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WO1999048919A1 - Antigene du h. pilori et ses utilisations - Google Patents

Antigene du h. pilori et ses utilisations Download PDF

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Publication number
WO1999048919A1
WO1999048919A1 PCT/GB1999/000885 GB9900885W WO9948919A1 WO 1999048919 A1 WO1999048919 A1 WO 1999048919A1 GB 9900885 W GB9900885 W GB 9900885W WO 9948919 A1 WO9948919 A1 WO 9948919A1
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WO
WIPO (PCT)
Prior art keywords
protein
antigenic
pylori
antigen
composition
Prior art date
Application number
PCT/GB1999/000885
Other languages
English (en)
Inventor
Christopher John Smith
David Robert Tyreman
Mark Wayne Smith
Original Assignee
Cortecs (Uk) Limited
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 Cortecs (Uk) Limited filed Critical Cortecs (Uk) Limited
Priority to AU30429/99A priority Critical patent/AU3042999A/en
Publication of WO1999048919A1 publication Critical patent/WO1999048919A1/fr

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    • 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 present invention relates to the use of an antigen common to both the spiral and coccoid form of Helicobacter pylori, or antigenic fragments thereof, in the preparation of a vaccine, as well as methods of treatment employing such a vaccine.
  • the use of the antigen or fragments thereof in detecting Helicobacter pylori and kits comprising them, are also provided.
  • H. pylori is a Gram negative bacteria that has been strongly implicated in chronic active gastritis and peptic ulcer disease (Marshall et al, Medical Journal of Australia, 142:439-444 (1985); Buck, G.E., Journal of Clinical Microbiology , 3:1- 12 (1990)).
  • H. pylori exists in two distinct morphological forms, the culturable spiral form and the non-culturable coccoid form (Marshall et al, Microbios letters, 25:83-88 (1984); Kung, J.S.L., and Ho, B., Workshop on Gastroduodenal Pathology and Campylobacter pylori (abstract P9), edited by F.
  • WO 97/12910 described studies on the life cycle of H.pylori and the importance of the coccoid form in ensuring survival of the organism, thus allowing for the possibility of reinfection.
  • the identification of the importance of the coccoid form means that any vaccine based therapy must include antigens which not only induce the desired immune response in the subject, but also are common to both forms of the organism to ensure complete eradication. Such common antigens would also find use in diagnostic tests where again it is essential to be able to detect both forms of H.pylori.
  • the present invention provides a vaccine composition 5 comprising a protein which is an H. pylori antigen, having an approximate molecular weight of 48kDa, as determined under native conditions and having the following N-terminal amino acid sequence:
  • the 5 present invention provides a vaccine composition comprising a protein which is the 50S L7/L12 ribosomal protein of H.pylori.
  • the vaccine compositions of the invention can include one or more adjuvants.
  • adjuvants well known in the art include inorganic gels such as o aluminium hydroxide or water-in-oil emulsions such as incomplete Freund's 3 adjuvant.
  • Other useful adjuvants will be well known to the skilled man.
  • the present invention provides the use of a protein being an H. pylori antigen, having an approximate molecular weight of 48kDa, as determined under native conditions and being the 50S L7/L12 ribosomal protein in the preparation of a medicament, preferably a vaccine, for the prophylaxis or treatment of H.pylori infection.
  • the protein will have the following amino acid sequence:
  • the protein of the invention finds use as an H.pylori antigen.
  • an antigen can be "antigenic” and/or “immunogenic”.
  • antigenic is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject.
  • immunogenic is taken to mean that the protein is capable of eliciting a protective immune response in a subject.
  • the protein may be capable of not only generating an antibody response and in addition non-antibody based immune responses.
  • homologues or derivatives of the protein of the invention will also find use in the context of the present invention, ie as antigenic/immunogenic material.
  • a protein which includes one or more additions, deletions, substitutions or the like are encompassed by the present invention.
  • a program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention.
  • homologues and derivatives the degree of identity with the protein described herein is less important than that the homologue or derivative should retain its antigenicity or immunogenicity to H.pylori.
  • homologues or derivatives having at least 60% similarity (as discussed above) with the protein described herein are provided.
  • homologues or derivatives having at least 70% similarity, more preferably at least 80% similarity are provided.
  • homologues or derivatives having at least 90% or even 95% similarity are provided.
  • the homologues or derivatives could be fusion proteins, incorporating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide. It may be necessary to remove the "tag” or it may be the case that the fusion protein itself retains sufficient antigenicity to be useful.
  • the present invention provides antigenic/immunogenic fragments of the protein described herein.
  • the antigenic fragment is 5 a peptide having the sequence:
  • antigenic fragments of the invention can also be used in the preparation of a medicament as described herein and such use forms a fifth aspect of the invention.
  • antigenic protein or fragments thereof, described herein can be provided alone, as a purified or isolated preparation, or as part of a mixture with other H. pylori antigenic proteins.
  • the invention provides an antigen composition comprising the protein described herein or one or more antigenic fragments thereof, together with at least one other H.pylori antigen, or one or more antigenic fragments thereof.
  • a composition can be used as a vaccine or for the detection and/or diagnosis of H. pylori.
  • the present invention provides a method of detecting and/or diagnosing H. pylori which comprises:
  • the protein, antigenic fragments thereof or antigen composition of the invention can be used to detect IgG antibodies.
  • the sample to be tested will be a biological sample, e.g. a sample of blood or saliva.
  • An example of a suitable method for detection of H.pylori using a sample of a mucous secretion is that described in WO-A-9322682.
  • the invention provides the use of the antigenic protein, antigenic fragment thereof or antigenic composition of the present invention in detecting and /or diagnosing H. pylori.
  • the detecting and/or diagnosing is carried out in vitro.
  • the antigenic protein, antigenic fragment thereof or antigen composition of the invention can be provided as part of a kit for use in in vitro detection and/or diagnosis of H.pylori.
  • the present invention provides a kit for use in the detection and/or diagnosis of H.pylori comprising the antigenic protein, antigenic fragment thereof or antigen composition of the invention.
  • the antigenic protein or antigenic fragments thereof can be used to induce an immune response against H. pylori.
  • the present invention provides the use of the antigen, a fragment thereof or an antigenic composition of the invention in medicine.
  • the present invention provides:
  • amino acid sequence described herein is coded for, for instance, by the following nucleotide sequence:
  • FIGURE 1 shows the results of an SDS Immunoblot of an acid-glycine extract from the coccoid form of H.pylori;
  • FIGURE 2 shows Native PAGE of the extracted protein mixture
  • FIGURE 3 shows SDS PAGE of the extracted protein mixture
  • FIGURE 4 shows the elution profile of the acid-glycine extract of a culture of coccoid H.pylori of single gel permeation chromatographic separation
  • FIGURE 5 shows overlays of various gel permeation chromatographic runs
  • FIGURE 6 shows native page analysis of fractions from the gel permeation column chromatography
  • FIGURE 7 (a) shows the results of immunoblotting carried out on the native electrophoresed samples with H.pylori positive sera while (b) shows the results of immunoblotting carried out on the native electrophoresed samples with H.pylori negative sera;
  • FIGURES 8-11 shows the results of native immunoblots using a selection of different human sera samples.
  • the NCTC 11637 strain of H. pylori was used throughout these studies.
  • the bacterium was grown in Brain Heart Infusion Broth supplemented with 10% v/v horse serum and 0.4% w/v yeast extract.
  • the optical density at 650nm (OD 650 ) and cell count of the inoculum was determined and a plate culture test for purity initiated. Flasks confirmed to have an adequate cell count ( > 10 9 cells per ml) were used to inoculate 5 litres of broth in the 10 litre fermenters. Fermenters were maintained at 37°C, with 5% oxygen and —95% nitrogen, CO 2 was supplied as a 5 second burst of 100% CO 2 at a flow rate of 0.51itres/min. each morning and evening. Culture OD was monitored daily and conditions maintained for 7 days, at which point the dissolved oxygen tension was allowed to rise above 10% and the CO 2 sparge was stopped.
  • Coccoid forms began to appear immediately and after 24 hours the ferments were deemed to consist entirely of coccoid forms when examined microscopically. Ferments were harvested by transferring the broth containing the cultures to centrifuge bottles and pelleting the coccoid forms by centrifugation at 5000 x g. The supernatant was decanted and the pellets washed in situ three times with phosphate buffered saline, pH 7.2. The final washed pellets were extracted with acid-glycine (25ml per gram wet weight of cells). The acid-glycine soluble material was recovered by centrifugation (lOmin, 10,000 x g). The supernatant was decanted, filtered (0.8 ⁇ m and 0.2 ⁇ m filters) and soluble supernatants subjected to further purification. 10
  • Acid-glycine soluble extracts of the coccoid material were transferred to 5 ammonium bicarbonate buffer by buffer exchange dialysis (2 x 5 litres for at least 3 hours each time, 4°C).
  • the protein concentration of each extract was determined (Bradford Dye Assay [1]) and then each extract was concentrated to approx. 18 mg ml "1 by means of Nivaspin 15ml and 500 ⁇ l units.
  • the samples were placed in the 15ml unit and centrifuged at 2,000 x g.
  • the concentrate was o recovered and transferred to the 500 ⁇ l unit which was centrifuged at 15,000 x g to achieve the final concentration.
  • Antigen purification was achieved by gel permeation chromatography.
  • a BioRad Mini Protean II electrophoresis apparatus was used as per the manufacturer's instructions.
  • BioRad 5 Ready Gels or self-cast gels (4-20% T) were used.
  • the samples were prepared by mixing each sample, 1:1 v/v, with 2 x native sample buffer (20mM Tris, 0.05% w/v Bromophenol Blue, 20% v/v Glycerol pH 8.8).
  • the gels were prepared as per the manufacturer's instructions and placed in the gel cassette holder.
  • the lower chamber of the electrophoresis tank was filled with Native o PAGE Electrode Buffer (25mM Tris, 0.192M Glycine pH 8.3).
  • the prepared 11 gel cassette was then placed in the tank, the well-forming comb removed and the upper electrode chamber filled with Native PAGE electrode buffer. Samples were loaded using an air-displacement pipette. For analytical studies a volume of lO ⁇ l of each sample was loaded, one sample per well. For immunoblot studies a single well across the full width of the gel was used and filled (50 ⁇ l) with a single protein sample. Following loading of the samples the chamber lid was put in position. A voltage of 200V was applied to the chamber for ⁇ 500Vh.
  • Gels were anlaysed using a gel scanner and PhoretixTM software and photographs were taken as a permanent record.
  • a Pharmacia Phast system was used as per the manufacturer's instructions.
  • the samples were either used per se or mixed, 1: 1 v/v, with 2 x native PAGE sample buffer.
  • the gels (8-25% T) were set up and run for 220 to 240 Vh as described in the Phast System Separation Technique File No. 120 supplied by the manufacturer.
  • the various sample applicators supplied by the manufacturer were used as appropriate to the technique and volume of sample.
  • the Titration curve sample applicator was employed with 3 to 6 ⁇ l of a single sample applied across the full width of the gel. 12
  • gels were stained, either automatically using the Pharmacia Phast Development Unit with Coomassie Blue R350 stain as described by Heukeshoven and Dernick (2), or manually for 3h using Coomassie Blue G250 (0.075% w/v in 1.5% v/v phosphoric acid, 11.25% w/v ammonium sulphate and 25 % v/v methanol) followed by destaining in 25 % v/v methanol, or silver stained manually at ambient temperature using a modification of the method as described by Heukeshoven and Dernick (3) involving the final clearing of the gel with Farmer's reagent after staining and destaining.
  • Sample preparation was in SDS sample buffer (2x SDS PAGE Sample Buffer, 125mM Tris, 0.05% w/v Bromophenol Blue, 20% v/v Glycerol, 4.0% w/v SDS, 0.32M DTT, pH 6.8). Samples were mixed, 1: 1 v/v, with the buffer and heated for 5 minutes at 100°C. Samples were allowed to cool then used for the experiments. Electrophoresis was achieved, using SDS PHAST buffer strips and
  • the gel was placed on the PVDF membrane and the combination sandwiched between supporting layers of filter paper, pre- wetted with transfer buffer (Bjerrum and Schafer-Nielsen buffer, for native gels, 48mM Tris, 39mM glycine, pH 9.2; for SDS gels, 48mM Tris, 39mM glycine, 20% v/v methanol, pH 9.2) to ensure complete contact without air bubbles between the layers.
  • Transfer buffer Bjerrum and Schafer-Nielsen buffer, for native gels, 48mM Tris, 39mM glycine, pH 9.2; for SDS gels, 48mM Tris, 39mM glycine, 20% v/v methanol, pH 9.2
  • Blotting was confirmed by the inclusion on the gel of prestained electrophoresis standards which could be seen to have transferred to the PVDF membrane or by including standard proteins in one of the gel lanes which were revealed by cutting off that lane (always at one side of the main experimental run) and staining this section with either Coomassie Blue R250 (0.025% w/v in 40% v/v Methanol) or silver (4).
  • the blocked PVDF membrane was immersed in the serum (diluted 1:60 v/v in 1 % w/v BSA in TTBS) for 2 hours and then the serum 14 was removed and the membrane washed twice with TTBS.
  • the bound H. pylori specific antibodies were visualised by immersion of the blot in rabbit anti-human
  • IgG peroxidase conjugate diluted 1:500 v/v in 1 % w/v BSA in TTBS for 2 hours followed by 2 washes in TTBS and 1 in TBS to ensure complete removal of any Tween before addition of the substrate solution (60mg 4-(chloro- l-napththol)[4CN] in 20 ml methanol plus 60 ⁇ l of 30% v/v ⁇ 2 0 2 in 100 ml TBS).
  • the membrane was cut in different ways; into sections before immunostaining treatment so that the section with prestained standards and a section for total protein staining were removed for separate staining; if analysis of gels with several different serum samples was required then strips were cut for each treatment. In the latter cases the use of the single well in the original electrophoresis process allowed identical protein patterns to be probed with different sera and by protein staining.
  • N-terminal amino acid sequence analysis of the protein selected by immunostaining was achieved by analysis of the single electrophoresis band directly from PVDF. Electrophoresis and blotting was carried out as described with the variation that the blot was stained with Coomassie Blue to reveal the bands. The stained blot was then subjected to solid phase amino acid sequencing by cutting out the region of interest and analysing the protein in that region. The amino acid sequencing was achieved using an Applied Biosystems 471 A pulsed liquid sequencer as described in the Manufacturer's instructions. 15
  • Samples of the isolated protein designated 48kDa fraction, prepared in carbonate bicarbonate buffer (0.5M, pH 8.9), were coated onto the surfaces of ELISA microtitre plate wells using conventional techniques. The surfaces were subsequently treated with casein (5 % w/v non-fat dried milk in PBS) to prevent non-specific binding before analyses were undertaken. The coated plates were dried and stored at 4°C until used. Serum samples were diluted 1:500 v/v in PBS and lOO ⁇ l aliquots added to individual wells. The binding reaction between antigen-specific serum antibodies and the antigen coated plate was allowed to occur over a period of 1 hour, after which the wells were washed with PBS buffer.
  • Goat anti-human IgG peroxidase conjugate (Dako, diluted 1:500 v/v in PBS) was then added to the wells and allowed to react for 1 hour.
  • the wells were washed again and substrate (lmg TMB in 0.1M citrate buffer (20ml), pH 5.0, plus 30 ⁇ l of 30% v/v H 2 0 2 ) added.
  • substrate lmg TMB in 0.1M citrate buffer (20ml), pH 5.0, plus 30 ⁇ l of 30% v/v H 2 0 2
  • the reaction was stopped after 15 minutes by the addition of 0.5M H 2 SO 4 .
  • the optical density of the solutions was measured at 450nm using an ELISA plate reader (Multiskan, Titertek).
  • HelisalTM ELISA were carried out according to the manufacturer's instructions.
  • the purification of the 48kDa antigenic protein was achieved by gel permeation chromatography of the acid-glycine extract of a culture comprising totally or almost totally of the coccoid form of Helicobacter pylori.
  • the elution profile of a single chromatographic separation is shown in Figure 4.
  • the specific peak selected for further examination is marked.
  • the reproducibility of the procedure is shown by the observation that repeated results can be overlaid ( Figure 5).
  • Electrophoretic analysis of the fractions eluted from the column shows a complex profile with proteins at different molecular weight eluted in sequence ( Figures 6a and 6b). It is believed that the high molecular weight materials observed in fractions 28-29 are the result of reaggregation of molecules separated during the chromatographic step. Selection of these fractions into those containing predominantly antigenic and those containing only non antigenic proteins was achieved by immunoblotting the electrophoresed samples with the same serum sample as used in the acid-glycine extract analysis ( Figures 7a [positive sample]). The positive identification of a single antigenic band with the H. pylori positive serum suggested that this could be H. pylori specific. The analysis was repeated using a selection of different human serum samples.
  • UBT data was determined by BSIA Ltd. n/a indicates not available.
  • Biotechnology Information (NCBI) server revealed correspondence with an H. pylori, strain 921023, putative protein recorded as 50S L7/L12 ribosomal protein.
  • the subsequent publication of the genome of H. pylori strain 26695 revealed considerable identity between the N-terminal sequence obtained in these studies and the protein derived from the L7/L12 gene of this strain (26695).

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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention porte sur un nouvel antigène du H. pylori, sur une molécule d'acide nucléique codant pour lui, et sur un vaccin et ses utilisations comme antigène/immunogène. Elle porte également sur des méthodes de diagnostic.
PCT/GB1999/000885 1998-03-20 1999-03-19 Antigene du h. pilori et ses utilisations WO1999048919A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30429/99A AU3042999A (en) 1998-03-20 1999-03-19 H.pylori antigen and its use

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GBGB9806039.5A GB9806039D0 (en) 1998-03-20 1998-03-20 Therapy
GB9806039.5 1998-03-20

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WO1999048919A1 true WO1999048919A1 (fr) 1999-09-30

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WO (1) WO1999048919A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6617116B2 (en) 2000-01-28 2003-09-09 Genelabs Diagnostics Pte. Ltd. Assay devices and methods of analyte detection

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033220A1 (fr) * 1995-04-21 1996-10-24 Csl Limited Antigenes protecteurs d'helicobacter
WO1997012910A1 (fr) * 1995-09-29 1997-04-10 Cortecs International Limited Proteine d'helicobacter pylori
WO1998049314A2 (fr) * 1997-04-25 1998-11-05 Genelabs Technologies, Inc. COMPOSITION ANTIGENIQUE ET METHODE DE DETECTION D'$i(HELICOBACTER PYLORI)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033220A1 (fr) * 1995-04-21 1996-10-24 Csl Limited Antigenes protecteurs d'helicobacter
WO1997012910A1 (fr) * 1995-09-29 1997-04-10 Cortecs International Limited Proteine d'helicobacter pylori
WO1998049314A2 (fr) * 1997-04-25 1998-11-05 Genelabs Technologies, Inc. COMPOSITION ANTIGENIQUE ET METHODE DE DETECTION D'$i(HELICOBACTER PYLORI)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HOCKING D ET AL.: "Recombinant Helicobacter pylori ribosomal protein of 13kDa mass, predicted to be a 50S ribosomal protein L7/L17", EMBL SEQUENCE DATABASE, 14 February 1997 (1997-02-14), HEIDELBERG DE, XP002113149 *
TOMB J F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GASTRIC PATHOGEN HELICOBACTER PYLORI", NATURE, vol. 388, 7 August 1997 (1997-08-07), LONDON GB, pages 539 - 547, XP002066695, ISSN: 0028-0836 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6617116B2 (en) 2000-01-28 2003-09-09 Genelabs Diagnostics Pte. Ltd. Assay devices and methods of analyte detection
US6849414B2 (en) 2000-01-28 2005-02-01 Genelabs Diagnostics Pte Ltd. Assay devices and methods of analyte detection

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GB9806039D0 (en) 1998-05-20
AU3042999A (en) 1999-10-18

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