EP0797586A1 - NOUVELLE PROTEINE MEMBRANAIRE p76 D'HELICOBACTER PYLORI - Google Patents

NOUVELLE PROTEINE MEMBRANAIRE p76 D'HELICOBACTER PYLORI

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Publication number
EP0797586A1
EP0797586A1 EP96933488A EP96933488A EP0797586A1 EP 0797586 A1 EP0797586 A1 EP 0797586A1 EP 96933488 A EP96933488 A EP 96933488A EP 96933488 A EP96933488 A EP 96933488A EP 0797586 A1 EP0797586 A1 EP 0797586A1
Authority
EP
European Patent Office
Prior art keywords
protein
recovered
nacl
subjected
centrifugation
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.)
Withdrawn
Application number
EP96933488A
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German (de)
English (en)
French (fr)
Inventor
Ling Lissolo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Pasteur SA
Original Assignee
Pasteur Merieux Serum et Vaccines SA
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Filing date
Publication date
Application filed by Pasteur Merieux Serum et Vaccines SA filed Critical Pasteur Merieux Serum et Vaccines SA
Publication of EP0797586A1 publication Critical patent/EP0797586A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the subject of the present invention is in particular a protein d! Helicobacter pylori newly obtained in substantially purified form, as well as the pharmaceutical compositions containing them.
  • Helicobacter is a bacterial genus characterized by gram negative spiral bacteria. Several species colonize the gastrointestinal tract of mammals. We cite in particular H. pylori, H. heilmanii, H. felis and H. mustelae. Although H. pylori is the species most commonly associated with human infections, in some admittedly rare cases, H. heilmanii and H. felis have been isolated from humans.
  • Helicobacter infects more than 50% of the adult population in developed countries and almost 100% of that of developing countries; making it one of the predominant infectious agents worldwide.
  • H. pylori is found exclusively to date on the surface of the stomach lining in humans and more particularly around crater lesions of gastric and duodenal ulcers. This bacterium is currently recognized as the etiological agent of antral gastritis and appears as one of the cofactors required for the development of ulcers. Furthermore, it seems that the development of gastric carcinomas may be associated with the presence of H. pylori.
  • H. proteins. pylori have been characterized or isolated to date. These include urease, composed of two subunits A and B of 30 and 67 kDa respectively ( ⁇ u & Mobley, Infect. Immun. (1990) 58: 992; Dunn et al. J. Biol. Chem (1990) 265: 9464; Evans et al. Microbial Pathogenesis (1991) K): 15; Labigne et al, J. Bact, (199) 173: 1920); 87 kDa vacuolar cytotoxin (VacA) (Cover & Blaser. J. Biol. Chem. (1992) 267: 10570: Phadnis et al, Infect. Immun.
  • urease composed of two subunits A and B of 30 and 67 kDa respectively ( ⁇ u & Mobley, Infect. Immun. (1990) 58: 992; Dunn et al. J. Biol. Chem (1990) 265:
  • HpaA fibrillar hemaglutinin
  • Hpn fibrillar hemaglutinin
  • a 15 kDa protein rich in histidine Hpn
  • a 30 kDa outer membrane protein Bolin et al, J. Clin. Microbiol. (1995) 33: 381
  • a 20 kDa lipoprotein associated with the membrane Kostrcynska et al, J. Bact.
  • urease is recognized as being a first choice antigen which can be used for this purpose (WO 94/9823; WO95 / 3824; WO 95/22987; Michetti et al, Gastroenterology (1994) 107: 1002).
  • WO 94/9823 WO95 / 3824
  • WO 95/22987 Michetti et al, Gastroenterology (1994) 107: 1002
  • the subject of the invention is in particular an H. protein. pylori in substantially purified form, capable of being obtained from a membrane fraction of H. pylori and whose molecular weight after electrophoresis on 10% polyacrylamide gel in the presence of SDS. appears on the order of 76 kDa.
  • substantially purified form is meant that the protein is separated from the environment in which it exists naturally. Among others, it may be a preparation in particular devoid of the cytoplasmic and periplasmic proteins of H. pylori.
  • the membrane protein whose apparent molecular weight is of the order of 76 kDa is capable of being obtained according to a process in which:
  • the membrane fraction consisting of the centrifugation pellet is recovered, which is resuspended in an aqueous medium, advantageously in a carbonate buffer pH 9.5 containing 5% of zwittergent 3-14;
  • the membrane fraction is subjected to anion exchange chromatography on a Q-Sepharose column in 0.1 - 0.5 M NaCl gradient, advantageously in carbonate buffer pH 9.5 at 0.1% of zwittergent 3-
  • the fraction eluted is recovered in 0.25 - 0.45 M NaCl, eg in 0.25 - 0.35 M NaCl or preferably, in 0.35 - 0.45 M NaCl, which is subjected to a cation exchange chromatography on an S-Sepharose column in NaCl gradient 0 - 1 M, advantageously in acetate buffer pH 5 at 0.1% zwittergent 3-14 (advantageously, the NaCl fraction 0.25 - 0, 45 M, eg in 0.25 - 0.35 M NaCl or 0.35 - 0.45 M NaCl, is first dialyzed against acetate buffer pH 5 at 0.1% of zwittergent 3-14); and
  • the fraction eluted is recovered in NaCl 0.15 - 0.2 M. preferably in NaCl 0.15 M.
  • a fraction eluted in 0.25-0.45 M NaCl should be understood as follows: a fraction eluted at an NaCl concentration of between 0.25 and 0, 45 M.
  • the fraction eluted can therefore include material eluted between 0.25 and 0.45 M or else material eluted between two concentrations in the range from 0.25 to 0.45 M. eg 0.35 - 0.45 M .
  • the NaCl gradient applied to the S-Sepharose column is preferably a step gradient.
  • 0.1 M 0.15 M, 0.2 M and 1 M NaCl steps are used.
  • the 76 kDa membrane protein has the presumed biological function of being a porin. probably located at the level of the external membrane. Exner et al, Infect. Immun. (Apr.1995) 63: 1567 characterizes a family of porins which each present in SDS-Page electrophoresis, a different migration profile depending on whether they were heated to 95 ° C (conventional process) or not. This is not the case with regard to the 76 kDa protein according to the invention; indeed its migration on gel is not modified with a sample heated to 60 ° C and not to 95 ° C.
  • the N-terminal sequence of the 76 kDa protein of an H. strain. pylori is as follows (one letter code): EDDGFYTSVGYQIGEAAQMV.
  • allelic differences in the amino acid sequence may consist of one or more substitutions, deletions or additions of amino acids, which do not alter the biological function.
  • biological function is meant the function of the protein which participates in the survival of cells in which the protein exists naturally (even if the function is not absolutely essential).
  • the function of a porine is to allow compounds present in the external environment to enter the interior of the cell.
  • the biological function is distinct from the antigenic function.
  • a protein can have more than one biological function.
  • the subject of the invention is also a protein in substantially purified form and capable of having been purified according to the method described above from a bacterium of the genus Helicobacter e.g., H. pylori. H. heilmanii, H. felis and H. mustelae.
  • Helicobacter e.g., H. pylori. H. heilmanii, H. felis and H. mustelae.
  • the subject of the invention is also any other protein or polypeptide. in substantially purified form, insofar as it is analogous in terms of antigenicity to a protein to 'Helicobacter capable of being purified according to the process described above.
  • polypeptides these are in particular polypeptides derived by fragmentation or by mutation of one or more amino acids, eg by deletion, addition or substitution, of a protein which exists in nature and whose purified form can be obtained according to the process described above.
  • Such polypeptides can in particular be obtained by enzymatic digestion using proteases such as pepsin or trypsin. It is not necessary that such polypeptides can be purified according to the method described above.
  • polypeptide regardless of the size of the molecules (length of the amino acid chain) and any post-translational modifications.
  • polypeptide is reserved to denote a product derived from a protein by fragmentation or mutation.
  • a protein or a polypeptide according to the invention must be capable of being recognized by monospecific antibodies established against a protein d! Helicobacter capable of being purified according to the process described above
  • This specific antigenicity can be revealed by a number of methods; for example by Western biot (Towbin et al, PNAS (1979) 76: 4350). dot biot and ELISA.
  • the product intended to be tested eg either in the form of a purified preparation, or in the form of a bacterial extract
  • SDS-Page gel polyacrylamide 10%
  • the latter is incubated with a hyperimmune monospecific serum diluted in the range of dilutions from 1: 50 to 1: 5000, preferably from 1: 100 to 1: 500
  • the specific antigenicity is demonstrated as soon as a band corresponding to the product tested exhibits reactivity to one of the dilutions included in the range established above.
  • the product to be tested is preferably used to cover the wells. It is preferred to use a purified preparation, although a total extract may also be used.
  • 100 ⁇ l of a preparation at 10 ⁇ g of protein / ml are distributed in the wells of a 96-well plate. The plate is incubated for 2 hrs at 37 ° C. and then overnight at 4 ° C. The plate is washed with PBS buffer (phosphate buffered saline) containing 0.05% of Tween 20 (PBS / Tween buffer). The wells are saturated with 250 ⁇ l of PBS containing 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the reaction is measured by colorimetry (absorbance measured by spectrophotometry). Under these conditions, a positive reaction is observed when an OD value of 1 is associated with a dilution of at least 1:50, preferably at least 1: 500.
  • the appropriate wavelength at which the optical density depends on the substrate.
  • a preparation of the product to be tested with 100 ⁇ g of protein / ml is diluted in series twice in 50 mM Tris-HCl pH 7.5.
  • 50 mM Tris-HCl pH 7.5 One hundred ⁇ l of each dilution is applied to a 0.45 ⁇ m nitrocellulose membrane in a 96-well biot dot apparatus (Biorad).
  • the buffer is removed by vacuum.
  • the wells are washed by adding 50 mM Tris-HCl pH 7.5 and the membrane is air dried.
  • the membrane is saturated with blocking buffer (Tris-HCl 50 mM pH 7.5, NaCl 0.15 M.
  • the reaction is revealed according to standard methods. For example, a rabbit anti-immunoglobulin goat peroxidase-immunoglobulin conjugate is added to the wells. The incubation is continued for 90 min at 37 ° C., then the plate is washed. The reaction is developed with the appropriate substrate. The reaction is measured by colorimetry or chemiluminescence.
  • a reaction is positive when a coloration is observed at the level of the deposition on the nitrocellulose sheet directly for revelation by colorimetry or on photographic film for revelation by chemiluminescence, associated with a dilution of at least 1:50, preferably at least 1: 500.
  • a protein according to the invention can in particular be obtained by purification from Helicobacter or expressed by recombinant way in a heterologous system (which can also be the case for a polypeptide according to the invention).
  • the protein may have post-translational modifications which are not identical to those of the corresponding protein derived from the original strain.
  • the therapeutic or prophylactic efficacy of a protein or a polypeptide according to the invention can be evaluated according to standard methods; for example by measuring the induction of a mucosal immune response or the induction of an immune response with a therapeutic or protective effect using e.g. the mouse / H. felis model and the procedures described in Lee et al, Eur. J. Gastroenterology & ⁇ epatology, (1995) 7: 303 or Lee et al, J. Infect. Say. (1995) 172: 161, provided that the following precaution is taken: When the protein originates from a species other than H felis, the strain of H.
  • felis must be replaced by a Helicobacler strain belonging to the species from which the protein originates and adapted for this purpose (the other experimental conditions remaining identical).
  • a Helicobacler strain belonging to the species from which the protein originates and adapted for this purpose (the other experimental conditions remaining identical).
  • the capacity of a polypeptide derived by fragmentation of an H. protein is tested.
  • pylori to induce a protective or therapeutic effect, by substituting a strain of H. pylori.
  • Such a strain is proposed by e.g. Kleanthous et al, Abstr. presented at the VlIIth International Workshop on Gastroduodenal Pathology 7-9th July 1995, Edinburgh, Scotland.
  • a protective effect is noted when an infection in the gastric tissue is less compared to a control group. Infection is assessed by testing urease activity. bacterial load or leukocyte infiltration. For example, when a reduction in urease activity in the gastric tissue is found after the test, even if it is
  • the invention also relates to (i) a composition of matter comprising a protein or a polypeptide according to the invention and a diluent or a support; in particular (ii) a pharmaceutical composition in particular intended for the prevention or treatment of a Helicobacter infection, which comprises, as active principle, a protein or a polypeptide according to the invention, in an amount effective from a prophylactic point of view or therapeutic; (iii) the use of a protein or a polypeptide according to the invention as a therapeutic or prophylactic agent; (iv) the use of a protein or a polypeptide according to the invention for the manufacture of a medicament intended for the prevention or the treatment of a Helicobacter infection; as well as (v) a method of inducing an immune response against Helicobacler eg, H.
  • a composition of matter comprising a protein or a polypeptide according to the invention and a diluent or a support
  • a mammal is administered an immunologically effective amount of a protein or polypeptide according to the invention in order to to develop an immune response; in particular (vi) a method of preventing or treating a Helicobacterllc infection, an individual is administered a prophylactically or therapeutically effective amount of a protein or a polypeptide according to the invention.
  • the methods and pharmaceutical compositions according to the invention can treat or prevent infections with Helicobacter and consequently, gastrointestinal diseases associated with such infections. These include, in particular, acute chronic atrophic gastritis; peptic ulcers e.g. gastric and duodenal ulcers; gastric cancers; chronic hyperopia; refractory non-ulcerative dyspepsia; intestinal metaplasia and certain lymphomas (e.g. low grade MALT lymphoma).
  • peptic ulcers e.g. gastric and duodenal ulcers
  • gastric cancers chronic hyperopia
  • refractory non-ulcerative dyspepsia e.g. low grade MALT lymphoma
  • intestinal metaplasia e.g. low grade MALT lymphoma
  • a composition according to the invention can be administered by any conventional route in use in the field of vaccines, in particular through a mucosal surface (eg ocular, nasal, oral, gastric, intestinal, rectal, vaginal, or of the urinary tract) or parenterally (eg subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal).
  • a mucosal surface eg ocular, nasal, oral, gastric, intestinal, rectal, vaginal, or of the urinary tract
  • parenterally eg subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal.
  • the choice of administration route depends on a certain number of parameters such as the adjuvant associated with the protein or the polypeptide according to the invention. For example, if a mucosal adjuvant is used, the nasal or oral route is preferred. If a lipid formulation is used, the parenteral route will be chosen. preferably the subcutaneous or intramuscular route.
  • a composition according to the invention can comprise, in addition to a protein or a polypeptide according to the invention, at least one other Helicobacler antigen such as the apoenzyme of urease, or a subunit, fragment, homologous, mutant or derivative. of this urease.
  • a protein or a polypeptide according to the invention can be formulated in or with liposomes, preferably neutral or anionic liposomes. microspheres. ISCOMs or viral pseudo-particles (VLPs), to promote targeting of the protein or polypeptide or to increase the immune response.
  • liposomes preferably neutral or anionic liposomes. microspheres. ISCOMs or viral pseudo-particles (VLPs)
  • VLPs viral pseudo-particles
  • Adjuvants other than liposomes can also be used. Many are known to those skilled in the art. Such adjuvants are referenced below
  • aluminum compounds such as aluminum hydroxide, aluminum phosphate and aluminum hydroxyphosphate are mentioned.
  • the antigen can be adsorbed or precipitated on an aluminum compound according to standard methods.
  • Other adjuvants such as the RIBI from ImmunoChem (Hamilton, MT) can be used for parenteral administration.
  • bacterial toxins e.g. cholera toxin (CT), toxin from E. coli. heat labile coli (LT) Clostridium difficile toxin and pertussis toxin (PT) as well as the detoxified forms (subunit, toxoid or mutant) of these toxins.
  • CT cholera toxin
  • LT heat labile coli
  • PT pertussis toxin
  • a preparation containing the CT subunit B (CTB) and a smaller amount of CT can be used.
  • Fragments, homologs and derivatives of these toxins are likewise suitable insofar as they retain an adjuvant activity.
  • a mutant having reduced toxicity is used.
  • Such mutants are described eg in WO 95/1721 1 (Arg-7-Lys CT mutant), WO 95/34323 (Arg-9-Lys Glu-129-Gly PT mutant) and WO 96/6627 (Arg-192- Gly LT mutant).
  • Other adjuvants such as the major bacterial lipopolysaccharide (MPLA) from e.g. E. coli. Salmonella minnesota, Salmonella typhimurium or Shigella flexneri, can be used for mucosal administration.
  • MPLA major bacterial lipopolysaccharide
  • Adjuvants useful for both mucosal and parenteral administration include in particular polyphosphazene (WO 95/2415).
  • DC-chol (3 beta-fN- (N'.N'-dimethyl aminomethane) -carbamoyl] cholesterol) (USP 5,283,185 and WO 96/14831) and QS-21 (WO 88/9336).
  • the administration can take place in single or repeated dose one or more times after a certain delay.
  • the appropriate dosage varies according to various parameters, for example, the individual treated (adult or child) of the vaccine antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and if present, the type of adjuvant and the desired effect (eg protection or treatment), as can be determined by those skilled in the art.
  • an antigen according to the invention can be administered in an amount ranging from 10 ⁇ g to 500 mg, preferably from 1 mg to 200 mg.
  • a parenteral dose should not exceed 1 mg, preferably 100 ⁇ g. Higher doses may be prescribed for eg oral use.
  • the amount of protein administered to humans by the oral route is, for example, of the order of 1 to 10 mg per dose, and at least 3 doses are recommended at 4-week intervals.
  • a composition according to the invention can be produced in a conventional manner.
  • a protein or a polypeptide according to the invention is combined with a diluent or a support which is acceptable from a pharmaceutical point of view, eg water or a saline solution such as a phosphate salt buffer (PBS), supplemented. optionally with a bicarbonate salt such as sodium bicarbonate eg 0.1 to 0.5 M when the composition is intended for oral or intragastric administration.
  • a diluent or carrier is selected based on the mode and route of administration and standard pharmaceutical practices. Pharmaceutically acceptable diluents and carriers and all that is necessary for their use in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, a standard reference text in this area and in the USP / NP.
  • a protein or a polypeptide according to the invention can be encapsulated alone or in the presence of other H. pylori proteins in gelatin capsules in order to protect the antigen against degradation by gastric juice or else administered in presence of sodium bicarbonate.
  • Such formulations have already been used for pharmaceutical compositions (Black et al, Dev. Biol. Stand. (1983) 5_3: 9).
  • the protein can also be encapsulated in PLGA microspheres (copolymers of glycolic acid and lactic acid) according to the protocol described elsewhere (Eldridge et al, Curr.
  • the protein can also be included in liposomes prepared according to conventional methods widely described ("Liposomes: a practical approach. Ed. RRC New. D. Rickwood & B.D. ⁇ ames,
  • a protein or polypeptide according to the invention can be administered parenterally.
  • a protein or polypeptide according to the invention is adsorbed on alumina gel in a completely conventional manner.
  • the protein in solution at 1 mg / ml in a buffer whose p ⁇ is close to 6.5 is brought into contact for 1 hour with aluminum hydroxide at 10 mg / ml measured at AL " 1 " * -1 '.
  • the final composition of the preparation is as follows: protein 50 ⁇ g / ml, AL +++ 250 ⁇ g / ml, merthiolate 1 / 10,000, all in PBS. As in the case of oral administration, 3 injections are recommended each spaced 4 weeks from the previous one.
  • a polypeptide according to the invention can also be useful as a diagnostic reagent, for example for detecting the presence of anti-Helicobacter antibodies in a biological sample e.g. a blood sample.
  • a polypeptide advantageously comprises 5 to 80 amino acids, preferably 10 to 50 amino acids.
  • a polypeptide reagent according to the invention may or may not be labeled, depending on the diagnostic method used. Diagnostic methods are described further in the text.
  • the invention provides a monospecific antibody capable of recognizing a protein or a polypeptide according to the invention.
  • an antibody capable of reacting mainly with a single Helicobacler protein. Such an antibody can only be obtained by using a substantially purified protein for immunogen.
  • An antibody according to the invention can be polyclonal or monoclonal; the monoclonals can be chimeric (for example, constituted by a variable region of murine origin associated with a constant human region) or humanized (only the hypervariable regions are of animal origin, for example of murine origin) and / or by a single chain.
  • the polyclonal as the monoclonal can also be in the form of fragments of immunoglobulins for example a fragment F (ab) '2 or Fab.
  • An antibody according to the invention can also be of any isotype, for example IgG or IgA; a polyclonal can be of a single isotype or a mixture of all or part of them.
  • An antibody which is directed against a protein according to the invention can be produced and subsequently identified using a standard immunoassay, for example analysis by Western biot, dot biot or ELISA (see for example Coligan et al Current Protocols in Immunology (1994) John Wiley & sons Inc., New York,
  • An antibody according to the invention can be useful in diagnosis, as well as in affinity chromatography for purifying on a large scale, a protein or a polypeptide according to the invention; such an antibody is also potentially useful as a therapeutic agent in a passive immunization procedure.
  • the invention also provides (i) a reagent for detecting the presence of Helicobacler in a biological sample, which comprises an antibody or a polypeptide according to the invention; and (ii) a diagnostic method for detecting the presence of Helicobacler in a biological sample, according to which the biological sample is brought into contact with an antibody or a polypeptide according to the invention, so that an immune complex is formed: optionally, the unbound material is eliminated, and the immune complex formed between the sample and the antibody or the polypeptide according to the invention is detected, as an indicator of the presence of Helicobacler in the sample or in the organ from which the sample was taken.
  • an antibody according to the invention makes it possible to test the presence of Helicobacler in a gastric extract.
  • the reagent to be presented in free form or immobilized on a solid support can be any support commonly used in this field, for example, a tube, a ball or a well.
  • Immobilization can be obtained by direct or indirect means.
  • the direct means include passive adsorption (non-covalent bond) or covalent bonds between the support and the reagent.
  • indirect means it is meant that an anti-reagent compound capable of interacting a reagent is first of all attached to a solid support. For example, if a polypeptide reagent is used.
  • an antibody capable of binding it can be used as an anti-reagent, provided that it can bind to an epitope of the polypeptide which is not involved in the recognition of the antibodies present in the biological samples.
  • the indirect means can also be implemented via a ligand-receptor system, for example by grafting a molecule, such as a vitamin, onto a polypeptide reagent and then immobilizing the corresponding receptor in solid form. This is illustrated eg by the biotin-streptavidin system.
  • indirect means are used for example by adding a peptide tail to the reagent eg by means chemical, and by immobilizing the grafted product by passive adsorption or by covalent bonding of the peptide tail.
  • the invention also relates to a method for purifying a protein or a polypeptide according to the invention from a biological sample, according to which the biological sample is subjected to an affinity chromatography using a monospecific antibody according to the invention.
  • the antibody can be polyclonal or monoclonal, preferably of the IgG type.
  • Purified IgGs can be prepared from an antiserum according to commonly practiced methods (see for example Coligan et al).
  • a biological sample preferably in a buffer solution
  • chromatography equipment preferably equilibrated with the buffer used for diluting the biological sample so that the protein or the polypeptide according to the invention (antigen ) can be adsorbed on the material.
  • the chromatography equipment such as a gel or a resin associated with an antibody according to the invention, can be in the form of a bath or a column.
  • the components which remain unbound are eliminated by washing and the antigen is then eluted in an appropriate elution buffer, such as for example, a glycine buffer or a buffer containing a chaotropic agent eg guanidine HCl, or a rich concentration. salt (for example, 3M MgCU).
  • the eluted fractions are recovered and the presence of the antigen is then demonstrated, for example by measuring the absorbance at 280 nm.
  • the subject of the invention is also (i) a composition of matter comprising a monospecific antibody according to the invention, and a diluent or a support; in particular, (ii) a pharmaceutical composition comprising a monospecific antibody according to the invention in an amount effective from a therapeutic or prophylactic point of view; (iii) the use of a monospecific antibody according to the invention in the preparation of a medicament to treat or prevent Helicobacter infection; as well as (iv) a method for treating or preventing a Helicobacter infection (for example, H. pylori, H. felis, H. musielae or H. heilmanii), according to which a therapeutically or prophylactically effective amount of an antibody is administered according to the invention, to an individual in need of such treatment.
  • a Helicobacter infection for example, H. pylori, H. felis, H. musielae or H. heilmanii
  • the monospecific antibody can be polyclonal or monoclonal, preferably of the IgA isotype (predominantly).
  • the antibody is administered mucosally to a mammal, for example in the gastric mucosa, either orally or intragastrically, advantageously in the presence of a bicarbonate buffer.
  • a monospecific antibody according to the invention can be administered as the sole active component or as a mixture comprising at least one monospecific antibody specific to each Helicobacler polypeptide.
  • the dose of antibody to be used in this method can be readily determined by those of skill in the art. For example, it is indicated that a dosage can be characterized by a daily administration of between 100 and 1000 mg of antibody for one week; or a dose comprising 100 to 1000 mg of antibody administered three times a day for two to three days.
  • a pharmaceutical composition comprising an antibody according to the invention can be produced according to the rules set out above for a composition comprising a protein or a polypeptide according to the invention. Likewise, identical medical indications apply.
  • FIG. 1 presents the analysis of the Cg2d P membrane fraction by electrophoresis on 10% polyacrylamide gel and staining with Coomassie blue. The molecular weight markers appear in column 1.
  • FIG. 3 shows, after electrophoresis on 10% polyacrylamide gel and staining with Coomassie blue, the electrophoretic profile of fraction D resulting from chromatography on a Q-Sepharose column of the membrane fraction C ⁇ 2d (column 3) and of the D fraction from chromatography on an S-Sepharose column from fraction D (column 4).
  • Column 1 corresponds to the molecular weight markers and column 2 to the membrane fraction Cg2d-
  • the H. pylori ATCC 43579 strain is cultured in a liquid medium in a 10 L fermenter.
  • a glycerol germ freeze is used to inoculate a 75 cm ⁇ flask containing so-called "biphasic" medium (a solid phase in Colombia agar containing 6% fresh sheep blood and a liquid phase in soy trypticase containing 20% fetal calf serum). After 24 hours of culture under microaerophilic conditions, at 37 ° C. the liquid phase of this culture is used to inoculate several 75 cm ⁇ flasks in a biphasic medium in the absence of sheep's blood. After 24 hours of culture, the liquid phase makes it possible to inoculate a biofermenter of 2 1 in a soy trypticase liquid medium containing beta cyclodextrin at 10 g / 1.
  • This culture with DO 1.5-1.8 is inoculated in a 10 1 fermenter in liquid medium. After 24 hours of culture, the bacteria are harvested by centrifugation at 4000 x g for 30 minutes at 4 ° C. A culture of 10 liters of H. pylori ATCC 43579 in a fermenter makes it possible to obtain approximately 20 to 30 g (wet weight) of bacteria.
  • the pellet of germs obtained previously is washed with 500 ml of PBS (phosphate buffered saline; NaCl 7.650 g, disodium phosphate 0.724 g monopotassium phosphate 0.210 g for one liter: p ⁇ 7.2) per liter of culture. Then the germs are again centrifuged under the same conditions.
  • the bacterial pellet obtained (Cj) is resuspended in a 1% solution of OG (Sigma) (30 ml / liter of culture). The bacterial suspension is incubated for 1 hour at room temperature with magnetic stirring, then centrifuged at 17,600 xg for 30 minutes at 4 ° C.
  • the precipitate formed during the dialysis is recovered by centrifugation at 2,600 x g for 30 minutes at 4 ° C.
  • the supernatant (S2d) is eliminated and the pellet (C ⁇ 2d) ⁇ contains membrane proteins, is stored at -20 ° C.
  • the resuspension of the pellet is carried out in 20 mM Tris-HCl buffer pH 7.5 and Pefabloc 100 ⁇ M (buffer A).
  • the membrane fraction C £ 2d is analyzed by electrophoresis on polyacrylamide gel in the presence of SDS according to the method of Laemmli (1970). The proteins are visualized after staining with Coomassie blue.
  • the protein profile of the membrane fraction shows the presence of 4 major bands at 76, 67, 50 and 30 kDa ( Figure 1. column 4).
  • Electrophoresis is carried out on polyacrylamide gel according to the method of Laemmli (1970) with a gel of concentration of 5% and a separation gel of 10%.
  • the membrane fraction is resuspended in buffer A, then diluted to half in the 2X sample buffer. The mixture is heated for 5 minutes to 95 ° C.
  • About 19 mg of protein are deposited on a gel with a dimension of 16 x 12 cm and a thickness of 5 mm.
  • Premigration is carried out at 50 V for 2 hours, followed by migration at 65 V overnight.
  • the coloring of the gel with Coomassie R250 blue (0.05% in ultrafiltered water) allows good visualization of the bands.
  • the 76 kDa major band is cut with a scalpel and ground with ultra-turrax in the presence of 10 or 20 ml of 25 mM Tris-HCl extraction buffer pH 8.8, 8 M urea, 10% SDS, phenyl methyl 100 ⁇ M fluoric sulfonyl (PMSF) and 100 ⁇ M Pefabloc (buffer C).
  • the filtrate is precipitated with 3 volumes of a 50:50 mixture of 75% methanol and 75% isopropanol, then ultracentrifuged at 240,000 x g for 16 hours at 10 ° C. on a 70 TFT rotor (J8-55, Beckman).
  • the pellet is taken up in 2 ml of 10 mM NaP04 solubilization buffer pH 7.0, 1 M NaCl, 0.1% Sarkosyl, 100 ⁇ M PMSF, 100 ⁇ M Pefabloc and 6 M urea (buffer D).
  • the solubilized sample is dialyzed successively against 100 ml of buffer D with 4 M urea and 0.1% of Sarkosyl, against 100 ml of buffer D with 2 M of urea and 0.5% of Sarkosyl and against 2 times 100 ml of buffer D without urea and 0.5% Sarkosyl. Dialysis is carried out for 1 hour with magnetic stirring at room temperature.
  • the final dialysate is incubated for 30 minutes in an ice bath, then centrifuged at low speed for 10 minutes at 4 ° C (Biofuge A. Heraeus Sepatech). The supernatant is recovered, filtered on a Millipore filter at 0.45 ⁇ m and stored at -20 ° C.
  • the protein balance shows that 55% of the proteins are eluted during the 0.1-0.5 M NaCl gradient, 15% are eluted during the washing with 0.1 M NaCl and 15% are eluted during the washing with NaCl. 1 M.
  • Fraction D or E dialyzed beforehand against the equilibration buffer (50 mM acetate pH 5.0, Pefabloc 100 ⁇ M and Zwittergent 3-14 0.1%) is deposited on the S-Sepharose column. The column is then washed with the same buffer until the absorbance at 280 nm is stabilized. About 3 column volumes are required to return to the baseline.
  • the proteins are eluted by a gradient of 0 to 0.5 M NaCl in equilibrium buffer (10 times VT), followed by washing with the equilibrium buffer containing 0.5 and 1 M NaCl (4 times VT). The collected fractions are analyzed, collected and stored as before.
  • the fraction D 'or E' leaving during the 0-0.5 M NaCl gradient, between 0.15 and 0.20 M NaCl, preferably at the concentration of 0.15 M NaCl, is recovered.
  • an analysis by SDS-PAGE and by Western biot is carried out. The results are shown in Figure 3.
  • EXAMPLE 4 Preparation of a hyperimmune serum against the 76 kDa membrane protein.
  • a polyclonal serum specific for the major membrane protein of H. pylori is obtained by hyperimmunization of rabbits respectively with the antigen purified by preparative SDS-PAGE.
  • the first OJ injection multisite and intramuscular subcutaneous is carried out with a preparation containing 50 ⁇ g of membrane protein emulsified in complete Freund's adjuvant, then the reminders J21 and J42 are made by injection of 25 ⁇ g of membrane protein in incomplete adjuvant Freund.
  • the animals are sacrificed on D60.
  • the sera obtained are decomplemented for 30 minutes at 56 ° C and sterilized by filtration on a membrane with a porosity of 0.22 ⁇ m (Millipore).
  • the antiserum reacts with the 76 kDa protein as isolated according to Example 3.
  • a monospecific serum can be obtained in an equivalent manner using a preparation purified according to the method described in 3.B from fraction E obtained after chromatography on Q-Sepharose (3. A) and corresponding to the fraction eluted in 0.15 M NaCl on S-Sepharosis.
  • a hyperimmune serum as prepared in Example 4 is placed on a Protein A Sepharose 4 Flast Flow column (Pharmacia) previously balanced in Tris- ⁇ Cl 100 mM p ⁇ 8.0.
  • the resin is washed with 10 column volumes of Tris- ⁇ CI 100 mM p ⁇ 8.0 then with 10 column volumes of Tris- ⁇ Cl 10 mM p ⁇ 8.0.
  • the IgGs are eluted in 0.1 M glycine buffer p ⁇ 3.0. IgGs are collected as 5 ml fractions to which 0.25 ml of 1 M Tris- ⁇ Cl p ⁇ 8.0 is added.
  • the optical density of the eluate is measured at 280 nm and the fractions containing the IgGs are combined and if necessary, frozen at -
  • CNBr - activated Sepharose 4B gel (knowing that 1 g of dry gel gives approximately 3.5 ml of hydrated gel and that the capacity of the gel is 5 to 10 mg of IgG per ml of gel) manufactured by Pharmacia ( ref: 17-0430-01) is suspended in 1 mM NaCl buffer. The gel is then washed using a buchner by adding small amounts of 1 mM HCl. The total volume of 1 mM HCl used is 200 ml per gram of gel.
  • the purified IgGs are dialyzed for 4 hrs at 20 + 5 ° C against 50 vol. 500 mM sodium phosphate buffer pH 7.5. They are then diluted in 500 mM sodium phosphate buffer pH 7.5 to a final concentration of 3 mg / ml.
  • the IgGs are incubated with the gel overnight at 5 + 3 ° C with rotary shaking.
  • the gel is placed in a chromatography column and washed with 2 vol. column of 500 mM phosphate buffer pH 7.5.
  • the gel is then transferred to a tube and incubated in 100 mM ethanolamine pH 7.5 at room temperature with stirring. It is then washed by 2 vol. PBS column.
  • the gel can be stored in PBS merthiolate 1 / 10,000.
  • the amount of IgGs coupled to the gel can be determined by measuring the difference in optical density at 280 nm from the initial solution of IgGs and the direct eluate plus washes .
  • a protein preparation of antigen in Tris-HCl 50 mM pH 8.0 EDTA 2 mM, for example the Cg2d fraction obtained in 1.B is filtered through a 0.45 ⁇ m membrane and is then deposited on the balanced column beforehand with 50 mM Tris-HCl pH 8.0 2 mM EDTA. at a flow rate of approximately 10 ml / hr.
  • the column is washed with 20 vol. 50 mM Tris-HCl pH 8.0 2 mM EDTA.
  • the adsorption can be carried out in a bath; incubation is continued at 5 + 3 ° C overnight and with shaking.
  • the gel is washed with 2 to 6 vol. 10 mM sodium phosphate buffer, pH 8.
  • the antigen is eluted with 100 mM glycine buffer pH 2.5.
  • the eluate is collected in 3 ml fractions to which are added 150 ⁇ l of 1 M sodium phosphate buffer, pH 8.0.
  • the optical density of each fraction is measured at 280 nm; the fractions containing the antigen are pooled together and stored at -70 ° C.
  • a 25 cm 2 bottle containing a two-phase medium is inoculated.
  • the biphasic medium comprises a solid phase consisting of 10 ml of Colombia agar (BioMérieux) supplemented with 6% of fresh sheep blood and a liquid phase consisting of 3 ml of Trypticase soy broth (Difco) containing 20% of fetal calf serum.
  • the bottles are placed in a waterproof bag called
  • This 48 hour culture is used to re-inoculate vials containing biphasic medium.
  • the initial absorbance of this culture at 600 nm must be between 0.15 and 0.2.
  • the flasks are incubated under conditions identical to those described above.
  • the bacterial suspension is transferred to a test tube.
  • the absorbance of this culture is measured and it must be between 3.0 and 3.5 at 600nm.
  • the appearance of the germs is checked under a microscope after Gram staining.
  • Example 4 An antiserum as obtained in Example 4 is filtered through a 0.45 ⁇ m membrane to remove the small aggregates if they exist before use.
  • the anti-76 kDa antiserum gives a very strong agglutination reaction. Under the conditions tested, H. bacteria. pylori quickly clump together and the reaction is complete after a minute. The results indicate that the 76 kDa protein is likely exposed on the surface of H. pylori.
  • mice Groups of ten Swiss Webster mice aged 6 to 8 weeks (Taconic Labs, Germantown, NY) are immunized intragastrically with 1, 5, 25. 50 or 100 ⁇ g of the 76 kDa antigen purified by chromatography as described in Example 3, and diluted in PBS or in PBS containing 0.24 M sodium bicarbonate.
  • the antigen is supplemented with 5 or 10 ⁇ g of cholera toxin (CT) (Calbiochem, San Diego) or heat-labile toxin (LT) (Berna Products. Coral Gables FL).
  • CT cholera toxin
  • LT heat-labile toxin
  • mice Four doses are administered to each mouse 7-10 days apart. Two weeks after the last antigen administration, the mice are tested by a single dose of the H. pylori strain ORV2002 (1 ⁇ 10 7 of live bacteria in 200 ⁇ l of PBS; OD 550 of approximately 0.5) administered by the route. intragastric. A group having received no dose of antigen and serving as a control is likewise tested. Two weeks after the test, the mice are sacrificed. The percentage of protection is determined either by measuring the urease activity or by evaluating the bacterial load by histology as described in Lee et al (supra) or directly by quantitative culture of H. pylori.
  • NAME Pasteur Merieux serums and vaccines
  • B STREET: 58 avenue Leclerc

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EP96933488A 1995-10-04 1996-10-04 NOUVELLE PROTEINE MEMBRANAIRE p76 D'HELICOBACTER PYLORI Withdrawn EP0797586A1 (fr)

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FR9511891A FR2739623B1 (fr) 1995-10-04 1995-10-04 Nouvelle proteine membranaire p76 d'helicobacter pylori
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PCT/FR1996/001551 WO1997012908A1 (fr) 1995-10-04 1996-10-04 NOUVELLE PROTEINE MEMBRANAIRE p76 D'HELICOBACTER PYLORI

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NZ333250A (en) 1996-06-10 2000-05-26 Thomas Boren Helicobacter pylori blood group antigen binding adhesion protein
DE19630390A1 (de) * 1996-07-26 1998-01-29 Chiron Behring Gmbh & Co Proteine, insbesondere Membranproteine von Helicobacter pylori, ihre Herstellung und Verwendung
CA2286893A1 (en) * 1997-04-01 1998-10-08 Merieux Oravax 76 kda, 32 kda, and 50 kda helicobacter polypeptides and corresponding polynucleotide molecules
JP3869556B2 (ja) * 1998-06-15 2007-01-17 勇 近藤 ヘリコバクター・ピロリ由来抗原及びそれを用いたヘリコバクター・ピロリ感染の診断方法
AU4718999A (en) * 1998-06-26 2000-01-17 American Cyanamid Company Novel antigens of (helicobacter pylori)
JP5250812B2 (ja) * 2006-04-27 2013-07-31 国立大学法人富山大学 ヘリコバクター・ピロリ菌由来の新規抗原、抗原組成物およびピロリ菌抗体の検出方法。

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