NZ319640A - protein obtained from the membrane of helicobacter pylori - Google Patents

Abstract

A Helicobacter pylori protein in a substantially purified form, capable of being obtained from a membrane fraction of H. pylori and of which the molecular weight after electrophoresis on polyacrylamide gel 10 % in the presence of SDS is 54 kDa is claimed. The protein can be obtained by a process as defined in the specification.

Description

319 64 0 WO 97/12909 PCT/FR96/01552 NEW HELICOBACTER PYLORI MEMBRANE PROTEINS The object of the present invention is 5 Helicobacter pylori proteins newly obtained m substantially purified form, as well as the pharmaceutical compositions containing them.

Helicobacter is a bacterial genus characterized by Gram-negative helical bacteria. Several species 10 colonize the gastrointestinal tract of mammals There may be mentioned m particxilar 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, it has been possible to 15 isolate in man H. heilmanii and H felis Helicobacter infects more than 50% of the adult population m developed countries and nearly 100% of that of developing countries, thereby making it one of the predominant infectious agents worldwide 20 H pylori is so far exclusively found at the surface of the mucous membrane of the stomach m man and more particularly around the crater lesions of gastric and duodenal ulcers This bacterium is cuirently recognized as the aetiologicai agent of 25 antral gastritis and appears as one of the cofactors required for the development of ulcers Moreover, it seems that the development of gastric carcinomas may be associated with the presence of H pylori It therefore appears to be highly desirable to 30 develop a vaccine intended to prevent or treat H pylori infections Such a vaccine would be most probably of a subunit nature.

Various H. pylori proteins have been characterized or isolated so far They are especially 35 urease, composed of two subunits A and B of 3 0 and 67 kDa respectively (Hu & Mobley, Infect Immun (1990) 58 992; Dunn et al , J Biol Chem. (1990) 265 • 9464; Evans et al. , Microbial Pathogenesis (1991) Ul • 15, Labigne et al , J Bact, (199) [sic] 173 1920), the 40 vacuole cytotoxm of 87 kDa (VacA) (Cover & Blaser, J. 319 64 0 Biol. Chem (1992) 267 : 10570, Phadnis et al., Infect Immun. (1994) 62 : 1557; WO 93/18150); an immunodominant antigen of 128 kDa associated with the cytotoxm (CagA, also called TagA) (WO 93/18150, USP 5 5 403 924), heat shock proteins HspA and HspB of 13 and 58 kDa respectively (Suerbaum et al., Mol. Microbiol (1994) 2A • 959, WO 93/18150), a catalase of 54 kDa (Hazell et al , J Gen Microbiol (1991) 137 • 57) ; a fibrillar haemaglutinin (HpaA) of 20 kDa; a histidme-10 rich protein of 15 kDa (Hpn) (Gilbert et al., Infect. Tmmun (1995) §2. 2682); an outer membrane protein of 30 kDa (Bolm ut al , J. Clin Microbiol (1995) 32. '■ 381), a membrane-associated lipoprotein of 20 kDa (Kostrcynska et al , J Bact (1994) 176 . 5938) as 15 well as a family of porins HopA, HopB, HopC and HopD, of molecular weight between 48 and 67 kDa (Exner et al , Infect Immun (1995) 1567) Some of these proteins have already been proposed as potential vaccinal antigens. In particular, urease is recognized as being a most preferred antigen which can be used for this purpose (WO 94/9823, WO 95/3824, WO 95/22987, Michetti et al , Gastroenterology (1994) 107 3 002) The fact remains that the search for new antigens must continue, 25 especially since it is envisaged that, in order to obtain an optimum vaccinal effect, several antigens will probably have to be incorporated into a vaccine In summary, it still appears necessary to identify additional antigens in order to incorporate 3 0 them into a vaccine of high efficacy Accordingly, the subject of the invention is especially an H pylori protein in a substantially purified form, capable of being obtained from an H. pylori membrane fraction, and whose molecular weight 3 5 after electrophoresis on a 10% polyacrylamide gel m the presence of SDS appears of the order of 54, 50, 32-35 or 3 0 kDa. When the protein has a molecular weight of about 54 kDa, it is specified, in addition, that it does not react with an77an£i^catSlage^antiserum.

* .... — SO J <.L' £IVrcD J 319 6 4 0 An anti-H pylori catalase antiserum may be especially prepared according to the immunization process described m Example 5 below, using a catalase preparation obtained by chromatography, as described m Example 6.

"Substantially purified form" is understood to mean that the protein is separated from the medium in which it exists naturally. Among others, it may be a preparation lacking especially the H. pylori cytoplasmic and periplasmic proteins.

The membrane protein whose apparent molecular weight is of the order of 54 kDa is capable of being obtained by a process m which (l) the H pylori bacteria are extracted with 1% n-octyl fe-D glucopyranoside, followed by centrlfugation; (n) a bacterial pellet is recovered and it is treated tfith lysozyme and subjected to sonication, followed by centrifugation; (in) a centrifugation pellet is recovered and it is subjected to washing with 20 mM Tris-HCl buffer pH 7 5, followed by centrifugation, (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended in aqueous medium, advantageously in carbonate buffer pH 9 5 containing 5% zwittergent 3-14, (v) the membrane fraction is subjected to an 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 0.1% zwittergent 3-14; (vi) the fraction eluted at the start of washing in 1 M NaCl is recovered and it is subjected to an anion-exchange chromatography on a DEAE-Sepharose column, m a—0 - 0.5 M NaCl 319 6 4 0 gradient, advantageously m Tris-HCl buffer pH 7.5 containing 0 1% zwittergent 3-14 (advantageously, the fraction m 1 M NaCl is first dialysed against Tris-HCl buffer pH 5 7.5 containing 0 1% zwittergent 3-14); and (vn) the fraction eluted in 0.1 - 0 25 M NaCl is recovered.

The membrane protein whose apparent molecular weight is of the order of 50 kDa is capable of being 10 obtained by a process m which* (l) the H pylori bacteria are extracted with 1% n-octyl £-D glucopyranoside, followed by centrifugation; (n) a bacterial pellet is recovered and it is 15 treated with lysozyme and subjected to sonication, followed by centrifugation; (in) a centrifugation pellet is recovered and it is subjected to washing with 20 mM Tris-HCl buffer pH 7 5, followed by centrifugation, 20 (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended i.4 aqueous medium, advantageously in carbonate buffer pH 9 5 containing 5% zwittergent 3-14; 25 (v) the membrane fraction is subjected to an 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 3-30 14, followed by washing in 1 M NaCl, advantageously in a carbonate buffer pH 9 5 containing 0 1% zwittergent 3-14; (vi) the fraction eluted at the start of washing in 1 M NaCl is recovered and it is subjected 35 to an anion-exchange chromatography on a DEAE-Sepharose column, m a 0 - 0 5 M NaCl gradient, advantageously in Tris-HCl buffer pH 7.5 containing 0.1% zwittergent 3-14 (advantageously, the fraction in 1 M NaCl is 31 9 6 4 0 first dialysed against Tris-HCl buffer pH 7.5 containing 0.1% zwittergent 3-14), and (vn) the fraction eluted in 0 3 - 0 4 M NaCl is recovered The membrane protein whose apparent molecular weight is of the order of 30 kDa is capable of being obtained by a process m which: (l) the H. pylori bacteria are extracted with 1% n-octyl S-D glucopyranoside, followed by 10 centrifugation, (n) a bacterial pellet is recovered and it is treated with lysozyme and subjected to sonication, followed by centrifugation; (in) a centrifugation pellet is recovered and it 15 is subjected to washing with 20 mM Tris-HCl buffer pH 7 5, followed by centrifugation, (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended in aqueous medium, advantageously in carbonate buffer pH 9 5 containing 5% zwittergent 3-14; (v) the membrane fraction is subjected to an anion-exchange chromatography on a Q-Sepharose column m a 0 - 0 5 M NaCl gradient, advantageously in a carbonate buffer pH 9 5 containing 0 1% zwittergent 3-14, (vi) the fraction eluted in 0 28-0 35 M NaCl is recovered and it is subjected to an anaon- exchange chromatography on a DEAE-Sepharose column, in a 0 - 0 5 M NaCl gradient, advantageously m Tris-HCl buffer pH 7 5 containing 0.1% zwittergent 3-14 (advantageously, the fraction m 1 M NaCl is 35 first dialysed against Tris-HCl buffer pH 7.5 containing 0 1% zwittergent 3-14), and (vii) the fraction corresponding to the direct eluate is recovered (absence of NaCl) '"TTCFBC' 3 0 ttW 1597 RECEIVED 31 9 64 (/) The membrane protein whose apparent molec/alar weight is of the order of 32-35 kDa is capable of being obtained by a process m which: (l) the H pylori bacteria are extracted wj/th 1% 5 n-octyl £-D glucopyranoside, followed by centrifugation; (n) a bacterial pellet is recovered and it is treated with lysozyme and subjected to sonication, followed by centnfugatio/i; (m) a centrifugation pellet is recoverec and it is subjected to washing with 20 mM Tris-HCl buffer pH 7 5, followed by centrifuc/ation; (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended in aqueous medium, advantageously m carbonate buffer )H 9 5; (v) the suspension obtained in (iv) is centrifuged at about 200,000 x g and the supernatant is recovered, (vi) the pH of the supernatant obtain/sd in (v) is reduced to about pH 7, advan ageously by dialysing against phosphate buf' er pH 7; (vn) the preparation obtained n (vi) is subjected to a c/it ion-exchange chromatography on an SP-Sepha/ose column m a 0 - 0.5 M NaCl gradient, advantageously in a phosphate buffer pH 7; and (vii) the fraction eluted m 0 26 -i 0 31 M NaCl is recovered The 54, 50, 32 and 30 kDa prote'ms according to the invention are probably intrinsic r/iembrane proteins or proteins associated with the memhr.'ane The 54 kDa protein does not redct with anti-cat/alase antibodies, nor in Western blotting, or m do, blotting The t 30 kDa protein does not react wi'th anti-urease A subunit antibodies, nor m Western tlottmg, or in dot blotting. The 32 kDa protein proves to be an alkaline protein; its molecular weight majy appear slightly f —\ 319 6 4 0 greater eg of the order of 35 kDa under certain experimental conditions.

The N-terminal sequence of the 50 kDa protein of an H. pylori strain (ATCC 43579) is as follows (one-5 letter code) : MKEKFNRTKPHVNIGTIGHVDH. This information does not exclude the fact that equivalent proteins capable of being purified according to the process indicated above can have a slightly different N-termmal sequence, since they may be derived from 10 another bacterial strain. Such a difference would indeed reflect the phenomenon of allelic variance commonly encountered within the same species. For example, a bacterial species is usually represented by a group of strains which differ from each other m 15 minor allelic characteristics A polypeptide which fulfils the same biological function m different strains may have an amino acid sequence which is not different for all the strains. Such an allelic variation also exists m DNA.

The allelic differences at the level of the amino acid sequence may consist of one or more substitutions, deletions or additions of ammo acids, which do not alter the biological function "Biological function" is understood to mean the 25 function of the protein wnich participates in the survival of the cells in which the protein exists naturally (even if the function is not absolutely essential) For example, the function of a porin is to allow compounds present m the external medium to enter 30 inside the cell. The biological function is distinct from the antigenic function A protein may have more than one biological function The subject of the invention is also a protein m a substantially purified form and which may have 35 been purified according to one of the processes described above from a bacterium of the genus Helicobacter, e.g H pylori, H heilmanii, H. felis and H. mustelae N Z Pt OFFlCEj 8 0 HAY 1997 ~~recbved" 319 6 4 0 The subject of the invention is also any protein or polypeptide, m a substantially purified form, insofar as it is analogous, m terms of antigenicity, to a Helicobacter protein capable of 5 being purified according to one of the processes described above. As regards the polypeptides, tuey are especially polypeptides derived by fragmentation or by mutation of one or more amino acids, e g by deletion, addition or substitution, of a protein which exists in 10 nature and whose purified form may be obtained according to one of the processes described above. Such polypeptides may be especially obtained by enzymatic digestion with the aid of proteases such as pepsin or trypsin It is not necessary for such 15 polypeptides to be purified according to one of the processes described above The present description uses the terms "protein" and "polypeptide" independently of the size of the molecules (length of the amino acid chain) and 20 of the possible ^ost-translational modifications. In the remainder of the description, the term "polypeptide" is reserved to designate a product derived from a protein by fragmentation or mutation.

A protein or a polypeptide according to the 25 invention should be capable of being recognized by monospecific antibodies raised against a Helicobacter protein capable of being purified according to one of the processes described above This specific antigenicity may be revealed according to a number of 30 methods; for example by Western blotting (Towbin et al , PNAS (1979) 2Sl - 43 50) , dot blotting and 5LISA.

In Western blotting, the product intended to be tested, e g either m the form of a purified preparation or in the form of a bacterial extract, is 35 subjected to an SDS-Page gel electrophoresis (10% polyacrylamide) as described by Laemmli U K , Nature (1970) 227 . 680 After transferring onto a nitrocellulose membrane, the latter is incubated with a monospecific hyperimmune serum diluted in the range of 319 6 4 0 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 test product exhibits a reactivity at one of the 5 dilutions included m the range established above.

In ELISA, the product intended to be tested is preferably used to coat wells. A purified preparation is preferably used although a total extract can also be used. Briefly, 100 pi of a preparation containing 10 10 pg of protein/ml are distributed into the wells of a 96-well plate. The plate is incubated for 2 h at 37°C and then overnight at 4°C The plate is washed with PBS (phosphate buffered saline) buffer containing 0 05% Tween 20 (PBS/Tween buffer). The wells are saturated 15 with 250 ill of PBS containing 1% bovine serum albumin (BSA) The whole is incubated for 1 h at 37°C and then the plate is washed with PBS/Tween buffer. A monospecific rabbit antiserum is serially diluted m PBS/Tween buffer containing 0.5% BSA One hundred vil 20 of a dilution are added to each well. The plate is incubated for 90 mm at 37°C and then washed. The plate is visualized according to standard methods For example, a conjugate peroxidase-goat immunoglobulin against rabbit immunoglobulins is added to the wells 25 The incubation is continued for 90 min at 37°C and then the plate is washed The reaction is developed with the appropriate substrate The reaction is measured by colorimetry (absorbance measured by spectrophotometry) Under these conditions, a positive reaction is observed 30 when an OD value of 1 is associated with a dilution of at least 1 50, preferably of at least 1 500 The appropriate wavelength at which tho optical density is measured depends on the substrate.

In dot blotting, a purified preparation of the 35 product to be tested is preferably used although a total extract can also be used Briefly, a preparation of the product to be tested containing 100 yg of ,__--;--r".^5^in/ml is serially diluted twice m 50 mM Tris-HCl pH~~T]\5 One hundred pi of each dilution are applied to ws J1 9 6 A 0 a 0 45 ym nitrocellulose membrane m a 96-well dot blot apparatus (Biorad). The buffer is removed by placing under 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 skimmed milk) and then incubated with a monospecific antiserum diluted m the range from 1 50 to 1 • 5000, preferably from 1 • 50 to 1 : 500. The reaction is visualized according to standard methods. For example, a conjugate peroxidase-goat immunoglobulin against rabbit immunoglobulins is added to the wells The incubation is continued for 90 mm at 37°C and then the plate is washed. The reaction is developed with the appropriate substrate. The reaction is measured by colorimetry or chemiluminescence. Under these conditions, a reaction is positive when a colour is observed at the level of the spot on the nitrocellulose sheet directly for visualization by colorimetry or on a photographic film for visualization by chemiluminescence, associated with a dilution of at least 1 50, preferably at least 1 • 500.

According to a specific embodiment, a protein according to the invention may be obtained especially by purification from Helicobacter or expressed by t-he recombinant route m 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 which 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 may be evaluated according to standard methods, for example by measuring the induction of a mucosal immune response or the induction of an immune response having a therapeutic or protective effect using e g the mouse/H. felis model and the procedures, described m Lee et al , Eur. J. Gastroenterology & Hepatology, v- I \ ' 319 6 4 0 - n - (1995) , 2 "• 303 or Lee et al , J. Infect. Dis (1995) 172 161, on condition that the following precaution is taken, when the protein is derived from a species other than H felis, the H. felis strain should be 5 replaced by a Helicobacter strain belonging to the species from which the protein is derived and adapted to this end (the other experimental conditions remaining identical). For example, the capacity of a polypeptide derived by fragmentation from an H pylori 10 protein to induce a protective or therapeutic effect is tested by substituting an H. pylori strain. Such a strain is proposed by e g. Kleanthous et al., Abstr. presented at the Vlllth International Workshop on Gastroduodenal Pathology 7-9th July 1995, Edinburgh, 15 Scotland. A protective effect is observed once an infection in the gastric tissue is reduced compared with a control group. The infection is evaluated by testing the urease activity, the bacterial load or the leucocyte infiltration For example, when a reduction 20 in the urease activity m the gastric tissue is observed after a challenge, even if it is not completely abolished it is reasonable to assert that there is partial protection.

Consequently, the invention also relates to (i) 25 a composition of material comprising a protein or a polypeptide according to the invention and a diluent or a carrier, in particular (n) a pharmaceutical composition intended especially for the prevention or treatment of a Helicobacter infection, which comprises 30 as active ingredient a protein or a polypeptide according to the invention, in a quantity effective from a prophylactic or therapeutic point of view, (in) the use of a protein or a polypeptide according to the invention as therapeutic or prophylactic agent; (iv) 35 the use of a protein or a polypeptide according to the invention for the manufacture of a medicament intended for the prevention or treatment of a Helicobacter infection, as well as to (v) a method for inducing an immune response against tieJLicobactS^^ie.g. H pylori, 319 6 4 0 H heilmanii, H. felis and H mustelae m a mammal, according to which an immunologically effective quantity of a protein or of a polypeptide according to the invention is administered to the said mammal so as 5 to develop an immune response, m particular (vi) a method for the prevention or treatment of a Hehcobacterlle [sic] infection, there is administered to an individual a prophylactically or therapeutically effective quantity of a protein or of a polypeptide 10 according to the invention.

The methods and pharmaceutical compositions according to the invention can treat or prevent Helicobacter infections and, consequently, gastrointestinal diseases associated with such 15 infections. They are m particular chronic and atrophic acute gastritis; peptic ulcers, e g. gastric and duodenal ulcers; gastric cancers; chronic dyspepsias; refractory non-ulcerous dyspepsias; intestinal metaplasias and certain lymphomas (e g low 20 grade MALT lymphoma) A composition according to the invention may be administered by any conventional route m use in the field of vaccines, in particular through a mucosal (e.g. ocular, nasal, oral, gastric, intestinal, rectal, 25 vaginal or the urinary tract) surface or by the parenteral (e.g subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal) route. The choice of the route of administration depends on a number of parameters such as the adjuvant associated 30 with the protein or the 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 35 chosen A composition according to the invention may comprise m addition to a protein or a polypeptide according to the invention, at least one other Helicobacter antigen such as the urease apoenzyme, or a 319 64 0 subunit, fragment, homologue, mutant or derivative of this urease For use m a composition according to the invention, a protein or a polypeptide according to the 5 invention may be formulated m or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS or virus-like particles (VLPs), so as to promote the targeting of the protein or polypeptide or to enhance the immune response Persons skilled in the 10 art obtain these compounds without any difficulty, for example see Liposomes A Practical Approach, RRC New ED, IRL press (1990).

Adjuvants other than liposomes may also be used A large number are known to persons skilled m 15 the art Such adjuvants are referenced below: For parenteral administration, there may be mentioned especially aluminium compounds such as aluminium hydroxide, aluminium phosphate and aluminium hydroxyphosphate. The antigen may be adsorbed or 20 precipitated onto an aluminium compound according to standard methods. Other adjuvants such as RIBI from ImmunoChem (Hamilton, MT) may be used for parenteral administration.

For mucosal administration, there may be 25 mentioned especially the bacterial toxins, e g. the cholera toxin (CT), the heat-labile E coll toxin (LT), the Clostridium difficile toxin and the pertussis toxin (PT) as well as the detoxified forms (subunit, toxoid or mutant) of these toxins For example, a preparation 30 containing the B subunit of CT (CTB) and a smaller quantity of CT may be used Fragments, homologues and derivatives of these toxins are likewise appropriate insofar as they retain an adjuvant activity. Preferably, a mutant having a reduced toxicity is used. 35 Such mutants are described e g. in WO 95/17211 (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) r-u£, n^C^ECy^S^, Salmonella 3 0 WW 319 64 0 minnesota, Salmonella typhimurium or Shigella flexneri, may be used for mucosal administration Adjuvants useful both for mucosal and parenteral administration include especially polyphosphazine (WO 95/2415), DC-chol (3-beta-[N-(N' ,N' -dimethylammomethane) carbamoyl] cholesterol (USP 5 283 185 and WO 96/14831) and QS-21 (WO 88/9336) .

The administration may be made as a single dose or as a dose repeated once or several times after a 10 certain period. The appropriate dosage varies according to various parameters, for example the individual treated (adult or child), the vaccinal antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and 15 if present, the type of adjuvant and the desired effect (e g protection or treatment), as can be determined by persons skilled in the art In general, an antigen according to the invention may be administered m a quantity ranging from 10 pg to 50C mg, preferably from 20 1 mg to 200 mg In particular, it is indicated that a parenteral dose should not exceed 1 mg, preferably 100 jig Higher doses may be prescribed for e.g. oral use Independently of the formulation, the quantity of protein administered to man by the oral route is for 25 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 may be manufactured m a conventional manner In particular, a protein or a polypeptide according to the invention 3 0 is combined with a diluent or a carrier which is pharmaceutically acceptable, e g. water or a saline solution such as a phosphate buffered saline (PBS) , optionally supplemented with a bicarbonate salt such as sodium bicarbonate, e.g. 0 1 to 0.5 M when the 3 5 composition is intended for oral or intragastric administration. In general, the diluent or carrier is selected on the basis of the mode and route of administration and of standard pharmaceutical practices Diluents and - carriers which are i \ - " 319 64 0 pharmaceutically acceptable, as well as all that is necessary for their use in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, a standard reference text in this field, and in USP/NP.

In a more detailed manner, it is proposed, by way of example, to administer a protein or a polypeptide according to the invention by the oral route To this end, a protein or a polypeptide according to the invention may be encapsulated alone or 10 in the presence of other H pylori proteins in gelatin capsules in order to protect the antigen against degradation by the gastric juice, or administered in the presence of sodium bicarbonate Such formulations have already been used for pharmaceutical compositions 15 (Black et al , Dev Biol. Stand (1983), ill • 9) The protein may also be encapsulated in PLGA microspheres (glycolic acid and lactic acid copolymers) according to the procedure described elsewhere (Eldridge et al , Curr. Top. Microbiol. Immuno. (1989) 146 . 59); the 20 protein may also be encapsulated m liposomes prepared according to widely-described conventional methods ("Liposomes : a practical approach, Ed RRC New, D. Rickwood & B.D Hames, 1990, Oxford University Press, ISBN 0-19-96307 7-1) Alternatively, a protein oi a polypeptide according to the invention may be administered by the parenteral route To do this, a protein or a polypeptide according to the invention is adsorbed onto alumina gel in a completely conventional manner. The 30 protein in solution at 1 mg/ml in a buffer whose pH is close to 6 5 is brought into contact, for 1 hour, with aluminium hydroxide at 10 mg/ml, measured at AL+++. The final composition of the preparation is the following: protein 50 pg/ml, AL+++ 250 yg/ml, merthiolate 1/10,000, 35 the whole in PBS. As in the case of oral administration, 3 injections are recommended, each at an interval of 4 weeks from the preceding one.

A polypeptide according to the invention may also be useful as diagnostic reagent, for example for 319 64 0 detecting the presence of anti-Helicobacter antibody m a biological sample, eg. a blood sample. To this end, such a polypeptide advantageously comprises 5 to 80 amino acids, preferably 10 to 50 amino acids. A 5 polypeptide reagent according to the invention may be labelled or otherwise, according to the diagnostic method used Diagnostic methods are described earlier in the text According to another aspect, the invention 10 provides a monospecific antibody capable of recognizing a protein or a polypeptide according to the invention.

"Monospecific antibody" is understood to mean an antibody capable of reacting predominantly with a single Helicobacter protein. Such an antibody can only 15 be obtained using a substantially purified protein as immunogen An antibody according to the invention may be polyclonal or monoclonal; the monoclonals may be chimeric (for example, consisting of a variable region of murine origin associated with a human constant 20 region) or humanized (only the hypervariable regions are of animal origin, for example of murine origin) and/or a single chain The polyclonals, like the monoclonals, may also be m the form of immunoglobulin fragments, for example an F(ab)'2 or Fab fragment. An 25 antibody according to the invention may also be of any isotype, for example IgG or IgA, a polyclonal may be of a single isotype or a mixture of all or some of them.

In the text which follows, the terms "monospecific antibody" and "monospecific antiserum" 3 0 are used interchangeably An antibody which is directed against a protein according to the invention may be produced and subsequently identified using a standard immunological assay, for example Western blot, dot blot or ELISA 3 5 analysis (see for example Coligan et al., Current Protocols m Immunology (1994) John Wiley & sons Inc., New York, NY), Antibodies * A laboratory Manual, D Lane, (1988) Harlow Ed ). ^7. 3 0 KM 1397 319 6 4 0 An antibody according to the invention may be useful m diagnosis, as well as m affinity chromatography for large-scale purification of a protein or a polypeptide according to the invention; such an antibody is also potentially useful as therapeutic agent m a passive immunization procedure.

Consequently, the invention also provides (i) a reagent for detecting the presence of Helicobacter m a biological sample, which comprises an antibody or a 10 polypeptide according to the invention; and (n) a diagnostic method for detecting the presence of Helicobacter m a biological sample, according to which the biological sample is brought into contact with an antibody or a polypeptide according to the invention, 15 so that an immune complex forms, optionally, the unbound material is removed 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 Helicobacter m the 20 sample or in the organ from which the sample was collected As can be easily understood, an antibody according to the invention makes it possible to test for the presence of Helicobacter in a gastric extract. 25 For use in a diagnostic test, the reagent for to be [sic] be provided m a free form or may be immobilized on a solid support; the latter may be any support commonly used in this domain, for example a tube, a bead or a well. The immobilization may be 30 obtained by direct or indirect means. The direct means comprise passive adsorption (noncovalent bonding) or covalent bonds between the support and the reagent. "Indirect means" means that an anti-reagent compound capable of interacting with a reagent is first attached 35 to a solid support. For example, if a polypeptide reagent is used, an antibody capable of binding it may be used as 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 319 64 0 biological samples. Indirect means may also be implemented through a ligand-receptor system, for example by grafting a molecule, such as a vitamin, onto a polypeptide reagent and then by immobilizing, m 5 solid form, the corresponding receptor. This is illustrated e g. by the biotm-strepcavidm system Alternatively, indirect means are used, for example, by adding a peptide tail to the reagent, e.g. by chemical means, and by immobilizing the grafted product by 10 passive adsorption or by covalent bonding of the peptide tail The invention also relates to a process for the purification of a protein or of a polypeptide according to the invention from a biological sample, according to 15 which the biological sample is subjected to an affinity chromatography using a monospecific antibody according to the invention.

To this end, the antibody may be polyclonal or monoclonal, preferably of the IgG type Purified IgGs 20 may be prepared from an antiserum according to methods which are commonly used (see for example Coligan et al.) .

Conventional chromatography supports, like standard methods of grafting antibodies, are described 25 for example m Antibodies : A Laboratory Manual, D. Lane, Harlow Ed (1988) Briefly, a biological sample, preferably a buffer solution, is applied to a chromatography material, preferably equilibrated with the buffer used 30 for the dilution of the biological sample so that the protein or the polypeptide according to the invention (antigen) can be adsorbed onto the material. The chromatography material, such as a gel or a resin associated with an antibody according to the invention, 35 may be provided m the form of a bath or a column The components which remain unbound are removed 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, e.g. guanidine- 319 6 4 0 HCl, or a salt-rich concentration (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 5 Such a purification process may, for example, be used to purify a protein from a total extract However, if the antibody is not perfectly monospecific, it is advisable to enrich beforehand the material intended to be subjected to the linmunoaf f mity 10 chromatography m terms of quantity of protein to be purified For example, such a process may be used to perfect the purification of the 32 kDa protein as obtained according to the process described above which comprises a step of purification on SP-Sepharose 15 The therapeutic or prophylactic usefulness of an antibody according to the invention may be demonstrated according to the protection test by Lee et al., proposed above for the proteins or polypeptides according to the indention. Thus, the subject of the 20 invention is also (i) a composition of material comprising a monospecific antibody according to the invention, and a diluent or a carrier, m particular, (ii) a pharmaceutical composition comprising a monospecific antibody according to the invention in an 25 effective quantity from a therapeutic or prophylactic point of view; (in) the use of a monospecific antibody according to the invention m the preparation of a medicament for treating or preventing a Helicobacter infection, as well as (iv) a method for treating or 30 preventing a Helicobacter infection (for example, H. pylori, H. felis, H. mustelae or H heilmanii), according to which a therapeutically or prophylactically effective quantity of an antibody according to the invention is administered to an 35 individual requiring such a treatment To this end, the monospecific antibody may be polyclonal or monoclonal, preferably of IgA isotype (predominantly) . In the context of a passive immunization method, the^antibody^r^^p^dministered by U i G 0 ^ 31 9 64 0 the mucosal route to a mammal, for example at the level of the gastric mucous membrane, either by the oral or intragastric route, advantageously m the presence of a bicarbonate buffer. A monospecific antibody according 5 to the invention may be administered as sole active component or as a mixture comprising at least one monospecific antibody specific to each Helicobacter polypeptide. The dose of antibody which should be used in this method can be easily determined by persons 10 skilled in the art. For example, it is suggested that a dosage may 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 per day for two to three days. 15 A pharmaceutical composition comprising an antibody according to the invention may be manufactured according to the rules stated above for a composition comprising a protein or a polypeptide according to the invention Likewise, identical medical indications 2 0 apply.

The invention is illustrated below with reference to the following Figures: Figure 1 is a summary of the procedure for the preparation of the H. pylori membrane fractions I, II 25 and III.

Figure 2 presents the analysis of the membrane fractions I, II and III by electrophoresis on a 10% polyacrylamide gel and staining with Coomassie blue The samples loaded are: membrane fraction I (lane 2), 30 membrane fraction II (lane 3), membrane fraction III (lane 4) and molecular weight markers (lane 1).

Figure 3 presents the analysis, by electrophoresis on a 10% polyacrylamide gel and staining with Coomassie blue, of the proteins purified 35 from a preparative gel (lanes 3 to 7) The samples loaded are. the HpPl fraction (lane 3), the HpP2 fraction (lane 4), the HpP4 fraction (lane 5), the HpP5 fraction (lane 6) , the HpP6 fraction (lane 7) , the 319 64 0 molecular weight markers (lanes 1 and 8) and the membrane fraction I (lane 2).

Figure 4 represents the analysis of the fractions obtained from the passage on DEAE-Sepharose 5 of the fractions 7 and 9 (obtained after elution on Q-Sepharose). The fractions were separated by electrophoreses on a 10% or 12 5% polyacrylamide gel and stained with Coomassie blue The samples loaded are: fraction 7 (lane 2A) , fraction 7.1 (lane 3A) , 10 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) (lane 1A and IB) Figure 5 presents, after electrophoresis on a 15 10% polyacrylamide gel and staining with Coomassie blue, the electrophoretic profile of the D fraction obtained from the chromatography on a Q-Sepharose column of the membrane fraction III (lane 3) and of the fraction D' obtained from the chromatography on an S-20 Sepharose column of the fraction D (lane 4) . Lane 1 corresponds to the molecular weight markers and lane 2 to the membrane fraction III.

EXAMPLE 1; Preparation of the membrane fractions 1A - Culture The H pylori strain ATCC 43579 is cultured m liquid medium in a 10 1 fermenter.

A frozen sample of microorganisms m glycerol 3 0 is used to inoculate a 75-cm2 flask containing a so-called "two-phase" medium (a solid phase in Colombia agar containing 6% fresh sheep blood and a liquid phase m soya bean trypcase containing 20% foetal calf serum). After 24 hours of culture 35 under microaerophilic conditions, the liquid phase of this culture is used to inoculate several 75-cm flasks m a two-phase medium m the absence of sheep blood. After 24 hours of culture, the liquid phase makes it possible to inoculate a 2-1 ]77 7. C--.TENT C 3 0 MW 15c 319 6 4 0 bioferrnenter m liquid soya bean trypcase medium containing beta-cyclodextrm at 10 g/1. This culture at OD 1.5-1.8 is inoculated into a 10-1 fermenter m liquid medium. After 24 hours of culture, the bacteria are harvested by centrifugation at 4000 x g for 30 minutes at 4°C. A 10-litre culture of H. pylori ATCC 43579 m a fermenter makes it possible to obtain about 20 to 30 g (wet weight) of bacteria.

IB - Extraction with n-octyl S-D-glucopyranoside (OG) The pellet of microorganisms which is obtained above is washed with 500 ml of PBS 15 (phosphate buffered saline; NaCl 7.650 g, disodium phosphate 0.724 g, monopotassium phosphate 0.210 g per litre; pH 7 2) per litre of culture. The microorganisms are then centrifuged agaan under the same conditions.

The bacterial pellet obtained (Cx) is resuspended m an OG solution (Sigma) at 1% (30 ml/litre of culture). The bacterial suspension is incubated for 1 hour at room temperature, with magnetic stirring, and then 25 centrifuged at 2 7,600 x g for 30 minutes at 4°C.

The pellet (C2) is stored for subsequent treatment.

The supernatant (S2) obtained is dialysed (MWCO = 10000 Da, Spectra/por) overnight at 4°C, 30 with magnetic stirring, against twice 1 litre of PBS diluted 1/2 The precipitate formed during the dialysis is recovered by centrifugation at 2600 x g for 3 0 minutes at 4°C The supernatant (S2d) is removed and the pellet (CS3a) which 35 contains membrane proteins is stored at -20°C. 1C of - Breaking of the microorganisms The pellet (Ca) obtained after centrifugation the microorganisms treated with OG is 319 64 0 - 23 resuspended in 20 mM tns-HCl buffer pH 7 5 and 100 pM Pefabloc (buffer A" and then homogenized by Ultra-turrax (3821, Jaike & Kungel). The homogenate obtained is exposed to lysozyme 5 (0.1 mg/ml final) and EDTA (1 mM final) The homogenate is sonicated for 3 times 2 minutes at 4°C (probe <p = 0 5 cm, power = 20%, Sonifier 450 Branson), and then ultracentrifuged at 210,000 x g for 30 minutes at 4°C. The 10 supernatant (S3) which contains cytoplasmic and periplasmic proteins is removed, while the pellet (C3) is recovered, wasned with buffer A, and then ultracentrifuged at 23 0,000 x g for 30 minutes at 4°C. After removal of the supernatant (S*) , the 15 pellet (C4) is stored at -20°C. This pellet contains intrinsic and peripheral membrane proteins.

The procedure may be continued by a double washing of the pellet C4 in order to remove 20 peripheral membrane proteins. The pellet C4 is resuspended m 50 mM NaC03 buffer pH 9.5, 100 pM Pefabloc (buffer B). The suspension is ultracentrifuged at 210, 000 x g for 30 minutes at 4°C. The supernatant (S5) is removed and then the 25 pellet (C5) is washed and ultracentrifuged under the same conditions as above. After removal of the supernatant (S6) , the pellet (C6) which contains essentially intrinsic membrane proteins is stored at -20°C.

The fractions C4, C6 and CS2a are called hereinafter membrane fractions I, II and III respectively.

ID - Analysis of the membrane fractions The various membrane fractions are analysed by polyacrylamide gel electrophoresis xn the presence of SDS according to the Laemmli method (1970).' The proteins are visualized after Coomassie blue staining 31 9 6 4 0 each fraction a 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 fraction II On the other hand, 5 these two differ substantially from membrane fraction III The profile of membrane fraction I shows 7 major protein bands of respective molecular weights 87, 76, 67, 54, 50, 47 and 32-35 kDa (lane 10 2) . By Western blotting m the presence of anti- ureB antibody or anti-catalase antibody, it was shown that the band at 67 kDa corresponded to the B subunit of urease and the band at 54 kD corresponded to catalase. These two proteins do 15 not exist m the profile of fraction II (lane 3) since the washing with carbonate buffer removes the proteins weakly associated with the membrane As for the protein profile of the membrane fraction III, it shows the presence of 4 major 20 bands at 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 is carried out on 25 polyacrylamide gel according to the Laemmli method (1970) with a 5% stacking gel and a 10% separating gel The membrane fraction is resuspended in buffer A and then diluted one half in 2X sample buffer. The mixture is heated for 5 minutes at 95 °C About 19 mg of 30 proteins are loaded onto a gel 16 x 12 cm m size and 5 mm thick. A premigration is performed at 50 V for 2 hours, followed by a migration at 65 V overnight. The staining of the gel with Coomassie blue R250 (0.05% m ultraflltered water) allows good visualization of the 3 5 bands.

The major bands HpPl, HpP2, HpP4, HpP5 and HpP6 are cut out with a scalpel and ground in an Ultra-turrax in the presence of 10 or 20 ml of extraction buffer containing 25 mM TriSnIffiT^f(^$f. 8, 8 M urea, 10% 319640 SDS, 100 pM phenylmethy 1 sulphonyl fluoric [sic] (PMSF) and 100 pM Pefabloc (buffer C). Each ground product is filtered on a Millipore AP20 prefliter (fouter = 4*7 cm' ^ora = 20 pm) with the aid of an extruder at a pressure 5 of 7 bar, at room temperature. Each ground product is washed with 5 to 10 ml of buffer C and filtered as above The two filtrates obtained from each corresponding ground product are combined Each filtrate is precipitated with 3 volumes of 10 a 50 50 mixture 75% methanol and 75% isopropanol, and then ultracentrifuged at 240,000 x g for 16 hours at 10°C on a 70 TFT rotor (J8-55, Beckman) Each pellet is taken up m 2 ml of solubilization buffer containing 10 mM NaP04 pH 7.0, 15 1 M NaCl, 0.1% sarcosyl, 100 pM PMSF, 100 pM Pefabloc and 6 M urea (buffer D) . The solubilized sample is dialysed successively against 100 ml of buffer D containing 4 M urea and 0.1% sarcosyl, against 100 ml of buffer D containing 2 M urea and 0 5% sarcosyl and 20 against twice 100 ml of buffer D without urea and containing 0.5% sarcosyl. The 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 and then centrifuged at low 25 speed for 10 minutes at 4°C (Biofuge A, Heraeus Sepatech). The supernatant is recovered, filtered on a 0 45 pm Millipore filter and stored at -20°C An SDS-PAGE analysis was carried out for each fraction (Figure 3).

Analysis of the electrophoretic profile of each fraction shows that the fractions HpPl, HpP2 and HpP4 are pure with a single gel band for each of these fractions (at 87, 76 and 54 kDa respectively) . The fraction HpP5 has a band of high intensity at 50 kDa 35 which is slightly contaminated with a band at 47 kDa, likewise the fraction HpP6 has a band of high intensity at 32 kDa which is slightly contaminated with a band at 35 kDa 319 64 0 Tgyawpr.E 3 : Purification of the membrane proteins of 30, 50 and 54 kD from the membrane fraction X 3A - Anion-exchange chromatography on Q-Sepharose A Q-Sepharose column of 40 ml (<f> =25 cm, h = 8 cm) is prepared according to the recommendations of the manufacturer (Pharmacia). The column is washed and then equilibrated with 10 the 50 mM NaC03 buffer pH 9.5 containing 100 pM Pefabloc and 0 1% zwittergent 3-14. The chromatography was monitored by UV detection at 280 nm at the outlet of the column One hundred and forty mg of previously 15 solubilized proteins of membrane fraction I are loaded onto the column which is then washed with the equilibrating buffer (50 mM NaC03 pH 9 5, 100 joM Pefabloc and 0.1% zwittergent 3-14) untj.1 the absorbance at 280 nm is stabilized. The 20 proteins are eluted by a 0 1 to 0 5 M NaCl gradient m the equilibration buffer (10 times VT) followed by washing m equilibration buffer containing 0.5 and 1 M NaCl (twice VT) The fractions collected are analysed by SDS-PAGE and 25 combined into different pools according to their electrophoretic profile, and then stored at -20°C. The fractions are as follows: Fractions Elution Fractions Elution 1 direct eluate 6 0 25-0 28 M NaCl 2 washing equilibration buffer 7 0.28-0 35 M NaCl 3 0-0.1 M NaCl 8 0 35-0 5 M NaCl 4 0 1-0.2 M NaCl 9 start of washing 1 M NaCl 0 2-0.25 M NaCli - !-rpMT C end of washing 1 M NaCl ' S 0 MRf ^ 319 64 0 The protein evaluation shows that 53% of the proteins are eluted m the 0-0.5 M NaCl gradient, 14% of the proteins are not attached to the column and 33% of the proteins are eluted during the washing m 1 M NaCl (Table 5) The proteins which are not bound to the column correspond to alkaline proteins which are positively charged at pH 7 5, whereas the proteins eluted in 1 M NaCl correspond to acidic proteins which are highly charged at this pH.

The purification of the fractions 7 and 9 is continued as follows. 3B - Separation of the proteir.3 of fractions 7 and 9 by anion-exchange chromatography on DEAE- Sepharose A DEAE-Sepharose column is prepared according to the recommendations of the manufacturer (Pharmacia) for a gel volume of about 10 ml ((J) = 1.5 cm, h = 5 cm) (maximum 10 mg protem/ml of gel) . The column is washed and then equilibrated with the 50 mM Tris-HCl buffer pH 7.5 containing 100 pM Pefabloc and 0 1% zwittergent 3-14. The chromatography is monitored as before by UV detection at 280 nm at the outlet of the column.

The fraction 7 dialysed beforehand against the equilibration buffer (50 mM Tris-HCl pH 7.5, 100 pM Pefabloc and 0.1% zwittergent 3-14) containing 10 mg of proteins is loaded onto the DEAE-Sepharose column The column is washed with equilibration buffer until the absorbance at 280 nm is stabilized. The proteins are eluted by a 0 to 0 5 M NaCl gradient m the equilibration buffer (10 times VT), followed by washing with equilibration buffer containing 1 M NaCl (twice VT). The fractions collected are analysed by SDS-PAGE and then, combined into different pools according to at 319 6 4 0 -20°C. By SDS-PAGE, it is shown that the fraction 7.1 (direct eluate) is of interest An identical purification is repeated with the fraction 9 containing 31 rng of protein. By 5 SDS-PAGE, it is shown that the 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 the fraction 7 (Figure 4A) , the results obtained show that only a protein of 30 kDa (lane 10 3; fraction 7.1) was enriched and partially separated after passage through the DEAE-Sepharose column, the other proteins were not separated. For the fraction 9 (Figure 4B) , the electrophoretic profiles show that two proteins of 15 54 and 15 kDa (lanes 3 and 5; fractions 9.1 and 9 3) were separated and a protein of 50 kDa was enriched (lane 4; fraction 9.2). The protein of 54 kDa of fraction 9 1 does not react with anti-catalase antibodies EXAMPLE 4 : Purification of the membrane protein of 32 kDa from the membrane fraction I The membrane fraction I is solub-ilized in 50 mM 25 NaC03 buffer pH 9 5 at room temperature for 30 mm, with stirring. The suspension is then centrifuged at 200,000 x g for 30 mm at +4°C The supernatant is dialysed against 50 mM NaP04 buffer pH 7.4 and then loaded onto an SP-Sepharose column previously 30 equilibrated with this same buffer. After washing the column with this same buffer, the column is subjected to a 0-0 5 M NaCl gradient The fraction eluted between 0 26 and 0 31 M contains the protein of 32 kDa.

EXAMPLE 5 : Preparation of hyperimmune sera against the fractions HpP5 and HpP6.

Polyconal sera specific for the H. pylori major membrane proteins are obtained by hyperimmunization of 319 64 0 rabbits respectively with the antigens purified by preparative SDS-PAGE HpP5 and HpP6. The first injection DO (subcutaneous multisite and intramuscular) is carried out with a preparation containing 50 yg of 5 emulsified membrane protein m complete Freund's adjuvant, and then the boosters D21 and D42 are made by injection of 25 jig of membrane protein m incomplete Freund's adjuvant The animals are sacrificed on D60. The sera obtained are decomplementized for 30 minutes 10 at 56°C and sterilized by filtration on a membrane with a porosity of 0 22 pm (Millipore) The anti-HpP5 antiserum reacts with the 50 kDa protein isolated in the fraction 9.2 obtained in Example 3 The anti-HpP6 antiserum reacts with the 15 32 kDa protein isolated m the fraction eluted between 0.26-0.31 M NaCl on SP-Sepharose, as obtained m Example 4.

Quite obviously, the immunization procedure described above may be used in a similar manner to 20 produce antisera against each of the proteins purified m Example 3. It will be possible for the preparations obtained m these Examples to be advantageously subjected to a preparative electrophoresis on an SDS-PAGE gel. The protein bands will be treated as above 25 so as to obtain a preparation intended for immunization.

EXAMPLE 6 : Purification of a catalase from H. pylori 3 0 A culture is performed as described in Example 1A. The washed bacterial pellet is resuspended in 50 mM sodivim phosphate buffer pH 7 5 containing 100 pM PMSF (phenylmethylsulphonyl fluoride Sigma) (buffer A) at a final concentration of 0.1 g (wet weight) per 35 rrullilitre. The suspension is homogenized with the aid of an Ultraturrax-type mixer. The bacterial cells are then broken by sonication with a Sonifler-type apparatus (Branson) equipped with a—-pxpbe with a diameter of 18 cm ation -is performed \ t \ 30 0 319 64 0 intermittently, 1 mm of somcation and 1 mm of rest on ice A 10-mm sonication is sufficient to break completely 5 g of microorganisms m suspension. The lysate thus obtained is centrifuged for 15 mm at 4°C 5 at 4000 g The supernatant is recovered and then again centrifuged at 100, 000 g for 30 min at 4°C. The supernatant from this second centrifugation (S2) is recovered for chromatographic purification. The fraction S2 prepared m this manner retains about 90% 10 of the total "catalase" enzymatic activity, as measured according to the Hazell et al. (supra) technique or Beers & Sizer, J. Biol. Chem. (1952) 195 . 133 technique.

The fraction S2 is loaded onto an S-Sepharose 15 column (Pharmacia) previously equilibrated with buffer A. The column is washed with the same buffer The chromatography is monitored with a UV detector at 280 nm for the proteins and by the enzymatac activity for the catalase. After removal of the unbound 20 proteins (absorption at 280 nm returned to the baseline), the column is then washed with a 0 to 1 M NaCl gradient in buffer A The fractions corresponding to the catalase activity peak are recovered, concentrated m an Amicon-type concentration cell 25 equipped with a membrane whose molecular weight cut-off is 100,000 Daltons. The concentrated fraction thus obtained is loaded onto a Sephacryl S-300 HR column previously equilibrated with PBS buffer. The fractions containing the catalase activity are combined, 30 concentrated to 1 mg/ml and dialysed against the PBS buffer The final solution is filtered on a membrane with a porosity of 0.22 ym and stored at -70°C.

The protein thus purified has the following characteristics (1) A typical catalase enzymatic activity, in the absence of peroxidase activity, (n) A visible spectrum typical of a haemoprotein, a Soret peak' at 406 nm and i l31 9 6 4 0 alpha and beta peaks between 520 nm and 550 nm (in) A rnonomeric form at 54 kDa m SDS-PAGE with the following N-termmal sequence.

NVNKDVKQTTAFGAPVWDDNNVITAGPRG. example 7 : Purification of the membrane protein of 50 kDa by immunoaf f inity 7.A - Purification of the IgGs A hyperimmune serum against fraction HpP5 as prepared m Example 5 is loaded onto a Protein A Sepharose 4 Fast Flow column (Pharmacia) previously equilibrated m 100 mM Tris-HCl pH 8.0. 15 The resin is washed with 10 column volumes of 100 mM Tris-HCl pH 8.0 and then with 10 column volumes of 10 mM Tris-HCl pH 8.0. The IgGs are eluted m 0 1 M glycine buffer pH 3 0. The IgGs are collected as 5-ml fractions to which 0.25 ml 20 of 1 M Tris-HCl pH 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 -70°C. 7.B - Preparation of the column An appropriate quantity of CNBr-activated Sepharose 4B gel (knowing that 1 g of dry gel gives about 3.5 ml of hydrated gel and that the capacity of the gel is 5 to 10 mg of IgG per ml of 30 gel) manufactured by Pharmacia (ref- 17-0430-01) is suspended m 1 mM NaCl buffer. The gel is then washed with the aid of a buchner by adding small quantities of 1 mM HC1 The total volume of 1 mM HCl used is 200 ml per gram of gel 35 The purified IgGs are dialysed for 4 h at 20 + 5°C against 50 vol. of 500 mM sodium phosphate buffer pH 7 5. They are then diluted m 500 mM sodium phosphate buffer pH 7.5 to a final concentration of 3 mg/ml, 319 6 4 0 The IgGs are incubated with the gel overnight at 5 + 3°C, with rotary stirring The gel is placed in a chromatography column and washed with 2 column vol of 500 mM phosphate buffer pH 7 5.

The gel is then transferred into a tube and incubated m 100 mM ethanolamine pH 7 5 at room temperature, with stirring It is then washed with 2 column vol. of PBS The gel may be stored in PBS merthiolate 1/10,000 The quantity of IgGs 10 coupled to the gel may 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 antigen m 50 mM Tris-HCl pH 8 0, 2 mM EDTA, for example the membrane fraction I or II (fraction C4 or C6 as obtained m Example 1C and solubilized in 20 zwittergent) is filtered through a 0.45 membrane and is then loaded onto the column previously equilibrated with 50 mM Tris-HCl pH 8 0, 2 mM EDTA, at a flow rate of about 10 ml/h The column is then washed with 20 vol of 50 mM 25 Tris-HCl pH 8.0, 2 mM EDTA Alternatively, the adsorption may take place in a bath; the incubation is continued at 5 + 3°C overnight and with stirring The gel is washed with 2 to 6 vol of 10 mM 30 sodium phosphate buffer pH 6 8. The antigen is eluted with 100 mM glycine buffer pH 2 5. The eluate is harvested m 3-ml fractions to which 150 (Xl of 1 M sodium phosphate buffer pH 8 0 are added. The optical density of each fraction is 35 measured at 2 80 nm; the fractions containing the antigen are combined and stored at -70°C. The analysis by electrophoresis on a 10% SDS-Pftge gel shows only one band at 50 kD^^^^^fp^f K'V ^ 319 64 0 EXAMPLE 8 : Purification of the membrane protein of 32 kDa by immunoaffinity Example 7 is repeated using the antiserum 5 against fraction HpP6, m order to continue the purification of the fraction eluted between 0.26 and 0 31 M NaCl as described m Example 4. The fractions collected after elution and containing the protein are combined into a single preparation; the latter is 10 analysed by SDS-Page electrophoresis on a 10% gel. A single band appears at 32 kDa.

EXAMPLE 9 : Agglutination test 9.A - Culture From a strain of H. pylori No. ATCC 43579 (available from ATCC, 12301 Parklawn Drive, Rockville MD - USA) stored m glycerol at -70°C, a 25 cm2 flask containing a two-phase medium is 20 inoculated. The two-phase medium comprises a solid phase consisting of 10 ml of Colombia agar (BioMerieux) supplemented with 6% fresh sheep blood and a liquid phase consisting of 3 ml of soya bean Trypcase broth (Difco) containing 20% 25 foetal calf serum. The flasks are placed m a sealed bag called "generbag" (BBL) and incubated with gentle rotary shaking at 37°C for 48 hours under microaerophilic condition (8-10% CO2, 5-7% O2 and 85-87% N2) obtained by the Microaer System 30 (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 flasks are 35 incubated under conditions identical to those described above.

After 48 hours, the bacterial suspension is transferred to a test tube. The absorbance of this culture is measured and itr s15ould be between 319 64 0 3.0 and 3.5 at 600 nm The appearance of the microorganisms is checked under a microscope after Gram staining. 9.B - Antisera An antiserum as obtained m Example 5 is filtered on a 0.45 ytm membrane so as to remove small aggregates, if they exist, before use 9.C - Agglutination test On a black-bottomed immunoprecipitation plate (Prolabo ref. 10050) , there are deposited 20 of physiological saline m the first well, 20 ul of serun, collected before immunization, in the 15 central well and 20 ul of antiserum m the third well Twenty pi of bacterial suspension of H. pylori are added to each of the three wells and the drops are then mixed with the aid of a Pasteur pipette with a sealed round tip. 20 The onset of agglutination is observed under a magnifying glass at most 5 minutes after the mixing The agglutination is complete when the mixture appears m the form of a clear solution comprising large aggregates The negative controls, either with physiological saline, or with the preimmunization serum, should remain cloudy, revealing that the bacterial suspension is intact The antiserum against fraction HpP5 and against fraction HpP6 give a very strong 30 agglutination reaction Under the conditions tested, the H. pylori bacteria aqglutmate rapidly and the reaction is complete after one minute. The results indicate that the proteins of 50 and 32 kDa are probably exposed at the surface of H. 35 pylori.

EXAMPLE 10 : Damons t rat ion of the protective effect of the membrane proteins of 54, 50, 30 and 32 kDa 319 64 0 Groups of about ten Swiss Webster mice aged from 6 to 8 weeks (Tacomc Labs. Germantown, NY) are immunized by the intragastric route with 1, 5, 25, 50 or 100 pg of the antigen of 54, 50 or 30 kDa purified 5 by chromatography as described m Example 3, or of the antigen of 32 kDa purified by chromatography as described m Example 4 or by immunoaf f mity as described m Example 8, or of the antigen of 50 kDa purified by immunoaffmity as described m Example 7 10 (preferred) . The antigen is diluted m PBS or m PBS containing 0.24 M sodium bicarbonate The antigen is supplemented with 5 or 10 pg of cholera toxin (CT) (Calbiochem, San. Diego) or with heat-labile toxin (LT) (Berna Products, Coral Gables FL) . The mice are first 15 anaesthetized with isoflurane and then the dose is administered m a volume of about 0 5 ml with the aid of a cannula Four doses are administered to each mouse at 7-10 day intervals. Two weeks after the last administration of antigen, the mice are challenged with 20 a single dose of H pylori ORV2 002 strain (1 x 1Q7 live bacteria m 200 pi of PBS; OD550 of about 0 5) administered by the intragastric route. A group having received no dose of antigen and serving as control is challenged likewise Two weeks after the challenge, 25 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 m Lee et al. (supra) or directly by quantitative culture of H. pylori. Under these 30 conditions, it is possible to observe for each of the proteins of 54, 50, 3 0 and 32 kDa, a substantial reduction in the infectious load m most of the mice immunized with 25 pg compared with the control group; this makes it possible to conclude that the H. pylori 35 antigens of 54, 50, 30 and 32 kDa are at least partially protective; the best results being obtained with the protein of 32 kDa (100% protection). - 36 -SEQUENCE LISTING 319 6 4 0 (1)GENERAL INFORMATION (x) APPLICANT (A) NAME: Pasteur Meneux Serums et Vaccms (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 (11) TITLE OF THE INVENTION New Helicobacter pylori 15 membrane proteins (in)NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM-20 (A) MEDIUM TYPE- Tape (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE- Patentln Release #1.0, Version #1.30 (EPO) (2)INFORMATION FOR SEQ ID NO- 1: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH 22 amino acids 30 (B) TYPE, amino acid (C) STRANDEDNESS single (D) TOPOLOGY: linear (li) MOLECULE TYPE: protein 35 , (in) HYPOTHETICAL NO (iv) ANTISENSE. NO (v) FRAGMENT TYPE: N-termmal 319 64 0 (XI) SEQUENCE DESCRIPTION. SEQ ID NO. 1 Met Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn lie Gly Thr 15 10 15 lie Gly His Val Asp His 5 20 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: 10 (A) LENGTH: 29 ammo acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY, linear (n) MOLECULE TYPE* protein (in)HYPOTHETICAL: NO (IV) ANTISENSE: NO (v) FRAGMENT TYPE: N-terminal (Xl) SEQUENCE DESCRIPTION. SEQ ID NO: 1: Met Val Asn Lys Asp Val Lys Gin Thr Thr Ala Phe Gly Ala Pro Val 15 10 15 Trp Asp Asp Asn Asn Val lie Thr Ala Gly Pro Arg Gly 25 20 25 ® - 38 - ~Z r ^ v* '

Claims (10)

Claims

1 Helicobacter pylon protein in a substantially purified form, capable of being obtained from H pylori membrane fraction, and whose molecular weight after electrophoresis on a 10% polyacrylamide gel in the presence of SDS appears of the order of 54 kDa and which is capable of being obtained by a process in which (I) the H pylori bacteria are extracted with 1% n-octyl £-D glucopyranoside, followed by c entrlfugation; (II) a bacterial pellet is recovered and it is treated with lysozyme and subjected to sonication, followed by centrifugation; (in) a centrifugation pellet is recovered and it is subjected to washing with 20 mM Tris-HCl buffer pH 7.5, followed by centrifugation; (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended m aqueous medium,- (v) the membrane fraction is subjected to an anion-exchange chromatography on a Q-Sepharose column m a 0 - 0.5 M NaCl gradient, followed by washing m 1 M NaCl; (vi) the fraction eluted at the start of washing in 1 M NaCl is recovered and it is subjected to an anion-exchange chromatography on a DEAE-Sepharose column, in a 0 - 0.5 M NaCl gradient, and (vii) the fraction eluted in 0 1 - 0 25 M NaCl is recovered.

2 Helicobacter pylori protein in a substantially purified form, capable of being obtained from H pylori membrane fraction, and whose molecular weight after electrophoresis on a 10% polyacrylamide gel in the presence of SDS appears of the order of 50 kDa and which is capable of beart^EUECiUML HHUPtRTY OFFlcT obtained by a process in which 3 0 SEP 1999 -RECEIVED T' ' - n • 39 - (i) the H. pylon bacteria are extracted with 1% n- octyl S-D glucopyranoside, followed by centrifugation; (11) a bacterial pellet is recovered and it is treated with lysozyme and subjected to sonication, followed by centrifugation; (ni) a centrifugation pellet is recovered and it is subjected to washing with 20 xnM Tris-HCl buffer pH 7.5, followed by centrifugation; (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended in aqueous medium; (v) the membrane fraction is subjected to an anion-exchange chromatography on a Q-Sepharose column m a 0 - 0.5 M NaCl gradient, followed by washing in 1 M NaCl; (vi) the fraction eluted at the start of washing m 1 M NaCl is recovered and it is subjected to an anion-exchange chromatography on a DEAE-Sepharose column, in a 0 - 0.5 M NaCl gradient; and (vn) the fraction eluted m 0.3 - 0 4 M NaCl is recovered.

3 Protein according to claim 2, which has as N-terminal sequence the amino acid sequence as shown in SEQ ID NO 1

4 Helicobacter pylori protein in a substantially purified form, capable of being obtained from H pylori membrane fraction, and whose molecular weight after electrophoresis on a 10% polyacrylamide gel in the presence of SDS appears of the order of 30 kDa and which is capable of being obtained by a process in which (i) the H. pylori bacteria are extracted with 1% n-octyl £-D glucopyranoside, followed by centrifugation; (ii) a bacterial pellet is recovered and it is treated with lysozyme and sub j ecfead««^to sonication, followed by centrifugation; f k^7i/AL property 1 OF biZ 3 0 SEP 1999 ■SgCE/yeo - 40 - I (111V a centrifugation pellet is recovered and it is subjected to washing with 20 mM Tris-HCl buffer pH 7.5, followed by centrifugation; (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it is resuspended in aqueous medium; (v) the membrane fraction is subjected to an anion-exchange chromatography on a Q-Sepharose column in a 0 - 0.5 M NaCl gradient; (vx) the fraction eluted in 0.28 - 0.35 M NaCl is recovered and it is subjected to an anion-exchange chromatography on a DEAE-Sepharose column, in a 0 - 0.5 M NaCl gradxent; and (vxi) the fraction corresponding to the direct eluate is recovered (absence of NaCl).

5 Helicobacter pylori protein in a substantially purified form, capable of being obtained from H pylori membrane fraction, and whose molecular weight after electrophoresis on a 10% polyacrylamide gel in the presence of SDS appears of the order of 32-35 kDa and which is capable of being obtained by a process in which (i) the H. pylori bacteria are extracted with 1% n- octyl S-D glucopyranoside, followed by centrifugation; (n) a bacterial pellet is recovered and it is treated with lysozyme and subjected to sonication, followed by centrifugation; (iii) a centrifugation pellet is recovered and it is subjected to washing with 20 mM Tris-HCl buffer pH 7.5, followed by centrifugation; (iv) the membrane fraction consisting of the centrifugation pellet is recovered and it xs resuspended in aqueous medium, advantageously in carbonate buffer pH 9.5; (v) the suspension obtained m (iv) xs centrifuged at about 200,000 x g and the supernatant is recovered; | 'n/euectUvL rnuptHTY OFFICE] OF NZ 30 SEP TO RECEIVED -41 - (vi) the pH of the supernatant obtained ^ (v)_ isj reduced to about pH 7, advantageously by dialysing against phosphate buffer pH 7, (vii) the preparation obtained m (vi) is subjected to a cation-exchange chromatography on an SP-Sepharose column in a 0 - 0.5 M NaCl gradient, advantageously in a phosphate buffer pH 7, and (vii) the fraction eluted m 0.26 - 0.31 M NaCl is recovered.

6. Helicobacter protein or a polypeptide derived from the protein by fragmentation and/or mutation, m a substantially purified form, which is capable of being recognized by an antiserum raised against a protein according to one of Claims 1 to 5 .

7. Pharmaceutical composition for the prevention or treatment of an H. pylori infection, which comprises as active ingredient a protein or a polypeptide according to one of Claims 1 to 6 .

8. Monospecific antibody capable of recognizing a protein or a polypeptide according to one of Claims 1 to 6.

9. Pharmaceutical composition intended for the prevention or treatment of an H. pylori infection, which comprises as active ingredient a monospecific antibody according to Claim 8

10- Diagnostic method which makes it possible to detect the presence of Helicobacter in a biological sample, according to which the biological sample is brought into contact with an antibody according to Claim 8 or a polypeptide according to Claim 6 so that an immune complex forms, the unbound material is optionally removed and the immune complex formed between the sample and the antibody or the polypeptide is detected. 11 Process for the purification of a protein or of a polypeptide according to one of Claims 1 to 6 , from a biological sample, according to which the bi sample is subjected to an affinity chromatograp a monospecific antibody according to Claim 8.