CA2164729A1 - Catalase-based anti-helicobacter pylori immunising composition - Google Patents
Catalase-based anti-helicobacter pylori immunising compositionInfo
- Publication number
- CA2164729A1 CA2164729A1 CA002164729A CA2164729A CA2164729A1 CA 2164729 A1 CA2164729 A1 CA 2164729A1 CA 002164729 A CA002164729 A CA 002164729A CA 2164729 A CA2164729 A CA 2164729A CA 2164729 A1 CA2164729 A1 CA 2164729A1
- Authority
- CA
- Canada
- Prior art keywords
- catalase
- pylori
- helicobacter pylori
- based anti
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000016938 Catalase Human genes 0.000 title claims abstract description 32
- 108010053835 Catalase Proteins 0.000 title claims abstract description 32
- 239000000203 mixture Substances 0.000 title claims abstract description 13
- 229940037467 helicobacter pylori Drugs 0.000 title 1
- 230000003053 immunization Effects 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 claims description 9
- 108010046334 Urease Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 239000002502 liposome Substances 0.000 claims description 3
- 230000035772 mutation Effects 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 2
- 229940037003 alum Drugs 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- 241000518994 Conta Species 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010050327 trypticase-soy broth Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- VVNCNSJFMMFHPL-VKHMYHEASA-N D-penicillamine Chemical compound CC(C)(S)[C@@H](N)C(O)=O VVNCNSJFMMFHPL-VKHMYHEASA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 206010019375 Helicobacter infections Diseases 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 231100000749 chronicity Toxicity 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229940075911 depen Drugs 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000001429 visible spectrum Methods 0.000 description 1
Classifications
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Communicable Diseases (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a composition for immunizing against H. pylori, comprising, as active principle, an H. pylori catalase, in substantially purified form.
Description
2~ 64729 .. _ '' ' ` ,' E, H~ ~ r-~ J~3 -~=~T ¦ L ~r;~
The subject of the present invention is a pharma-ceutical composition intended for the treatment or pre-vention of Helicobacter pylori infections.
N. pylori is a gram-negative bacterium to date found exclusively at the surface of the mucous membrane of the stomach in man, and more particularly around the crater lesions of gastric and duodenal ulcers. This bacterium was initially called Campylobacter pyloridis (Warren & Marshall (1983) Lancet 1 : 1273-1275).
H. pylori is currently recognized as being the aetiological agent of antral gastriti8, and appears to be one of the cofactors required for the development of ulcers. Moreover, it appears that the development of gastric carcinomas may be associated with the presence of H. pylori.
Various factors contribute to the infection. This bacterium is endowed in particular with high urease activity imparted by a urease which allows it to survive in the acidic environment of the stomach. Moreover, the flagellae make it mobile, allowing it to cross the mucus in order to reach its ecological niche and to adhere to the mucous m~hrane via adhesins.
In spite of an appreciable serous immune response and a very pronounced inflammatory response, the bacterial infection is persistent. One very likely hypothesis gives a catalase produced by the pathogenic agent a central role in the chronicity of the infection.
The reason for this is that catalases (EC 1.11.1.6) are, in general, important enzymes for the survival of bacteria in the presence of H2O2. Thus, in the present case, a catalase would allow H. pylori to escape the toxic ef$ects of the oxygen radicals produced during the inflammatory ph~om~no~.
One catalase has already been characterized in H. pylori (Hazell et al., J. Gen. Microbiol. (1991) 137 : 57). This is a tetramer whose subunit has a molecular _ - 2 -weight of about 50-60 kD. The N-terminal sequence of such a catalase has also been established (Westblom et al., Eur. J. Clin. Microbiol. Inf. Dis. (1992) 11 (6) : 522).
However, it is not impossible for this bacterium to possess several catalases, which are slightly different from one another.
It has now been found that an N. pylori catalase could be used as a substance for immunizing against the pathogenic agent itself, and could be useful in particu-lar for prophylactic or therapeutic purposes.
Accordingly, the subject of the invention is acomposition for immunizing against N. pylori which com-prises, as active principle, an N. pylori catalase in substantially purified form.
The term "substantially purified catalase" means a catalase preparation devoid of the majority of the H. pyl ori cell constituents.
A catalase which is useful for the purposes of the present invention may either be obtained by extrac-tion from H. pylori or may be obtained via a recombinantroute, by expression of the correspo~;n~ DNA fragment, in a heterologous system, bacteria, yeasts or mammalian cells.
To this end, it is shown that a catalase may be purified from N. pylori according to a technique which is identical to or similar to that described by Hazell et al., J. of Gen. Microbiol. (1991) 137 : 57. Similarly, it is noted that a DNA fragment coding for this protein has been cloned and sequenced by Newell et al., Micro-biol. Ecology in Health and Disease (1991) 4 : Supplement S120, H3-1.
A catalase which is useful for the purposes of the present invention may have retained its catalase activity or may have lost it by mutation, preferably by point mutation.
A catalase which is useful for the purposes of the present invention may be encapsulated in a delivery vector such as liposomes; this is particularly advant-ageous when the catalase is intended for oral _ - 3 -administration.
A composition according to the invention may also contain an H. pylori antigen other than a catalase; for example a urease.
A composition according to the invention may be manufactured conventionally. In particular, the catalase is combined with an adjuvant, a diluent or a vehicle which is pharmaceutically acceptable. A composition according to the invention may be administered by any conventional route in use in the field of vaccine~, in particular via the oral or parenteral route. The adminis-tration may take place in a ~ingle dose or repeated one or more times after a certain delay interval. The appro-priate dosage varies depen~;ng on variou~ parameters, for example the individual treated or the mode of administra-~ tion.
Example 1 : Purification of a catalase from H. pylori Culture:
A 25 cm2 fla~k contA;n;ng a two-pha~e medium i8 seeded with a strain of H. pylori No. ATCC 43579 (avail-able from the ATCC, 12301 Parklawn Drive, Rockville MD -USA) stored in glycerol at -70C. The two-phase medium comprises a solid phase consisting of 10 ml of Trypticase soya agar (Difco) supplemented with 6% fresh sheep blood and a liquid pha~e consisting of 3 ml of Trypticase ~oya broth (Difco) contA;n;ng 20% foetal calf serum. The flasks are placed in a leaktight sack known as a "gener-bag" (BBL) and are incubated with gentle rotary stirring at 37C for 48 hours under microaerophilic conditions (8-10% CO2, 5-7% 2 and 85-87% N2) obtained by the Microaer System (BBL). After culturing for 48 hours, a sub-culture is prepared under the same conditions, in order to increase the biomass. The bacteria are harvested by centrifugation and are washed with PBS (Biomérieux) in order to remove the residual culture medium.
Purification:
The washed bacterial pellet is resuspended in 50 mM pH 7.5 sodium phosphate buffer cont~;n;ng 100 ~M
2~ 64729 _ -- 4 PMSF (phenylmethylsulphonyl fluoride, Sigma) (buffer A) to a final concentration of 0.1 g (wet weight) per millilitre. The 6uspension is homogenized u~ing an Ultraturrax-type mixer. The bacterial cells are then ruptured by sonication with a Sonifier-type machine (Branson) fitted with a probe 1.8 cm in diameter. The sonication i8 carried out intermittently, sonicating for 1 min and 8tAn~; ng on ice for 1 min. A 10-min sonication i8 sufficient to rupture completely 5 g of suspended bacteria. The lysate thus obtained i8 centrifuged for 15 min at 4C and at 4000 g. The supernatant is recovered and then recentrifuged at 100,000 g for 30 min at 4C.
The supernatant of this second centrifugation (S2) i8 recovered for chromatographic purification. The fraction S2 prepared in this way retains about 90% of the total "catalase" enzyme activity, as measured according to the technique of Hazell et al. (above) or Beers & Sizer, J. Biol. Chem. (1952) 195 : 133.
The fraction S2 is loaded onto an S-Sepharose column (Pharmacia) preequilibrated with buffer A. The column is washed with the same buffer. The chromatography is monitored by a W detector at 280 nm for the proteins and by the enzyme activity for the catalase. After removal of the non-bound proteins (the absorption at 280 nm returns to the base line), the column i8 then washed with a 0 to lM gradient of NaCl in buffer A. The fractions correspon~i ng to the peak of the catalase activity are collected and concentrated in an Amicon-type concentration cell fitted with a membrane whose cutoff threshold i8 100~000 daltons. The concentrated fraction thus obtained is loaded onto a Sephacryl S-300 HR column preequilibrated with PBS buffer. The fractions cont~in;ng the catalase activity are collected, concentrated to 1 mg/ml and dialysed against the PBS buffer. The final solution is filtered on a membrane of porosity 0.22 ~m and stored at -70C.
The protein thus purified has the following characteristics:
(1) A typical catalase enzyme activity, in the absence of peroxidase activity.
(2) A visible spectrum typical of a haemoprotein, a Soret peak at 406 nm and alpha and beta peaks between 520 nm and 550 nm.
The subject of the present invention is a pharma-ceutical composition intended for the treatment or pre-vention of Helicobacter pylori infections.
N. pylori is a gram-negative bacterium to date found exclusively at the surface of the mucous membrane of the stomach in man, and more particularly around the crater lesions of gastric and duodenal ulcers. This bacterium was initially called Campylobacter pyloridis (Warren & Marshall (1983) Lancet 1 : 1273-1275).
H. pylori is currently recognized as being the aetiological agent of antral gastriti8, and appears to be one of the cofactors required for the development of ulcers. Moreover, it appears that the development of gastric carcinomas may be associated with the presence of H. pylori.
Various factors contribute to the infection. This bacterium is endowed in particular with high urease activity imparted by a urease which allows it to survive in the acidic environment of the stomach. Moreover, the flagellae make it mobile, allowing it to cross the mucus in order to reach its ecological niche and to adhere to the mucous m~hrane via adhesins.
In spite of an appreciable serous immune response and a very pronounced inflammatory response, the bacterial infection is persistent. One very likely hypothesis gives a catalase produced by the pathogenic agent a central role in the chronicity of the infection.
The reason for this is that catalases (EC 1.11.1.6) are, in general, important enzymes for the survival of bacteria in the presence of H2O2. Thus, in the present case, a catalase would allow H. pylori to escape the toxic ef$ects of the oxygen radicals produced during the inflammatory ph~om~no~.
One catalase has already been characterized in H. pylori (Hazell et al., J. Gen. Microbiol. (1991) 137 : 57). This is a tetramer whose subunit has a molecular _ - 2 -weight of about 50-60 kD. The N-terminal sequence of such a catalase has also been established (Westblom et al., Eur. J. Clin. Microbiol. Inf. Dis. (1992) 11 (6) : 522).
However, it is not impossible for this bacterium to possess several catalases, which are slightly different from one another.
It has now been found that an N. pylori catalase could be used as a substance for immunizing against the pathogenic agent itself, and could be useful in particu-lar for prophylactic or therapeutic purposes.
Accordingly, the subject of the invention is acomposition for immunizing against N. pylori which com-prises, as active principle, an N. pylori catalase in substantially purified form.
The term "substantially purified catalase" means a catalase preparation devoid of the majority of the H. pyl ori cell constituents.
A catalase which is useful for the purposes of the present invention may either be obtained by extrac-tion from H. pylori or may be obtained via a recombinantroute, by expression of the correspo~;n~ DNA fragment, in a heterologous system, bacteria, yeasts or mammalian cells.
To this end, it is shown that a catalase may be purified from N. pylori according to a technique which is identical to or similar to that described by Hazell et al., J. of Gen. Microbiol. (1991) 137 : 57. Similarly, it is noted that a DNA fragment coding for this protein has been cloned and sequenced by Newell et al., Micro-biol. Ecology in Health and Disease (1991) 4 : Supplement S120, H3-1.
A catalase which is useful for the purposes of the present invention may have retained its catalase activity or may have lost it by mutation, preferably by point mutation.
A catalase which is useful for the purposes of the present invention may be encapsulated in a delivery vector such as liposomes; this is particularly advant-ageous when the catalase is intended for oral _ - 3 -administration.
A composition according to the invention may also contain an H. pylori antigen other than a catalase; for example a urease.
A composition according to the invention may be manufactured conventionally. In particular, the catalase is combined with an adjuvant, a diluent or a vehicle which is pharmaceutically acceptable. A composition according to the invention may be administered by any conventional route in use in the field of vaccine~, in particular via the oral or parenteral route. The adminis-tration may take place in a ~ingle dose or repeated one or more times after a certain delay interval. The appro-priate dosage varies depen~;ng on variou~ parameters, for example the individual treated or the mode of administra-~ tion.
Example 1 : Purification of a catalase from H. pylori Culture:
A 25 cm2 fla~k contA;n;ng a two-pha~e medium i8 seeded with a strain of H. pylori No. ATCC 43579 (avail-able from the ATCC, 12301 Parklawn Drive, Rockville MD -USA) stored in glycerol at -70C. The two-phase medium comprises a solid phase consisting of 10 ml of Trypticase soya agar (Difco) supplemented with 6% fresh sheep blood and a liquid pha~e consisting of 3 ml of Trypticase ~oya broth (Difco) contA;n;ng 20% foetal calf serum. The flasks are placed in a leaktight sack known as a "gener-bag" (BBL) and are incubated with gentle rotary stirring at 37C for 48 hours under microaerophilic conditions (8-10% CO2, 5-7% 2 and 85-87% N2) obtained by the Microaer System (BBL). After culturing for 48 hours, a sub-culture is prepared under the same conditions, in order to increase the biomass. The bacteria are harvested by centrifugation and are washed with PBS (Biomérieux) in order to remove the residual culture medium.
Purification:
The washed bacterial pellet is resuspended in 50 mM pH 7.5 sodium phosphate buffer cont~;n;ng 100 ~M
2~ 64729 _ -- 4 PMSF (phenylmethylsulphonyl fluoride, Sigma) (buffer A) to a final concentration of 0.1 g (wet weight) per millilitre. The 6uspension is homogenized u~ing an Ultraturrax-type mixer. The bacterial cells are then ruptured by sonication with a Sonifier-type machine (Branson) fitted with a probe 1.8 cm in diameter. The sonication i8 carried out intermittently, sonicating for 1 min and 8tAn~; ng on ice for 1 min. A 10-min sonication i8 sufficient to rupture completely 5 g of suspended bacteria. The lysate thus obtained i8 centrifuged for 15 min at 4C and at 4000 g. The supernatant is recovered and then recentrifuged at 100,000 g for 30 min at 4C.
The supernatant of this second centrifugation (S2) i8 recovered for chromatographic purification. The fraction S2 prepared in this way retains about 90% of the total "catalase" enzyme activity, as measured according to the technique of Hazell et al. (above) or Beers & Sizer, J. Biol. Chem. (1952) 195 : 133.
The fraction S2 is loaded onto an S-Sepharose column (Pharmacia) preequilibrated with buffer A. The column is washed with the same buffer. The chromatography is monitored by a W detector at 280 nm for the proteins and by the enzyme activity for the catalase. After removal of the non-bound proteins (the absorption at 280 nm returns to the base line), the column i8 then washed with a 0 to lM gradient of NaCl in buffer A. The fractions correspon~i ng to the peak of the catalase activity are collected and concentrated in an Amicon-type concentration cell fitted with a membrane whose cutoff threshold i8 100~000 daltons. The concentrated fraction thus obtained is loaded onto a Sephacryl S-300 HR column preequilibrated with PBS buffer. The fractions cont~in;ng the catalase activity are collected, concentrated to 1 mg/ml and dialysed against the PBS buffer. The final solution is filtered on a membrane of porosity 0.22 ~m and stored at -70C.
The protein thus purified has the following characteristics:
(1) A typical catalase enzyme activity, in the absence of peroxidase activity.
(2) A visible spectrum typical of a haemoprotein, a Soret peak at 406 nm and alpha and beta peaks between 520 nm and 550 nm.
(3) A monomer form at 54 kD in SDS-PAGE with the following N-terminal sequence:
MVNRDVRQTTAFGAPVWDDNNVITAGPRG
Example 2 : Pharmaceutical composition intended for oral administration The protein obtained in Example 1 may be encap-sulated alone or in the presence of other H. pylori proteins, in gelatin capsules 80 as to protect the anti-gen against degradation by the gastric juices, or may be administered in the presence of sodium bicarbonate. Such formulations have already been used for pharmaceutical compositions (Black et al., Dev. Biol. Stand. (1983) 53).
The protein may also be encapsulated in PLGA microspheres (copolymers of glycolic acid and lactic acid) according to the procedure described elsewhere (Eldridge et al., Curr. Top. Microbiol. Immunol. (1989) 146 : 59-66). The protein may also be included in liposomes prepared according to the widely-described conventional methods ( nr-; po~omes: a practical approach, ed. RRC New, D. Rickwood & B.D. Hames, l990, Oxford Press, ISBN 0-19-963077-1).
Independently of the formulation, the amount of protein ~; n; stered orally to humans is of the order of from 1 to 10 mg per dose, and at least 3 doses taken with a 4-week interval between each are recommended.
Exam~le 3 : Pharmaceutical composition intended for parenteral administration The protein as in Example 1 is adsorbed onto alumina gel in an entirely conventional manner. The protein, in solution at a concentration of 1 mg/ml in a buffer whose pH is in the region of 6.5, is placed in contact for 1 hour with aluminium hydroxide at a concen-tration of 10 mg/ml measured with respect to Al+~. The final composition of the preparation is as follows:
_ - 6 -protein having catalase acti~ity 50 ~g/ml, A1+++
- 250 ~g/ml, merthiolate 1/1000, all in PBS.
AB in the case of the oral administration, 3 injections are recommended, each with a 4-week delay after the previous.
MVNRDVRQTTAFGAPVWDDNNVITAGPRG
Example 2 : Pharmaceutical composition intended for oral administration The protein obtained in Example 1 may be encap-sulated alone or in the presence of other H. pylori proteins, in gelatin capsules 80 as to protect the anti-gen against degradation by the gastric juices, or may be administered in the presence of sodium bicarbonate. Such formulations have already been used for pharmaceutical compositions (Black et al., Dev. Biol. Stand. (1983) 53).
The protein may also be encapsulated in PLGA microspheres (copolymers of glycolic acid and lactic acid) according to the procedure described elsewhere (Eldridge et al., Curr. Top. Microbiol. Immunol. (1989) 146 : 59-66). The protein may also be included in liposomes prepared according to the widely-described conventional methods ( nr-; po~omes: a practical approach, ed. RRC New, D. Rickwood & B.D. Hames, l990, Oxford Press, ISBN 0-19-963077-1).
Independently of the formulation, the amount of protein ~; n; stered orally to humans is of the order of from 1 to 10 mg per dose, and at least 3 doses taken with a 4-week interval between each are recommended.
Exam~le 3 : Pharmaceutical composition intended for parenteral administration The protein as in Example 1 is adsorbed onto alumina gel in an entirely conventional manner. The protein, in solution at a concentration of 1 mg/ml in a buffer whose pH is in the region of 6.5, is placed in contact for 1 hour with aluminium hydroxide at a concen-tration of 10 mg/ml measured with respect to Al+~. The final composition of the preparation is as follows:
_ - 6 -protein having catalase acti~ity 50 ~g/ml, A1+++
- 250 ~g/ml, merthiolate 1/1000, all in PBS.
AB in the case of the oral administration, 3 injections are recommended, each with a 4-week delay after the previous.
Claims (5)
1. A composition for immunizing against H. pylori comprising, as active principle, an H. pylori catalase, in substantially purified form.
2. A composition according to Claim 1, in which the said catalase has lost its enzyme activity by mutation.
3. A composition according to Claim 1 or 2, in which the said catalase is encapsulated in a delivery vector such as liposomes.
4. A composition according to one of Claims 1 to 3, which also comprises an adjuvant such as alum.
5. A composition according to one of Claims 1 to 4, which also comprises an H. pylori antigen other than a catalase, for example urease.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9404172A FR2719998B1 (en) | 1994-04-08 | 1994-04-08 | Anti-helicobacter pylori immunizing composition based on catalase. |
| FR94/04172 | 1994-04-08 | ||
| PCT/FR1995/000383 WO1995027506A1 (en) | 1994-04-08 | 1995-03-27 | Catalase-based anti-helicobacter pylori immunising composition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2164729A1 true CA2164729A1 (en) | 1995-10-19 |
Family
ID=9461897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002164729A Abandoned CA2164729A1 (en) | 1994-04-08 | 1995-03-27 | Catalase-based anti-helicobacter pylori immunising composition |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0702565B1 (en) |
| JP (1) | JPH08511282A (en) |
| AT (1) | ATE187339T1 (en) |
| CA (1) | CA2164729A1 (en) |
| DE (1) | DE69513760T2 (en) |
| DK (1) | DK0702565T3 (en) |
| ES (1) | ES2140673T3 (en) |
| FR (1) | FR2719998B1 (en) |
| GR (1) | GR3032649T3 (en) |
| PT (1) | PT702565E (en) |
| WO (1) | WO1995027506A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AUPM612494A0 (en) | 1994-06-08 | 1994-06-30 | Csl Limited | Treatment or prevention of helicobacter infection |
| EP0745674B1 (en) * | 1995-06-02 | 2006-07-05 | Eisai Co., Ltd. | Helicobacter polypeptides, their production and use |
| US6551779B1 (en) | 1996-05-31 | 2003-04-22 | Tosiro Sugiyama | Helicobactor catalase nucleotide sequences, their production and use |
| AU3762397A (en) * | 1996-08-15 | 1998-03-06 | Csl Limited | Treatment and prevention of helicobacter infection |
| BR0015145A (en) * | 1999-10-29 | 2002-07-16 | Wakamoto Pharma Co Ltd | Monoclonal antibody, hybridoma, immunoassay method and diagnostic kit |
| WO2002088737A1 (en) * | 2001-04-23 | 2002-11-07 | Wakamoto Pharmaceutical Co., Ltd. | Immunochromatographic test piece and diagnosis kit |
| JP4763149B2 (en) * | 2001-05-10 | 2011-08-31 | わかもと製薬株式会社 | Test method to determine infection with Helicobacter pylori |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2637612B1 (en) * | 1988-10-06 | 1993-09-10 | Pasteur Institut | NUCLEOTIDE SEQUENCES ENCODING A PROTEIN WITH UREASIC ACTIVITY |
-
1994
- 1994-04-08 FR FR9404172A patent/FR2719998B1/en not_active Expired - Fee Related
-
1995
- 1995-03-27 CA CA002164729A patent/CA2164729A1/en not_active Abandoned
- 1995-03-27 WO PCT/FR1995/000383 patent/WO1995027506A1/en not_active Application Discontinuation
- 1995-03-27 DK DK95914428T patent/DK0702565T3/en active
- 1995-03-27 ES ES95914428T patent/ES2140673T3/en not_active Expired - Lifetime
- 1995-03-27 JP JP7526111A patent/JPH08511282A/en not_active Ceased
- 1995-03-27 PT PT95914428T patent/PT702565E/en unknown
- 1995-03-27 DE DE69513760T patent/DE69513760T2/en not_active Expired - Fee Related
- 1995-03-27 EP EP95914428A patent/EP0702565B1/en not_active Revoked
- 1995-03-27 AT AT95914428T patent/ATE187339T1/en not_active IP Right Cessation
-
2000
- 2000-02-14 GR GR20000400347T patent/GR3032649T3/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| GR3032649T3 (en) | 2000-06-30 |
| DE69513760D1 (en) | 2000-01-13 |
| JPH08511282A (en) | 1996-11-26 |
| PT702565E (en) | 2000-05-31 |
| FR2719998B1 (en) | 1996-09-27 |
| FR2719998A1 (en) | 1995-11-24 |
| WO1995027506A1 (en) | 1995-10-19 |
| DK0702565T3 (en) | 2000-04-25 |
| EP0702565B1 (en) | 1999-12-08 |
| ATE187339T1 (en) | 1999-12-15 |
| DE69513760T2 (en) | 2000-06-21 |
| EP0702565A1 (en) | 1996-03-27 |
| ES2140673T3 (en) | 2000-03-01 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |