EP0977864A2 - Composition antigenique et methode de detection d'helicobacter pylori - Google Patents

Composition antigenique et methode de detection d'helicobacter pylori

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Publication number
EP0977864A2
EP0977864A2 EP98918806A EP98918806A EP0977864A2 EP 0977864 A2 EP0977864 A2 EP 0977864A2 EP 98918806 A EP98918806 A EP 98918806A EP 98918806 A EP98918806 A EP 98918806A EP 0977864 A2 EP0977864 A2 EP 0977864A2
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EP
European Patent Office
Prior art keywords
seq
cluster
pylori
antigen
lys
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.)
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EP98918806A
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German (de)
English (en)
Inventor
Theresa P. Chow
Kirk E. Fry
Moon Y. Lim
C. P. Mcatee
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Genelabs Technologies Inc
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Genelabs Technologies Inc
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Publication of EP0977864A2 publication Critical patent/EP0977864A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to one or more antigens of H. pylori, to polynucleotide sequences coding for the antigens, and to diagnostic and therapeutic methods employing such antigens and polynucleotides.
  • Mullis, K.B. U.S. Patent No. 4,683,202, issued 28 July 1987.
  • H. pylori is a human gastric pathogen associated with chronic superficial gastritis, peptic ulcer disease, and chronic atrophic gastritis leading to gastric adenocarcinoma, although for most of this century, peptic ulcer disease was thought to be stress-related rather than caused by H. pylori infection.
  • Acceptance for the causal role of H. pylori in peptic ulcer disease and gastric inflammation developed when studies showed that human subjects who ingested H. pylori developed gastritis, a condition that was resolved after the infection was eliminated by antibiotic treatment (Warren and Marshall, 1983).
  • H. pylori is a micro-aerophilic, Gram negative, slow-growing, flagellated organism with a spiral or S-shaped morphology which infects the lining of the stomach.
  • H. pylori was originally cultured from gastric biopsy in 1982 and was placed in the Campylobacter genus based upon gross morphology.
  • the new genera Helicobacteracea was proposed and accepted, with H. pylori being its sole member (Blaser, 1996).
  • 13 members of the Helicobacter genus are recognized.
  • urease is one of the most abundant surface proteins produced by the bacteria.
  • This enzyme is a multisubunit urease which functions to hydrolyse urea into carbon dioxide and ammonia (Cover, 1995). The resulting ammonia molecules surround the bacteria, thereby neutralizing the acid in the immediate vicinity of the bacteria.
  • urease is crucial for the survival of H. pylori at acidic pH and for its successful colonization of the gastric environment.
  • H. pylori is one of the most common chronic bacterial infections in humans. H. pylori infection is found in over 90% of patients with active gastritis, and the presence of H. pylori in the gastric mucosa has been associated with mucosa-associated lymphoid tissue lymphomas (Cover, et al, 1996). In developed countries, about half of the population has been colonized with H. pylori by age 50, and in developing countries, colonization is common even among children. Further, one to two out of ten infected individuals will develop peptic ulcer disease in the course of a lifetime. Current approaches for assessing H.
  • Non-invasive approaches include the urea breath test, UBT (Atherton, et al, 1994) and serological tests which utilize various H. pylori antigens for detecting anti-H. pylori antibodies.
  • the urea breath test relies upon the presence of the urease enzyme from H. pylori to convert isotopic urea to isotopic carbon dioxide (the analyte) and ammonia.
  • the reagents for such an assay should be readily and reproducibly prepared, in addition to being highly selective and specific for H. pylori.
  • the method should be accurate and exhibit high sensitivity, in addition to being simple, convenient and cost-effective.
  • the invention pertains to the discovery and characterization of new, highly immunogenic polypeptide antigens of H. pylori. Also forming part of the invention are 69 heretofore unrecognized immunogenic cluster families. The sequence and location of these cluster families within the H. pylori genome were determined on the basis of the over 250 disclosed DNA replicas of portions of the genome of H. pylori discovered to encode highly immunogenic antigens. Also disclosed are native antigenic proteins recovered from H. pylori using a proteomics methodology. The invention further provides methods employing one, several, many or each of the above-described antigens. Also forming part of the invention is a diagnostic kit and method employing one, several, many or each of the herein described antigenic proteins to detect H. pylori infection, where the assay is effective for detecting active infective status H. pylori.
  • the present invention includes H. pylori genomic polynucleotides encoding one or more of the polypeptide antigens described herein.
  • some aspects of the invention include H. pylori derived RNA and DNA polynucleotides, recombinant H. pylori polynucleotides, a recombinant vector including any of the above polynucleotides, and a host cell transformed with any of these vectors.
  • polypeptides encode H. pylori-specific polypeptide antigens.
  • the corresponding coding sequences allow for the production of polypeptides which are useful, for example, as reagents in diagnostic tests and/or as components of vaccines.
  • Preferred polynucleotides are H. pylori antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment identified by SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs: 169, 172 (Y261A), SEQ ID NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID NOs:51 , 54 (B2), SEQ ID NO: 60 (Y104B), and SEQ ID NO: 98 (Y128D).
  • Polynucleotides encoding these antigens are particularly preferred due to the high sensitivity and specificity exhibited by the resulting antigens.
  • polynucleotides contemplated by the invention are antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment (typically at least about 18 nucleotides in length) spanning one of the following DNA fragment clusters corresponding to SEQ ID NOs:469-547:
  • a H. pylori polynucleotide is one that is capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:24.
  • a H. pylori antigen coding polynucleotide is one that is capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NOs:94, 95
  • a H. pylori antigen coding polynucleotide is composed of at least 18 contiguous nucleotides spanning a cluster region selected from the group consisting of SEQ ID NOs: 469-547.
  • a H. pylori antigen coding polynucleotide according to the invention is composed of at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO.51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:264, SEQ ID NO:270, SEQ ID NO.271, SEQ ID NO:
  • a H. pylori antigen coding polynucleotide is composed of at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs: 169, 172 (Y261A), SEQ ID NO:253 (c5), SEQ ID NO:20 ⁇ Cl), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID NO:98 (Y128D).
  • the invention includes a H. pylori polypeptide antigen, in substantially purified form, characterized by immunoreactivity with H. pylori positive anti-sera.
  • An antigen in accordance with the invention is, in one embodiment, encoded by a polynucleotide that is typically at least 18 nucleotides in length, having the features described above. More specifically, the antigen is encoded by all or a portion of a polynucleotide sequence at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
  • Additional antigens according to the invention are encoded by H. pylori antigen coding sequences as described above.
  • an antigen is encoded by a polynucleotide at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl
  • the invention encompasses a H. pylori antigen comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID NOS: 340-468, where the antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
  • a H. pylori antigen comprises at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS: 2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and 550-554.
  • a H. pylori antigen comprises at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS: 555-602.
  • a H. pylori antigen comprises a polypeptide sequence selected from the group consisting of SEQ ID NOs: 555-602.
  • a H. pylori antigen is one comprising at least 6 contiguous amino acids contained within a sequence selected from the group consisting of SEQ ID NO:44 (A22), SEQ ID NO:39 (Cl), SEQ ID NO:568 (Y124A), SEQ ID NO:557 (Y261A), SEQ ID NO:254 (c5), SEQ ID NO:21 (C7), SEQ ID NO:55 (B2), SEQ ID NO:61 (Y104B), SEQ ID NO:573 (Y128D).
  • kits for use in screening a biological fluid such as sera for the presence of anti-H. pylori antibodies.
  • the kit includes a substantially purified H. pylori antigen of the type characterized above that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of the antibody to the antigen.
  • the polypeptide antigen may be attached to a solid support, and the kit may further include a non-attached reporter-labelled anti-human antibody, where binding of the anti-H. pylori antibodies to the polypeptide antigen can be detected by binding of the reporter-labelled antibody to the anti-H. pylori antibodies.
  • the kit includes at least two H. pylori antigens having different antibody specificities.
  • the invention includes a method of detecting H. pylori infection in a subject, or detecting the eradication of the bacteria in a previously infected subject. The method involves reacting a biological fluid sample from a subject with a purified H. pylori polypeptide antigen of the type described above, and examining the antigen for the presence of bound antibody.
  • Preferred antigens for use in the method correspond to one of the following polypeptide sequences or a contiguous region contained therein: SEQ ID NO:44 (A22), SEQ ID NO:39 (Cl), SEQ ID NO:568 (Y124A), SEQ ID NO:557 (Y261A), SEQ ID NO:254 (c5), SEQ ID NO:21 ⁇ Cl), SEQ ID NO:55 (B2), SEQ ID NO:61 (Y104B), SEQ ID NO:573 (Y128D).
  • the invention includes a H. pylori vaccine composition containing a H. pylori polypeptide antigen of the type described above.
  • the antigen is characterized by its ability to reduce the level of H. pylori infection in a mammalian model system, such as a mouse or rhesus monkey challenged with the peptide, and then infected with H. pylori.
  • Preferred antigens for use in a vaccine are those which invoke a long-lasting antigenic response, as evidenced by the persistance of antibodies for an extended period of time subsequent to antimicrobial treatment.
  • Representative antigens for use in a vaccine composition are selected from the group consisting of SEQ ID NO:565 (Y139), SEQ ID NO:575 (Y146B), SEQ ID NO:555 (Y175A), SEQ ID NO:44 (A22), SEQ ID NO:569 (Y184A), SEQ ID NO:578 (Z9A), SEQ ID NO:557 (Y261A) and SEQ ID NO:575 (Y146B).
  • Fig. 1 is a computer scanned image of a Western blot of a 2-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis (high pH conditions) of native H. pylori antigens blotted with Roost pooled sera ⁇ H. pylori positive);
  • Fig. 2 is a computer scanned image illustrating a Western blot of a sodium dodecyl sulfate polyacrylamide gel 2-dimensional electrophoretic (SDS-PAGE) analysis (low pH conditions) of native H. pylori antigens blotted with Roost pool;
  • Fig. 3 shows a schematic representation of the amino acid translation of ORF3 of clone Y104- l .asm (299 amino acids). Regions of sequence indicated in the figure were confirmed by amino acid sequence analysis of the Y104-l .asm protein expressed in E. coli strain XLlBlue, and correspond to
  • Fig. 4 is a schematic representation of the in vivo processing pathway of the 36 kD protein of H. pylori and its relationship to the "spot 15" antigen disclosed herein;
  • Fig. 5 is a reverse phase HPLC peptide profile corresponding to the "spot 15" antigen isolated from H. pylori (ATCC 43504) which illustrates the presence of numerous identifiable protein peaks;
  • Fig. 6 presents the amino acid sequence of the 36-kD protein of //, pylori, where underlining indicates the 28 kD protein region;
  • Fig. 7 is a schematic representation of the proteome methodology employed to identify several native antigens of H. pylori;
  • Fig. 8 is a graphical representation summarizing percent sensitivity of various clones against representative H. Py/ori-immunopositive sera panels;
  • Fig. 9 is a linear representation of the H. pylori genome, indicating the approximate positions of immunogenic cluster regions forming one aspect of the present invention.
  • Figs. 10-63 are linear maps indicating the relative positions of immunogenic subclones within the clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18),
  • Figs. 64A-D present a summary of the immunogenic clone clusters of the present invention including (i) cluster number, (ii) clones defining the start and end regions of each cluster, coordinates of the cluster consensus region within the H. pylori genome, and expression data;
  • Fig. 65 is a graphical representation of comparative sensitivities of H. pylori recombinant antigens against various sera panels. Sensitivity values were calculated using various gold reference standards as described in Example 6B;
  • Fig. 66 is a graphical representation of comparative specificities of H. pylori recombinant antigens against various sera panels, computed against gold standards as indicated; and Figs. 67A and 67B provide a tabular summary of (i) immunopositive clones forming the basis of the invention, (ii) their corresponding cluster numbers (indicating the relative position of each clone and cluster within the H. pylori genome), and relative (iii) sensitivity and (iv) specificity values.
  • a polypeptide sequence or fragment is "derived" from another polypeptide sequence or fragment when it has the same sequence of amino acid residues as the corresponding region of the fragment from which it is derived.
  • a polynucleotide sequence or fragment is "derived" from another polynucleotide sequence or fragment when it has the same sequence of nucleic acid residues as the corresponding region of the fragment from which it is derived.
  • a first polynucleotide fragment is "selectively-hybridizable" to a second polynucleotide fragment if the first fragment or its complement can form a double-stranded polynucleotide hybrid with the second fragment under selective (stringent) hybridization conditions.
  • the first and second fragments are typically at least 15 nucleotides in length, preferably at least 18-20 nucleotides in length.
  • Selective (stringent) hybridization conditions are defined herein as hybridization at ⁇ 45 °C in ⁇ 1.
  • Two or more polynucleotide or polypeptide fragments have at least a given percent "sequence identity” if their nucleotide bases or amino acid residues are identical, respectively, in at least the specified percent of total base or residue position, when the two or more fragments are aligned such that they correspond to one another using a computer program such as ALIGN.
  • ALIGN The ALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs, Pearson and Lipman, 1988; Pearson, 1990).
  • H. pylori polynucleotide refers to a polynucleotide sequence derived from the genome of H. pylori and variants thereof.
  • H. pylori polynucleotides of the type disclosed herein encode H. pylori polypeptide antigens, where the resulting antigen is characterized by immunoreactivity with H. pylori positive anti-sera.
  • a H. pylori polynucleotide of the invention will be at least about 18 or more nucleotides in length ⁇ i.e. , encoding a 6 peptide-antigen).
  • the pylori polynucleotide will be at least about 24 nucleotides in length (i.e. , encoding an 8 peptide-antigen). In yet another embodiment, the H. pylori polynucleotide will be at least about 30 nucleotides in length. In some instances, the H. pylori polynucleotide will range from about 45 to 75 nucleotides in length, but may of course be longer.
  • the polynucleotides of the invention may be obtained from natural or synthetic sources, or, may be prepared recombinantly.
  • the polynucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, by deletions, by insertions and inversions, to a particular naturally-occurring sequence, provided that the subject polynucleotide is capable of expressing a H. pylori antigen as described herein.
  • the polynucleotide sequence may optionally contain expression control sequences (typically from a heterologous source) positioned adjacent to the coding region.
  • nucleotide sequences described herein are meant to encompass variants possessing essentially the same "sequence identity" as defined above. Nucleotide sequences having essentially the same sequence identity are typically selectively hybridizable to one another under selective (stringent) hybridization conditions. This is to say, a nucleic acid fragment is considered to be selectively hybridizable to a H. pylori polynucleotide if it is capable of specifically hybridizing to the H. pylori polynucleotide sequence or a variant thereof (e.g. , a probe that hybridizes to a H. pylori polynucleotide but not to polynucleotides from other members of the Helicobacter family) under stringent hybridization and wash conditions.
  • H. pylori antigenic polypeptide is meant to encompass immunoreactive variants of the polypeptide, or regions, or parts thereof, provided that the variant is immunogenic.
  • a suitable variant is defined as any polypeptide having a sequence that is identical ⁇ i.e. , shares sequence identity) to that of a H. pylori polypeptide.
  • an antigenic polypeptide that is essentially identical to a H. pylori polypeptide antigen is (i) encoded by a nucleic acid that selectively hybridizes to sequences of H. pylori or its variants or (ii) is encoded by H. pylori or its variants.
  • a sequence comparison may also be employed for the purpose of determining "polypeptide homology", e.g., by using the local alignment program LALIGN.
  • a polypeptide sequence is typically compared against a selected H. pylori amino acid sequence or any of its variants, as defined above, using the LALIGN program with a ktup of 1, default parameters and the default PAM.
  • Any polypeptide with an optimal alignment longer than about 6 to 8 amino acids and greater than 70%, or more preferably 75% to 80% of identically aligned amino acids is considered to be a "homologous polypeptide.”
  • the LALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs (Pearson, etal. , 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, VA). Sequence variations among antigens will depend upon a number of factors, such as the strain of //. pylori, the location of the gene or gene family encoding the antigen within the H.
  • An immunogenic polypeptide or polypeptide "fragment” is one that is (i) encoded by an open reading frame of a H. pylori polynucleotide, or (ii) displays sequence identity to H. pylori polypeptides as defined above, and is immunoreactive with a H. pylori immunopositive sample, such as sera.
  • the immunogenic fragment will comprise at least about 6 to 8 amino acids, and preferably at least about 10 to 12 contiguous amino acid residues of a particular antigen.
  • immunoreactive variant of a H. pylori polypeptide antigen is meant an amino acid substitution, deletion, and/or addition variant of a particular H. pylori polypeptide antigen sequence disclosed herein, having substantially the same or increased binding affinity to a given antibody as the particular polypeptide antigen, as determined by conventional methods, e.g. , a competition assay or a two antibody sandwich assay.
  • a representative H. pylori antigen is composed of at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID NOS:340-468, where the antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
  • Cluster sequences correspond to regions within the H. pylori genome encoding highly immunogenic polypeptides. The cluster sequences were determined on the basis of DNA sequence information for the over 250 immunogenic clones described herein. As a result, 69 unique clusters were identified, and antigens in accordance with the invention are those encoded by a contiguous series of nucleotides contained within any of the 69 clusters. Representative antigen coding sequences falling within each of the clusters are summarized in Figs. 64A-D. The cluster regions are referred to herein as clusters 1 to 69, corresponding to SEQ ID NOs: 340-468.
  • each of the cluster regions within the H. pylori genome is llustrated pictorially in Fig. 9.
  • a cluster is defined by various regions within the H. pylori genome rather than by a single sequence.
  • a particular cluster may be defined by a "start” sequence, an "end” sequence, and perhaps an invervening "middle” sequence, and thus may correspond to more than one sequence contained in the Sequence Listing.
  • the descriptor for the cluster sequence will indicate its relationship to a given cluster (e.g. , start, middle, end).
  • a defining cluster sequence will code for one or more antigens, and the remainder of the sequence for a particular cluster, if not explicitly provided, can be readily determined based upon the information provided herein, when considered along with the information, e.g. , provided in Tomb, et al, 1997.
  • Cluster sequences defined by more than one representative sequence are cluster 1 (SEQ ID NOs: 469, 470), cluster 5 (SEQ ID NOs:474,475), cluster 7 (SEQ ID NO:477, end), cluster 15 (SEQ ID NOs:485, 486), cluster 35 (SEQ ID NOs:506-508), cluster 40 (SEQ ID NOs:513, 514), cluster 41 (SEQ ID NO:515, end), cluster 43 (SEQ ID NOs:517, 518), cluster 47 (SEQ ID NOs:522, 523), cluster 49 (SEQ ID NO:525, end), cluster 58 (SEQ ID NOs:534, 535), and cluster 59 (SEQ ID NO:536, 537).
  • substantially purified and “in substantially purified form” are used in several contexts and typically refer to at least partial purification of a H. pylori polynucleotide or polypeptide away from unrelated or contaminating components ⁇ e.g. , serum, cells, proteins, non-H. pylori polynucleotides, etc.) by at least one purification or isolation step. Methods and procedures for the isolation or purification of compounds or components of interest are described herein (e.g. , SDS-PAGE, affinity purification of fusion proteins, blotting, and recombinant production of H. pylori polypeptides).
  • An antigen is "specifically immunoreactive" with H. pylori positive anti-sera or a biological fluid sample when, under optimal conditions, the antigen binds to antibodies present in the H. pylori infected sample but does not bind to antibodies present in the majority (greater than about 60 to 65% , preferably from about 70% to 80% , even more preferably greater than about 85 %) of fluid samples from subjects who are not or have not been infected with H. pylori.
  • "Specifically immunoreactive" antigens may be immunoreactive with monoclonal or polyclonal antibodies generated against specific H. pylori antigens.
  • biological fluid any fluid derived from the body of a mammal, particularly a human.
  • Representative biological fluids include blood, serum, plasma, urine, faeces, mucous, gastric secretions, dental plaques, or saliva.
  • Immunologically effective amount refers to an amount administered to a mammalian host, either as a single dose or as part of a series, that is effective for treatment or prevention of infection by H. pylori.
  • the amount will vary depending upon the health and physical condition of the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, and the like. Such an amount will typically fall within a relatively broad range, and can be determined in routine trials.
  • the present invention is based on the identification and isolation of a number of highly immunogenic H. pylori polypeptides, resulting from the screening of over a million individual H. pylori compositions.
  • the antigens of the present invention were either produced recombinantly, or, were separated from a mixture of soluble proteins obtained from pelleted and lysed H. pylori.
  • disparate sequence information corresponding to all of the disclosed immunogenic clones was compiled to provide a collection of heretofore unrecognized antigenic cluster regions contained within the genome of H. pylori.
  • the H. pylori antigens of the invention can be obtained from phage libraries using conventional screening methods described below. Unless otherwise stated, the DNA lambda libraries described herein have been deposited in the Genelabs Technologies, Inc. Culture Collection, 505 Penobscot Drive, Redwood City, CA, 94063, or in the Genelabs Diagnostics PTE LTD Culture Collection, 85 Science Park Drive #04-01, The Cavendish, Singapore Science Park, Singapore 118259.
  • E. coli XL-1 Blue MRF plasmids containing inserts corresponding to the following H. pylori clones were accepted for deposit by the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852 on September 9, 1997 and assigned the following designation numbers: clone dHClS.
  • the antigen-encoding DNA fragments of the invention can be identified by (i) immunoscreening, as described above, and/or (ii) computer analysis of coding sequences using an algorithm (such as, "ANTIGEN,” Intelligenetics, Mountain View, CA) to identify potential antigenic regions.
  • an antigen-encoding DNA fragment is subcloned, and the subcloned insert is then fragmented by partial DNase digestion to generate random fragments or by specific restriction endonuclease digestion to produce specific subfragments.
  • the resulting DNA fragments are then inserted into an expression vector, such as the lambda gtl l vector, and subjected to immunoscreening in order to provide an epitope map of the cloned insert.
  • DNA fragments of the type described herein can be employed as probes in hybridization experiments to identify overlapping H. pylori sequences, and these in turn can be further used as probes to identify additional sets of contiguous clones.
  • any of the herein-described clone sequences can be used to probe a DNA library, generated in a vector such as lambda gtlO or "LAMBDA ZAP II" (Stratagene, La Jolla, CA). Specific subfragments of known sequence may be isolated using the polymerase chain reaction or after restriction endonuclease cleavage of vectors carrying such sequences. The resulting DNA fragments can be used as radiolabelled probes against any selected library. In particular, the 5' and 3' terminal sequences of the clone inserts are useful as probes to identify additional clones.
  • sequences provided by the 5' end of cloned inserts are useful as sequence specific primers in first-strand DNA synthesis reactions (Maniatis, et al, 1982; Scharf, et al, 1986).
  • specifically primed H. pylori DNA libraries can be prepared by using specific primers derived from one of the cloned DNA sequences described herein as a template.
  • the second-strand of the new DNA is synthesized using RNase H and DNA polymerase I.
  • the above procedures identify or produce DNA molecules corresponding to nucleic acid regions that are 5' adjacent to the known clone insert sequences. These newly isolated sequences can in turn be used to identify further flanking sequences, and so on.
  • the polynucleotides can be cloned and immunoscreened to identify specific sequences encoding H. pylori antigens.
  • DNA libraries were prepared from commercially available strains of H. pylori (American Type Culture Collection, Rockville, MD; ATCC Designation No. 43504, ATCC Designation No. 43526) in either the expression vector lambda gtl l or "ZAPII" (Stratagene, La Jolla, CA; Example 1). Polynucleotide sequences were then selected for the expression of peptides which were immunoreactive with pooled sera obtained from 11 patients identified by endoscopy as H. />y/ ⁇ ' -positive, herein identified as "Roost pool” sera, or another pool of 4 H. j py/ori-irnmunopositive sera samples identified as "SFA001 ".
  • the samples may be individual samples, i.e. , derived from a single subject, or may be pooled samples, such as those described herein.
  • Recombinant proteins identified by this approach provided candidates for polypeptides that can serve, either singly or in combination, as substrates in diagnostic tests for detecting infection by H. pylori. Further, the corresponding nucleic acid coding sequences serve as useful hybridization probes for the identification of additional H. pylori antigen coding sequences.
  • the H. pylori strains described above were used to generate DNA libraries in lambda vectors
  • Example 1 Other commonly available strains of H. pylori include, for example, H. pylori samples identified as strain # 29995 and J-170. Alternatively, libraries can be constructed from H. pylori isolated from a sample confirmed as //. /o ⁇ ' -positive. In the method illustrated in Example 1, the libraries were generated from genomic DNA isolated from the pelleted bacteria. Alternatively, centrifugation can be used to pellet bacteria from infected biological specimens such as gastric mucosa.
  • H. pylori DNA libraries were generated using DNase-digested genomic DNA fragments isolated from H. pylori as starting material.
  • the resulting molecules were ligated to Sequence Independent Single Primer
  • SISPA SISPA
  • Reyes, et al, 1991 linker primers and expanded in a non-selective manner, and then cloned into a suitable vector, for example, lambda gtl l or ZAPII, for expression and screening of peptide antigens.
  • a suitable vector for example, lambda gtl l or ZAPII
  • the libraries disclosed herein have been designated as the "short antigen clone” library, typically designated by a prefix beginning with the letters Y or Z; and the "long antigen clone” library, designated as libraries 1 and 2.
  • the ZAPII libraries 1 and 2 were similarly constructed, with the following exceptions.
  • the ZAPII libraries 1 and 2 were generated from longer H. pylori DNA fragments, i.e., either EcoRI or Z/m- ⁇ II-digested genomic DNA which had not undergone Sequence Independent Single Primer Amplification.
  • Library 1 clones (designated herein by upper case letters) were generated by ligating EcoRI-digested H. pylori DNA directly into the EcoRI sites of the lambda "ZAPII" vector.
  • Library 2 clones (lower case designations) were obtained by digesting H. pylori genomic DNA with Hindlll, then blunt ended with ⁇ .
  • Lambda gtl 1 is a particularly useful expression vector for producing H. pylori antigens.
  • the vector contains a unique EcoRI insertion site, located 53 base pairs upstream of the translation termination codon of the /3-galactosidase gene.
  • an inserted sequence is expressed as a ⁇ - galactosidase fusion protein which contains the N-terminal portion of the 3-galactosidase gene product, the heterologous peptide, and optionally the C-terminal region of the /3-galactosidase peptide (the C ⁇ terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon).
  • the lambda gtll vector also produces a temperature-sensitive repressor (cI857) which causes viral lysogeny at permissive temperatures, e.g., 32°C, and leads to viral lysis at elevated temperatures, e.g., 42°C.
  • Advantages of lambda gtll include: (1) highly efficient recombinant clone generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) production of recombinant fusion protein.
  • phage containing a heterologous insert produces an inactive / 3-galactosidase enzyme, phage with inserts are typically identified using a colorimetric substrate conversion reaction employing ⁇ - galactosidase.
  • E. coli expression vectors are useful for expression of antigens.
  • Alternative microbial hosts suitable for expression include bacilli, such as B. subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • the expression vectors will typically contain control sequences compatible with the host cell.
  • Other known promoter sequences may be present in the expression vector, such as the lactose promoter system, a tryptophan promoter system, or a promoter system from phage lambda.
  • An amino terminal methionine can be provided, if necessary, by insertion of a Met codon in-frame with the antigen.
  • the carboxy terminal extension of an antigen can be removed by conventional mutagenesis procedures.
  • yeast expression systems can be used, such as the Saccharomyces cerevisiae pre- pro-alpha-factor leader region used to direct protein expression from yeast.
  • the antigen coding sequence can be fused in frame to the leader region. This construct is then typically put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen sequence is followed by a translation termination codon which is followed by transcription termination signals.
  • the antigen coding sequences can be fused to a second protein coding sequence, such as / 3-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography.
  • protease cleavage sites may be inserted to facilitate separation of the fusion protein components.
  • mammalian cells can be used for expression of the antigens of the invention.
  • Vectors useful for expression in mammalian cells are typically characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation (poly A) signal.
  • the vectors may optionally include selectable marker genes, such as those conferring antibiotic resistance.
  • Suitable host cells include Chinese hamster ovary cells (CHO) cell lines, HeLa cells, myeloma cells, Jurkat cell lines, and the like.
  • the expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer, information processing sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • expression control sequences such as an origin of replication, a promoter, an enhancer, information processing sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • the DNA sequences are expressed in hosts after the sequences of interest have been operably linked to (i.e., positioned to enable the functioning of) an expression control sequence.
  • Example 1 describes the preparation of a DNA library for H. pylori (ATCC No. 43504).
  • the library was immunoscreened using H. pylori-positive pooled sera (Example 2).
  • a number of lambda clones were identified which were immunoreactive with anti-//. pylori antibodies present in the pooled sera.
  • Selected immunopositive clones were plaque-purified and their immunoreactivity retested.
  • the immunoreactivity of the clones with normal human sera (control, H. >y/or.-negative) was also tested.
  • Numerous clones were identified by immunosceening and further characterized, as described in Examples 3 and 4.
  • Immunoreactive clones further described in Example 3 and referred to herein as the Y and Z families of clones include clone Y-104-1, and the clones summarized in Table 2, corresponding to SEQ ID NOs: 70-230.
  • the DNA inserts of the immunoreactive recombinant lambda clones were PCR amplified, using primers corresponding to lambda arm sequences flanking the EcoRI cloning site of the vectors, and utilizing each immunoreactive clone as template.
  • gtl 1 clones gtl IF (S ⁇ Q ID NO: 65) and gtl 1 R (S ⁇ Q ID NO:66) primers were used.
  • T3 S ⁇ Q ID NO:67
  • T7 S ⁇ Q ID NO:68
  • the resulting amplification products were agarose gel purified and eluted from the gel (Ausubel, et al, 1988) to remove primers and other components.
  • the purified insert DNA was then subjected to direct sequencing. In some cases, the insert DNA was first subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and then sequenced. Clones exhibiting immunoreactivity against
  • the genomic clones were digested by restriction enzyme treatment, and the resulting subfragments were inserted into a suitable expression vector.
  • the resulting subclones, containing the specific digested DNA fragments, were then screened for immunogenicity. Clones identified as immunoreactive towards //. /ry/ ⁇ -positive pooled sera were plaque purified. Plasmid DNA containing inserts obtained by recovery from phage were sequenced as described above and also in Example 4.
  • H. pylori Antigens ZAPII Libraries 1 and 2.
  • Library 1 and library 2 clones isolated by immunoscreening with H. pylori immunopositive pooled sera include the following: LIBRARY 1: A3, A22, B2, B9, B17, B23, Cl, C3, C7, DH, and LIBRARY 2: al, a3, a5, b5, b8c7, c2, c5, cl3, d5, d6, dll, d7, e6, f3, f8, fll, g2, g9, gll, k4.
  • Clone Gla was isolated by screening with monoclonal antibody 1G6 from Biogenesis, Inc. (Bournemouth, England). Sequence data for these clones is presented in the sequence listing herein.
  • Clone d7 and its Relationship to the 36 kD Protein of H. pylori.
  • Clone d7 another immunoreactive clone of the present invention, is nHindl clone that was blunt-ended, ligated to A/B linkers, digested with EcoRI, and subcloned into lambda vector ZAPII to produce a beta- galactosidase fusion protein.
  • the nucleotide sequence is presented as S ⁇ Q ID NO:ll. Polynucleotides and polypeptides derived from this clone represent preferred embodiments of the invention.
  • this clone codes for about 70% of the carboxy- end of the 36 Kd native protein of H. pylori, while clone Y104 codes for the entire 36 K protein.
  • clone Y104 codes for the entire 36 K protein.
  • portions of the amino acid sequence of the native 36 Kd protein of H. pylori have been determined.
  • the 36 kD protein (encoded in part by clone d7) appears to be a precursor to a highly antigenic H. pylori protein referred to herein as the "spot 15" protein (Examples 8,9, to be described in more detail below).
  • the native H. pylori translation product a 34 kD protein (i.e, calculated molecular weight) composed of 299 amino acids, appears to be cleaved in vivo at amino acid position 23. This results in a 31.6 kD cleavage product commonly referred to as "the 36 kD protein" of //. pylori.
  • the differences in molecular weight terminology in referring to this protein arise due to the following: 36-kD is observed from SDS-PAGE; 34-kD is calculated from the corresponding DNA sequence (Fig. 6); and 31 kD is determined from experimental sequence data, as determined starting from residue 23 of SEQ ID NO:60.
  • a post translation modification of the 36 kD protein i.e., acetylation at the amino acid terminus (position 23) and cleavage at the carboxy end, results in the "spot 15" antigen, having a molecular weight of 28 kD.
  • "X"s correspond to positions where deamidations (i.e. , asparagine or glutamine) or point mutations have occurred.
  • Further proposed modifications leading to the minor spot 15 protein are also indicated.
  • the antigenic polypeptide corresponding to spot 15, and sequences coding for this protein e.g. , clones Y-104 and d7, represent one particularly preferred embodiment of the present invention. This is due to the strong antigenicity of the peptide.
  • the spot 15 peptide has been detected in various strains of //, pylori, as indicated in Table 9 and in Example 10.
  • Genome The antigenic sequences described herein were determined based upon the screening of over a million discrete H. pylori antigenic compositions. DNA sequencing was carried out for H. pylori immunopositive clones, and open reading frames coding for antigenic proteins were identified. Nucleic acid sequences coding for the thus-identified antigens were then inserted into expression vectors and expression of the desired antigenic protein was confirmed. As a result of this work, over 250 antigenic clone sequences have been identified and are disclosed herein.
  • the antigenic polynucleotide fragments isolated herein map into 69 clusters which are identified herein as clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69).
  • Fig. 9 The position of these antigenic clusters within the complete H. pylori genome is shown in Fig. 9. As can be seen from Fig. 9, the locations of the clusters within the genome (and clones within the clusters) are highly random, and represent only a small portion (i.e. , approximately 2-3 %) of the overall nucleotides contained within the entire genome. Prior to this work, a comprehensive guide to the unique and highly antigenic regions within the genome of H. pylori was unknown.
  • Cluster analysis all of the immunoclone sequences were combined into a FASTA database as defined by Pearson and Lipman (1988). This database was converted to a BLAST database for high speed searching (Altschul, et al, 1990). The sequence of each immunoclone was then searched against this database using the program BLASTN to define clusters. Clusters are an assembly of clones that contain identical sequences with other clones in the group. The sequences in each group or cluster were then combined in separate database files and formatted for entry in the GEL program of IG-Suite (Oxford Molecular). GEL then assembled the sequences and suggested a consensus sequence with ambiguities. A non-ambiguous sequence was determined for each cluster by editing the consensus sequence.
  • Clusters 1 to 69 correspond to SEQ ID NO:s 469-547. Open reading frames for the antigens were then determined from the cluster consensus sequence. Single clone sequences were translated directly to provide antigen sequences. Antigenic regions contained within each of the clusters as determined by translation of cluster open reading frames are provided as SEQ ID NO:s 340-468. Figs.
  • 10 to 63 are linear maps indicating the relative positions of immunogenic subclones within the clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (23), (25), (27), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (53), (54), (58), (59), (61), (62), (68), and (69).
  • Clusters not shown on the linear maps are clusters defined by one clone, i.e., clusters (22), (24), (26), (31), (34), (52), (55), (56), (57), (60), (63), (64), (65), (66), (67).
  • Figs. 64A-D present a tabular summary of clusters 1-69, clones contained within each cluster, and coordinates of each cluster consensus region within the H. pylori genome.
  • H. pylori possesses a circular genome of 1,667,867 base pairs and 1590 predicted coding sequences.
  • the genome sequence reported on the TIGR Web site was used as a reference for reporting nucleotide positions of the clusters and immunogenic clones of the invention. Based upon the present work, it can be seen that a relatively small number of the predicted open reading frames reported for the H. pylori genome encode the antigenic proteins or cluster regions forming the basis of the present invention.
  • Each cluster defines a continuous DNA sequence that spans, i.e., extends, from the 5' end of the most upstream clone in the cluster to the 3' end of the most downstream clone in the cluster.
  • the spanning sequence is incomplete, it has been filled in with sequence from the reported H. pylori genomic sequences (TIGR Web site).
  • the sequence defined by cluster 1 includes the sequence beginning at the 5' end of clone Y92 (SEQ ID NO: 222) and ending at the 3' end of clone Y92 (SEQ ID NO:223), including the short (about 730 bases) genomic sequence connecting the two clone sequences.
  • the positions of the individual clones in each cluster are shown in Figs. 10-63, along with corresponding SEQ ID NOs.
  • the invention includes antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment spanning one of the DNA fragment clusters: (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69).
  • the antigen-coding regions are the spanning sequences themselves from the clusters.
  • Preferred polynucleotides are H. pylori antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs: 169, 172 (Y261A), SEQ ID NO:253 (c5), SEQ ID NO:20 (Cl), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID NO:98 (Y128D).
  • the clusters disclosed herein provide a non-random collection of H. pylori antigen coding sequences and resulting antigens which have been shown to react with H. pylori immunopositive samples and are useful in a variety of diagnostic applications.
  • Fig. 7 An overview of the proteome methodology disclosed herein and utitilized to separate and identify over twenty H. pylori antigens is provided in Fig. 7 (right-hand side).
  • bacterial proteins are separated by two-dimensional (2D) electrophoresis.
  • Immunoreactive spots ⁇ i.e. , reactive in Western blots with pooled sera from H. pylori infected patients) are then selected and subsequently characterized by endoproteolytic digestion, chromatography, and mass spectrometry (e.g. , matrix assisted laser desorption time of flight mass spectrometry, MALDI-TOF).
  • Box A indicates the use of electrospray mass spectrometry to determine the mass of the intact protein.
  • Box B indicates the use of MALDI-TOF mass spectrometry to evaluate the number and mass of Lys-C peptides.
  • Box C indicates the use of MALDI-MS to evaluate chromatographically separated Lys-C peptides and provide sequence information by post source decay (when peptides are pure).
  • Box D indicates the use of electrospray mass spectrometry to evaluate and sequence peptides through collision induced dissociation.
  • Antigens are generally obtained from whole lysates as follows. Fractionation of H. pylori soluble proteins is carried out by SDS-PAGE, preferably utilizing 2-dimensional electrophoresis (O'Farrell, 1975; O'Fanell, et al , 1977). In performing a typical 2-dimensional electrophoretic separation, an isoelectric focusing gel separation is first carried out (first dimension gel). Isoelectric focusing is carried out, e.g. , on a acrylamide/bisacrylamide gel, for an appropriate number of volt-hrs, determined as described in "CURRENT PROTOCOLS IN PROTEIN SCIENCE” . Units 10.46, John Wiley and Sons, Inc., New York (1996).
  • the final tube gel pH gradient is then measured by a surface pH electrode.
  • Protein components in a sample mixture undergo a first separation according to pi value, as indicated on the horizontal axes of Figs. 1 and 2.
  • the first dimensional separation can be carried under either acidic (Fig. 2) or basic (Fig. 1) conditions, depending upon the nature of the proteins to be separated.
  • a second dimensional, sized-based polyacrylamide slab gel separation is then carried out, to further separate the proteins on the basis of molecular weight.
  • Suitable molecular weight markers are typically added to the gel, for determining corresponding molecular weights of eluted proteins.
  • Detection is typically carried out by Coomassi blue staining or by silver staining. Silver staining may be preferred, in some cases, since silver staining methods are considerably more sensitive and can be used to detect smaller amounts of protein.
  • Figs. 1 and 2 illustrate H. pylori antigens obtained generally as described above, which have been Western blotted with Roost H. pylori-positive serum pool and a negative serum pool, respectively. The numbers in each figure correspond to spots representing H.
  • immunoreactive spots were excised from the gels, digested, and sequenced.
  • the native H. pylori antigens were further characterized by a combination of sequencing methodologies, including N-terminal sequencing, liquid-chromatography- mass spectrometry, and determination of internal sequences by amino-acid specific chemical cleavage, followed by Edman sequencing (Example 9).
  • Internal amino acid sequences can be determined by utilizing a combination of various site- specific cleavage reagents, such as ortho-pthalaldehyde (OPA)/cyanogen bromide (CNBr), hydroxy lamine, formic acid, and BNPS-skatole, which cleave as follows: CNBr (cleaves at C- terminus of methionine), BNPS-skatole (cleaves at C-terminus of tryptophan), formic acid (cleaves at Asp-Pro peptide bond), hydroxylamine (cleaves at Asn-Gly bond), and OPA, which distinguishes between secondary and primary amines; and enzymatic reagents such as Endo Protease Lvs-C, Endoproteinase ASP-N, Endoproteinase GLU-C.
  • OPA ortho-pthalaldehyde
  • CBr cyanogen bromide
  • CNBr cleaves at C-terminus of methionine
  • Endo Protease Lvs-C cleaves at C-terminus of lysine
  • Endoproteinase ASP-N cleaves at N-terminus of aspartic acid and cysteic acid
  • Endoproteinase GLU-C cleaves at C-terminus of glutamic acid
  • Trvsin cleaves at C-terminus of arginine and lysine
  • spots 9, 11, 12, 13, 15, 16 (major and minor), and 17 represent unique antigens.
  • Spot 9 represents the native H. pylori antigen corresponding to the recombinant protein expressed by ORF2 of clone al
  • spot 12 represents the native antigen that corresponds to the antigenic protein encoded by clone a5.
  • spots 9 (major and minor) 10, 12, 13, and
  • Mass spectral profiles of selected Lys-C digested H. pylori proteins corresponding to Western positive spots are provided in Tables 11 and 12.
  • the mass "fingerprints" of the peptide digests were then used as a basis for comparison to proteins predicted from various genomics databases (details are provided in Example 12) to further confirm the identify of selected H. pylori antigens described herein.
  • proteomic approach is useful for analyzing the genetic diversity of H. pylori, examining antigen-antibody responses during acute and chronic infection, and with particular gastroduodenal pathologies and possible autoimmune components to //. y/o ⁇ -associated disease.
  • 2-D gel electrophoresis and in-situ proteolytic digestion in conjunction with MALDI-TOF MS provides an extremely sensitive technique for the rapid identification of//, pylori antigens and for rapid screening for preferred vaccine and diagnostic candidates.
  • the recombinant antigenic peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.
  • H. pylori antigens in accordance with the invention comprise at least 6 contiguous amino acids contained within one of the following cluster antigen sequences: SEQ ID NOS: 340-468.
  • a H. pylori antigen may comprise at least 6 contiguous amino acids contained within a polypeptide sequence selected from one of the following SEQ ID NOS: 2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265- 269, 323, 324, and 550-554.
  • a H. pylori antigen corresponds to at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS: 555-602, where these sequences represent illustrative expression proteins corresponding to H. pylori antigens. Even more preferably, a H.
  • pylori antigen is identified by at least 6 contiguous amino acids contained within one of the following sequences: SEQ ID NO:44 (A22), SEQ ID NO:39 (Cl), SEQ ID NO:568 (Y124A), SEQ ID NO:557 (Y261A), SEQ ID NO:254 (c5), SEQ ID NO:21 (C7), SEQ ID NO:55 (B2), SEQ ID NO:61 (Y104B), SEQ ID NO:573 (Y128D).
  • Polynucleotide sequences encoding the antigens of the present invention have been cloned in the plasmid p-GEX (Example 5) or various derivatives thereof (pGEX-del65).
  • the plasmid pGEX (Smith, et al. , 1988) and its derivatives express the polypeptide sequences of a cloned insert fused in- frame to the protein glutathione-S-transferase (sj26).
  • plasmid pGEX-hisB an amino acid sequence of 6 histidines, is introduced at the carboxy terminus of the fusion protein.
  • the various recombinant pGEX plasmids can be transformed into appropriate strains of E. coli and fusion protein production can be induced by the addition of IPTG (isopropyl-thio galactopyranoside) as described in Example 5.
  • Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using Ni-NTA+ + affinity chromatography (Example 5).
  • Insoluble fusion protein expressed by the plasmids can be purified by means of immobilized metal ion affinity chromatography (Porath, 1992) in buffers containing 6 M Urea or 6 M guanidinium isothiocyanate, both of which are useful for the solubilization of proteins.
  • insoluble proteins expressed in pGEX-GLI or derivatives thereof can be purified using combinations of centrifugation to remove soluble proteins followed by solubilization of insoluble proteins and standard chromatographic methodologies, such as ion exchange or size exclusion chromatography, and other such methods are known in the art.
  • the fused protein can be isolated readily by affinity chromatography, or by passing cell lysis material over a solid support having surface-bound anti- / 3-galactosidase antibody.
  • an expression vector such as the lambda gtl 1 or pGEX vectors described above, containing H. pylori antigen coding sequences and expression control elements which allow expression of the coding regions in a suitable host.
  • the control elements generally include a promoter, a translation initiation codon, translation and transcription termination sequences, and an insertion site for introducing the insert into the vector.
  • the DNA encoding the desired antigenic polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system.
  • These systems include, but are not limited to the following: baculovirus expression (Reilly, et al, 1992; Beames, et al, 1991; Pharmingen, San Diego, CA; Clontech, Palo Alto, CA), vaccinia expression (Earl, et al, 1991; Moss, et al, 1991), expression in bacteria (Ausubel, et al, 1988; Clontech), expression in yeast (Gellissen, etal , 1992; Romanos, etal , 1992; Goeddel, 1990; Guthrie and Fink, 1991), expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, NY), e.g.
  • Example 5 Expression of large polypeptide antigens is described in Example 5.
  • Several of the long antigen clone sequences were cloned into expression vectors and successfully expressed in E. coli, as described in Example 5 and indicated in Tables 3 a and 3b.
  • Expression in yeast systems has the advantage of commercial production.
  • Recombinant protein production by vaccinia and CHO cell line have the advantage of being mammalian expression systems.
  • vaccinia virus expression has several advantages including the following: (i) a wide host range; (ii) faithful post-transcriptional modification, processing, folding, transport, secretion, and assembly of recombinant proteins; (iii) high level expression of relatively soluble recombinant proteins; and (iv) a large capacity to accommodate foreign DNA.
  • the recombinant expressed polypeptide-produced H. pylori polypeptide antigens are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated based on the H. pylori antigens identified by the methods of the present invention.
  • the resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides.
  • amino acid sequences can be readily chemically synthesized using commercially available synthesizer (Applied Biosystems, Foster City, CA) or "PIN” technology (Applied Biosystems). Antigens obtained by any of these methods can be used for antibody generation, diagnostic tests and vaccine development.
  • the invention includes specific antibodies directed against the polypeptide antigens of the present invention. Antigens obtained by any of these methods may be directly used for the generation of antibodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e.g. , Pierce, Rockford, IL). Typically, to prepare antibodies, a host animal, such as a rabbit or a goat, is immunized with the purified antigen or fused protein antigen.
  • Hybrid or fused proteins may be generated using a variety of coding sequences derived from other proteins, such as glutathione-S-transferase or /3-galactosidase.
  • the host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen.
  • the gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate precipitation or DEAE Sephadex chromatography, affinity chromatography, or other techniques known to those skilled in the art for producing polyclonal antibodies.
  • purified antigen or fused antigen protein may be used for producing monoclonal antibodies.
  • the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art.
  • a human lymphocyte donor is selected.
  • a donor known to be infected with a H. pylori may serve as a suitable lymphocyte donor.
  • Lymphocytes can be isolated from a peripheral blood sample.
  • Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a suitable fusion partner can be used to produce human-derived hybridomas.
  • Primary in vitro sensitization with viral specific polypeptides can also be used in the generation of human monoclonal antibodies.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al, 1988).
  • H. pylori antigens can then be used in any of a number of standard immunoassay formats to detect the presence of antigen, such as described in Harlow, et al, 1988.
  • One representative assay format is an antigen capture sandwich assay.
  • antibody is immobilized on a solid support.
  • H. pylori infected samples e.g. , feces, dental plaque, gastric biopsies, culture suspension from a biopsy sample
  • H. pylori infected samples e.g. , feces, dental plaque, gastric biopsies, culture suspension from a biopsy sample
  • H. pylori infected samples e.g. , feces, dental plaque, gastric biopsies, culture suspension from a biopsy sample
  • a different antibody directed against H. pylori e.g.
  • H. pylori antigen in a test sample is indicative of the presence of H. pylori antigen in a test sample.
  • the above-described assay is representative of any of an antigen-based assay based on antibodies prepared as described above, useful for the early detection of H. pylori antigens in a sample suspected of infection by H. pylori.
  • H. pylori antigens are first identified, typically through plaque immunoscreening as described above, and expressed and purified (as previously described). The antigens are then screened rapidly against a large number of suspected H. pylori positive anti-sera using alternative immunoassays, such as, ELISAs or Protein Blot Assays (Western blots) employing the isolated antigen peptide.
  • the antigen polypeptide fusion protein is then isolated as described above, usually by affinity chromatography to the fusion partner such as /3-galactosidase or glutathione-S-transferase. Alternatively, the antigen itself is purified using antibodies generated against it (see below).
  • a general ELISA assay format may be employed, such as those described in Harlow, et al. (1988).
  • the purified antigen polypeptide or fusion polypeptide containing the antigen of interest is attached to a solid support, for example, a multiwell polystyrene plate.
  • Biological fluid e.g., sera
  • Biological fluid e.g., sera
  • the sera are washed out of the wells.
  • a labelled reporter antibody is added to each well along with an appropriate substrate: wells containing antibodies bound to the purified antigen polypeptide or fusion polypeptide containing the antigen are detected by a positive signal.
  • a typical format for protein blot analysis using one, any, several, or each of the polypeptide antigens of the present invention is presented in Example 6. General protein blotting methods are described by Ausubel, et al. (1988).
  • Example 6A the antigenic protein expressed by clone dHA22.8 (A22) was used to screen a number of sera samples (both pooled sera and discrete samples). The high percentage of sera reacting to antigen produced from recombinant clone dHA22.8 indicates that it is a dominant epitope, and that it is a suitable infection marker for H. pylori. The results presented in Example 6 A demonstrate that several different source H. pylori-positive anti-sera are immunoreactive with this representative polypeptide antigen. Similar results are described for recombinant protein expressed by clone dHClS.il (Cl).
  • Example 6B Additional experiments carried out in support of the invention as described in Example 6B reveal, on the basis of sera paneling data using both single antigens and antigen combinations, that preferred antigens for use in reliably and universally detecting H. pylori infection include but are not limited to the following: A22, Cl, Y124A, Y261A, c5, C7, B2, Y104B, and Y128D. These antigens are effective as serological markers for detecting active infection by H. pylori, based upon favorable sensitivity and selectivity features. In Example 8, native proteins from H. pylori are shown to be immunoreactive with anti-/ . pylori primary antibodies obtained from "Roost" pooled serum.
  • Protective Antibodies can be identified using, for example, an animal model system (DuBois, et al. , 1996). To identify protective antibodies, polyclonal or monoclonal antibodies are generated against the antigens of the present invention, where the antigens may be used as the immune-stimulation component arm in conjunction with cholera toxic (CT). Antibodies thus generated are then used to pre-treat an infectious H. py /on-containing inoculum ⁇ e.g. , serum) before infection of cell cultures or animals. The ability of a single antibody or mixtures of antibodies to protect the cell culture or animal from infection is evaluated.
  • CT cholera toxic
  • the absence of antigen and/or nucleic acid production serves as a screen.
  • the absence of H. pylori disease symptoms e.g. , elevated carbon dioxide/ammonia levels in a urea breath test (UBT) is also indicative of the presence of protective antibodies.
  • UBT urea breath test
  • the urea breath test takes advantage of the action of the urease enzyme of H. pylori to decompose ingested 13 C or 14 C urea to radioactive carbon dioxide and ammonia, and radioactive carbon dioxide is then measured.
  • Animal models for investigating H. infection include: (i) gnotobiotic newborn piglets (easily infected by H. pylori of human origin, but preferred for short term studies), (ii) mice and ferrets, which can be colonized for months (mice) or years (ferrets), and (iii) certain domestic cats, which can carry H. pylori (DuBois, et al, 1996).
  • H. pylori causes ulcers in gnotobiotic piglets, and in mice using "THE SYDNEY STRAIN" of H. pylori, and gastritis in mouse strains SJL, C3H/HZ, DBA, C56BL b, and Balb/C.
  • a rhesus monkey infection model has also been developed (DuBois, et al, 1996).
  • convalescent sera can be screened for the presence of protective antibodies and then these sera used to identify H. pylori antigens that bind with the antibodies.
  • the identified H. pylori antigen is then recombinantly or synthetically produced. The ability of the antigen to generate protective antibodies is tested as above.
  • the antigen or antigens identified as capable of generating protective antibodies can be used as a vaccine to inoculate test animals (to be described in greater detail below).
  • the animals are then challenged with infectious H. pylori. Protection from infection indicates the ability of the animals to generate antibodies that protect them from infection. Further, use of the animal models allows identification of antigens that activate cellular immunity.
  • a protective immune response in response to challenge by a bacterial preparation ⁇ e.g. , infected serum
  • a bacterial preparation e.g. , infected serum
  • Vaccines can be prepared from one or more of the immunogenic polypeptides identified herein.
  • Numerous H. pylori polypeptides of the invention e.g. , spot 15
  • the intensity of color development is representative of the strength (binding affinity) of the antigen-anti-/ . pylori antibody interaction.
  • Representative serum paneling results for proteins expressed by two of the clones, dHA22.8 (A22) and dHClS.ll (Cl), indicate that both of these recombinant proteins are highly immunogenic.
  • Protein produced by clone dHA22.8 reacted with antibodies present in both of the H. pylori-positive pooled sera sources, Roost pool and SFA 001, and exhibited no cross reactivity with antibodies present in H. /ry/o ⁇ ' -negative samples. Similar results were observed for protein produced by clone dHClS.ll (Cl).
  • antigenic protein expressed by each of the clones dHA22.8 and dHClS.11 reacted with anti-/ . pylori antibodies in 100% and 95% of the samples, respectively, indicating the ability of the antigens of the present invention to detect H. pylori infection, and to provide components for vaccines against H. pylori.
  • antigens for use in vaccine compositions are described in Example 13.
  • Exemplary antigens are those capable of invoking a long-lasting antigenic response, as evidenced by the persistant presence of antibodies whose titre remains high for an extended period of time subsequent antimicrobial treatment for H. pylori.
  • particularly preferred antigens for use in vaccine compositions include Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261Ains and Y146B.
  • H. pylori peptides can be identified as useful in a vaccine for H. pylori as follows.
  • the individual test peptide is formulated in a suitable carrier, e.g. , adjuvant, at a concentration suitable for injection, e.g., 5-500 mg/ml.
  • a suitable carrier e.g. , adjuvant
  • One suitable test animal for vaccination is the Rhesus monkey, as detailed in DuBois (DuBois, et al., 1996).
  • the animal is vaccinated, e.g. , by oral, intramuscular or intravenous injection, in an amount typically between 0.2 to 2.0 mg/kg body weight. After a suitable period, e.g.
  • the animal may be given a booster by the same route and typically in the same amount.
  • the animal is challenged with H. pylori in a known manner, e.g. , as described in DuBois (DuBois, et al, 1996).
  • H. pylori e.g. , a suspension of approximately 10 8 -10 9 CFU of H. pylori, 1 ml
  • the subject is typically monitored by endoscopy, histologic examination, microbiological methods, and/or measurement of H. pylori- specific plasma IgG, as described in DuBois (DuBois, et al, 1996).
  • the level of infection in the vaccinated animal is then compared with that of a control animal to assess the degree of protection.
  • Those peptides which provide a measurable degree of protection against H. pylori infection are suitable for vaccine use, either alone or in combination with other peptide vaccine agents , such as the above noted dH A22.8 (A22) , dHC 1 S .11 (C 1 ) and spot 15 peptides .
  • the selected peptide is formulated according to known vaccine formulations.
  • the peptide is conjugated to a carrier protein, e.g. , keyhole limpet hemocyanin or human serum albumen, and/or suspended in a suitable adjuvant, such as Freund's adjuvant.
  • a carrier protein e.g. , keyhole limpet hemocyanin or human serum albumen
  • a suitable adjuvant such as Freund's adjuvant.
  • the vaccine is administered by conventional routes, typically IM or IV routes, as above, at peptide levels preferably in the range of 0.2 to 2.0 mg/kg. If necessary, one or more booster injections is given.
  • the specificity of a putative immunogenic fragment can be assessed by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other related bacteria.
  • Synthetic Peptides Using the coding sequences of H. pylori polypeptide antigens disclosed herein, synthetic peptides can be generated which correspond to these polypeptides. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA).
  • oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea, 1989; Yoshio, et al, 1989; Eaton, et al, 1988). Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis, et al, 1982; Ausubel, et al, 1988).
  • antigens herein are their use as diagnostic reagents for the effective and reliable detection of antibodies present in the sera of test subjects infected with H. pylori, to thereby provide an indication of infection in a test subject.
  • Preferred antigens which can be employed either singly or in combination in such a method include, e.g.
  • antigens identified by or derived from SEQ ID NO:44 A22
  • SEQ ID NO:39 Cl
  • SEQ ID NO:568 Y124A
  • SEQ ID NO:557 Y261A
  • SEQ ID NO:254 c5
  • SEQ ID NO:21 C7
  • SEQ ID NO:55 B2
  • SEQ ID NO:61 Y104B
  • SEQ ID NO:573 Y128D
  • preferred antigens are defined in terms of their DNA coding sequences, corresponding to or derived from SEQ ID NO:43 (A22), SEQ ID NO:38 (Cl), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs: 169, 172 (Y261A), SEQ ID NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), or SEQ ID NO:98 (Y128D).
  • the antigen used for the detection of antibodies present in the sera of test subjects infected with H. pylori is encoded by a DNA fragment spanning one of the DNA fragment clusters: (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69), or immunoreactive variants thereof.
  • the antigens of the present invention can be used singly, or in combination with each other, in order to detect H. pylori, as illustrated by the results in Example 6B.
  • the antigens of the present invention may also be coupled with diagnostic assays for other infectious agents.
  • test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention, e.g. , the "spot 15" antigen.
  • a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention e.g. , the "spot 15" antigen.
  • Exemplary antigens are A22 and Cl, which both show high sensitivity (as indicated in Figs. 65 and 66, and in Example 6B).
  • the reagent is reacted with reporter-labelled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-//. pylori antibody on the solid support.
  • the reagent is again washed to remove unbound labelled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or color-metric substrate, e.g. , 5-bromo-4-chloro-3-indoyl-phosphate (BCIP) and nitroblue tetrazolium (NBT). (Sigma, St. Louis, MO).
  • BCIP 5-bromo-4-chloro-3-indoyl-phosphate
  • NBT nitroblue tetrazolium
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material.
  • attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group binding to a chemically reactive group on the solid support, e.g. , an activated carboxyl, hydroxyl, or aldehyde group.
  • streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
  • an assay system or kit for carrying out this diagnostic method generally includes a support with surface-bound recombinant antigen (e.g. , antigens such as those described in Tables 3a and 3b, or encoded by the representative clones summarized in Table 2) or native H. pylori antigen (such as those identified in Table 6 and in Fig. 1 and Fig. 2, as above), and a reporter-labelled anti-human antibody for detecting surface-bound anti-//. pylori antigen antibody.
  • surface-bound recombinant antigen e.g. , antigens such as those described in
  • homogeneous assay In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected.
  • Known general types of homogeneous assays proposed heretofore include (a) spin-labelled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency or polarization, (c) enzyme reporters, where antibody binding causes enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
  • spin-labelled reporters where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks)
  • fluorescent reporters where binding is detected by a change in fluorescence efficiency or polarization
  • enzyme reporters where antibody binding causes enzyme/substrate interactions
  • liposome-bound reporters where binding leads to lip
  • the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
  • the examining may involve attaching a labelled anti-human antibody to the subject antibody (for example from acute, chronic or convalescent phase) and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
  • an antigen capture assay as previously described in Section D.2.
  • Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligonucleotide synthesizers . Alternatively, custom designed synthetic oligonucleotides may be purchased from commercial suppliers.
  • H. pylori strains corresponding to ATCC Designation Nos. 43504 (short antigen clone set) and 43526 (Libraries 1 and 2) were used to generate the DNA libraries. //. pylori strain ATCC 43504 was used for isolation of native proteins produced by H. pylori. Escherichia coli strain(s) Y1088, Y1089,and XLI-Blue for libraries 1 and 2 (Stratagene, La Jolla, CA) was the host used for phage infection. E. coli strains XLI-Blue, XLOLR (Stratagene, La Jolla, CA) were used for protein expression of cloned genes.
  • H. pylori American Type Culture Collection, Rockville, MD; ATCC Designation Nos. 43504 was streaked on blood agar plates and incubated in a microaerophile environment at room temperature for 7 days. Cells were harvested by scraping the bacterial cells from 10 plates, followed by washing once with phosphate buffered saline (Dulbecco's, Gibco BRL, Gaithersburg, MD).
  • Genomic DNA was prepared as described in Ausubel et al. 1988, with minor modifications.
  • Cell pellets from 5 plates were resuspended in 510 ⁇ l of TE (Ausubel et al, 1988), to which was added 60 ⁇ l of 10 % SDS and 30 ⁇ l of 20 mg/ml of proteinase K.
  • the suspension was mixed, followed by incubation for a period from 4 - 8 hours at 37 °C.
  • To the suspension was added 80 ⁇ l of CTAB/NaCl (10% hexadecyltrimethyl ammonium bromide in 0.7 M NaCl), and the resulting solution was then mixed and incubated for 10 minutes at 65 °C.
  • the solution was then extracted with an equal volume of chloroform/ isoamyl alcohol and spun in a microcentrifuge for 5 minutes. The separated aqueous phase was transferred to a new tube, and the DNA was precipitated by addition of 0.6 volumes of isopropanol, followed by centrifugation. The DNA pellet was washed with 70% ethanol, dried briefly under vacuum and solubilized in 100 ⁇ l of distilled water. The DNA solution was then treated with DNase-free Rnase (Boehringer Mannheim, Indianapolis, IN) (Ausubel, et al, 1988; Maniatis, et al., 1982) to selectively degrade any RNA present in the sample. 2. DNase Digestion and DNA Amplification a. Short Antigen Clone Libraries.
  • H. pylori genomic DNA as described above was digested with pancreatic DNase I (Boehringer Mannheim) essentially as described in Ausubel, et al. (1988) and Sambrook, et al (1989). Aliquots of the digested DNA were taken at various time points. The DNA digests were resolved by preparative agarose gel electrophoresis. Product bands containing the desired size range of DNA (200-2000 base pairs) were excised from the gel, and recovered using the "GENE CLEAN II" kit (Bio 101 Inc., La Jolla, CA) or the "MERMAID” kit (Bio 101 Inc., La Jolla, CA), according to the manufacturer's instructions.
  • pancreatic DNase I Boehringer Mannheim
  • the recovered DNA fragments were incubated with E. coli Klenow fragment of DNA polymerase (Ausubel, et al, 1988; Sambrook, et al, 1989). The reaction mixture was incubated at room temperature for 30 minutes, followed by extraction with phenol/chloroform.
  • phosphorylated SISPA Sequence-Independent Single Primer Amplification
  • linker AB a double strand linker comprised of SEQ ID NO: 63 and SEQ ID NO: 64, where SEQ ID NO: 64 is in a 3' to 5' orientation relative to SEQ ID NO: 63 as a partially complementary sequence to SEQ ID NO:63
  • the DNA and linker were mixed at a 1: 100 ratio.
  • the linker ligated DNAs were then amplified by SISPA (Reyes, et al , 1991).
  • SISPA Reyes, et al , 1991.
  • 10 mM Tris-Cl buffer, pH 8.3, containing 1.5 mM MgCl 2 and 50 mM KCl (Buffer A) was added about 1 ⁇ l of the linker-ligated DNA preparation, 2 ⁇ M of a primer having the sequence shown as SEQ ID NO:63, 200 ⁇ M each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of Amplitaq DNA polymerase (Applied Biosystems Division, Perkin Elmer, Foster City, CA).
  • the reaction mixture was heated to 94°C for 30 seconds for denaturation, allowed to cool to 50°C for 30 seconds for primer annealing, and then heated to 72 °C for 0.5-3 minutes to allow for primer extension by Taq polymerase.
  • the amplification reaction involving successive heating, cooling, and polymerase reaction, was repeated an additional 25-40 times with the aid of a Perkin-Elmer Cetus DNA thermal cycler (Mullis, 1987; Mullis, et al, 1987; Reyes, et al, 1991; Perkin-Elmer Cetus, Norwalk, CT).
  • the ligated DNA was packaged by standard procedures using a lambda DNA packaging system (GIGAPAK, Stratagene, La Jolla), and then plated at various dilutions to determine the titer.
  • the titer of the DNA-insert phage libraries and percent recombination were determined by a standard X-gal blue/white assay (Miller, 1994; Maniatis et al, 1982).
  • the titer of the recombinant libraries ranged from 1.5 x 10 4 to 3 x 10 6 PFU/ml.
  • Percent recombination in each library can also be confirmed by selecting a number of random clones and isolating the corresponding phage DNA.
  • Polymerase chain reaction (Mullis, 1987; Mullis, et al, 1987) is then performed using isolated phage DNA as template and lambda DNA sequences, derived from lambda sequences flanking the EcoRI insert site for the DNA molecules, as primers. The presence or absence of insert is then evident from gel analysis of the polymerase chain reaction products.
  • the lambda gtl l and ZAPII phage libraries described in l.C. above were immunoscreened for the production of antigens recognizable by a pool of sera from 11 patients (designated herein as
  • the fusion proteins expressed by the recombinant lambda phage clones were screened with serum antibodies essentially as described by Ausubel, et al. (1988).
  • Each library was plated at approximately 1.5 to 2 x 10 4 phages per 150 mm plate. Plates were overlaid with nitrocellulose filters overnight. Filters were washed with TBS (10 mM, Tris pH 7.5; 150 mM NaCl), blocked with AIB (TBS buffer with 1 % gelatin) and incubated with a primary antibody diluted 100 times in AIB.
  • TBS 10 mM, Tris pH 7.5; 150 mM NaCl
  • AIB TBS buffer with 1 % gelatin
  • SFA001 is a pool of 4 donor sera (plasma packs) showing strong reactive bands on Western blot format with a crude lysate antigen preparation from Helicobacter lysate using in HelicoBlot 2.0.
  • NAA121 is a pool of 4 donor sera (plasma packs) showing no reactive bands on Western blot format with the crude lysate antigen preparation from Helicobacter lysate used in HelicoBlot 2.0.
  • H. pylori cloned families were isolated by PCR amplification of representative clones. Cloned sequences having an .ASM extension were sequenced completely; sequences with a .SEQ extension were partially sequenced.
  • the DNA inserts of the immunoreactive recombinant lambda clones were PCR amplified using primers corresponding to lambda arm sequences flanking the EcoRI cloning site of the vectors. Amplification was carried out by polymerase chain reactions utilizing each immunoreactive clone as template.
  • gtl IF S ⁇ Q ID NO:65
  • gtll R S ⁇ Q ID NO:66
  • primers T3 S ⁇ Q ID NO:67
  • T7 S ⁇ Q ID NO:68
  • the resulting amplified fragments were then agarose gel purified and eluted from the gel (Ausubel, et al, 1988).
  • the PCR products were further purified by "WIZARD PCR PR ⁇ PS” (Promega, Madison, WI) or "CHROMASPIN” columns (Clonetech, Mountain View, CA) to remove primers and other ingredients.
  • the purified insert DNA was then subjected to direct sequencing. In some cases, the insert DNA was first subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and then sequenced.
  • Sequence determination for the DNA inserts was carried out using a Perkin Elmer Applied Biosystems 373A DNA sequencer (Perkin Elmer, Applied Biosystems Division, Foster City, CA) according to the manufacturer's protocol (dideoxy chain terminator sequencing methodology. Sanger, et al , 1977).
  • Clone Y104-1 (SEQ ID NO:60) contains the entire d7 clone, and encodes all of the 36K peptides and all of the spot 15 peptides, as indicated in Fig. 3.
  • Example 5A and 5B The production of expressed antigenic proteins and their subsequent purification is generally described in Examples 5A and 5B.
  • a tabular summary indicating clone name, expression, purification, and panelling data is provided in Table 3b, and a summary of the immunoreactivy of various recombinant antigens is provided in Table 5b.
  • Expression profiles were obtained, and the immunoreactivity of various clones to H. pylori positive pooled sera such as "Roost" was confirmed.
  • amplified products corresponding to a particular ORF were typically cloned into a pGEXhisB vector and expressed in E. coli. The size of the expression product was then determined, followed by confirmation of immunoreactivity (e.g., with Roost pool sera).
  • Example 3 Additional immunopositive clones, as described in Example 2 above, were purified and analyzed for DNA insert size and expressed protein size as in Example 3 above.
  • the ZAPII clones were rescued into plasmids from phagemids (as per protocol in Stratagene, La Jolla, U.S.A.); the resulting insert DNA was excised with EcoRI and ran on agarose gel to determine size.
  • Slightly longer versions of T3 and T7 (GD 60 and GD 61, corresponding to SEQ ID NO:231 and SEQ ID NO:232 respectively) were then used as primers for sequencing.
  • libraries correspond to the "library 1 " series (EcoRI-cut), i.e., clones A3, A22, B2, B9, B17, B23, Cl, C3, C7, and "library 2" series clones (Hind cut), i.e., clones al, a3, a5, b5, b8c7, c2, c5, cl3, d5, d6, d7, dll, fll, e6, f3, f8, g2, g9, gll, and k4; and also clone Gla from the EcoRL/Xbal cut library. Many clones were determined to be larger than the predicted coding regions of the proteins.
  • the specific antigen coding sequences from H. pylori library 1 and library 2 clones were subcloned.
  • the subclones were typically prepared by fragmenting the corresponding genomic clone by specific restriction endonuclease digestion to produce a specific subfragment or subfragments, which were purified using "G ⁇ N ⁇ CLEAN II" kit (Bio 101 Inc., La Jolla, CA) or the "MERMAID” kit (Bio 101 Inc., La Jolla, CA).
  • the resulting DNA fragments were then inserted into a suitable expression vector, i.e., the PB/Bluescript (SK) vector (Stratagene, La Jolla, CA).
  • Immunoreactive DNA regions were then sequenced and locations of the open reading frames were determined. Unless otherwise indicated as a 0-galactoside fusion product, for each of the clones in Tables 3a and 3b below, expression of coding regions was determined to be driven by the corresponding H. pylori promoter rather than by the /3-galactosidase gene promoter in lambda gtl 1. LIBRARY 1 CLONES 2. Clone A3
  • the corresponding nucleotide sequence (1878 bp) is presented herein as SEQ ID NO: 16.
  • the open reading frame (ORF) extends from nucleotides 399-1743, and codes for a putative protein containing 448 amino acids (443 amino acids when calculated from the first methionine).
  • the protein sequence corresponding to the translation of open reading frame (ORF) 399-1743 is presented herein as SEQ ID NO: 17.
  • Subclone A22210DMIC was obtained from an original genomic clone having an insert size of about 2.2 kb, to produce a /3-galactosidase fusion protein in E. coli.
  • the open reading frame extends from nucleotides 12-599 of SEQ ID NO:43, where bases 12-121 correspond to the /3-galactosidase fusion peptide and bases 122-599 code for a unique A22 antigenic sequence.
  • the translation sequence for the corresponding protein is presented as SEQ ID NO:44.
  • B3C19 is a subclone obtained from the 5.0 kb insert in genomic clone B3 as follows.
  • the insert was excised, digested with DNase, followed by treatment with T4 DNA ligase and Klenow enzyme to produce blunt-ends.
  • the blunt-ended short DNA pieces were then ligated to kinased A/B linkers (linker A, SEQ ID NO:63, corresponding to the top strand of AB SISPA linker; linker B, SEQ ID NO:64, corresponding to the bottom strand ot AB SISPA linker), PCR amplified with primer A, and the amplified products digested with EcoRI.
  • the digested products were then ligated into EcoR ⁇ - digested lambda vector ZAPII.
  • the DNA sequence of B3C19 is presented as S ⁇ Q ID NO:51.
  • primers GD77 (5' primer, S ⁇ Q ID NO:52) and GD80 (3' primer, S ⁇ Q ID NO:53) were designed to walk back and forward the sequence of B3C19 using genomic B2 DNA as the template.
  • the sequence of clone B3C19 was extended in both the 5' and 3' directions, resulting in B2 extension clone, B2197780 (S ⁇ Q ID NO:54).
  • a computer-generated protein translation of the B2197780 sequence resulted in a corresponding 291 amino acid putative protein, with a predicted transmembrane segment from amino acids 9-25 (predictions obtained using "SOAP" program from "PCG ⁇ N ⁇ ").
  • Subclone B9.4C is a 1.2 kb Hindlll fragment obtained from original genomic clone B9 (4.5 kb insert) as follows.
  • Subclone B9.4C corresponding to a 1.2 kb Hindlll subfragment of genomic clone B9, produced an immunoreactive protein of 32 kd.
  • the nucleotide sequence of subclone B9.4C is presented as SEQ ID N0:47; the translated protein sequence ⁇ i.e. , nucleotides 230-931 of SEQ ID N0:47) is presented as SEQ ID NO:48.
  • SEQ ID NO:47 an AGGA Shine-Dalgarno sequence occurs at nucleotide 218, and the first amino acid of the translated protein sequence begins with a GTG codon for valine at position 230.
  • the predicted molecular weight of the corresponding protein is 25.2 kd, with a pi of 10.35.
  • a potential cleavage site occurs between amino acid positions 225 and 226.
  • clone B9 appears to code for the 50A LI protein of H. pylori.
  • Clone B17 B17CON4 (2006 bases) was subcloned from a genomic clone having an insert size of about
  • the corresponding nucleotide sequence is presented as SEQ ID NO: 24.
  • the open reading frames (ORF) correspond to the following regions of SEQ ID NO:24: ORF1 (nucleotides 500-700); ORF2 (nucleotides 870-1406); ORF3 (nucleotides 1410-2000); and ORF4 (nucleotides 142-705).
  • the corresponding translated protein sequences are presented herein as SEQ ID NO: 25 (B170RF4), SEQ ID NO:26 (B170RF1), SEQ ID NO:27 (B170RF2) and SEQ ID NO:28 (B170RF3). Based upon nested deletion experiments, it appears that the immunoreactive protein corresponds to B170RF4.
  • B170RF4, B170RF2 and B170RF3 encode putative proteins having predicted sizes of 20.9 kd, 20 kd and 22.2 kd respectively.
  • the predicted size of the immunoreactive genomic protein is a doublet of 22 kd and 23 kd. 7.
  • Immunoreactivity was determined to reside in a 3.5 kb Pstl subclone (one Pstl site is derived from vector pBSK) of original genomic clone B23 (5.5 kb).
  • the 3.5 kb subclone was further sequenced, to determine the immunoreactive coding sequence presented in SEQ ID NO: 13.
  • the nucleotide 1078 base pair sequence (SEQ ID NO: 13) was determined to be open all the way (bases
  • the translated protein sequence is presented as SEQ ID NO: 14.
  • C1CON6V2 was subcloned from an original genomic clone having an insert size of about 4.0 kb.
  • the sequence information obtained from exo-mung deletion clones is presented as SEQ ID NO:38.
  • the open reading frame (ORF) extends from nucleotides 868-1926.
  • the subcloning of the antigen coding sequence from the Cl clone into an expression vector, and characteristics of the corresponding protein product are presented in Example 5 below.
  • the translated protein sequence is presented herein as SEQ ID NO:39.
  • C3 (2.9 kb insert size)
  • several different subfragments of the genomic clone were obtained and sequenced.
  • the immunoreactive clone was determined to reside in an EcoRI/Kpn 2.2 kb subclone. Based upon a number of subcloning experiments and subfragments, the C3 DNA sequence is presented as S ⁇ Q ID
  • Clone C7.2C is a EcoRI/ Hindlll subclone obtained from genomic clone C7.
  • the original genomic C7 clone is a EcoRI clone of approximately 3.8kb size that produces an immunoreactive protein having a molecular weight of approximately 30 kd.
  • Subclone C7.2C was obtained as follows. The genomic C7 clone was digested into individual fragments using Hindlll. Each DNA restriction fragment was then subcloned into the pBKS vector via either the Hindlll site, or alternatively, for end-piece fragments, via the EcoRI/ 'Hindlll sites. The corresponding nucleotide sequence of C7.2C (616 base pairs) is presented as S ⁇ Q ID NO: 20. Subclone C7.2C produces an immunoreactive protein which is the same size as that produced by the genomic clone. The translated protein sequence for bases 1-561 of clone C7.2C is presented herein as S ⁇ Q ID NO :21.
  • Clone B8 Clone B8 (SEQ ID NO:549) is a genomic clone with an insert size of 3.5 kb that produces an immunoreactive protein of about 50 kd on SDS Western blot.
  • the DNA sequence of B8 overlaps with that of clone C7 (clone C7.2C) which was a fusion protein with beta-galactosidase.
  • Clone B8 added on 266 additional amino acids 5' (upstream) to C7 and contains the beginning of the gene. However, B8 ends 64 amino acids before the C-terminus of the gene, which is also encoded by C7. Based on the above, the complete sequence of the full gene was compiled (SEQ ID NO:549).
  • the corresponding protein sequence from the compiled gene sequence codes for a putative protein of 48.9 kd, and is presented herein as SEQ ID NO: 550.
  • Clone al is a Hindlll clone that produces an immunoreactive protein of 28-30 kd on SDS gel.
  • the corresponding DNA sequence encoding an antigenic protein contains 1208 nucleotides (SEQ ID NO:35). There are two open reading frames.
  • ORFl extends from bases 53 - 801 of SEQ ID NO: 35, and encodes a putative protein containing 249 amino acids (SEQ ID NO:36, translated protein).
  • ORFl contains a Shine-Dalgarno sequence extending from nucleotides 43-46 ("AGGA" sequence). The predicted molecular weight of the protein is 27.5 kd, which is in agreement with the expected protein size based upon SDS-PAGE (above).
  • the putative protein for ORFl has a calculated pK of 8.42 and a predicted transmembrane region extending from amino acids 1-19.
  • the second ORF contained in clone al extends from nucleotides 880-1206 and ends at one of the Hindlll sites, which indicates that clone al is a partial clone.
  • the predicted size of the partial protein is 109 amino acids (SEQ ID NO: 37).
  • Immunoreactive clone a3 (not to be confused with clone A3 from library 1) is a ⁇ - galactosidase fusion clone with an insert size of 1975 base pairs. The corresponding nucleotide sequence corresponds to SEQ ID NO: 248.
  • the clone contains two open reading frames (ORFs): ORFl (nucleotides 3-608, SEQ ID NO:249) and ORF2 (nucleotides 613-1266, SEQ ID NO:250). Based upon the observed size of the immunoreactive protein, i.e. , 30 kd, the expected immunoreactive-expressing open reading frame is ORFl . Expression of a3 protein is described in the following section.
  • Clone a5 is an original Hindlll/ Eco ⁇ l genomic clone (1 kb) that produces a 25 kd immunoreactive protein.
  • the corresponding nucleotide sequence is presented as SEQ ID NO: 3.
  • the clone contains two open reading frames, (ORFs): ORFl (nucleotides 3-545, SEQ ID NO: 5) which codes for a beta-galactosidase fusion protein, and ORF2 (nucleotides 569-1021, SEQ ID NO:4).
  • ORFs open reading frames
  • Clone b5 contains a double insert with an internal EcoRI site.
  • the individual inserts are about 0.3 and 0.2 kb in length.
  • the combined nucleotide sequence for clone b5 corresponds to S ⁇ Q ID NO:6, with the open reading frame (ORF) extending from nucleotides 1 through 414.
  • the corresponding protein sequence translation is presented as S ⁇ Q ID NO:7.
  • Clone c2 is a library 2 clone with an insert size of 2077 nucleotides (S ⁇ Q ID NO:251).
  • the size of the immunoreactive protein on SDS-gel is 30kd.
  • the corresponding protein sequence translation is presented as S ⁇ Q ID NO:253.
  • the protein has a predicted pi of 9.54, with a potential transmembrane region between amino acids 2 through 24.
  • the predicted molecular size of this c2 protein is 30.2kd, which agrees with the observed size on SDS gel.
  • Clone c5 is a 650 base pair Hindllll Hindlll clone (S ⁇ Q ID NO:253). Clone c5 is a partial clone which is not in phase with the /3-galactosidase gene, and contains a 29 amino acid stretch extending into pBluescript at the N-terminus.
  • the ORF extends from bases 2-604, with a predicted protein size of 23.5kd (S ⁇ Q ID NO:254).
  • the observed protein size on SDS-gel is 30kd.
  • the putative protein has a pi of 9.6.
  • Clone c!3 Clone cl3 is a Hindlll/ Hindlll clone with an insert size of 1742 b.p. (S ⁇ Q ID NO:255). It is a /3-galactosidase fusion protein with an observed size of 55kd on SDS gel. The ORF extends from bases 2 to 1420. The corresponding protein possesses a predicted molecular weight of 54.6kd (S ⁇ Q ID NO:256). A sequence search using SWISS pro database indicates that the sequence possesses homologies with threonyl-tRNA synthetase of various organisms, from bacteria to yeast and human. 19. Clone d5
  • Clone d5 is an EcoRI clone that produces an immunoreactive protein (/3-galactosidase fusion protein) of about 70 kd on SDS-PAG ⁇ gel.
  • the size of the cloned insert was determined to be 1795 bases (S ⁇ Q ID NO:58), with an ORF from extending from nucleotides 1-1704.
  • the open reading frame codes for a putative protein composed of 568 amino acids (S ⁇ Q ID NO:59), and having a predicted molecular weight of 62.1 kd.
  • the putative protein has a predicted pK value of 5.1, and the predicted antigenic determinant lies in the N-terminus region of the protein.
  • Clone d7 is a Hindlll clone that was blunt-ended, ligated to A/B linkers, digested with EcoRI, and subcloned into lambda vector ZAPII as described above for Example 4.1.
  • the product is a ⁇ - galactosidase fusion protein.
  • the nucleotide sequence is presented as SEQ ID NO:ll.
  • the induced protein was determined to have sizes of 40 kd and 35 kd.
  • the corresponding protein sequence translated from SEQ ID NO: 11 is presented as SEQ ID NO: 12.
  • Clone O is a EcoRI clone having an insert size of 2274 nucleotides (S ⁇ Q ID NO:257).
  • Clone f3 produces a /3-galactosidase fusion protein.
  • the ORF extends from nucleotides 1-1788 and codes for a putative protein having a predicted molecular size of 67 kd (excluding the /3-galactosidase portion) (S ⁇ Q ID NO:258).
  • the calculated pi of the protein is 9.66.
  • Streptococcus pneumoniae A search against the ⁇ MBL DNA data base reveals a 56% homology with penicillin binding proteins of Haemophilus influenza.
  • the size of the protein observed on SDS-gel is 25kd (major band), with a minor band of around 67kd. This size discrepancy may possibly be due to cleavage of the protein to produce a smaller fragment.
  • F8CON1 is a Hindlll subclone that produces an immunoreactive protein of 33-35 kd on SDS gel.
  • the DNA sequence corresponding to the f 8 subclone is presented herein as S ⁇ Q ID NO: 1.
  • the cloned 1459 base DNA subfragment contains an open reading frame (ORF) from nucleotides 134 - 1042.
  • the putative protein encoded by the f8 subclone contains 303 amino acids, with a predicted molecular weight of 33.9 kd (which is in agreement with the expected protein size).
  • the predicted antigenic determinant is at the 3' end of the putative protein - extending from amino acid 270 to amino acid 275 (nucleotides 941-958).
  • the putative protein has a pK of 9.75.
  • the corresponding protein sequence based upon a translation of SEQ ID NO:l is presented as SEQ ID NO:2.
  • Clone g2 is a Eco /Hindlll clone with an insert size of 2474 nucleotides (S ⁇ Q ID NO:259).
  • ORFl extends from nucleotide 2 to 445 and codes for the carboxyl end of a putative protein of 148 amino acids (S ⁇ Q ID NO:260).
  • ORF2 extends from bases 461-1156, coding for a putative protein of molecular size 25.4 kd (S ⁇ Q ID NO:261).
  • ORF3 extends from bases 1156 to 1776 and codes for a protein having a size of about 22.2 kd (S ⁇ Q ID NO:262).
  • ORF4 (nucleotides 1798-2472) codes for the amino terminus of a putative protein of 24.4 kd (S ⁇ Q ID NO:263). The predicted pis of ORF2 and ORF3 are 7.82 and 5.26, respectively. While ORF2 and ORF3 putative protein does not contain predicted transmembrane regions, ORF4 putative protein contains 3 predicted transmembrane regions with 6 predicted transmembrane helices.
  • Clone g9 possesses an insert size of 4292 bases. Two subclones, k4 and gll, are contained within clone g9. Clone g9 produces an immunoreactive protein band of about 85kd, whilst k4 has immunoreactive bands of 55 kd, 46 kd, 35 kd and 30 kd and gl 1 produces a weakly immunoreactive band of 22 kd.
  • the complete sequence of g9 was obtained by walking in the 5' direction from the gll C-terminus sequence, and walking in the 3' direction from k4 N-terminus sequence (S ⁇ Q ID NO:264).
  • Clone g9 has 5 ORFs.
  • ORFl encodes a partial protein in the region extending from bases 2-349 (S ⁇ Q ID NO:265). The corresponding predicted molecular size of the protein is 12.3 kd.
  • ORF2 extends from bases 495-1403 (S ⁇ Q ID NO:266), and the corresponding predicted protein possesses a molecular size of 33.8 kd.
  • ORF2 is followed by a "AGGA" Shine-Dalgarno sequence, positioned in front of the ATG of ORF3 (bases 1418-2209).
  • the predicted molecular size for the putative protein encoded by ORF3 is 29.7 kd (S ⁇ Q ID NO:267).
  • ORF4 corresponds to bases 2223-3719.
  • the protein encodes by ORF4 has a predicted molecular size of 56.8 kd (SEQ ID NO:268).
  • ORF5 extends from nucleotides 3133 to 4236, with a predicted protein molecular size of 19.9 kd (SEQ ID NO:269).
  • H. pylori antigen coding regions Amplified products from various clone families, e.g., A3, A22, B2, B9, Cl, C5, C7, and Gla, were cloned into pGEX vectors (Pharmacia). The cloned constructs were expressed in E. coli strain (Stratagene, LaJolla, CA).
  • the protein was purified by affinity purification chromatography, employing Ni-NTA+ + resin from Qiagen (Chatsworth, CA).
  • the purified protein was paneled against various sources of H. pylori-ir ⁇ ecte ⁇ sera.
  • the serum paneling was carried out twice, with differing results: (i) 75% sensitivity, based upon 35 sera samples, and (ii) sensitivity of about 30% , based upon 183 sera.
  • the serum panel was performed using protein that had undergone further SDS gel electroelution following affinity chromatography purification.
  • Amplified products from clone C1CON6V2 were cloned into expression vector pGEXdl65 using primers BF (SEQ ID NO:40) and BG (SEQ ID NO:41) as outlined in Table 5 below.
  • the expressed protein was confirmed to be immunoreactive; however, it was cleaved beyond the transmembrane region to form a smaller immunoreactive band. Based upon this observation, a new forward primer was designed for the expression of the smaller protein, primer BO (SEQ ID NO:42), as indicated in Table 5.
  • the resulting protein was determined to be immunoreactive, and formed in much higher yields than in the previous construct.
  • Expression primers CA forward primer, SEQ ID NO:29
  • CB reverse primer, SEQ ID NO:30
  • Expression primers CC forward primer, SEQ ID NO:31
  • CD reverse primer, SEQ ID NO:32
  • Amplified products from the genomic A3 DNA were digested with NcollBamHl restriction enzymes and subcloned into the Ncol and BamHl sites of expression vector pGEXdl65 polyHis, using primers BP (SEQ ID NO: 18) and BQ (SEQ ID NO: 19) respectively.
  • Expression of protein was induced at 0.5 mM IPTG (isopropylthiogalacto-pyranoside). The expressed protein was of the expected size, and was confirmed to be immunoreactive against Roost pooled sera.
  • PCR amplified products from H. pylori genomic DNA produced a DNA fragment of the expected size, which was then subcloned into the NcollBamHl sites of expression vector pGEXdel65 using primers GD100 (SEQ ID NO:22) and GD101 (SEQ ID NO:23) respectively.
  • the expressed protein was of the expected size, and was highly insoluble. The protein was readily purifiable by Ni-NTA column chromatography as described above. Immunogenic screening confirmed the expressed protein to be immunoreactive against Roost pooled sera.
  • PCR amplified products were subcloned into expression vector pdell65polyHis using primers BW (SEQ ID NO:33) and BX (SEQ ID NO:34) as outlined in Table 5 below.
  • the expressed protein was confirmed to be immunoreactive. However, the expression product was internally cleaved, with cleavage most likely occurring at the transmembrane region.
  • a pellet from a shake flask was spun and then submitted to differential solubilization. Briefly, the pellet was homogenized in a solution containg PBS/5mM PMSF, and spun for 60 minutes at 4°C, 30k. Subsequent rounds of homogenization/centrifugation were as follows: (i) 100 mM Tris/2% Triton/2M Urea/5mM EDTA/0.5 mM DTT (homogenization step)/60 minutes at 4°C, 30k (centrifugation); (ii) PBS pH 7.8/2M Urea/0.5 mM DTT/ 60 minutes at 4°C, 30k; (iii) PBS pH 7.8/4M Urea/0.5 mM DTT/60 minutes at 4°C, 30k, (iv) PBS pH 8.0/6M Urea/0.5 mM DTT/60 minutes at 4°C, 30k; (v) PBS pH 8.0/6M urea/2M guanidine HCL/2mM BME /60 minutes
  • step (iv) above was then dialyzed into PBS pH 8.0/6M Urea/2mM BME, followed by chromatographic separation using a pre-packed column of Chelating Sepharose
  • 8.0/6M Urea/2mM BME and Nickel IMAC Buffer B contained Buffer A and 250 mM imidazole.
  • Nickel IMAC fractions were pooled and dialyzed overnight into PBS pH 8.0/6M Urea/0.5mM DTT, and the final product was then vialed and stored at -80°C.
  • glutathione-sepharose packed columns (Sigma) were utilized.
  • Protein expressed by clone dHA22.8 (corresponding to clone A22) was isolated and purified as follows. 2000 ml of culture pellet was suspended in 200 ml of Buffer B (48g urea, 1.2 g NaH 2 P0 4 ,
  • the resin was washed with 50 ml of Buffer C (48 g urea, 1.2 g NaH 2 P0 4 , 0.12 Tris-HCl and 90 ml deionized water, adjusted to pH 6.3 and brought to a total volume of 100 ml), centrifuged, and the supernatant discarded. The resin was then loaded into a disposable column, and the wash step repeated with remaining Buffer C. The protein was then eluted with 50 ml of Buffer III (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 250 imidazole). Fractions were collected (2 ml) and analyzed on 12% SDS-PAGE gel.
  • Buffer C 48 g urea, 1.2 g NaH 2 P0 4 , 0.12 Tris-HCl and 90 ml deionized water, adjusted to pH 6.3 and brought to a total volume of 100 ml
  • the resin was then loaded into a disposable column, and the wash step repeated with remaining
  • the purified protein was then screened with serum antibodies using conventional techniques (Ausubel, et al, 1988).
  • the purified protein was slotted at concentrations of 0.1-20 ⁇ g/ml 0.1M carbonate buffer, blocked with 5 % skim milk in TBS buffer, and reacted with H. pylori positive and H. pylori negative sera diluted 100 times.
  • a serum panel consisting of 36 total sera was used as indicated above. Positive sera was from Roost pool #2 or SFA 001 pool; negatives were from donor packs.
  • the strips were washed 3 times with TBS buffer, and incubated with alkaline phosphatase-conjugated anti-human Ig secondary antibodies (Promega Biotech, Madison, WI) diluted 1000 times in 5% skim milk in TBS buffer. The strips were then washed 3 times with TBS wash buffer. Immunoreactive proteins were developed with a substrate, e.g. , BCIP, (5-bromo-4-chloro-3-indolyl-phosphate), and NBT (nitro blue tetrazolium salt (Sigma)). The development of color indicated the highly antigenic nature of the purified A22 protein (i.e. , reactive with H. pylori-positive sera).
  • a substrate e.g. , BCIP, (5-bromo-4-chloro-3-indolyl-phosphate)
  • NBT nitro blue tetrazolium salt (Sigma)
  • the optimum concentration of antigen was determined to be 2.0 mg/ml.
  • the dHA22.8 (A22) expression product reacted with anti-H. pylori antibodies present in both pooled sera sources (Roost pool #2 and SFA 001 pool), and with 100% of the individual H. pylori-positive sera samples tested.
  • the antigen exhibited no detectable cross reactivity with normal sera.
  • Protein expressed by clone dHClS. l l (Cl) was isolated and purified according to the following protocol.
  • Protein was extracted from a culture pellet by differential solubilization using a series of homogenization/centrifugation steps.
  • the pellet was (i) homogenized in PBS/1 mM PMSF and centrifuged for 30 minutes at 4°C, 19,000 rpm, followed by separation of the supernatant, (ii) homogenized using 1 M urea/ 10 mM Tris at pH 8.0/10 mM DTT, followed by centrifugation as in (i) and separation of the supernatant; (iii) homogenized in 4 M urea/10 mM Tris pH 8.0/2 mM BME, followed by centrifugation as in (i) and separation of the supernatant; and finally, (iv) homogenization of the pellet with 6 M urea/lOmM Tris pH 8.0/2 mM BME, followed by centrifugation as described above.
  • the protein was then separated from the combined supernatants by immobilized metal adsorption chromatography (IMAC) using a 5 ml prepacked column containing chelating sepharose (Pharmacia, Chelating Sepharose Fast Flow) loaded with 2 C.V. (column volumes) 0.2 M NiCl 2 .
  • the protein was eluted from the column using a 20 C.V. gradient of Buffer A (4 M urea/10 mM Tris at pH 8.0/0.2mM BME/150 mM NaCl) into 50% Buffer B (Buffer A to which was added 0.5 M imidazole). Fractions were collected (2 ml) and analyzed on 12% SDS-PAGE gel.
  • the optimal concentration for immunoscreening was determined essentially as described above. Serum panelling as described above was repeated for the purified Cl protein expressed by clone dHClS.il .
  • the expressed protein was reactive with both sources of pooled H. pylori-positive sera, and showed no signs of cross reactivity with control sera (H. pylori negative samples, 1 sample of pooled sera and 13 individual samples). Additionally, as can be seen in panels
  • the recombinant protein exhibited immunoreactivity with 95 % of the H. pylori- positive samples.
  • Gold standard tests were employed as reference standards to compute the sensitivities and specificities of each individual antigen.
  • Standards used were conventional gold standard tests including histology, CLO test, and the UBT test.
  • UBT is the current most widely used gold standard for indication of "active infection” by non-invasive methods. Histology, CLO (rapid urease test) and UBT are all indicate "active infection” since these tests depend on the presence of bacteria to produce a positive result. Since serology measures both past and present infection, the aim of this study was to (i) identify serological markers that would correlate with UBT results for use as "active infection markers", and (ii) to explore both single and muliple antigen combinations to effectively screen for active infection by H. pylori.
  • POW Panel Based upon this paneling study, the best single antigen selection appeared to be Y128D12S and Y124A, where sensitivity and specificity were about 75-80% for Y128D12S, and both about 80% for Y124A. Another preferred antigen is Y261A, which exibited a sensitivity of 70% and a specificity of 80% . For this paneling series, preferred clones A22 and Cl were found to be about 95-98% sensitive, and about 60% specific.
  • the 2-4 antigen format was based on the criteria that, for any 2 -3 highly sensitive antigens, at least both or all 3 have to be positive with respect to the criteria to increase the specificity.
  • This "2 antigen both positive” criteria was applied to a selected 12 antigen set, where the 12 antigens were selected for a sensitivity of at least 40-50% for consideration.
  • the sensitivities and specificities of the 2 antigen both positive criterion is computed, and the resulting table is examined for good performers. Additionally, 2 antigen combinations with high specificity and lower selectivity is run against the entire two antigen combination matrix to provide a result indicating "2 antigen both positive or 2 other antigen both positive”. The final analysis then provides a selection of commonly occurring antigens that provide good results across the board between the USC and POW panels.
  • an antigen combination of Cl and c5 was preferred, and for the POW panel, antigens combinations Y261A and Y124A or antigens C7 and B2 also were preferred.
  • preferred antigens for use in reliably and universally detecting H. pylori infection include but are not limited to the following: A22, Cl , Y124A, Y261A, c5, C7, B2, Y104B, and Y128D.
  • H. pylori ATCC No. 43504
  • Prefractionation of soluble H. pylori lysate supernatant was carried out only when enriching for spot 15 (high pH), for subsequent sequence/structural investigations (e.g. , mass spectrometry, peptide map).
  • Fractionation of the spot 15 antigen was carried out in a similar fashion.
  • the sample was passed over Sephacryl S-100 ion exchange resin (Pharmacia Biotechnology, Piscataway, NJ) (to 10 mM phosphate buffer, 50 mM NaCl, pH 8.0), followed by further fractionation by gradient elution over Resource S cation exchange resin (Pharmacia Biotechnology, Piscataway, NJ).
  • the fraction containing spot 15 antigen was analyzed by SDS-PAGE and Western blot, and was similarly confirmed to be highly antigenic in nature, as indicated by Western blot and immunoreactivity with Roost pooled sera.
  • Two-dimensional electrophoresis was performed essentially as described by O'Farrell (1975). Isoelectric focusing was carried out in glass tubes having an inner diameter of 2.0 mm, using 2% ampholines (BDH, Hofer Scientific Instruments, San Francisco, CA) for 9600 volt-hrs. The final tube gel pH gradient as measured by a surface pH electrode is on the enclosed pH gradient form.
  • the following proteins were added as molecular weight markers to the agarose which sealed the tube gel to the slab gel: mysin (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydrase (29 kD), and lysozyme (14 kD). These standards appear as horizontal lines on the silver-stained (Oakley, et al, 1980) 10% acrylamide slab gels. The silver stained gel was dried between sheets of cellophane paper with the acid edge to the left.
  • the blot was blocked for 2 hours in 2% bovine serum albumin (BSA) in TTBS (Tween-Tris-Buffered Saline), rinsed in TTBS, incubated in primary antibody from Roost pool or negative serum pool and diluted 1:2500 in 1 % BSA/TTBS for 2 hours, rinsed in TTBS and placed in a solution containing secondary antibody (antihuman IgG horse radish peroxidase, 1:5000 diluted in TTBS) for 1 hour.
  • the blot was rinsed with TTBS, treated with ECL (Amersham Corporation, Arlington Heights, IL), and exposed to X-ray film.
  • FIG. 2 A computer-generated photograph of an exemplary stained membrane containing antigenic proteins from H. pylori as described above is shown in Figure 2.
  • the "normal” human sera was confirmed to be H. pylori-negative using "HELICOBLOT 2.0" (Genelabs Diagnostics (PTE) Ltd.,
  • the tube gel was sealed to the top of the stacking gel, which was placed on top of a 10% acrylamide slab gel (0.75 mm thick), and SDS slab gel electrophoresis was carried out for 4 hours at 12.5 mA/gel.
  • the slab gels were fixed in a solution of 10% acetic acid/50% methanol overnight.
  • the following proteins were added as molecular weight standards to the agarose which sealed the tube gel to the slab gel: myosin (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydrase (29 kD) and lysozyme (14 kD). These standards appear as horizontal lines on the silver stained (Oakley, et ⁇ l, 1980) 10% acrylamide slab gels. The silver stained gel was dried between sheets of cellophane paper with the acid edge to the left.
  • a duplicate gel was transblotted onto PVDF paper and Western blotting carried out as described in 8.1.b. above.
  • the PVDF blots from Example 8 above were stained with Coomassie brilliant blue. Spots corresponding to Western positive bands were excised by scalpel and sequenced directly using a Hewlett-Packard G 1005 A N-terminal sequencer with conventional sequencing techniques ⁇ e.g. , Miller, 1994; Spiecher, 1989). The sequencing techniques employed gave high repetitive yields (typically ranging from 93-98%), with a detection limit of approximately 100-200 fmol.
  • Lys-C peptide map and sequencing were utilized to obtain internal sequence information for the above- described isolated antigens of H. pylori (Allen, 1981): Lys-C peptide map and sequencing, CNBr peptide map and sequencing, OPA/CHBr peptide map and sequencing, and LC-MS/MS sequencing of Lys-C digests.
  • Cyanogen bromide (CNBr) cleavage was performed on PVDF membranes in 70% formic acid (Crimmins and Mische, 1996). The cyanogen bromide digested peptides were then either repurified by capillary HPLC and sequenced directly, or subjected to ortho-phthalaldehyde (OP A) modification.
  • OP A ortho-phthalaldehyde
  • LC-MS Liquid chromatography-mass spectrometry
  • a Carlo Erba Phoenix 20 CU pump was used to deliver a mixture of methoxy ethanol and isopropanol (1:1, v/v) at a rate of 50 microliters per minute, which was combined with the column eluent in a post column mixing chamber.
  • An in-line flow splitter was used to restrict flow to the mass spectrometer to approximately 10 microliters/minute. Detection was performed immediately following elution from the column at 214 nanometers using an ABI 759 variable wavelength detector. Mass spectrometric detection was achieved following post column solvent addition and flow splitting by a VG BioQ triple quadruple mass spectrometer with a nano-electrospray ion source.
  • Spectra were recorded in the positive ion mode using electrospray ionization. Calibration of the instrument was performed in the range m/z 500-2000 by using direct injection analysis of myoglobin. Spectra were recorded at 1.5 seconds intervals and a drying gas of nitrogen was used to aid evaporation of the solvent. The capillary voltage was maintained at approximately 4 kV with a source temperature of 60°C.
  • spots 9, 11, 12, 13, 15, 16 (major and minor), and 17 represent unique antigens.
  • Spot 9 represents the native H. pylori antigen corresponding to the recombinant protein expressed by ORF2 of clone al
  • spot 12 represents the native antigen that corresponds to the antigenic protein encoded by clone a5.
  • spots 9 major and minor
  • 10, 12, 13, and 15 represent unique antigens.
  • the N-terminus of the spot 15 antigen high pH
  • 2-dimensional gel analysis was later shown to be inaccurate.
  • the H. pylori spot 15 antigen as described above was isolated from 3 different sources of H. pylori as described in Examples 8 and 9.
  • the H. pylori samples used were the following: H. pylori, ATCC 43504 (Australian type strain); H. pylori, strain # 26695, and H. pylori, J-170, (both obtained from Washington University School of Medicine, St. Louis, MO).
  • the corresponding protein isolated from each source was then analyzed by reverse phase high performance liquid chromatography (HPLC) as described in Example 9.
  • the spot 15 antigen appears to be a processed product of the putative 36 kD protein, as shown in Fig. 4.
  • H. pylori strains tested were the following: Chico (clinical isolate from Oroville
  • J170, A-lc Lithuanian isolate, which contains at least 6 kB of DNA referred to as "X" segment as an insertion near cag region and not present in C-3c
  • Rus-95 isolated from Russian immigrant to the United States
  • #9 Peruvian isolate
  • C-3c Lithuanian isolate
  • ATCC 45304 are highly conserved between various strains of H. pylori.
  • Western blot results further supported the highly antigenic nature of this protein.
  • Gel pieces stained with Coomassie or compatible silver stain as described in Example 9 were transferred to a microcentrifuge tube and rehydrated with 10 microliters of water. The gel pieces were then washed 3 times with 500 microliters 50% acetonitrile/0.05 M Tris-HCl, pH 8.5 for 20 minutes.
  • the supernatants were discarded and the washed pieces were dried for 30 minutes in a Speed- Vac concentrator.
  • Five microliters of a solution containing 0.05 micrograms Lys-C was added to the tube and incubated for 20 hours at 32°C. Following digestion, the gel pieces were extracted three times with 30 microliters 50% acetonitrile/0.1 % TFA.
  • the supernatants were transferred to a 0.5 ml microcentrifuge tube and dried.
  • the extracted peptides were redissolved in 4 microliters 4-hydroxy- alpha-cyano cinnamic acid and a 0.8 microliter sample was spotted onto a MALDI sample plate.
  • Antigens for Use in Vaccines Representative antigens described herein were evaluated as vaccine candidates on the basis of Western blot analyses (as described above) of sera obtained from patients prior to and after antimicrobial treatment.
  • the Greenberg panel was obtained from male and female patients in California, also aged 18- 70, who were diagnosed as having H. pylori infection, as confirmed in antibody tests. Prior to antimicrobial treatment and 24 months after treatment, serum was collected from the patients.
  • Table 13 summarizes the data obtained from the Gasbarrinni panel. The numbers and percentages of patients who exhibited high antibody titre against the indicated antigens at 12 months are indicated therein. Twelve months after treatment, a high percentage of patients continued to exhibit high antibody titre against clones Y139, Y146B, Y175A, and A22.
  • Table 14 summarizes the data obtained from the Greenberg panel. The numbers and percentages of patients who exhibited high antibody titre against the indicated antigens at 24 months are shown. Twenty four months after treatment, a high percentage of patients continued to exhibit high antibody titre against clones Y184A, Z9A, Y261Ains and Y146B. Since antigens which invoke a long-lasting antigenic response are considered to be good vaccine candidates, and based upon the results provided in the Tables below, the following antigens are considered to be preferred vaccine candidates: Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261Ains and Y146B.
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  • TCTTTATCCC ACAAGCTCAT CTAAAACCAC ACCCGCTAAA AACTAAAATT AACAAAAACT 1260 AAAATCTTTT TTAAGAGCCT ACACGAGCGA GCAAAAAGAA TGACAATCAA TAAAAACGAA 1320
  • Met Lys Lys lie lie Leu Ala Cys Leu Met Ala Phe Val Gly Ala Asn
  • Glu lie Lys Asn Ala Leu lie Ser Ala Tyr Ala Arg Val Leu Thr Pro
  • Asn Lys Asn Phe Ala lie Thr Arg Leu Gin Ser Leu Leu Tyr Lys Glu 275 280 285 Leu Lys Asp Tyr Ala Asn Lys Glu Gly Gin Gly Asn Thr Gly Leu 290 295 300
  • AAAGAAGAAA AATTGGCGTG CATGACAATG AAGTCTTTCA AACCTTGTAT TATGAAGCGA 360
  • Glu Lys lie Val Phe Asp Leu Pro Lys Thr lie lie Glu Gin Glu Met
  • Ala Met lie Glu Asp Arg Val Leu Ala Tyr Leu Leu Asp Lys Asn Leu 145 150 155 160
  • TTTAGAAAAA CCTTTAAAAA AACCACACAA ACACAGCTTT TTAGCCGCTT CAAAAGCGTT 300 AGAAGAGAGC AAACGGCAGG CCTTAAAAGT CGCAAGCACG GACGCTAATG TCATGCTATT 360
  • GGTTCAGACT GCACCTGTTA CTACAGAACC AGCTCCAGAG AAAGAAGAGC CTAAACAAGA 1200 GCCAGCTCCA GTGGTTGAAG AAAAGCCGGC TATTGAAAGC GGGACTATCA TCGCTTCTAT 1260
  • TTTAGTCATT AAAGGGGTAG AAAAAGATAT GATCAAAACC ATCAGTTTTG GTGAAACCAA 1500 ACCCAAATGC GCCCAAAAAA CTAGAGAATG TTACAAAGAA AACAGAAGAG TGGATGTCAA 1560
  • Asp Asn Lys Ser Val Lys lie Asp Val Arg Phe lie Ser Ala Thr Asn 210 215 220
  • Lys lie Gin Ala Phe Asp Trp 305 310
  • GGAAATTCCA AAAGAACCAA ATGGCTCCGG ATTTTTCTAA AGCCGCTTTC GCTTTAACTT 480 CTGGGGATTA CACTAAAACC CCTGTTAAAA CAGAGTTTGG TTATCATATT ATCTATTTGA 540
  • CTGCAGCAGG CAATATTGGT GGTGGAGGTT TTGCGGTTAT CCATTTGGCT AATGGTGAAA 60 ATGTTGCCTT AGATTTTAGA GAAAAAGCCC CCTTGAAAGC CACTAAAAAC ATGTTTTTAG 120
  • AACTTTATAA TCTACCAATC CATCGCATGA CTTTTAAAAT ACTCAAAGAT CCTAGATGAG 1440 AGCTTGAGTT GGATTGACTT TAGTTTATTT TAATTTTTCT TTATTTTGAA ATATCTTGAA 1500
  • GGGATTTAGT CAATAACAGC GTGCTTTTAG TGGAAAATGA GCATAAAGAA AAATTAAAAG 780

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne la caractérisation et l'isolement d'antigènes polypeptidiques de H. pylori récemment découverts. L'invention a pour objet des familles groupées de répliques d'ADN de parties du génome d'antigènes codant H. pylori hautement immunogènes. L'invention porte également sur de nouvelles protéines antigéniques obtenues à partir de H. pylori. Cette invention concerne enfin des méthodes utilisant les antigènes décrits ci-dessus.
EP98918806A 1997-04-25 1998-04-25 Composition antigenique et methode de detection d'helicobacter pylori Withdrawn EP0977864A2 (fr)

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US4510797P 1997-04-25 1997-04-25
US45107P 1997-04-25
US6195897P 1997-10-14 1997-10-14
US61958P 1997-10-14
PCT/US1998/008487 WO1998049314A2 (fr) 1997-04-25 1998-04-25 COMPOSITION ANTIGENIQUE ET METHODE DE DETECTION D'$i(HELICOBACTER PYLORI)

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EP0977864A2 true EP0977864A2 (fr) 2000-02-09

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EP (1) EP0977864A2 (fr)
JP (1) JP2001517091A (fr)
AU (1) AU7166098A (fr)
CA (1) CA2288211A1 (fr)
WO (1) WO1998049314A2 (fr)

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GB9806039D0 (en) * 1998-03-20 1998-05-20 Cortecs Ltd Therapy
WO2000046242A2 (fr) * 1999-02-04 2000-08-10 American Cyanamid Company PROTEINE DE 19 KILODALTONS PRODUITE PAR LA BACTERIE $i(HELICOBACTER PYLORI)
US6316205B1 (en) 2000-01-28 2001-11-13 Genelabs Diagnostics Pte Ltd. Assay devices and methods of analyte detection
ATE345354T1 (de) * 2000-04-27 2006-12-15 Max Planck Gesellschaft Methode zur identifizierung von helicobacter antigenen
WO2002040516A2 (fr) * 2000-11-15 2002-05-23 Ludwig Deml Hcpa (helicobacter cystein rich protein a) et ses utilisations
SE0101030D0 (sv) * 2001-03-23 2001-03-23 Nordic Bio Ab Immunogenic cell surface proteins of helicobacter pylori
WO2003085378A2 (fr) * 2002-04-03 2003-10-16 Syngenta Participations Ag Detection de pathogenes fongiques de ble et d'orge resistant a certains fongicides au moyen de la reaction en chaine de la polymerase
JP3823268B2 (ja) * 2002-05-30 2006-09-20 独立行政法人科学技術振興機構 ヘリコバクター・ピロリの細胞死誘導剤
GB0718966D0 (en) * 2007-09-28 2007-11-07 Liverpool School Of Tropical M Bacterial vaccine
MY182584A (en) * 2011-08-03 2021-01-25 Univ Sains Malaysia Helicobacter pylori proteins for diagnostic kit and vaccine
CN113694191A (zh) * 2014-06-30 2021-11-26 默多克儿童研究所 螺杆菌治疗剂

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WO1996040893A1 (fr) * 1995-06-07 1996-12-19 Astra Aktiebolag Sequences d'acide nucleique et d'acides amines concernant helicobacter pylori, utilisees a des fins diagnostiques et therapeutiques
FR2739622B1 (fr) * 1995-10-04 1997-12-05 Pasteur Merieux Serums Vacc Nouvelles proteines membranaires d'helicobacter pylori

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Title
See references of WO9849314A3 *

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WO1998049314A3 (fr) 1999-01-14
CA2288211A1 (fr) 1998-11-05
JP2001517091A (ja) 2001-10-02
WO1998049314A8 (fr) 1999-04-08
AU7166098A (en) 1998-11-24
WO1998049314A2 (fr) 1998-11-05

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