salmonella and Listeria.
  • recombinant viruses such as, for example, recombinant vaccinia viruses (for example MVA, NY-VAC, bird pox viruses), recombinant adenoviruses, recombinant rabies viruses, recombinant adeno-associated alphaviruses, recombinant viruses, recombinant viruses Forest viruses, but also recombin
  • Particularly suitable vectors for the transfer of the polynucleotides or polynucleotide combinations according to the invention are DNA vaccines, for example ring-shaped plasmid DNA or linear plasmids (MIDGES) vectors which can ensure sufficient and even better efficient expression of the desired polypeptides in individuals and are used for immunotherapy and immunoprophylaxis become.
  • Vectors based on plasmid DNA are preferably administered by injection by means of a needle, but preferably, but not exclusively, into the muscle or under or into the skin, or by injection of DNA bound to gold digels, preferably into the dermis, using a particle gun or "powder gun" become.
  • vectors described can also be used for nucleic acid transfer.
  • These vaccines contain one of the vaccine combinations already described, usually in combination with "pharmaceutically usable carrier substances", which include any carrier and which do not induce any antibodies which are harmful to the recipient of the vaccine composition.
  • Suitable carrier substances are, for example, large, slowly metabolizable macromolecules such as proteins, Polysacchari.de, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles.
  • other published carrier substances which correspond to the state of the art can also be used.
  • these vaccine combinations already described can be administered either alone or in combination with immunostimulating agents ("adjuvants").
  • adjuvants for increasing the effectiveness of the vaccine / vaccine combinations described include, for example: (1) gel-like adjuvants such as aluminum salts (aluminum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate and calcium phosphate; (2) microbial adjuvants such as bacterial nucleic acids with CpG motifs, endotoxins such as monophosphoryl lipid A, exotoxins such as the diphtheria, cholera, tetanus toxoid, the heat-labile enterotoxin from E.
  • gel-like adjuvants such as aluminum salts (aluminum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate and calcium phosphate
  • microbial adjuvants such as bacterial nucleic acids with CpG motifs, endotoxins such as monophosphoryl lipid A, exotoxins such as the dip
  • coli and Muramyl dipeptides such as MDP
  • oil emulsions and emulsion based vaccines such as incomplete Freund's adjuvant (IFA), MF59, SAF and Ribi TM adjuvant system (RAS), (Ribi Lnmunochem, Hamilton, Mont);
  • particulate adjuvants such as immunostimulatory complexes (ISCOMs), liposomes, PLG polymers, biodegradable microspheres and saponins (QS-21), and synthetic adjuvants such as nonionic block polymers, muramypeptide analogs, polyphosphazenes and synthetic polynucleotides and (5) such as interleukins (D -1, IL-2, IL-12, etc.), granulocyte macrophage cloning-stimulating factor (GM-CSF) or macrophage cloning-stimulating factor (M-CSF), and the tumor necrosis factor (TNF).
  • IFA incomplete Freund's adju
  • the vaccine combinations described e.g. one or more polypeptides and / or polynucleotides in combination with a pharmaceutically acceptable carrier component and / or an adjuvant
  • a pharmaceutically acceptable carrier component and / or an adjuvant will also contain dilution solutions, such as water, salt solutions, glycerin, ethanol, or others.
  • additional accessory components such as moisturizing and emulsifying agents, pH-buffering substances and similar components, can be present in these compositions.
  • these vaccine combinations are in injectable form, either as liquid solutions or suspensions; or be in solid form and dissolved before injection.
  • the immunogenic composition can also be emulsified or incorporated into liposomes in order to achieve increased adjuvant properties in the sense of a pharmaceutically acceptable carrier component.
  • the vaccine composition can be administered in suitable administration routes. Oral, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal or intradermal routes of administration are possible.
  • the vaccine composition is used in the doses appropriate for the indication. Oral administration is most suitable for the administration of vaccine compositions based on polypeptides for the treatment and prevention of Helicobacter infections.
  • the vaccine combinations described can be used either prophylactically (to prevent infection) or therapeutically (to treat an achievement after infection).
  • the HcpA polypeptide can also be used therapeutically for the treatment of diseases and infections, preferably in diseases in which interferons, in particular IFN- ⁇ , TL6 and IL12, but also IFN- ⁇ and IFN-ß have a therapeutic effect exhibit.
  • Preferred fields of application are diseases in which interferons are used clinically as antiviral agents, e.g. in the treatment of HIV infections, viral hepatitis including chronic hepatitis B and C, papilloma viral infections, herp it viral
  • Infections from cytomegalovirus infections, viral encephalitis and in the Rhinitisprophylaxe.
  • Preferred areas of application are also diseases in which interferons as therapeutic agents for the treatment of parasite infections, for example in Cryptosporidium parvum infections, for the treatment of parasite infections, for example in Cryptosporidium parvum infections, as therapeutic agents against bacteria, for example in the treatment of multi-resistant pulmonary overcolosis, for the therapy of various types of cancer, e.g.
  • hairy cell leukemia acute myeloid leukemia, osteosarcoma, basal cell carcinoma, glioma, renal cell carcinoma, in the case of multiple myeloma, melanoma and Hodgkin's lymphoma, in the prevention of rejection reactions or the progression block of autoimmune diseases such as arthritis, diabetes, multiple sclerosis, and treatment used to reinforce or stimulate the immune response in prophylactic or therapeutic vaccinations.
  • the HcpA polypeptides, fragments, variants, derivatives, mutants or analogs thereof, and polynucleotides encoding HcpA, and vectors containing these polynucleotides, and vectors containing these polynucleotides, can therefore be used clinically to stimulate interferon production in mammals, in particular in humans.
  • the polypeptides of the invention can be administered to patients to stimulate IFN- ⁇ , IFN- ⁇ , IFN-ß, IL-6, and IL-12 production.
  • the polypeptides according to the invention have prophylactic and therapeutic properties for antiviral, antibacterial or antiparasitic therapy.
  • polypeptides according to the invention can also be used as an adjuvant or coadjuvant for enhancing or stimulating an immune response in prophylactic or therapeutic vaccination.
  • the present invention relates to a method for the treatment of infections by administering a suitable dose of the polypeptide described in the present invention, a fragment, of variants, derivatives or analogs thereof, as well as of these polypeptides encoding to patients who receive an anti-infection treatment, for example a viral one , bacterial or parasitic infections that need cancer-fighting treatment or that require immunotherapy.
  • the polypeptides according to the invention, polynucleotides and vectors containing these polynucleotides can also be administered in combination with a suitable carrier.
  • Carrier substances include salt solutions, buffered salt solutions, dextrose, water, glycerin, ethanol, oils, ethyl oleates and liposomes and combinations thereof.
  • the carrier substances are selected according to the dosage form.
  • the polypeptides according to the invention, polynucleotides and vectors containing these polynucleotides can be used in combination with other pharmaceuticals.
  • polypeptides according to the invention can be administered in suitable administration routes.
  • suitable administration routes Oral, topical, intravenous, intraperitoneal, intramuscular, intra-articular, subcutaneous, intranasal or intradermal routes of administration are preferred.
  • polypeptides, polynucleotides and vectors containing these polynucleotides according to the invention can also be administered by continuously releasing systems.
  • appropriately shaped (films or microcapsules) semi-peninable polymer matrices are used.
  • the polypeptides, polynucleotides and vectors containing these polynucleotides according to the invention can be present in solution or in lyophilized form.
  • the lyophilized form can be reconstituted with sterile water to form an aqueous solution.
  • the medicaments containing the polypeptides, polynucleotides and vectors containing these polynucleotides according to the invention can have different routes of administration: oral, rectal, parenteral, intradermal, intravaginal, intraperitoneal, topical (as powder, plaster, Gel, cream, drops or transdermal stain), buccally or as an oral or nasal spray.
  • a pharmacologically recognized carrier material is understood to mean a non-toxic, solid, semi-solid or liquid filler, a solvent, a shell or another auxiliary.
  • Parenteral refers to the route of administration and includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal and intraarterial hijections and infusions.
  • Pofynucleotides encoding HcpA polypeptides can also be used in gene therapy for the treatment of patients in whom increased interferon production is to be induced. This group of patients includes those who receive anti-viral, anti-bacterial, anti-parasitic, anti-cancer therapy, or immunotherapy.
  • cells of patients are to be transformed ex vivo with the polynucleotides (DNA or RNA) coding for the HcpA polypeptides according to the invention, and the type of changed cells are to be reinfused in the patients / test subjects to be treated with the polypeptide.
  • cells can be transformed using a variety of viral and non-viral vectors containing the polynucleotides encoding HcpA polypeptides.
  • cells can be transformed in vivo using a variety of viral and non-viral vectors containing the polynucleotides encoding HcpA polypeptides that they express the polypeptides of the invention.
  • Suitable veto systems can be derived, for example, from lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, pox viruses, alpha viruses, baculoviruses, rabies viruses or herpes viruses. Suitable vectors can also be based on bacteria such as Listeria, Shigella or Salmonella.
  • non-viral vectors are often used in combination with particle-mediated gene transfer or with viral vector systems. These methods can be used in the fields of gene therapy, therapy and prevention.
  • These vectors can also be coupled to synthetic gene transfer molecules such as, for example, polymeric DNA-binding cations such as polylysine, protamine and albumin, or can be bound to ligands which mediate specific customs targeting, such as, for example, asialoorosomucoid, insulin, galactose, lactose or transferrin.
  • synthetic gene transfer molecules such as, for example, polymeric DNA-binding cations such as polylysine, protamine and albumin
  • ligands which mediate specific customs targeting, such as, for example, asialoorosomucoid, insulin, galactose, lactose or transferrin.
  • the efficiency of the absorption of naked DNA can also be increased by using biodegradable latex beads be increased.
  • compositions of liposomes and immunostimulatory reconstituted influenza virosomes are also suitable as vehicles for the transfer of the vectors according to the invention into mammalian and human cells (US Pat. No. 5,879,685).
  • the gene transfer systems described can be used for the genetic manipulation of isolated human or mammalian cells, but also tissues.
  • the present invention further relates to antibodies against the polypeptides according to the invention, in the form of a serum or preferably in the form of polyldonal or monoclonal antibodies. Furthermore, the present invention relates to antibodies in the form of a serum or preferably in the form of polyldonal or monoclonal antibodies as a diagnostic agent and for therapy and prevention.
  • HcpA polypeptides can be used in immunoassays for the detection of specific antibodies (or conversely, HcpA-specific antibodies can be used for the detection and concentration determination of HcpA polypeptides thereof).
  • the titer corrects HcpA-specific antibodies with various diseases associated with H pylori.
  • immunoassays that are suitable for replacing the diagnostic methods that are common today. These methods can be used to detect antibodies to the HcpA protein in biological samples, including, for example, blood, serum, plaque or stool.
  • methods for immunoassay can be based on the principle of competition, or the direct reaction, or the sandwich assay. The methods can also be based, for example, on the use of solid surfaces or on the principle of immunoprecipitation.
  • Most test systems use labeled antibodies or labeled polypeptides and the labels can be, for example, fluorescence, chemiluminescence, radioactive or color molecules.
  • kits which amplify the signals of a probe
  • examples are methods which contain biotin and avidin, and enzyme-labeled and mediated immunoassays, such as the ELISA assay.
  • the patent also includes the production and use of HcpA polypeptide-specific sera and monoclonal antibodies for use in diagnostics, therapy and prevention.
  • HcpA or HcpA expressing H. pylori isolates can be demonstrated by detection of a HcpA specific nucleic acid can be determined. Since HcpA is a species-specific marker for H. pylori, the detection of hcpA enables the detection of H. pylori. The specificity of the nucleic acid can be ensured by database research (e.g. with GenBank, EMBL).
  • the nucleic acid can be detected by hybridization or enzymatic amplification methods such as polymerase or ligase chain reactions.
  • PCR enzymatic amplification methods
  • Acp - ⁇ - specific primers are used, which can only bind to hcpA polynucleotides.
  • the detection of an amplificate is therefore an indicator of the presence of the DNA encoding HcpA.
  • the amphicates can be sequenced directly and their identity can be determined by comparing databases.
  • Corresponding nucleic acids can also be detected by direct hybridization with complementary gene probes. Depending on the stringency, nucleic acids with 70%, 80%, 90%, 95% or 100% sequence identity hybridize.
  • Test kits for human and nucleic acid diagnostics include suitable labeled reagents, including the reagents of the invention and additional reagents and materials e.g. Suitable buffers, salt solutions, etc., which also contain suitable containers for packaging the reagents for carrying out the assay.
  • the present invention further relates to genetically modified strains of H pylori that do not express a functional HcpA polypeptide.
  • These mutated strains can be distinguished from naturally occurring isolates of H. pylori strains, for example by the presence or absence or the mutation of the hcpA gene.
  • a mutant H. pylori strain can be produced by its mutation in the coding sequence of the HcpA protein.
  • other methods for mutagenesis of the coding sequence of the HcpA protein can also be used.
  • the present invention preferably relates to an HcpA hiock out of H pylori 2802. In addition to artificially HcpA deficient H.
  • the present invention also relates to the use of naturally occurring isolates which have no HcpA expression or secretion.
  • additional Mutants can be generated, for example, by substitution mutagenesis in the coding region for the HcpA protein or by deletion of a region of the hcpA gene, with the aim of changing the coding sequence of the HcpA protein in such a way that a functional HcpA protein is no longer produced.
  • These mutations are said to produce Helicobacter strains that are either no longer infectious, no longer pathogenic, less pathogenic or attenuated.
  • Targeted point mutations, insertions and deletions within the coding region for the hcpA gene or within the regulatory region for expression of the hcpA gene or in both sequences can lead to the generation of such Helicobacter isolates.
  • the present invention further relates to the use of mutated H. pylori isolates which, in addition to mutations, deletions or insertions in the hcpA gene, also have mutations, deletions or insertions in one or more further genes.
  • these mutated Helicobacter isolates can be used, for example, as live attenuated vaccines, as apathogenic commensals for colonizing the stomach or as vectors for the expression of foreign polypeptides.
  • HcpA deficient Helicobacter strains can thus be used as medicines, gene transfer vectors or food additives.
  • the promoter region of the hcpA gene or another gene can be replaced by an efficient He / zcob ⁇ cter-specific promoter or a suitable foreign promoter including suitable regulatory elements which enable strong expression of the foreign protein in Helicobacter.
  • the proteins to be expressed can also be provided with a Helicobacter-specific secretion signal. Suitable secretion signals from other organisms can also be used.
  • the present invention further relates to the production and use of HcpA-specific aptamers, primers and probes.
  • the invention is illustrated by the following examples, but is not limited to these:
  • Example 1 Sequence and genome organization of the HcpA open reading frame. According to Karita et al. (Karita et al., Infect. Immun. (1997), 65: 4158-64) (Genbank Accession No. AF008565) the HcpA ORF, which includes nucleotides 3703 (start codon) up to and including nucleotide 4455 stop codon, lies in the H. pylori strain 60190 together with the GidA and DapE ORF in one operon. Cao and co-workers (Cao et ah, Infect. Immun.
  • HcpA As an immunogenic protein secreted into the culture supernatant with a molecular weight of 27 kDa. Secretion may occur through the constitutive sec secretion apparatus.
  • HcpA has an N-terminal sec-dependent signal sequence.
  • the hcpA reading frame corresponds to ORF 211 (Tomb et ah, Nature (1997), 388: 539-47).
  • the hcpa gene used in the experiments on which the invention is based comes from the hitherto unpublished H. pylori strain 60190RE.
  • the hcpa sequence of the H pylori strain 60190RE has at least 22 nucleotide exchanges in comparison with all hcpa sequences published to date.
  • the hcpA sequence of the H pylori 60190RE isolate determined by us differs from the hcpA sequences (gb AF053708 and gb AF008565) deposited by other groups from H pylori 60190 in the gene bank (gb), as well all other HcpA sequences published so far.
  • our investigations also described a previously unknown potential internal translation start with a start codon from nucleotide 70 and a ribosome binding site of Nt 60 -65 relative to the translation start of the full length protein.
  • the HcpA ORF could be detected in every H pylori strain examined.
  • the DNA amphicates obtained in the PCR were separated on an agarose gel. After the run, the gel was briefly rinsed with water and the DNA was denatured by shaking in 0.5 M NaOH and 1.5 M NaCl for 30 min. After briefly rinsing again in water, the mixture was neutralized in 1 M Tris-HCl pH 8.0 and 1.5 M NaCl with shaking for 30 min. The gel was rinsed briefly with water. The blot was carried out by vacuum transfer using a VacuGene XL blot apparatus from Pharmacia according to the manufacturer's instructions.
  • a positively charged nylon membrane from Boehringer was used as the membrane, which was briefly soaked before blotting and then swung in 20 x SSC.
  • the UV fixation was carried out on both sides by means of an autocrosslink in a Stratali ⁇ ker (Stratagene).
  • incubation was carried out in hybridization buffer at 42 ° C. for at least 1 h. It was hybridized with 3 ml hybridization buffer (50% formamide) with 25 ng / ml digoxigenin-labeled probe for at least 24 h at 42 ° C.
  • Post-hybridization was carried out twice in 5 minutes in 2x SSC with 0.1% SDS and twice in 15 minutes in 0.1 x SSC with 0.1% SDS.
  • the chemiluminescence detection was carried out using CSPD substrate (Boehringer) according to the manufacturer's instructions.
  • the probe labeling with digoxigenin was carried out by PCR with 200 ⁇ M dA C / GTP, 150 ⁇ M dTTP and 50 ⁇ M 11 -digoxigenin dUTP with AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, NJ, USA) under the same reaction conditions as with normal amphfication.
  • the probe for the detection of the hcpA operon was created using the parameters listed in the table below.
  • HcpA protein Expression of the HcpA protein using the pET system and refolding from inclusion bodies.
  • Native HcpA protein in its pure form is the basic prerequisite for the characterization of the biochemical and immunological properties. Since the HcpA protein is only weakly expressed in H. pylori, various bacterial systems for the expression of the HcpA protein were established in the required yields.
  • the hcpA gene was PCR by means of the oligonucleotides 5'-AGA GGG TTA CAT ATG GAG CCA GAC GCT AAA GAG C-3 '(SEQ TD NO: 17) and 5'-TTG CTC GAG AAG TTC TAT TTT CAA TTC C -3 '(SEQ ID NO: 17) from genomic DNA of the H pylori strain 60190RE and was cloned into the expression vector pET 21 a (+) (Novagene, Heidelberg) via the restriction sites Ndel and JXJl ⁇ oI. Then 11 LB (100 mg / 1 ampicillin) were inoculated 1: 100 with a pre-culture overnight of an E.
  • coli BL21DE3 transformed with pET21a-HcpA and incubated at 37 ° C. with shaking (200 rpm).
  • OD600 0.5
  • the culture was induced with 400 ⁇ M ff TG and incubated for a further 4 h at 37 ° C. with shaking.
  • the cells were centrifuged (10 min, 6000 ⁇ m, 4 ° C harvested), washed with 5 ml of cold PBS, resuspended in 25 ml of lysis buffer (10 mM Tris-HCl pH 7.5 1 mM EDTA) and disrupted by ultrasound treatment ( Branson Sonifier 450, 30 impacts, 70% duration, level 8, 4 ° C).
  • the lysate was pelleted at 14,000 ⁇ m at 4 ° C. for 30 min, the supernatant was discarded and the pellet was resuspended in 20 ml of 6 M urea in 100 mM sodium phosphate buffer pH 8.0 and incubated at 4 ° C. in an overhead shaker. Insoluble cell components were separated by centrifugation (30 min, 14000 ⁇ m, 4 ° C). The HcpA protein is in the supernatant. The protein was purified by metal affinity chromatography using an LKB-GP 10 FPLC from Pharmacia according to the manufacturer's instructions.
  • 1 ml of the urea-denatured HcpA was applied at a flow rate of 1 ml / min to a Hitrap Chelating 1 ml column (Pharmacia) and until the flow rate OD280 decreased to below 0.1 with 6 M urea in 100 mM sodium phosphate buffer pH 8, 0 washed.
  • the washing step was repeated with 6 M urea in 100 mM sodium phosphate buffer pH 6.3.
  • Elution was carried out with 6 M urea in 100 mM sodium phosphate buffer pH 4.0.
  • the column material was freshly loaded with NiCl2 before each run and regenerated by rinsing with 50 mM EDTA after the run.
  • the HcpA protein can be expressed in high yield in the pET21a (+) system.
  • the protein carries a Hisg tag at the C-terminal.
  • Both the use of full-length clones of HcpA and of HcpA variants with a deleted endogenous signal sequence leads to expression of the protein in inclusion bodies.
  • the signal sequence functional in H pylori alone is not sufficient for efficient protein translocation into the periplasm of E. coli BL21-DE3.
  • the yield of the full length contract is about 10 mg of denatured HcpA / 1 culture and that of the deletion variant is about 50 mg / 1.
  • HcpA was finally purified from the 6 M urea fraction using metal affinity chromatography ( Figure 2).
  • Figure 2 The amino acid sequence of the HcpA protein expressed in the pET 21 a (+) system is shown in FIG. 1B. Compared to the wild-type HcpA protein, additional amino acids are printed in bold. A His tag (6 x His) was fused to the C-terminus of the HcpA protein to facilitate purification.
  • Refolding of the HcpA protein For the functional characterization of the protein, the protein has to be folded back into the native form. Due to the large number of cysteines that are potentially disulfide-bridged in an extracellular protein, refolding conditions were chosen, which include folding mediators such as L-arginine for the efficient formation of disulfide bridges as well as a GSH-GSSG equilibrium with intermediate redox potential. Refolding was carried out in a batch process by diluting the denatured, completely reduced protein in refolding buffer.
  • HcpA 6 mg HcpA were completely reduced by incubation with 300 M DTE (Sigma) for 15 min at RT and 1: 200 in 0.4 M L-arginine (Sigma), 100 mM Tris-HCl pH 8.5 and 4 for refolding mM GSSG (Sigma) diluted and incubated for 3 days at 25 ° C.
  • the renatured protein was dialyzed against PBS.
  • the kinetics of the in vitro refolding of the HcpA protein can be followed by fluorescence spectroscopy.
  • 100 ⁇ l of the refolding batch at 20 ° C. were 3 d in the Fluoromax-2
  • HcpA contains only tyrosine and phenylalanine as fluorochromes
  • the excitation took place at 275 nm (excitation of tyrosine)
  • the emission at 305 nm wavelength of the maximum emission of tyrosine
  • the changed solvent exposure or molecular environment of the fluorochromes leads to a changed emission at 305 nm in the case of tyrosine, but the wavelength of the emission maximum per se is not shifted. Therefore, no difference spectra between native and denatured protein were determined to determine the optimal difference wavelength.
  • the refolding takes place in two phases. First of all, the fluorescence kinetics decrease exponentially in a fast kinetics.
  • the refolding of recombinant HcpA from inclusion bodies therefore leads to the formation of a stable conformation.
  • the analysis of the refolded protein according to Ellmann shows a complete disulfide bridging of all cysteines. However, there is no intermolecular linkage, since the apparent molecular weight is 24 kDa even in the non-reducing SDS-PAGE. The cysteines therefore probably do not have a catalytic effect, but rather stabilize the tertiary structure of the monomeric protein.
  • HcpA in the pET system provides high yields of denatured protein. Since refolding is complex and only practical with small amounts of protein, an alternative expression system for the native expression of HcpA in the periplasm of E. coli has been established. In addition, the protein should be purified without any structure and function-influencing day.
  • the vector system used is pRBI-PDI (Wunderlich et al., J Biol Chem. (1993), 268: 24547-50, the disulfide isomerase DsbA being deleted by HindJUI and BamHI and replaced by DsbC from E. coli using the same interfaces) especially suitable for the expression of strongly disulfide-bridged extracellular proteins.
  • DsbC disulfide isomerase DsbC is transcribed in a bicistronic operon.
  • DsbC catalyzes a shuffeling of Disulfide bridges. Incorrect disulfide bridges can therefore be released again, while correct disulfide bridges are stabilized by the conformation of the protein. Due to the localization of the expressed protein in the periplasm and the constant new formation of the disulfide bridges, the protein is in native form.
  • the gene from H. pylori 60190RE was cloned without the endogenous signal sequence as a translational fusion with the OMP signal sequence in pRBI-PDI.
  • the HcpA gene was PCR by means of the oligonucleotides 5'-CCG AGC CAG ACG CTA AAG AGC-3 '(SEQ ID NO: 19) and 5'-TTT AAG CTT TCA GAG AAG TTC TAT TTT CAA TTC C-3' (SEQ ID NO: 20) amplified from genomic DNA of the H pylori strain 60190RE.
  • the vector pRBI-PDI was cut with the restriction enzymes StuI and HindIII, overhanging ends were filled in by treatment with T4-DNA polymerase and dephosphorylated with alkaline phosphatase.
  • the PCR product was kinased by treatment with T4 polynucleotide kinase and blunt-ended cloned into the vector pRBI-PDI. The correct insertion was checked by sequencing.
  • the contract was transformed into E. coli JM 83.
  • Three L LB 100 mg / 1 ampicillin
  • the entire pellet was washed with ice-cold Polymyxin B buffer and carefully taken up on ice in 4 ml Polymyxin B (1 mg / ml in Polymyxin B buffer, Fluka, Neu Ulm) per 1 culture and OD600 unit and incubated on ice for 1 h.
  • the cytoplasm was separated by centrifugation (30 min, 15000 ⁇ m, 4 ° C) and the supernatant was incubated for 30 min at 40 ° C in a water bath. After centrifugation as above, the supernatant was heat precipitated at 50 ° C and 60 ° C.
  • HcpA was isolated from the dialysate by cation exchange chromatography with a BioCAD 700E FPLC from PerSeptive Biosystems, Rothstadt, over a Porös CM column (PerSeptive Biosystems) at a flow rate of 10 ml / min with 50 mM MES pH 6.4 as running buffer. Elution was carried out in a salt gradient from 0 to 1 M NaCl at about 0.5 M NaCl. The protein was then dialyzed against 20 mM Tris-HCl pH 7.5, 50 mM NaCl.
  • the expression yield is best for hiduction OD600 between 1.0 and 1.5. Induction was carried out with 1 mM IPTG (final concentration). In order to establish an intermediate redox potential, N-acetyl-L-cysteine ad 5 mM was added to the culture during induction. The periplasm digestion was carried out with Polymyxin B. Polymyxin B also inactivates the LPS from E. coli. Due to the strong disulfide bridging, native HcpA is very heat-stable and can be purified by sequential heat precipitation for 30 min at 40 ° C, 50 ° C and 60 ° C. Higher molecular weight proteins from E. coli precipitate during heat precipitation.
  • HcpA Since incompletely folded HcpA aggregates very easily, non-native HcpA is also separated by the heat precipitation.
  • HcpA can be isolated in pure form via cation exchange chromatography (FIGS. 3A and B). The protein was dialyzed against 20 mM Tris-HCl pH 7.5, 50 mM NaCl. The yield is about 10 mg of pure HcpA in pure form / 1 culture. The correct processing was checked by N-terminal sequencing. The amount and purity were sufficient for the protein to crystallize. Ellmann analysis, non-reducing SDS PAGE and UV-visible spectrum (FIG. 3 C) are analogous to the refolded protein.
  • HcpA is present as a monomer in solution (FIG. 3D).
  • the natively expressed protein was also used for functional assays.
  • the amino acid sequence of the HcpA protein expressed in the pRBI-PDI system is shown in FIG. IC.
  • the HcpA protein has a modular structure. A sequence motif with a length of approximately 36 amino acids is repeated seven times within the protein. To identify functionally important regions within the protein, several fragments of the HcpA protein comprising these sequence motifs were expressed.
  • Example 2C shows the expression of the C-terminal fragment consisting of the last 4 sequence motifs as an example.
  • the expression vector pRBI was obtained by PCR with the oligonucleotides 5'-GGC CTG CGC TAC GGT AG-3 '(SEQ ID NO: 21) and 5'-AAA GCC TCA CAA TAC TAC TCC AA-3' (SEQ ID NO: 21) -PDI -HcpA (see Example 2B) linearized excluding the N-terminal three sequence motifs.
  • the amplificate was purified by agarose gel electrophoresis and kinased with the T4 polynucleotide kinase.
  • the linearized vector was generated using the T4 ligase ligated and transformed into E. coli XL1 Blue.
  • Example 3 Expression of the HcpA protein by means of stably transfected Drosophila Schneider-2 cells
  • the starting plasmid pMta (Johansen et ah, Genes Dev. (1989), 3: 882-9) used for the cloning rank of the Drosophila transfer vector comprises 3.4 kBp and contains a heavy metal inducible Drosophila metallothionein (Mtn) promoter in connection with a early SV40 polyadenylation signal (PolyA) and was developed by Dr. Rosenberg provided.
  • the gene for the HcpA polypeptide was amphfected by PCR, using genomic DNA from the H. pylori 60190RE as a template.
  • the interfaces (Agel / BgUI) required for cloning the amphfected gene segment were introduced at the same time.
  • the DNA fragment coding for the HcpA polypeptide was then introduced into the vector pMta cut with Agel / BgUI.
  • the expression plasmid pMta-HcpA and the resistance plasmid pA5c-DHFR were used together in co-transfection experiments to transfect DS-2 cells.
  • the plasmids were introduced by means of cathionic liposomes (hivitrogen Inc., San Diego) by lipofection into serum-free growing Drosophila Schneider-2 (DS-2) cells.
  • the transfections were carried out in 25 cm 2 bottles ( Falcon® , Becton Dickinson, Heidelberg).
  • 6 ⁇ 10 6 Drosophila Schneider-2 (DS-2) cells were sown per small bottle (approximately 80% confluent). Afterwards the cells had stopped, the transfection with liposomes was carried out according to a modified protocol of the manufacturer. By simply shaking the liposome dispersion, the negatively charged plasmid DNA can be attached to the positively charged liposomes.
  • the manufacturer's information was modified as follows: First, the transfection mixture consisting of 4 ⁇ g of high-purity plasmid DNA, 1 ml of serum-free hisect Express High Five Medium (PAA Laboratories, Linz, Austria) with 1% kanamycin (10 mg / ml) (PAN) and 20 ⁇ l of well resuspended cationic liposomes pipetted together in an autoclaved Eppendorf tube.
  • the plasmids pMta-HcpA and pA5c-DHFR were used proportionately in a molar ratio of 20: 1. These approaches were 10 sec.
  • the cells were split 1: 2 into 25 cm 2 culture bottles, the cell poison methotrexate (MTX) being added to the culture medium in slowly increasing concentrations (0.5 ⁇ g / ml to 4 ⁇ g / ml) in this and the subsequent dilutions of the cell culture has been.
  • the selection medium (with MTX) was changed every 7 days until the formation of MTX-resistant cell lines (after 3 to 6 weeks, depending on the expression rates of the dihydrofolate reductase).
  • the plasmids pMta-HcpA and pA5c-DHFR were transfected by lipofection using DOTAP liposomes (Röche Diagnostics, Mannheim) into serum-free DS-2 cells.
  • the transfection with DOTAP liposomes was carried out according to the same principle.
  • 4 ⁇ g of highly pure plasmid DNA (pMta-HcpA and pA5c-DHFR in a molar ratio of 20: 1) were diluted with 50 ⁇ l of HBS buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) and with 15 ⁇ l of one DOTAP liposome dispersion added.
  • HcpA protein produced by means of stably transfected DS-2 cells After 8 weeks of selection with methotrexate, the expression rates of the HcpA polypeptide from the supernatant and cells of the stably transfected DS-2 cell lines were determined. For this purpose, 5 x 10 cells were sown in 5 ml of serum-free induction medium (Insect Express High Five Medium with 1% Kanamycin and 200 ⁇ M CuSO 4 ). After 8 days of incubation at 26 ° C., the cells were detached from the bottom of the cell culture flasks by tapping them gently.
  • serum-free induction medium Insect Express High Five Medium with 1% Kanamycin and 200 ⁇ M CuSO 4
  • the cell-containing culture broth obtained was then centrifuged for 10 min at 1200 ⁇ m in a Hettich Rotixa / P centrifuge and the supernatant was separated from the cell pellet. The remaining cell residues were then sedimented by repeated centrifugation of the supernatant for 30 min at 4000 ⁇ m in a Hettich Rotixa P centrifuge. An aliquot of the supernatant obtained from the induced HcpA-producing DS-2 cell line, and - for comparison - of induced, non-transfected DS-2 cells, acetone was precipitated and the pellet obtained was analyzed by means of an HcpA-specific rabbit serum in a Western blot.
  • a polypeptide with the molecular mass of 24 kD corresponding to the HcpA protein could be detected from the supernatant of the stably transfected DS-2 cell line, but not from the supernatant of the non-transfected DS-2 cell line.
  • the HcpA polypeptide was then purified by heat precipitation and ion exchange chromatography in the same way as the HcpA protein produced in E. coli. Using the described methods, the HcpA protein could be prepared with a purity of> 90%.
  • Example 4 Strain-dependent secretion of HcpA by H. pylori. Due to the strong association of increased serotiter against HcpA with inflammatory H. pylori-induced diseases and the immense HcpA-induced IFN- ⁇ release from murine spleen cells, the question arises of differences in the HcpA expression of clinical isolates from patients with different disease courses.
  • the hcpA gene can be detected genomically by Southern blot in all strains examined, regardless of the cag status of the isolate. HcpA can also be detected without exception in strains of American origin. With ORC primers, the ORF from each strain could be amphfected. HcpA belongs to a multi-gene family.
  • FIG. 4 shows the expression and secretion of HcpA in the culture supernatant detected in the culture supernatant in the Western blot.
  • HcpA is expressed in almost every strain regardless of cag status.
  • the gene product of ORF1098 has strong homology to HcpA and also reacts with the polyldonal ⁇ HcpA serum.
  • the band in question is not an alternative form of HcpA, since the band is retained in the case of a genomic hiock out of HcpA (FIG. 8).
  • the recombinant expression of ORF1098 in the pET system yields a 29 kDa protein which reacts with the anti-HcpA serum.
  • the molecular weight of HcpA in the total lysate and in the culture supernatant is 24 kDa and corresponds to that of the processed form.
  • pylori Lyl27 is 27 kDa and corresponds to that of the unprocessed form.
  • the apparent molecular weights correspond to those of the HcpA variants produced recombinantly in the pET system with and without a signal sequence.
  • Sequencing of hcpA from H. pylori Lyl27 showed no alternative translation start before ORF, which would result in an extended protein. Suppressed tennination is also unlikely, since in all reading patterns several stop codons are directly connected to the coding area. Alternatively, post-translational modifications can lead to increased molecular weight. In H. pylori OM1651, neither HcpA nor ORF1098 can be detected. Sequencing of hcpA showed a mutation in the start codon from ATG to ATA (isoleucine), the cause of the lack of expression of ORF 1098 is unclear.
  • HcpA may be regulated at the level of replication by slipped beach mispairing.
  • HcpA from H. pylori 26695 contains a five repetition of the dinucleotide AT and is a candidate for phase variation. The dinucleotide repeat is mutated in all strains examined in the context of this work, so that no slipped beach mispairing within the motif is possible with these strains. In contrast to H. pylori 26695, this is also the case with the fully sequenced isolate H. pylori J99.
  • HcpA forms an operon with gidA and dapE in some strains, with hcpA being the last ORF of the operon.
  • the release into the culture supernatant shows a strong strain dependency, but is independent of the Cag status and the extent of the IL-8 release from AGS cells.
  • the ⁇ cpA content in 100 ⁇ l culture supernatant was determined after 24 h of growth.
  • H. pylori Lyl27 does not secrete detectable amounts of ⁇ cpA in the supernatant.
  • the serum of the corresponding patient was also negative in serology. Obviously, the non-processing of the protein prevents its export.
  • the amount released in these strains was determined to be about 50 ng / ml culture. This amount causes the release of about 3000 pg IFN- ⁇ / ml culture supernatant in the spleen model.
  • Periplasma is contaminated with cytoplasm, ie the fractionation conditions sometimes led to cell lysis, it can be assumed that all cells have been spheroblasted. The cytoplasmic fraction should therefore not be contaminated by periplasm. The detection of a significant amount of HcpA in processed form in the cytoplasm is therefore not a disruptive artifact. Since HcpA is secreted into the supernatant regardless of the presence of a functionally active Cag PAI, transport via the Cag apparatus is ruled out. Serologically no correlation of the anti-HcpA titer with the cag status of the corresponding isolate is detectable.
  • HcpA postulates a coiled-coil domain at the C-terminus of the protein, which can be detected by sequencing in all strains examined.
  • the coiled-coil domain does not dimerize HcpA. It may be involved in the interaction with receptors or transport factors.
  • Sequence analysis of the ORF postulates an internal translation start near the cleavage site of the signal peptidase.
  • the internal translation start has a strong Shine-Dalgarno sequence (GAGG) at a suitable distance from the start codon ATG.
  • GGG Shine-Dalgarno sequence
  • the protein expressed via the internal translation start would have a molecular weight of 24 kDa and would be located in the cytoplasm due to the lack of a signal sequence.
  • the H pylori OM1651 (mutation in the start codon) corresponding seram is positive for HcpA.
  • the mutation Apart from a mixed infection with other H. pylori, the mutation only occurred secondarily or the strain expressed HcpA in the course of the infection via the internal translation start.
  • the HcpA ORF from H. pylori 60190RE is shown schematically in FIG. 5.
  • HcpA Sequence studies of HcpA from various Helicobacter strains investigated show sequence differences at the DNA level of up to 10%. Therefore, in addition to the export behavior of the strains examined, the specific DNA sequence of the respective hcpA genes may also be of importance for the course of the disease.
  • ORF 1098 has an internal start codon near the postulated cleavage site of the signal peptidase. However, there is no classic Shine Dalgarno sequence in front of the start codon (corresponding section from H.
  • pylori 26695 TTA GGG GGG CTA ATG, internal start codon printed in bold), so that there is probably no alternative internal translation start.
  • secretion into the culture supernatant is strain-dependent (FIG. 4).
  • Predict Protein does not postulate a coiled-coil region at the C-terminus in ORF 1098.
  • the exchange of glutamate for glycine in ORF 1098 with an otherwise largely unchanged amino acid sequence in the postulated coiled-coil region is decisive for this (FIG. 6).
  • HcpA the majority of the strains examined express HcpA.
  • secretion into the culture supernatant is strain-dependent. Secretion does not take place via the postulated sec-dependent route and not via the Cag apparatus.
  • the release of the protein is serologically corrected with the antibody titer.
  • HcpA is only strongly secreted in isolates from ulcer patients.
  • the C-terminal coiled-coil domain may be an effector for export.
  • Example 5 Serology.
  • HcpA protein HcpA with signal sequence: 10 ng / well, HcpA without signal sequence: 100 ng / well
  • HcpA without signal sequence: 100 ng / well
  • the plates were then washed five times in a washing machine (SLT 96 PW, SLT Labinstruments) with PBS, 3%> FKS, 2%> Tween 20 and blocked for 1 h at 37 with this buffer. Blocked plates were kept at -20 ° C for several months.
  • the raw data show a significant difference in seroreactivity against HcpA between H. pylori infected and uninfected patients.
  • H. pylori infected patients the titer in patients with ulcer is significantly higher than in patients with non-inflammatory H. pylori-induced diseases such as carcinoma and MALToma.
  • the titres of the ulcer patients are not nomially distributed but show a second cluster with OD492 of more than 0.7. This is less pronounced in patients with gastritis, the titer in patients with carcinoma or MALToma is largely normally distributed (data not shown).
  • an OD492 of more than 0.7 had a positive predictive value of 56%> for the appearance of an ulcer and a predictive value of 78%> for the absence of a carcinoma or lymphoma.
  • the predictive value of the CagA serology for the appearance of an ulcer in the examined group is only 29%.
  • the measured HcpA titers show no correlation to the CagA status of the isolate.
  • a cut-off value In order to establish a test that can be used diagnostically, a cut-off value must be defined that classifies the sera as positive or negative. Since the measured values are not normally distributed, the cut off cannot be calculated from the mean and standard deviation of negative controls. In the case of non-normally distributed clinical data, the cut is often determined empirically. For arbitrary cut-off values, sensitivity and specificity are determined from the raw data. B. shown in a receiving operating characteristics. For each cut off, the percentage of the correctly positively recognized versus the false positively recognized samples is plotted. FIG. 9 shows the receiving operating characteristics of the HcpA ELISA. The HcpA ELISA shows a bent course typical of clinical tests in the receiving operating characteristics. Cut off ' values from the area of the kink represent useful values for setting up a test system. The evaluation of the test results with the cut off determined in the receiving operating characteristics is shown in Table 1.
  • Table 1 shows the evaluation of the HcpA ELISA of the patient group examined.
  • the cross-table shows the reactivity of the sera in the examined test (rows) and in the gold standard (columns).
  • Performance of the HcpA ELISA An OD492 of 110 was used as the cut off.
  • the key figures of the test which result without consideration of patients with MALToma or carcinoma, are noted in brackets (cut of OD / ⁇ 2 of 120).
  • HcpA shows cross-reaction with sera H. pylori negative patients.
  • the origin of the cross reaction is unclear.
  • some sera from infected people are classified as false negative.
  • carcinoma and MALToma also occur very rarely in H. pylori infected people (around 6 in 10,000), the test results were only evaluated taking into account H. pylori negatives, ulcer and gastritis patients.
  • ⁇ cpA is therefore also an antigen in the human system and is suitable for the serological diagnosis of an H. pylori infection.
  • the titer is significantly increased in patients with inflammatory diseases compared to non-inflammatory H. pylori. High titers have strong predictive values for the absence of carcinoma and MALToma.
  • Example 6 HcpA mediated immune modulation in the spleen model.
  • HcpA shows a priori interactions with the immune system. Since patients with inflammatory H. pylori-induced diseases show a greater reactivity to HcpA and HcpA is actively secreted by H. pylori, HcpA could act not only as an antigen, but also as an immune modulator and thus be directly involved in the extent and persistence of the inflammation.
  • the murine spleen model was chosen to investigate the role of HcpA as an immunomodulator. All relevant cell types of the immune system are represented in the spleen, in particular an efficient antigen presentation is guaranteed. Spleens from Balb / c mice were also used.
  • cytokines 100 ml protein solution was added to each 1 ml cell suspension, sown in 24 well plates and incubated for 36 h at 37 ° C. and 5% CO2. The cytokines from the cell culture supernatant were determined after 36 hours of incubation according to the manufacturer's instructions using kits from PharMingen, San Diego, USA. The culture supernatant was centrifuged for 10 min at 3000 ⁇ m and 4 ° C. Maxiso ⁇ plates from Nunc, Wiesbaden, were used as ELISA plates. used. An immune modulation by HcpA manifests itself in a changed cytokine pattern of the spleen cells, hi FIG. 10 shows the release of the leading cytokine IFN- ⁇ from murine milk cells after incubation with HcpA or periplasm from H. pylori.
  • H. pylori 60190RE periplasm Incubation of murine spleen cells with H. pylori 60190RE periplasm leads to a weak release of IFN- ⁇ with a bell-shaped dose dependence. H. pylori 60190RE periplasm contains HcpA. Incubation of the spleen cells with recombinant HcpA leads to an enormously strong release of IFN- ⁇ . HcpA itself shows no cross reaction in the ELISA for the determination of IFN- ⁇ . The bell-shaped course of IFN- ⁇ release by Periplasma may be caused by an inhibitor overlaying the HcpA-mediated effect. According to Sutton et al. Incubation of spleen cells from CBA mice (impaired Th-1 response) with H. pylori antigens leads to a strong release of IL-10. EL-10 is a potent suppressor of IFN- ⁇ release.
  • Immunomodulation is caused by a proteinaceous substance. Digestion of the protein with chymotrypsin leads to the loss of induction ability. Thus, the induction of IFN- ⁇ is not caused by contamination of the protein with non-proteinaceous contaminants such as LPS from E. coli, a very strong immune stimulator.
  • HcpA has a variety of interfaces for chymotrypsin. No chymotrypsin activity was measurable in the culture supernatant of the spleen cells (detection by chromogenic substrate). The chymotrypsin was inactivated after digestion by freezing the mixture.
  • the FCS contained in the culture medium contains chymotrypsin inhibitors.
  • IFN- ⁇ is specifically induced by HcpA, since the effect can be inhibited against HcpA by incubating the protein with a polyldonal seram. Since the seram was obtained by immunization with refolded HcpA (isolation from inclusion bodies), a possible interaction with contaminations in the native purified protein (isolation from the periplasm) is very unlikely. Despite the heterologous origin (rabbit), the seram itself shows no induction of IFN- ⁇ . However, it may act synergistically with HcpA and thus lead to the relatively strong IFN- ⁇ release in the falling branch of the neutralization curve (FIG. 10 C). In addition to IFN- ⁇ , HcpA also influences the expression of other cytokines. FIG.
  • IFN- ⁇ is the leading cytokine of the Th-1 immune response that is typical of the whole H. pylori bacterium in vivo. IFN- ⁇ leads to the activation of macrophages and the increased expression of MHC molecules and costimulators on APCs. It causes a Th-1 polarization of T-helper cells and the expression of immunoglobulins of the IgG2a type in mice. It is striking that the cytokine TNF- ⁇ , which is also typical for Th-1 responses, is not induced.
  • HcpA induces IFN- ⁇ in a highly specific manner.
  • the lack of induction of TNF- ⁇ is an indication of the LPS freedom of the protein fraction used or the inactivation of LPS by binding to polymyxin B during cleaning.
  • IL-4 and IL-5 as lead cytokines of a Th-2 directed immune response are also not induced.
  • IL-5 is an inducer of IgA release in mucosal lymphoid tissue and would therefore be a prerequisite for an efficient immune response against H pylori.
  • IL-6 is the second most strongly induced cytokine. IL-6 causes the proliferation of differentiated, antibody-secreting B-lymphocytes.
  • Th-2 cytokine IL-10 which acts as a suppressor of the IFN- ⁇ release, is only released in insignificant amounts.
  • EL-10 inhibits the expression of IL-12 and MHC-II in macrophages.
  • IL-12 is an antagonist to EL-10 and induces IFN- ⁇ release from T and NK cells.
  • incubation with HcpA does not lead to induction of IFN- ⁇ but, depending on the dose, to a decrease below the spontaneous IFN- ⁇ secretion.
  • HcpA protein in the murine spleen model were tested for immunomodulation.
  • the C-terminal HcpA fragment described in Example 2C qualitatively induces the same cytokine pattern as the HcpA full-length protein.
  • the C-terminal fragment induces a strong secretion of IFN- ⁇ quasi-monospecifically.
  • the Th-2 cytokines IL-4 and -5 are not induced. This fragment therefore contains the areas relevant for immunododulation and can therefore be used in analogy to the HcpA protein.
  • the HcpA protein and in particular the C-terminal region of the HcpA has strong immunomodulatory properties.
  • the cytokine pattern induced by HcpA is strictly directed to Th-1.
  • the very strong induction of IFN- ⁇ is striking.
  • Example 7 Immunogenicity of the HcpA protein in different animal models
  • A Immunization of mice with the HcpA protein in the presence and absence of additional adjuvants. Due to the strong immune modulation by HcpA and the resulting possible importance of HcpA for the course of infection, the long-term goal is to investigate the effect of vaccination with HcpA on colonization, persistence and degree of inflammation of the H pylori infection in the mouse model.
  • mice could also lead to neutralization of HcpA.
  • the basic prerequisite for vaccination against extracellular proteins is generally the generation of a sufficient antibody response in combination with a Th-2 polarization of the T helper cells.
  • the immunogenicity of the purified HcpA protein was tested in Balb / c mice. The immunization was carried out without adjuvation or with adjuvation with aluminum (Th-2 directed) or CpG oligonucleotide (Th-1 directed).
  • 8-week-old female Balb / c mice (Charles River, Kissleg) were immunized subcutaneously on the tail root of 5 mice two weeks after delivery in grapples.
  • 3 grappes were immunized with 10 ⁇ g HcpA in PBS per mouse, one control group with PBS. Adjuvation was omitted, with alum (Alhydrogel, Superfos Biosector, Frederikssund, DK) or 50 ⁇ g LPS-free CpG oligonucleotide in PBS (5'-TCA TTG GAA AAC GTT CTT CGG GGC G-3 '; SEQ ED NO: 25). 3 and 8 weeks after the primary immunization ranked according to the same protocol.
  • FIG. 12 shows the development of the anti-HcpA titer when immunizing Balb / c mice with HcpA.
  • Boost injections increased the titer to over 1: 5,000,000.
  • the titer continued to increase to a maximum of 1: 40,000,000.
  • Adjuvation with CpG oligonucleotides led to a similar immune response, which was about 10 times weaker.
  • the titer rose only slightly, but was nevertheless stable at a high level. Immunization without adjuvation leads to the weakest antibody response.
  • the distribution is very heterogeneous. In fact, three of the five animals were non-responders. The reason for the heterogeneous distribution is unclear.
  • the polarization of the T helper cells is reflected in the antibody isotype ratio.
  • a Th-1 directed immune response is expressed in high levels of IgG2a, a Th-2 directed immune response in high levels of IgG1.
  • the antibody isotype ratio of the immunizations carried out is shown in FIG.
  • Adjuvation with alum leads to a strongly Th-2 polarized immune response throughout the course of the immunization.
  • adjuvation with CpG oligonucleotides leads to an approximately equilibrium Th-1 / Th-2 response. Only in the case of antibody isotype ratios of IgG2a to IgGl of more than 1 would the immune response be considered directed as Th-1.
  • the immune response of the non-adjuvanted immunization is directed to Th-2, whereby the meaningfulness is limited due to the strong fluctuation of the titers within the group.
  • Systemic Th-2 polarization is the normal case for an extracellular protein antigen, but contradicts the local Th-1-directed immune response when stimulating spleen cells.
  • the immune response can be polarized in the direction of Th-2 or Th-1 by adjuvation with alum or CpG oligonucleotides.
  • Adjuvation was carried out with Titermax (Sigma-Aldrich). 6 weeks after grand immunization was boosted using the same protocol. After a further 6 weeks, the rabbits were bled by cardiac puncture after an overdose of pentob arbitutrate (250 mg of nenbutanol). The whole blood was coagulated at RT for 1 h after collection. The mixture was then centrifuged three times for 20 minutes at 2000 ⁇ g and the supernatant was stored at ⁇ 20 ° C.
  • Titermax Sigma-Aldrich
  • HcpA with signal sequence: 10 ng / well, HcpA without signal sequence: 100 ng / well
  • 100 mM sodium carbonate buffer pH 9.5 100 mM sodium carbonate buffer pH 9.5
  • the plates were then washed five times in a washing machine (SLT 96 PW, SLT Labinstruments) with PBS, 3% FKS, 2% Tween 20 and blocked with this buffer at 37 for 1 h. Blocked plates were kept at -20 ° C for several months.
  • the color reaction was stopped by adding 100 ⁇ l of 1N sulfuric acid and the absorption at 492 nm was determined using a reference measurement at 620 nm in an ELISA reader (SLT Spectra, SLT Labinstruments).
  • the sera obtained showed a boost titer of more than 1: 1,000,000 and can be used in Western blot with dilutions between 1: 200,000 and 1: 500,000. If the total IgG from the serum is purified by affinity chromatography using ProteinG, the IgG denature during acidic elution from the column and can only be used in dilutions of 1: 50,000.
  • Example 8 Generation and characterization of an isogenic HcpA knock-out mutant.
  • An isogenic HcpA knock out mutant is the prerequisite for examining the importance of HcpA in the H. pylori mouse model in terms of colonization, persistence and degree of inflammation. The characterization of the immunomodulation of cytoplasm and periplasm as well as of culture supernatant with the influence of HcpA is made possible. By complementing the hiock out mutant with wild-type or mutated HcpA, the level of expression can be regulated or effector domains for export and function can be identified. Karita et al. (Karita et al., Infect. Immun. (1997), 65: 4158-64) generated an isogenic HcpA mutant of H.
  • H. pylori 60190RE by insertion mutagenesis of HcpA with a kanamycin cassette.
  • the mutant showed a normal phenotype in vitro. Since the H. pylori 60190RE used in these studies, despite the presence of Cag PAI, no EL-8 release from AGS cells is induced and is difficult to transform, an HcpA knock out mutant was generated in H. pylori 2802.
  • H. pylori 2802 is a Cag PAI positive clinical isolate from a patient with an ulcer and easily transformable.
  • the hiock out of ⁇ cpA was carried out using conventional molecular biological methods by insertion mutagenesis with the cat cassette from p ⁇ el2 ((company, or ref. ??)).
  • the resistance cassette has its own promoter and terminator. To produce the hcpa knockout mutant of H.
  • the hcpA ORF was amplified by PCR and cloned into the pUC 18 vector by blunt end using standard molecular biological methods (Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION (1989)) , The amplification was carried out with the oligonucleotides 5'-GAC ACA GGC ATA TGC TAG GAA ACG TTA AAA AAA CCC-3 '(SEQ ID NO: 26) and 5'-GGA CTC GAG AAG TTC TAT TTT TAA TTC CTT G-3' ( SEQ ED NO: 27) from 100 ng genomic DNA from H. pylori 2802.
  • the PCR mixture also contained 400 nM each of the oligonucleotides, 200 ⁇ M dNTP each, 1 unit Pwo polymerase (Boehringer) in the buffer supplied by the manufacturer.
  • the reaction comprised 2 min denaturation at 94 ° C and 27 cycles of 30 s 94 ° C, 30 s 53 ° C and 1 min 72 ° C. This was followed by 7 minutes of polymerisation at 72 ° C.
  • the PCR product was purified with Qiaex II (Qiagen) according to the manufacturer's instructions and cloned into the Smal interface of pUC18 using standard methods.
  • the vector contract was generated by PCR with the oligonucleotides 5'-TAA ATC TTG TGA ATT GAA CCA TGC-3 '(SEQ ID NO: 28) and 5'- GAG TAA TAT TGT GAG GCT TTT TTG G-3' (SEQ ID NO: 29) linearized in the middle of hcpA.
  • the reaction mixture contained 400 nM each of the oligonucleotides, 200 ⁇ M each of dNTP, 1 unit of Pwo polymerase in the buffer supplied by the manufacturer.
  • the reaction included 2 min denaturation at 94 ° C and 14 cycles of 30 s 94 ° C, 30 s 55 ° C and 8 min 72 ° C.
  • the hcpAr.cat contract was generated by PCR with the oligonucleotides 5'-GAC ACA GGC ATA TGC TAG GAA ACG TTA AAA CCC-3 '(SEQ ID NO: 26) and 5'-GGA CTC GAG AAG TTC TAT TTT TAA TTC CTT G- 3 '(SEQ ED NO: 27).
  • the PCR approach contained 20 ng DNA of the pUC18-hcpAr.cat construct, 400 nM each of the oligonucleotides, 200 ⁇ M dNTP each, 1 unit Pwo polymerase (Boehringer) in the buffer supplied by the manufacturer.
  • the reaction included 2 min denaturation at 94 ° C, 27 cycles of 30 s 94 ° C, 30 s 53 ° C and 1.5 min 72 ° C. This was followed by 7 minutes of polymerisation at 72 ° C.
  • the amphibians were cleaned with Qiaex II (Qiagen) according to the manufacturer's instructions.
  • Qiaex II Qiagen
  • logarithmically growing H pylori ad OD 6 oo 0.2 were resuspended in 1 ml Brucella broth (Difco, Detroit, MI, USA) with 10% fetal calf serum and 5 ⁇ g amplicon three times at intervals of 2 h added. The bacteria were incubated at 37 ° C.
  • HcpA The inactivation of HcpA reproducibly leads to an almost complete loss of IFN- ⁇ induction in murine spleen cells by an aqueous extract of the corresponding strains compared to the aqueous extract of the corresponding wild-type H. pylori extract (FIG. 14D).
  • the importance of the HcpA protein for the H. pylori-mediated EFN- ⁇ stimulation would also be confirmed in co-cultivation studies of murine spleen cell cultures with the living wild-type H. pylori isolate and the corresponding hcpA hiock out mutant.
  • pylori 2802hcpA cat (hiock out) growing on aga plates were resuspended in single vaccine medium, washed three times with PBS and resuspended in RPMI medium. The cell number was determined by measuring the optical density.
  • the isogenic ⁇ cpA hiock out mutant induced a significantly lower IFN- ⁇ secretion than the wild-type bacterium ( Figure 15).
  • ⁇ cpA in H. pylori 2802 is the main inducer of IFN- ⁇ in the murine spleen cell model.
  • the strong IFN- ⁇ secretion from spleen cells after stimulation with recombinant ⁇ cpA protein from E. coli is therefore an original, ⁇ cpA specific effect.
  • the insertion of the cat cassette is stable even after approx. 15 passages without antibiotics.
  • ORF 1098 is not affected by the lenock out of the strongly homologous ⁇ cpA. Changes could not be detected either in the Western blot (FIG. 14) or in the PCR (data not shown).
  • the hiock out mutant shows weakly attenuated growth in liquid medium. The growth behavior under stress conditions (high salt, high iron, low iron, increased oxygen partial pressure, increased temperature and acid stress) is unchanged compared to the wild type.

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Abstract

La présente invention concerne des polynucléotides comprenant une séquence d'acide nucléique correspondant au SEQ ID NO: 1, 3, 5, 7 ou 9, ou bien un fragment ou dérivé de cette séquence, ou bien un polynucléotide qui s'hybride avec la séquence d'acide nucléique correspondant au SEQ ID NO: 1, 3, 5, 7, 9 ou 11, codant la HcpA (Helicobacter cystein rich Protein A). La présente invention concerne en outre les polypeptides qui sont codés par la séquence d'acide nucléique, ou un fragment ou un dérivé de la séquence d'acide nucléique correspondant au SEQ ID NO: 1, 3, 5, 7, 9 ou bien 11. De plus, la présente invention concerne l'utilisation desdits polynucléotides et polypeptides.
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