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WO1998056815A1 - VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI Pfr POLYPEPTIDE - Google Patents

VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI Pfr POLYPEPTIDE Download PDF

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Publication number
WO1998056815A1
WO1998056815A1 PCT/SE1998/001092 SE9801092W WO9856815A1 WO 1998056815 A1 WO1998056815 A1 WO 1998056815A1 SE 9801092 W SE9801092 W SE 9801092W WO 9856815 A1 WO9856815 A1 WO 9856815A1
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Prior art keywords
helicobacter pylori
pfr
polypeptide
mammal
infection
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PCT/SE1998/001092
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French (fr)
Inventor
Thomas Berglindh
Björn MELLGÅRD
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Astra Aktiebolag
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Priority to AU80486/98A priority Critical patent/AU8048698A/en
Publication of WO1998056815A1 publication Critical patent/WO1998056815A1/en

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to vaccine compositions useful for inducing a protective immune response to Helicobacter pylori infection.
  • the invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection.
  • H. pylori The gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen, involved in several gastroduodenal diseases. Colonization of gastric epithelium by the bacterium leads to active inflammation and progressive chronic gastritis, with a greatly enhanced risk of progression to peptic ulcer disease. A lifelong inflammation of the gastric mucosa is very closely correlated with a significantly enhanced risk for gastric cancer.
  • H. pylori In order to colonize the gastric mucosa, H. pylori uses a number of virulence factors. Such virulence factors comprise several adhesins, with which the bacterium associates with the mucus and /or binds to epithelial cells; urease which helps to neutralize the acid environment; and proteolytic enzymes which makes the mucus more fluid.
  • H. pylori has developed a number of specific mechanisms for the survival in the hostile gastric environment. One such mechanism could be to trap excess iron by the protein Pfr, in order to avoid toxic iron effects. Despite a strong apparent host immune response to H.
  • the pathogen persists in the gastric mucosa, normally for the life of the host.
  • the reason for this is probably that the spontaneously induced immune-responses are inadequate or directed towards the wrong epitopes of the antigens.
  • the immune response could be of the wrong kind, since the immune system might treat H. pylori as a commensal (as indicated from the life-time host/bacteria relationship).
  • the H. pylori cell transforms from a bacillary to a coccoid form.
  • the H. pylori cell is much less sensitive to antibiotics and other anti-bacterial agents.
  • Circumstantial evidence indicate the H. pylori might be transmitted between individuals in this form, possibly via water or direct contact (oral-oral; feacal-oral).
  • An efficient vaccine composition should therefore elicit an immune response towards both the coccoid and the bacillary form of H. pylori. Since systemic immunity probably only plays a limited role in protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach.
  • the 19. 3 kDa polypeptide Pfr has been identified as an intracellular, cytosolic protein of H. pylori by Doig et al. (Journal of Bacteriology 175(2), 557-560, 1993) and by Frazier et al. (Journal of Bacteriology 174 (4), 966-972, 1993).
  • the location of the Pfr polypeptide has been identified both by antibody staining as well as by direct electron microscopy using ammonium molybdate as a marker. These studies have shown that the Pfr polypeptide is an iron binding protein of paracristalline nature. The gastric environment is directly exposed to large concentrations of iron.
  • the assumed function of the Pfr polypeptide is therefore to serve as a sink for intracellular iron, to avoid toxicity.
  • Sequence homology of the H. pylori Pfr to other known proteins show 42% identity to a 165 amino acid E. coli protein, with known similarities to human ferritin H subunit.
  • Fig. 3 Antibodies (mucosal total Ig and IgA) against strain 244 or specific antigen (Pfr) gastric and duodenal mucosa.
  • A, B and C as for fig. 2.
  • Mucosal samples from 5 animals were pooled and mucosal antibodies determined.
  • Total mucosal antibodies (Ig) as well as specific antibodies (IgA) against the infecting strain 244 could be detected both in stomach and duodenum(PBS and CT).
  • Specific antibodies to Pfr was detected following immunization with Pfr + CT. From left to right for each group the bars represent: Ig gastric; Ig duodenum; IgA gastric; and IgA duodenum.
  • the purpose of this invention is to provide an antigenic H. pylori polypeptide which can be useful for eliciting a protective immune response against, and for diagnosis of, H. pylori infection.
  • This purpose has been achieved by the recombinant cloning of an H. pylori gene which encodes a well conserved abundantly present cytosolic protein.
  • the nucleic acid sequence of this gene is similar to the sequence of the pfr gene as published by Frazier et al. (Journal of Bacteriology 174 (4), 966-972, 1993).
  • the pfr gene is expressed by all H. pylori strains tested Qiang et al., Molecular Biology 20(4), 833-842, 1996).
  • H. pylori Pfr polypeptide in spite of being a mainly intracellular protein, can serve as a therapeutic antigen in an H. pylori infected mouse model, when given together with the adjuvant cholera toxin.
  • the experimental data below thus indicates that the H. pylori Pfr polypeptide, when used as an oral immunogen, acts as a stimulator of an immune response leading to a significant reduction of colonization of H. pylori in mice which were infected with H. pylori one month prior to immunization.
  • H. pylori Pfr polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections.
  • the Pfr polypeptide will be useful both for the detection of H. pylori infections as well as for the manufacture of vaccine compositions, which when given in an appropriate pharmaceutical formulation will elicit a protective or therapeutic immune response against such infections.
  • the present invention provides a Helicobacter pylori Pfr polypeptide for use in inducing a protective immune response to Helicobacter pylori infection.
  • the term "Helicobacter pylori Pfr polypeptide” is intended to mean the polypeptide disclosed by Frazier et al., Journal of Bacteriology 175(4), 966-972, 1993, and which is encoded by the gene whose sequence is set forth as SEQ ID NO: 1, or can be obtained from the National Center for Biotechnology Information (Accession number S54729), or a substantially similar modified form thereof retaining functionally equivalent antigenicity.
  • protective immune response is to be understood as an immune response which makes the composition suitable for therapeutic and /or prophylactic purposes.
  • the term "functionally equivalent antigenicity" is to be understood as the ability to induce a systemic and mucosal immune response while decreasing the number of H. pylori cells associated with the gastric mucosa.
  • the skilled person will be able to identify modified forms of the Pfr polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies.
  • the Helicobacter pylori Pfr polypeptide for use in inducing a protective immune response to Helicobacter pylori infection, has substantially the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing, or is a modified form thereof retaining functionally equivalent antigenicity.
  • the definition of the Helicobacter pylori Pfr polypeptide is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of the Helicobacter pylori Pfr polypeptide and is retaining functionally equivalent antigenicity.
  • Helicobacter pylori Pfr polypeptide includes amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing.
  • the invention provides a vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori Pfr polypeptide as defined above, optionally together with a pharmaceutically acceptable carrier or diluent.
  • an immunologically effective amount is to be understood as an amount which elicits a significant protective Helicobacter pylori response, which will eradicate a H. pylori infection in an infected mammal or prevent the infection in a susceptible mammal.
  • an immunologically effective amount will comprise approximately 1 ⁇ g to 1000 mg, preferably approximately 10 ⁇ g to 100 mg, of H. pylori antigen for oral administration, or approximately less than 100 ⁇ g for parenteral administration.
  • the vaccine composition comprises optionally in addition to a pharmaceutically acceptable carrier or diluent one or more other immunologically active antigens for prophylactic or therapeutic use.
  • Physiologically acceptable carriers and diluents are well known to those skilled in the art and include e.g. phosphate buffered saline (PBS), or, in the case of oral vaccines, HCO3" based formulations or enterically coated powder formulations.
  • the vaccine composition can optionally include or be administered together with acid secretion inhibitors, preferably proton pump inhibitors (PPIs), e.g. omeprazole.
  • PPIs proton pump inhibitors
  • the vaccine can be formulated in known delivery systems such as liposomes, ISCOMs, cochleates, etc. (see e.g. Rabinovich et al. (1994) Science 265, 1401-1404) or be attached to or incorporated into polymer microspheres of degradable or non-degradable nature.
  • the antigens could be associated with live attenuated bacteria, viruses or phages or with killed vectors of the same kind.
  • the antigens can be chemically or genetically coupled to carrier proteins of inert or adjuvantic types (i.e Cholera B subunit).
  • the invention provides in a further aspect a vaccine composition according to above, in addition comprising an adjuvant, such as a pharmaceutically acceptable form of cholera toxin.
  • an adjuvant such as a pharmaceutically acceptable form of cholera toxin.
  • Such pharmaceutically acceptable forms of cholera toxin are known in the art, e.g. from Rappuoli et al. (1995) Int. Arch. Allergy & Immunol. 108(4), 327-333; and Dickinson et al. (1995) Infection and Immunity 63(5), 1617-1623.
  • a vaccine composition according to the invention can be used for both therapeutic and prophylactic purposes. Consequently, the invention includes a vaccine composition according as defined above, for use as a therapeutic or a prophylactic vaccine in a mammal, including man, which is infected by Helicobacter pylori.
  • the term "prophylactic purpose” means to induce an immune response which will protect against future infection by Helicobacter pylori
  • therapeutic purpose means to induce an immune response which can eradicate an existing Helicobacter pylori infections.
  • the vaccine composition according to the invention is preferably administered to any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the gastric, the intestinal (small and large intestine), the rectal and the vaginal mucosa.
  • the mucosal vaccines can be given together with for the purpose appropriate adjuvants.
  • the vaccine can also be given orally, or parenterally, by the subcutaneous, intracutaneous or intramuscular route, optionally together with the appropriate adjuvant.
  • the vaccine composition can optionally be given together with antimicrobial therapeutic agents.
  • the invention proivides the use of a Helicobacter pylori Pfr polypeptide, as defined above, in the manufacture of
  • compositions for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection (i) a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection;
  • a vaccine for use in eliciting a protective immune response against Helicobacter pylori (ii) a vaccine for use in eliciting a protective immune response against Helicobacter pylori; and (iii) a diagnostic kit for diagnosis of Helicobacter pylori infection.
  • the invention provides a method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori Pfr polypeptide as defined above, optionally labelled or coupled to a solid support, is used.
  • the said method could e.g. comprise the steps (a) contacting a said Helicobacter pylori Pfr polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and (b) detecting antibodies from the said body fluid binding to the said Pfr polypeptide.
  • Preferred methods of detecting antibodies are ELISA (Enzyme linked immunoabsorbent assay) methods which are well known in the art.
  • the invention provides a diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method of in vitro diagnosis as described above to be carried out.
  • the said diagnostic kit could e.g. comprise: (a) a Helicobacter pylori Pfr polypeptide; and (b) reagents for detecting antibodies binding to the said Pfr polypeptide.
  • the said reagents for detecting antibodies could e.g. be an enzyme-labelled anti- immunoglobulin and a chromogenic substrate for the said enzyme.
  • the invention provides a method of eliciting in a mammal, including humans, a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a Helicobacter pylori Pfr polypeptide as defined above, or alternatively administering to the said mammal an immunologically effective amount of a vaccine composition as defined above.
  • standard protocols and “standard procedures”, when used in the context of molecular cloning techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989.
  • Sequences were cloned from the J99 strain of H. pylori by amplification cloning using the polymerase chain reaction (PCR).
  • Synthetic oligonucleotide primers (see below) specific for the 5'- and 3'-ends of open reading frames of genes were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA).
  • Forward primers (specific for the 5'-end of the sequence) were designed to include an Ndel cloning site at the extreme 5'-terminus, while reverse primers included a EcoRI site at the extreme 5'-terminus to permit cloning of each H. pylori sequence into the reading frame of the pET28b vector.
  • Inserts cloned into the Ndel-EcoRI site of pET-28 are fused to a DNA sequence encoding a hexa His-Tag (six histidine residues) located at the extreme 3'-terminus of the recombinant protein.
  • Genomic DNA prepared from the J99 strain of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, editors F. Ausubel et al, John Wiley and Sons, Inc. 1994).
  • genomic DNA 50 ng was introduced into a reaction vial containing 2 mM MgCl 2 , 1 ⁇ M synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H.
  • pylori ORF 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP, and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 ⁇ l.
  • thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Per kin Elmer Cetus/ Gene Amp PCR System 9600 thermal cycler: Denaturation at +94°C for 2 min; 2 cycles at +94°C for 15 sec, +30°C for 15 sec and +72°C for 1.5 min; 23 cycles at +94°C for 15 sec, +58°C for 15 sec and +72°C for 1.5 min; Reactions were concluded at +72°C for 6 minutes.
  • each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). Amplified DNA samples were subjected to digestion with the restriction endonucleases Ndel and EcoRI according to standard procedures. DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave UN irradiation. D ⁇ A contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).
  • the pET-28b vector was prepared for cloning by digestion with Ndel and EcoRI according to standard procedures. Following digestion, D ⁇ A inserts were cloned according to standard procedures into the previously digested pET-28b expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli as described below. Transformation of competent bacteria with recombinant plasmids
  • Competent bacteria E. coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods. Briefly, 1 ⁇ l of ligation reaction was mixed with 50 ⁇ l of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgS0 4 and 20, mM glucose) at +37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 ⁇ g/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below.
  • the pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5 ⁇ , etc. for the purpose of cloning or plasmid preparation.
  • Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase.
  • T7 RNA polymerase is induced by addition of isopropyl- ⁇ -D- thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest.
  • Strains used in our laboratory include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Methods Enzymol. 185, 60-89).
  • H. pylori sequences 50 ng of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 ⁇ g/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 ⁇ g/ml) to an optical density at 600 ran of 0.5 to 1.0 O.D. units, at which point, 1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
  • the concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. (1986) Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry et al. (1951) J. Biol. Chem. 193, 265-275, using bovine serum albumin as a standard.
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gels (12% or 4 to 25 % gradient acrylamide) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie Brilliant Blue. Molecular mass markers included rabbit skeletal muscle myosin (200 kDa), E.
  • coli ⁇ -galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
  • the resultant homogenate was made 0.1 % Brij 35, and centrifuged (100,000 g x 1 hour) to yield a clear supernatant (crude extract). Following filtration through a 0.8 ⁇ m Supor filter (Gelman Sciences, FRG), the crude extract was loaded directly onto a 5-ml Ni 2+ -nitrolotriacetate-agarose (NTA) column (Hochuli,E., Dobeli, H., and Schacheer, A. (1987) J. Chromatography 411, 177-184) equilibrated in lysis buffer containing 10 % glycerol, 0.1 % Brij 35 and 1 mM PMSF.
  • NTA Ni 2+ -nitrolotriacetate-agarose
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10% glycerol, 0.1 % Brij 35, and developed with sequential steps of Lysis Buffer containing 10% glycerol, 0.05% Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole. Fractions were monitored by absorbance at OD 2 80 an d peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant proteins eluted at 100 mM imidazole.
  • the Pfr polypeptide eluted as a sharp peak at 325 mM NaCl. Fractions containing the recombinant protein were pooled and dialyzed against Tris Buffered Saline (TBS; 10 mM Tris pH 7.4, 150 mM NaCl). Recombinant Pfr polypeptide was subjected to electrophoresis by SDS-PAGE and visualized by Coomassie blue staining. The recombinant polypeptide was determined to be greater than 95% pure.
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding centre (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
  • H. pylori strain 244, originally isolated from an ulcer patient. This strain has earlier proven to be a good colonizer of the mouse stomach. Bacteria from a stock kept at -70°C were grown overnight in Brucella broth supplemented with 10% fetal calf serum, at +37°C in a microaerophilic atmosphere (10% C0 2 , 5% 0 2 ). The animals were given an oral dose of omeprazole (400 ⁇ mol/kg) and after 3-5 h an oral inoculation of H. pylori (approximately 10 7 -10 8 CFU/animal). Infection was checked in control animals 2-3 weeks after the inoculation.
  • mice were immunized 4 times over a 34 day period (day 1, 15, 25 and 35).
  • Purified recombinant Pfr dissolved in PBS was given at a dose of 100 ⁇ g/mouse to group 3.
  • CT cholera toxin
  • Omeprazole 400 ⁇ mol/kg was given orally to all animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Animals were sacrificed 1-2 weeks after final immunization.
  • Group 3 300 ⁇ l PBS containing 100 ⁇ g Pfr and 10 ⁇ g CT
  • mice were sacrificed by C0 2 and cervical dislocation.
  • the abdomen and chest cavity was opened and blood sampled by heart puncture.
  • the stomach and the upper part of the small intestine was removed.
  • After cutting the stomach along the greater curvature it was rinsed in saline and subsequently cut into two identical pieces.
  • An area of 25 mm 2 of the mucosa from the antrum and corpus was scraped separately with a surgical scalpel.
  • the mucosa scraping was suspended in Brucella broth, diluted and plated onto Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted.
  • the identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
  • Mucosal antibodies were collected by the following technique. One half of the rinsed stomach was placed mucosal side up on a piece of paper. Likewise the duodenum was cut open and placed mucosal side up. One standardized round filter paper (30.4 mm 2 ) was placed on the antrum and one on the corpus musosa. After 10 minutes both papers were transferred to one tube with 200 ⁇ l special buffer containing protease inhibitors. A paper-strip, 4.8x19 mm (91.2 mm 2 ) was in the same way placed on the duodenum mucosa and was subsequently placed in a separate tube with buffer. After a minimum of one hour extraction of the filter papers, the buffer solutions from the 10 mice within each group was pooled. The pooled solutions were either used directly for ELISA measurements of antibody concentration or keep frozen at -20°C.
  • Serum antibodies were collected from blood drawn by heart-puncture under anaesthesia. Prior to centrifugation, the blood was diluted with equal amount of PBS. The serum was kept at -20°C until analysis.
  • Mucosal antibodies were measured using an ELISA were plates were coated with Pfr polypeptide followed by addition of mucosal extract.
  • the ELISA was developed with alkaline phosphatase-labelled anti-mouse Ig or anti-mouse IgA antibodies.
  • the anti-Ig antibodies were of an anti-heavy /anti-light chain type, which should detect all types of antibodies .
  • Standard curves were created by coating known amounts of mouse IgA and Ig.
  • Serum antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with Pfr polypeptide followed by addition of different dilutions of serum.
  • the ELISA was developed with alkaline phosphatase-labelled anti-mouse-Ig-antibodies as described above.
  • mice in this study were infected with H. pylori strain 244 one month prior to immunizations. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant Pfr polypeptide. Control animals received vehicle only (PBS). Two weeks after the final immunization, the animals were sacrificed and CFU (colony-forming units) was determined (Fig.l.).
  • CT cholera toxin
  • PBS vehicle only
  • ORGANISM Helicobacter pylori
  • ATC AGC GAG TCT ATT AAT AAT ATC GTC GAT CAC GCC ATA AAA GGC AAA 632 lie Ser Glu Ser lie Asn Asn lie Val Asp His Ala lie Lys Gly Lys 100 105 110
  • GGT AAT GAA AAC CAT GGC TTG TAT TTG GCT GAT CAG TAT GTC AAA GGG 776 Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gin Tyr Val Lys Gly 145 150 155 160

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Abstract

The present invention relates to vaccine compositions useful for inducing a protective immune reponse to Helicobacter pylori infection. The invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection.

Description

VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI Pfr POLYPEPTTDE
VACCINE COMPOSITIONS IV
TECHNICAL FIELD
The present invention relates to vaccine compositions useful for inducing a protective immune response to Helicobacter pylori infection. The invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection.
BACKGROUND ART
Helicobacter pylori
The gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen, involved in several gastroduodenal diseases. Colonization of gastric epithelium by the bacterium leads to active inflammation and progressive chronic gastritis, with a greatly enhanced risk of progression to peptic ulcer disease. A lifelong inflammation of the gastric mucosa is very closely correlated with a significantly enhanced risk for gastric cancer.
In order to colonize the gastric mucosa, H. pylori uses a number of virulence factors. Such virulence factors comprise several adhesins, with which the bacterium associates with the mucus and /or binds to epithelial cells; urease which helps to neutralize the acid environment; and proteolytic enzymes which makes the mucus more fluid. In addition H. pylori has developed a number of specific mechanisms for the survival in the hostile gastric environment. One such mechanism could be to trap excess iron by the protein Pfr, in order to avoid toxic iron effects. Despite a strong apparent host immune response to H. pylori, with production of both local (mucosal) as well as systemic antibodies, the pathogen persists in the gastric mucosa, normally for the life of the host. The reason for this is probably that the spontaneously induced immune-responses are inadequate or directed towards the wrong epitopes of the antigens. Alternatively the immune response could be of the wrong kind, since the immune system might treat H. pylori as a commensal (as indicated from the life-time host/bacteria relationship).
In order to understand the pathogenesis and immunology of H. pylori infections, it is of great importance to define the antigenic structure of this bacterium. In particular, there is a need for characterization of surface-exposed, surface associated as well as secreted proteins which, in many bacterial pathogens, have been shown to constitute the main virulence factors, and which can be useful for the diagnosis of H. Pylori and in the manufacture of vaccine compositions.
Whenever stressed or threatened, the H. pylori cell transforms from a bacillary to a coccoid form. In the coccoid form, the H. pylori cell is much less sensitive to antibiotics and other anti-bacterial agents. Circumstantial evidence indicate the H. pylori might be transmitted between individuals in this form, possibly via water or direct contact (oral-oral; feacal-oral). An efficient vaccine composition should therefore elicit an immune response towards both the coccoid and the bacillary form of H. pylori. Since systemic immunity probably only plays a limited role in protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach.
The 19.3 kDa polypeptide Pfr ofH. pylori
The 19. 3 kDa polypeptide Pfr has been identified as an intracellular, cytosolic protein of H. pylori by Doig et al. (Journal of Bacteriology 175(2), 557-560, 1993) and by Frazier et al. (Journal of Bacteriology 174 (4), 966-972, 1993). The location of the Pfr polypeptide has been identified both by antibody staining as well as by direct electron microscopy using ammonium molybdate as a marker. These studies have shown that the Pfr polypeptide is an iron binding protein of paracristalline nature. The gastric environment is directly exposed to large concentrations of iron. The assumed function of the Pfr polypeptide is therefore to serve as a sink for intracellular iron, to avoid toxicity. Sequence homology of the H. pylori Pfr to other known proteins show 42% identity to a 165 amino acid E. coli protein, with known similarities to human ferritin H subunit.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l Therapeutic immunization of BALB/c mice infected with H. pylori strain 244
(n=10/group). Results are given as the geometrical mean of the total CFU (colony- forming units) in corpus + antrum. Abbreviations: A=phosphate buffered saline (PBS); B=cholera toxin (CT); C=recombinant Pfr H. pylori polypeptide + CT. All animals in PBS and CT control groups were well infected in corpus and antrum mucosa. The Pfr + CT treated group had significantly lower CFU than the PBS or the CT group. Numbers denote number of animals free of H. pylori in different parts of the stomach, i.e. 3 animals each were H. pylori free in corpus and antrum respectively. In 2/10 animals the H. pylori infection was eradicated. ** p<0.01; (Wilcoxon-Mann-Whittney sign rank test).
Fig. 2
Serum antibody response (IgG titer) against membrane proteins of Helicobacter pylori strain 244 (unshaded bars) or against Pfr (shaded bars). All animals showed Ig antibodies to the infecting strain. Only Pfr immunized animals have systemic IgG antibodies to this protein. Geometrical mean values given; n=10/group. A and B as for fig. 1; C=Pfr.
Fig. 3 Antibodies (mucosal total Ig and IgA) against strain 244 or specific antigen (Pfr) gastric and duodenal mucosa. A, B and C as for fig. 2. ELISA antigen: A and B - strain 244; C - Pfr. Mucosal samples from 5 animals were pooled and mucosal antibodies determined. Total mucosal antibodies (Ig) as well as specific antibodies (IgA) against the infecting strain 244 could be detected both in stomach and duodenum(PBS and CT). Specific antibodies to Pfr was detected following immunization with Pfr + CT. From left to right for each group the bars represent: Ig gastric; Ig duodenum; IgA gastric; and IgA duodenum.
DISCLOSURE OF THE INVENTION
The purpose of this invention is to provide an antigenic H. pylori polypeptide which can be useful for eliciting a protective immune response against, and for diagnosis of, H. pylori infection. This purpose has been achieved by the recombinant cloning of an H. pylori gene which encodes a well conserved abundantly present cytosolic protein. The nucleic acid sequence of this gene is similar to the sequence of the pfr gene as published by Frazier et al. (Journal of Bacteriology 174 (4), 966-972, 1993). The pfr gene is expressed by all H. pylori strains tested Qiang et al., Molecular Biology 20(4), 833-842, 1996).
It has surprisingly been found that the recombinant H. pylori Pfr polypeptide, in spite of being a mainly intracellular protein, can serve as a therapeutic antigen in an H. pylori infected mouse model, when given together with the adjuvant cholera toxin. The experimental data below thus indicates that the H. pylori Pfr polypeptide, when used as an oral immunogen, acts as a stimulator of an immune response leading to a significant reduction of colonization of H. pylori in mice which were infected with H. pylori one month prior to immunization.
These results strongly support the use of the H. pylori Pfr polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections. As such, the Pfr polypeptide will be useful both for the detection of H. pylori infections as well as for the manufacture of vaccine compositions, which when given in an appropriate pharmaceutical formulation will elicit a protective or therapeutic immune response against such infections.
Consequently, in one aspect the present invention provides a Helicobacter pylori Pfr polypeptide for use in inducing a protective immune response to Helicobacter pylori infection. The term "Helicobacter pylori Pfr polypeptide" is intended to mean the polypeptide disclosed by Frazier et al., Journal of Bacteriology 175(4), 966-972, 1993, and which is encoded by the gene whose sequence is set forth as SEQ ID NO: 1, or can be obtained from the National Center for Biotechnology Information (Accession number S54729), or a substantially similar modified form thereof retaining functionally equivalent antigenicity.
The term "protective immune response" is to be understood as an immune response which makes the composition suitable for therapeutic and /or prophylactic purposes.
The term "functionally equivalent antigenicity" is to be understood as the ability to induce a systemic and mucosal immune response while decreasing the number of H. pylori cells associated with the gastric mucosa. The skilled person will be able to identify modified forms of the Pfr polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies. In a preferred form of the invention, the Helicobacter pylori Pfr polypeptide, for use in inducing a protective immune response to Helicobacter pylori infection, has substantially the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing, or is a modified form thereof retaining functionally equivalent antigenicity.
It is thus to be understood that the definition of the Helicobacter pylori Pfr polypeptide is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of the Helicobacter pylori Pfr polypeptide and is retaining functionally equivalent antigenicity. Included in the definition of the Helicobacter pylori Pfr polypeptide are consequently polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing.
In another aspect, the invention provides a vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori Pfr polypeptide as defined above, optionally together with a pharmaceutically acceptable carrier or diluent.
In the present context the term "immunologically effective amount" is to be understood as an amount which elicits a significant protective Helicobacter pylori response, which will eradicate a H. pylori infection in an infected mammal or prevent the infection in a susceptible mammal. Typically an immunologically effective amount will comprise approximately 1 μg to 1000 mg, preferably approximately 10 μg to 100 mg, of H. pylori antigen for oral administration, or approximately less than 100 μg for parenteral administration.
The vaccine composition comprises optionally in addition to a pharmaceutically acceptable carrier or diluent one or more other immunologically active antigens for prophylactic or therapeutic use. Physiologically acceptable carriers and diluents are well known to those skilled in the art and include e.g. phosphate buffered saline (PBS), or, in the case of oral vaccines, HCO3" based formulations or enterically coated powder formulations.
The vaccine composition can optionally include or be administered together with acid secretion inhibitors, preferably proton pump inhibitors (PPIs), e.g. omeprazole. The vaccine can be formulated in known delivery systems such as liposomes, ISCOMs, cochleates, etc. (see e.g. Rabinovich et al. (1994) Science 265, 1401-1404) or be attached to or incorporated into polymer microspheres of degradable or non-degradable nature. The antigens could be associated with live attenuated bacteria, viruses or phages or with killed vectors of the same kind. The antigens can be chemically or genetically coupled to carrier proteins of inert or adjuvantic types (i.e Cholera B subunit). Consequently, the invention provides in a further aspect a vaccine composition according to above, in addition comprising an adjuvant, such as a pharmaceutically acceptable form of cholera toxin. Such pharmaceutically acceptable forms of cholera toxin are known in the art, e.g. from Rappuoli et al. (1995) Int. Arch. Allergy & Immunol. 108(4), 327-333; and Dickinson et al. (1995) Infection and Immunity 63(5), 1617-1623.
A vaccine composition according to the invention can be used for both therapeutic and prophylactic purposes. Consequently, the invention includes a vaccine composition according as defined above, for use as a therapeutic or a prophylactic vaccine in a mammal, including man, which is infected by Helicobacter pylori. In this context the term "prophylactic purpose" means to induce an immune response which will protect against future infection by Helicobacter pylori, while the term "therapeutic purpose" means to induce an immune response which can eradicate an existing Helicobacter pylori infections.
The vaccine composition according to the invention is preferably administered to any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the gastric, the intestinal (small and large intestine), the rectal and the vaginal mucosa. The mucosal vaccines can be given together with for the purpose appropriate adjuvants. The vaccine can also be given orally, or parenterally, by the subcutaneous, intracutaneous or intramuscular route, optionally together with the appropriate adjuvant. The vaccine composition can optionally be given together with antimicrobial therapeutic agents.
In a further aspect, the invention proivides the use of a Helicobacter pylori Pfr polypeptide, as defined above, in the manufacture of
(i) a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection;
(ii) a vaccine for use in eliciting a protective immune response against Helicobacter pylori; and (iii) a diagnostic kit for diagnosis of Helicobacter pylori infection.
In yet a further aspect, the invention provides a method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori Pfr polypeptide as defined above, optionally labelled or coupled to a solid support, is used. The said method could e.g. comprise the steps (a) contacting a said Helicobacter pylori Pfr polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and (b) detecting antibodies from the said body fluid binding to the said Pfr polypeptide. Preferred methods of detecting antibodies are ELISA (Enzyme linked immunoabsorbent assay) methods which are well known in the art.
In another aspect the invention provides a diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method of in vitro diagnosis as described above to be carried out.
The said diagnostic kit could e.g. comprise: (a) a Helicobacter pylori Pfr polypeptide; and (b) reagents for detecting antibodies binding to the said Pfr polypeptide. The said reagents for detecting antibodies could e.g. be an enzyme-labelled anti- immunoglobulin and a chromogenic substrate for the said enzyme.
In yet a further aspect, the invention provides a method of eliciting in a mammal, including humans, a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a Helicobacter pylori Pfr polypeptide as defined above, or alternatively administering to the said mammal an immunologically effective amount of a vaccine composition as defined above.
EXPERIMENTAL METHODS
Throughout this description the terms "standard protocols" and "standard procedures", when used in the context of molecular cloning techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989.
Preparation of recombinant H. pylori Pfr polypeptide DNA sequence information
Gene sequence information for the iron binding polypeptide Pfr was obtained from the National Center for Biotechnology Information (Accession number S54729; SEQ ID NO: 1).
PCR Amplification and cloning of DNA sequences containing ORF'sfor membrane and secreted proteins from the J99 Strain of Helicobacter pylori.
Sequences were cloned from the J99 strain of H. pylori by amplification cloning using the polymerase chain reaction (PCR). Synthetic oligonucleotide primers (see below) specific for the 5'- and 3'-ends of open reading frames of genes were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). Forward primers (specific for the 5'-end of the sequence) were designed to include an Ndel cloning site at the extreme 5'-terminus, while reverse primers included a EcoRI site at the extreme 5'-terminus to permit cloning of each H. pylori sequence into the reading frame of the pET28b vector. Inserts cloned into the Ndel-EcoRI site of pET-28 are fused to a DNA sequence encoding a hexa His-Tag (six histidine residues) located at the extreme 3'-terminus of the recombinant protein.
Forward primer (SEQ ID NO: 3): 5'-AGG AGA TCA TAT GTT ATC AAA AGA C-3' Reverse primer (SEQ ID NO: 4): 5--TGA ATT CAA GAT TTC CTG CTT TTA G-3'
Genomic DNA prepared from the J99 strain of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, editors F. Ausubel et al, John Wiley and Sons, Inc. 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 ng) was introduced into a reaction vial containing 2 mM MgCl2, 1 μM synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP, and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 μl. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Per kin Elmer Cetus/ Gene Amp PCR System 9600 thermal cycler: Denaturation at +94°C for 2 min; 2 cycles at +94°C for 15 sec, +30°C for 15 sec and +72°C for 1.5 min; 23 cycles at +94°C for 15 sec, +58°C for 15 sec and +72°C for 1.5 min; Reactions were concluded at +72°C for 6 minutes.
Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). Amplified DNA samples were subjected to digestion with the restriction endonucleases Ndel and EcoRI according to standard procedures. DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave UN irradiation. DΝA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).
Cloning ofH. pylori DNA sequences into the pET-28b prokaryotic expression vector.
The pET-28b vector was prepared for cloning by digestion with Ndel and EcoRI according to standard procedures. Following digestion, DΝA inserts were cloned according to standard procedures into the previously digested pET-28b expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli as described below. Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E. coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods. Briefly, 1 μl of ligation reaction was mixed with 50 μl of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl2, 10 mM MgS04 and 20, mM glucose) at +37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 μg/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below.
Identification of recombinant pET expression plasmids carrying H. pylori sequences
Individual BL21 clones transformed with recombinant pET-28b H. pylori genes were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the expression vector according to standard procedures.
Isolation and Preparation of lasmid DNA from BL21 transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H. pylori ORFs were picked and incubated in 5 ml of LB broth plus 25 μg/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA). Expression of recombinant H. pylori sequences in E. coli
The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5α, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-β-D- thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest. Strains used in our laboratory include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Methods Enzymol. 185, 60-89).
To express recombinant H. pylori sequences, 50 ng of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 μg/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 μg/ml) to an optical density at 600 ran of 0.5 to 1.0 O.D. units, at which point, 1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 ml cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at +4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification. Analytical Methods
The concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. (1986) Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry et al. (1951) J. Biol. Chem. 193, 265-275, using bovine serum albumin as a standard.
Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels (12% or 4 to 25 % gradient acrylamide) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie Brilliant Blue. Molecular mass markers included rabbit skeletal muscle myosin (200 kDa), E. coli β-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
Purification of recombinant proteins
All steps were carried out at +4°C. Frozen cells were thawed, resuspended in 5 volumes of lysis buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM imidazole) with 10% glycerol, 0.1 % β-mercaptoethanol, 200 μg/ml lysosyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 μg/ml each of leupeptin, aprotinin, pepstatin, L-l-chloro-3-[4-tosylamido]-7-amino-2-heptanone (TLCK), L-l-chloro-3- [4-tosylamido]-4-phenyl-2-butanone (TPCK), and soybean trypsin inhibitor), and ruptured by several passages through a small volume microfluidizer (Model M- 110S, Microfluidics International Corporation, Newton, MA). The resultant homogenate was made 0.1 % Brij 35, and centrifuged (100,000 g x 1 hour) to yield a clear supernatant (crude extract). Following filtration through a 0.8 μm Supor filter (Gelman Sciences, FRG), the crude extract was loaded directly onto a 5-ml Ni2+ -nitrolotriacetate-agarose (NTA) column (Hochuli,E., Dobeli, H., and Schacheer, A. (1987) J. Chromatography 411, 177-184) equilibrated in lysis buffer containing 10 % glycerol, 0.1 % Brij 35 and 1 mM PMSF. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10% glycerol, 0.1 % Brij 35, and developed with sequential steps of Lysis Buffer containing 10% glycerol, 0.05% Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole. Fractions were monitored by absorbance at OD280 and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant proteins eluted at 100 mM imidazole.
Fractions from the Ni2+ -NTA-agarose column were pooled and dialyzed overnight against 1 liter of dialysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM EGTA, 0.02% Brij 35 and 1 mM PMSF. In the morning, a fine white precipitate was removed by centrifugation (10,000 g x 30 min) and the resulting supernatant was loaded onto an 8 ml (8 x 75 mm) Mono Q high performance liquid chromatography column (Pharmacia Biotechnology, Inc., Piscataway, NJ, USA) equilibrated in Buffer B (10 mM Tris, pH 8.5, 0.1 mM EGTA) containing 50 mM NaCl. The column was washed with five bed volumes of buffer B containing 50 mM NaCl, and developed with a 50-ml linear gradient of increasing NaCl (50 to 500 mM).
The Pfr polypeptide eluted as a sharp peak at 325 mM NaCl. Fractions containing the recombinant protein were pooled and dialyzed against Tris Buffered Saline (TBS; 10 mM Tris pH 7.4, 150 mM NaCl). Recombinant Pfr polypeptide was subjected to electrophoresis by SDS-PAGE and visualized by Coomassie blue staining. The recombinant polypeptide was determined to be greater than 95% pure. EXAMPLES OF THE INVENTION
EXAMPLE 1: THERAPEUTIC IMMUNIZATION
1. Materials & Methods
1.1 Animals
Female SPF BALB/c mice were purchased from Bomholt Breeding centre (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
1.2. Infection
After a minimum of one week of acclimatization, the animals were infected with a type 2 strain of H. pylori (strain 244, originally isolated from an ulcer patient). This strain has earlier proven to be a good colonizer of the mouse stomach. Bacteria from a stock kept at -70°C were grown overnight in Brucella broth supplemented with 10% fetal calf serum, at +37°C in a microaerophilic atmosphere (10% C02, 5% 02). The animals were given an oral dose of omeprazole (400 μmol/kg) and after 3-5 h an oral inoculation of H. pylori (approximately 107-108 CFU/animal). Infection was checked in control animals 2-3 weeks after the inoculation.
1.3. Immunizations
One month after infection, 3 groups of mice (10 mice/group) were immunized 4 times over a 34 day period (day 1, 15, 25 and 35). Purified recombinant Pfr dissolved in PBS was given at a dose of 100 μg/mouse to group 3. As an adjuvant, the animals in groups 2 and 3 were also given 10 μg/mouse of cholera toxin (CT) with each immunization. Omeprazole (400 μmol/kg) was given orally to all animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Animals were sacrificed 1-2 weeks after final immunization.
Group 1: 300 μl PBS
Group 2: 300 μl PBS containing 10 μg CT
Group 3: 300 μl PBS containing 100 μg Pfr and 10 μg CT
1.4. Analysis of infection
The mice were sacrificed by C02 and cervical dislocation. The abdomen and chest cavity was opened and blood sampled by heart puncture. Subsequently the stomach and the upper part of the small intestine was removed. After cutting the stomach along the greater curvature, it was rinsed in saline and subsequently cut into two identical pieces. An area of 25 mm2 of the mucosa from the antrum and corpus was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth, diluted and plated onto Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
1.5 Antibody measurements
Mucosal antibodies were collected by the following technique. One half of the rinsed stomach was placed mucosal side up on a piece of paper. Likewise the duodenum was cut open and placed mucosal side up. One standardized round filter paper (30.4 mm2) was placed on the antrum and one on the corpus musosa. After 10 minutes both papers were transferred to one tube with 200 μl special buffer containing protease inhibitors. A paper-strip, 4.8x19 mm (91.2 mm2) was in the same way placed on the duodenum mucosa and was subsequently placed in a separate tube with buffer. After a minimum of one hour extraction of the filter papers, the buffer solutions from the 10 mice within each group was pooled. The pooled solutions were either used directly for ELISA measurements of antibody concentration or keep frozen at -20°C.
Serum antibodies were collected from blood drawn by heart-puncture under anaesthesia. Prior to centrifugation, the blood was diluted with equal amount of PBS. The serum was kept at -20°C until analysis.
Mucosal antibodies were measured using an ELISA were plates were coated with Pfr polypeptide followed by addition of mucosal extract. The ELISA was developed with alkaline phosphatase-labelled anti-mouse Ig or anti-mouse IgA antibodies. The anti-Ig antibodies were of an anti-heavy /anti-light chain type, which should detect all types of antibodies . Standard curves were created by coating known amounts of mouse IgA and Ig.
Serum antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with Pfr polypeptide followed by addition of different dilutions of serum. The ELISA was developed with alkaline phosphatase-labelled anti-mouse-Ig-antibodies as described above.
2. Results
2.1. Therapeutic immunization: effects on colony-forming units (CFU)
The animals in this study were infected with H. pylori strain 244 one month prior to immunizations. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant Pfr polypeptide. Control animals received vehicle only (PBS). Two weeks after the final immunization, the animals were sacrificed and CFU (colony-forming units) was determined (Fig.l.).
All control animals, as well as those immunized with only CT, were infected both in corpus and antrum. Animals actively immunized with recombinant H. pylori Pfr polypeptide and CT, had significantly decreased CFU values (p<0.05; Wilcoxon- Mann-Whittney sign rank test) compared with the controls. Three animals from each group had no detectable H. pylori infection in corpus and in antrum, respectively. Two out of 10 animals were totally free of H. pylori.
2.2. Therapeutic immunization: effects on antibody formation and secretion
All animals responded with an increased serum titer against membrane proteins of the infecting H. pylori strain 244 as measured by ELISA. Only in animals given Pfr + CT a serum IgG titer against the Pfr polypeptide could be measured. Based on the standard curve, using known amounts of mouse IgG antibody, a mean response level of 770 μg/ml was calculated (Fig. 2).
Total mucosal Ig against Pfr showed highly significant, 4-fold increases, following immunization with Pfr + CT for both gastric and duodenal samples. IgA in gastric samples showed a minor increase, whereas the level in duodenum increased 3 times following Pfr + CT immunization (Fig. 3).
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Astra AB
(B) STREET: Vastra Malarehamnen 9
(C) CITY: Sδdertalje
(E) COUNTRY: Sweden
(F) POSTAL CODE (ZIP) : S-151 85
(G) TELEPHONE: +46 8 553 260 00 (H) TELEFAX: +46 8 553 288 20
(ii) TITLE OF INVENTION: Vaccine Compositions IV
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 981 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 297..800
(D) OTHER INFORMATION: /product= "Pfr protein"
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Frazier, B.A.
Pfeifer, J.D. Russell, D.G. Fal , P. Olsen, A.N. Hammar , M . Westblom, T.U. Normark, S.J
(B) TITLE: Paracrystalline Inclusions of a Novel ferritin Containing Nonheme Iron, Produced by the Human Gastric Pathogen Helicobacter pylori : Evidence for a Third Class of Ferritins
(C) JOURNAL: J. Bacteriol .
(D) VOLUME: 175
(E) ISSUE: 4
(F) PAGES: 966-972
(G) DATE: 1993 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1:
CTTTCAAAGC ATGTAATGCC TTCATTTAAG TAACATGTAG CATTTAGATG AGATGCTTTT 60
ATATATTATA TAAAAATATC CCTTTTAATC CCCCCCTATT GATACCAGCC CCCTTTTTTT 120
GACCTAATTC TCATTAAAAG CACTTTTATG ATAAAATCTA AGCTTTATCA AGCCATTAGC 180
TGGCGTTCTT TCTCATTTTT TGCAAGTTTT TAAAAATTTT CATACTCTTG TTTACTTTTT 240
CATTATCATT TATGCTATAA TTATGGGACA ACTTAAACCA ACACAAAGGA GATACT 296
ATG TTA TCA AAA GAC ATC ATT AAG TTG CTA AAC GAA CAA GTG AAT AAG 344 Met Leu Ser Lys Asp lie lie Lys Leu Leu Asn Glu Gin Val Asn Lys 1 5 10 15
GAA ATG AAC TCT TCC AAC TTG TAT ATG AGC ATG AGT TCT TGG TGC TAT 392 Glu Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr 20 25 30
ACC CAT AGC TTA GAC GGC TCG GGG CTT TTC TTG TTT GAC CAT GCG GCT 440 Thr His Ser Leu Asp Gly Ser Gly Leu Phe Leu Phe Asp His Ala Ala 35 40 45
GAA GAA TAC GAG CAT GCT AAA AAG CTT ATC GTC TTC TTG AAT GAA AAC 488 Glu Glu Tyr Glu His Ala Lys Lys Leu lie Val Phe Leu Asn Glu Asn 50 55 60
AAT GTG CCT GTG CAA TTG ACT AGC ATC AGC GCG CCT GAG CAT AAG TTT 536 Asn Val Pro Val Gin Leu Thr Ser lie Ser Ala Pro Glu His Lys Phe 65 70 75 80
GAA GGT TTG ACT CAA ATT TTC CAA AAA GCC TAT GAA CAT GAG CAA CAC 584 Glu Gly Leu Thr Gin lie Phe Gin Lys Ala Tyr Glu His Glu Gin His 85 90 95
ATC AGC GAG TCT ATT AAT AAT ATC GTC GAT CAC GCC ATA AAA GGC AAA 632 lie Ser Glu Ser lie Asn Asn lie Val Asp His Ala lie Lys Gly Lys 100 105 110
GAT CAT GCG ACT TTC AAT TTC TTG CAA TGG TAT GTG TCT GAA CAG CAT 680 Asp His Ala Thr Phe Asn Phe Leu Gin Trp Tyr Val Ser Glu Gin His 115 120 125
GAA GAA GAA GTG CTT TTC AAG GAT ATT TTG GAT AAA ATT GAG TTG ATT 728 Glu Glu Glu Val Leu Phe Lys Asp lie Leu Asp Lys lie Glu Leu lie 130 135 140
GGT AAT GAA AAC CAT GGC TTG TAT TTG GCT GAT CAG TAT GTC AAA GGG 776 Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gin Tyr Val Lys Gly 145 150 155 160
ATC GCT AAA AGC AGG AAA TCT TAA TTTTAGGGTC ATTGAGTGCA AAAACTAGCC 830 lie Ala Lys Ser Arg Lys Ser * 165
GTTTTTGATT TTGACTCCAC GCTAGTCAAT GCTGAGACGA TTGAGTCTTT AGCGAGGGCG 890
TGGGGGTGTT TGATGAAGGT GAAACAATCA CTTCACAAGC CATGAATGCA GACAGATTTC 950
ATAAAGTCTA TTGAGGTTCA CTAACGTCCG T 981
( 2 ) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 168 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Ser Lys Asp lie lie Lys Leu Leu Asn Glu Gin Val Asn Lys 1 5 10 15
Glu Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr 20 25 30
Thr His Ser Leu Asp Gly Ser Gly Leu Phe Leu Phe Asp His Ala Ala 35 40 45
Glu Glu Tyr Glu His Ala Lys Lys Leu lie Val Phe Leu Asn Glu Asn 50 55 60
Asn Val Pro Val Gin Leu Thr Ser lie Ser Ala Pro Glu His Lys Phe 65 70 75 80
Glu Gly Leu Thr Gin lie Phe Gin Lys Ala Tyr Glu His Glu Gin His 85 90 95 lie Ser Glu Ser lie Asn Asn lie Val Asp His Ala lie Lys Gly Lys 100 105 110
Asp His Ala Thr Phe Asn Phe Leu Gin Trp Tyr Val Ser Glu Gin His 115 120 125
Glu Glu Glu Val Leu Phe Lys Asp lie Leu Asp Lys lie Glu Leu lie 130 135 140
Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gin Tyr Val Lys Gly 145 150 155 160 lie Ala Lys Ser Arg Lys Ser * 165
(2) INFORMATION FOR SEQ ID NO : 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3: AGGAGATCAT ATGTTATCAA AAGAC 25
(2) INFORMATION FOR SEQ ID NO : 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGAATTCAAG ATTTCCTGCT TTTAG 25

Claims

1. A Helicobacter pylori Pfr polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, for use in inducing a protective immune response to Helicobacter pylori infection.
2. A Helicobacter pylori Pfr polypeptide according to claim 1 which has substantially the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing, for use in inducing a protective immune response to Helicobacter pylori infection.
3. A vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori Pfr polypeptide as defined in claim 1 or 2, optionally together with a pharmaceutically acceptable carrier or diluent.
4. A vaccine composition according to claim 3 in addition comprising an adjuvant.
5. A vaccine composition according to claim 4 wherein the adjuvant is a pharmaceutically acceptable form of cholera toxin.
6. A vaccine composition according to any one of claims 3 to 5 for use as a therapeutic vaccine in a mammal, including man, which is infected by Helicobacter pylori.
7. A vaccine composition according to any one of claims 3 to 5 for use as a prophylactic vaccine to protect a mammal, including man, from infection by Helicobacter pylori.
8. Use of a Helicobacter pylori Pfr polypeptide as defined in claim 1 or 2 in the manufacture of a composition for the treatment, prophylaxis or diagnosis of Helicobacter pylori infection.
9. Use of a Helicobacter pylori Pfr polypeptide as defined in claim 1 or 2 in the manufacture of a vaccine for use in eliciting a protective immune response against Helicobacter pylori.
10. Use of a Helicobacter pylori Pfr polypeptide as defined in claim 1 or 2 in the manufacture of a diagnostic kit for diagnosis of Helicobacter pylori infection.
11. A method of in vitro diagnosis of Helicobacter pylori infection comprising at least one step wherein a Helicobacter pylori Pfr polypeptide as defined in claim 1 or 2, optionally labelled or coupled to a solid support, is used.
12. A method according to claim 11 comprising the steps
(a) contacting a said Helicobacter pylori Pfr polypeptide, optionally bound to a solid support, with a body fluid taken from a mammal; and
(b) detecting antibodies from the said body fluid binding to the said Pfr polypeptide.
13. A diagnostic kit for the detection of Helicobacter pylori infection in a mammal, including man, comprising components which enable the method according to claim 11 or 12 to be carried out.
14. A diagnostic kit according to claim 13, comprising:
(a) a Helicobacter pylori Pfr polypeptide; and
(b) reagents for detecting antibodies binding to the said Pfr polypeptide.
15. A method of eliciting in a mammal a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a Helicobacter pylori Pfr polypeptide as defined in claim 1 or 2.
16. A method of eliciting in a mammal a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a vaccine composition according to any one of claims 3 to 7.
17. A method according to claim 15 or 16 wherein the said mammal is a human.
PCT/SE1998/001092 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI Pfr POLYPEPTIDE WO1998056815A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU80486/98A AU8048698A (en) 1997-06-12 1998-06-08 Vaccine compositions comprising the (helicobacter pylori) pfr polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9702241-2 1997-06-12
SE9702241A SE9702241D0 (en) 1997-06-12 1997-06-12 Vaccine compositions IV

Publications (1)

Publication Number Publication Date
WO1998056815A1 true WO1998056815A1 (en) 1998-12-17

Family

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Country Link
AU (1) AU8048698A (en)
SE (1) SE9702241D0 (en)
WO (1) WO1998056815A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6617116B2 (en) 2000-01-28 2003-09-09 Genelabs Diagnostics Pte. Ltd. Assay devices and methods of analyte detection

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038475A1 (en) * 1995-06-01 1996-12-05 Astra Aktiebolag Helicobacter pylori antigens and vaccine compositions

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1996038475A1 (en) * 1995-06-01 1996-12-05 Astra Aktiebolag Helicobacter pylori antigens and vaccine compositions

Non-Patent Citations (2)

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Title
JOURNAL OF BACTERIOLOGY, Volume 174, No. 8, April 1992, PETER DOIG et al., "Production of a Conserved Adhesin by the Human Gastroduodenal Pathogen Helicobacter Pylori", pages 2539-2547. *
JOURNAL OF BACTERIOLOGY, Volume 175, No. 4, 1993, B.A. FRAZIER et al., "Paracrystalline Inclusions of a Novel Ferritin Containing Nonheme Iron, Produced by the Human Gastric Pathogen Helicobacter Pylori: Evidence for a Third Class of Ferritins", pages 966-972. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6617116B2 (en) 2000-01-28 2003-09-09 Genelabs Diagnostics Pte. Ltd. Assay devices and methods of analyte detection
US6849414B2 (en) 2000-01-28 2005-02-01 Genelabs Diagnostics Pte Ltd. Assay devices and methods of analyte detection

Also Published As

Publication number Publication date
AU8048698A (en) 1998-12-30
SE9702241D0 (en) 1997-06-12

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