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WO2000000614A9 - NOUVEAUX ANTIGENES D'$i(HELIOBACTER PYLORI) - Google Patents

NOUVEAUX ANTIGENES D'$i(HELIOBACTER PYLORI)

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Publication number
WO2000000614A9
WO2000000614A9 PCT/US1999/014375 US9914375W WO0000614A9 WO 2000000614 A9 WO2000000614 A9 WO 2000000614A9 US 9914375 W US9914375 W US 9914375W WO 0000614 A9 WO0000614 A9 WO 0000614A9
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WO
WIPO (PCT)
Prior art keywords
polypeptide
acid sequence
kda
seq
nucleic acid
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.)
Ceased
Application number
PCT/US1999/014375
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English (en)
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WO2000000614A2 (fr
WO2000000614A3 (fr
Inventor
James Peter Fulginiti
Michael James Fiske
Deborah Ann Dilts
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.)
Wyeth Holdings LLC
Original Assignee
American Cyanamid Co
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Filing date
Publication date
Application filed by American Cyanamid Co filed Critical American Cyanamid Co
Priority to AU47189/99A priority Critical patent/AU4718999A/en
Priority to CA002329264A priority patent/CA2329264A1/fr
Publication of WO2000000614A2 publication Critical patent/WO2000000614A2/fr
Publication of WO2000000614A9 publication Critical patent/WO2000000614A9/fr
Publication of WO2000000614A3 publication Critical patent/WO2000000614A3/fr
Anticipated expiration legal-status Critical
Ceased 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel nucleic acids and polypeptides relating to Helicobacter pylori.
  • the nucleic acid sequences and polypeptides are useful for diagnostic and therapeutic purposes.
  • H. pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen infection. [Warren, J.R. et al., Lancet, 1: 1273-1275, (1983) ; and Marshall et al., Microbios Lett, 25: 83-88, (1984)]. H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. [Rathbone et al., Gut, 27: 635-641, (1986)]. Additional evidence has developed for an etiological role of H.
  • H. pylori in non- ulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. [Blaser, M.J., Trends Microbiol, 1: 255-260, (1993)].
  • H. pylori colonizes the human gastric mucosa, establishing an infection that usually persists for many years. About 30-50% of the human population appear to be chronically infected. [Rainer, H. et al, Biologicals, 25: 175-177, (1997)].
  • the current recommended treatment for chronic H. pylori infection is multiple antibiotic treatment combined with a proton pump inhibitor, or with bismuth salts. However, this treatment may not fully resolve the infection and resistance to antibiotics can occur.
  • Proteins were selected for use as an antigen and/or vaccine candidate based on the following criteria: (i) the antigen is located on the bacterial surface, (ii) the antigen is conserved among H pylori clinical isolates, (iii) the antigen elicits functional antibodies, (iv) the antigen is able to confer protection to vaccinated mice from challenge with a live organism.
  • This invention relates to novel H. pylori bacterial surface proteins and nucleic acid sequences encoding therefor, in particular novel H. pylori bacterial surface proteins having molecular weights of approximately 75, 77, and 79 kilo daltons (kDa).
  • novel H. pylori bacterial surface proteins having molecular weights of approximately 75, 77, and 79 kilo daltons (kDa).
  • the mature processed forms of these proteins share a common amino-terminal amino acid sequence.
  • the proteins and nucleic acid sequences of the present invention have diagnostic and therapeutic utility for H pylori and other Helicobacter species. They can be used to detect the presence of H. pylori and other Helicobacter species in a sample, and to screen compounds for the ability to interfere with the H. pylori life cycle or to inhibit H. pylori infection.
  • this invention includes embodiments relating to isolated nucleic acid sequences corresponding to the entire coding sequences of H. pylori surface proteins or portions thereof, nucleic acids capable of binding mRNA from H. pylori surface proteins and methods for producing H. pylori surface proteins or portions thereof using peptide synthesis and recombinant techniques. Additional embodiments are also directed to antigenic and vaccine compositions based on agents prepared from the proteins and nucleic acids of this invention and methods for treatment and prevention of H. pylori infections employing such compositions.
  • Figure 1 Figure 1(A) depicts an SDS-PAGE gel with bands showing the 75kDa and 77kDa proteins in lane 3 in comparison to ZwittergentTM 3-14 crude extract of H pylori outer membrane proteins in lane 2 and molecular weight standards in lane 1.
  • Figure 1 (B) depicts a Western blot of monoclonal antibody 64-27 with the co-purified 75kDa and 77kDa proteins from ATCC 43579, as described in Example 1. Lane 1 is the molecular weight markers, lane 2 is the crude extract and lane 3 is the co-purified proteins.
  • Figure 2 depicts an electron micrograph of the surface labeling of H. pylori strain PBCC 1105 with mouse polyclonal antisera to a co-purified mixture of 75/77 kDa proteins, as described in Example 2.
  • Figure 3 depicts two graphs of Flow Cytometry analysis of strain PBCC 1105 with labeled an anti-75/77 mouse polyclonal antibody, at day 0 ( Figure 3A) and day 49 (Figure 3B) following injection of mice with a mixture of 75kDa and 77kDa proteins, as described in Example 2.
  • Profilel is ATCC 43579 (homologous strain); profile 2 is strain PBCC 1105; profile 3 is strain ATCC 43504; profile 4 is strain SS-1 and profile 5 is a urease-negative strain.
  • Figure 4 is a graph depicting the bactericidal activity of anti-75/77kDa polyclonal mouse sera, as described in Example 3, with either incomplete Freund's adjuvant or MPLTM.
  • FIG. 5 This Figure depicts the mouse protection data (in colony forming units) from the H. pylori SSI experimental challenge following vaccination of mice with a mixture of the co-purified 75kDa/77kDa proteins, as described in Example 4.
  • Figure 6 This Figure depicts a DNA (SEQ ID NO 19)for the 75kDa gene from strain ATCC 43579.
  • Figure 7 This Figure depicts the predicted translated protein sequence for the DNA sequence (SEQ ID NO 21)for the 75kDa gene from strain ATCC 43579 in figure 6.
  • Figure 8 This Figure depicts a DNA sequence (SEQ ID NO 20)for the 79kDa gene from strain ATCC 43579.
  • Figure 9 This Figure depicts the predicted translated protein sequence (SEQ ID NO 22)for the DNA sequence for the 79kDa gene from strain ATCC 43579 in figure 8.
  • Figure 10 This Figure depicts SDS-PAGE gel bands from comparison expression experiments from Example 13 for recombinant 75kDa protein expressed from a low copy number plasmid_pBAD24 and the high copy number T7 expression plasmid pRSETb. The bands show the increased amounts of protein 75 kDa expressed.
  • FIG 11 This Figure depicts SDS-PAGE gel bands from comparison expression experiments from Example 13 for recombinant 77kDa protein expressed from a low copy number plasmidjpET17 and the high copy number T7 expression plasmid pRSETb. The bands show the increased amounts of protein 77 kDa expressed.
  • Figure 12 This Figure depicts SDS-PAGE gel bands from comparison expression experiments from Example 13 for recombinant 79kDa protein expressed from a low copy number plasmid pBAD24 and the high copy number T7 expression plasmid pRSETb. The bands show the increased amounts of protein 79 kDa expressed.
  • Figure 13 This Figure depicts mouse protection data (in colony forming units) from the H. pylori SSI experimental challenge following vaccination of mice with a mixture of the co-purified 75kDa/77kDa proteins
  • Figure 13 This Figure depicts therapeutic effect (in colony forming units) of a mixture of the co-purified 75kDa/77kDa proteins when vaccinating mice after infection with H. pylori SSI Experiments included intragastric vaccination and a subcutaneous vaccination.
  • One aspect of the present invention provides an isolated, substantially purified H. pylori polypeptide selected from the group consisting of (i) a polypeptide having a molecular weight of about 75 kDa; (ii) a polypeptide having a molecular weight of about 77 kDa; and (iii) a polypeptide having a molecular weight of about 79 kDa; wherein the mature processed form of each polypeptide has a starting sequence consisting essentially of EDDGFYTSVGYQIGEAAQMV (SEQ. ID NO.7).
  • the present invention also relates to isolated polypeptides.
  • Preferred embodiments of the invention relate to an isolated polypeptide of H pylori selected from the group consisting of (i) a polypeptide having a molecular weight of about 75 kDa and having the amino acid sequence of SEQ. ID NO.l or SEQ ID NO. 19; (ii) a polypeptide having a molecular weight of about 77 kDa and having the amino acid sequence of SEQ. ID NO.2; and (iii) a polypeptide having a molecular weight of about 79 kDa and having the amino acid sequence of SEQ. ID NO.3 or SEQ ID NO. 20.
  • the polypeptides of this invention have antigenic properties, such as being reactive with H. pylori antibodies.
  • Antigens can be based on the isolated polypeptides sequences, or allelic or other variants thereof, which are biological equivalents. Suitable biological equivalents have about 70 to about 80%, and most preferably at least about 90%, similarity to one of the amino acid sequences referred to above, or to a portion thereof, provided the equivalent is capable of eliciting substantially the same antigenic properties as the isolated polypeptide sequences specified hereinabove.
  • the biological equivalents are obtained by generating variants and modifications to the isolated polypeptides of this invention. These variants and modifications to the isolated polypeptides are obtained by altering the amino acid sequences by insertion, deletion or substitution of one or more amino acids.
  • the polypeptides are then selected for use as an antigen and/or vaccine candidate based on the following criteria: (i) the antigen is located on the bacterial surface, (ii) the antigen is conserved among H pylori clinical isolates, (iii) the antigen elicits functional antibodies, (iv) the antigen is able to confer protection to vaccinated mice from challenge with a live organism.
  • Modifying the amino acid for example by substitution, the amino acids of the protein to create a polypeptide having substantially the same or improved qualities.
  • the amino acid changes are achieved by changing the codons of the nucleic acid sequence. It is known that such polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve antigenic or immunogenic activity (see, e.g. Kyte and Doolittle, 1982, Hopp, US Patent 4,554,101, each incorporated herein by reference). For example ,through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide which result in increased activity or enhanced immune response.
  • amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide-molecule conjugates which retain sufficient antigenic properties of the starting polypeptide to be useful for other purposes.
  • a selected polypeptide of the present invention may be bound to a solid support in order to have particular advantages for diagnostic applications.
  • hydropathic index of amino acids in conferring interactive biological function on a polypeptide, as discussed by Kyte and Doolittle (1982), wherein it is found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity.
  • substitution of like amino acids may be made on the basis of hydrophilicity, particularly where the biological function desired in the polypeptide to be generated is intended for use in immunological embodiments.
  • US Patent 4,554,101 which states that the greatest local average hydrophilicity of a "protein,” as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid.
  • hydrophilicity index or hydropathic index which assigns values to each amino acid
  • Preferable characteristics of the polypeptides of this invention include one or more of the following: (a) being a membrane protein or being a protein directly associated with a membrane; (b) capable of being separated as a protein using an SDS acrylamide (10%>) gel; (c) generating antibody which exhibits bactericidal activity upon injection in a mouse; and/or (d) reducing the colonization of H. pylori in mice upon delivery thereto.
  • the isolated polypeptides having molecular weights of about 75, 77, and 79 kDa are particular useful in antigenic compositions for several reasons.
  • the polyclonal antibodies generated by injecting mice with a mixture of the purified polypeptides are bactericidal.
  • CT cholera toxin
  • an antigenic composition comprising (i) at least one isolated polypeptide as disclosed above and (ii) a pharmaceutically acceptable buffer, diluent, adjuvant or carrier.
  • the antigenic composition may comprise a carrier, which in turn may be conjugated to said polypeptide.
  • the antigenic composition may further comprise an adjuvant.
  • these compositions have therapeutic and prophylatic applications as vaccines in preventing and/or ameliorating H. pylori infection. In such applications immunologically effective amount (as discussed herein) of at least one polypeptide of this invention is employed.
  • Antigenic compositions of the invention containing antigenic components preferably include a pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • pharmaceutically acceptable carrier refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered.
  • Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the antigenic compositions of the present invention is contemplated.
  • Such antigenic compositions are conventionally administered parenterally, e.g, by injection, either subcutaneously or intramuscularly. Methods for intramuscular immunization are described by Wolff et al. and by Sedegah et al. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori (See Czinn et al.). Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the antigenic compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; aluminum phosphate; StimulonTM QS-21 (Aquila Biopharmaceuticals, Inc, Worcester, MA); MPLTM (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Hamilton, MT), IL-12 (Genetics Institute, Cambridge, MA) N-acetyl-muramyl ⁇ L-theronyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl- sn-glycero-3-hydroxyphos-phoryloxy ⁇ ethylamine (CGP 19835
  • Non-toxic derivatives of cholera toxin including its B subunit, and/or conjugates or genetically engineered fusions of the H. pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-H. pylori bacterial lysates, block polymers or saponins.
  • this antigenic composition or an isolated polypeptide of this invention is used in a vaccine composition for oral administration which includes a mucosal adjuvant.
  • a vaccine composition for oral administration which includes a mucosal adjuvant.
  • an oral vaccine composition comprising an antigenic composition in association with a mucosal adjuvant, is used for the treatment or prevention of H. pylori infection in a human host.
  • the mucosal adjuvant can be cholera toxin; however, preferably, mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivatives of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. These may be added to, or conjugated with, the Helicobacter antigenic composition. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin (LT).
  • CTB B sub-unit
  • LT heat labile toxin
  • mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces.
  • Other mucosal ly active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine.
  • QS-21, MPLTM, and IL-12 which described above, may also be used
  • the Helicobacter antigenic composition of this invention may be delivered in the form of ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co- glycoside) to form microspheres of a size suited to adsorption by M cells.
  • ISCOMS immune stimulating complexes
  • ISCOMS containing CTB liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co- glycoside) to form microspheres of a size suited to adsorption by M cells.
  • micro or nanoparticles may be covalently attached to molecules such as vitamin B12 which have specific gut receptors.
  • the Helicobacter isolated polypeptides of this invention may also be incorporated into oily emulsions.
  • the Helicobacter isolated polypeptides of the present invention may be administered as the sole active immunogen in an antigenic composition.
  • the antigenic, or vaccine, composition may include other active immunogens, including other Helicobacter antigens such as urease, lipopolysaccharide, Hsp60, VacA, CagA or catalase, as well as immunologically active antigens against other pathogenic species.
  • Helicobacter antigens such as urease, lipopolysaccharide, Hsp60, VacA, CagA or catalase, as well as immunologically active antigens against other pathogenic species.
  • One of the important aspects of this invention relates to a method of inducing immune responses in a mammal comprising the step of providing to said mammal an antigenic composition of this invention.
  • Immunologically effective amount means the administration of that amount to a mammalian host, either in a single dose or as part of a series of doses, sufficient to at least cause the immune system of the individual treated to generate a response that reduces the clinical impact of the bacterial infection. This may range from a minimal decrease in bacterial burden to prevention of the infection. Ideally, the treated individual will not exhibit the more serious clinical manifestations of the Helicobacter infection.
  • the dosage amount can vary depending upon specific conditions of the individual. This amount can be determined in routine trials by means known to those skilled in the art.
  • Another specific aspect of the present invention relates to using a vaccine vector expressing an isolated Helicobacter polypeptide, or an immunogenic fragment thereof.
  • this invention provides a method of inducing an immune response in a mammal, which comprises providing to a mammal a vaccine vector expressing at least one, or a mixture of isolated Helicobacter polypeptides of this invention, or an immunogenic fragment thereof.
  • the isolated polypeptides of the present invention can be delivered to the mammal using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the an antigenic polypeptide or immunogenic fragment as a foreign polypeptide.
  • bacteria that colonize the gastrointestinal tract such as Salmonella, Shigella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are discussed by Holmgren et al. (1992) and McGhee et al. (1992).
  • An additional embodiment of the present invention relates to a method of inducing an immune response in a human comprising administering to said human an amount of a DNA molecule encoding an isolated polypeptide of this invention, optionally with a transfection-facilitating agent, where said polypeptide retains immunogenicity and, when incorporated into an antigenic composition or vaccine and administered to a human, provides protection without inducing enhanced disease upon subsequent infection of the human with Helicobacter pathogen, such as H pylori.
  • Transfection-facilitating agents are known in the art.
  • the present invention also relates to an antibody, which may either be a monoclonal or polyclonal antibody, specific for antigenic polypeptides as described above.
  • an antibody which may either be a monoclonal or polyclonal antibody, specific for antigenic polypeptides as described above.
  • Such antibodies may be produced by methods which are well known to those skilled in the art.
  • the antibodies of this invention can be employed in a method for the treatment or prevention of Helicobacter infection in mammalian hosts, which comprises administration of an immunologically effective amount of antibody, specific for antigenic polypeptide as described above.
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and western blot methods, as well as other procedures which may utilize antibodies specific to H pylori proteins. While ELISAs are preferred, it will be readily appreciated that such assays include RIAs and other non-enzyme linked antibody binding assays or procedures. Additionally, it is proposed that monoclonal antibodies specific to the particular H. pylori protein or polypeptides may be utilized in other useful applications. For example, their use in immunoadsorbent protocols may be useful in purifying native or recombinant H pylori proteins or variants thereof.
  • H pylori polypeptides of the invention will find use as antigens for raising antibodies and in immunoassays for the detection of anti- 75/77 kDa antigen-reactive antibodies.
  • samples suspected of containing H. pylori may be screened, in immunoassay format, for reactivity against antibodies specific for 75, 77 and 79 kDa polypeptides of this invention. Results from such analyses may then be used to determine the presence of H. pylori and potential infection.
  • Diagnostic immunoassays include direct culturing of bodily fluids or tissue, either in liquid culture or on a solid support such as nutrient agar.
  • a typical assay involves collecting a sample of bodily fluid from a patient and placing the sample under conditions optimum for growth of the pathogen. The determination can then be made as to whether the microbe exists in the sample. Further analysis can be carried out to determine the hemolyzing properties of the microbe.
  • Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot), which U.S. Patents are incorporated herein by reference.
  • Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
  • the anti-75/77 kDa antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label.
  • ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing an H. pylori polypeptide are immobilized onto the well surface and then contacted with the anti- 75/77 kDa antibodies. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the primary antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed.
  • This system may provide for signal amplification if desired.
  • Competition ELISAs are also possible in which test samples compete for binding with known amounts of labeled antigens or antibodies.
  • the amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells.
  • Antigen or antibodies may also be linked to a solid support, such as in the form of beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody).
  • the presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely absorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the layered antisera is then allowed to incubate for from 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C. Following incubation, the anti sera-contacted surface is washed so as to remove non-immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first.
  • the second antibody will preferably be an antibody having specificity in general for human IgG.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase- conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2.2'-azino-di-(e- ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H 2 0 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. Alternatively, the label may be a chemilluminescent one. The use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605.
  • nucleic acid sequences encoding the polypeptides of this invention, in particular the nucleic acid sequences as set forth in SEQ. ID NO.4, 5, and 6, or being substantially similar to all or a portion thereof.
  • substantially similar means having a least 50-70%, more preferably 70-80%, and most preferably 80 or 90% identity to one of said sequences.
  • Such substantially similar nucleic acid sequences hybridize under high stringency southern hybridization conditions with at least one of the nucleic acid sequences set forth in SEQ ID Nos. 4, 5 or 6.
  • the nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form.
  • RNA or DNA single stranded or double stranded, in linear or covalently closed circular form.
  • sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturally occurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a Helicobacter antigen as broadly described above.
  • the nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences usually being derived from a heterologous source.
  • This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter sequences and initiator sequences and a nucleotide sequence which codes for a Helicobacter antigen, the nucleotide sequence being located 3' to the promoter and initiator sequences.
  • the invention provides a recombinant DNA cloning vehicle capable of expressing a Helicobacter antigen comprising an expression control sequence having promoter sequences and initiator sequences, and a nucleotide sequence which codes for a Helicobacter antigen, the nucleotide sequence being located 3' to the promoter and initiator sequences.
  • Cloning vehicles can be any plasmid (or vector) known in the art, including viral vectors, such as alphavirus pox viruses.
  • a host cell containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.
  • Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989).
  • One embodiment of this invention relates to expression systems that employ the use of plasmids.
  • Preferred embodiments of the invention employ plasmids which exhibit high copy number upon replication in a transformed host cell.
  • Copy number refers to number of copies of the plasmid, or the genes contained therein, which replicate in a host cell upon induction of the plasmid.
  • the copy number within a cell determines the copies of the desired nucleotide sequence (or gene). This copy number equates to the gene dosage.
  • Certain plasmids are characterized as high copy number.
  • high copy number refers to a plasmid which generates at least about 100 copies per cell and preferably generating from about 100 to about 700 or 1,000 copies per cell.
  • the selection of the appropriate copy number for the expression of the nucleotides sequences of this invention when combined with the selection of a strong promoter improves the amount of desired polypeptide that is generated in a host cell.
  • the nucleic acid sequence encoding a selected polypeptide is operably linked to a strong promoter (Hannig et al.). Plasmid copy number is determined by the nature the origin of replication of the plasmid and corresponding cis acting control elements, together these genetic elements are defined as a replicon.
  • Plasmids which carry these replicons maintain about 15-20 copies per cell under normal growth conditions.
  • a small protein encoded by the rop gene adjacent to the origin of replication negatively regulates pMBl/ColEl plasmid replication.
  • Plasmids with deletions in the rop gene or mutations in the site of rop action have increased replication and higher copy number.
  • One example of this is in the pUC plasmids whose copy number is increased to 500-700 copies per cell.
  • Other lower copy number replicons are pi 5 A (10-12 copies/cell), and pSClOl (5-10 copies per cell).
  • the moderate copy number of pMBl and ColEl plasmids can be modified to high copy numbers by inhibiting the E. coli protein synthesis.
  • the copy number exhibited in a specific host is the proper measurement of plasmid copy number.
  • Plasmids which normally do not reside in gram negative hosts or E. coli hosts may not be regulated for replication when introduced into E. coli hosts. When such promoters are not regulated, they can exhibit high copy number in the selected host.
  • An example of this is pNG2, a plasmid from the gram positive bacteria Corynebacterium diphtheriae.
  • the normal copy number for this plasmid in C. dipththeriae is 1-2 copies per cell
  • transformants of the plasmid in E. coli are estimated to have >100 copies of the plasmid (Serwold-Davis, T.et al,PNAS, 84: 4964-8, 1987).
  • Strong promoters are selected such they are easily regulated in order that they may be repressed during culture growth "towards maximal cell numbers.
  • the strong promoters can also be induced so that the host cell overproduces the recombinant polypeptide at a desired level, usually in excess of about 10 to about 30% total cellular protein.
  • the promoter is selected to maintain the desired level of polypeptide expression.
  • Exemplary promoters are T7 promoter from T7 bacteriophage, arabinose promoter for the araBAD operon, lambda phage promoters (such as PL and PR) he trc and tac promoters.
  • the T7 promoter is controlled by the T7 RNA polymerase gene which is a very active enzyme; it elongates RNA chains five times faster than the E. coli RNA polymerase.
  • the polymerase is very selective for specific promoter sequences and termination signals so that the action of the enzyme is targeted specifically to the gene of interest.
  • the T7 polymerase gene has been integrated into the host chromosome under the control of the lac promoter. Upon induction with IPTG, T7 RNA polymerase is produced and acts to transcribe a T7 promoter gene housed on an expression plasmid in the cell.
  • a second plasmid expressing T7 lysozyme is included to control the background expression of the T7 polymerase.
  • an alternative induction system for the expression of genes by the T7 promoter is to grow E. coli cells containing the T7 recombinant plasmid to the desired density and then introduce the T7 polymerase gene by infecting with a bacteriophage which carries that gene.
  • Expression can also be enhanced based on the choice of cellular compartmentalization. Outer membrane proteins are often difficult to overexpress recombinantly because of the requirement for transport to the outer membrane and correct insertion into that membrane. Overexpression of full length outer membrane proteins which contain the leader sequences will many times overcome the host cell export machinery leading to cessation of growth and low recombinant protein yield. Cloning the mature sequences of an outer membrane protein behind translation start signals provided by the expression vector can eliminate the need for the host cell to transport the protein to the membrane and allow the cell to overexpress the recombinant protein as inclusion bodies in the cytosol which are relatively stable and resistant to proteolysis.
  • One or more of the above considerations are included when selecting a desired host.
  • factors include growth and induction conditions, mRNA stability, codon usage, translational efficiency and the presence of transcriptional terminators to minimize promoter read through.
  • the polypeptides of the invention contain cysteine bonds. Accordingly, one can aid cysteine bond formation, for example through the use of trx hosts, which can help with the proper folding in the
  • Suitable host cell or host strains for the practice of this invention are omp Tlon host strains such as BL21, BLR, B834; trx ⁇ host strains such as AD494, Ion clpA mutant strains such as KY 42263 and Ion clpA hslVU mutant strains such as KY 2266 (for the latter two strains the Kanemori, M. et al. J. Bact. 1997. 79:7219-7225).
  • the polypeptide is expressed as inclusion bodies.
  • the nucleotide sequence selected for forming inclusion bodies is the nucleotide sequence corresponding to the mature portion of the polypeptide.
  • the signal sequence of the desired polypeptide is not included in this nucleotide sequence for the mature polypeptide.
  • pylori protein can be obtained by a method comprising: (a) transforming a selected host cell with at least one high copy number plasmid, which comprises the nucleotide sequence of interest operably linked to a strong promoter, and (b) growing the transformed host cell in culture media.
  • an inducible promoter to control the timing of the expression of the nucleotide sequence. Basically, when the promoter is inducible, the rate of transcription increases in response to the inducing agent or inducing conditions for the promoter.
  • a selectable marker can used in the plasmid in order to grow the transformed host in the presence of a selecting agent that works in combination with the chosen marker. The H.
  • pylori polypeptide produced by the above method is expressed as inclusion bodies and is also soluble in detergent extractions, without the requiring denaturants, such as urea or guanidine for solubilizing the inclusion bodies.
  • denaturants such as urea or guanidine for solubilizing the inclusion bodies.
  • fused polypeptides comprising a Helicobacter polypeptide of this invention and an additional polypeptide, for example a polypeptide coded for by the DNA of a cloning vehicle, fused thereto.
  • fused polypeptide can be produced by a host cell transformed or infected with a recombinant DNA cloning vehicle as described above and it can be subsequently isolated from the host cell to provide the fused polypeptide substantially free of other host cell proteins.
  • nucleic acid sequences encoding the polypeptides of this invention due to the degeneracy of the genetic code.
  • Amino acids and their codons are well- known. Accordingly, using site-directed mutagenesis of one polypeptide of H. pylori, one can generate additional nucleic acid sequences, as desired. These methods of generating nucleic acid sequences and fragments thereof provide a convenient manner in which to generate portions of the polypeptides for fusion molecules.
  • polypeptides displaying the antigenicity of & Helicobacter isolated polypeptide of this invention.
  • synthetic means that the polypeptides have been produced by chemical or biological means, such as by means of chemical synthesis or by recombinant DNA techniques leading to biological synthesis.
  • Such polypeptides can, of course, be obtained by cleavage of a fused polypeptide as described above and separation of the desired polypeptide from the additional polypeptide coded for by the DNA of the cloning vehicle by methods well known in the art.
  • the polypeptide may be produced synthetically, for example by the well known Merrifield solid-phase synthesis procedure.
  • Helicobacter polypeptide of this invention have been constructed by transforming or transfecting such cloning vehicles or host cells with plasmids containing the corresponding Helicobacter nucleic acid sequence, cloning vehicles or host cells are cultured under conditions such that the polypeptides are expressed. The polypeptide is then isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art. In a preferred embodiment for purifying the desired polypeptide one can first follow the standard techniques of lysing the host cells and then isolating the inclusion bodies by removing soluble proteins and other contaminants potentially. In a second step, it is preferred to solubilize the inclusion bodies in a zwitterionic detergent, which are well-known in the art.
  • the detergent may be used with a denaturant; however, surprisingly, no denaturant is required.
  • the solubilized inclusion body material is purified using cationic exchange gel chromatography, followed by eluting with a salt solution to collect the purified polypeptide. This method generates a polypeptide which is at least about 75 or 80% pure, preferably at least about 90%.
  • This invention also provides for a method of diagnosing an H pylori infection comprising the step of determining the presence, in a sample, of an amino acid sequence EDDGFYTSVGYQIGEAAQMV (SEQ ID No.: 7), or preferably any of the isolated H pylori polypeptides of this invention.
  • Any conventional diagnostic method may be used. These diagnostic methods can easily be based on the presence of an amino acid sequence or polypeptide.
  • such a diagnostic method matches for a polypeptide having at least 10, and preferably at least 20, amino acids which are common to the polypeptides of this invention.
  • the present invention also relates to nucleic acid sequences encoding H pylori polypeptides.
  • the nucleic acid sequences disclosed herein can also be used for a variety of diagnostic applications. These nucleic acids sequences can be used to prepare relatively short DNA and RNA sequences that have the ability to specifically hybridize to the nucleic acid sequences encoding the polypeptides of this invention.
  • Nucleic acid probes are selected for the desired length in view of the selected parameters of specificity of the diagnostic assay. The probes can be used in diagnostic assays for detecting the presence of pathogenic organisms in a given sample.
  • nucleic acid sequences can be inserted into an expression construct for the purpose of screening the corresponding oligopeptides and polypeptides for reactivity with existing antibodies or for the ability to generate diagnostic or therapeutic reagents.
  • the nucleic acid sequences employed for hybridization studies or assays include sequences that are complementary to a nucleotide stretch of at least about 10 to about 20 nucleotides, although at least about 10 to 30, or about 30 to 60 nucleotides can be used. Nucleotide stretches of at least 10 nucleotides are beneficial for providing stability and selectivity when testing a clinical sample for Helicobacter infection.
  • a variety of known hybridization techniques and systems can be employed for practice of the hybridization aspects of this invention, including diagnostic assays such as those described in Falkow et al, US Patent 4,358,535. Depending on the application, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward a target sequence.
  • hybridization conditions For some applications, if one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent hybridization conditions are called for in order to allow formation of the heteroduplex.
  • the conditions may be altered by using 0.15M-0.9M salt, at temperatures ranging from about 20°C to about 55°C.
  • conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature.
  • hybridization conditions can be readily manipulated, and the method of choice will generally depend on the desired results.
  • mutant clone colonies growing on solid media which contain variants of an H. pylori polypeptide sequence could be identified on duplicate filters using hybridization conditions and methods, such as those used in colony blot assays, to obtain hybridization only between probes containing sequence variants and nucleic acid sequence variants contained in specific colonies. In this manner, small hybridization probes containing short variant sequences of the H.
  • pylori genes of the invention may be utilized to identify those clones growing on solid media which contain sequence variants of the entire genes encoding polypeptides of 75, 77 and 79 kDa as discussed herein. These clones can then be grown to obtain desired quantities of the variant nucleic acid sequences or the corresponding antigen.
  • nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase.
  • the test DNA (or RNA) from suspected clinical samples such as exudates, body fluids (e.g., amniotic fluid, middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is absorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, et.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
  • the nucleic acid sequences which encode for the H pylori polypeptides of the invention, or their variants, may be useful in conjunction with PCRTM technology as set out, e.g., in U.S. Patent 4,603,102, one may utilize various portions of any of H.
  • pylori sequences of this invention as oligonucleotide probes for the PCRTM amplification of a defined portion of an H. pylori (75, 77 kDa) sequence may then be detected by hybridization with a hybridization probe containing a complementary sequence. In this manner, extremely small concentrations of//, pylori nucleic acid may be detected in a sample utilizing the nucleic acid sequences of this invention.
  • H. pylori strains PBCC 1 101, PBCC 1102, PBCC 1103, PBCC 1105, and PBCC 1107 were isolated from human gastric biopsies obtained from the University of Rochester School of Medicine and Dentistry (Rochester, NY).
  • H pylori strains LET 13 and RSD 14 were isolated from human gastric biopsies obtained from the Syracuse Veterans Administration Medical Center (Syracuse, NY).
  • H. pylori strains MH 60 EG 52, RJ 17, LJ 63, and MJ 34 were obtained as frozen stocks from the Clement J. Zablocki VA Medical Center (Milwaukee, WI).
  • H. pylori strain SSI was originally isolated from a human gastric biopsy, and subsequently adapted to infect mice (obtained from A. Lee, University of New South Wales, Sydney, Australia).
  • H. pylori strains obtained from American Type Culture Collection were ATCC 43504 and ATCC 43579.
  • the H.felis strain was obtained from T. Blanchard, Case Western Reserve University, Cleveland, OH. 1.2 Culturing of Helicobacter strains. Cultures of H. pylori and Hfeli were grown at 37°C on Columbia broth agar plates with 10% defibrinated horse blood and 10 ⁇ g/ml vancomycin in a microaerophilic chamber.
  • Liquid cultures of// pylori strains were grown at 37°C in BHI medium with 4% fetal calf serum and 10 ⁇ g /ml vancomycin in flasks infused with a gas mixture of 10% C0 2 / 6% 0 2 / 84% N 2 (vol/vol/vol).
  • Strain PBCC 1 103 was grown as above. The cell pellet was washed twice in a phosphate buffered saline (PBS) solution, and resuspended in a 0.3% formaldehyde solution for 1 hr. The fixed cells were then washed in and resuspended in PBS to an O.D. o00 of 0.1 (approximately 10 cfu).
  • PBS phosphate buffered saline
  • mice Ten 6 to 8 wk old female ⁇ BALB/c mice were primed by interperitoneal injection with ca. 10 formalin fixed PBCC 1103 cells at weeks 0, and boosted at weeks 2, 4 and 8. After a 34 week rest ⁇ period a pre- fusion boost of approximately 10 formalin fixed PBCC 1103 was given interperioneally at week 42.
  • mice sera were obtained (week 6, 10) and tested for antibody activity by ELISA by using air-dried, formalin fixed PBCC 1103 cells, 0.1 ml per well at an absorbance of 0.100 (A 62 o) as the coating antigen.
  • Spleens were recovered from five immunized mice about 72 hours after the last injection, and were combined with nonsecreting X63Ag8.653 mouse (BALB/c) myeloma cells in 5:1 ratio (splenocytes:myeloma).
  • the cells were fused for four minutes in 50% (v/v) polyethylene glycol 1500 and 10% dimethylsulfoxide in Dulbecco's Modified Eagle medium (D-MEM).
  • D-MEM Dulbecco's Modified Eagle medium
  • the fused cells were diluted in selection medium, D-MEM supplemented with hypoxanthine, aminopterin, thymidine, 10% fetal bovine serum and 10% NCTC-109 media supplement (Gibco-BRL).
  • the fusion efficiency (wells with colony growth vs. number of wells seeded) was 100% (900/900).
  • Monoclonal antibodies were provided as tissue culture supernatant (TCS), concentrated by 50% saturated ammonium sulfate precipitation (SAS-TCS) or ascites.
  • Membranes were probed with the indicated antisera followed by goat anti-mouse or anti-rabbit alkaline phosphatase conjugate as the secondary antibody (BioSource International, Camarillo, CA). Western blots were developed using the BCIP/ ⁇ BT Phosphatase Substrate System from Kirkegaard and Perry Laboratories (Gaithersburg, MD).
  • Tissue culture supernatants from hybridomas were analyzed by whole cell ELISA and antigen ELISA, followed by Western analysis using whole cell lysates of a number of H. pylori strains.
  • Parent hybridoma designated ⁇ py 64 was determined by these methods to react to proteins in the range of approximately 75-79 kDa.
  • Subsequent cloning produced the monoclonal antibody (MAb) designated ⁇ py 64-27, which has reactive epitopes in 10 of 12 H. pylori strains tested, but not in H. fells (Table 1).
  • Bacterial cells (ca. 10 g wet wt of H pylori ATCC 43579) were resuspended in 70 ml of 0.05 M ⁇ EPES / 10 mM EDTA / 1.0 mM PMSF (p ⁇ 7.0) by homogenization using a Tekmar Ultra- Turrex tissue homogenizer. The cells were disrupted by sonication using a Branson Sonifier Cell Disrupter. The disrupted cells, including the membrane fraction, were pelleted by centrifugation at 42,000 rpm using a Beckman 70Ti rotor for 40 min at 4°C.
  • the pellet was resuspended in 70 ml of 0.01 M ⁇ EPES / 1.0 mM MgCl 2 / 1.0 mM PMSF / 1.0% TX-100 (p ⁇ 7.4) and stirred for 1 hr at room temp.
  • the suspension was centrifuged at 42,000 rpm using a Beckman 70Ti rotor for 40 min at 4°C.
  • the pellet was resuspended in 70 ml of 0.05 M Tris- ⁇ Cl / 10.0 mM EDTA / 1.0% ZWITTERGENTTM3-14 (p ⁇ 7.4) and stirred for 1 hr at room temp.
  • the suspension was then centrifuged at 42,000 rpm using a Beckman 70Ti rotor for 40 min at 4°C. Following centrifugation, the supernatant containing the 75 and 77 kDa proteins was collected and stored at -20°C for further purification.
  • the ZWITTERGENTTM 3-14 crude extract was buffer exchanged by passage over a 250 ml Sephadex G-25 (coarse) column (Pharmacia) equilibrated in 0.02 M Tris- HC1 / 5.0 mM EDTA / 1.0% ZWITTERGENTTM3-14 (pH 8.0).
  • Fractions were screened for the 75 and 77 kDa proteins by SDS-PAGE / Western and pooled. Twelve ml of pooled co-eluting 75 and 77 kDa was subsequently applied to a 500 ml Pharmacia Superose-12 column equilibrated in phosphate buffered saline (PBS) / 1.0% ZWITTERGENTTM 3-14. Fractions were screened for the co-eluting 75 and 77 kDa proteins by SDS-PAGE / Western and pooled. Material from two independent SP Sepharose / Superose-12 runs was combined and the co-eluted protein was precipitated by the addition of 9 volumes of ethanol overnight at -20°C.
  • PBS phosphate buffered saline
  • Protein concentration was estimated by the BCA assay from Pierce (Rockford, IL) using BSA as a standard.
  • Grids were then incubated on a fifty-fold dilution of Nanogold (Nanoprobes, Inc, Stony Brook, NY), washed in buffer, and fixed with a solution of 1% gluteraldehyde in PBS. The fixative was removed from the grids with deionized water.
  • the HQ silver enhancement kit (Nanoprobes, Inc.) was used to nucleate the Nanogold.
  • the grids were then stained with Nanovan (Nanoprobes, Inc.) and viewed on a Zeiss 10C transmission electron microscope operating at 100 Kv.
  • the polyclonal antiserum obtained from injecting mice with live strain PBCC 1103 was able to label the surface of heterologous strain PBCC 1105. As seen in Figure 2, the 75/77 kDa proteins are surface localized and that the reactive epitopes are present in both H pylori strains.
  • H. pylori strain PBCC 1 105 for the BC assay were provided on the morning of the assay.
  • the optical density of the liquid culture at 600 nm was approximately 0.1.
  • Cells pelleted from 10 ml of this liquid culture were used to adsorb any nonspecific bactericidal activity in the complement source (human complement).
  • Cells and serum were allowed to react for at least 1 hour on ice with occasional agitation. After incubation, the serum and cell mixture was centrifuged, and the serum was removed and placed on ice until needed.
  • the BC assay was performed when the liquid culture attained an O.D. 6 o 0 of approximately 0.3. These cells were diluted in PCM buffer to a concentration of 10 cfu per ml .
  • the reaction consisted of 10 ⁇ l strain PBCC 1105 at a concentration of 10 cfu per ml, 10 ⁇ l adsorbed serum as a complement source, 5 ⁇ l of diluted polyclonal antisera (heat inactivated 60°C for 10 min), and 25 ⁇ l of PCM.
  • the reaction was incubated at 36°C in a 10% C0 chamber for 30 min. Two hundred ⁇ l of PCM was then added to the reaction and duplicate 50 ⁇ l aliquots were plated on Columbia agar with 10% defibrinated horse blood and 10 ⁇ g/ml vancomycin. Plates were incubated in a microaerophilic chamber for at least 72 hours, and counted on an automated plate reader.
  • Hp H. pylori
  • CT cholera toxin
  • the challenge inocula contained a suspension of/ pylori at a concentration of 2 x 10 cfu per ml.
  • the amount of viable H pylori in the stomachs of mice was determined 2, 4 and 8 weeks following challenge (days 50 and 64).
  • 5 mice were sacrificed by cervical dislocation, their stomachs harvested and split into 2 longitudinal sections.
  • One section from each mouse was homogenized and the homogenate diluted and plated on Columbia agar containing defibrinated horse serum and the appropriate antibiotics. Plates were incubated at 37 C in a microaerophilic incubator for 5 days.
  • the peptides were eluted with a linear gradient from 0-100% Solvent B in 30 min and detected by absorbance at 220 nm. Suitable fractions were collected, dried down in a Speed-Vac concentrator (Jouan Inc, Winchester, VA) and subsequently resuspended in distilled water. The fractions were subjected to SDS-PAGE using 10-18% (w/v, acrylamide) gradient gels (Owl Separation Systems) in a Tris-Tricine buffer system (Sch ⁇ gger and von Jagow). The fractions exhibiting a single peptide band were submitted for N-terminal sequence analysis.
  • MALDI-TOF analysis using 3,5-dimethoxy-4-hydroxy- cinnamic acid matrix in presence of 70% (v/v) aqueous acetonitrile / 0.1% TFA resulted in the identification of predominantly two species with average molecular weights of 75,572 kDa and 77,633 kDa.
  • N-terminal Sequence Analysis 6.1 Amino Acid Sequence Analysis. N-terminal sequence analysis was carried out using an Applied Biosystems Model 477A Protein/Peptide Sequencer equipped with an on-line Model 120A PTH Analyzer (Applied Biosystems, Foster City, CA). The phenylthiohydantoin (PTH) derivatives were identified by reversed-phase HPLC using a Brownlee PTH C-18 column (particle size 5 mm, 2.1 mm i.d. x 22 cm 1.; Applied Biosystems).
  • PTH phenylthiohydantoin
  • Example 7 Identification of the 75 kDa, 77 kDa and 79 kDa Genes in the Chromosome of H. pylori.
  • DNA sequencing was obtained by asymmetric PCR amplification using the fluorescent dye-labeled dideoxynucleotide terminator method (Gyllensten et al, 1988). The dye-labeled single stranded DNA fragments were separated and identified with an Applied Biosystem model 373 A automatic sequencing apparatus. Primary sequence information was analyzed using MacVector DNA analysis program (IBI, New Haven CT).
  • PCR amplifications were performed in 500 ⁇ l tubes containing 100 ⁇ l reaction volume overlaid with mineral oil. Each reaction contained 10 mM Tris-HCL, pH 8.3, 50 mM KC1 and 1.5 mM MgCl 2 ; 2.5 units Taq polymerase (Boehringer Mannheim), 200 mM dNTPs; 20 mM oligonucleotide primers and 1-2 ⁇ g chromosomal DNA. Templates were amplified for 30 cycles with a 1 second extension on the annealing time of each cycle in a Perkin-Elmer Cetus thermocycler.
  • DNA alignment searches were performed using the MacVector program (IBI, New Haven CT). Protein homology searches were performed using either MacVector or DNASTAR (DNASTAR, Inc.) clustal alignment programs.
  • the H. pylori genome (unannotated) was downloaded from TIGR (The Institute for Genomic Research) ftp file and stored as a MacVector file.
  • the N-terminal amino acid sequence of the 77 kDa H. pylori protein was entered into MacVector program and reverse translated to give the degenerate DNA oligonucleotide which was aligned to the genome.
  • pylori genome had the following sequence: 5'GARGAYGAYGGNTTYTAYACNWSNGTNGGNTAYCARATHGGNGAR GCNGCNCARATGGTN 3' [SEQ. ID NO. 33]. Three matches were observed, corresponding to nucleotide positions 1404865-1404807, 722946-722888 and 352616-352558, all on the negative strand of the genome. MacVector was used to outline open reading frames which would overlap the above regions in the genomic DNA. Three open reading frames based on translational start and stop codons were found. An open reading frame between nucleotides 1402991-1404925 (negative strand) predicted a protein of 81 , 157 kDa.
  • the three proteins have identical 20 amino acids at the N-terminus and share internal peptide residues empirically determined from purified protein preparations (compare SEQ ID Nos. 1, 2, 3 and 7).
  • Example 8 8.1 PCR Amplification and Cloning of the 77 kDa Gene.
  • PCR primers were synthesized which hybridized to the translational start and stop codons of the ORF.
  • the PCR primers synthesized to amplify the 77 kDa gene from the chromosome of//, pylori had the following sequences: Forward Primer: 5' GGC CAT ATG AAA AAA CAC ATC CTT TCA TTA GCT TTA GGC TCG 3' (SEQ ID No.: 8) and Reverse Primer: 5' GGC AAG CTT GGG AGT TTC ACA AAA AGC TTA GTA AGC GAA CAC 3 ' (SEQ ID No.: 9).
  • E. coli strain BLR(DE3) pLysS was obtained from Novogen.
  • E. coli strains TOP 10 and Topi OF' were obtained from Invitrogen.
  • E. coli strains were grown in HYSOY buffer containing 1% glucose or glycerol. Amplicilin (100 ⁇ g/mL) and chloramphenicol (30 ⁇ g/mL) were added when appropriate.
  • a primer corresponding to the 75 kDa gene start region could not be designed because a stretch of 11 CT dinucleotide repeats exists 10 bp downstream from the ATG start codon. CT dinucleotide repeats are also found in other H pylori genes, and therefore a primer containing the repeats would not be specific for the 75 kDa gene.
  • DNA primers were designed based on the predicted sequences in and surrounding the 75 kDa gene sequence as published by TIGR. A primer was designed to anneal upstream from the 75 kDa coding region (Table 3, primer C).
  • the mature gene fragment was PCR amplified using primer J and K (Table 3) with pPX7768 as template DNA.
  • the fragment was cloned into pRSETb at the NdelmECoRV sites to produce plasmid pPX7811.
  • This plasmid was transformed into the expression strain BLR(DE3)pLysS, selecting for ampicilin and chloroamphenicol resistance.
  • the strain was grown to mig-logarithmic phase in HYSOY broth( Difco) containing ampicilin and chloroamphenicol and expression was induced by the addition of ImM isopropanol beta-D-thiogalactopyranoside (IPTG).
  • Bacterial cells (ca. 15 g wet wt of E. coli BLR(DE3)pLysS / pPX781 1) were resuspended in 75 ml of 10 mM NaP04 / 150 mM NaCl / 5.0 mM EDTA / 1.0 mM Pefabloc (pH 7.2) by homogenization using a Ultra-Turrex tissue homogenizer (IKA Works, Wilmington, NC). The cells were disrupted by sonication using a Branson Sonifier Cell Disrupter.
  • Inclusion bodies containing the recombinant 75 kDa protein were isolated by centrifugation at 10,000 rpm using a Sorvall SLA- 1500 rotor for 30 min at 10°C. Following centrifugation, the pellet was resuspended by homogenization in 75 ml of 10 mM NaP04 / 150 mM NaCl / 5.0 mM EDTA / 4.0% TX-100 (pH 7.2) and stirred for 1.5 hr at 4°C. The suspension was centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at 10°C.
  • the pellet was resuspended by homogenization in 75 ml of 10 mM NaP ⁇ 4 / 150 mM NaCl / 5.0 mM EDTA / 1.0% Zwittergent 3-14 (Z 3-14) (pH 7.2) and stirred for 1.5 hr at room temp.
  • the suspension was centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at 10°C.
  • the pellet was resuspended by homogenization in 75 ml of 10 mM NaP04 / 150 mM NaCl / 5.0 mM EDTA / 1.0% Zwittergent 3-16 (Z 3-16) (pH 7.2) and stirred for 2 hr at room temp.
  • the suspension was centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at 10°C.
  • the Z 3-14 and Z 3-16 supernatants containing the recombinant 75 kDa protein were collected and stored at -20°C for further purification.
  • the Z 3-14 extract was buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column (Pharmacia Biotech Inc, Piscataway, NJ) equilibrated in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3- 14 (pH 8.0).
  • the buffer exchanged Z 3-14 crude extract preparation was loaded onto a 10 ml SP-Sepharose Fast Flow column (Pharmacia Biotech Inc.) equilibrated with 0.02 M Tris- HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0). Unbound protein was washed through the column with an additional 2 column volumes of equilibration buffer.
  • the recombinant 75 kDa protein was eluted using a linear NaCl gradient (0-0.5 M NaCl) in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 7.0) over 8 column volumes. Fractions were screened for recombinant 75 kDa protein by SDS-PAGE and Western analysis and pooled. Pooled fractions were dialyzed into PBS / 1.0% Z 3- 14 overnight at 4°C. The Z 3- 16 extract was processed as described for the Z 3-14 extract.
  • DNA sequences of the 77 kDa genes from strains ATCC 43504, PBCC 1103 and PBCC 1105 were only partial but showed more homology to the 77 kDa gene from TIGR than to the 75 kDa or 79 kDa genes.
  • the 77 kDa gene from strain ATCC 43579 was completely sequenced (plasmid pPX7760b).
  • the DNA sequence of the complete coding region is shown in SEQ ID NO. 5.
  • the predicted protein translation from this sequence is shown in SEQ ID NO. 2.
  • the 77 kDa gene was cloned into expression vector pRSETb which directs expression of the foreign protein by the T7 promoter.
  • the mature gene fragment was PCR amplified using primers J and K (Table 3) with pPX7760b as template DNA.
  • the band was cloned into PCR2.1 (pPX7792).
  • pPX7792 was cleaved with Ndel and EcoRV, and the 2 kb gene fragment was ligated into pRSETb cut with the same enzymes.
  • the resulting plasmid was transformed into the expression strain BLR(DE3)pLysS, selecting for ampicillin and chloramphenicol resistance.
  • the strain was grown to mid-logarithmic phase in HYSOY broth (Difco, Detroit, Mich.) containing ampicillin and chloramphenicol, and expression was induced by the addition of 1 mM IPTG. Expression proceeded for 2 hours, after which the cells were harvested by centrifugation. Whole cells lysates were run on 10-12% SDS-PAGE. The 77 kDa protein band was overexpressed as determined by coomassie stained gels, and reacted with polyclonal serum raised against the native protein.
  • Bacterial cells (ca. 32 g wet wt of E. coli BLR(DE3)pLysS (pPX7796) were resuspended in 150 ml of 10 mM NaP04 / 150 mM NaCl / 5.0 mM EDTA / 1.0 mM Pefabloc (pH 7.2) by homogenization using a Ultra-Turrex tissue homogenizer. The cells were disrupted using a Microfluidizer Model 110Y.
  • Inclusion bodies containing the recombinant 77 kDa protein were isolated by centrifugation at 10,000 rpm using a Sorvall SLA-1500 rotor for 30 min at 10°C. Following centrifugation, the pellet was resuspended by homogenization in 150 ml of lOmM NaPO 4 / 150mM NaCl / 5.0 mM EDTA / 1.0% TX-100 (pH 7.2) and stirred for 2 hr at 4°C. The suspension was centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at 10°C.
  • the pellet was resuspended by homogenization in 75 ml of lOmM NaP ⁇ 4 / 150mM NaCl / 5.0 mM EDTA / 1.0% Z 3-16 (pH 7.2) and stirred overnight at room temp.
  • the suspension was centrifuged at 10,000 m using a Sorvall SS- 34 rotor for 30 min at 10°C.
  • the supernatant containing the recombinant 77 kDa protein was collected and stored at -20°C for further purification.
  • the Z 3-16 extract was buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column equilibrated in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0).
  • the buffer exchanged Z 3-16 crude extract preparation was loaded onto a 10 ml SP-Sepharose Fast Flow column equilibrated 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0). Unbound protein was washed through the column with an additional 2 column volumes of equilibration buffer.
  • the recombinant 77 kDa protein was eluted using a linear NaCl gradient (0-0.5 M NaCl) in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 7.0) over 8 column volumes. Fractions were screened for recombinant 77 kDa protein by SDS-PAGE and Western analysis and pooled. Pooled fractions were dialyzed into PBS / 1.0% Z 3-14 overnight at 4°C.
  • Example 11 11.1 Cloning and expression of the 79 kDa gene from H. pylori strain PBCC 1107
  • the 79 kDa gene was cloned form 3 strains of H. pylori , PBCC 1 107, Let 13, and SSI .
  • Primers J and K (Table 3) were used to amplify the mature coding region for cloning into pRSETb following the same protocol as for the 75 and 77 kDa constructs.
  • the recombinant plasmid pPX5048 directed the overexpression of the 79 kDa protein.
  • the recombinant protein was easily detected in coomassie stained gels and reacted with both polyclonal sera raised against native 75, and 77 kDa genes (due to its homology) and with peptide-conjugate antisera directed against a sequence unique to the 79 kDa protein (described below).
  • Bacterial cells (ca. 15 g wet wt of E. coli BL21(DE3)pLysS / pPX5048) are resuspended in 75 ml of 10 mM NaP04 / 150 mM NaCl / 5.0 mM EDTA / 1.0 mM Pefabloc (pH 7.2) by homogenization using a Ultra-Turrex tissue homogenizer. The cells are disrupted by sonication using a Branson Sonifier Cell Disrupter. Inclusion bodies containing the recombinant 79 kDa protein are isolated by centrifugation at 10,000 m using a Sorvall SS- 31 rotor for 30 min at 10°C.
  • the pellet is resuspended by homogenization in 75 ml of lOmM NaP04 / 150mM NaCl / 5.0 mM EDTA / 4.0% TX-100 (pH 7.2) and stirred for 1.5 hr at 4°C.
  • the suspension is centrifuged at 10,000 ⁇ m using a Sorvall SS-31 rotor for 30 min at 10°C.
  • the pellet is resuspended by homogenization in 75 ml of lOmM NaP ⁇ 4 / 150mM NaCl / 5.0 mM EDTA / 1.0% Z 3- 14 (pH 7.2) and stirred for 1.5 hr at room temp.
  • the suspension is centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at 10°C. Following centrifugation, the pellet is resuspended by homogenization in 75 ml of 10 mM NaP ⁇ 4 / 150 mM NaCl / 5.0 mM EDTA / 1.0% Z 3-16 (pH 7.2) and stirred for 2 hr at room temp.
  • the suspension is centrifuged at 10,000 ⁇ m using a Sorvall SS-31 rotor for 30 min at 10°C. Following centrifugation, the Z 3-14 and Z 3-16 supernatants containing the recombinant 79 kDa protein are collected and stored at -20°C for further purification.
  • the Z 3-14 extract is buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column equilibrated in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0).
  • the buffer exchanged Z 3-14 crude extract preparation is loaded onto a 10 ml SP-Sepharose Fast Flow column equilibrated 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0). Unbound protein is washed through the column with an additional 2 column volumes of equilibration buffer.
  • the recombinant 79 kDa protein is eluted using a linear NaCl gradient (0-0.5 M NaCl) in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 7.0) over 8 column volumes. Fractions are screened for recombinant 79 kDa protein by SDS-PAGE and Western analysis and pooled. Pooled fractions are dialyzed into PBS / 1.0% Z 3-14 overnight at 4°C. The Z 3-16 extract is processed as described for the Z 3-14 extract.
  • the DNA sequence of the 75 kDa gene from strain ATCC 43579 corresponding to the mature protein is shown in Figure 6, SEQ ID NO. 21.
  • the open reading frame is 2070 bp long, the same as the TIGR predicted protein.
  • the translated protein is 689 aas long containing 6 cysteine residues with an estimated pi of 8.88 ( Figure 7, SEQ ID NO. 19). Alignment of the 75 kDa proteins from TIGR strain 26695 and ATCC 43579 reveals that 26 amino acid residues are not conserved.
  • the DNA sequence of the 77 kDa gene from strain ATCC 43579 is shown in SEQ ID NO. 5.
  • the open reading frame corresponding to the mature protein is 2166 bp long, the same as the TIGR predicted protein.
  • the translated protein is 721 aas long containing 6 cysteine residues with an estimated pi of 8.89 (SEQ ID NO. 2).
  • Alignment of the 77 kDa proteins from TIGR strain 26695 and ATCC 43579 reveals substitutions in 67 aas.
  • An additional 3 aas are unique to the TIGR protein, and an additional 11 aas are unique to the ATCC 43579 sequence.
  • the DNA sequence of the 79 kDa gene from strain PBCC 1107 is shown in Figure 8, SEQ ID NO. 22.
  • the open reading frame corresponding to the mature protein is 2157 bp long, 21 bp shorter than the TIGR predicted protein.
  • the translated protein from strain PBCC 1107 is 718 aas long containing 8 cysteine residues with an estimated pi of 6.99 which is considerably less than the pi of 8.68 predicted by the TIGR sequence ( Figure 9, SEQ ID NO. 20).
  • Alignment of the 79 kDa proteins from the TIGR strain 26695 and PBCC 1107 revealed 99 amino acid residue changes. There are 6 aas unique to the TIGR 79 kDa sequence, and 1 aa unique to the PBCC 1107 79 kDa sequence.
  • a third construct cloned the mature 77K gene to an ompT leader in pET12b which could enhance the translation and signal processing of the mature 77K gene (pPX7782). None of the constructs containing the gene with a signal sequence were overexpressed in excess of 1% total cellular protein (Table 4).
  • Primers was made corresponding to the start of the mature protein and the end of the gene (Table 3, primers D and E).
  • DNA sequence corresponding to the mature protein was PCR amplified using the pPX7760b as template DNA and cloned into PCR2.1 (pPX7769).
  • the gene was cleaved from pPX7769 by digestion with Asp718 and Spel and inserted into the T7 promoted pET17b (Novagen, Madison, Wisc.)at the Asp718 and Spel sites.
  • the resulting plasmid fused 18 amino acid residues from the vector to the mature 77 kDa gene.
  • Figure 11 shows that the 77K protein expressed from pE717b was not above 1% of the total cellular protein.
  • the pRSETb based clone, pPX7796 was able to overexpress the protein greater than 10-20% of total cellular protein.
  • Analysis of the differences between pET17b and pRSETb reveals that both vectors have identical promoter sequences. A significant difference between the two vectors is in the copy number.
  • pRSET b is based on the pUC replicon which maintains 100-700 copies per cell whereas pET vectors are based on a pMBl/ColEl replicon maintaining 25-30 copies per cell.
  • a strong promoter coupled with a relatively high gene dosage resulted in overproduction of the recombinant mature 77K protein.
  • Example -75K The mature gene (pPX7768) was amplified with primers D and E (Table 3) to obtain a fragment with appropriate restriction site ends for cloning into the arabinose expression vector pBAD24 (Guzman et al. 1995. J. Bacteriol. 177:4121- 4130), fusing the vector ATG start codon and two additional vector encoded amino acids to the mature gene (pPX7794).
  • the 75 kDa gene was expressed by this vector following induction with arabinose, but the level was approximately 1% of cellular protein and the recombinant protein was prone to proteolytic cleavage.
  • the mature coding region was cloned into the T7 polymerase expression system pRSETb
  • Example -79K The mature 79K gene from strain PBCC 1 107 was cloned into the arabinose vector pBAD24 fusing the vector ATG and 3 other amino acids to the start of the 75K gene (pPX5043, Table 4) and to the pRSET vector, fusing the vector ATG to the start of the mature 75K gene (pPX5048, Table). Comparison of recombinant protein yields after induction reveals that pPX5048 was superior in overexpressing the 79K mature protein ( Figure 12).
  • Plasmid Gene Vector Cell line Description pPX7762 full 77K pET17b (T7) BL21(DE3) pLysS full length 77K gene fused to vector ATG pPX7772 signal-77K pET17b (T7) BL21(DE3) pLysS mature 77K gene N terminal fusion to T7Tag pPX7779 full 77K pBAD24 (arabinose) TOP 10 full length 77K gene fused to vector ATG pPX7782 signal-77K pBAD24 (arabinose) TOP 10 mature 77K gene fused to vector ATG, 3 aas on N terminus
  • CRM197 is a well known carrier protein as described in Uchida, T. et al, 1971 Nature New Biology, Vol. 233, 8-1 1. All of the conjugates were characterized by SDS-PAGE and amino acid analysis. The soluble conjugates were characterized by MALDI-TOF mass spectrometry analysis. An animal study was performed using the peptide conjugates as the antigen in order to produce antisera that would specifically recognize either the 77 or 79 kDa protein of H. pylori by western blot for purification protocols.
  • TNBS 2,4,6-trinitrobenzenesulfonic acid
  • the resulting material was gently mixed overnight at 4°C in the dark followed by dialysis in the dark against two 1 L changes of PBS, pH 7.2. Each conjugate was then transferred into a sterile 15 ml polypropylene tube, wrapped in aluminum foil and stored at 4°C.
  • the experimental mol. wt. of activated CRMi 97 determined by MALDI-TOF mass spectrometry was 61,208 for activated CRMi 97 useo ⁇ f° r me 7 k peptide conjugates and 61,009 for activated CRMi 97 use d f° r the 79k peptide conjugates.
  • the degree of conjugation for each conjugate was calculated by subtracting the mass value of activated CRMi 97 from the mass value of each conjugate and dividing by the mass of the peptide used to prepare the conjugate.
  • the degree of conjugation for the soluble 77k and 79k peptide conjugates is described in the table 8. The mass values for heavily precipitated conjugates could not be determined.
  • peptide ELISAs were run using Nunc Maxisorp 96-well plates. Weeks 0 and 6 peptide conjugate antisera were all titered against homologous peptides. Plates were coated with 100 ⁇ l of peptides diluted to 1.0 ⁇ g/ml with 50 mM sodium bicarbonate (pH 9.0) and incubated at 37°C overnight. Plates were washed and blocked for 1 hour at 37°C with 250 ⁇ l of 3% BSA in 137 mM tris buffered saline (TBS), pH 7.6.
  • TBS tris buffered saline
  • Peptide conjugate antisera were serially diluted into 0.3% BSA in TBS containing 0.05%) Tween-20, 100 ⁇ l of each dilution was added to the plates and incubated at 37°C for 1 hour. The plates were washed and subsequently incubated for 1 hour at 37°C with 100 ⁇ l of alkaline phosphatase conjugated goat anti mouse secondary antibody (Zymed) diluted 1 : 1500 in 0.3% BSA in TBS containing 0.05% Tween-20.
  • C57BL/6 mice were vaccinated intragastrically with 100 ⁇ g native 75.77 kDa proteins admixed with 10 ⁇ g CT, 10 ⁇ g CT-E29H (an attenuated cholera toxin mutant as disclosed in U.S. Provisional Application No. 60/102,430 which is hereby inco ⁇ orated herein), 100 ⁇ g CpG20mer (a CpG oligonucleotide sequence, see Davis et al, 1998. J. Immunol. 160:870-876), or 10 ⁇ g CT-E29H + 100 ⁇ g CpG20mer on days 0, 2,14, and 16.
  • mice were challenged intragastrically with 2 x 10°* cfu H pylori strain SSI on day 31.
  • Organisms recovered on days 58 and 59 are expressed as logio cn ⁇ P er gram of stomach tissue +/- 1 standard error of the mean.
  • the number of colony forming units recovered was reduced by approximately 1-2 logs in mice vaccinated with 75/77 kDa protein admixed with CT, CT-E29H, and with CT-E29H + CpG20mer (Table 13).
  • Pre-challenge pooled sera (day 30) from mice vaccinated as above were analyzed for the presence of antibodies to the native 75/77 kDa proteins by standard ELISA (Table 11). Samples containing mucosal antibodies were also screened for specific antibodies to the 75/77 kDa proteins (Table 12).
  • mice were infected intragastrically with 2 x 10& cfu H pylori strain SSI on day 0.
  • mice received native 100 ⁇ g 75/77 kDa proteins, recombinant urease, or KLH admixed with CT on days 31, 33, 45, and 47.
  • mice were injected with 10 mg native 75/77 proteins admixed with AIPO4 on days 32 and 60.
  • a challenged but unvaccinated control group was included to assess the role of nonspecific clearance from CT in the KLH/CT group. Recovery of organisms 2 and 4 wks after the last vaccination is expressed in log 10 cfu per gram of stomach tissue +/- 1 standard deviation.
  • the number of colony forming units recovered was significantly reduced at the 2 wk time point for mice receiving native 75/77 kDa proteins intragastrically admixed with CT, and at the 4 wk time point for mice receiving native 75/77 kDa proteins subcutaneously admixed with AIPO4 ( Figure 14).
  • Table 11 Serum antibody responses to Hp n75/77kDa proteins administered intragastrically with various adjuvants in C57BL/6 mice
  • n 12 per group.
  • b 100 delivered IG in NaHC0 3 buffer on days 0, 2, 14, and 16..
  • c adjuvanted with 10 ⁇ g CT, 10 ⁇ g E29H, 100 ⁇ g CpG20mer or 10 ⁇ g E29H + 100 ⁇ g CpG20mer.
  • Table 12 Mucosal antibody responses to Hp n75/77kDa proteins administered intragastrically with various adjuvants in C57BL/6 mice
  • n 12 per group.
  • b 100 ⁇ g delivered IG in NaHC0 3 buffer on days 0, 2, 14, and 16.
  • c adjuvanted with 10 ⁇ g CT, 10 ⁇ g E29H, 100 ⁇ g CpG20mer or 10 ⁇ g E29H + 100 ⁇ g CpG20mer.
  • gram (g) amounts noted above are micrograms.

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Abstract

Cette invention concerne de nouveaux acides nucléiques ainsi que de nouveaux polypeptides apparentés à l'Heliobacter pylori. Cette invention concerne plus particulièrement de nouvelles protéines de surface bactérienne d'H. pylori qui possèdent des poids moléculaires d'environ 75, 77 et 79 kilodaltons (kDa). Ces séquences d'acides nucléiques et ces polypeptides sont utiles à des fins diagnostiques et thérapeutiques.
PCT/US1999/014375 1998-06-26 1999-06-25 NOUVEAUX ANTIGENES D'$i(HELIOBACTER PYLORI) Ceased WO2000000614A2 (fr)

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AU47189/99A AU4718999A (en) 1998-06-26 1999-06-25 Novel antigens of (helicobacter pylori)
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FR2739623B1 (fr) * 1995-10-04 1997-12-05 Pasteur Merieux Serums Vacc Nouvelle proteine membranaire p76 d'helicobacter pylori
WO1997037044A1 (fr) * 1996-03-29 1997-10-09 Astra Aktiebolag Acide nucleique et sequence d'acides amines pour lutter contre helicobacter pylori et compositions de vaccins
WO1997047646A1 (fr) * 1996-06-10 1997-12-18 Boren Thomas Adhesine d'helicobacter pylori se fixant a un antigene de groupe sanguin
WO1998043479A1 (fr) * 1997-04-01 1998-10-08 Merieux Oravax POLYPEPTIDES D'HELICOBACTER DE 76 kDa, 32 kDa ET 50 kDa ET MOLECULES DE POLYNUCLEOTIDES CORRESPONDANTES

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