WO2000003730A1 - VACCIN A BASE D'UREASE CONTRE LES INFECTIONS A $i(HELICOBACTER) - Google Patents

VACCIN A BASE D'UREASE CONTRE LES INFECTIONS A $i(HELICOBACTER) Download PDF

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Publication number
WO2000003730A1
WO2000003730A1 PCT/KR1998/000216 KR9800216W WO0003730A1 WO 2000003730 A1 WO2000003730 A1 WO 2000003730A1 KR 9800216 W KR9800216 W KR 9800216W WO 0003730 A1 WO0003730 A1 WO 0003730A1
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Prior art keywords
urease
vaccine according
fermentum
preparation comprises
subunit
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PCT/KR1998/000216
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English (en)
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Jong Beak Park
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Cheil Jedang Corporation
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Priority to PCT/KR1998/000216 priority Critical patent/WO2000003730A1/fr
Priority to AU84643/98A priority patent/AU8464398A/en
Publication of WO2000003730A1 publication Critical patent/WO2000003730A1/fr

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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route

Definitions

  • the present invention relates to the prevention and treatment of gastric infection in mammals, including humans. More particularly, the present invention relates to a vaccine suitable for use in the prevention and treatment of Helicobacter infection in mammals, including humans, and to a method of treatment of humans suffering gastric infection, its consequences such as chronic gastritis or peptic ulcer, and prevention of gastric cancer.
  • Helicobacter infection of human gastric epithelium cause gastritis. Helicobacter infection is also a leading cause of peptic ulcers and gastric lymphoma and may be a risk factor in the development of gastric cancer (Blaser, M.J.,
  • H. pylori a slender, S-shaped gram-negative microorganism, which is routinely recovered from gastric biopsies of adults and children with histologic evidences of gastritis or peptic ulceration. Evidence for a causal relationship between H.
  • pylori and gastroduodenal disease comes from studies in human volunteers, patients with ulcers and gastric cancer, gnotobiotic pigs, and germ-free rodents.
  • Koch's postulates were satisfied by creating histologically confirmed gastritis in previously uninfected individuals following consumption of viable microorganisms (Marshall, B.J. et al., Med. J. Aust., 1985, 142, 436-439; Morris, A, et a ⁇ , Am. J. Gastroenterology, 1987, 82, 192-199; Engstrand, L. et al, Infect, Immun., 1990, 53, 1763-1768; Fox, J.G. etal, Infect.
  • Ig immunoglobulin
  • the distribution of immunoglobulin (Ig) producing cells in the normal gastric antrum indicates that IgA plasma cells make up 80% of the total plasma cell population.
  • the number of plasma IgA cells present in the gastral antrum is comparable to other mucous membranes (Brandtzaeg, P., Scan. J. Immunol, 1985, 22, 111-146; and Brandtzaeg, P., Ann. Allergy, 1987, 59, 21-39).
  • a number of studies in humans (Brandtzaeg, P., Ann. Allergy, 1987, 59, 21-39) and in animal models (Fox, J. G.
  • Davin showed that some monoclonal antibodies of //, felis are cross- reactive with the urease of H. pylori (Davin, C, et al., Proceedings of the DDW, American Gastroenterology Association, May 16-19, 1993, 1213, A-304). This demonstrates that an antigenic cross reaction is generated between H. pylori urease and other Helocobacter ureases.
  • H. pylori urease or other related ureases may be used as a vaccine for H. pylori infection. However, it did not contain any evidence of the vaccination of any mammal with urease against any Helicobacter infection.
  • H. pylori is able to transport the urease out of the cell and to release the urease from its surface (Evans, D. J. et al, Infect. Immun., 1992, 60, 2125-2127 and Ferrero, R. L. and Lee, A., Microb. Ecol HealthDis., 1991, 4, 121-134).
  • urease may not represent an appropriate target for the development of a protective mucosal immune response.
  • mucosal immune protection is thought to be mainly mediated by secretory IgA, the agglutinating activity of which would be impaired when the recognised antigen is shed by the target pathogen and thereby serve as a decoy for the protective antibody.
  • urease appears to be toxic for epithelial cells in culture, and has been suspected to play a role in mucous degradation and in peptic ulceration in vivo. Thus, its use as an antigen may be toxic.
  • the present invention provides a method of inducing immunity to Helicobacter infection by administering to a mucosal surface of a mammal a polyaminoacid preparation, i.e. a mixture of peptides and/or proteins together with an appropriate adjuvant.
  • the polyaminoacid preparation presents a plurality of epitopes characteristic of and exhibited by a urease enzyme endogenous to infecting Helicobacter organism.
  • the polyaminoacid preparation may be orally administered.
  • the active component of the polyaminoacid preparation may comprise natural or biosynthetic epitopes and may take various forms.
  • a non exhaustive list of possible preparations includes purified, naturally occurring or recombinantly produced urease preparations of bacterial or other origin, digests of urease, fusion proteins comprising urease epitopes, truncated forms of urease enzyme, or peptides homologous with the amino acid sequence of urease. Since development of immunity depends on induction of humoral and/or cellular immune responses which bind to the infecting Helicobacter organism, preferred preparations are those which most closely duplicate the epitopes of the urease endogenous to the infecting organism.
  • a method of eliciting in a mammalian host a protective immunity to Helicobacter infection wherein an immunologically effective amount of a urease antigen capable of eliciting such a protective immune response, preferably L. reuteri urease A subunit or L. fermentum urease A subunit, is administered to a mucosal surface of the host.
  • a urease antigen capable of eliciting such a protective immune response, preferably L. reuteri urease A subunit or L. fermentum urease A subunit
  • a vaccine composition for prevention of Helicobacter infection comprising an effective amount of a urease antigen, preferably L. reuteri urease A subunit or L. fermentum urease A subunit, capable of eliciting in a host a protective immune response to Helicobacter infection, in association with a pharmaceutically acceptable carrier or diluent.
  • a urease antigen preferably L. reuteri urease A subunit or L. fermentum urease A subunit
  • a method of imparting to a mammalian host passive protection to Helicobacter infection comprising administering to a mucosal surface of the host an immunologically effective amount of a urease antigen, preferably L. reuteri urease A subunit or L. fermentum urease A subunit, capable of eliciting a protective immune response to Helicobacter infection.
  • a urease antigen preferably L. reuteri urease A subunit or L. fermentum urease A subunit
  • a method of treating a mammalian host, including humans, infected with Helicobacter or preventing the host from Helicobacter infection which comprises administering to a mucosal surface of the host an immunologically effective amount of a urease antigen, capable of elicting a protective immune response to Helicobacter infection.
  • the preferred urease includes L. reuteri urease A subunit and L. fermentum urease A subunit, and can be administered singly or in combination with hydroxy calcium phosphate such as hydroxy apatite.
  • H. pylori urease is administered together with mucosal adjuvant, B subunit of cholera toxin, muramyl dipeptide or other adjuvants.
  • Figure 1 shows the results obtained when mice were orally immunized with sonicates of L. fermentum, purified L. fermentum urease or cholera toxin and hydroxy apatite (control).
  • Figure 2 shows the results obtained when mice were orally immunized with sonicates of L. reuteri, purified L. reuteri urease or cholera toxin and hydroxyapatite
  • L. fermentum ATCC 11739 and L. reuteri ATCC 23272 were anaerobically cultured on MRS medium.
  • Urease was purified according to a general method as described in Kakimoto et al., Agric. Biol Chem 53, 1119-1125 and Kakimoto et al., Appl. Microbiol. Biotechnol.32, 538-543.
  • the strains were cultured in a bottle at 37 ° C for 2 days. After completion of the culture, the strains were introduced in TEA buffer solution (1 mM 2-mercaptoethanol, 50 mM Tris-HCl, pH 7.0) and the solution was stirred.
  • Fractions with high urease activity were pooled, dialyzed overnight, and subjected to chromatography on DEAE-Sepharose CL-6B column. After elution was conducted with a concentration gradient of NaCl buffer solution, highly active fractions were pooled, dialyzed overnight against 5 mM phosphate buffer solution (pH 7.0), and were subjected to affinity chromatography on Affi-Gel 202 column on which hydroxyurea was immobilized.
  • L. fermentum or L. reuteri ureases obtained as described above.
  • urease or its subunit obtained by a DNA recombinant technique digests of natural or synthetic urease, fusion proteins including whole urease or its fragments, end-excised urease structures, other peptide or protein preparations with urease epitopes which are able to induce protective immune response against Helicobacter infection can be used.
  • the urease which is substantially homologous with L. fermentum or L. reuteri ureases and induces cross-protective immune response against Helicobacter infection.
  • any polyaminoacid preparations which include urease epitopes and effectively induce protective immune response against Helicobacter infection in host, can be used as an antigenic substance.
  • Examples of potentially effective urease preparation include, but not limited to this, peptides obtained by digesting purified urease with physical and/or chemical treatment (e.g., CNBr) and/or proteolysis (e.g., V8-protease, trypsin or others), or chemically synthesized peptides having the same as epitope of urease.
  • physical and/or chemical treatment e.g., CNBr
  • proteolysis e.g., V8-protease, trypsin or others
  • chemically synthesized peptides having the same as epitope of urease e.g., V8-protease, trypsin or others
  • epitopes identified by their cross-reactivity with urease as the result of screening with anti-urease antibodies include epitopes identified by their cross-reactivity with urease as the result of screening with anti-urease antibodies.
  • peptides can be naturally occurring peptides or peptides resulting from chemical synthesis. Furthermore such peptides can result from the expression of recombinant random oligonucleotide.
  • Another source of potentially useful epitopes includes epitopes similar to urease as a result of the generation of anti-idiotype antibodies to urease.
  • Such anti- idiotypic antibodies generated in any immunocompetent host, are obtained by immunization of this host with anti-urease antibodies, with the goal of generating antibodies directed against anti-urease antibodies, which share structural homologies with urease.
  • Tables 1 to 4 below and Figures 1 and 2 describe the results obtained when mice were orally immunized with purified L. fermentum or L. reuteri ureases . In this experiment, the antigen administered was the L. fermentum urease or L.
  • mice were each orally immunized with 30 ug of purified L. fermentum urease or L. reuteri urease coupled to 1 mg of hydroxyapatite plus 10 ug of cholera toxin adjuvant at day 0, 7, 14 and 21. At day 28, 30 and 32, mice were challenged with 10 8 H. felis. For comparison purpose, similar female SPF B ALB/c mice were orally immunized with whole L.
  • the fermentum or L. reuteri lysate (sonicate) and 10 ug of cholera toxin at day 0, 7, 14 and 21.
  • the mice were challenged with H. felis at day 28, 30 and 32.
  • the L. fermentum or L. reuteri lysate was prepared by centrifuging the recovered L. fermentum or L. reuteri from cell cultures and resuspending the resulting pellet in 0.9% sodium chloride, followed by sonication.
  • mice Female SPF B ALB/c mice were orally sham-immunized with 10 ug of cholera toxin and 1 mg of hydroxyapatite at day 0, 7, 14 and 21. All mice were housed, immunized and challenged in parallel. All mice were sacrificed on day 39.
  • A, B and C subunits of L. fermentum urease and L. reuteri urease were prepared by the method as described in Kakimoto et al., Appl. Microbiol. Biotechnol. 32, 538-543. Purified ureases were subject to chromatography on Micropak Protein
  • the present invention includes the use of the gene encoding the urease which is substantially homologous with L. fermentum urease or L. reuteri urease and induce a cross-protective immune response against Helicobacter infection.
  • recombinant ureases which has 70 to 95% homologies with L. fermentum urease or L. reuteri urease can be used.
  • A, B and C subunits of purified L. fermentum urease or L. reuteri urease are representatively used in the present invention. However, it should be understood that any urease or its subunit or structure capable of inducing the desired protective immune response can be produced using DNA recombinant technique and procaryotic or eucaryotic expression vectors.
  • mice were orally immunized with purified subunits of I. fermentum or L. reuteri ureases are shown in Tables 5 to 8 below.
  • mice were challenged with 10 8 H felis.
  • female SPF BALB/c mice were orally sham-immunized with 10 ug of cholera toxin and 1 mg of hydroxyapatite at day 0, 7, 14 and 21. All mice were immunized and challenged in parallel. They were sacrificed on day 46 (14 days after challenge).
  • gastric biopsies from each animal were screened for the presence of H. felis by assessing urease activity by the CLO test according to the supplier's directions (Delta West Pty Ltd). Gastric biopsies were impregnated in CLO test gel using 19 gauge injection syringes. While the tissue was kept at 37°C for 3 hours and subsequently moved to room temperature, the colorimetric change of the gel was observed. After 24 hours, when the colour of the tissue remained unchanged and was yellowish, it was determined as negative.
  • gastric biopsies of each animal described in section B also were cultured under anaerobic conditions on 7% horse serum-containing Brucella agarose plate supplemented with Campylobacter selective adjuvant (Mikrobiologie,
  • Tables 1 and 2 and Figure 1 describe the results obtained when mice were orally immunized with purified L. fermentum urease and were challenged with H felis.
  • Tables 3 and 4, and Figure 2 describe the results obtained when mice were orally immunized with purified L. reuteri urease and were challenged with H felis.
  • Tables 5 and 6 show the results obtained when mice were orally immunized with purified A,
  • a "urease + HF” means that mice were orally immunized with urease coupled to hydroxyapatite and cholera toxin and then challenged with H. felis.
  • a "urease” means that mice were orally immunized with urease coupled to hydroxyapatite and cholera toxin but were not challenged.
  • a "CT + HF” means that mice were orally sham-immunized with cholera toxin and then challenged with //. felis.
  • a "LF sonification + HF” means that mice were orally immunized with L. fermentum sonicate and cholera toxin and then challenged with H felis.
  • a "LF sonicate” means that mice were orally immunized with L. fermentum sonicate and cholera toxin but was not challenged.
  • a "LR sonicate + HF” means that mice were orally immunized with L. reuteri sonicate and cholera toxin and then challenged with
  • a "LR sonicate" means that mice were orally immunized with L. reuteri sonicate and cholera toxin but were not challenged.
  • mice The results of the Section B experiment obtained by CLO test on gastric biopsies and Gram staining of H. felis culture are shown in Table 2.
  • the infection of mice was defined as having at least one colony and label by H. felis, in addition to the urease test or the Gram staining of cultures.
  • mice with L. fermentum urease affords a statistically significant protection, compared to the immunization of mice with L. fermentum sonicate or cholera toxin.
  • Table 2 it can be seen from Table 2 that whereas 7 of 10 mice immunized with L. fermentum sonicate and all of 10 mice immunized with cholera toxin were infected with H. felis, only 4 of 10 mice immunized with L. fermentum urease were infected with H. felis. That is, whereas 30% of the mice immunized with L. fermentum sonicate and challenged with
  • H felis were protected and all of mice immunized with cholera toxin and challenged with H. felis were not protected, 60% of the mice immunized with L. fermentum urease were protected from H felis challenge.
  • 100% of the control mice exposed on H. felis were infected with the pathogenic agent, but the infection rate of mice immunized with L. fermentum urease 28 days before exposure to H. felis was only 40%.
  • the protection obtained by using L. fermentum urease was not expected and could never be foreseen from the results obtained by using L fermentum sonicate.
  • mice immunized withZ. reuteri sonicate and challenged with H felis and none of the mice immunized with cholera toxin and challenged with H felis were protected
  • 50% of the mice immunized with L. reuteri urease were protected from an H. felis challenge. That is, 100% of the control mice exposed to H. felis were infected with the pathogenic agent, but the infection rate of mice immunized with L. reuteri urease 28 days before exposure to H. felis was 50%.
  • Tables 5 to 8 show the results of the above Section C experiment obtained by gastric urease test (purified urease subunit) .
  • a "CT” means cholera toxin.
  • a "urease A” means purified urease A subunit.
  • a “urease B” means purified urease B subunit.
  • a “urease C” means purified urease C subunit.
  • a “HAP” means hydroxyapatite.
  • mice with purified A subunit of L. fermentum urease affords a statistically significant protection, compared to the immunization of mice with purified B or C subunits of L. fermentum urease or cholera toxin.
  • Table 6 it can be seen from Table 6 that whereas all of 10 mice immunized with purified C subunit of L. fermentum urease and 9 of 10 mice immunized with purified B subunit of L fermentum urease were infected 14 days after challenge with H. felis, only 4 of 10 mice immunized with purified A subunit of L. fermentum urease were infected with H. felis.
  • mice immunized with purified C subunit of L. fermentum urease and cholera toxin and challenged with H. felis were protected and 10% of the mice immunized with purified B subunit of I. fermentum urease and challenged with H. felis were protected
  • 60% of mice immunized with purified A subunit of L. fermentum urease were protected from H felis challenge.
  • mice with purified A subunit of X. reuteri urease affords statistically significant protection, compared to the immunization of mice with purified B or C subunits of L. reuteri urease or cholera toxin.
  • mice immunized with purified B or C subunit of L. reuteri urease were infected 14 days after challenge with H felis, only 5 of 10 mice immunized with purified A subunit of L. reuteri urease were infected with H. felis. That is, 100% of the control mice challenged with H.
  • the present invention provides a vaccine composition for the prevention of Helicobacter infection which comprises an immunologically effective amount of urease antigen, preferably purified L. fermentum urease, purified L. reuteri urease, purified L. fermentum urease subunit, purified L. reuteri urease subunit, to induce a protective immune response in the host against Helicobacter infection in combination with a pharmaceutically acceptable carrier or diluent.
  • urease antigen preferably purified L. fermentum urease, purified L. reuteri urease, purified L. fermentum urease subunit, purified L. reuteri urease subunit, to induce a protective immune response in the host against Helicobacter infection in combination with a pharmaceutically acceptable carrier or diluent.
  • the vaccine of the present invention can be administrated in an amount to be determined by one skilled in the art.
  • the appropriate dose for adults is between 10 ug and 100 mg, preferably between 50 ug and 50 mg.
  • a similar dose can be administrated to young children.
  • the carrier system includes an inactivated form of urease in enteric releasing capsule or fusion protein for protecting the antigen from a gastric acidic environment.
  • the vaccine of the present invention is used as a primary prophylactic for uninfected adult or young children.
  • the vaccine of the present invention can be administrated as a secondary prophylactic for an infected host once H pylori is removed from the host.
  • the vaccine of the present invention can be used as a therapeutic agent which induces an immune response in infected host to eliminate
  • a suitable mucosal adjuvant is cholera toxin.
  • B subunit or recombinant conjuate thereof can be used as mucosal adjuvant.
  • Other examples of carrying the urease antigen include a biodegradable microcapsule or immune stimulatory complex (ISCOM'S) or liposome, genetically engineered attenuated viral or bacterial biovector and recombinant (chimeric) viral particle such as bluetongue.
  • the amount of mucosal adjuvants used depends on the type of the mucosal adjuvant used. For example, cholera toxin is used in amount of 5 ug to 35 ug. An appropriate amount of mucosal adjuvants to be used can be easily determined by those skilled in the art.
  • Suitable examples of carriers to be used for the vaccine of the present invention include an enteric coated capsule and polyactide-glycolide granule. Examples of diluent include 0.2 N NaHCO 3 and/or saline.
  • An especially suitable carrier to coat L. fermentum urease or L reuteri urease on a mucosal surface is particulate hydroxy calcium phosphate.
  • the L. fermentum urease-hydroxy calcium phosphate conjugate or the L reuteri urease-hydroxy calcium phosphate conjugate appear to move across the epithelium in which poly Ig immune response takes place.
  • the hydroxy calcium phosphate is preferably in a microparticle form, so it can traverse the epithelium cell by an M cell differentiated so as to traverse the epithelium cell.
  • the preferred form of hydroxy calcium phosphate is hydroxyapatite, commercially available crystalline hydroxy calium phosphate Ca 10 (PO 4 ) 6 (OH) 2 .
  • hydroxyapatite is classified into inorganic hydroxyapatite present in normal bony tissue and chemically and physically similar plate type crystal. As verified from the fact that bone-derived calcium/phosphorous supplementary nutrient is currently available, hydroxyapatite designed to be ingested is safe. The size of commercially available highly degradable hydroxyapatite crystal
  • hydroxyapatite (CalBioChecm) is varied. Crystal with a dimeter of 1 um cannot be adsorbed into an M cell. Thus, the commercial hydroxyapatite crystal is homogeneously crushed by sonication before it is used for the vaccine of the present invention.
  • the preferred size of hydroxyapatite according to the present invention is in the range of 0.01 um to 0.05 um. The size of hydroxyapatite can be measured using an electron microscope or light dispersion.
  • L. fermentum urease or L. reuteri urease antigens is oral, intranasal, rectal or intraocular.
  • the oral administration leads to the delivery of the vaccine into gastrointestinal mucosa.
  • the vaccine of the present invention to be administrated to mucosal surface can be formulated into aerosol, suspension, capsule and/or suppository.
  • the administration method will be apparent to those skilled in the art.
  • the present invention is directed to a passive immunization of mammals including humans against Helicobacter infection.
  • the passive immunization can be obtained by administrating an effective amount of urease-specific antibody, preferably L. fermentum urease or ,, reuteri urease specific IgA monoclonal antibody, to the mucosal surface of an animal.
  • the present invention is also directed to use of//, pylori urease as diagnostics to measure the immune response of the human body to which a urease-based vaccine is administrated or to determine immunity and sensitivity of an individual (i.e., whether the individual needs to be vaccinated)
  • the present invention is directed to use of urease or urease specific antibodies in the assay or the construction of a kit to diagnose immunity against Helicobacter infection, to assess sensitivity against Helicobacter infection, or to assay immune response to the vaccine.
  • H. felis, L. fermentum and L. reuteri were, as standard strains, obtained from the American Type Culture Collection, USA without any restriction.
  • H. felis Culture The bacteria was cultured under anaerobic conditions at 37°C on agar plate containing trypticase soy II and 5% sheep blood for three days. Also, the bacteria was anaerobically cultured under shaking at 37°C in tripticase soy II liquid medium containing 10% fetus calf serum for 2 to 3 days.
  • L. fermentum or L. reuteri was collected from liquid cultures in 0.15 M NaCl and was centrifuged at 3000g, 4 ° C for 5 minutes. The resulting pellet was suspended in 10 ml of NaCl and was ultrasonicated for 3 minutes. The amount of the protein was assessed by the Lowry method.
  • the fractions with strong urease activity were pooled, and 14 ml of total pools was dialysed against 2 L of TEA buffer (1 mM 2- mercaptoethanol, 5 mM potassium phosphate buffer, pH 7.0) at 4"C overnight and then subjected to chromatography on Affi-Gel 202 column (Bio-Rad) on which hydroxyapatite was immobilized.
  • the fractions were eluted with 100 mM of TEA buffer to obtain 15 ml of pool with strong urease activity.
  • the pool was dialysed against 1 L of 25 mM imidazole-hydrochloride buffer solution (pH 7.4) overnight and then subjected to chromatofocusing on PBE 94 (Pharmacia) column with a mobile phase of 25 mM imidazole-hydrochloride buffer solution (pH 7.4).
  • the fractions were eluted with 9 x diluted polybuffer 74 (Pharmacia) to obtain 4 ml of the pool with strong urease activity at pH 4.
  • the pool was dialysed against 1 L of TEA buffer solution overnight, subjected to chromatography on Affi-Gel 202 column, and stained with Coomassie Brilliant Blue. Five mM fractions showing three distinct bands corresponding to 68 kDa, 16kDa and 8.8kDa on the gel were pooled.
  • mice were lightly anaesthetized with ether prior to immunization. Sonicate or purified urease, hydroxyapatite and cholera toxin were suspended in PBS and 200 ml of the suspension was delivered into stomach of each mouse using oral cannula. Three oral immunization protocols were assessed as described below.
  • mice Twenty Female BALB/c 5-week old mice were orally immunized with 30 ug of purified H. pylori urease, 1 mg of hydroxyapatite and 10 ug of cholera toxin at day 0, 7, 14 and 21. Ten immunized mice were challenged with 5 x 10 7 and 10 8 H felis at day 28, 30 and 32.
  • mice Twenty female BALB/c 5-week old mice were orally immunized with 2 mg of L. fermentum sonicate or 2 mg of L. reuteri sonicate at day 0, 7, 14 and 21. Ten immunized mice were challenged with 5 x 10 7 and 10 8 H. felis at day 28, 30 and 32.
  • Protocol B3 Control Protocol
  • mice Twenty female 5-week old BALB/c mice were orally immunized with 1 mg of hydroxyapatite and 10 ug of cholera toxin at day 0, 7, 14 and 21. Ten immunized mice were challenged with 5 x 10 7 and 10 8 / . felis at day 28, 30 and 32. All mice were sacrificed on day 39 and gastric biopsies were taken from each animal.
  • the tissue was impregnated in the CLO test gel using 19 guage injection syringes. After the tissue was incubated at 37 ° C for 3 hours, the colorimetric change of the tissue was observed at room temperature. After 24 hours, when the colour of the tissue remained unchanged and was yellowish, the tissue was determined as negative. Additionally, to confirm the presence of//, felis, the gastric biopsy was cultured and assessed by Gram staining. The tissue was homogenized, and the homogenate was diluted in 0.15 M NaCl (1:10 and 1 : 1 ,000). The dilution was smeared on blood plate and cultured under anaerobic conditions at 37 ° C for 4 to 10 days.
  • mice were lightly anaesthetized with ether prior to immunization.
  • Purified L. fermentum urease A, B or C subunit or purified L. reuteri urease A, B or C subunit, hydroxyapatite and cholera toxin were suspended in PBS and 200 ul of the suspension was delivered into stomach of each mouse using oral cannula.
  • Three oral immunization protocols were assessed as described below. Protocol Cl - Vaccination with purified urease A subunit
  • mice Female BALB/c 5-week old mice (20) were orally immunized with 30 ug of purified L. fermentum urease A subunit or purified L. reuteri A subunit, 1 mg of hydroxyapatite and 10 ug of cholera toxin at day 0, 7, 14 and 21. Ten immunized mice were challenged with 5 x 10 7 and 10 8 H. felis at day 28, 30 and 32.
  • mice Female BALB/c 5-week old mice (20) were orally immunized with 50 ug of recombinant H. pylori urease B subunit, 1 mg of hydroxyapatite and 10 ug of cholera toxin at day 0, 7, 14 and 21. Ten immunized mice were challenged with 5 x 10 7 and
  • mice Female BALB/c 5-week old mice (20) were orally immunized with 1 mg of hydroxyapatite and 10 ug of cholera toxin at day 0, 7, 14 and 21. Ten immunized mice were challenged with 5 x 10 7 and 10 8 /Z felis at day 28, 30 and 32. All the mice were sacrificed on day 46 and gastric biopsies were taken from each animal.
  • the urease activity of the gastroc biopsy was detected by the CLO test (Delta West Pty Ltd).
  • the tissue was impregnated in the CLO test gel using 19 gauge injection syringes. After the tissue was incubated at 37 ° C for 3 hours, the colorimetric change of the tissue was observed at room temperature. After 24 hours, when the colour of the tissue remained unchanged and was yellowish, the tissue was determined as negative. Additionally, to confirm the presence of H. felis, the gastric biopsy was cultured and assessed by Gram staining method. The tissue was homogenized, and the homogenate was diluted in 0.15 M NaCl (1 :10 and 1 : 1 ,000). The dilution was smeared on blood plate and cultured under anaerobic condition at 37°C for 4 to 10 days.

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  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention a trait à un vaccin destiné à générer une immunité protectrice contre les infections à Helicobacter chez des mammifères comprenant une préparation de polyaminoacides contenant des épitopes présentés par une uréase dérivée de Lactobacillus en combinaison avec un excipient ou un diluant acceptable sur le plan pharmaceutique. La préparation de polyaminoacides comprend une Lactobacillus fermentum uréase, une Lactobacillus reuteri uréase purifiée ou des sous-unités de celle-ci. De plus, la présente invention a trait à un procédé de dosage de la réponse immune protectrice chez des mammifères infectés par un organisme à Helicobacter consistant à déterminer la présence d'anticorps réactif avec des épitopes mis en lumière par une uréase endogène audit organisme Lactobacillus, dans un échantillon prélevé dans une voie gastrique desdits mammifères.
PCT/KR1998/000216 1998-07-16 1998-07-16 VACCIN A BASE D'UREASE CONTRE LES INFECTIONS A $i(HELICOBACTER) WO2000003730A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/KR1998/000216 WO2000003730A1 (fr) 1998-07-16 1998-07-16 VACCIN A BASE D'UREASE CONTRE LES INFECTIONS A $i(HELICOBACTER)
AU84643/98A AU8464398A (en) 1998-07-16 1998-07-16 Urease based vaccine against (helicobacter) infection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR1998/000216 WO2000003730A1 (fr) 1998-07-16 1998-07-16 VACCIN A BASE D'UREASE CONTRE LES INFECTIONS A $i(HELICOBACTER)

Publications (1)

Publication Number Publication Date
WO2000003730A1 true WO2000003730A1 (fr) 2000-01-27

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PCT/KR1998/000216 WO2000003730A1 (fr) 1998-07-16 1998-07-16 VACCIN A BASE D'UREASE CONTRE LES INFECTIONS A $i(HELICOBACTER)

Country Status (2)

Country Link
AU (1) AU8464398A (fr)
WO (1) WO2000003730A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10800462B2 (en) 2016-07-21 2020-10-13 Zephyros, Inc. Reinforcement structure

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009823A1 (fr) * 1992-11-03 1994-05-11 Oravax, Inc. Vaccin a base d'urease contre l'infection due a l'helicobacter
EP0654273A1 (fr) * 1993-11-18 1995-05-24 Harry H. Leveen Produit pharmaceutique et méthode pour traitement
WO1996033732A1 (fr) * 1995-04-28 1996-10-31 Oravax, Inc. Vaccin d'urease recombinante et multimere
EP0835928A1 (fr) * 1996-10-11 1998-04-15 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Vaccin vivant d'hélicobacter pylori
WO1998028002A1 (fr) * 1996-12-20 1998-07-02 Research Foundation Of The City University Of New York Nouvel organisme pathogene cocciforme, staphylococcus leei, avec trophisme de la mucine gastrique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009823A1 (fr) * 1992-11-03 1994-05-11 Oravax, Inc. Vaccin a base d'urease contre l'infection due a l'helicobacter
EP0654273A1 (fr) * 1993-11-18 1995-05-24 Harry H. Leveen Produit pharmaceutique et méthode pour traitement
WO1996033732A1 (fr) * 1995-04-28 1996-10-31 Oravax, Inc. Vaccin d'urease recombinante et multimere
EP0835928A1 (fr) * 1996-10-11 1998-04-15 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Vaccin vivant d'hélicobacter pylori
WO1998028002A1 (fr) * 1996-12-20 1998-07-02 Research Foundation Of The City University Of New York Nouvel organisme pathogene cocciforme, staphylococcus leei, avec trophisme de la mucine gastrique

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10800462B2 (en) 2016-07-21 2020-10-13 Zephyros, Inc. Reinforcement structure

Also Published As

Publication number Publication date
AU8464398A (en) 2000-02-07

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