FR2724936A1 - Protein isolated from Helicobacter pylori membrane - Google Patents
Protein isolated from Helicobacter pylori membrane Download PDFInfo
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- FR2724936A1 FR2724936A1 FR9411323A FR9411323A FR2724936A1 FR 2724936 A1 FR2724936 A1 FR 2724936A1 FR 9411323 A FR9411323 A FR 9411323A FR 9411323 A FR9411323 A FR 9411323A FR 2724936 A1 FR2724936 A1 FR 2724936A1
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- helicobacter
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- lactoferrin
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
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- 241000590002 Helicobacter pylori Species 0.000 title 1
- 229940037467 helicobacter pylori Drugs 0.000 title 1
- 235000018102 proteins Nutrition 0.000 claims abstract description 26
- 102000010445 Lactoferrin Human genes 0.000 claims abstract description 8
- 108010063045 Lactoferrin Proteins 0.000 claims abstract description 8
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims abstract description 8
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Abstract
Description
La présente invention concerne la prévention et le traitement des infections gastriques dues à la bactérie
Hélicobacter. Elle a pour objet une protéine de surface nouvellement identifiée et purifiée à partir d'Hélicobacter ainsi que son usage à titre de vaccin. Cette protéine a la capacité de se lier à la lactoferrine d'origine humaine.The present invention relates to the prevention and treatment of gastric infections due to the bacteria
Helicobacter. It relates to a newly identified surface protein purified from Helicobacter as well as its use as a vaccine. This protein has the ability to bind to lactoferrin of human origin.
Hélicobacter pylori est une bactérie gramnégative retrouvée à la surface de la muqueuse gastrique chez l'homme. Cette bactérie est associée à un certain nombre de pathologies gastroduodénales dont elle serait, du moins pour certaines, l'agent pathogène responsable. I1 s'agit en particulier des gastrites aiguës ou chroniques (inflammation de la muqueuse), des ulcères (destruction de la muqueuse), des dyspepsies et de certains cancers tel que 1' adénocarcinome gastrique. Hélicobacter pylori is a gram-negative bacterium found on the surface of the gastric mucosa in humans. This bacterium is associated with a certain number of gastroduodenal pathologies of which it is, at least for some, the pathogenic agent responsible. These are in particular acute or chronic gastritis (inflammation of the mucosa), ulcers (destruction of the mucosa), dyspepsia and certain cancers such as gastric adenocarcinoma.
A cet égard, il est déjà apparu hautement souhaitable de mettre au point un vaccin pour prévenir et lutter contre les infections à Hélicobacter. In this regard, it has already appeared highly desirable to develop a vaccine to prevent and combat Helicobacter infections.
Différents antigènes ont déjà été proposés comme agent vaccinal potentiel : il s'agit notamment de l'uréase (WO 90/4030), de la cytotoxine et de la protéine heat-shock (WO 93/18150) et des adhésines. Different antigens have already been proposed as a potential vaccine agent: these include urease (WO 90/4030), cytotoxin and heat-shock protein (WO 93/18150) and adhesins.
Néanmoins, on prévoit qu'un vaccin anti
Hélicobacter, pour atteindre un maximum d'efficacité, devra contenir plus d'un antigène. Il est donc nécessaire de poursuivre l'identification des protéines d'Hélicobacter susceptibles d'être employées à cet effet.However, it is expected that a vaccine against
Helicobacter, to reach maximum efficiency, must contain more than one antigen. It is therefore necessary to continue the identification of the Helicobacter proteins likely to be used for this purpose.
On a maintenant mis en évidence dans un extrait membranaire d'Hélicobacter, la présence d'une protéine capable de se lier à la lactoferrine humaine. De manière surprenante, cette protéine est constituée de deux sousunités et peut être reconnue par des anticorps monoclonaux initialement dressés contre le récepteur transferrine de
Neisseria meningitidis.We have now demonstrated in a membrane extract of Helicobacter, the presence of a protein capable of binding to human lactoferrin. Surprisingly, this protein consists of two subunits and can be recognized by monoclonal antibodies initially raised against the transferrin receptor of
Neisseria meningitidis.
C'est pourquoi l'invention a pour objet
(i) une protéine d'Hélicobacter sous forme substantiellement purifiée, ladite protéine ayant un poids moléculaire apparent d'environ 98 kD ou d'environ 70 kD, tel que défini après migration sur gel de SDS-PAGE à environ 10 % de polyacrylamide et susceptible d'être substantiellement purifiée à partir d'un extrait membranaire d'Hélicobacter par chromatographie d'affinité sur une colonne de lactoferrine ; ainsi
(ii) qu'une composition pharmaceutique destinée au traitement ou à la prévention d'une infection à
Hélicobacter qui comprend à titre de principe vaccinant, au moins une protéine selon l'invention.This is why the subject of the invention is
(i) a helicobacter protein in substantially purified form, said protein having an apparent molecular weight of approximately 98 kD or approximately 70 kD, as defined after migration on SDS-PAGE gel to approximately 10% polyacrylamide and capable of being substantially purified from a membrane extract of Helicobacter by affinity chromatography on a lactoferrin column; so
(ii) a pharmaceutical composition intended for the treatment or prevention of an infection with
Helicobacter which comprises, as a vaccinating principle, at least one protein according to the invention.
Par "protéine de 98 ou 70 kD substantiellement purifiée", on entend une préparation de cette protéine dépourvue de la majeure partie des constituants cellulaires d'Hélicobacter. Bien évidemment, des contaminants mineurs ne sont pas exclus. By "substantially purified 98 or 70 kD protein" is meant a preparation of this protein devoid of most of the cellular constituents of Helicobacter. Obviously, minor contaminants are not excluded.
Une protéine selon l'invention peut être obtenue à partir d'Hélicobacter ou être obtenue par voie recombinante par expression du fragment d'ADN correspondant, dans un système hétérologue, bactéries, levures ou cellules de mammifères. A protein according to the invention can be obtained from Helicobacter or can be obtained by recombinant route by expression of the corresponding DNA fragment, in a heterologous system, bacteria, yeasts or mammalian cells.
De manière avantageuse, une protéine selon l'invention est susceptible d'être obtenue à partir d'H. Advantageously, a protein according to the invention can be obtained from H.
pylori.pylori.
Une composition selon l'invention peut être fabriquée de manière conventionnelle. En particulier, on associe une protéine selon l'invention avec un adjuvant, un diluant ou un support acceptable d'un point de vue pharmaceutique. Une composition selon l'invention peut être administrée par n'importe quelle voie conventionnelle en usage dans le domaine des vaccins, en particulier par voie orale ou parentérale. L'administration peut avoir lieu en dose unique ou répétée une ou plusieurs fois après un certain délai d'intervalle. Le dosage approprié varie en fonction de divers paramètres, par exemple, de l'individu traité ou du mode d'administration. A composition according to the invention can be produced in a conventional manner. In particular, a protein according to the invention is combined with an adjuvant, a diluent or a support which is acceptable from a pharmaceutical point of view. A composition according to the invention can be administered by any conventional route in use in the field of vaccines, in particular by oral or parenteral route. The administration can take place in single dose or repeated one or more times after a certain interval of interval. The appropriate dosage will vary depending on various parameters, for example, the individual being treated or the mode of administration.
L'invention est illustrée ci-après, en se référant à la Figure 1 qui présente le profil électrophorétique sur gel de SDS-PAGE (polyacrylamide 10 %) d'une préparation éluée d'une colonne de Sépharose 4Blactoferrine, après application d'un extrait membranaire d'H. pylori, tel que révélé au bleu de Coomassie (colonne
A) et au nitrate d'argent (colonne B). Les étalons des poids moléculaires sont la phosphorylase B (94 kD), l'albumine bovine (67 kD), l'ovalbumine (43 kD), l'anhydrase carbonique (30 kD), l'inhibiteur de la trypsine isolé du soja (soybean trypsin inhibitor) (20,1 kD) et l'alpha-lactalbumine (14,4 kD).The invention is illustrated below, with reference to FIG. 1 which shows the electrophoretic profile on SDS-PAGE gel (polyacrylamide 10%) of a preparation eluted from a Sepharose 4Blactoferrin column, after application of a membrane extract of H. pylori, as revealed in Coomassie blue (column
A) and silver nitrate (column B). The molecular weight standards are phosphorylase B (94 kD), bovine albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor isolated from soybeans ( soybean trypsin inhibitor) (20.1 kD) and alpha-lactalbumin (14.4 kD).
Exemple 1 : Purification du récepteur de la lactoferrine à partir d'H. pylori. Example 1: Purification of the lactoferrin receptor from H. pylori.
1.1 Culture. 1.1 Culture.
A partir d'une souche d'H. pylori n ATCC 43579 (disponible auprès de l'ATCC, 12301 Parklawn Drive,
Rockville MD - U.S.A) conservée en glycérol à - 70 C, on ensemence un flacon de 25 cm2 contenant un milieu biphasique. Le milieu biphasique comprend une phase solide constituée de 10 ml de gélose Colombia (bioMérieux) additionnée de 6 % de sang de mouton frais et une phase liquide constituée de 3 ml de bouillon Trypticase soja (Difco) contenant 20 % de sérum de veau foetal. Les flacons sont placés dans un sac étanche dit "generbag" (BBL) et incubés sous agitation rotative douce à 37 C pendant 48 heures en condition de microaérophilie (8-10 % C02, 5-7 %
O2 et 85-87 % N2) obtenu par le System Microaer (BBL).From a strain of H. pylori n ATCC 43579 (available from ATCC, 12301 Parklawn Drive,
Rockville MD - USA) stored in glycerol at - 70 C, a 25 cm2 bottle containing a two-phase medium is inoculated. The biphasic medium comprises a solid phase consisting of 10 ml of Colombia agar (bioMérieux) supplemented with 6% of fresh sheep blood and a liquid phase consisting of 3 ml of Trypticase soy broth (Difco) containing 20% of fetal calf serum. The bottles are placed in a waterproof bag called "generbag" (BBL) and incubated under gentle rotary shaking at 37 C for 48 hours in microaerophilic conditions (8-10% C02, 5-7%
O2 and 85-87% N2) obtained by the System Microaer (BBL).
Après 48 heures de culture, une sous-culture est réalisée en ensemençant à partir de cette culture liquide, des boîtes de Pétri contenant un milieu solide gélosé (gélose Colombia additionnée de 6 % de sang de mouton frais). A partir d'une culture obtenue dans un flacon de 25 cm2, on ensemence environ 50 boîtes de Pétri. Ces boites sont placées dans des jarres d'anaréobiose en conditions de microaérophilie obtenues par le système Anaérocult C (Merck). Les boîtes de Pétri sont ainsi incubées pendant 4 jours à 37 C. After 48 hours of culture, a subculture is produced by seeding from this liquid culture, Petri dishes containing a solid agar medium (Colombia agar supplemented with 6% fresh sheep blood). From a culture obtained in a 25 cm 2 flask, approximately 50 Petri dishes are inoculated. These boxes are placed in anaerobic jars under microaerophilic conditions obtained by the Anaerocult C system (Merck). The petri dishes are thus incubated for 4 days at 37 C.
Les nappes bactériennes sont récoltées par raclage en présence d'un faible volume de PBS (bioMérieux) et par centrifugation. Les germes sont ensuite lavés par du
PBS dans le but d'éliminer les résidus de milieu de culture.The bacterial layers are harvested by scraping in the presence of a small volume of PBS (bioMérieux) and by centrifugation. The germs are then washed with
PBS for the purpose of removing residues from the culture medium.
1.2 Purification. 1.2 Purification.
Le culot bactérien lavé est mis en suspension dans de l'eau distillée et est soumis à une agitation vigoureuse. Cette suspension est centrifugée à 18 000 x g pendant 30 min. On rajoute au surnageant recueilli un certain volume de N-Lauroyl Sarkosine à 25 % (p/v) de façon que la concentration finale en Sarkosyl soit de 0,1 % (p/v). The washed bacterial pellet is suspended in distilled water and is subjected to vigorous stirring. This suspension is centrifuged at 18,000 x g for 30 min. A certain volume of 25% (w / v) N-Lauroyl Sarkosine is added to the collected supernatant so that the final concentration of Sarkosyl is 0.1% (w / v).
La fraction est placée dans un boyau de dialyse (Spectrum, seuil de coupure 10 000 daltons) et dialysée contre 10 volumes du tampon Tris-HCl 50 mM pH 8,0 contenant
NaCl 0,15M, EDTA lOmM, Sarkosyl 0,1 %.The fraction is placed in a dialysis hose (Spectrum, cutoff threshold 10,000 daltons) and dialyzed against 10 volumes of 50 mM Tris-HCl buffer pH 8.0 containing
0.15M NaCl, 10MM EDTA, 0.1% Sarkosyl.
En parallèle, on prépare une colonne de Sépharose 4B (Pharmacia) sur lequel a été immobilisée de la lactoferrine humaine (Sigma). Le greffage de la lactoferrine sur la résine de Sépharose 4B CNBr se fait selon les recommandations du fabricant. La densité de ligand est d'environ 5 mg de lactoferrine par ml de gel. Le mélange est incubé une nuit à +4'C sous agitation rotative. In parallel, a Sepharose 4B column (Pharmacia) is prepared on which human lactoferrin (Sigma) has been immobilized. The grafting of lactoferrin onto Sepharose 4B CNBr resin is carried out according to the manufacturer's recommendations. The ligand density is approximately 5 mg of lactoferrin per ml of gel. The mixture is incubated overnight at + 4 ° C. with rotary shaking.
Le gel est conditionné dans une colonne de 10 ml; après décantation, le gel est lavé par environ 20 volumes de colonne de tampon A (Tris-HCl 50 mM pH 8,0 contenant EDTA 10 mM, Sarkosyl 0,1 %, PMSF 100MM (phényl méthyl sulfonyl fluoride Sigma)) contenant du NaCl 0,15M, puis environ 6 volumes de colonne de tampon A contenant du NaCl 0,5M, enfin par environ 6 volumes de colonne de tampon A contenant du NaCl 1M.The gel is packaged in a 10 ml column; after decantation, the gel is washed with approximately 20 column volumes of buffer A (50 mM Tris-HCl pH 8.0 containing 10 mM EDTA, 0.1% Sarkosyl, 100MM PMSF (phenyl methyl sulfonyl fluoride Sigma)) containing NaCl 0.15M, then approximately 6 column volumes of buffer A containing 0.5M NaCl, finally with approximately 6 column volumes of buffer A containing 1M NaCl.
L'élution est réalisée en appliquant à la colonne le tampon Tris-HCl 50 mM pH 8,0 contenant EDTA 10 mM,
Sarkosyl 0,05 %, PMSF 100 M et un gradient de guanidine
HCl de 0 à 2M.The elution is carried out by applying to the column the 50 mM Tris-HCl buffer pH 8.0 containing 10 mM EDTA,
0.05% Sarkosyl, 100 M PMSF and a guanidine gradient
HCl from 0 to 2M.
Les fractions éluées dans la gamme de guanidine de 0,75 à 2M sont regroupées. Cette préparation est dialysée contre le tampon Tris-HCl 50mM pH 8,0 et concentrée par évaporation rotative sous vide. The fractions eluted in the guanidine range from 0.75 to 2M are pooled. This preparation is dialyzed against the 50 mM Tris-HCl buffer pH 8.0 and concentrated by rotary evaporation under vacuum.
1.3 Analyse de la fraction purifiée. 1.3 Analysis of the purified fraction.
La préparation obtenue après chromatographie d'affinité sur colonne de Sépharose 4B-lactoferrine humaine est analysée par électrophorèse sur gel de SDS-PAGE à 10 % d'acrylamide, selon la technique de Laemmli, Nature (1970), 227 : 680. The preparation obtained after affinity chromatography on a Sepharose 4B-human lactoferrin column is analyzed by electrophoresis on SDS-PAGE gel containing 10% acrylamide, according to the technique of Laemmli, Nature (1970), 227: 680.
Après migration, le gel a été coloré au bleu de
Coomassie. Cette coloration comprend plusieurs étapes : (1) étape de fixation : incubation du gel dans une solution de méthanol (50 ml), acide acétique (25 ml), acide trichloroacétique (25 ml) et eau distillée (900 ml) ; (2) étape de coloration : incubation du gel dans un mélange de méthanol : acide acétique : eau (30:10:60) contenant du bleu de Coomassie R250 0,25% (p/v) ; (3) étape de décoloration dans le mélange méthanol : acide acétique eau (30:10:60).After migration, the gel was stained with blue
Coomassie. This staining comprises several steps: (1) fixing step: incubation of the gel in a solution of methanol (50 ml), acetic acid (25 ml), trichloroacetic acid (25 ml) and distilled water (900 ml); (2) staining step: incubation of the gel in a mixture of methanol: acetic acid: water (30:10:60) containing Coomassie blue R250 0.25% (w / v); (3) bleaching step in the methanol: acetic acid water mixture (30:10:60).
Tel que montré à la figure 1, colonne A, cette coloration révèle un certain nombre de bandes, aucune n'étant révélée aux alentours de 100 kD. As shown in FIG. 1, column A, this coloration reveals a certain number of bands, none being revealed around 100 kD.
La préparation obtenue a été analysée par la méthode de coloration au nitrate d'argent. The preparation obtained was analyzed by the staining method with silver nitrate.
Cette coloration s'effectue sur les protéines transférées préalablement sur nitrocellulose (Schleicher and Schuell BA 0,45), selon la méthode décrite par Kovarik et al, J. Folia Biologica (Praha) 1987, 33 : 253. This coloration is carried out on the proteins previously transferred to nitrocellulose (Schleicher and Schuell BA 0.45), according to the method described by Kovarik et al, J. Folia Biologica (Praha) 1987, 33: 253.
Tel que montré à la figure 1, colonne B, la révélation au nitrate d'argent a permis de mettre en évidence deux bandes supplémentaires, non révélées par la coloration au bleu de Coomassie. Néanmoins, parmi ces bandes, il n'était pas possible d'indiquer si l'une d'entre elles correspondait au récepteur lactoferrine. As shown in FIG. 1, column B, the revelation with silver nitrate made it possible to highlight two additional bands, not revealed by the staining with Coomassie blue. However, among these bands, it was not possible to indicate whether one of them corresponded to the lactoferrin receptor.
Pour surmonter cette déficience, on a eu l'idée à tout hasard, de soumettre une préparation telle que précédemment obtenue à une révélation par immunoaffinité en utilisant des anticorps monoclonaux initialement dressés contre le récepteur transferrine de N. meningitidis. Pour ce faire, une préparation a été soumise à une électrophorèse sur gel de SDS-PAGE, puis transférée sur nitrocellulose. To overcome this deficiency, we had the idea, by any chance, to subject a preparation as previously obtained to revelation by immunoaffinity using monoclonal antibodies initially raised against the transferrin receptor of N. meningitidis. To do this, a preparation was subjected to an electrophoresis on SDS-PAGE gel, then transferred to nitrocellulose.
Un gel de SDS-PAGE est préparé comme précédemment et, après électrophorèse, transféré sur nitrocellulose. La nitrocellulose est saturée dans un tampon bloquant (lait écrémé 1% (p/v), NaCl 0,9 % (p/v) dans Tris-HCl 50 mM pH 8), puis incubée dans une solution d'anticorps antirécepteur de la transferrine de N. meningitidis (25 Hg
IgG/ml dans du tampon bloquant) pendant 1 heure à 37'C sous agitation, puis lavée 3 fois avec du tampon bloquant et enfin incubée avec un deuxième anticorps (anti-espèce du premier) conjugué à la peroxydase. La nitrocellulose est enfin révélée avec un subtrat précipitant de la peroxydase : le 4-chloro-1-naphtol en présence de H202.An SDS-PAGE gel is prepared as above and, after electrophoresis, transferred to nitrocellulose. The nitrocellulose is saturated in a blocking buffer (skim milk 1% (w / v), NaCl 0.9% (w / v) in 50 mM Tris-HCl pH 8), then incubated in a solution of anti-receptor antibodies. N. meningitidis transferrin (25 Hg
IgG / ml in blocking buffer) for 1 hour at 37 ° C. with stirring, then washed 3 times with blocking buffer and finally incubated with a second antibody (anti-species of the first) conjugated to peroxidase. Nitrocellulose is finally revealed with a substrate which precipitates peroxidase: 4-chloro-1-naphthol in the presence of H2O2.
De manière surprenante, deux bandes ont été ainsi révélées : l'une ayant un poids moléculaire apparent de 98 000 daltons, l'autre de 70 000 daltons. L'étude aux anticorps monoclonaux permet d'indiquer que ces deux bandes correspondent à deux sous-unités d'un même récepteur alors qu'il est connu que le récepteur lactoferrine de N. Surprisingly, two bands were thus revealed: one having an apparent molecular weight of 98,000 daltons, the other of 70,000 daltons. The study of monoclonal antibodies makes it possible to indicate that these two bands correspond to two subunits of the same receptor whereas it is known that the lactoferrin receptor of N.
meningitidis est un récepteur simple chaîne (Schryvers & BR<
Morris, Infect. Immun. (1988) 56:1144).meningitidis is a single chain receiver (Schryvers & BR <
Morris, Infect. Immun. (1988) 56: 1144).
Exemple 2 : Composition pharmaceutique destinée à une administration orale. Example 2: Pharmaceutical composition intended for oral administration.
La protéine de 98 ou 70 kD susceptible d'être obtenue comme dans l'exemple 1 peut être encapsulée seule ou en présence d'autres protéines de H. pylori dans des capsules de gélatine afin de protéger l'antigène contre la dégradation par le suc gastrique ou bien administrée en présence de bicarbonate de sodium. De telles formulations ont déjà été utilisées pour des compositions pharmaceutiques (Black et al, Dev. Biol. Stand. (1983) 53). The 98 or 70 kD protein capable of being obtained as in Example 1 can be encapsulated alone or in the presence of other H. pylori proteins in gelatin capsules in order to protect the antigen against degradation by the juice. gastric or administered in the presence of sodium bicarbonate. Such formulations have already been used for pharmaceutical compositions (Black et al, Dev. Biol. Stand. (1983) 53).
La protéine peut également être encapsulée dans des microsphères de PLGA (copolymères d'acide glycolique et acide lactique) selon le protocole décrit ailleurs (Eldridge et al, Curr. Top. Microbiol. Immunol. (1989) 146:59). La protéine peut également être incluse dans desliposomes préparés selon les méthodes conventionnelles largement décrites ("Liposomes: a practical approach, ed.The protein can also be encapsulated in microspheres of PLGA (copolymers of glycolic acid and lactic acid) according to the protocol described elsewhere (Eldridge et al, Curr. Top. Microbiol. Immunol. (1989) 146: 59). The protein can also be included in liposomes prepared according to conventional methods widely described ("Liposomes: a practical approach, ed.
RRC New, D. Rickwood & B.D. Hames, 1990, Oxford University
Press, ISBN 0-19-963077-1).RRC New, D. Rickwood & BD Hames, 1990, Oxford University
Press, ISBN 0-19-963077-1).
Indépendamment de la formulation, la quantité de protéine administrée à l'homme par voie orale est de l'ordre de 1 à 10 mg par prise, et l'on préconise au moins 3 prises à intervalle de 4 semaines. Regardless of the formulation, the amount of protein administered to humans by the oral route is of the order of 1 to 10 mg per dose, and at least 3 doses are recommended at intervals of 4 weeks.
Exemple 3 : Composition pharmaceutique destinée à une administration parentérale. Example 3: Pharmaceutical composition intended for parenteral administration.
La protéine de 98 ou 70 kD susceptible d'être obtenue comme dans l'exemple 1, est adsorbée sur gel d'alumine de façon tout à fait conventionnelle. La protéine en solution à 1 mg/ml dans un tampon dont le pH est voisin de 6,5 est mise en contact pendant 1 heure avec de l'hydroxyde d'aluminium à 10 mg/ml mesuré en Al+++. La composition finale de la préparation est la suivante : une protéine selon l'invention 50 pg/ml, Al+++ 250 Hg/ml, merthiolate 1/1000, le tout en PBS. The 98 or 70 kD protein capable of being obtained as in Example 1, is adsorbed on alumina gel in a completely conventional manner. The protein in solution at 1 mg / ml in a buffer the pH of which is close to 6.5 is brought into contact for 1 hour with aluminum hydroxide at 10 mg / ml measured in Al +++. The final composition of the preparation is as follows: a protein according to the invention 50 pg / ml, Al +++ 250 Hg / ml, merthiolate 1/1000, all in PBS.
Comme dans le cas de l'administration orale, 3 injections sont préconisées, chacune espacée de 4 semaines de la précédente. As in the case of oral administration, 3 injections are recommended, each spaced 4 weeks from the previous one.
Claims (3)
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| FR9411323A FR2724936A1 (en) | 1994-09-22 | 1994-09-22 | Protein isolated from Helicobacter pylori membrane |
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| FR9411323A FR2724936A1 (en) | 1994-09-22 | 1994-09-22 | Protein isolated from Helicobacter pylori membrane |
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| FR2724936A1 true FR2724936A1 (en) | 1996-03-29 |
| FR2724936B1 FR2724936B1 (en) | 1997-02-07 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1997013784A1 (en) * | 1995-10-09 | 1997-04-17 | Pasteur Merieux Serums Et Vaccins | Helicobacter lactoferrin receptor |
| WO1998004702A2 (en) * | 1996-07-26 | 1998-02-05 | Chiron Behring Gmbh & Co. | Proteins, in particular membrane proteins, of helicobacter pylori, their preparation and use |
| US6709656B1 (en) * | 1996-06-10 | 2004-03-23 | Thomas Boren | Helicobacter pylori adhesin binding group antigen |
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| WO1992003467A1 (en) * | 1990-08-23 | 1992-03-05 | The University Of North Carolina Of Chapel Hill | Transferrin binding proteins from neisseria gonorrhoeae and neisseria meningitidis |
| EP0586266A1 (en) * | 1992-06-19 | 1994-03-09 | Pasteur Merieux Serums Et Vaccins | DNA fragments coding for the Neisseria meningitidis receptor fragments |
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1994
- 1994-09-22 FR FR9411323A patent/FR2724936A1/en active Granted
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992003467A1 (en) * | 1990-08-23 | 1992-03-05 | The University Of North Carolina Of Chapel Hill | Transferrin binding proteins from neisseria gonorrhoeae and neisseria meningitidis |
| EP0586266A1 (en) * | 1992-06-19 | 1994-03-09 | Pasteur Merieux Serums Et Vaccins | DNA fragments coding for the Neisseria meningitidis receptor fragments |
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| ILLINGWORTH D S ET AL: "THE EFFECT OF IRON LIMITATION ON THE COMPOSITION OF THE OUTER MEMBRANE PROTEINS OF HELICOBACTER-PYLORI", SOMED, 4 (SPEC. ISSUE). 1991. S134. * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997013784A1 (en) * | 1995-10-09 | 1997-04-17 | Pasteur Merieux Serums Et Vaccins | Helicobacter lactoferrin receptor |
| US6086893A (en) * | 1995-10-09 | 2000-07-11 | Pasteur Merieux Serums & Vaccins | Helicobacter lactoferrin receptor |
| US6709656B1 (en) * | 1996-06-10 | 2004-03-23 | Thomas Boren | Helicobacter pylori adhesin binding group antigen |
| US7271251B2 (en) | 1996-06-10 | 2007-09-18 | Thomas Boren | Helicobacter pylori adhesin binding group antigen |
| WO1998004702A2 (en) * | 1996-07-26 | 1998-02-05 | Chiron Behring Gmbh & Co. | Proteins, in particular membrane proteins, of helicobacter pylori, their preparation and use |
| WO1998004702A3 (en) * | 1996-07-26 | 1998-04-23 | Chiron Behring Gmbh & Co | Proteins, in particular membrane proteins, of helicobacter pylori, their preparation and use |
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| FR2724936B1 (en) | 1997-02-07 |
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