WO1998004702A2 - Proteines, notamment proteines membranaires, d'helicobacter pylori, leur preparation et utilisation - Google Patents

Proteines, notamment proteines membranaires, d'helicobacter pylori, leur preparation et utilisation Download PDF

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Publication number
WO1998004702A2
WO1998004702A2 PCT/IB1997/000981 IB9700981W WO9804702A2 WO 1998004702 A2 WO1998004702 A2 WO 1998004702A2 IB 9700981 W IB9700981 W IB 9700981W WO 9804702 A2 WO9804702 A2 WO 9804702A2
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gly
seq
protein
leu
asn
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PCT/IB1997/000981
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WO1998004702A3 (fr
Inventor
Bernhard Knapp
Erika Hundt
Karl-Heinz Schmidt
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Chiron Behring Gmbh & Co.
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Priority to CA002259924A priority Critical patent/CA2259924A1/fr
Priority to EP97932967A priority patent/EP0918864A2/fr
Priority to JP10508651A priority patent/JP2001502886A/ja
Publication of WO1998004702A2 publication Critical patent/WO1998004702A2/fr
Publication of WO1998004702A3 publication Critical patent/WO1998004702A3/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • Proteins in particular membrane proteins, of Hel ⁇ cobacter pylori , their preparation and use
  • the present invention relates to novel proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, which are derived from Helicobacter pylori (H. pyl ori ) and which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five a ino acids, and to their preparation and use as pharmaceutical compositions, in particular as vaccines, or as a diagnostic agent. Based on these data, genes coding for these and related proteins were also isolated as shown in SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27.
  • Helicobacter pylori is a Gram-negative, microaerophilic, spiral bacterium which colonizes the mucosa of the human stomach.
  • the bacterium is the cause of chronic active gastritis and of peptic ulcer, in particular duodenal ulcer, and plays a role in the development of carcinomas of the stomach; consequently, Helicobacter pylori is an important human pathogen.
  • Immunization is the conventional way of preventing infectious diseases. It is therefore important to examine this option with regard to controlling an H. pylori infection.
  • Bacteriology 175, 674-683) and 63,000 Da are located in the outer membrane, which adhesins are candidates for an experimental vaccine which has the aim of inducing antibodies which prevent adhesion of the bacterium to the mucosal surface.
  • the outer membrane possesses porins of 30,000 Da (M.A. Tufano et al., 1994, Infection and Immunity 62, 1392-1399), 48,000 Da, 49,000 Da, 50,000 Da, 67,000 Da (M.M. Exner et al., 1995, Infection and Immunity 63, 1567-1572) and 31,000 Da (P.
  • a protein from Helicobacter pyl ori (H. pylori ) containing one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 and 19 according to Tables la-lc, or parts or homologues thereof having a minimum length of five amino acids.
  • the peptide sequences of the protein are N-terminal sequences.
  • the protein according to the first aspect of the present invention preferably contains a peptide sequence having the SEQ ID NO: 1 according to Table la and has a molecular weight of approx. 250 kD, or preferably contains a peptide sequence having the SEQ ID NO: 2 according to Table la and has a molecular weight of approx. 110 kD, or preferably contains a peptide sequence having the SEQ ID NO: 3 according to Table la and has a molecular weight of approx. 100 kD, or preferably contains a peptide sequence having the SEQ ID NO: 6 according to Table la and has a molecular weight of approx.
  • 60 kD or preferably contains a peptide sequence having the SEQ ID NO: 10 according to Table lb and has a molecular weight of approx. 42 kD, or preferably contains a peptide sequence having the SEQ ID NO: 11 according to Table lb and has a molecular weight of approx. 42 kD, or preferably contains a peptide sequence having the SEQ ID NO: 12 according to Table lb and has a molecular weight of from approx. 32 to approx. 36 kD, or preferably contains a peptide sequence having the SEQ ID NO: 14 according to Table lc and has a molecular weight of approx.
  • 30 kD or preferably contains a peptide sequence having the SEQ ID NO: 15 according to Table lc and has a molecular weight of approx. 28 kD, or preferably contains a peptide sequence having the SEQ ID NO: 16 according to Table lc and has a molecular weight of approx. 28 kD, or preferably contains a peptide sequence having the SEQ ID NO: 17 according to Table lc and has a molecular weight of approx. 25 kD, or preferably contains a peptide sequence having the SEQ ID NO: 18 according to Table lc and has a molecular weight of approx.
  • the protein according to the first aspect of the present invention is preferably a membrane protein or a protein which is firmly associated with the membrane. More preferably said protein is an integral membrane protein, in particular a Sarkosyl ® -insoluble integral membrane protein.
  • step (b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods;
  • step (c) isolating the proteins, which have been separated in accordance with step (b) .
  • the proteins according to the second aspect of the present invention can be obtained by means of differential solubilization using Sarkosyl ® .
  • the proteins can also be obtained by means of separation by one or more SDS polyacrylamide gel electrophoreses, preferably by means of several SDS polyacrylamide gel electrophoreses having different polyacrylamide contents, more preferably wherein the polyacrylamide content of said gel electrophoreses is approximately 8%, 10% or 16%.
  • an antibody against one or more proteins according to the first or second aspects of the present invention and/or against one or more peptides according to the third aspect of the present invention is provided.
  • a polynucleotide encoding one or more proteins according to the first or second aspects of the present invention or one or more peptides according to the third aspect of the present invention.
  • a process for preparing the proteins according to the first or second aspects of the present invention characterized in that the following procedural steps are carried out: (a) isolating the proteins, by means of differential solubilization;
  • step (b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods; and (c) isolating the proteins, which have been separated in accordance with step (b) .
  • the process is characterized in that the proteins are isolated in accordance with step (a) using Sarkosyl ® .
  • a seventh aspect of the present invention there is provided a process for preparing the peptides according to the third aspect of the present invention, characterized in that a chemical peptide synthesis is carried out.
  • an eighth aspect of the present invention there is provided a process for preparing the proteins according to the first or second aspects of the present invention or the peptides according to the third aspect of the present invention, characterized in that a polynucleotide according to the fifth aspect of the present invention is expressed.
  • a ninth aspect of the present invention there is provided the use of one or more proteins according to the first or second aspects of the present invention, one or more peptides according to the third aspect of the present invention, one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention for preparing a pharmaceutical composition or a diagnostic agent.
  • a pharmaceutical composition comprising one or more proteins according to the first or second aspects of the present invention and/or one or more peptides according to the third aspect of the present invention or one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention or their expression products.
  • said pharmaceutical composition is used as a vaccine.
  • a diagnostic agent comprising one or more proteins according to the first or second aspects of the present invention and/or one or more peptides according to the third aspect of the present invention or one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention or their expression products.
  • a protein from H. pylori containing one of the peptide sequences deduced from SEQ ID NO: 21, 22, 23, 24, 25, 26 and 27, or parts or homologues thereof having a minimum length of five amino acids.
  • a peptide selected from the C-terminal region of the peptide sequence of SEQ ID NO: 20 or homologue thereof.
  • said peptide is selected from RDPKFNLAHIEKEFEVWNWDYRA and EKHQKMMKDMHGKDMHHTKKKK, or parts or homologues thereof.
  • an antibody against one or more proteins according to the twelfth aspect of the present invention and/or against one or more peptides according to the thirteenth or fourteenth aspects of the present invention is provided.
  • a sixteenth aspect of the present invention there is provided a polynucleotide encoding one or more proteins according to the twelfth aspect of the present invention or one or more peptides according to the thirteenth or fourteenth aspects of the present invention.
  • a host cell transformed with the polynucleotide according to the fifth or sixteenth aspects of the present invention.
  • an expression product expressed from the host cell according to the seventeenth aspect of the present invention is provided.
  • a pharmaceutical composition comprising one or more proteins according to the twelfth aspect of the present invention and/or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products.
  • said pharmaceutical composition is used as a vaccine. More preferably, when the pharmaceutical composition comprises a nucleotide sequence, said pharmaceutical composition is used as a DNA vaccine.
  • a diagnostic agent comprising one or more proteins according to the twelfth aspect of the present invention and/or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products.
  • a twenty-first aspect of the present invention there is provided the use of one or more proteins according to the twelfth aspect of the present invention or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products for preparing a pharmaceutical composition or a diagnostic agent.
  • the present application describes the isolation and determination of, in all, 19 proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, especially integral membrane proteins, which proteins are in a molecular weight range of from 17 kD to approx. 250 kD (Tables la-lc) .
  • membrane protein is generally understood to mean integral and peripheral membrane proteins and transmembrane proteins. Integral membrane proteins are proteins which are partially or entirely inserted into the cytoplasmic membrane. By contrast, peripheral membrane proteins only adhere to the surface of the membrane. Transmembrane proteins pass completely through the membrane (see, for example, B. Alberts et al. (eds).
  • the invention describes proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, especially integral membrane proteins, in particular Sarkosyl ® -insoluble integral membrane proteins of H. pylori , which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five, preferably six amino acids, with these peptide sequences preferably constituting N-terminal sequences of the said proteins.
  • the novel peptides are particularly preferred which exhibit at least ten consecutive amino acids selected from the sequences having the SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16 and 19.
  • those said parts are in particular preferred which contain an uninterrupted sequence of unambiguously specified amino acids.
  • part in the context of "part(s) of a sequence” in the present invention is defined herein as meaning a sequence of amino acids which can form a T-cell or B-cell epitope. Such an amino acid sequence is usually of a minimum of approximately four to eight amino acids.
  • homologue (s) in the context of the present invention is defined herein as meaning the same protein or peptide of a different strain of H. pylori but exhibiting the same function.
  • the actual amino acid sequences may not be identical between homologous proteins or peptides from different strains of H. pylori , the differences between the amino acid sequences merely represent strain-specific differences; the function of the homologues is identical.
  • the protein containing a peptide sequence having the SEQ ID NO: 1 according to Table la has a molecular weight of approx. 250 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 2 according to Table la has a molecular weight of approx. 110 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 3 according to Table la has a molecular weight of approx. 100 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 6 according to Table la has a molecular weight of approx.
  • the protein containing a peptide sequence having the SEQ ID NO: 10 according to Table lb has a molecular weight of approx. 42 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 11 according to Table lb has a molecular weight of approx. 42 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 12 according to Table lb has a molecular weight of from approx. 32 to approx. 36 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 14 according to Table lc has a molecular weight of approx.
  • the protein containing a peptide sequence having the SEQ ID NO: 15 according to Table lc has a molecular weight of approx. 28 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 16 according to Table lc has a molecular weight of approx. 28 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 17 according to Table lc has a molecular weight of approx. 25 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 18 according to Table lc has a molecular weight of approx. 25 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 19 according to Table lc has a molecular weight of approx. 17 kD.
  • H. pylori strain No. ATCC 43504 is used, for example, as the starting material when isolating the proteins, with it being possible, in particular, to carry out the following procedural steps:
  • step (b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods, preferably by means of SDS polyacrylamide gel electrophoresis, with use being made, in particular, of polyacrylamide gels having differing polyacrylamide contents, in particular containing approx. 8, 10 or 16% polyacrylamide, and
  • step (c) isolating the proteins, which have been separated in accordance with step (b) , by means of known methods, for example by elution or by isolation on a membrane .
  • the proteins were first of all obtained using the method of Blaser et al. (see above).
  • the bacteria which had been disrupted in a glass bead homogenizer, were freed of intact bacteria by centrifugation at 5000 g; the supernatant was then centrifuged at 100,000 g.
  • the pellet was dissolved in Sarkosyl ® , and the Sarkosyl ® -insoluble fraction, which contains the integral membrane proteins in particular, was centrifuged off.
  • the pellet was resuspended in distilled water and fractionated by SDS polyacrylamide gel electrophoresis- (PAGE) .
  • the amino acids which are labelled Xaa in the sequence listing can be explained as follows:
  • the non-identifiable amino acids can be caused by interference due to impurities in the first sequencing step, a non-analysable amino acid, such as Cys or Trp, a modifiable amino acid which is missing in the elution programme, or an amino acid, such as Ser or Thr, which is difficult to determine, basically due to low sequence yields.
  • Different bands can also contain two proteins of very similar molecular weights in different quantities. This then results in two sequences which then also have to be assigned unambiguously on account of the different frequency of the individual amino acids.
  • the present invention also describes the peptides which are designated by the sequences according to SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 16, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five amino acids, in particular of six amino acids, which can be prepared, for example, by well-known chemical peptide synthesis
  • Bodanszky M. & Bodanszky, A. "The practice of peptide synthesis”. Springer Verlag, 1984) .
  • the novel peptides are particularly preferred which possess at least ten consecutive amino acids selected from the sequences having the SEQ ID NO: 1 , 2 , 3, 6, 10, 11, 12, 14, 15, 16 and 19.
  • those said peptides are, in particular, preferred which contain an uninterrupted sequence of unambiguously determined amino acids, as is the case with the sequences from SEQ ID NO: 12, 14 and 15.
  • the present application also describes antibodies which can also be prepared by methods which are well known to the skilled person (see, for example, B.A. Diamond et al. (1981), The New England Journal of Medicine, 1344-1349) and which are directed against one or more of the novel proteins or peptides.
  • the skilled person is also familiar, from
  • nucleotide sequences which encode the peptides according to the sequence listing.
  • nucleotide sequences are preferred which occur most frequently in accordance with the rules for the frequency of use of the different codons in Helicobacter pylori .
  • These nucleotide sequences can be prepared, for example, by means of chemical polynucleotide synthesis (see, for example, E. Uhlmann & A. Peyman (1990), Chemical Reviews, 543-584, Vol. 90, No. 4) .
  • oligodeoxynucleotides which have been prepared in accordance with these rules can be employed for screening Helicobacter pylori gene libraries using known methods (J. Sambrook et al., 1989, "Molecular Cloning, A Laboratory Manual", 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Furthermore, taking the sequence data as a basis, peptides can be synthesized which are employed for obtaining antisera. Gene expression libraries can then be screened using these antisera. The clones resulting from these different screening methods can then be employed, by isolating and sequencing the inserted DNA fragments, for identifying DNA sequence segments which encode the N-terminally sequenced protein segments of the proteins.
  • DNA fragments do not contain the complete gene encoding any particular protein, these DNA fragments can be used to isolate the complete genes by screening other gene libraries. The genes which have been completely isolated in this manner can then be expressed, in accordance with the state of the art, in various well- known systems in order to obtain the corresponding protein.
  • oligonucleotides deduced from the N- terminal sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15, the genes corresponding to the SEQ ID NOS: 5, 8, 10, 12 and 15 were isolated and are specified as SEQ ID NOS: 20 (catalase) , 24 (50 kD membrane protein), 25 (42 kD protein), 26 (36/35/32 kD protein) and 23 (28 kD protein) .
  • the gene coding for Hop C could not be isolated using oligonucleotide 7.
  • oligonucleotide 7 hybridizes with an homologous gene specified as SEQ ID NO: 21 (Hop X) .
  • Two additional genes which belong to this family were able to be isolated and are specified as SEQ ID NO: 21 (Hop Y) and SEQ ID NO: 22 (Hop Z) .
  • Such vaccines may either be prophylactic (to prevent infection) or therapeutic (to treat disease after infection) .
  • These vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants") .
  • the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
  • Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum) , such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components) , such as for example (a) those formulations described in
  • microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA) )
  • SAF containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion
  • MT monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon) , macrophage colony stimulating factor (M-CSF) , tumour necrosis factor (TNF)
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA)
  • muramyl peptides include, but a r e n o t l i m i t e d t o , N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) , N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP) , N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2- (1 ' -2 * -dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy) - ethyla ine (MTP-PE), etc.
  • the immunogenic compositions typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the vaccine may be administered in conjunction with other immunoregulatory agents .
  • compositions in particular vaccines, and diagnostic agents which comprise one or more of the novel proteins and/or one or more of the novel peptides or one or more of the novel antibodies or one or more of the novel polynucleotides or one or more expression products of the novel polynucleotides.
  • a DNA vaccine can be prepared on the basis of the polynucleotides, or a diagnostic agent can be prepared on the basis of the polymerase chain reaction (PCR diagnosis), or an immunotest, for example a Western blot test or an enzyme immunotest (ELISA) can be prepared on the basis of the antibodies.
  • novel proteins or peptides, or their immunogenic moieties in particular when they contain an uninterrupted sequence of unambiguously determined amino acids, having a minimum length of five amino acids, preferably six amino acids and, in particular, in the case of the novel peptides having the SEQ ID NOS: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16 and 19 and peptides or proteins encoded by the DNA sequences of SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27, at least ten consecutive amino acids, can be used as antigens for immunizing mammals.
  • the two C-terminal regions Cl and C2 specific for H. pylori catalase (c.f. Example 6) can also be used as immunogens .
  • the antibodies which are formed by the immunization, or antibodies which are prepared by means of recombinant DNA methods can, inter alia, prevent adhesion of the bacteria to the mucosal surface, attract macrophages for the purpose of eliminating bacteria, and activate the complement system for the purpose of lysing the bacteria.
  • the following examples are intended to clarify the invention.
  • the H. pylori stain ATCC 43504 was passaged under microaerophilic conditions (BBL Jar/Campy Pak Plus, from Becton & Dickinson) on Columbia Agar plates containing 5% horse blood (incubation 48 h, 37°C) . Three plates were rinsed off when inoculating a 500 ml flow-spoiler flask (100 ml of Columbia broth, 7% FCS) ; during the incubation
  • the bacterial cultures are harvested in the phase of late logarithmic growth, washed in 10 mM Tris buffer (pH 7.4) and disrupted with glass beads in a ho ogenizer (Institut fiir Molekularbiologie und Analytik (IMA), Germany) at 4°C and 4000 rpm for 15 min. After that, the glass beads are removed by filtration and the bacterial suspension is centrifuged at 5000 g for 20 min in order to remove intact cells.
  • IMA Institute fiir Molekularbiologie und Analytik
  • the cell walls are pelleted out of the supernatant by centrifuging at 100,000 g for 60 minutes and at 4°C.
  • the resulting pellet is resuspended with a 1% solution of Sarkosyl ® in 7 mM EDTA, and the suspension is incubated at 37°C for 20 min.
  • the Sarkosyl ® -insoluble fraction which contains the integral membrane proteins, is pelleted by centrifugation at 50,000 g for 60 minutes and at 4°C and the pellet is resuspended in sterile distilled water; the suspension is then stored at -20°C.
  • the membrane protein fraction starting material is dissolved in 1.5% SDS, 2.5% mercaptoethanol, 5% glycerol and bromophenol blue in 63 mmol/1 Tris buffer, pH 6.8, and fractionated by SDS polyacrylamide gel electrophoresis.
  • Protein transfer from the SDS gel to the PVDF membrane is carried out in a BioRad (Munich) Trans Blot SD apparatus, under modified conditions.
  • the number of sequencing steps was 5 to 25 (depending on the quantity of substance available for sequencing) .
  • the oligonucleotides were deduced using the species-specific codon usage of Helicobacter pylori , which had been determined from 19 known H. pylori genes, and using the base inosine (I), which is capable of undergoing stable base pairing with the bases adenine (A) , cytosine (C) and thymine (T) with, in each case, two hydrogen bridges.
  • the degeneracy of the codon was kept as low as possible.
  • oligonucleotides which had been deduced from the peptide sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15 were labelled with digoxigenin (DIG) using a kit manufactured by Boehringer Mannheim (DIG Oligonucleotide 3'-End Labelling Kit) and employed for screening a H. pylori gene library which had been prepared using a kit manufactured by Stratagene (Predigested ZAP ExpressTM BamHI/CIAP Vector Cloning Kit) at 32°C under standard conditions.
  • DIG Oligonucleotides 1, 3 and 6 it was possible to identify clones which carry DNA fragments containing sequences which encode the peptide sequences of SEQ ID NOS: 5, 8 and 15.
  • Oligonucleotide 2 hybridized with a DNA fragment which encodes an homologous sequence of SEQ ID NO: 7.
  • oligonucleotides 4 and 5 it was only possible to isolate clones whose DNA fragments did not encode SEQ ID NOS: 10 and 12. This is why these oligonucleotides and the clones which had been isolated from the ⁇ ZAP Express gene library were employed in a Southern Blot analysis, which permitted the unequivocal identification of DNA fragments which hybridized with the oligonucleotides, but not with the DNA fragments resulting from the screening. With these DNA fragments, in each case one sub-gene library was prepared in the ⁇ ZAP Express vector, and each sub-gene library was screened with oligonucleotides 4 and 5. This allowed the identification of clones which carry DNA fragments encoding the sequences of SEQ ID NOS: 10 and 12.
  • SEQ ID NO: 20 describes the DNA sequence which encodes the catalase of H. pylori .
  • the nucleotide region 337 to 378 describes the hybridization site with oligonucleotide 1.
  • the catalase gene of H. pylori has been described in 1996 by Stefan Odenbreit, Bj ⁇ rn Wieland and Rainer Haas (J. Bacteriol. 178, 6960-6967) and is therefore not new. However, when comparing the amino acid sequences of the catalases of Escheri chia coJi, Bacillus firm ⁇ s, B. subtilis A, B.
  • subtilis B rats, mice, cattle, humans, Staphylococcus violaceus, Haemophilus infl ⁇ enzae, B. fragilis, Pseudomonas mirabilis, B. pertussis and P. syringae with the amino acid sequence of H. pylori , it is possible to identify two C-terminal regions Cl (RDPKFNLAHIEKEFEVWNWDYRA) and C2 (EKHQKMMKDMHGKDMHHTKKKK) , which are specific to H. pyl ori catalase. These two peptides were synthesized using standard techniques, coupled to KLH and used for immunizing rabbits.
  • H. pylori catalase which had been produced by recombinant technique.
  • H. pyl ori- catalase-specific regions may conceivably be used for developing a vaccine which avoids the problem complex of autoimmune reactions or for the development of a diagnostic which reacts specifically with H. pylori catalase.
  • SEQ ID NO: 21 describes a nucleotide sequence which was identified by hybridization with the oligonucleotide 2.
  • the oligonucleotide hybridized with the sequence of nucleotide 1240 to 1284. This encodes a sequence which is homologous to the porin Hop C (Exner et al., 1995) and is identical with the published amino- terminal sequence EDDGGFFTVGYQLGQVMQDVQNPG in positions 1, 2, 3, 4, 9, 10, 11, 12, 14, 18 and 22.
  • the porins Hop A, Hop B, Hop C and Hop D have identical amino acids in 9 positions of the 20 N-terminal amino acids (Exner et al . , 1995). In 8 of these positions, there are identical positions also in the sequence described in the present publication; in the 9th position, a conserved amino acid exchange is present (Val - lie) . It can thus be assumed that the protein described in the present publication is equally part of this group of the porins; it was therefore termed Hop X.
  • the mature protein with 428 amino acids has a molecular weight of 47.3 kD and an isoelectric point of 10.0.
  • a further open reading frame was found upstream of the gene which encodes Hop X.
  • This further open reading frame encodes a protein which is homologous to Hop X (34% identity) and which was therefore termed Hop Y.
  • the gene region found to date encodes the 361 C- terminal amino acids of the protein. The gene region as yet outstanding is currently being isolated using stan- dard techniques .
  • SEQ ID NO: 22 describes a nucleotide sequence which was concomitantly isolated and sequenced during the screening process .
  • the amino acid sequence deduced encodes the 392 C-terminal residues of a protein which shows a high homology with Hop X (33% identity) and Hop Y (28% identity) and which was therefore termed Hop Z.
  • Hop Z The gene region which encodes the N-terminal portion of the protein is currently being isolated.
  • SEQ ID NO: 23 describes a DNA sequence which encodes a hitherto undescribed protein.
  • the nucleotide region 696 to 767 describes the hybridization site with the oligonucleotide 6.
  • the molecular weight is quite close to the molecular weight of 28 kD which had been determined by SDS gel electrophoresis.
  • the amino acid sequence deduced is homologous with the sequences of the proteins Hop X, Hop Y and Hop Z, for which the GCG Bestfit Programme determined identity values of 41%, 38% and 41%, respectively.
  • the 28 kD protein thus also seems to be part of the family of the porins or porin-like proteins.
  • SEQ ID NO: 24 describes a DNA sequence which encodes the non-heat-modifiable 50 kD membrane protein. This protein was first described by Exner et al., 1995, and an N-terminal sequence of the protein was determined. Using the approach described by us, we were then able to describe, with SEQ ID NO: 8, an N-terminal sequence which is identical to the sequence described by Exner et al. (1995) . With the aid of the oligonucleotide 3, which had been deduced using the method illustrated in Example 5 and had been used for screening a H. pylori gene library using the above-described methods, it was then possible to identify a DNA fragment which encodes the 50 kD membrane protein.
  • the amino acid residues 236 to 254 contain a hydrophobic region which is large enough to act as a transmembrane region. Based on such data and using standard methods for epitope analysis, it is possible to identify regions which might be presented on the surface of bacteria. Such regions might be used for developing a vaccine or a diagnostic.
  • SEQ ID NO: 25 describes a DNA sequence 2825 bp in size which was identified by means of hybridization with oligonucleotide 4, which was deduced from SEQ ID NO: 10. Oligonucleotide 4 hybridized with the nucleotide region 897 to 923 of the described sequence of SEQ ID NO: 25. The protein has no signal sequence.
  • the encoding region of SEQ ID NO: 25 codes for a protein of 399 amino acids with a molecular weight of 43.6 kD and an isoelectric point of 5.0.
  • a search for homologous sequences using the BLASTP program (S. F. Altschul et al., 1990, J. Mol. Biol. 215, 403-410) identified the 42 kD antigen of H.
  • elongation factor TU The maximum percentage of identity (89%) was found with the elongation factor TU from Wolinella succinogenes (W. Ludwig et al . , 1993, Antonie van Leeuwenhoek 64, 285- 305 ) .
  • SEQ ID NO: 26 describes a DNA sequence 2182 bp in size which hybridizes with oligonucleotide 5, which had been deduced from SEQ ID NO: 12. Oligonucleotide 5 hybridized with a Sau3AI fragment (position 1 to 575) of the gene library starting from position 524. The screening of different DNA libraries with specific oligonucleotides allowed the isolation of the complete gene described in SEQ ID NO: 26. An amino acid sequence which is identical to the one from SEQ ID NO: 12 can be deduced from SEQ ID NO: 26. Both protein sequencing and the hydrophobicity of the N-terminal sequence deduced allow the conclusion that the antigen has a signal sequence.
  • the mature protein consists of 328 amino acid residues with a molecular weight of 36.1 kD and an isoelectric point of 9.95. No homologous proteins were identified using the BLASTP program (S. F. Altschul et al., 1990) .
  • sequences described in SEQ ID NOS: 20 to 26 indicate nucleotide sequences which encode antigens of the H. pylori strain ATCC 43504. However, it is known for H. pylori that heterogeneity between identical antigens may exist amongst various strains. We therefore claim not only the sequences described in SEQ ID NOS: 21 to 26, but in addition also the sequences of other H. pylori strains which are homologous with the sequences described herein.
  • SEQ ID NO: 19 aligned with a very similar sequence using the TBLASTN program.
  • SEQ ID NO: 27 describes the nucleic acid sequence and deduced amino acid sequence from the coding region of a H. pylori gene (strain 26695) localised between position 843212 and 843691 of the genomic sequence. The protein has no signal sequence.
  • the N-terminal sequence of SEQ ID NO: 19 is highly homologous to the N-terminal region of the deduced amino acid sequence from amino acid residue 1 to 15. Only one different amino acid residue is present at position 4: the nucleotide sequence found by the alignment encodes a Ser residue in this position instead of an Asn residue determined by N-terminal sequencing. This can be explained by strain specific differences.
  • the identified nucleic acid sequence in SEQ ID NO: 27 codes for a protein of 159 amino acid residues with a molecular weight of 18.2 kD and an isoelectric point of 7.2. The molecular weight is very close to that of 17 kD determined from SDS polyacrylamide gel electrophoresis.
  • a search for homologous sequences using the BLASTP program shows that the
  • 17 kD antigen is very homologous to "hydroxymyristol-
  • Proteins in particular membrane proteins, of Heli cobacter pylori , their preparation and use
  • Xaa at positions 1, 5, 12, 14 and 16 are unknown amino acids. At position 8, Xaa is probably Gin, while at position 10 it is probably Ser, at position 11 it is probably Tyr and at position 15 it is probably Thr. Identification: unknown
  • Xaa is an unknown amino acid.
  • Xaa is He or Thr and at position 7 it is Ala or Lys.
  • Identification: unknown (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Xaa Lys Leu Xaa Pro Gin Xaa Gly Tyr Val Leu Met Tyr 1 5 10
  • Xaa at positions 1 and 10 are unknown ammo acids, and at position 4, Xaa is He or Thr and at position 7 it is Ala or Lys . Identification: unknown
  • Xaa at positions 1, 5 and 9 are unknown ammo acids, and at position 7 Xaa is Ala or Leu. Identification: unknown
  • Xaa at positions 1 and 11 are unknown, while at position 6, Xaa is probably Asn or Gin, at position 13 it is probably Thr and at position 14 it is probably He. Identification: unknown
  • Xaa at positions 1 and 4 are unknown, while at position 6 Xaa is Asn or Gin and at position 8 it is probably Pro. Identification: unknown
  • Xaa at positions 1 and 13 are unknown amino acids. Identification: unknown
  • Xaa at position 1 is an unknown ammo acid and at position 2 it is probably Trp. Identification: unknown
  • Xaa at positions 1, 2, 3, 6, 10 and 14 are unknown ammo acids, while at position 5, Xaa is Pro or Val and at position 7 it is probably Lys. Identification: unknown
  • Xaa at position 1 is an unknown ammo acid, while at position 5 Xaa is Pro or Lys.
  • Identification: unknown (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17; Xaa Gin Arg Met Xaa Gin Val Gly 1 5
  • Xaa at position 1 is an unknown amino acid, while at position 5 Xaa is Pro or Lys. Identification: unknown
  • Xaa at positions 1, 5 and 10 are unknown amino acids, while at position 11 Xaa is probably Gin and at position 15 it is probably Lys. Identification : unknown
  • PROPERTIES catalase from Heli cobacter pylori
  • GAG AAA GAG TTT GAA GTG TGG AAT TGG GAT TAC AGA
  • GCT GAT GAT AGC 1602 Glu Lys Glu Phe Glu Val Trp Asn Trp Asp Tyr Arg Ala Asp Asp Ser 410 415 420 425
  • PROPERTIES coding region coding for related proteins of
  • PROPERTIES protein HopZ of Helicobacter pylori
  • PROPERTIES 28 kD protein from Helicobacter pylori
  • AAA ATG GTG AAT GAC AAC GGC TTG CAA AAG CCT TTG ATA AAG TTC CCG 800
  • PROPERTIES 50 kD membrane protein from Helicobacter pylori
  • GCT ACT AAA ATG TGG CAA CAA TCA GGG CCA GGT GGT GTC ATT AAC CCT 1574 Ala Thr Lys Met Trp Gin Gin Ser Gly Pro Gly Gly Val He Asn Pro 100 105 110
  • PROPERTIES 42 kD protein from Helicobacter pylori
  • GCTAGAGCAT CAGCCTTCCA AGCTGAGGGT CGCGGGTTCG AGTCCCGTTT CCCGCTCCAT 660
  • GCA AAA GAA AAG TTT AAC AGA ACT AAG CCG CAT GTT AAT ATT GGA ACC 941
  • GGT AGG ACC GTT GGT GCT GGT GTT GTG AGC AAT ATT ATT GAA TAA 2090
  • PROPERTIES 36/35/32 kD protein from Helicobacter pylori
  • ATC GGT AAA AAA AGG TGG TTT GGG TTG CGC TAT TAC GGC TTT TTT GAT 883 He Gly Lys Lys Arg Trp Phe Gly Leu Arg Tyr Tyr Gly Phe Phe Asp 110 115 120
  • PROPERTIES 17 kD protein from Helicobacter pylori

Abstract

L'invention concerne de nouvelles protéines, notamment des protéines membranaires ou protéines solidement associées à la membrane, lesquelles sont dérivées d'hélicobacter pilori (H. pilori) et lesquelles contiennent une des séquences peptidiques sélectionnées par les SEQ ID NO:1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 ou 19 selon les tableaux 1a-1c, ou des parties ou homologues de celles-ci, présentant une longueur minimum de 5 acides aminés, ainsi que leur préparation et utilisation en tant que composition pharmaceutique, notamment en tant que vaccins, ou en tant qu'agents diagnostiques. Sur la base de ces données, on a également isolé des gènes codant pour ces protéines et des protéines apparentées, telles que l'illustrent les SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 et 27.
PCT/IB1997/000981 1996-07-26 1997-07-25 Proteines, notamment proteines membranaires, d'helicobacter pylori, leur preparation et utilisation WO1998004702A2 (fr)

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CA002259924A CA2259924A1 (fr) 1996-07-26 1997-07-25 Proteines, notamment proteines membranaires, d'helicobacter pylori, leur preparation et utilisation
EP97932967A EP0918864A2 (fr) 1996-07-26 1997-07-25 Proteines, notamment proteines membranaires, d'helicobacter pylori, leur preparation et utilisation
JP10508651A JP2001502886A (ja) 1996-07-26 1997-07-25 Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉのタンパク質、特に膜タンパク質、それらの調製および使用

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DE19630390A DE19630390A1 (de) 1996-07-26 1996-07-26 Proteine, insbesondere Membranproteine von Helicobacter pylori, ihre Herstellung und Verwendung

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WO1998004702A3 (fr) 1998-04-23
JP2001502886A (ja) 2001-03-06
DE19630390A1 (de) 1998-01-29
EP0918864A2 (fr) 1999-06-02
CA2259924A1 (fr) 1998-02-05

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