WO1998056412A1 - VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI 26kDa POLYPEPTIDE - Google Patents

VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI 26kDa POLYPEPTIDE Download PDF

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WO1998056412A1
WO1998056412A1 PCT/SE1998/001091 SE9801091W WO9856412A1 WO 1998056412 A1 WO1998056412 A1 WO 1998056412A1 SE 9801091 W SE9801091 W SE 9801091W WO 9856412 A1 WO9856412 A1 WO 9856412A1
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helicobacter pylori
pylori
kda polypeptide
kda
polypeptide
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PCT/SE1998/001091
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French (fr)
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Thomas Berglindh
Björn MELLGÅRD
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Astra Aktiebolag
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Priority to AU79457/98A priority Critical patent/AU7945798A/en
Publication of WO1998056412A1 publication Critical patent/WO1998056412A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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 ⁇ ylori infection.
  • the invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment or prophylaxis of Helicobacter ⁇ ylori 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 for a protein to work as scavenger against oxygen free radicals. This property has been ascribed to the 26 kDa Helicobacter pylori polypeptide (O'Toole et al, Journal of Bacteriology, 173(2), 505-513, 1991).
  • H. pylori 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).
  • 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; faecal-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.
  • a 26 kDa polypeptide has been identified as an intracellular, cytosolic, yet surface associated protein of H. pylori by O'Toole et al. (Journal of Bacteriology 173(2), 505- 513, 1991). This confusing localization relates to the findings that 26 kDa polypeptide can be extracted from whole cells with very mild methods. However, fractionation of the cells reveal that the 26 kDa polypeptide is associated with the supernatant and not the particulate fraction. It is thus possible that 26 kDa polypeptide belongs to a group of H. pylori proteins, which either can leak out from the cells or which can be taken up from lysed dead cells. The latter process has been termed altruistic lysis (Phadnis et al. Infection and Immunity, 64(3) 905- 912, 1996.) Helicobacter seems to have a unique ability to bind/adsorb protein to its surface.
  • Fig. 1 Therapeutic immunization of Balb/c mice infected with H. pylori strain 244
  • H.pylori status in gastric mucosa i.e. 3 and 2 animals respectively were free of H. pylori in corpus and antrum. In 1/10 animals the H. pylori infection was totally eradicated. * p ⁇ 0.05; (Wilcoxon-Mann-Whittney sign rank test) Fig. 2:
  • H. pylori strain 244 or the H. pylori 26 kDa polypeptide (ELISA antigen: A and B - strain 244; C - the 26 kDa polypeptide).
  • ELISA antigen A and B - strain 244; C - the 26 kDa polypeptide.
  • Gastric and duodenal mucosal Ig and IgA antibodies against membrane proteins of H. pylori were found in the infected animals (PBS and CT).
  • Ig duodenum IgA gastric; and IgA duodenum.
  • group B Ig gastric; Ig duodenum; IgA gastric; and IgA duodenum.
  • group C Ig duodenum; and
  • 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 gene coding for the 26 kDa protein as disclosed by O'Toole et al. (Journal of Bacteriology 173(2), 505-513, 1991).
  • the gene coding for the 26 kDa polypeptide is expressed by all H. pylori strains tested (O'Toole et al., supra).
  • the recombinant H. pylori 26 kDa 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 26 kDa 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.
  • monoclonal antibodies directed against the native 26 kDa polypeptide was shown to partially prevent H. pylori infection in mice.
  • the H. pylori 26 kDa polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections.
  • the 26 kDa 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 vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori 26 kDa polypeptide.
  • the term "protective immune response” is to be understood as an immune response which makes the composition suitable for therapeutic and /or prophylactic purposes.
  • Helicobacter pylori 26 kDa polypeptide is intended to mean a polypeptide disclosed by O'Toole et al, Journal of Bacteriology 173(2), 505-513, 1991, and which is encoded by the gene whose nucleotide sequence is set forth as SEQ ID NO: 1, or can be obtained from the National Center for Biotechnology Information (Accession number M55507), or a substantially similar modified form thereof retaining functionally equivalent antigenicity.
  • 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 26 kDa polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies.
  • an immunologically effective amount is to be understoood 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.
  • 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 provides the use of the Helicobacter pylori 26 kDa polypeptide in the manufacture of a composition for the treatment or prophylaxis of Helicobacter pylori infection, and in the manufacture of a vaccine for use in eliciting a protective immune response against Helicobacter pylori.
  • the invention provides a method of eliciting in a mammal, including man, 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 as defined above.
  • the Helicobacter pylori 26 kDa polypeptide 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 26 kDa 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 26 kDa polypeptide and is retaining functionally equivalent antigenicity.
  • Helicobacter pylori 26 kDa 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.
  • 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-EcoKl site of pET-28 are fused to a DNA sequence encoding a hexa His-Tag (six histidine
  • 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 M Cl 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
  • 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 UV irradiation. DNA 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, DNA inserts were cloned according to standard procedures into the previously digested pET-28b expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli as described below.
  • 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 nm of 0.5 to 1.0 O.D. units, at which point, 1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
  • 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, O.H., Rosebrough, N., Farr, A.L. & Randall, R.J. (1951) , 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).
  • 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 280 and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant proteins eluted at 100 mM imidazole.
  • the 26 kDa polypeptide eluted as a sharp peak at 100 mM NaCl.
  • Fractions containing the recombinant protein were pooled and dialyzed against Tris Buffered Saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) with 0.1 % Deoxycholate (DOC).
  • Recombinant 26 kDa polypeptide was subjected to electrophoresis by SDS- PAGE and visualized by Coomassie blue staining. The recombinant protein was determined to be greater than 95% pure.
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding centre
  • H.p. 26 kDa polypeptide was purified according to O'Toole et al. ( j. Bacteriology 173(2), 505- 513, 1991). Production of monoclonal antibodies against H.p. 26 kDa polypeptide was done by standard procedures. Briefly, purified H.p. 26 kDa polypeptide 5-10 ⁇ l, was injected i.p. and i.v. in Balb/c mice with and without Freund's complete adjuvance 5 times during 109 days. Spleen cells are prepared and fused with myeloma cells by standard procedures.
  • the resulting hybrids were analysed by ELISA as Described (Lopez- Vidal et al., J Clin Microbiol. 26, 1967-1972; 1988) using purified 26 kDa polypeptide for coating.
  • the antibody producing hybridomas having the highest ELISA titers were cloned and expanded. Culture fluids from established hybridomas were harvested and frozen at -20°C and the correponding antibody producing cells were frozen in liquid nitrogen for long term storage.
  • the monoclonal anti 26 kDa polypeptide antibody used in the ex vivo neutralization studies was denoted HP26:26.
  • 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% CO2, 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 -10 8 CFU/animal). Infection was checked in control animals 2-3 weeks after the inoculation.
  • mice 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 26 kDa polypeptide dissolved in PBS was given at a dose of 100 ⁇ g/mouse to group 3.
  • mice in groups 2 and 3 were also given 10 ⁇ g/mouse of cholera toxin (CT) with each immunization.
  • 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 1 300 ⁇ l PBS
  • Group 2 300 ⁇ l PBS containing 10 ⁇ g CT
  • Group 3 300 ⁇ l PBS containing 100 ⁇ g 26 kDa polypeptide 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 kept 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 26 kDa 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 Ig antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with 26 kDa 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 immunization. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant 26 kDa polypeptide. Control animals received only vehicle (phosphate-buffered saline). Two weeks after the final immunization, the animals were sacrificed and CFU (colony-forming units) was determined.
  • CT cholera toxin
  • Fig. 2 shows the serum antibody response to the infecting H. pylori strain (strain 244 as membrane proteins) 11 weeks after infection, and the response to 26 kDa polypeptide after 4 immunizations during 6 weeks. All animals had serum antibodies to the infecting strain (244) as measured by ELISA. This response was significantly increased in the 26 kDa + CT treated group (£; p ⁇ 0.02 against PBS group; $; p ⁇ 0.03 against CT group). Only in animals given 26 kDa + CT, a serum IgG titer against the 26 kDa polypeptide could be detected.
  • the objective of this study was to investigate whether specific monoclonal antibodies against the 26 kDa polypeptides could bind or interfere with H. pylori in such a way that oral inoculation with such a mixture would decrease or prevent infection.
  • mice 10 animal/group
  • One group was challenged with a mixture of freshly grown H. pylori, strain 244, and the monoclonal antibody HP26:26 at approximately 80 ⁇ g/ml. The mixture was incubated 10 minutes at room temperature before inoculation of the animals.
  • H. pylori strain 244 alone.
  • Ten days after challenge the mice were sacrificed and analyzed for presence of gastric H. pylori as described in Example 1.
  • ORGANISM Helicobacter pylori
  • Lys Asn Gly Val lie Leu Phe Phe Trp Pro Lys Asp Phe Thr Phe Val 35 40 45

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Abstract

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 or prophylaxis of Helicobacter pylori infection.

Description

VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI 26kDa POLYPEPΗDE
TECHNICAL FIELD
The present invention relates to vaccine compositions useful for inducing a protective immune response to Helicobacter γylori infection. The invention furthermore relates to the use of Helicobacter pylori polypeptides in the manufacture of compositions for the treatment or prophylaxis of Helicobacter γylori 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 for a protein to work as scavenger against oxygen free radicals. This property has been ascribed to the 26 kDa Helicobacter pylori polypeptide (O'Toole et al, Journal of Bacteriology, 173(2), 505-513, 1991).
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. If such proteins in addition to being surface associated also are essential for survival and /or colonization their usefulness as a target for vaccine mediated immunotherapy targets increase.
Whenever stressed or threatened, the H. pylori cell transforms from a bacillary to a coccoid form. In the coccoid form, the H. pylori cell is much less sensitive to 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; faecal-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 26 kDa polypeptide (O'Toole) ofH. pylori
A 26 kDa polypeptide has been identified as an intracellular, cytosolic, yet surface associated protein of H. pylori by O'Toole et al. (Journal of Bacteriology 173(2), 505- 513, 1991). This confusing localization relates to the findings that 26 kDa polypeptide can be extracted from whole cells with very mild methods. However, fractionation of the cells reveal that the 26 kDa polypeptide is associated with the supernatant and not the particulate fraction. It is thus possible that 26 kDa polypeptide belongs to a group of H. pylori proteins, which either can leak out from the cells or which can be taken up from lysed dead cells. The latter process has been termed altruistic lysis (Phadnis et al. Infection and Immunity, 64(3) 905- 912, 1996.) Helicobacter seems to have a unique ability to bind/adsorb protein to its surface.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1: Therapeutic immunization of Balb/c mice infected with H. pylori strain 244
(n=10/group). Results are given as the geometrical mean of CFU (colony-forming units) in corpus (shaded bars) and in antrum (unshaded bars). Abbreviations: A=PBS, phosphate buffered saline; B=CT, cholera toxin; C=recombinant H. pylori 26 kDa polypeptide. The asterisk (*) indicates p<0.05. All animals in PBS and CT control groups were infected in antrum and corpus mucosa. The 26 kDa polypeptide + CT treated group had significantly lower CFU than the PBS or the CT group in the antrum mucosa . Numbers denote H.pylori status in gastric mucosa, i.e. 3 and 2 animals respectively were free of H. pylori in corpus and antrum. In 1/10 animals the H. pylori infection was totally eradicated. * p<0.05; (Wilcoxon-Mann-Whittney sign rank test) Fig. 2:
IgG concentration in serum against H. pylori strain 244 membrane proteins (unshaded bars) and the H. pylori 26 kDa polypeptide (shaded bar)(mean ± SEM). A, B and C as for fig. 1 and n=10 for all groups. All animals had serum antibodies to the infecting strain (244) as measured by ELISA. This response was significantly increased in the 26 kDa + CT treated group (£; p< 0.02 against PBS group; $; p<0.03 against CT group). Thus immunization with 26 kDa apparently increased the general immune response towards H. pylori. Only 26 kDa immunized animals had systemic antibodies to this protein.
Fig. 3:
Mucosal total Ig and IgA from gastric and duodenal mucosa. A and B as for fig. 1;
C=26 kDa protein. Antibodies were against H. pylori strain 244 or the H. pylori 26 kDa polypeptide (ELISA antigen: A and B - strain 244; C - the 26 kDa polypeptide). Gastric and duodenal mucosal Ig and IgA antibodies against membrane proteins of H. pylori were found in the infected animals (PBS and CT).
Specific Ig and IgA antibodies against 26 kDa could only be detected in the duodenum. Thus immunization with 26 kDa could induce a specific mucosal immune response. From left to right, the bars represent: for group A - Ig gastric;
Ig duodenum; IgA gastric; and IgA duodenum. For group B - Ig gastric; Ig duodenum; IgA gastric; and IgA duodenum. For group C - Ig duodenum; and
IgA duodenum.
Fig. 4:
"Ex vivo neutralisation of H. pylori by 26 kDa monoclonal antibodies": Colonization of H. pylori in gastric mucosa following oral inoculation of mice with H. pylori with and without the addition of specific monoclonal antibodies against the 26 kDa polypeptide. The results are given as CFU in antrum (unshaded bars) and corpus (shaded bars) (mean ± SEM). The presence of antibodies against the 26 kDa polypeptide inhibited the degree of colonization in the stomach 10 days after inoculation. The decrease was significant in the antrum. (**p<0.01; Wilcoxon- Mann-Whittney sign rank test). A=control; B=26 kDa polypeptide.
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 gene coding for the 26 kDa protein as disclosed by O'Toole et al. (Journal of Bacteriology 173(2), 505-513, 1991). The gene coding for the 26 kDa polypeptide is expressed by all H. pylori strains tested (O'Toole et al., supra).
It has surprisingly been found that the recombinant H. pylori 26 kDa 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 26 kDa 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. In addition, monoclonal antibodies directed against the native 26 kDa polypeptide was shown to partially prevent H. pylori infection in mice.
Taken together, these results strongly support the use of the H. pylori 26 kDa polypeptide in an oral vaccine formulation for the use in humans to treat and prevent H. pylori infections. As such, the 26 kDa 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, the present invention provides a vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount of a Helicobacter pylori 26 kDa polypeptide. 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 "Helicobacter pylori 26 kDa polypeptide" is intended to mean a polypeptide disclosed by O'Toole et al, Journal of Bacteriology 173(2), 505-513, 1991, and which is encoded by the gene whose nucleotide sequence is set forth as SEQ ID NO: 1, or can be obtained from the National Center for Biotechnology Information (Accession number M55507), or a substantially similar modified form thereof retaining functionally equivalent antigenicity.
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 26 kDa polypeptide retaining functionally equivalent antigenicity, by use of known methods, such as epitope mapping with in vivo induced antibodies.
In the present context the term "immunologically effective amount" is to be understoood 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. 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 provides the use of the Helicobacter pylori 26 kDa polypeptide in the manufacture of a composition for the treatment or prophylaxis of Helicobacter pylori infection, and in the manufacture of a vaccine for use in eliciting a protective immune response against Helicobacter pylori.
In yet another aspect, the invention provides a method of eliciting in a mammal, including man, 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 as defined above.
In preferred forms of the above aspects of the invention, the Helicobacter pylori 26 kDa polypeptide 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 26 kDa 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 26 kDa polypeptide and is retaining functionally equivalent antigenicity. Included in the definition of the Helicobacter pylori 26 kDa 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.
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, ]., 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 26 kDa polypeptide
DNA sequence Information
Gene sequence information for the 26 kDa Helicobacter polypeptide was obtained from the National Center for Biotechnology Information (Accession number M55507; 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-EcoKl site of pET-28 are fused to a DNA sequence encoding a hexa His-Tag (six histidine
10 residues) located at the extreme 3'-terminus of the recombinant polypeptide.
Forward primer (SEQ ID NO: 3): 5'-AGG AGT TGC ATA TGT TAG TTA CAA-3' Reverse primer (SEQ ID NO: 4): l s 5'-ATG AAT TCT GTT CAT AAA AAC CCC T-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
20 amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 ng) was introduced into a reaction vial containing 2 mM M Cl2, 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
25 (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 Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler: Denaturation at +94°C for 2 min;
30 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 UV irradiation. DNA 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, DNA inserts were cloned according to standard procedures into the previously digested pET-28b expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli as described below.
Transformation of competent bacteria with recombinant plasmids
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 plasmid 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 nm of 0.5 to 1.0 O.D. units, at which point, 1 mM IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 ml cold 10 mM Tris-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, O.H., Rosebrough, N., Farr, A.L. & Randall, R.J. (1951) , 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
Soluble 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 phenylmethyisulfonyl 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 0J % Brij 35, and centirifuged (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., Dδbeli, 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, 0J 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 26 kDa polypeptide eluted as a sharp peak at 100 mM NaCl. Fractions containing the recombinant protein were pooled and dialyzed against Tris Buffered Saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) with 0.1 % Deoxycholate (DOC). Recombinant 26 kDa polypeptide was subjected to electrophoresis by SDS- PAGE and visualized by Coomassie blue staining. The recombinant protein was determined to be greater than 95% pure.
Production of Helicobacter pylori 26 kDa monoclonal antibodies
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 wee 4-6 weeks old at arrival. Native H.p. 26 kDa polypeptide was purified according to O'Toole et al. ( j. Bacteriology 173(2), 505- 513, 1991). Production of monoclonal antibodies against H.p. 26 kDa polypeptide was done by standard procedures. Briefly, purified H.p. 26 kDa polypeptide 5-10 μl, was injected i.p. and i.v. in Balb/c mice with and without Freund's complete adjuvance 5 times during 109 days. Spleen cells are prepared and fused with myeloma cells by standard procedures.
The resulting hybrids were analysed by ELISA as Described (Lopez- Vidal et al., J Clin Microbiol. 26, 1967-1972; 1988) using purified 26 kDa polypeptide for coating. The antibody producing hybridomas having the highest ELISA titers were cloned and expanded. Culture fluids from established hybridomas were harvested and frozen at -20°C and the correponding antibody producing cells were frozen in liquid nitrogen for long term storage. The monoclonal anti 26 kDa polypeptide antibody used in the ex vivo neutralization studies was denoted HP26:26.
EXAMPLES OF THE INVENTION
EXAMPLE 1: THERAPEUTIC IMMUNIZATION
1.1. Materials & Methods
1.1.1. 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.
1.1.2. 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% CO2, 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 10 -108 CFU/animal). Infection was checked in control animals 2-3 weeks after the inoculation.
1.1.3. 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 26 kDa polypeptide 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 26 kDa polypeptide and 10 μg CT
1.1.4. 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.
1.1.5. 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 kept 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 26 kDa 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 Ig antibodies were measured using an ELISA where plates were coated either with a particulate fraction of H. pylori strain 244 or with 26 kDa polypeptide followed by addition of different dilutions of serum. The ELISA was developed with alkaline phosphatase-labelled anti-mouse-Ig-antibodies as described above.
1.2. Results
1.2.1. Therapeutic immunization: effect on colony -forming units (CFU)
The animals in this study were infected with H. pylori strain 244 one month prior to immunization. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant 26 kDa polypeptide. Control animals received only vehicle (phosphate-buffered saline). Two weeks after the final immunization, the animals were sacrificed and CFU (colony-forming units) was determined.
All control animals, as well as those immunized with only CT, were infected both in corpus and antrum. Animals actively immunized with recombinant 26 kDa polypeptide and CT had decreased CFU values (Fig. 1). In the antrum mucosa this decrease was significant compared to both control and CT treated animals (p<0.05; Wilcoxon-Mann-Whittney sign rank test). Following 26 kDa + CT immunization, 3 animals had no detectable H. pylori infection in corpus, while 2 animals had no detectable infection in antrum. 1 out of 10 animals were totally free of H. pylori.
2.2.2. Therapeutic immunization: effects on antibody formation and secretion
Fig. 2 shows the serum antibody response to the infecting H. pylori strain (strain 244 as membrane proteins) 11 weeks after infection, and the response to 26 kDa polypeptide after 4 immunizations during 6 weeks. All animals had serum antibodies to the infecting strain (244) as measured by ELISA. This response was significantly increased in the 26 kDa + CT treated group (£; p< 0.02 against PBS group; $; p<0.03 against CT group). Only in animals given 26 kDa + CT, a serum IgG titer against the 26 kDa polypeptide could be detected.
Gastric and duodenal mucosal total Ig and IgA anti H. pylori antibodies were found in the infected animals (PBS and CT). Total Ig and IgA antibodies against 26 kDa protein could only be found in duodenum (Fig. 3). Consequently, it was shown that systemic immunological response to H. pylori is enhanced by 26 kDa + CT immunization, and that 26 kDa-specific mucosal antibody response is induced by immunization with 26 kDa + CT.
EXAMPLE 2: EX VIVO NEUTRALIZATION
2.1. Objective
The objective of this study was to investigate whether specific monoclonal antibodies against the 26 kDa polypeptides could bind or interfere with H. pylori in such a way that oral inoculation with such a mixture would decrease or prevent infection.
Two groups of mice, 10 animal/group, were used in this experiment. One group was challenged with a mixture of freshly grown H. pylori, strain 244, and the monoclonal antibody HP26:26 at approximately 80 μg/ml. The mixture was incubated 10 minutes at room temperature before inoculation of the animals. As a control, one group was inoculate with H. pylori strain 244 alone. Ten days after challenge the mice were sacrificed and analyzed for presence of gastric H. pylori as described in Example 1.
2.2. Results All control animals were well infected. In animals given H. pylori together with a specific monoclonal antibody against the 26 kDa polypeptide, the degree of colonization was decreased in both antrum and corpus, and significantly decreased (** p<0.01; Wilcoxon-Mann-Whittney sign rank test) in the antrum (Fig. 4)-
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 III
(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: Patentln Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 601 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
(E) LOCATION: 8..601
(D) OTHER INFORMATION: /product= "26 kDa protein"
(x) PUBLICATION INFORMATION:
(A) AUTHORS: O'Toole, Paul J.
Logan, Susan M. Kostrzynska, Magdalena Wadstrδm, Torkel Trust, Trevor J.
(B) TITLE: Isolation and Biochemical and Molecular
Analyses of a Species-Specific Protein Antigen from the Gastric Pathogen Helicobacter pylori
(C) JOURNAL: J. Bacteriol .
(D) VOLUME: 173
(E) ISSUE: 2
(F) PAGES: 505-513
(G) DATE: 1991
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATAGAAG ATG TTA GTT ACA AAA CTT GCC CCC GAT TTT AAA GCG CCT GCC 49 Met Leu Val Thr Lys Leu Ala Pro Asp Phe Lys Ala Pro Ala 1 5 10
GTT TTA GGA AAC AAT GAG GTG GAT GAA CAC TTT GAG CTT TCT AAA AAT 97 Val Leu Gly Asn Asn Glu Val Asp Glu His Phe Glu Leu Ser Lys Asn 15 20 25 30
TTA GGC AAA AAT GGT GTG ATC CTT TTC TTT TGG CCA AAA GAT TTT ACT 145 Leu Gly Lys Asn Gly Val lie Leu Phe Phe Trp Pro Lys Asp Phe Thr 35 40 45
TTT GTA TGC CCT ACA GAG ATC ATT GCG TTT GAC AAA AGA GTG AAA GAC 193 Phe Val Cys Pro Thr Glu lie lie Ala Phe Asp Lys Arg Val Lys Asp 50 55 60
TTC CAC GAA AAA GGC TTT AAT GTG ATT GGC GTG TCT ATT GAC AGC GAG 241 Phe His Glu Lys Gly Phe Asn Val lie Gly Val Ser lie Asp Ser Glu 65 70 75
CAA GTG CAT TTC GCA TGG AAA AAC ACC CCT GTG GAA AAA GGC GGT ATC 289 Gin Val His Phe Ala Trp Lys Asn Thr Pro Val Glu Lys Gly Gly lie 80 85 90
GGT CAA GTG TCT TTC CCT ATG GTG GCT GAT ATT ACT AAG AGC ATT TCT 337 Gly Gin Val Ser Phe Pro Met Val Ala Asp lie Thr Lys Ser lie Ser 95 100 105 110
AGA GAC TAT GAT GTG CTG TTT GAA GAA GCG ATC GCT TTG AGA GGT GCT 385 Arg Asp Tyr Asp Val Leu Phe Glu Glu Ala lie Ala Leu Arg Gly Ala 115 120 125
TTT TTG ATT GAC AAA AAC ATG AAA GTA AGA CAT GCA GTG ATC AAT GAC 433 Phe Leu lie Asp Lys Asn Met Lys Val Arg His Ala Val lie Asn Asp 130 135 140
TTG CCA TTA GGT AGG AAT GCA GAT GAA ATG CTT CGC ATG GTA GAC GCT 481 Leu Pro Leu Gly Arg Asn Ala Asp Glu Met Leu Arg Met Val Asp Ala 145 150 155
CTC TTA CAC TTT GAA GAA CAT GGT GAA GTA TGC CCA GCA GGT TGG AGA 529 Leu Leu His Phe Glu Glu His Gly Glu Val Cys Pro Ala Gly Trp Arg 160 165 170
AAA GGC GAT AAA GGG ATG AAA GCA ACC CAC CAA GGC GTT GCA GAA TAT 577 Lys Gly Asp Lys Gly Met Lys Ala Thr His Gin Gly Val Ala Glu Tyr 175 180 185 190
CTT AAA GAA AAT TCC ATT AAG CTT 601
Leu Lys Glu Asn Ser lie Lys Leu 195
(2) INFORMATION FOR SEQ ID NO : 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Val Thr Lys Leu Ala Pro Asp Phe Lys Ala Pro Ala Val Leu 1 5 10 15
Gly Asn Asn Glu Val Asp Glu His Phe Glu Leu Ser Lys Asn Leu Gly 20 25 30
Lys Asn Gly Val lie Leu Phe Phe Trp Pro Lys Asp Phe Thr Phe Val 35 40 45
Cys Pro Thr Glu lie lie Ala Phe Asp Lys Arg Val Lys Asp Phe His 50 55 60
Glu Lys Gly Phe Asn Val lie Gly Val Ser lie Asp Ser Glu Gin Val 65 70 75 80
His Phe Ala Trp Lys Asn Thr Pro Val Glu Lys Gly Gly lie Gly Gin 85 90 95
Val Ser Phe Pro Met Val Ala Asp lie Thr Lys Ser lie Ser Arg Asp 100 105 110
Tyr Asp Val Leu Phe Glu Glu Ala lie Ala Leu Arg Gly Ala Phe Leu 115 120 125 lie ASΌ Lys Asn Met Lys Val Arg His Ala Val lie Asn Asp Leu Pro 130 135 140
Leu Gly Arg Asn Ala Asp Glu Met Leu Arg Met Val Asp Ala Leu Leu 145 150 155 160
His Phe Glu Glu His Gly Glu Val Cys Pro Ala Gly Trp Arg Lys Gly 165 170 175
Asp Lys Gly Met Lys Ala Thr His Gin Gly Val Ala Glu Tyr Leu Lys 180 185 190
Glu Asn Ser lie Lys Leu 195
(2) INFORMATION FOR SEQ ID NO : 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 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: AGGAGTTGCA TATGTTAGTT ACAA 24
(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: ATGAATTCTG TTCATAAAAA CCCCT 25

Claims

1. A vaccine composition for inducing a protective immune response to Helicobacter pylori infection, comprising an immunogenically effective amount
5 of the Helicobacter pylori 26 kDa polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, optionally together with a pharmaceutically acceptable carrier or diluent.
2. A vaccine composition according to claim 1 in addition comprising an o adjuvant.
3. A vaccine composition according to claim 2 wherein the adjuvant is a pharmaceutically acceptable form of cholera toxin.
s 4. A vaccine composition according to any one of claims 1 to 3 for use as a therapeutic vaccine in a mammal, including man, which is infected by Helicobacter pylori.
5. A vaccine composition according to any one of claims 1 to 3 for use as a o prophylactic vaccine to protect a mammal, including man, from infection by
Helicobacter pylori.
6. A vaccine composition according to any one of claims 1 to 5 wherein the said Helicobacter pylori 26 kDa polypeptide has substantially the amino acid 5 sequence set forth as SEQ ID NO: 2 in the Sequence Listing.
7. Use of a Helicobacter pylori 26 kDa polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, in the manufacture of a composition for the treatment or prophylaxis of Helicobacter pylori infection.
8. Use of a Helicobacter pylori 26 kDa polypeptide, or a modified form thereof retaining functionally equivalent antigenicity, in the manufacture of a vaccine for use in eliciting a protective immune response against Helicobacter pylori.
9. The use according to claim 7 or 8 wherein the said Helicobacter pylori 26 kDa polypeptide has substantially the amino acid sequence set forth as SEQ ID
NO: 2 in the Sequence Listing.
10. 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 26 kDa polypeptide, or a modified form thereof retaining functionally equivalent antigenicity,
11. 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 1 to 5.
12. A method according to claim 10 or 11 wherein the said mammal is a human.
13. The method according to claim 10 or 12 wherein the said Helicobacter pylori 26 kDa polypeptide has substantially the amino acid sequence set forth as SEQ ID NO: 2 in the Sequence Listing.
PCT/SE1998/001091 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI 26kDa POLYPEPTIDE WO1998056412A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU79457/98A AU7945798A (en) 1997-06-12 1998-06-08 Vaccine compositions comprising the (helicobacter pylori) 26kda polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9702240A SE9702240D0 (en) 1997-06-12 1997-06-12 Vaccine compositions III
SE9702240-4 1997-06-12

Publications (1)

Publication Number Publication Date
WO1998056412A1 true WO1998056412A1 (en) 1998-12-17

Family

ID=20407349

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1998/001091 WO1998056412A1 (en) 1997-06-12 1998-06-08 VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI 26kDa POLYPEPTIDE

Country Status (3)

Country Link
AU (1) AU7945798A (en)
SE (1) SE9702240D0 (en)
WO (1) WO1998056412A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020843A1 (en) * 1992-04-13 1993-10-28 Steven James Czinn Oral treatment of helicobacter infection
WO1996033220A1 (en) * 1995-04-21 1996-10-24 Csl Limited Protective helicobacter antigens
WO1997037044A1 (en) * 1996-03-29 1997-10-09 Astra Aktiebolag Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020843A1 (en) * 1992-04-13 1993-10-28 Steven James Czinn Oral treatment of helicobacter infection
WO1996033220A1 (en) * 1995-04-21 1996-10-24 Csl Limited Protective helicobacter antigens
WO1997037044A1 (en) * 1996-03-29 1997-10-09 Astra Aktiebolag Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABSTRACT OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY, Vol. 97, 1997, A. LUNDSTROM et al., "B-71. Expression of a 26 kDa Protein Gene in Helicobacter Species", Sid 41. *
DIALOG INFORMATION SERVICES, File 155, Medline, Dialog Accession No. 06690990, Medline Accession No. 91100336, O'TOOLE P.W. et al., "Isolation and Biochemical and Molecular Analyses of a Species-Specific Protein Antigen from the Gastric Pathogen Helicobacter Pylori"; & J. BACTERIOL., Jan. 1991, 173(2), p. 505-13. *

Also Published As

Publication number Publication date
SE9702240D0 (en) 1997-06-12
AU7945798A (en) 1998-12-30

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