EP1360193A1 - Verfahren zur reinigung des adhesin-like proteins a (alpa) aus helicobacter - Google Patents

Verfahren zur reinigung des adhesin-like proteins a (alpa) aus helicobacter

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Publication number
EP1360193A1
EP1360193A1 EP02701347A EP02701347A EP1360193A1 EP 1360193 A1 EP1360193 A1 EP 1360193A1 EP 02701347 A EP02701347 A EP 02701347A EP 02701347 A EP02701347 A EP 02701347A EP 1360193 A1 EP1360193 A1 EP 1360193A1
Authority
EP
European Patent Office
Prior art keywords
alpa
guanidine
solubilization
urea
point
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02701347A
Other languages
English (en)
French (fr)
Inventor
Laurence Fourrichon
Ling Lissolo
Olivier Pitiot
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merieux Oravax
Original Assignee
Merieux Oravax
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merieux Oravax filed Critical Merieux Oravax
Publication of EP1360193A1 publication Critical patent/EP1360193A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the subject of the invention is a process for the purification of the protein AlpA with Helicobacter, which has the characteristics required for implementation on an industrial scale.
  • Helicobacter is a bacterial genus characterized by gram-negative spiral bacteria. Several species colonize the gastrointestinal tract of mammals. We cite in particular H. pylori, H. heilmanii, H. felis and H. mustelae. Although H. pylori is the species most commonly associated with human infections, in some rare cases, H. heilmanii and H. felis have been isolated from humans. A Helicobacter type bacterium, Gastrospirillum hominis has also been described in humans.
  • Helicobacter infects more than 50% of the adult population in developed countries and almost 100% of that in developing countries; making it one of the predominant infectious agents worldwide.
  • H. pylori is found exclusively to date on the surface of the stomach lining in humans and more particularly around crater lesions of gastric and duodenal ulcers. This bacterium is currently recognized as the etiological agent of antral gastritis and appears as one of the cofactors required for the development of ulcers. Furthermore, it seems that the development of gastric carcinomas may be associated with the presence of H. pylori.
  • the lipoprotein AlpA Adhesin-like lipoprotein A
  • AlpA and the gene encoding it were originally disclosed in WO 96/41880. It therefore remained to develop a production and purification process which made it possible to produce batches of pharmaceutical quality.
  • the main objective of the present invention is to provide a simple and robust process which can be implemented on an industrial scale. This process must in particular make it possible to produce a large quantity of antigen without having to make costly investments.
  • E. coli is par excellence the bacterium used as a host organism to produce all kinds of heterologous proteins by the recombinant route. When these proteins are not soluble or somehow excreted, they can accumulate as inclusion bodies. These inclusion bodies are intra-cytoplasmic corpuscles specific to E. coli. The appearance of these inclusion bodies is favored by a high level of expression (over-expression). The purification of the recombinant proteins from the inclusion bodies requires in particular that these inclusion bodies be dissolved beforehand. This solubilization is obtained in the usual way with a chaotropic agent (urea, guanidine) or a detergent.
  • a chaotropic agent urea, guanidine
  • the recombinant protein rAlpA deliberately produced in a non-lipid form in E. coli, still retains a high hydrophobicity. This strong intrinsic hydrophobicity makes its purification difficult by conventional means.
  • the rAlpA protein is found in the inclusion bodies of E. coli and the presence of a chaotropic agent at high concentration is almost compulsory throughout the purification.
  • hydrophobic interaction chromatographies are commonly used in order to purify proteins having a marked hydrophobic character.
  • a protein occurs in a medium with a high concentration of guanidine
  • the use of this type of chromatography becomes very problematic because the presence of guanidine must normally prevent adsorption.
  • a very large number of proteins capable of being adsorbed under conventional conditions on a CIH support would be eluted at a high concentration of guanidine.
  • AlpA we have now found that there is a narrow guanidine concentration window which allows both the maintenance of AlpA in soluble form and its adsorption on a CIH support. This window is between approximately 2.5 and 3.5 M.
  • the subject of the invention is a method of purifying the adhesin-like protein A (AlpA) from Helicobacter eg H. pylori, according to which (i) a preparation containing AlpA and 2.5 to 3.5 M of guanidine with a hydrophobic interaction chromatography material so that AlpA is adsorbed on the material and (ii) AlpA is eluted with a solution containing 3.5 to 4.5 M of guanidine.
  • AlphaA adhesin-like protein A
  • the preparation brought into contact with the chromatography material contains 3 M of guanidine and AlpA is eluted with 4 M of guanidine at neutral pH (6 - 8; e.g. 6.5 - 7.5).
  • a preparation containing AlpA in the presence of guanidine can in particular be obtained from a production process according to which (a) a Gram-negative bacterium, in particular E. coli, is capable of expressing AlpA in recombinant form, (b) the bacterial cells from the culture are recovered and lysed, (c) the bodies of inclusions contained in the cells are recovered, they are washed and solubilized, and (d) optionally, they are precipitated the inclusion bodies, the supernatant is removed and the precipitate is solubilized.
  • a Gram-negative bacterium in particular E. coli
  • the bacteria harvested after culture are first lysed.
  • Cell lysis can be carried out in various ways, in particular by microfluidization.
  • the cells can also be treated with benzonase during the lysis, so that the DNA of E. coli be digested.
  • the inclusion bodies are then subjected to successive washings; preferably in a buffer containing a high concentration of salt in order to eliminate the nucleic acids and to best dissociate rAlpA from the contaminants; suitably, it can be a sodium buffer eg a buffer containing 0.5 M NaCl.
  • the inclusion bodies are finally dissolved in highly concentrated guanidine eg guanidine 5.5 to 6.5 M, preferably 6 M.
  • the ammonium sulphate is 0.4 to 0.6 M ammonium sulphate, eg 0.5 M.
  • the precipitate containing rAlpA is again dissolved in highly concentrated guanidine eg 5.5 to 6.5 M guanidine, preferably 6 M.
  • the preparation is diluted with Vz and then loaded onto an appropriate column.
  • Chromatography of hydrophobic interactions can be carried out in a conventional manner using supports such as Sepharose TM (Pharmacia) and Fractogel TM (Merck) or else supports equivalent to the latter such as those found at Tosohaas or Biorad .
  • the ligand can be butyl, octyl or phenyl.
  • the ICIDH makes it possible in particular to separate the AlpA protein from its degradation products.
  • the eluate originating from the ICIDH can be concentrated in different ways and in particular by ultrafiltration. It is also advisable to eliminate guanidine which is a toxic product.
  • a diafiltration step eg tangential diafiltration, can be implemented, against several volumes of neutral buffer eluate containing urea with a molarity greater than or equal to 4 M, preferably greater than or equal to 6 M, of preferably 8 M urea; urea being present to maintain rAlpA in soluble form.
  • 6 to 10 volumes of urea 7.5 to 8.5 M are used.
  • a “polishing” can be carried out by ion exchange chromatography, in particular by anion exchange chromatography in order to remove the residual contaminants coming from E. coli.
  • this last step is carried out in the presence of 8 M urea.
  • the preparation of rAlpA resulting from the diafiltration is then applied to anion exchange chromatography equipment; the contaminants are adsorbed on this material while rAlpA does not adsorb and ends up in the filtrate (also called direct eluate).
  • the presence of 8 M urea is essential for the following reasons: AlpA is a positively charged protein (very high pi) and therefore should not normally be adsorbed on anion exchange chromatography equipment. But its hydrophobic character is so marked that it would still be absorbed if 8 M urea was absent from the preparation.
  • the invention also relates to a process for the purification of AlpA according to which (i) a preparation containing AlpA and urea 7.5 to 8.5 M, preferably 8 M, is brought into contact with material. anion exchange chromatography so that the contaminants adsorb on this material and (ii) AlpA is recovered in the filtrate.
  • Anion exchange chromatography can be carried out in a conventional manner using supports such as Sepharose (Pharmacia) and Fractogel (Merck) or else supports equivalent to the latter such as those found at Tosohaas or Biorad.
  • the ligand can be a tertiary amine such as DEAE (diethyleaminoethyl) or quaternary amine such as Q (quaternary, hydroxypropylediethyleaminoethyl) and TMAE (trimethyleaminoethyl).
  • DEAE diethyleaminoethyl
  • quaternary amine such as Q (quaternary, hydroxypropylediethyleaminoethyl) and TMAE (trimethyleaminoethyl).
  • the AlpA protein characterized by a degree of purity greater than or equal to 90% of monomer, as measured by SDS-PAGE, staining with Coomassie blue and reading of densitometry.
  • the remaining material other than the monomeric form consists of AlpA protein in multimeric form.
  • the overall degree of purity being greater than or equal to 99%.
  • this preparation must constitute a batch; that is to say, it was obtained from a single culture and the singular implementation of a purification process.
  • the inclusion bodies can be dissolved by replacing guanidine with urea 7.5 to 8.5, preferably 8 M in a buffer with basic pH, preferably greater than or equal at 10.5, eg at pH 11.
  • a phosphate buffer is used.
  • the invention also relates to a process for the purification of the adhesin-like protein A (AlpA) Hél 'Helicobacter according to which (i) a preparation containing AlpA and 4.5 to 5.5 M is brought into contact. of urea with hydrophobic interaction chromatography equipment such that AlpA is adsorbed on the equipment and (ii) AlpA is eluted with a solution containing 7.5 to 8.5 M of urea.
  • AlphaA adhesin-like protein A
  • An inducible expression system has been constructed to produce the AlpA protein of Helicobacter pylori in E. coli.
  • the vector that was used is derived from the plasmid pET28c (Novagen). This vector comprises: an expression cassette under the control of the promoter of bacteriophage T7,
  • a transcription terminator also derived from bacteriophage T7, and
  • the promoter is upregulated in the presence of T7 RNA polymerase.
  • strains of E. genetically modified coli can be used, among which the strains BL21 ⁇ D ⁇ 3 (Studier, F.W. et al 1990 Meth. Enzymol 185, 60-69.). These strains contain the gene coding for the RNA polymerase of phage T7 under the control of the lac UV5 promoter, inducible by addition of IPTG. In addition, the BL21 ⁇ D ⁇ 3 strain is deficient for the OmpT and Lon protease activities.
  • a 1.6 Kb DNA fragment containing the sequence coding for the mature AlpA protein was obtained by PCR amplification using the strain of Helicobacter pylori X47-2 (ORV 2001) as a source of DNA.
  • the Ncol and Xh ⁇ l restriction sites used for cloning are included in the 5 '(HP3 ⁇ co C-) and 3' HP3 (Xho) PCR primers respectively.
  • the lipidation site [Cysteine codon (TGC codon)] has been replaced by an ATG initiation codon (Met).
  • the PCR amplified fragment was first cloned into the Topo TA shuttle vector (Invitrogen), then transferred into p ⁇ T28c using the Ncol and Xh ⁇ l sites.
  • the PCR amplification conditions were as follows: 97 ° C / 30 s; 55 ° C / 1 min; 72 ° C / 50 s; Wind DNA polymerase - 25 cycles.
  • Ncol and Xhol sites are indicated in italics and the ATG initiation and TAA termination codons (opposite strand) are in bold underlined characters.
  • Erlens of 2 liters each containing 500 mL of Luria Broth (LB) 2X medium modified (peptone replaced by yeast extract 30 g / L) and supplemented with kanamycin 50 ⁇ g / mL, are inoculated with 500 ⁇ L of a batch of E. coli BL21D ⁇ 3 / pMHp3.1 seed and are incubated for 15-18 hrs at 37 ° C with shaking 175 rpm.
  • This preculture is used to seed a 30 L fermenter (B Braun) containing the SKY ⁇ 4 medium (for one liter: yeast extract 40 g; MgCl 2 1 M 3 ml; K 2 SO 530 mg; NaCl 1 g; K 2 HPO 4844 g) supplemented with glucose monohydrate 44 g / L and kanamycin 50 mg / mL.
  • the inoculum corresponds to 5% of the total volume.
  • the culture parameters are as follows: pH: 7.00; regulation H3PO4 10% and NH4OH 28%; temperature: 37 ° C; initial agitation:
  • the culture is continued for more than 3 hrs.
  • the expression of AlpA is induced by adding the volume of IPTG (at 200 mM) necessary to obtain a final concentration equal to 1 mM.
  • the culture is immediately cooled.
  • the cells are harvested by centrifugation 20 min at 7,000 g and the pellets stored at - 35 ° C.
  • a 2.5 IU / ⁇ L benzonase solution is prepared extemporaneously from a 250 IU / ⁇ L stock solution stored at ⁇ 20 ° C., ie 20 ⁇ L + 1980 ⁇ L of 50 mM Tris buffer pH 8.0. Then 1700 ⁇ L of this solution is added in 42.5 ml of 100 mM MgCl 2 . All of this preparation is poured into the germ suspension. The mixture is incubated at 5 ⁇ 3 ° C with magnetic shaking for at least 30 minutes.
  • the cells thus homogenized are broken using a PANDA cell disintegrator at a pressure set at 1000 bars, with 3 breaking cycles and at a temperature not exceeding 15 ° C, using the cooling system ( cryostat set at + 5 ° C).
  • the ratio DO before breaking / DO after the last breaking must be> 4.5.
  • the suspension of the inclusion bodies after 3 breaking cycles is centrifuged at 10,000 g for 30 min. at 5 ⁇ 3 ° C to remove contaminants (DNA, RNA and lipopolysaccharides ).
  • the pellet After centrifugation, the pellet is recovered and then resuspended in 4.25 L of 50 mM sodium phosphate buffer, 0.5 M NaCl pH 7.0 cooled to 5 ⁇ 3 ° C.
  • the protein concentration is around 8 g / L.
  • the suspension is homogenized with Ultraturrax in an ice bath of so as to maintain the suspension at 5 ⁇ 3 ° C. and then left under magnetic stirring for 30 min at 5 ⁇ 3 ° C.
  • the suspension thus washed is centrifuged at 10,000 g for 30 min at 5 ⁇ 3 ° C.
  • the washing operation is repeated twice. Approximately 110 g of washed inclusion bodies are thus obtained, ie approximately 0.3 g of proteins per gram of washed inclusion bodies. Storage is possible at - 20 ° C.
  • the inclusion bodies are then dissolved in 50 mM Tris buffer, 6 M Guanidine pH 7.5.
  • the final protein concentration to be obtained is 20 g / L.
  • the volume of buffer to be added is therefore approximately 1.65 L.
  • This suspension is homogenized with Ultraturrax at 16,000 rpm and then with magnetic stirring until complete solubilization, at a temperature maintained at 22 ⁇ 3 ° C.
  • the mixture is centrifuged at 10,000 g for 30 minutes at 22 + 3 ° C minutes in order to clarify the solution; then the solution is sterile filtered on Spiral Cap 0.8 / 0.2 ⁇ m.
  • the volume of the filtered supernatant is approximately 1.6 L
  • the supernatant is diluted by half with a 1 M ammonium sulphate solution, 50 mM Tris pH 7.5 with magnetic stirring.
  • the solution thus obtained is added in 30 minutes using a peristaltic pump at a slow and regular rate to obtain a solution with 10 g / L of proteins containing 0.5 M of (N ⁇ ) 2 SO 4 in the end.
  • the mixture is left under magnetic stirring for 30 minutes at 22 ⁇ 3 ° C. Then the incubation is continued without shaking overnight (15 hours) at laboratory temperature to allow the precipitate to mature.
  • the mixture is centrifuged for 30 min at 10,000 g at 22 ⁇ 3 ° C.
  • the precipitate is collected and then solubilized at 10 g / L of proteins with 50 mM Tris buffer, 6 M Guanidine pH 7.5 with magnetic stirring (The forecast volume after solubilization is close to 3.2 L).
  • This suspension is homogenized with Ultraturrax at 16,000 rpm and then with magnetic stirring until complete solubilization, at a temperature maintained at 22 ⁇ 3 ° C.
  • This suspension is then diluted to 1/2 by addition of 50 mM Tris buffer, 1.4 M NaCl pH 7.5 with magnetic stirring.
  • This buffer is added in 30 minutes using a peristaltic pump at a slow and regular flow to obtain a 5 g / L solution of proteins containing 50 mM Tris, 0.7 M NaCl, 3M Guanidine pH 7.5 in the end. .
  • the mixture is left under magnetic stirring for 15 minutes at 22 ⁇ 3 ° C.
  • the mixture is centrifuged at 10,000 g for 30 minutes at 22 ⁇ 3 ° C in order to clarify the solution; then it is sterile filtered on Supor Cap 0.8 / 0.2 ⁇ m.
  • the volume of the filtered supernatant is approximately 6 L.
  • the column is prepared as follows:
  • the gel is washed and then equilibrated at a flow rate close to 60 mL / cm 2 .h (19 L / h) with (i) 5 volumes of column of ultrafiltered water, then with (ii) 3 volumes of column of Tris 50 buffer mM, Guanidine 3 M, NaCl 0.7 M pH 7.5.
  • the column is loaded by injection of the filtrate (corresponding to 22 - 25 g of total proteins) at a flow rate close to 60 ml / cm 2 .h (19 L / h).
  • the column is then rinsed with 50 mM Tris buffer, 3M guanidine, 0.7 M NaCl pH 7.5 at a flow rate close to 90 ml / cm 2 .h (28 L / h) until the return to the baseline (2 column volumes).
  • AlpA is then eluted in 50 mM Tris buffer, 4 M Guanidine pH 7.5 at a flow rate of 90 mL / cm 2 .h (28 L / h) until the return to the baseline (1 column volume).
  • the eluate volume is approximately 8 L. Concentration and diafiltration on 30 kDa
  • the ultrafiltration module is prepared as follows:
  • the rAlpA eluate is concentrated in 50 mM Tris buffer, 4 M Guanidine pH 7.5, around 5 g / L of protein (concentration factor ⁇ 2.5).
  • the ultrafiltration conditions are as follows: (i) inlet pressure close to 1 bar, and (ii) flow rate of the ultrafiltrate at half that of the retentate.
  • the volume of the concentrated eluate is approximately 3.5 L.
  • the concentrated rAlpA eluate is diafiltered against 10 volumes of 50 mM Tris, 8 M urea pH 7.5 (35 L) under the same conditions as the concentration, at a flow rate of approximately 30 L / h, until obtaining:
  • the volume of the diafiltered concentrated eluate is approximately 3.7 L.
  • the column used has the following characteristics: Column (EMD TMAE Merck) with a diameter of 7 cm and a surface of 38.5 cm 2 ; Frost height close to 18 cm; Gel volume close to 700 mL.
  • the chromatography column is prepared as follows:
  • the gel is washed and then equilibrated at a flow rate close to 4 L / h with (i) 5 volumes of column of ultrafiltered water, then with (ii) 3 volumes of column of 50 mM Tris buffer, 8 M urea pH 7.5.
  • the chromatography is carried out as follows: The column is loaded by injection of the diafiluted eluate corresponding to 15 - 17 g of total proteins, at a flow rate of 100 ml / cm 2 .h (4 L / h) then rinsed with 50 mM Tris buffer, 8 M urea, pH 7.5, same flow rate, until returning to the baseline (6 column volumes). The filtrate thus collected (approximately 4 L) contains the pure rAlpA protein at 90 ⁇ 5% in the absence of endotoxins.
  • the purified rAlpA eluate is filtered through a 0.8 / 0.2 ⁇ m Supor Cap 100 type filter with a surface area of 1000 cm 2 under a laminar flow hood.
  • the volume of the sterile filtered concentrated bulk is approximately 4 L.
  • the degree of purity of the batch of AlpA thus obtained is evaluated in a conventional manner by SDS-PAGE, staining with Coomassie blue and reading of densitometry, as being greater than 90%.
  • the overall yield is estimated at around 50%.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Biochemistry (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
EP02701347A 2001-02-05 2002-01-30 Verfahren zur reinigung des adhesin-like proteins a (alpa) aus helicobacter Withdrawn EP1360193A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0101499 2001-02-05
FR0101499A FR2820424B1 (fr) 2001-02-05 2001-02-05 Procede de purification de alpa
PCT/FR2002/000355 WO2002064622A1 (fr) 2001-02-05 2002-01-30 Procede de purification de la proteine adhesine-like a (alpa) d'helicobacter

Publications (1)

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EP1360193A1 true EP1360193A1 (de) 2003-11-12

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EP02701347A Withdrawn EP1360193A1 (de) 2001-02-05 2002-01-30 Verfahren zur reinigung des adhesin-like proteins a (alpa) aus helicobacter

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US (1) US20040058402A1 (de)
EP (1) EP1360193A1 (de)
JP (1) JP2004520403A (de)
CA (1) CA2436889A1 (de)
FR (1) FR2820424B1 (de)
WO (1) WO2002064622A1 (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100378223C (zh) * 2003-04-17 2008-04-02 南方医院 具有粘附功能的幽门螺杆菌膜孔素
EP1498475A1 (de) * 2003-07-18 2005-01-19 Meristem Therapeutics S.A. Bioreaktor und Verfahren zur kontinuierlichen Züchtung von Pflanzenzellen
WO2011081598A1 (en) * 2009-12-28 2011-07-07 Mivac Development Aktiebolag New antigens for use in a vaccine against h.pylori infection

Family Cites Families (14)

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Publication number Priority date Publication date Assignee Title
US6258359B1 (en) * 1993-05-19 2001-07-10 Institut Pasteur Immunogenic compositions against helicobacter infection, polypeptides for use in the compositions, and nucleic acid sequences encoding said polypeptides
DE19521314A1 (de) * 1995-06-12 1996-12-19 Max Planck Gesellschaft Adhärenzgen aus Helicobacter pylori und davon codiertes Polypeptid
DE19535321A1 (de) * 1995-09-22 1997-03-27 Max Planck Gesellschaft Neues Adhäsin aus Helicobacter pylori
AU3657995A (en) * 1995-10-09 1997-04-30 Pasteur Merieux Serums Et Vaccins Helicobacter lactoferrin receptor
SE9504019D0 (sv) * 1995-11-13 1995-11-13 Pharmacia Ab Method for production of proteins
US20020161192A1 (en) * 1996-10-11 2002-10-31 Meyer Thomas F. Helicobacter pylori live vaccine
US20030069404A1 (en) * 1996-11-14 2003-04-10 Rainer Haas Helicobacter antigens and corresponding DNA fragments
US20010019834A1 (en) * 1997-03-31 2001-09-06 Byung-O Kim Recombinant microorganisms expressing antigenic proteins of helicobacter pylori
US20020160456A1 (en) * 1997-06-24 2002-10-31 Harold Kleanthous Identification of polynucleotides encoding novel helicobacter polypeptides in the helicobacter genome
FR2748478B1 (fr) * 1997-07-01 2001-07-13 Pasteur Merieux Serums Vacc Nouvelle proteine membranaire d'helicobacter pylori
US20030158396A1 (en) * 1997-07-29 2003-08-21 Harold Kleanthous Identification of polynucleotides encoding novel helicobacter polypeptides in the helicobacter genome
US20020151462A1 (en) * 1998-02-19 2002-10-17 Ling Lissolo Helicobacter pylori membrane proteins
EP1301204A1 (de) * 2000-07-05 2003-04-16 Merieux OraVax SNC Immunologische kombinationen zur prophylaxe und therapie einer helicobacter pylori infektion
US6931325B2 (en) * 2001-02-07 2005-08-16 Regents Of The University Of Michigan Three dimensional protein mapping

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Title
See references of WO02064622A1 *

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Publication number Publication date
US20040058402A1 (en) 2004-03-25
WO2002064622A1 (fr) 2002-08-22
FR2820424A1 (fr) 2002-08-09
FR2820424B1 (fr) 2004-01-02
JP2004520403A (ja) 2004-07-08
CA2436889A1 (en) 2002-08-22

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