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WO1998006853A1 - Traitement et prevention des infections a helicobacter - Google Patents

Traitement et prevention des infections a helicobacter Download PDF

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Publication number
WO1998006853A1
WO1998006853A1 PCT/AU1997/000515 AU9700515W WO9806853A1 WO 1998006853 A1 WO1998006853 A1 WO 1998006853A1 AU 9700515 W AU9700515 W AU 9700515W WO 9806853 A1 WO9806853 A1 WO 9806853A1
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WIPO (PCT)
Prior art keywords
lys
helicobacter
asp
catalase
host
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PCT/AU1997/000515
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English (en)
Inventor
Christopher Vincent Doidge
Adrian Lee
Fiona Jane Radcliff
Stuart Lloyd Hazell
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Csl Limited
The University Of New South Wales
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Publication date
Priority claimed from US08/695,987 external-priority patent/US6005090A/en
Application filed by Csl Limited, The University Of New South Wales filed Critical Csl Limited
Priority to AU37623/97A priority Critical patent/AU3762397A/en
Publication of WO1998006853A1 publication Critical patent/WO1998006853A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • 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

  • This invention relates to protective Helicobacter antigens, especially H. pylori antigens, and to the use of these antigens for the treatment and prevention of gastroduodenal disease associated with H. pylori infection in humans.
  • Helicobacter pylori is a bacterium that infects the stomach lining (or gastric mucosa) of perhaps half the world's population. Spiral organisms were first microscopically observed in human gastric mucosa in 1906. However, H. pylori was not successfully cultured until 1982. Infection with the organism is usually chronic, and results in continuing inflammation of the gastric mucosa. The infection is often asymptomatic. However, in association with other cofactors, a proportion of infected people go on to develop sequelae including peptic ulceration of the stomach or duodenum, gastric adenocarcinomas and gastric lymphomas. Peptic ulcer treatment studies have shown that cure of H.
  • H. pylori infection is associated with a dramatic reduction in the relapse rate of this usually chronic disease.
  • Long term infection with H. pylori leads to the development of chronic atrophic gastritis, which has long been recognised as a precursor lesion in the development of gastric cancer.
  • H. pylori infection With an increased risk of developing gastric cancer. Therefore eradication of current infection and prevention of new infection with this organism has the potential to significantly reduce the incidence of diseases that result in considerable morbidity and mortality 1 2 .
  • H. pylori Infection with H. pylori is difficult to treat. Current experimental therapies for treating the infection have problems with efficacy and significant levels of adverse effects. There are no prophylactic measures available.
  • a solution to both the prevention and treatment of H. pylori infection would be the development of an immunogenic preparation that, as an immunotherapeutic, treated established infections, and as a vaccine, prevented the establishment of new or recurrent infections. Such a preparation would need to induce effective immune responses to protective antigens, while avoiding inducing responses to self antigens or other potentially harmful immune responses. This may be achieved by identifying the specific protective component or components and formulating immunotherapeutic or vaccine preparations including these component(s).
  • H. pylori did not naturally infect laboratory animals.
  • an animal model of human H. pylori infection has been developed using a closely related organism, H. felis, and specific pathogen free (SPF) mice 3 . These organisms are able to colonise the gastric mucosa of SPF mice, where they establish a chronic infection with many of the features of H. pylori infection in humans.
  • H. felis infection in the mice induces a chronic gastritis and a raised immune response. As in the human case, this response is not effective in curing the infection.
  • these cross- reactive antigens were recognised by performing a Western blot using H. pylori disrupted cells as the antigen, and probing the blot with serum from mice immunised with H. felis and cholera toxin adjuvant Sections of membrane containing proteins recognised as cross-reactive were removed from the membrane, the proteins bound to them were eluted, and their N-terminal amino acid sequence determined by microsequencing.
  • N-terminal amino acid sequence of one of the two proteins that successfully yielded sequence data closely matched the previously published sequence of the microbial enzyme, urease 5 .
  • This enzyme has already been shown to be a protective antigen when used in a vaccine to prevent infection.
  • N-terminal amino acid sequence of the other protein closely matched the previously published N-terminal sequence of the microbial enzyme, catalase 6 .
  • This enzyme has not previously been shown to be a protective antigen of H. pylori.
  • the present invention provides an antigenic preparation for use in the treatment or prevention of Helicobacter infection, which comprises an at least partially purified preparation of the catalase of Helicobacter bacteria.
  • the term "at least partially purified” as used herein denotes a preparation i n which the catalase content is greater, preferably at least 30% and more preferably at least 50% greater, than the catalase content of a whole cell sonicate of Helicobacter bacteria.
  • the preparation is one in which the catalase is "substantially pure", that is one in which the catalase content is at least 80%, more preferably at least 90%, of the total Helicobacter antigens in the preparation.
  • the present invention provides an antigenic preparation for use in treatment or prevention of Helicobacter infection, which comprises substantially pure catalase of Helicobacter bacteria.
  • a preparation may be prepared as a recombinant catalase by techniques described hereinafter.
  • the present invention provides an isolated Helicobacter antigen for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises the catalase of Helicobacter bacteria, or an immunogenic fragment thereof.
  • isolated denotes that the antigen has undergcr.e at least one purification or isolation step, and preferably is in a form suitable for e . in a vaccine composition.
  • the present invention extends not only to an antigenic preparation or isolated antigen comprising the catalase of Helicobacter bacteria, but also to antigenic preparations comprising immunogenic fragments of this catalase, that is catalase fragments which are capable of eliciting a specific protective immune response in a mammalian host.
  • immunogenic fragments may also be recognised by He/Zcobacter-specific antibodies, particularly monoclonal antibodies which have a protective or therapeutic effect in relation to Helicobacter infection or polyclonal antibodies contained in immune sera from mammalian hosts which have been vaccinated against Helicobacter infection.
  • the present invention provides a vaccine composition for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an immunological ly effective amount of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, together with one or more pharmaceutically acceptable carriers and/or diluents.
  • the present invention provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of an immunologically effective amount of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant.
  • this invention provides the use of a vaccine composition comprising an immunologically effective amount of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, for the treatment or prevention of Helicobacter infection in a mammalian host.
  • the invention provides the use of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, in the manufacture of a vaccine composition for the treatment or prevention of Helicobacter infection in a mammalian host.
  • an antigenic preparation or isolated antigen of this invention is orally administered to the host, and is administered in association with a mucosal adjuvant.
  • the invention also extends to parenteral administration of this antigenic preparation or isolated antigen.
  • the term “immunologically effective amount” herein in the context of treatment of Helicobacter infection it is meant that the administration of that amount to an individual infected host, either in a single dose or as part of a series, that is effective for treatment of Helicobacter infection.
  • the term “immunologically effective amount” herein in the context of prevention of Helicobacter infection it is meant that the administration of that amount to an individual host, either in a single dose or as part of a series, that is effective to delay, inhibit or prevent Helicobacter infection.
  • the effective amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine- trials.
  • the catalase antigen above comprises an amino acid sequence substantially corresponding to the deduced sequence of the catalase gene from isolate RU1 or isolate 921023 hereinafter (SEQ ID NO.2 or 4), or allelic or other variants thereof.
  • Suitable variants may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to one of the amino acid sequences referred to above, or to a region or part thereof, provided the variant is capable ol eliciting a specific protective immune response in a mammalian host.
  • the present invention extends not only to the particular catalase antigen of Helicobacter bacteria as described above, but also to immunogenic fragments of the particular antigen, that is fragments of the antigen which are capable of eliciting a specific protective immune response in a mammalian host.
  • the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular antigen.
  • Such immunogenic fragments may also be recognised by He/Zcobacter-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to Helicobacter infection.
  • the present invention also extends to an antibody, which may be either a monoclonal or polyclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above.
  • an antibody which may be either a monoclonal or polyclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above.
  • Such antibodies may be produced by methods which are well known to persons skilled in this field.
  • the invention further provides a method for the treatment or prevention oi Helicobacter infection in a mammalian host, which comprises passive immunisation of said host by administration of an effective amount of an antibody, particularly a monoclonal antibody, specific for an antigenic preparation or an isolated
  • the Helicobacter antigenic preparation or isolated antigen of this invention may be prepared by purification or isolation from natural sources, such as a whole cell sonicate of Helicobacter bacteria. Alternatively, however the antigenic preparation or isolated antigen may be prepared by synthetic, preferably recombinant, techniques.
  • the antigen When prepared by recombinant techniques, the antigen may have an amino acid sequence substantially identical to the naturally occurring sequence or may contain one or more amino acid substitutions, deletions and/or additions thereto provided that following such alterations to the sequence, the molecule is still capable of eliciting a specific protective immune response against the naturally occurring Helicobacter antigen. A similar immunogenic requirement is necessary for any fragments or derivatives of the antigen whether made from the recombinant molecule or the naturally occurring molecule.
  • a Helicobacter antigen is considered reference to the naturally occurring molecule, its recombinant form and any mutants, derivatives, fragments, homologues or analogues thereof provided that such molecules elicit a specific protective immune response against the naturally occurring Helicobacter antigen.
  • fusion molecules between two or more Helicobacter antigens or with other molecules including fusion molecules with other molecules such as glutathione-S-transferase (GST) or ⁇ -galactosidase.
  • the present invention also extends to an isolated nucleic acid molecule encoding a Helicobacter catalase antigen and preferably having a nucleotide sequence as set forth in SEQ ID NO. 1 or 3, or being substantially similar to all or a part thereof.
  • substantially similar means having at least 40-50%, more preferably at least 60-70%, and most preferably at least 80% identity.
  • a "part” in this context means a contiguous series of at least 1 5 nucleotides, and more preferably at least 25 nucleotides.
  • nucleic acid molecule comprising a sequence of nucleotides which encodes a Helicobacter catalase antigen and hybridises under low stringency conditions to all or part of a nucleic acid sequence set forth in SEQ ID NO. 1 or 3, or to a complementary form thereof.
  • this invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO. 1 or 3, or a part thereof.
  • the nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form.
  • level of stringency reference can conveniently be made to Sambrook et ⁇ l. (1989) at pp 387-389 which is herein incorporated by reference where the washing step at paragraph 1 1 is considered high stringency.
  • a low stringency is defined herein as being in 0.1-0.5 w/v SDS at 37-45°C for 2-3 hours.
  • conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at ⁇ 45 °C for 2-3 hours or high stringent conditions as disclosed by Sambrook et al. (1989).
  • sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturally-occurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a Helicobacter antigen as broadly described above.
  • the nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences usually being derived from a heterologous source.
  • This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter sequences and initiator sequences and a nucleotide sequence which codes for a Helicobacter catalase antigen, the nucleotide sequence being located 3' to the promoter and initiator sequences.
  • the invention provides a recombinant DNA cloning vehicle capable of expressing a Helicobacter catalase antigen comprising an expression control sequence having promoter sequences and initiator sequences, and a nucleotide sequence which codes for a Helicobacter catalase antigen, the nucleotide sequence being located 3' to the promoter and initiator sequences.
  • a host cell containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.
  • fused polypeptides comprising a Helicobacter catalase antigen of this invention and an additional polypeptide, for example a polypeptide coded for by the DNA of a cloning vehicle, fused thereto.
  • fused polypeptide can be produced by a host cell transformed or infected with a recombinant DNA cloning vehicle as described above, and it can be subsequently isolated from the host cell to provide the fused polypeptide substantially free of other host cell proteins.
  • the present invention also extends to synthetic polypeptides displaying the antigenicity of a Helicobacter catalase antigen of this invention.
  • synthetic means that the polypeptides have been produced by chemical or biological means, such as by means of chemical synthesis or by recombinant DNA techniques leading to biological synthesis.
  • Such polypeptides can, of course, be obtained by cleavage of a fused polypeptide as described above and separation of the desired polypeptide from the additional polypeptide coded for by the DNA of the cloning vehicle by methods well known in the art.
  • the polypeptide may be produced synthetically, for example by the well-known Merrifield solid-phase synthesis procedure.
  • Helicobacter catalase antigen of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.
  • Isolated polypeptides comprising, or containing in part, amino acid sequences corresponding to a Helicobacter catalase antigen may be used to raise polyclonal antisera by immunising rabbits, mice or other animals using well established procedures.
  • polypeptides may be used in the preparation of monoclonal antibodies by techniques well known in the art.
  • polypeptides in accordance with this invention including fused polypeptides may be used as an active immunogen in the preparation of single or multivalent vaccines by methods well known in the art of vaccine manufacture for use in the treatment or prevention of Helicobacter infection in a mammalian host.
  • polypeptides in accordance with the present invention including fused polypeptides may be used as antigen in a diagnostic immunoassay for detection of antibodies to Helicobacter in a sample, for example, a serum sample from a human or other mammalian patient.
  • immunoassays are well known in the art, and include assays such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).
  • the present invention also extends to delivery to the host using a vector expressing the catalase of Helicobacter bacteria, or an immunogenic fragment thereof. Accordingly, in a further aspect this invention provides a preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof.
  • the invention extends to a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof.
  • the invention extends to the use of a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof, for the treatment or prevention of Helicobacter infection in a mammalian host.
  • a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof for the treatment or prevention of Helicobacter infection in a mammalian host.
  • the antigenic preparation or isolated antigen of this invention comprises the catalase of H. pylori or H. felis, most preferably H. pylori catalase.
  • this antigenic preparation or isolated antigen is used in a vaccine composition for oral administration which includes a mucosal adjuvant.
  • an oral vaccine composition comprising substantially pure H. pylori catalase, more preferably recombinant H. pylori catalase, in association with a mucosal adjuvant is used for the treatment or prevention of H. pylori infection in a human host.
  • the mucosal adjuvant which is optionally, and preferably, administered with the Helicobacter catalase preparation or antigen to the infected host is preferably cholera toxin.
  • Mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivatives of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. These may be added to, or conjugated with, the Helicobacter catalase preparation or antigen.
  • CTB B sub-unit
  • the same techniques can be applied to other molecules with mucosal adjuvant or deliver/ properties such as Escherichia coli heat labile toxin.
  • mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE- dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces.
  • Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine.
  • the Helicobacter catalase preparation or antigen may be delivered in accordance with this invention in ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells.
  • ISCOMS immune stimulating complexes
  • ISCOMS containing CTB liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells.
  • micro or nanoparticles may be covalently attached to molecules such as vitamin B12 which have specific gut receptors.
  • the Helicobacter catalase preparation or antigen may also be incorporated into oily emulsions and delivered orally. An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter 7 .
  • the vaccine composition may, for example, be formulated in enteric coated gelatine capsules including sodium bicarbonate buffers together with the Helicobacter catalase preparation or antigen and cholera toxin mucosal adjuvant.
  • Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the catalase or an immunogenic fragment thereof may be delivered to the host using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the catalase or immunogenic fragment as a foreign antigen.
  • a live vaccine vector in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the catalase or immunogenic fragment as a foreign antigen.
  • bacteria that colonise the gastrointestinal tract such as Salmonella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are discussed by Holmgren et al. 6 and McGhee et al. 9 .
  • the Helicobacter catalase preparation or antigen of the present invention may be administered as the sole active immunogen in a vaccine composition or expressed by a live vector.
  • the vaccine composition may include or the live vector may express other active immunogens, including other Helicobacter antigens such as urease or the lipopolysaccharide (LPS) of Helicobacter bacteria (see International Patent Application No. PCT/AU95/00077), as well as immunologically active antigens against other pathogenic species.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent.
  • the specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
  • H. pylon catalase is recognised by the serum of mice vaccinated with an H. felis antigen preparation (plus cholera toxin adjuvant). These mice can be shown to be protected against H. felis infection.
  • This data indicates the use of H. pylori catalase as a protective antigen in human H. pylori infection, and purified or recombinant catalase may be used as an antigenic component of a therapeutic or prophylactic vaccine, either on its own, or in combination with other antigens, carriers, adjuvants, delivery vehicles or excipients.
  • Sonicated H. pylori cells (strain HP 921023) were separated in a 12% discontinuous (i.e. homogeneous) SDS-PAGE gel under denaturing conditions using a Mini-Protean II apparatus (Bio-Rad). Proteins were transferred from the gel to ProBlott (Applied Biosciences PVDF-polyvinylidene difluoride) membrane using CAPS buffer (3-(cyclohexylamino)-1-proanesulphonic acid buffer) in a Mini transblot system (Bio-Rad).
  • ProBlott Applied Biosciences PVDF-polyvinylidene difluoride
  • CAPS buffer 3-(cyclohexylamino)-1-proanesulphonic acid buffer
  • PVDF strips were removed from the ends of the PVDF and reacted with immune sera from mice vaccinated with H. felis plus cholera toxin and traced with an HRP labelled anti-mouse sera and developed using 4-chloro-1-naphthol as per standard Western blot methods.
  • the remainder of the PVDF was stained with Coomassie blue (Bio-Rad) to visualise the protein bands.
  • Coomassie blue Bio-Rad
  • Six proteins recognised by the immune sera were selected and the corresponding Coomassie stained bands on the PVDF were carefully excised for sequencing.
  • the six excised bands of PVDF were cut into small pieces (approx.0.5 cm long) and placed into the reaction cartridge of an Applied Biosystems Model 476A Protein Sequencer System. All chemistry, HPLC separations, data quantitation and protein sequencing reporting is automatically carried out in this system.
  • sample 3 corresponds closely, but not exactly, with the previously published N-terminal sequence for the enzyme urease 5 .
  • This enzyme has been shown to be a protective antigen in studies using the H. felis/ mouse model.
  • the sequence data of sample 5 corresponds closely, with one difference, to the previously published N-terminal sequence of the enzyme catalase 6 .
  • This enzyme has not previously been shown to be a protective antigen however the fact that the enzyme is recognised by the immune serum of mice vaccinated with an H. felis antigen preparation to protect against H. felis infection, combined with the fact that mice vaccinated with an H. pylori antigen preparation are protected against H. felis infection, indicates the H. pylori catalase as a protective antigen in H. pylori infection in humans.
  • H. pylori (clinical strain 921023) were grown in 10% C0 2 at 37°C for 48 hours. All following steps until loading on the column were undertaken on ice.
  • the H. pylori cells were harvested in 0.1 M sodium phosphate buffer pH 7.2 and the suspension spun down gently and resuspended in no more than 5 mL of 0.1 M sodium phosphate buffer. The suspension was then sonicated at 6 kHz 40% duty cycle for 5 minutes. Following this, the sonicate was spun for 5 minutes at 10,000 g, the supernatant collected and passed through a 0.22 ⁇ m filter into a sterile container.
  • the filtrate was loaded onto a K26/100 gel filtration column of Sephacryl S-300 HR and eluted using sodium phosphate buffer at a flow rate of 1.0 mL min " ⁇
  • the eluate was collected into fractions (100 drops/fraction) and those containing catalase identified by testing for catalase activity (1 drop of the fraction placed in H 2 0 2 diluted 1 :10 in distilled water and examined for bubbling).
  • Fractions containing the strongest catalase activity were pooled then diluted 1 :10 in 0.01 M sodium phosphate (filtered).
  • the fractions were then run through a MEMSEP 1000 cm ion exchange capsule. 100 mL of the 0.01 M sodium phosphate buffer was then run through the ion exchange capsule to remove any excess proteins.
  • Catalase positive fractions were identified by their strong yellow colour and confirmed by testing for a bubbling reaction in H 2 0 2 .
  • the catalase positive fractions were stored at 4°C and protected from light. Each fraction was tested for protein concentration using the Bio-Rad DC protein assay, and selected for immunising mice if it contained over 1.5 mg/mL of protein. Prior to immunising mice the purified catalase was checked for contaminants using 12% SDS- PAGE. Proteins were visualised by staining with Coomassie Blue, which indicated that the catalase preparation was at least 95% pure. Image analysis indicated that the catalase's molecular weight was 52-53 kDa. The purified catalase was also strongly recognised by a catalase monoclonal antibody.
  • mice Sufficient purified catalase for immunising 10 mice was obtained and pooled. Mice were given 0.2 mg purified catalase + 10 ⁇ g cholera toxin (CT) 4 times on days 0, 7, 14 and 21. Control groups were given cholera toxin alone or PBS buffer alone. The dose size was 150 ⁇ for all groups. On the day of each immunising dose, the catalase was checked for activity using 2 and for any signs of degradation using SDS- PAGE and Coomassie Blue staining. No signs of declining activity or any degradation was observed throughout the immunisation course. Three weeks after the last immunising dose all groups were challenged twice with ⁇ 10 8 H. felis. Three weeks later mice were euthanased and samples (sera, saliva, bile and the stomach - half for histology and half the antrum for the direct urease test) were collected.
  • CT cholera toxin
  • the catalase gene has been cloned from each of two different isolates of H. pylori, isolate RU1 and isolate 921023.
  • Helicobacter pylori strain HP921023 was used as the DNA donor for preparing the gene library.
  • Escherichia coli strain ER1793 New England
  • strain ER1793 cells were initially grown in Luria-Bertani (LB) broth supplemented with 0.2% w/v maltose and 10mM MgS0 4 at 20°C. Following infection, cells were maintained in LB broth at 37°C for 15 minutes and then plated on NZY agar medium and incubated at 42°C for 4 hours then at 37°C overnight.
  • LB Luria-Bertani
  • MgS0 4 10mM MgS0 4
  • E. coli strains XLI-Blue and XLOLR were grown in LB broth at 37°C, and transformed XLOLR cells selected on LB/Kanamycin plates (50 ⁇ g/mL) at 37°C.
  • H. pylori expression library was constructed using standard procedures 12 , in the Lambda ZAP express vector (Stratagene) which had been predigested with BamHI and the terminal 5' phosphates removed with calf intestinal phosphatase. Genomic DNA partially digested with
  • Sau3AI was fractionated by gel electrophoresis and DNA fragments between 6 to 12kb were isolated. This DNA was ligated with 1.0 /g of BamHI-digested lambda arms. Recombinant phage DNA was packaged in vitro using Gigapack II extracts (Stratagene). The library was titred by infecting E. coli strain ER1793 or XL1-Blue MRF' cells with aliquots of packaged phage and plated onto indicator plates containing IPTG and
  • the ratio of non-recombinant phage to recombinant phage was 1 :5.
  • the titre of the recombinant library was calculated to be 1 x 10 6 pfu per yg of lambda DNA.
  • H. pylori gene library was screened by DNA hybridization techniques using a cloned probe comprising approximately 200 bp from the H. pylori catalase coding sequence beginning at nucleotide 410.
  • a total of 8000 plaques were plated (4000 bacteriophage plaques per plate) and lifted onto nitrocellulose filters for DNA hybridization analysis with a P-labelled probe.
  • an agar plug containing the plaque was picked and phage eluted into SM buffer. To obtain plaque purity the processes of infecting bacteria, replating and hybridization were repeated.
  • ExAssist helper phage M13 Excised phagemids were packaged as filamentous phage particles and secreted from host cells, which were subsequently heat killed. The phagemids were rescued by infecting XLOLR cells and plating onto LB/Kanamycin (50 g/mL) plates. Bacterial colonies appearing on plates contained pBK-CMV double-stranded phagemid with the cloned DNA insert from H. pylori. These colonies were then analysed for catalase expression.
  • the proteins produced by these potentially H. pylori catalase clones in E. coli XLOLR were analysed by standard SDS-PAGE and Western Blot techniques 12 13 .
  • 10 mL cultures of XLOLR containing expression plasmid were grown in supermedium at 37°C overnight. Cultures were induced with IPTG to a final concentration of 1 mM, with continued incubation for 2-4h. Aliquots of 1 mL were collected, cells pelleted by centrifugation and resuspended in 10mM Tris-HCI (pH 8). Cells were mixed with equal volume of SDS sample reducing buffer and boiled for 10 minutes.
  • Proteins were resolved by electrophoresis on 4-20% gradient Tris- glycine gels (Novex) and electrotransferred onto nitrocellulose membrane (BioRad) for detection of immunoreactive proteins of H. pylori using anti-H. pylori mouse sera as described above.
  • Plasmid DNA sequence analysis was performed by manual sequencing on both strands of plasmid DNA by primer walk using the dideoxynecleotide chain termination method 14 .
  • a genomic library of strain HP921023 was constructed in the lambda expression vector lambda ZAP Express. This library was screened with a radioactively labelled probe representing around 200 bp of the catalase sequence. Approximately 8000 plaques were screened resulting in the detection of eight positive clones. These were picked, purified and the expression plasmid pBK-CMV excised for further characterisation. The proteins expressed by the recombinant plasmids were analysed by Western blotting. Of the eight clones tested, five possessed an extremely immunoreactive band at approximately 50,000 Da (the size expected for catalase).
  • the DNA sequence of the 200 bp probe fragment was obtained and useful restrictions sites were identified for mapping (Hind ⁇ , BstXI, PflM ⁇ ). From this data both the direction and approximate position of the gene could be deduced. DNA sequence analysis confirmed the mapping data and the catalase sequence coding for a protein of predicted molecular weight of 58,650 Da is shown in SEQ ID NO. 1 and 2.
  • genomic DNA from H. pylori isolate RU1 was partially digested with Sau3A and cloned into the ⁇ -ZAP Express vector (Stratagene). This genomic library was probed with a 71 1 bp fragment of the H. pylori catalase ORF, which was generated by PCR using primers starting at nucleotide 1 (21 mer) and terminating at nucleotide 71 1 (18mer), and labelled with dioxigenin-dUTP (Boehringer Mannheim).
  • Catalase positive clones were excised into phagemid form and introduced into the XLOLR f. coli strain (Stratagene). Clones which produced a functional catalase were selected by placing the cells into 30% H 2 0 2 and checking for the rapid formation of oxygen. The selected strain was maintained on Luria agar containing 50mg/L Kanamycin sulfate (Gibco BRL). For recombinant catalase purification, the recombinant E. coli strain was grown in Luria broth plus Kanamycin, and purified using the method of Hazell et a/ 10
  • DNA sequence analysis was performed by manual sequencing on both strands of plasmid DNA by primer walk using the deoxynucleotide chain termination method 14 .
  • DNA sequence analysis identified a sequence coding for a protein of predicted molecular weight of 58,650 Da, which is shown in SEQ ID NO. 3 and 4.
  • mice were challenged with two doses of ⁇ 10 8 H. pylori (Sydney strain), given 48 hours apart. Three weeks later mice were euthanased and samples (sera, saliva, bile and the stomach - half for histology and half the antrum for the direct urease tests 3 ) were collected. The histology samples were fixed in 10% buffered formalin, paraffin embedded and stained using the May-Gr ⁇ nwald Giemsa stain. Stomach sections were scanned for H. pylori using light microscopy (1000x magnification) and scored as infected if one or more organisms were detected in either the gastric body or antrum.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Heliobacter pylori
  • AGG ATC CCT GAA AGG GTG GTG CAT GCT AAA GGA
  • ORGANISM Heliobacter pylori
  • AAA GCT CTT GAA AAA CAC CAA AAG ATG ATG AAA GAC ATG CAT GGA AAA 1488 Lys Ala Leu Glu Lye His Gin Lys Met Met Lys Asp Met His Gly Lys 485 490 495

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Abstract

Une préparation antigénique pouvant être utilisé pour le traitement et la prévention des infections à Hélicobacter chez un hôte mammifère comprend un enzyme catalase recombiné de la bactérie Helicobacter, plus particulièrement un enzyme catalase recombiné de Hélicabacter pylori ou de Helicobacter felis, ou un fragment immunogène dudit enzyme.
PCT/AU1997/000515 1996-08-15 1997-08-14 Traitement et prevention des infections a helicobacter WO1998006853A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029198A1 (fr) * 1999-10-15 2001-04-26 Csl Limited Fragments polypeptidiques comportant une partie c-terminale de helicobacter catalase
WO2002014541A1 (fr) * 2000-08-11 2002-02-21 Wakamoto Pharmaceutical Co., Ltd. Methode d'examen d'une infection a $m(f)i$m(g)helicobacter pylori$m(f)/i$m(g) et kit de diagnostic
AU772513B2 (en) * 1999-10-15 2004-04-29 Csl Limited Polypeptide fragments comprising C-terminal portion of helicobacter catalase
EP1227159A4 (fr) * 1999-10-29 2004-12-29 Wakamoto Pharma Co Ltd Anticorps monoclonal, hybridome, immunoessai et necessaire a diagnostic

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7260694A (en) * 1993-07-27 1995-02-28 Csl Limited Treatment of (h. pylori) associated gastroduodenal disease
WO1995027506A1 (fr) * 1994-04-08 1995-10-19 Pasteur Merieux Serums Et Vaccins Composition immunisante anti-helicobacter pylori a base de catalase
AU2609195A (en) * 1994-06-08 1996-01-04 Csl Limited Treatment and prevention of helicobacter infection

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Publication number Priority date Publication date Assignee Title
AU7260694A (en) * 1993-07-27 1995-02-28 Csl Limited Treatment of (h. pylori) associated gastroduodenal disease
WO1995027506A1 (fr) * 1994-04-08 1995-10-19 Pasteur Merieux Serums Et Vaccins Composition immunisante anti-helicobacter pylori a base de catalase
AU2609195A (en) * 1994-06-08 1996-01-04 Csl Limited Treatment and prevention of helicobacter infection

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Title
EMBL DATABASE, Accession No. g1561776, Submitted 21 August 1996. *
EUR. J. CLIN. MICROBIOL. INFECT DIS., Vol. 11, No. 6, June 1992, T.U. WESTBLOM et al., "Catalase Negative Mutants of Helicobacter Pylori", pages 522-526. *
J. BACTERIOLOGY, Vol. 178, No. 23, December 1996, S. ODENBREIT et al., "Cloning and Genetic Characterization of Helicobacter Pylori Catalase and Construction of a Catalase-Deficient Mutant Strain", pages 6960-6967. *
J. GEN. MICROBIOLOGY, Vol. 137, Part 1, January 1991, S.L. HAZELL et al., "Helicobacter Pylori Catalase", pages 57-61. *
NATURE, Vol. 388, 7 August 1997, J.F. TOMB et al., "The Complete Genome Sequence of the Gastric Pathogen Helicobacter Pylori", pages 539-547. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029198A1 (fr) * 1999-10-15 2001-04-26 Csl Limited Fragments polypeptidiques comportant une partie c-terminale de helicobacter catalase
AU772513B2 (en) * 1999-10-15 2004-04-29 Csl Limited Polypeptide fragments comprising C-terminal portion of helicobacter catalase
US7786260B1 (en) 1999-10-15 2010-08-31 Csl Limited Polypeptide fragments comprising c terminal portion of helicobacter catalase
EP1227159A4 (fr) * 1999-10-29 2004-12-29 Wakamoto Pharma Co Ltd Anticorps monoclonal, hybridome, immunoessai et necessaire a diagnostic
US6849419B1 (en) * 1999-10-29 2005-02-01 Wakamoto Pharmaceutical Co., Ltd. Monoclonal antibody hybridoma immunoassay method and diagnosis kit
KR100756117B1 (ko) * 1999-10-29 2007-09-05 와카모토 세이야꾸 가부시끼가이샤 모노클로날 항체, 하이브리도마, 면역분석 방법 및 진단킷트
WO2002014541A1 (fr) * 2000-08-11 2002-02-21 Wakamoto Pharmaceutical Co., Ltd. Methode d'examen d'une infection a $m(f)i$m(g)helicobacter pylori$m(f)/i$m(g) et kit de diagnostic

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