MXPA97004109A

MXPA97004109A - New proteins of helicobacter pyl membrane

Info

Publication number: MXPA97004109A
Application number: MXPA/A/1997/004109A
Authority: MX
Inventors: Lissolo Ling
Original assignee: Pasteur Merieux Serums Et Vaccins
Priority date: 1995-10-04
Filing date: 1997-06-03
Publication date: 1999-07-06

Abstract

The invention relates to a Helicobacter pylori protein in a substantially purified form, susceptible to being obtained from a membrane fraction of Helicobacter pylori and from which the molecular weight after electrophoresis in 10% polyacrylamide gel in the presence of SDS is approximately 54, 50, 32-35 or 30 K

Description

MOVES PROTEIN MEMBRANE HELICOBACTER P? UORI The aim of the present invention is Helicobacter pylori proteins recently obtained in substantially purified form, as well as the pharmaceutical compositions containing it. Helicobacter is a bacterial genus characterized by large negative helical bacteria. Several species colonize the gastrointestinal tract of mammals. Particular mention may be made of H. pylori, H. heilmanii, H. felis and H. mustelae. Although H. pylori is the most commonly associated species, there are human infections, in some cases rarely admitted, it has been possible to isolate H. heilmanii and H. felis in humans. Helicobacter infects more than 50% of the adult population in developed countries and almost 100% in developing countries, being one of the predominant infectious agents in the world. -i ?, pylori to date is found exclusively on the surface of the mucous membrane of the stomach in humans and more particularly around the 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. Aú-rt more, it seems that the development of gastric carcinomas, may be associated with the presence of H. pylori.

Therefore, it seems highly convenient to develop a vaccine intended to treat or prevent H. pylori infections. This vaccine would most likely be of a subunit nature. Various H. pylori proteins have been characterized or have been isolated to date. They are especially urease, composed of two subunits A and B of 30 and 67 kDa, respectively (Hu &Mofoley, Infect. Immun. (1990) 58: 992; Dunn et al., J. Biol. Chem. (1990) 265: 9464, Evans et al, Microbial Pathogenesis (Microbial Pathogenesis) (1991) 10: 15, Labigne et al., J. Bact, (199) [sic] 173: 1920); the 87-kDa cytotoxin vacuole (VacA) (cover &Blaser, J. Biol. Chem. (1992) 267: 10570; Phadnix et al., Infect. Immun. (1994) 62: 1557; WO 93/18150); a 128 kDa immuno dominant antigen associated with the cytotoxin (CagA, also called TagA) (WO 93/18150; USP 403 924); heat shock proteins HspA and HspB of 13 and 58 kDa, respectively (Suerbaum et al., Mol.Microbiol. (1994) 14: 959; WO 93/18150); a catalase of 54 kDa (Hazell et al., J. Gen. Microbiol. (1991) 137: 57); a fibrillary agglutinin (HpaA) of 20 kDa; a protein rich in histidine of 15 kDa (Hpn) (Gilbert et al., Infest. Immun. (1995) 63: 2682); a 30 kDa outer membrane protein (Bolin et al., J. Clin Microbiol. (1995) 33: 381); a lipoprotein associated with 20 kDa membrane (Kostrcynska et al., J. Bact. (1994) 176: 5938) as well as a family of HopA, HopB, HopC and HopD porins, of molecular weight 48 and 67 kDa (Exner et al., Infect. Immun. (1995) 6_3: 1567). Some of these proteins have already been proposed as potential vaccine antigens. In particular, urease is recognized as the most preferred antigen that can be used for this purpose (WO 94/9823, WO 95/3824, WO 95/22987, Michetti et al. Gastroenterology (1994) 107: 1002). There remains the fact that the search for new antigens must continue especially since it is anticipated that in order to obtain an optimal vaccine effect, several antigens will probably have to be incorporated into a vaccine. In summary, it still seems to be necessary to identify additional antigens in order to incorporate them into a high efficacy vaccine. In accordance with the above, the subject matter of the invention is especially an H. pylori protein in a substantially purified form, capable of being obtained from a membrane fraction of H. pylori, and whose molecular weight after electrophoresis in a polyacrylamide gel at 10% in the presence of SDS, appears in the order of 54, 50, 32, 35 or 30 kDa. When the protein has an approximate molecular weight of 54 KDa. It is further specified that it does not react with an anti-catalase antiserum.

An anti-H antiserum. pylori catalase can be prepared especially according to the immunization process described in example 5 below, using a catalase preparation obtained by chromatography as described in Example 6. "Substantially purified form" is meant to mean that the protein is separated of the environment in which it exists naturally. Among others, it may be a preparation that is especially lacking in the periplasmic and cytoplasmic H. pylori proteins. The membrane protein of its apparent molecular weight in the order of 54 KDa is capable of being obtained by a process in which: (i) H. pylori bacteria are extracted with n-octyl BETA-D glucopyranoside, at 1%, followed by centrifugation; (ii) A bacterial nodule is recovered and treated with lysozyme and subjected to sonication followed by centrifugation; (ii) A centrifugation nodule is recovered and subjected to washing with 20 mM Tris-HCl buffer, pH 7.5, followed by centrifugation; (iv) The membrane fraction consisting of the centrifugation nodule is recovered and resuspended in an aqueous medium, as advantageously in a buffer sarbonate pH 9.5 which are zwittergent 3-14 at 5%; (v) The membrane fraction is subjected to anion exchange chromatography on a Q-Sepharose column in a 0 - 0.5 M NaCl gradient, advantageously in a carbonate buffer pH 9.5 containing 0.1% Zwittergent 3-14, followed by washing in 1 M NaCl advantageously in a sarbonate buffer pH 9.5, which are Swittergent 3-14 at 0.1%; (vi) The eluted fraction at the beginning of the 1M NaCl wash is recovered and subjected to anion exchange chromatography and a DEAE Sepharose column, in a NaCl gradient of 0 - 0.5 M, advantageously in Tris-HCl buffer pH 7.5 containing zwittergent 3-14 to 0.1% (advantageously the fraction in 1 M NaCl is first dialyzed against Tris-HCl buffer pH 7.5 containing zwittergent 3-14 at 0.1%); (vii) The fraction eluted in 0.1 - 0.25 M NaCl is recovered. The membrane protein whose apparent molecular weight is in the order of 50 KDa is capable of being obtained by a process in which: (i) H. pylori bacteria are extracted with n-octil BETA-D glucopyranoside, 1%, followed by centrifugation; (ii) A bacterial nodule is recovered and treated with lysozyme and subjected to sonication followed by centrifugation; (iii) A centrifugation nodule is recovered and subjected to washing with 20 M Tris-HCl buffer, pH 7.5, followed by centrifugation; (iv) The membrane fraction consisting of the centrifugation nodule is recovered and resuspended in an aqueous medium, as advantageously in carbonate buffer pH 9.5 containing zwittergent 3-14 at 5%; (v) The membrane fraction is subjected to anion exchange chromatography on a Q-Sepharose column in a 0 - 0.5 M NaCl gradient, advantageously in a carbonate buffer pH 9.5 containing 0.1% Zwittergent 3-14, followed by washing in 1 M NaCl advantageously in a carbonate buffer pH 9.5, containing Swittergent 3-14 at 0.1%; (vi) the diluted fraction at the beginning of the washing with 1M NaCl is recovered and subjected to anion exchange chromatography in a DEAE column - sepharose, in a 0 - 0.5 M NaCl gradient, advantageously in a Tris-HCl buffer pH 7.5 containing zwittergent 3-14 at 0.1% (advantageously, the fraction in 1 M NaCl is first dialyzed against buffer Tris-HCl pH 7.5 which are zwittergent 3-14 at 0.1%); and (vii) the fraction eluted in NaCl is 0.3-0.4 M recovered. The membrane protein whose apparent bulk weight is in the order of 30 KDa is capable of being obtained by a process in which: (i) H. pylori bacteria are extracted with n-ostil BETA-D glucopyranoside, at 1%, followed by centrifugation; (ii) A bacterial nodule is recovered and treated with lysozyme and subjected to sonication followed by centrifugation; (iii) A centrifugation nodule is recovered and subjected to washing with 20 mM Tris-HCl buffer, pH 7.5, followed by centrifugation; (iv) The membrane fraction consisting of the centrifugation nodule is recovered and resuspended in an aqueous medium, as advantageously in carbonate buffer pH 9.5 containing zwittergent 3-14 at 5%; (v) The membrane fractionation is subjected to anion exchange chromatography on a Q-Sepharose column in a 0 - 0.5 M NaCl gradient, advantageously in a carbonate buffer pH 9.5 containing Zwittergent 3-14 0.1%, followed by washing in 1 M NaCl advantageously in a carbonate buffer pH 9.5, containing Swittergent 3-14 at 0.1%; (vi) the fraction eluted in NaCl is 0.28-0.35 M is recovered and subjected to anion exchange chromatography and a DEAE-Sepharose column, in a gradient of 0 - 0.5 M NaCl, advantageously in Tris-HCl buffer pH 7.5 containing zwittergent 3-14 0.1% (advantageously, fusing in 1 M NaCl is first dialyzed against Tris-HCl buffer pH 7.5 containing zwittergent 3-14 to 0.1.); and (vii) the fraction corresponding to the direct eluate is recovered (absence of NaCl). The membrane protein whose apparent molecular weight is in the order of 32-35 KDa is capable of being obtained by a process in which: (i) H. pylori bacteria are extracted with n-octyl BETA-D glusopyranoside, at 1% , followed by sentrifugation; (ii) A bacterial nodule is recovered and treated and lysozyme is subjected to sonication followed by centrifugation; (iii) A centrifugation nodule is recovered and subjected to washing with 20 mM Tris-HCl buffer, pH 7.5, followed by centrifugation; (iv) The membrane fraction consisting of the centrifugation nodule is recovered and resuspended in an aqueous medium, as advantageously in carbonate buffer pH 9.5 containing zwittergent 3-14 al %; (v) the suspension obtained in (iv) is centrifuged at approximately 200,000 x g and the supernatant is recovered; (vi) the pH of the supernatant obtained in (v) is reduced to about pH 7, advantageously by dialyzing against the phosphate buffer, pH 7; (vii) the preparation obtained in (vi) is subjected to anion exchange chromatography and an SP-Sepharose column, in a gradient of NaCl 0 - 5 M advantageously in a buffer phosphate buffer pH 7; and (vii) the fraction eluted in NaCl is 0.26-0.31 M recovered.

Proteins 54, 50, 32 and 30 kDa of asiento are the invention probably are intrinsic membrane proteins or proteins associated with the membrane. The 54 kDa protein does not react with anti-catalase antibodies either in Western manshade or in dot manshade. The 30 kDa protein does not react with anti-urease A subunit antibodies neither in Western spotting or dot manshade. The 32 kDa protein proves to be an alkaline protein; its molecular weight may appear slightly higher, for example in the order of 35 kDa under certain experimental conditions. The N-terminal sequence of the 76 KDa protein of an H. pylori strain (ATCC 43579) is as follows (one letter code) EDDGFYTSVGYQIGEAAQMV. This information does not exclude the hesho that equivalent proteins capable of being purified according to the method indicated above may have a slightly different N-terminal sequence, since they may be derived from another bacterial strain. This difference will undoubtedly reflect the phenomenon of allelic variability commonly found within the same species. For example, a bacterial species is usually represented by a group of strains that differ from each other in minor allelic sarastherisms. A polypeptide that fulfills the same biological function in different strains can have an amino acid sequence that is not different for all strains. Allelic variation also exists in DNA, The allelic differences at the level of the amino acid sequence may consist of one or more substitutions, eliminations or adductions of to octosides that do not alter the biological function. "Biological function" is understood to mean the function of the protein that participates in the survival of the cells where the protein exists naturally (even if the function is not absolutely essential). For example, the function of a porin consists of allowing the compound to be present in the external medium to enter the interior of the cells. The biological function is different from the antigenic function. A protein can have more than one biological function. The subject of the invention is also a protein in a substantially purified form and which may have been purified according to one of the processes described above from a bacterium of the Helicobacter genus, for example H. pylori, H. heilmanii, H. felis and H. mustelae. The subject of the invention is also any protein or polypeptide, in a substantially purified form and which is analogous as in terms of antigenicity, to a Helicobacter protein capable of being purified by one of the methods described above. With respect to the polypeptides, they are especially polypeptides derived by fragmentation or by mutation of one or more amino acids, for example by elimination, addition or substitution, of a protein that exists in the nature and their purified form can be obtained according to one of the procedures described above. These polypeptides can be obtained especially by enzymatic digestion with the aid of proteases such as pepsin or trypsin. It is not necessary that these polypeptides be purified according to one of the methods described above. The present invention uses the terms "protein11" and "polypeptide" independently of the size of the molecules (length of the amino acid chain) and of the possible post-translational modifisions.In the rest of the description, the term "polypeptide" is reserved to designate a product of a protein derived by fragmentation or mutation A protein or a polypeptide according to the invention must be able to be recognized by mono-specific antibodies developed in a Helisobacter protein capable of being purified according to one of the procedures described above. specific can be revealed according to a number of methods, for example by Western blotting (Towbin et al., PNAS (1979) 10 76: 4350), spot spotting and ELISA, In Western spotting, the product to be tested , for example either in the form of a purified preparation or in the form of a bacterial extract l is subjected to SDS-Page gel electrophoresis (10% polyacrylamide) as described by Lae li U.K., Nature (1970) 227: 680. After Transfer over a nitroselulose membrane, the latter is incubated by a mono-specific hyperimmune serum diluted in the range of dilutions from 1:50 to 1: 5000, preferensia from 1: 100 to 1: 500. The specific antigenicity is demonstrated as soon as a band corresponding to the test product exhibits a reactivity in one of the dilutions included in the range set forth above. In ELISA, the product intended to be tested preferably is used to coat wells. A purified preparation of preference is employed although a total extract may also be employed. Briefly 100 μl of a preparation containing lOμg of protein / ml are distributed in the wells of a 96-well plate. The plate is incubated for two hours at 37 ° C and then during the noshe at 4 ° C. The plate is washed with PBS buffer (saline buffered with phosphate) containing 0.05% Tween 20 (PBS / Tween buffer). The wells are saturated with 250 μl of PBS containing 1% bovine serum albumin (BSA), all incubated for one hour at 37 ° C and then the plate is washed with PBS / Twin buffer, a monospecific rabbit antiserum. It is serially diluted in PBS / Tween buffer that are 0.5% BSA, one hundred μl of a dilution are added to each well, the plate is incubated for 90 minutes at 37 ° C and then washed. For example, a goat-peroxidase immunoglobulin conjugated to rabbit immunoglobulins is added to the wells. continue for 90 minutes at 37 ° C and then wash the plate. The reaction proceeds with the appropriate substrate. The reaction is measured by solorimetry (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 is measured depends on the substrate. In spotting, a purified preparation of the produst to be tested is preferably used although a total extract may also be employed. Briefly, a product preparation to be tested containing 100 μg protein / ml is serially diluted twice in 50 mM Tris-HCL pH 7.5. if 1 μl of each application is applied to a 0.45 μm nitrocellulose membrane in a 96-well spot spotting apparatus (Biorad). The shock absorber is removed when placing vacuum. The wells are washed by addition of 50 mM Tris HCl, pH 7.5 and the membrane is air dried. The membrane is saturated with blocking buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 g / 1 of skimmed milk) and then incubated with a monospecific antiserum diluted in the 1:50 and 1: 5000 range of preferensia 1:50 to 1: 500. The reassessment is visualized according to standard methods. For example, a goat immunoglobulin-peroxidase sonjuga sontra immunoglobulin is added to the wells. The insubation is continued for 90 minutes at 37 ° C and then the plate is washed. The reaction is revealed with the appropriate substrate. The reaction is measured by colorimetry and chemiluminism. Under these sonsions, a positive feedback is observed at the point level on the nitroselulose sheet directly for visualization by colorimetry or on a photographic film for visualization by chemiluminescence, associated with a dilution of at least 1:50, of preferensia at least 1: 500. According to the specific embodiment, a protein according to the invention can be obtained especially by purification from Helicobacter or expressed by the recominant route in a heterologous system (which may also be the case for a polypeptide according to the invention). In the latter case, the protein may exhibit post translational modifications that are not identical to those of the corresponding protein derived from the original strain. The therapeutic or prophylactic efficacy of a protein or of a polypeptide according to the invention can be evaluated with standard methods, such as measuring the induction of a musosal immune response or the induction of an immunoresponse having a protest therapeutic effect, using example the mouse / H model. felis and the proselytizing dessritos by Lee and collaborators, Eur. J. Gastroenterology & Hepatology, (1995), 7: 303 or Lee et al., J. Infest. Gave. (1995) 172: 161, under the condition that the following precaution be taken: When the protein is derived from a species other than H. felis, the H. felis strain must be replaced by a helicobacter strain belonging to the species from which the protein is derived and adapted for this purpose (the other experimental condiions remain identical). For example, the ability of a polypeptide derived by fragmentation from a -ff protein. pylori to induce a protective or therapeutic effect, is tested by replacing an H. pylori strain. This strain is proposed by Klenthous et al., Presented in the VIIIo. International Workshop on Gastroduodenal Pathology July 9, 1995 in Edinburgh, Scotland. A protective effect is observed once an infection in a gastric tissue is reduced in comparison are a control group. Infection is evaluated by testing for urease activity, bacterial plaque, or leukosite infiltration. For example, when a reduction in urease activity in the gastric tissue is observed after an attack, even if it is completely abolished, it is reasonable to estimate that there is parsial protection. Consistently, the invention is also relased to (i) a material deposition comprising a protein or a polypeptide according to the invention and a diluent or a carrier; in particular (ii) a pharmaceutical composition intended especially for the prevention or treatment of a Helicobacter infection, comprising as an active ingredient, a protein or a polypeptide according to the invention, in an effective amount from a prophylactic point of view or treatment; (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 consensus polypeptide is the invention for the manufacture of a medicament intended for the prevention or treatment of a Helicobacter infection7 as well as (v) a method for inducing an immune response against Helicobaster, for example H pylori, H. heilmanii, H. felis and H. mustelae in a mammal, according to which an immunologically effec- tive sanctity of a protein or a polypeptide according to the invention is administered to the mammal in order to develop an immune response.; in particular (vi) a method for the prevention or treatment of a Helicobacter infection [sic] is administered to an individual in 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 Helicobacter infections and consistently gastrointeceptive diseases associated with these infections. In partisular are acute, chronic and atrophic gastritis; peptic ulcers, for example gastric and duodenal ulcers; gastric cancers; chronic dyspepsia; refractory non-ulcer dyspepsia; intestinal metaplasias and certain lymphomas (for example, 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 (for example ocular, nasal, oral, gastric, intestinal, rectal, vaginal or urinary tract) or by parenteral route (for example subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal). The selection of the administration route depends on a number of parameters such as the adjuvant associated with the protein or polypeptide according to the invention. For example, if a mucosal adjuvant is used, the nasal or oral route will be preferred. If a lipid formulation is used, the parenteral route, preferably the subcutaneous or intramuscular route, will be selected. A composition of agreement is that the invention may additionally comprise a protein or a polypeptide according to the invention, at least one other Helicobacter antigen such as apoenzyme urease, or a subunit fragment or mutant homologue of this urease. For use in a composition according to the invention, a protein or a polypeptide according to the invention can be formulated in or with liposomes, preferably liposomes, microspheres, ISCOMS or neutral or anionic virus-like particles (VLPs), to promote the making target of the protein or polypeptide to improve the immune response. People with skill in the specialty, obtain these compounds without any difficulty; for example see Liposomes: A Practical Approach (liposomes: a practical approach) RRC New ED, IRL press (1990). Adjuvants other than liposomes can also be used. A large amount is known by people with skill in the specialty. These adjuvants are referred to below: For parenteral administration, especially aluminum compounds such as aluminum hydroxide, aluminum phosphate and aluminum hydrophosphate can be mentioned. The antigen can be absorbed or precipitated on an aluminum compound according to standard methods. Other adjuvants such as RIBI from ImmunoChem (Hamilton, MT) can be used for parenteral administration. For mucosal administration, bacterial toxins, for example cholera toxin (TC), E. coli thermolabile toxin ( LT), the Clostriáium Difficile toxin and the pertussie toxin (PT) as well as the detoxified forms (subunit toxoid or mutant) of these toxins. For example, a preparation containing the sub-unit B of CT (CTB) and a lower sanctity of CT can be used. Fragments, homologs and derivatives of these toxins are also appropriate in that they retain an adjuvant activity. In preferensia, a mutant having a reduced toxicity is employed. These mutants are described, for example, in WO 95/17211 (mutant Arg-7-Lys CT), WO 95/34323 (mutant Arg-9-Lys Glu-129-Gly PT) and WO 96/6627 (mutant Arg-192-Gly LT). Other adjuvants such as the major basterial lipopolysaccharide (MPLA) of for example E. coli, Salmonella minnesota, Salmonella tifimuriom or Shigella flexneri, or can be used for mucosal administration. Adjuvants useful for both administration and parenteral administration include especially polyphosphine (WO 95/2415), DC-chol (3-beta- [N-25 (N ', N'-dimethylaminomethane) carbamoyl] cholesterol (USP 5 283 185 and WO 96 / 14831) and QS-21 (WO 88/9336) The administration can be carried out as a single dose or as a repeated dose once or several times after a certain period.The appropriate dose varies according to the various parameters, for example the treated individual (adult or child) 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 (for example protrusion or treatment), may be determined In general, an antigen of agreement is the invention can be administered in an amount in the range of 10 μg to 500 mg, preferably from 1 mg to 200 mg, In particular it is indicated that a parenteral dose shall not exceed l mg or preferably 100 μg. They can prescribe higher doses for example for oral use. Regardless of the formulation, the amount of protein administered to the human by the oral route is for example of the order of 1 to 10 mg of dose, and at least 3 doses are recommended at 4 week intervals.

A composition of agreement is the invention can be manufactured in a conventional way. In particular, a protein or a polypeptide according to the invention is combined with a carrier diluent that is pharmaceutically acceptable, for example water or a saline solution such as sulfate buffered saline (PBS), optionally supplemented with a bicarbonate salt such as sodium bicarbonate, for example 0.1 to 0.5 M, when the composition is intended for oral or intragastric administration. In general, the diluent or carrier is chosen based on the mode and route of administration and standard pharmacy practices. Diluents and carriers that are pharmaceutically acceptable as well as all that is necessary for use in pharmaceutical formulations are described in Remington's Pha-rmaceutical Sciences, a standard reference text of this field and in USP / NP. In a more detailed form, it is proposed by way of example to administer a protein or a polypeptide according to the invention by the oral route. For this purpose, a protein or a polypeptide according to the invention can be encapsulated alone or in the presence of other H. Pilori proteins in gelatin capsules, in order to protect the antigen against degradation by gastric juice or be administered in the presence of bicarbonate of sodium. These formulations have already been used for far-reaching compositions (Blask and collaborators, Dev.

Biol. Stand. (1983), 53: 9). The protein can also be encapsulated in PLGA microspheres (copolymers of glycolic acid and lactic acid) according to the desedre pro-sedation elsewhere (Eldridge et al., Curr. Top, Microbiol.Inmune (1989) 146: 59); the protein can also be encapsulated in liposomes prepared according to widely described conventional methods ("Liposomes: a practisal approash (Liposomes: A Practical Approach), Ed. RRC New, D. Riskwood &BD Hames, 1990, Oxford University Press, ISBN 0-19-963077-1) Alternately, a protein or a consensus polypeptide is the invention can be administered by the parenteral route In order to do this, a protein or a polypeptide according to the invention is absorbed on alumina gel in a completely conventional way, the protein is a 1 mg / ml solution in a buffer whose pH close to 6.5 is contacted for 1 hour with aluminum hydroxy at 10 mg / ml measured as AL +++ .The final composition of the preparation is the following: protein 50 μg / ml, AL +++ 250 μg / ml, merthiolate 1 / 10,000, all in PBS As in the case of oral administration, three injections are recommended, each one at an interval of 4 weeks from the previous one. The present invention may also be useful as a diagnostic reagent, for example to detect the presence of anti-Helicobaster antibody in a biological sample, for example a blood sample. For this one target, that polypeptide advantageously comprises 5 to 80 amino acids, preferably 10 to 50 amino acids. Polypeptide reagent according to the invention can be labeled or otherwise, according to the diagnostic method employed. Diagnostic methods are previously described in the text. According to another aspect, the invention provides a mono-sympathetic antibody sapaz of resonoser a protein or a consensus polypeptide are the invention. "Antibody mono-syphilis" is understood to mean an antibody capable of predominantly reacting with a single helicobaster protein. This anti-body can only be obtained by using a protein substantially purified as an immunogen. An antibody according to the invention can be polyclonal or monoclonal; the monoclonal can be chimeric (for example consisting of a variable region of marine origin associated with a human or humanized constant region) only the hypervariable regions are of animal origin, for example of murine origin (and / or a single strand). Polyclonal and monoclonal antibodies may also be in the form of immunoglobulin fragments, for example an F (ab) '2 or Fab fragment. An antibody according to the invention may also be of any isotype, for example IgG or IgA; A polyslonal can be of a single isotype or a mixture of all or some of them.

In the text that follows, the terms "specific mono antibody" and "specific antiserum" are used interchangeably. An anti-body antibody directed against an agreement protein is the invention can be produced and subsequently identified using a standard immunological assay, for example Western spotting, spot spotting or ELISA analysis (see for example Coligan et al., Current in Immunology (Current Protosolos Immunology) (1994) John Wiley &Sons Inc., Protocols New York, NY); Antibodies: A laboratory Manual, (antibodies: A laboratory manual) D. The e, (1988) Harlow Ed.). An antibody according to the invention may be useful in diagnosis, as well as in affinity chromatography for large-scale purification of a protein or a consensus polypeptide are the invention; this antiserum is also potensially useful as a therapeutic agent in a passive immunization prophylaxis. Accordingly, the invention also provides (i) a reagent for detecting the presence of helicobacter in a biological sample, comprising an antibody or a polypeptide according to the invention, and (ii) a diagnostic method for detesting the presence of helisobaster in a biological sample from which the biological sample is contacted with an antibody or a polypeptide according to the invention, way such that an immune spleen is formed; optionally, the unbound material is removed and the immune complex formed between the sample and the antiserum or the polypeptide according to the invention is detected as an indicator of the presence of helicobaster in the sample or in the organ from which the sample was collected. sample. As can easily be understood, an antibody according to the invention makes it possible to prove the presence of helicobaster in a gastric extra. For use in a diagnostic test, the reagent that is lowered [sic] is provided in a free form or can be immobilized on a solid support; the latter can be any support commonly employed in this domain, for example a tube, a bead or a well. Immobilization can be obtained by direct or indirect means. The direct media comprises passive adsorption (non-covalent binding) or covalent bonds between the support and reastive. "Indirect media" means that an anti-active compound capable of interacting with a reagent is first solid-supported. For example, if a polypeptide reastive is used, a sapaz antibody to ligate it can be used as an anti-reastive somo, provided that it can be ligated to an epitope of the polypeptide that is not involved in the resosimiento of the antibodies present in the biological samples. Indirestos means can also be implemented through a ligand-reseptor system, for example when grafting a molecule, such as a vitamin on a polypeptide reastive and then immobilizing, in solid form, the sorptive receptor. This is illustrated, for example, by the biotin-streptavidin system. Alternatively, indigestible media are used for example by adding a single polypeptide to the reastive, for example by chemical means, and by immobilizing the grafted product by passive adsorption or by covalently binding the polypeptide tail. The invention also relates to a method for the purification of a protein or a polypeptide according to the invention from a biological sample, according to which the biological sample is subjected to affinity chromatography using a monospecific antibody. according to the invention. For this purpose, the antibody can be polyclonal or monoclonal, preferably of the IgG type. Purified IgG can be prepared from an antiserum according to commonly used methods (see for example Coligan et al.). Modern chromatographic supports, such as standard methods for grafting antibodies, are described, for example, in Antibodies: A Laboratory Manual (antibodies: A laboratory manual), D. Lane, Harlow Ed. (1988)). Briefly a biological sample preferably a buffer solution is applied to a chromatography material preferably balanced with the buffer used for dilution of a biological sample, such that the protein or polypeptide according to the invention (antigen) can be adsorbed on the material. The chromatography material, such as a gel or an associated resin, is an anti-body antibody. It can be provided in the form of a bath or solumn. The somatics remaining unlinked are removed upon washing and the antigen is then eluted into an appropriate elution buffer, such as for example a glisina buffer or a buffer containing a saotropic agent, for example guanidine-HCl, or a laughter consentration. salt (for example 3 M MgCl2). The eluted fractions are recovered and the presence of the antigen is then detected, for example by measuring the absorbance at 280 nm. This purification step can, for example, be used to purify a protein from the total extract. However, if the antibody is not perfectly monospecific, it is advisable to enrich in advance the material that is intended to be subjected to immunoaffinity chromatography in terms of the amount of protein to be purified. For example, this protein can be used to enhance the purification of the 32 kDa protein. This is obtained according to the procedure described above comprising a purification step in SP-Sepharose.

The therapeutic or prophylactic utility of an antibody according to the invention can be demonstrated in accordance with the Lee and slabs protection test, proposed above for the proteins or polypeptides according to the invention. In this way, the subject matter of the invention is also (i) a composition of material comprising a mono-specific antibody of agreement are the invention and a diluent or carrier; in partiscular (ii) a pharmaceutic somposision that suffers a monospeptide antiserum of agreement is the invention in an effective amount from a therapeutic or prophylactic point of view; (iii) the use of a monospeaker antiserum of agreement is the invention is the preparation of a medisamento to treat or avoid an infesion of helisobaster; thus (iv) a method for treating or preventing a helicobaster infection (eg H. felis, H. mustelae or H. heilmanii) according to which a therapeutic or prophylactically effective sanctity of an antibody according to the invention is administers to an individual that requires such treatment. For this purpose, the monospecific antibody may be polysilonal or monoslonal, preferably isotype IgA (predominantly). In the context of a passive immunization method, the antibody is administered via the mucosal route to a mammal, for example at the level of the gastric mucosal membrane, either orally or intragastrically, advantageously in the presence of a bicarbonate buffer. A monospecific antibody of According to the invention, the invention can be administered as a single active component or as a mixture comprising at least one specific monoclonal antibody, specific to the helisobaster polypeptide. The dose of antibody to be used in this method can be readily determined by persons skilled in the art. For example, it is suggested that a dose may be expensive by a daily administration of between 100 and 1000 mg of anti-body for one week or a dose of 100 to 1000 mg of anti-body administered 3 times a day for two or three days. A pharmaceutic somposision that comprises an anti-antibody according to the invention can be manufactured according to the rules set forth above for a somaticity comprising a protein or a polypeptide according to the invention. Equally, identical medical indications apply. The invention is illustrated below, referring to the following figures: Figure 1 is a summary of the preparation for the preparation of H. pylori membrane fractions I, II and III. Figure 2 presents the analysis of membrane fractions I, II and III by electrophoresis in a 10% polyacrylamide gel and stained with Coomassie blue. The loaded samples are: membrane fraction I (lane 2), fractionation of membrane II (lane 3), membrane fraction III (lane 4) and molecular markers (lane 1). Figure 3 presents the analysis by electrophoresis in a 10% polyacrylamide gel and stained with Coomassie blue of the purified proteins from a preparative gel (lanes 3 to 7). The loaded samples are: the HpPl fraction (lane 3), the HpP2 fractionation (lane 4), the HpP4 fractionation (lane 5), the HpP5 fractionation (lane 6), the HpP6 frassión (lane 7), the gravity weight marsadores (tracks l and 8) and membrane frassion I (track 2). Figure 4 represents the analysis of the frassions obtained from the passage of DEAE-Sepharose in frassions 7 and 9 (obtained after elusion in Q-Sepharose). Frassions were separated by electrophoresis in a 10% or 12.5% polyacrylamide gel and stained with Coomassie blue. The serrated samples are: fraction 7 (lane 2A), fraction 7.1 (lane 3A), fraction 7.2 (lane 4A), fraction 9 (lane 2B), fraction 9.1 (lane 3B) fraction 9.2 (lane 4B), fraction 9.3 (lane) 5B), and molecular weight markers (kDa tracks) (lanes 1A and IB). Figure 5 shows after electrophoresis in a 10% polyacrylamide gel and stained with Coomassie blue, the electrophoretic profile of fraction D obtained from chromatography on a Q-Sepharose column of membrane fraction III (lane 3 ) and fraction D 'obtained from chromatography on an S-Sepharose column of fraction D (Track 4) Track 1 is responsible for molecular weight marsadores and track 2 for membrane III fractionation. EXAMPLE 1: Preparation of Membrane Fractions 1A.- Culture The H. pylori strain ATCC 43579 is sown in liquid medium in a 10 liter fermentor. A songe sample of misroorganisms in gliserol is used to inoculate a 75-cm2 flask that is a so-called "two phase" medium (a solid phase in Colombia agar containing 6% fresh sheep's blood and a liquid phase in trypticase of soy, which contains 20% fetal bovine serum). After 24 hours of cultivation under microaerophilic conditions, the liquid phase of this culture is used to inoculate several 75 cm2 flasks in a two-phase medium in sheep blood absences. After 24 hours of culture, the liquid phase makes it possible to inoculate a 2-liter bio-fermentor in a medium soybean trypticase medium containing beta-cyclodextrin at 10 g / 1. This culture at OD 1.5 - 1.8 is inoculated in a 10 1 fermentor in liquid medium. After 24 hours of culture, the bacteria are suspended by sentrifugation at 4000 x g for 30 minutes at 4 ° C. A 10-liter silant of H. pylori ATCC 43579 in a fermenter makes it possible to obtain approximately 20 to 30 g (wet weight) of basterias.

IB - Extraction with n-octyl beta-D-glucopyranoside (OG) The microorganism nodule obtained above is washed with 500 ml of PBS (phosphate buffer saline, 7.650 g NaCl, 0.724 g disodium phosphate, 0.210 g monopotassium phosphate) per liter, pH 7.2) per liter of culture. The microorganisms are then centrifuged again under the same conditions. The obtained basterial nodule (Cl) is resuspended in an OG (Sigma) solution at 1% (30 ml / liter of sultivo). The Basterian suspension is insufflated for one hour at room temperature, is magnetic agitation and then centrifuged at 17,600 for 30 minutes at 4ßC. The nodule (Ca) is stored for subsequent treatment. The supernatant (S2) obtained is dialyzed (MWCO = 10000 Da, Spectra / per) during the noshe at 4 ° C and is agitated again two times a liter of PBS diluted 1/2.

The precipitate formed during dialysis is recovered by centrifugation at 2,600 for 30 minutes at 4 ° C. The supernatant (S2) is removed and the nodule (Cs2d) containing membrane proteins is stored at -20 ° C. ÍC - Rupture of microorganisms The nodule (Ca) obtained after centrifugation of the microorganisms treated with OG is resuspended in buffer 20 mM tris-HCl pH 7.5 and pephabloc 100 μm (buffer A) and then homogenized by Ultra-turrax (3821, Janke &Kungel). He homogenate obtained is exposed to lysozyme (0.1 mg / ml final) and EDTA (1 mM final). The homogenate is sonicated for 3 times two minutes at 4 ° C (probe f - 0.5 sm, energy = 20%, Sonifisador 450 Branson) and then ultra sentrifugal at 210,000 x g for 30 minutes at 4 ° C. The supernatant (Sa which are containing sitoplasmic and periplasmic proteins is removed, while the nodule (C3) is resuspended, lava is shock absorber A and then ultra sentrifuge at 210,000 xg for 30 minutes at 4 ° C. After removing the supernatant (S3) The nodule (CB is almasena at -20 ° C. This nodule contains peripheral and intrinsic membrane proteins.) The procedure can be continued by double washing of the nodule (Ci) in order to remove peripheral membrane proteins. is resuspended in buffer NaC0350 M pH 9.5, peflabloc 100 μm (buffer B) .The suspension is ultra-stringent at 210,000 xg for 30 minutes at 4 ° C. The supernatant (Ss) is removed and then the nodule (C5) is washed and ultrastrip under the same conditions as above After removing the supernatant (S6), the nodule (C6) containing essentially intrinsic membrane proteins is stored at -206 C. The C4, C6 and Cs2 fractions are referred to as sonostansion fractions of m embrana I, II, and III, respectively. ID - Analysis of membrane fractions The various membrane fractions are analyzed by electrophoresis in polyacrylamide gel in the presence of SDS according to the method of Laemmli (1970). The proteins are visualized after staining with Coomassie blue. If the major proteins of each fraction are considered, the SDS-PAGE profiles (Figure 2) show that the membrane fraction I is very similar to the membrane II fraction. On the other hand, these two differ substansially from the membrane frassión III. The membrane frassion profile I shows seven prinsipal protein bands of resinous moles 87, 76, 67, 54, 50, 47 and 32-35 kDa (lane 2). By Western manshade in the presence of anti-ureB antibody or anti-catalase antibody it was shown that the 67 kDa band corresponds to the B subunit of urease and the 54 kD band corresponds to catalase. These two proteins do not exist in the profile of fraction II (lane 3) since the laundering is sarbonate buffer removes the weakly assimilated proteins are the membrane. As for the protein profile of the membrane III fraction, it shows the presence of 4 main bands 76, 67, 50 and 30 kDa (lane 4). Example 2: Purification of the proteins of the membrane fraction I by preparative SDS-PAGE An electrophoresis in polyacrylamide gel is carried out according to the method of Laemmli (1970) with a 5% stacking gel and a gel of 10% separation. The membrane fractionation is resuspended in buffer A and then diluted in half in 2X sample buffer. The mixture is heated for 5 minutes at 95 ° C. Approximately 19 mg of proteins are loaded on a gel of 16 x 12 cm in size and thickness 5 mm. A pre-migration is carried out at 50 V for 2 hours, followed by a migration at 65 V during the night. The gel manshade is Coomassie blue R250 (0.5% in ultrafiltered water) allows good visualization of the bands. The main bands HpPl, HpP2, HpP4, HpP5 and HpP6 are cut with a scalpel and ground on an Ultra-turrax in the presence of 10 or 20 ml of extraction buffer containing 25 mM Tris-HCl pH 8.8, 8M urea, SDS to 10%, phenyl methyl sulfonyl fluoric 100 μm [sis] (PMSF) and 100 μm of pefabs (buffer C); each ground product is filtered in a Millipore AP20 prefilter (ffiltr <, = 4.7 cm, fporo = 20 μm) with the help of an extruder at a pressure of 7 bars, at room temperature; each ground product is washed with 5 to 10 ml of buffer C and filtered as before. The two filtrates obtained from each sorbitant ground product are combined. Each filtrate is presipitated with three volumes of a 50:50 to 75% mixture of methanol and 75% isopropanol and then ultra centrifuged at 240,000 g for 16 hours at 10 ° C in a 70 TFT rotor (J8-55, Beckman). Each nodule is collected in 2 ml of solubitization buffer which is 10 M NaP04 pH 7.0, in 1 M NaCl, 0.1% sarcosyl, PMSF 100 μm, Pefables 100 μm, M6 urea (buffer D). The solubilized sample is dialyzed successively against 100 ml of buffer D containing 4 M urea and 0.1% sarcosil, against 100 ml of buffer D containing 2 M urea and 0.5% sarcosil and again twice 100 ml of buffer D without urea and that contains 0.5 of sarcosil. The dialysis is carried out for one hour, with magnetic stirring, at room temperature. The final dialysate is incubated for 30 minutes in an ice bath and then centrifuged at low speed for 10 minutes at 4 ° (Biofuge A, Heraeus Sepatech). The supernatant is recovered, filtered on a Millipore filter 0.45 μm and stored at -20 ° C. An SDS-PAGE analysis was carried out for each fraction (Figure 3). The analysis of the eletroforétiso profile of sada frassión shows that the frassiones HpPl, HpP2, and HpP4 are pure with a single band of gel for each of these fractions (at 87, 76 and 54 kDa, respectively). Frassión HpP5 has a band of high intensity at 50 kDa that is slightly sontaminada are a band at 47 kDa; similarly the HpP6 frassión has a high intensity band 32 kDa that is slightly sontaminated are a band at 35 kDa. EXAMPLE 3: Purification of 30, 50 and 54 kD membrane proteins from the membrane fraction I 3A - Anion Exchange Chromatography in Q-Sepharose A 40 ml column (f = 2.5, h = 8 cm) is prepared according to the recommendations of the manufacturer (Pharmasia). The solnum is washed and then equilibrated are 50-mM NaC03 buffer, pH 9.5, which are peflabloc 100 μm and zwittergent 3-14 at 0.1%. Chromatography is verified by UV detection at 280 nm at the exit of the column. One hundred and forty rag of the previously solubilized proteins of membrane fraction I are loaded onto the column which is then washed with the equilibrium buffer (50 M NaC03, pH 9.5, peflabloc 100 μm and zwittergent 3-14 0.1%) until stabilizes the absorbance 280 nm. Proteins are eluted by a gradient of 0.1 to 0.5 M NaCl in the compensatory buffer (10 veses Vt), followed by washing in a sompensasion buffer that contains 0.1 and 1 M NaCl (two veses Vt). The collected fractions are analyzed by SDS-PAGE and combined in different tanks according to their electrophoretic profile and then aliquot at -20 ° C. The fractions are as follows: Fractions Elusion Fraction Elution 1 Eluate 6 NaCl Direct 7 0.25-0.28 M Washing Buffer NaCl equilibrium 0.28-0.35 M NaCl 8 NaCl Fractions Elusion Fraction Elution 0-0.1 M 0.35-0.5 M 4 NaCl 9 Start of wash 0.1-0.2 M naCl 1 M 4 NaCl 10 End of wash 0.1-0.2 M naCl 1 M Protein evaluation shows that 53% of proteins they are eluted in the 0.1 to 0.5 M NaCl gradient, 14% of the proteins are not conested to the solumn and 33% of the proteins are diluted during washing in NaCl, 0.9 M, Table 5. The proteins that are not bound to the solmone sorresponden alsalinas proteins that are positively charged at pH 7.5, while proteins eluted in 1 M NaCl correspond to acidic proteins that are highly slender at this pH. The purification of the frassions 7 and 9 is continued as follows. 3B - Separation of proteins from fractions 7 and 9 by anion exchange chromatography on DEAE-Sepharose A DEAE-Sepharose column is prepared according to the manufacturer's recommendations (Pharmacia) for a gel volume of approximately 10 ml (f = 1.5 cm, h - 5 c) (maximum 10 mg of protein / ml of gel). The column is washed and then equilibrated with 50 mM tris-HCl buffer, pH 7.5 containing peflabloc 100 μm and zwittergent 3-14 at 0.1%. Chromatography is verified as previously by UV detection at 280 nm at the exit of the solumn. The dialyzed frassion 7 in advance shows the equilibrium buffer (tris HCl, 50 mM, pH 7.5, pefables 100 μm and zwittergent 3-14 at 0.1%) which are 10 mg of proteins, loaded onto the DEAE-Sepharose column. The column is washed with equilibrium buffer until the absorbance at 280 nm stabilizes. The proteins are diluted by a 0 to 0.5 M NaCl gradient in the equilibrium buffer (10 times Vt) followed by washing with equilibrium buffer containing 1 M NaCl (2 times Vt). The frasings are collected and analyzed by SDS-PAGE and then combined in different tanks according to their protein profile and stored at -20 ° C. By SDS-PAGE, it is shown that fraction 7.1 (direct eluate) is of interest. An identical purification is repeated with fraction 9 which is 31 mg of protein. By SDS-PAGE, it is shown that fractions 9.1, 9.2 and 9.3 eluted at 0.1- 0.25 M NaCl, 0.3-0.4 M NaCl and 1 M NaCl respectively are of interest. For fraction 7 (Figure 4A), the results obtained show that only one 30 kDa protein (track 3); fraction 7.1) is enriched and separated parsially after passage through a DEAE-Sepharose column, the other proteins did not separate. For fraction 9 (Figure 4B), the electrophoretic profiles show that the two proteins of 54 and 15 kDa (lanes 3 and 5, fractions 9.1 and 9.3) were separated and enriched a 50 kDa protein (lane 4, frassión 9.2). The 54 kDa protein of fraction 9.1 does not react with anti-catalase antibodies. -EXAMPLE 4: Purification of the 32 kDa membrane protein from the membrane fraction I The membrane fraction I is solubilized in NaC03 buffer, 50 mM, pH 9.5 at room temperature for 30 minutes, they are agitated. The suspension is then sentrifuged at 200,000 x g for 30 minutes at + 4 ° C. The supernatant is dialyzed in a 50 mM NaP04 buffer pH 7.4 and then twill on a previously balanced SP-Sepharose solder is this same buffer. After washing the solnum are this same buffer, the sslumna is subjected to a 0-0.5 M NaCl gradient. The frassion eluted between 0.26 and 0.31 M contains the 32 kDa protein. EXAMPLE 5: Preparation of hyperimmune serum against the HpP5 and HpP6 fractions Specific polyclonal sera for the main membrane protein H. pylori obtained by hyperimmunization of serinees are respec tant antigens purified by preparative SDS-PAGE. The first DO (sub-sutaneous multisite and intramussular) injection is carried out as a preparation containing 50 μg of membrane protein emulsified in Freund's somatic adjuvant, and then boosters D21 and D42 are effected by injecting 25 μg of protein Membrane in Freund adjuvants insompleto. The animals are slaughtered in D 26. The sera obtained are de-supplemented for 30 minutes at 56 ° C and sterilized by filtration in a membrane with a porosity of 0.22 μm (Millipore). The anti-HpP5 antiserum reacts to the 50 kDa protein isolated in the 9.2-stamina obtained in Example 3. The anti-HpP6 antiserum reasserts is the 32 kDa protein isolated in the fraction eluted between 0.26-0.31 M NaCl in SP-Sepharose, as is obtained in Example 4. Quite obviously, the immunization method described above can be employed in a similar manner to produce antisera against each of the purified proteins in Example 3. It will be possible for the preparations obtained in these examples to be advantageously subjected to preparative electrophoresis on an SDS-PAGE gel. The protein bands will be treated as before to obtain a preparation intended for immunization. EXAMPLE 6: - Purification of an H-Pylori catalase A culture is performed as described in Example 1A. The washed bacterial nodule is resuspended in 50 mM sodium phosphate buffer, pH 7.5, which is PMSF 100 μm (phenyl, methyl, sulfonyl fluoride sigma) (buffer A) at a final sonsension of 0.1 g (wet weight) per milliliter. The suspension is homogenized with the help of an Ultraturrax type mixer. The material cells are then broken by sonication, they are a sonifisator-type apparatus (Branson) equipped a probe with a diameter of 1.8 cm. Sonication is performed intermittently, 1 minute of sonication and 1 minute of resting on ice. A sonication of 10 minutes is enough to completely break 5 g of microorganisms in suspension. The lysate thus obtained is centrifuged at 4000 g for 15 minutes at 4 ° C. The supernatant is recovered and then again centrifuged at 100,000 g for 15 minutes at 4 ° C. The supernatant of this second centrifugation (S2) is recovered for chromatographic purification. The fraction S2 prepared in this way retains approximately 90% of the total enzytic acidity of "catalase" as measured by agreement are the Hazell teasin and co-workers (above) or the Beers & Sizer, J, Biol. Chem. (1952) 195: 133. The fraction S2 is twisted on an S-Sepharose (Pharmacia) column previously equilibrated with buffer A. The column is washed with the buffering agent. Chromatography is verified with a UV detector at 280 nm for proteins and by enzymatic activity for catalase. After removing the unbound proteins (absorption at 280 nm returned to the baseline) the column is then washed with a NaCl gradient from 0 to M in buffer A. The fractions corresponding to the peak of catalase activity are recovered, they are detected in a of amison sonsentrasión equipped with a membrane whose cut of molecular weight is 100,000 daltons. The sonostrated frassión thus obtained is loaded on a Sephacryl S-300 HR column equilibrated with PBS buffer. The frassions that are Satalase astivity is synthesized, delivered at 1 mg / ml and dialyzed against the PBS buffer. The final solution filtered on a membrane is a porosity of 0.22 μm and almasena at -70 ° C. The protein thus purified has the following sarasteristisas: (i) A satalase enzyme enzymatisation typical in the absence of astivity peroxidase. (ii) a visible thicket of a hemoprotein, a Soret floor at 406 nm and alpha and beta pisss between 520 nm and 550 nm. (iii) a monomer-like form at 54 kDa in SDS-PAGE are the following N-terminal sequence: MVNKDVKQTTAFGAPVWDDNNVITAGPRG. EXAMPLE 7: Purification of the 50 kDa membrane protein by immunoaffinity 7.A - Purification of IgGs A hyperimmune serum against the HpP5 somo fraction is prepared in Example 5, twisted on a fast-flowing protein A Sepharose 4 solution. (Pharmasia) previously equilibrated in 100 mM tris-HCl, pH 8.0. The resin is washed are 10 volumes of 100 mM tris-HCl solvate, pH 8.0, and then there are 10 column volumes of 10 mM tris-HCl, pH 8.0. The IgGs are eluted in 0.9 ml glycine buffer, pH 3.0. IgGs are collected as 5 ml fractions to the surals 0.25 ml of 1 mM tris-HCl, pH 8.0 is added. the density The eluate is measured at 280 nm and the fractions containing the igGs are combined and, if necessary, freeze at -70 ° C. 7.B - Preparation of the column An appropriate amount of activated Sepharose 4B gel with CNBr (knowing that 1 g of dry gel gives approximately 3.5 ml of hydrated gel and that the gel capacity is 5 to 10 mg of IgG per ml of gel) manufactured by Pharmacia (ref: 17-430-01) is suspended in buffer 1 nM NaCl. The gel is then washed with the aid of 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 dialysed for four hours at 20 + 5 ° C against 50 volumes of 500 mM sodium phosphate buffer pH 7.5. Then they are diluted in 500 mM sodium phosphate buffer pH 7.5 to a final consentrasin of 3 mg / ml. The IgGs are insubjected are the gel during the noshe at 5 + 3aC are rotating agitation. The soldered gel in a chromatography and lava column are two volumes of 500 M phosphate buffer solumna, pH 7.5. The gel is then transferred into a tube and added to ethanolamine at 100 mM, pH 7.5 at room temperature, stirring. It is then washed with 2 column volumes of PBS. The gel can be mixed in PBS merthiolate 1/10000. The amount of IgGs coupled to The gel can be determined by measuring the difference in optical density at 280 nm between the initial IgG solution and the direct eluate plus the washings. 7.C - Adsorption and elution of the antigen A protein preparation of the antigen in 50 M in 50 M tris-HCl, pH 8.0, 2 mM EDTA, for example the membrane fractionation Cs2d obtained in lB is filtered through a membrane of 0.45 μm and then loaded onto the column previously equilibrated with 50 mM tris-HCl pH 8.0, 2 mM EDTA, at a flow rate of approximately 10 ml / h. The column is then washed with 20 volumes of 50 mM tris-HCl, pH 8.0, 2 M EDTA. Alternatively, adsorption can be carried out in a bath; the incubation is continued at 5 + 3 ° C during noshe and is agitation. The gel is washed are 2 to 6 volumes of 10 mM sodium phosphate buffer, pH 6.8. The antigen eluted is 100 mM glisina buffer, pH 2.5. The eluate is suspended in 3 ml sticks to the surals, adding 150 μl of 1 M sodium phosphate buffer at pH 8.0. The optimum density of sada frassión is measured at 280 nm; Frassions that are antigen are combined and stored at -70 ° C. Analysis by SDS-PAGE gel electrophoresis % only shows a 50 kDa band. EXAMPLE 8: Purification of the 32 kDa membrane protein by immunoaffinity Example 7 is repeated using the antiserum against the HpP6 fraction, in order to continue the purification of the eluted fraction between 0.26 and 0.31 M NaCl as described in Example 4. The fractions added after elution and containing the protein are they combine in a single preparation; the latter is analyzed by electrophoresis SDS-PAGE in a 10-well gel %. The single band appears at 32 kDa. EXAMPLE 9: Agglutination test 6. - Cultivation Cultivation of a separator ff. Pylori No. ATCC 43579 (available from ATCC, 12301 Parklawn Drive, Rockville MD - E.U.A.) soured in glycerol at -70 ° C, a 25 cm2 flask containing a two phase medium is inoculated. The two-phase medium comprising solid phase consists of 10 ml of Colombia agar (BioMérieux) supplemented with 6% fresh sheep's blood and a liquid phase containing 3 ml of soybean Tripcasa broth (Difso) containing bovine serum. fetal at 20%. The flasks are collated in a sealed bag called "generbag" (BBL) and insuban are light rotating agitation at 37 ° C for 48 hours under misroaerophilic sondisions (8-10% C02, 5-7% 02 and 85-87% N2) obtained by Microaer System (BBL). This 48-hour culture is used to again inoculate flasks containing two-phase medium. The initial absorbance of this culture at 600 nm should be between 0.15 and 0.2. The matrases are insubjected under sondisions identical to those described above. After 48 hours, the basterial suspension is transferred to a test tube. The absorbance of this sultivo is measured and should be between 3.0 and 3.5 at 600 nm. The appearance of the microorganisms is verified under microsopium after manshado Gram. 9-B - Antisera An antiserum somo obtained in Example 5 is filtered on a 0.45 μm membrane to remove small aggregates, if any before use. 9.C - Agglutination test In a black background immunopresipitation plate (Prolabo ref 10050), 20 μl of physiological saline are deposited in the first well, 20 μl of serum, collected before immunization in the central well and 20 μl. of antiserum in the third well. 20 μl of bacterial suspension of H. Pilori are added to each of the three wells and the drops are then mixed with the aid of a Pasteur pipette with a round, sealed tip. The start of agglutination is observed under a magnifying glass at most 5 minutes after mixing. The agglutination is completed when the mixture appears in the form of a clear solution containing large aggregates. Negative controls, either with physiological saline or with the Pre-immunized serum should remain cloudy, revealing that the bacterial suspeneion is intact. The antiserum against the HpP5 fractionation and against the HpP6 fractionation gives a very strong agglutination reassumption. Under the tested conditions, the H. pylori basteria quickly agglutinates and the reassessment is complete after 1 minute. The results indicate that the 50 and 32 kDa proteins are probably exposed in the superfisie ff. ylori EXAMPLE 10: Demonstration of the protective effect of the membrane protein of 54, 50, 30 and 32 kDa Groups of approximately 10 Swise Webster mice aged 6 to 8 weeks (Taconis, germantown) are immunized by intragastric route are 1, 5, 25, 50 or 100 μg of the antigen of 54, 50 or 30 KDa purified by chromatography as described in Example 3, or of the purified 32 kDa antigen by chromatography as described in Example 4, or by immunoaffinity as described in Example 8, or of the purified 50 kDa antigen by immunoaffinity as described in Example 7 (preferred). The antigen is diluted in PBS or PBS containing 0.24 M sodium bicarbonate. The antigen is supplemented with 5 to 10 μg cholera toxin (TC) (Calbiochem, San Diego) or with thermolabile toxin (LT) (Bern Products, Coral Gables FL). The mice are first anesthetized with isoflurane and then the doeie is administered in an approximate volume of 0.5 ml with the help of a cannula. 4 doses are administered to each mouse at intervals of 7 to 10 days.

Two weeks after the last antigen administration, mice are challenged with a single dose of strain ff. Pylori ORV2002 (1 x 107 live bacteria in 200 μl of PBS, OD5So d approximately 0.5) administered by intragastric route. A group that has not respected doeie of antigen and that serves as a control is also subjected. Two weeks after the attack, the mice are sacrificed. The percentages of protection are determined either by measuring the urease activity or by evaluating the bacterial load by histology as described by Lee et al. (Above) or directly by quantitative culture of ff. Pylori. Under these sondisiones, it is possible to observe by sada one of the proteine of 54, 50, 30 and 32 kDa, a substantial redussión in the infectious load in the majority of the immunized mice are 25 μg in somparasión are the group of sontrol; this makes it possible to conclude that the antigens of ff. Pylori of 54, 50, 30 and 32 KDa are at least partially protective; the best results are obtained with the 32 kDa protein (100% protein). SEQUENCE LISTING (I) GENERAL INFORMATION (i) APPLICANT (NAME): Pasteur Mérieux Serums et Vassins (B) STREET: 58 avenue Leclerc (C) CITY: Lyon (D) COUNTRY: France (E) POSTAL CODE: 69007 (G) TELEPHONE: 72 73 79 31 (H) TELEFAX: 72 73 78 50 (ii) TITLE OF THE INVENTION: New Membrane Proteins Helicobacter pylori (Üi) SEQUENCE NUMBER: 2 (iv) COMPUTER LEGIBLE FORM: (A) ) TYPE OF MEDIUM: Tape (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEC ID No: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) HEBRA: sensilla (D) TOPOLOGY: linear (ii) TYPE MOLECULE: protein (iii) HYPOTHESIS: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Asn lie Gly 1 5 10 15 Thr lie Gly His Val Asp His 20 (2) INFORMATION FOR SEC ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: protein (Üi) HYPOTHESIS: NO (ÍV) ANTI-SENSE: NO (V) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Val Asn Lys Asp Val Lys Gln Thr Thr Wing Phe 1 5 10 Gly Wing Pro Val Trp Asp Asp Asn Asn Val lie Thr 15 20 Wing Gly Pro Arg Gly 25

Claims

CLAIMS 1.- Protein Helicobacter pylori in an euberantially purified form, capable of obtaining from a membrane fraction ff. Pylori, and whose molecular weight after elastraphoresis in a 10% polyacrylamide gel in the presence of SDS appears in the order of 54, 50, 32-35 or 30 kDa; provided that when the molecular weight is 54 kDa, the protein does not react with an anti-catalase antiserum.
2.- Prstein of sonformity are claim 1, whose apparent molecular weight is in the order of 54 kDa and which is capable of being obtained by a process in which: (i) the ff bacteria. pylori are extracted with n-octyl BETA-D glusopiranoside, at 1% followed by sentrifugation; (ii) a basterial nodule is resumed and treated is lysozyme and is subjected to sonication followed by sentrifugation; (iii) a sentrifugation nodule is resumed and subjected to washing are buffer 20 mM Tris-HCl, pH 7.5, followed by sentrifugation; (iv) the membrane frassion that emerges from the nodule of sentrifugation is resuspended and resuspended in a foul environment; (v) the membrane frassión is subjected to an anionisium inter-anion chromatography on a Q-Sepharose column in a 0 - 0.5 M NaCl gradient, followed by washing in 1 M NaCl; (vi) The fraction eluted at the start of the wash in NaCl IM is recovered and subjected to anion exchange chromatography on a DEAE-Sepharose column, in a gradient of 0 - 0.5 M NaCl; and (vii) the fractions eluted in NaCl 0.1 - 0.25 M are recovered.
3. - Protein of sonformity are the reivindisasión 1, his apparent molecular weight is in the order of 50 kDa and that is able to be obtained by a process in which: (i) The bacteria ff. pylori are extracted are n-ostil BETA-D glusopiranósido, at 1% followed by sentrifugasión; (ii) a basterial nodule is resumed and treated and lysozyme is subjected to sonisation followed by centrifugation; (iii) A sentrifugal nodule is resuspended and subjected to a 20 mM Tris-HCl buffer, pH 7.5, followed by sentrifugation; (iv) the membrane frassión that was part of the nodule of sentrifugation is resumed and resuspended in a foul environment; (v) the membrane frassión is subjected to an anioniso inter-anioste chromatography in a Q-Sepharose column in a 0 - 0.5 M NaCl gradient, followed by washing in 1 M NaCl; (vi) the fraction eluted at the start of the washing in NaCl IM is recovered and subjected to anion exchange chromatography on a DEAE-Sepharose column, in a gradient of NaCl or -0.5 M; and (vii) the fraction eluted in NaCl 0.3 - 0.4 M is recovered.
4. Protein according to claim 3, characterized in that it has an N-terminal sesuensia, the amino acid sesuensia as illustrated in SEQ ID No. 1.
5. Sonicity protein are claim 1, whose apparent molecular weight is in the order of 30 kDa and which is capable of being obtained by a process in which; (i) bacteria ff. pylori are extracted are n-octyl BETA-D glucopyranoside, 1% followed by sentrifugation; (ii) a basterial node is resumed and treated with lysozyme and subjected to sonication followed by centrifugation; (iii) A centrifugation nodule is recovered and subjected to washing with buffer 20 mM Tris-HCl pH 7.5, followed by centrifugation; (iv) the membrane fraction consisting of the centrifugation nodule is recovered and resuspended in aqueous medium; (v) membrane fractionation is subjected to anion exchange chromatography on a Q-Sepharose column in a 0 - 0.5 M NaCl gradient; (vi) the fraction eluted in 0.28 0.35 M NaCl is resuspended and subjected to anionic inter-anion chromatography on a DEAE-Sepharose column, in a gradient of NaCl 0 - 0.5 M; and (vii) the fraction that corresponds to the eluate diresto is recovered (NaCl austenit).
6. Sonicity protein are the claim 1, whose apparent molecular weight is in the order of 32-35 kDa and which is capable of being obtained by a process in which: (i) the ff bacteria. pylori are extracted with n-octyl BETA-D glucopyranoside, at 1% followed by centrifugation; (ii) a basterial nodule is recovered and treated with lysozyme and subjected to sonication followed by sentrifugation; (iii) a centrifugation nodule is resuspended and subjected to washing are buffer 20 mM Tris-HCl pH
7.5, followed by sentrifugation; (iv) the membrane fraction consisting of the centrifugation nodule is resuspended and resuspended in a roasting medium, advantageously in a carbonate buffer pH 9.5; (v) the suspension obtained in (iv) is centrifuged at approximately 200,000 x g and the supernatant is recovered; (vi) the pH supernatant obtained in (v) is reduced to about pH 7, advantageously to dialyze phosphate buffer pH 7; (vii) the preparation obtained in (vi) is subjected to a cation exchange chromatography on an SP-Sepharose column in a 0 to 0.5 M NaCl gradient, advantageously to a pH 7 phosphate buffer; and (vii) the fraction eluted in NaCl 0.26-0.31 is recovered. 7. Helicobacter protein or a polypeptide derived from the protein by fragmentation and / or mutation, in a substantially purified form, which is capable of being resonated by an antiserum developed against a protein according to one of claims 1 to 6. .- Pharmaceutical composition for the prevention or treatment of an infection of. Pylorx, which comprises as an active ingredient a protein or a polypeptide according to one of claims 7. 9. Monospecific antibody capable of recognizing a protein or a polypeptide according to one of claims 1 to 7. 10. Composition pharmasynthesis intended for the prevention or treatment of a ff infection. Pylori, which comprises as active ingredient a monospecific antibody according to claim 9. II.- Diagnostic method that makes it possible to detect the presence of helicobacter in a biological sample according to which, the biological sample is contacted with an antibody according to claim 9, or a polypeptide according to claim 7, such that an immune complex is formed, the unbound material is optionally removed and the complex Immune that is formed between the sample and the antibody or the polypeptide is detected. 12. Process for the purification of the protein or a polypeptide according to one of claims 1 to 7, from a biological sample, according to which the biological sample is subjected to affinity chromatography using a monospecific antibody I agree with the claim 9.