Helicobacter pylori diagnostics Number:6,902,903 from the United States Patent and Trademark Office (PTO) owispatent Novel methods, membrane supports and immunodiagnostic test kits for diagnosing Helicobacter pylori infection, are disclosed. The methods can also be used to monitor the progress of treatment of an infection. The methods, supports and kits employ both type-common and type-specific H. pylori antigens and can conveniently be performed in a single-step assay format. The methods provide for highly accurate results and discriminate between H. pylori Type I and H. pylori Type II infection so that an accurate diagnosis can be accomplished. Helicobacter, diagnostics, Helicobacter, py, 6902903.html, Patent, pto, 6902903

Title: Helicobacter pylori diagnostics

Abstract: Novel methods, membrane supports and immunodiagnostic test kits for diagnosing Helicobacter pylori infection, are disclosed. The methods can also be used to monitor the progress of treatment of an infection. The methods, supports and kits employ both type-common and type-specific H. pylori antigens and can conveniently be performed in a single-step assay format. The methods provide for highly accurate results and discriminate between H. pylori Type I and H. pylori Type II infection so that an accurate diagnosis can be accomplished.

Patent Number: 6,902,903 Issued on 06/07/2005 to Quan, et al.

Inventors: Quan; Stella (Emeryville, CA); Valenzuela; Pablo (Berkeley, CA); Polito; Alan (Emeryville, CA)

Assignee: Chiron Corporation (Emeryville, CA)

Appl. No.: 993010

Filed: December 18, 1997

Current U.S. Class: 435/7.32; 435/5; 435/6; 435/7.2; 435/7.4; 435/7.92; 435/7.93; 435/34; 435/94; 435/252.1; 436/172; 436/518; 436/530; 536/22.1; 536/23.1; 536/24.3; 536/24.32

Intern'l Class: G01N 033/55.4

Field of Search: 435/732,792,34,72,252.1,79,742,74,793,5,6,94,32 536/231,221,243,242.3 436/172,518,530

References Cited [Referenced By]

U.S. Patent Documents

5262156	Nov., 1993	Alemohammad.
5403924	Apr., 1995	Cover et al.
5420014	May., 1995	Cripps et al.
5567594	Oct., 1996	Calenoff.
5610060	Mar., 1997	Ward et al.
5733740	Mar., 1998	Cover et al.
5814455	Sep., 1998	Pronovost et al.
5846751	Dec., 1998	Pronovost et al.
5859219	Jan., 1999	Cover et al.
Foreign Patent Documents
90/03575	May., 1990	WO.
93/16723	Feb., 1993	WO.

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Primary Examiner: Smith; Lynette R. F.
Assistant Examiner: Portner; Ginny Allen
Attorney, Agent or Firm: Pasternak; Dahna S., Hale; Rebecca M., Harbin; Alisa A.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No. 60/033,707, filed Dec. 19, 1996, from which priority is claimed under 35 USC §119(e) (1) and which is incorporated herein by reference in its entirety.

Claims

1. A method of detecting Helicobacter pylori antibodies associated with infection in a human subject comprising:

(a) reacting a biological sample from the subject with one or more H. pylori type-common antigens provided in an H. pylori lysate and with one or more purified type-specific H. pylori Type I antigens, wherein the type-specific antigens are H. pylori vacuolating cytotoxin (VacA) and cytotoxin associated antigen (CagA), under conditions which allow H. pylori antibodies, when present in the biological sample, to specifically bind with said type-common antigens or said type-specific antigen(s);

(b) removing unbound antibodies;

(c) providing one or more moieties comprising a detectably labeled anti-human immunoglobulin antibody which bind to said bound antibodies;

(d) detecting the presence or absence of said one or more moieties;

(e) correlating the presence of antibodies that specifically bind to the type-specific antigens to infection with Type I H. pylori; and

(f) correlating the absence of antibodies that specifically bind to the type-specific antigens and the presence of antibodies that specifically bind to the type-common antigens to infection with Type II H. pylori.

2. The method of claim 1, wherein said H. pylori infection is H. pylori Type I.

3. The method of claim 1, wherein said H. pylori infection is H. pylori Type II.

4. The method of claim 1, wherein the detectable label is a fluorescer or an enzyme.

5. The method of claim 1, wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on a solid support or are immobilized on different solid supports.

6. The method of claim 5 wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on the same solid support.

7. The method of claim 5 wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on different solid supports.

8. The method of claim 5 wherein the solid support is a nitrocellulose strip.

9. The method of claim 1, wherein said one or more type-common antigens comprises an H. pylori urease.

10. The method of claim 1, wherein said biological sample is a serum sample.

11. A method for distinguishing between Helicobacter pylori Type I and Helicobacter pylori Type II antibodies associated with infection in a human serum sample, said method comprising:

(a) immobilizing an H. pylori lysate comprising one or more H. pylori type-common antigens and one or more purified type-specific H. pylori Type I antigens on at least one nitrocellulose strip, wherein the type-specific antigens are H. pylori vacuolating cytotoxin (VacA) and cytotoxin associated antigen (CagA);

(b) contacting said nitrocellulose strip from step (a) with said human serum sample under conditions which allow anti-H. pylori Type I and anti-H. pylori Type II antibodies, when present in the sample, to specifically bind with H. pylori type-common and type-specific H. pylori Type I antigens present in said lysate;

(c) removing unbound antibodies;

(d) providing a detectably labeled anti-human immunoglobulin antibody;

(e) detecting the presence or absence of bound anti-human immunoglobulin antibodies to said at least one nitrocellulose strip;

(f) correlating the presence of antibodies that specifically bind to the type-specific antigens to infection with Type I H. pylori; and

(g) correlating the absence of antibodies that specifically bind to the type-specific antigens and the presence of antibodies that specifically bind to the type-common antigens to infection with Type II H. pylori.

12. The method of claim 11 wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on the same nitrocellulose strip.

13. The method of claim 11, wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on different nitrocellulose strips.

14. A method of monitoring a human subject undergoing therapy for an Helicobacter pylori infection comprising:

(a) providing a biological sample from the human subject;

(b) immobilizing an H. pylori lysate comprising one or more H. pylori type-common antigens and one or more purified type-specific H. pylori Type I antigens on at least one nitrocellulose strip, wherein the type-specific antigens are H. pylori vacuolating cytotoxin (VacA) and cytotoxin associated antigen (CagA);

(c) contacting said nitrocellulose strip from step (b) with said biological sample under conditions which allow anti-H. pylori Type I and anti-H. pylori Type II antibodies, when present in the biological sample, to specifically bind with H. pylori type-common and type-specific H. pylori Type I antigens present in said lysate;

(d) removing unbound antibodies;

(e) providing a detectably labeled anti-human immunoglobulin antibody;

(f) detecting the presence or absence of bound anti-human immunoglobulin antibodies in said biological sample;

(g) correlating the presence of antibodies that specifically bind to the type-specific antigens to infection with Type I H. pylori; and

(h) correlating the absence of antibodies that specifically bind to the type-specific antigens and the presence of antibodies that specifically bind to the type-common antigens to infection with Type II H. pylori,

thereby monitoring the course of treatment of the infection.

15. The method of claim 14 wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on the same nitrocellulose strip.

16. The method of claim 14, wherein said one or more type-common antigens and said one or more type-specific antigens are immobilized on different nitrocellulose strips.

17. The method of claim 14, wherein said biological sample is a serum sample.

Description

BACKGROUND OF THE INVENTION

TECHNICAL FIELD

The present invention pertains generally to bacterial diagnostic techniques. In particular, the invention relates to methods for accurately detecting Helicobacter pylori infection in a biological sample and for monitoring the course of antibiotic treatment in a patient with an H. pylori infection.

BACKGROUND OF THE INVENTION

Helicobacter pylori, originally named Campylobacter pylori, is a curved, microaerophilic, gram-negative bacterium that exhibits high urease and catalase activity. Recent studies suggest that H. pylori infection may be either a cause of, or a cofactor in, type B gastritis, peptic ulcers, and gastric tumors. See, e.g., Blaser, Gastroenterology (1987) 93:371-383; Dooley et al., New Eng. J. Med. (1989) 321:1562-1566; Personnet et al., New Eng. J. Med. (1991) 325:1127-1131. In this regard, H. pylori colonizes the human gastric mucosa and causes an infection that can persist for decades. Many people with this condition are asymptomatic but are nonetheless at a considerable risk of developing peptic ulcers and/or gastric adenocarcinomas. For a review of H. pylori and its role in gastric disease, see, Telford et al., Trends in Biotech. (1994) 12:420-426 and Blaser, M. J., Scientific American (February 1996):104-107.

H. pylori bacteria are divided into two groups, Type I and Type II, based on the presence or absence of specific proteins. In this regard, H. pylori produces several factors that function to establish and maintain infection. For example, both Type I and Type II bacteria include flagella that aid in mobility in the viscous mucus layer of the stomach. Both types of bacteria also produce ureases, presumably to neutralize the acid environment of the stomach. Additionally, the two types of bacteria produce a number of adhesins for tissue-specific colonization. On the other hand, only H. pylori Type I strains produce a potent cytotoxin, known as VacA or CT, as well as a surface-exposed immunodominant antigen which is associated with cytotoxin expression, known as CagA, CAI antigen or tagA. For descriptions of VacA and CagA, see, e.g., International Publication No. WO 93/18150, published 16 Sep. 1993.

Patients with duodenal ulcers have been shown to produce antibodies to VacA and CagA and antibody titers appear to correlate with the severity of the disease. For example, in one study, more than 95% of patients with duodenal ulcer or duodenitis, and more than 70% of patients suffering from gastric ulcer, were found to be CagA seropositive. Telford et al., Trends in Biotech. (1994) 12:420-426. Furthermore, a correlation has been shown between CagA serum response and gastric adenocarcinoma. Telford et al., supra. Additionally, only cytotoxic strains are able to induce gastric lesions in a laboratory animal model. See, e.g., Telford et al., J. Exp. Med. (1994) 179:1653-1658. Thus, it is believed that only individuals infected with H. pylori Type I strains develop severe disease.

Several assays have been developed for the diagnosis of H. pylori infection. These assays, unfortunately, suffer from several drawbacks. For example, bacterial culture assays have been described for the detection of H. pylori. U.S. Pat. No. 5,498,528 describes such a method for detecting H. pylori in saliva. The assay requires incubating the test sample with a culture medium that supports the selective growth of H. pylori. The presence of the bacterium is detected by the activity of the enzyme urease which, as described above, is produced by H. pylori. Urease catalyzes the conversion of urea to ammonium causing an increase in the pH of the culture medium. The pH change can be detected by a color change to the medium due to the presence of a pH sensitive indicator. However, the assay is time consuming since the bacteria require a number of days for growth. The assay is also inconvenient and bacterial samples may degrade or become contaminated during transport to the laboratory.

Antibody detection tests provide an alternative to bacterial culture. In this regard, subjects colonized with H. pylori mount a humoral immune response and produce antibodies to the bacterium that can be used as a basis for diagnosis. IgA antibodies are found in gastric fluid while IgG antibodies are found in the circulation. However, such tests can suffer from a lack of specificity since H. pylori appears to be antigenically cross-reactive with Campylobacter jejuni and C. coli.

U.S. Pat. No. 4,882,271 describes an H. pylori assay that utilizes high molecular weight cell-associated proteins, on the order of 300 kDa to 700 kDa, having urease activity, in an enzyme-linked immunosorbent assay (ELISA), in an attempt to circumvent the problems with cross-reactivity.

International Publication No. WO 96/12965, published 2 May 1996, describes an immunoblot assay where a serological sample is reacted with two antigen components having molecular weights of 19.5 kDa, 26.5 kDa or 30 kDa, or alternatively, any one antigen component corresponding to a molecular weight of 35 kDa, 89 kDa, 116 kDa or 180 kDa. It is postulated by the inventors that the 19.5 kDa protein is a ferritin-like protein, the 26.5 and 30 kDa proteins are ureases, the 89 kDa protein is VacA, and that the 116 kDa protein is CagA. The 35 kDa and 180 kDa were uncharacterized.

Finally, European Patent Publication 329,570, published 23 Aug. 1989, describes immunoassays for H. pylori infection using pooled suspensions of sonicates of several H. pylori strains, as well as immunoassays using purified H. pylori flagellae.

Although faster and more sensitive than bacterial culture, antibody detection tests, such as those described above, can give false positive and negative results and generally do not distinguish between H. pylori Type I and Type II infection. Thus, an additional test must be conducted to determine whether the infection is due to H. pylori Type I or Type II.

Accordingly, the wide spread availability of an accurate and efficient assay for H. pylori infection that readily distinguishes between Type I and Type II infection, would be important for the diagnosis of infection in both symptomatic and asymptomatic individuals.

SUMMARY OF THE INVENTION

The present invention provides a simple, extremely accurate and efficient method for diagnosing H. pylori infection, as well as for distinguishing between H. pylori Type I and H. pylori Type II infections. Thus, the method provides a technique for screening for individuals with H. pylori Type I infection. If Type I infection is detected, the individual can be given antibiotics to treat or prevent type B gastritis, peptic ulcers, and gastric tumors. The method is also useful for monitoring the course of treatment in a patient with an H. pylori infection. The assay method utilizes both type-common antigens, as well as particular type-specific antigens from the bacterium.

Accordingly, in one embodiment, the subject invention is directed to a method of detecting H. pylori infection comprising:

(a) providing a biological sample;
(b) reacting the biological sample with one or more H. pylori type-common antigens and reacting the biological sample with one or more purified type-specific H. pylori Type I antigens, under conditions which allow H. pylori antibodies, when present in the biological sample, to bind with the H. pylori type-common antigens and/or the type-specific antigens,
thereby detecting the presence or absence of H. pylori infection.

In other embodiments, the invention is directed to a method for distinguishing between H. pylori Type I and H. pylori Type II infection in a biological sample, or a method of monitoring a subject undergoing therapy for an Helicobacter pylori infection, the methods comprising:

(a) immobilizing one or more H. pylori type-common antigens, e.g., an H. pylori lysate and/or H. pylori urease, and immobilizing one or more purified type-specific H. pylori Type I antigens, e.g., H. pylori VacA and/or H. pylori CagA, on a nitrocellulose strip;
(b) contacting the nitrocellulose strip from step (a) with the biological sample under conditions which allow anti-H. pylori Type I and anti-H. pylori Type II antibodies, when present in the biological sample, to bind with H. pylori type-common antigens present in the lysate and/or the type-specific H. pylori Type I antigens;
(c) removing unbound antibodies;
(d) providing a detectably labeled anti-human immunoglobulin antibody; and
(e) detecting the presence or absence of bound anti-human immunoglobulin antibodies in the biological sample,
thereby detecting the presence or absence of H. pylori Type I or Type II infection.

In particularly preferred embodiments, the biological sample is a human serum sample.

In yet further embodiments, the invention is directed to membrane supports comprising one or more H. pylori type-common antigens and one or more purified type-specific H. pylori Type I antigens, discretely immobilized thereon.

In another embodiment, the invention is directed to a nitrocellulose support comprising:

(a) an H. pylori Type I VacA polypeptide;
(b) an H. pylori Type I CagA polypeptide;
(c) an H. pylori urease; and
(d) a human IgG,
wherein the H. pylori polypeptides and urease, and the human IgG, are immobilized as discrete bands on said nitrocellulose support.

In a further embodiment, the invention is directed to a nitrocellulose support comprising:

(a) an H. pylori Type I VacA polypeptide;
(b) an H. pylori Type I CagA polypeptide;
(c) an H. pylori lysate; and
(d) a human IgG,
wherein the H. pylori polypeptides and lysate, and the human IgG, are immobilized as discrete bands on said nitrocellulose support.

In other embodiments, the invention is directed to immunodiagnostic test kits for detecting H. pylori infection. The kits comprise (a) one or more H. pylori type-common antigens; (b) one or more purified type-specific H. pylori Type I antigens; and (c) instructions for conducting the immunodiagnostic test.

In still further embodiments, the invention is directed to an immunodiagnostic test kit for distinguishing between H. pylori Type I and H. pylori Type II infection in a biological sample, or for monitoring a subject undergoing therapy for an Helicobacter pylori infection. The test kit comprises (a) one or more H. pylori type-common antigens immobilized on a nitrocellulose strip, e.g., an H. pylori lysate and/or H. pylori urease; (b) one or more purified type-specific H. pylori Type I antigens, e.g., H. pylori VacA and/or H. pylori CagA, immobilized on a nitrocellulose strip; and (c) instructions for conducting the immunodiagnostic test.

These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a representative test strip for use in a strip immunoblot assay (SIA). Human IgG is used as an internal control at two different levels (Level I, low control; and Level II, high control). CagA and VacA are used as the type-specific H. pylori Type I antigens and HP CE denotes the H. pylori lysate which contains type-common antigens.

FIG. 2 shows another representative test strip for use in an SIA. As above, human IgG is used as an internal control at two different levels (Level I, low control; and Level II, high control). CagA and VacA are used as the type-specific H. pylori Type I antigens and are also used at two different levels to enhance the sensitivity of the assay as well as to monitor response to treatment. Urease is used as the type-common antigen.

FIGS. 3A-3B (SEQ ID NOS: 1 and 2) show the nucleotide sequence and corresponding amino acid sequence for the H. pylori VacA antigen used in the SIAs described in the examples.

FIG. 4 (SEQ ID NO: 3 and 4) shows the nucleotide sequence and corresponding amino acid sequence for the H. pylori CagA antigen used in the SIAs described in the examples.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. Additionally, standard abbreviations for nucleotides and amino acids are used in this specification.

I. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By "an H. pylori lysate" is meant an extract or lysate derived from an H. pylori Type I or Type II whole bacterium which includes one or more H. pylori polypeptides, as defined below, that reacts with antibodies generated against both of H. pylori Type I and H. pylori Type II. Such polypeptides are termed "type-common" antigens herein. Thus the term "lysate" as used herein refers to crude extracts that contain several H. pylori antigens, so long as at least one of the antigens present in the lysate is a type-common antigen. The lysate can be augmented with additional purified type-common and/or type-specific antigens. The term also denotes relatively purified compositions derived from such crude lysates which include only one or few such type-common antigens. Such lysates are prepared using techniques well known in the art, described further below.

Representative antigens that may be present in such lysates, either alone or in combination, include one or more type-common epitopes derived from the H. pylori adhesins such as, but not limited to, a 20 kDa N-acetyl-neuraminillactose-binding fibrillar haemagglutinin (HpaA), a 63 kDa protein that binds phosphatidylethanolamine and gangliotetraosyl ceramide, and a conserved fimbrial pilus-like structure. See, e.g., Telford et al., Trends in Biotech. (1994) 12:420-426 for a description of these antigens. Other type-common antigens that may be present in the lysate include one or more type-common epitopes derived from any of the various flagellins such as the major flagellin, FlaA and the minor flagellin, FlaB. In this regard, the flagella of H. pylori are composed of FlaA and FlaB, each with a molecular weight of approximately 53 kDa. Either or both of FlaA and/or FlaB may be used as a source of type-common antigens for use with the present invention. Another representative type-common antigen includes H. pylori urease which is associated with the outer membrane and the periplasmic space of the bacterium. The holoenzyme is a large complex made up of two subunits of 26.5 kDa (UreA) and 61 kDa (UreB), respectively. Type-common epitopes derived from the holoenzyme, either of the subunits, or a combination of the three, can be present as the type-common antigen(s). Another representative type-common antigen that may be present in the lysate or used in further purified form includes the an H. pylori heat shock protein known as "hsp60." The DNA and corresponding amino acid sequences for hsp60 are known. See, e.g., International Publication No. WO 93/18150, published 16 Sep. 1993. The full-length hsp60 antigen shown has about 546 amino acids and a molecular weight of about 58 kDa. It is to be understood that the lysate can also include other type-common antigens not specifically described herein.

By a "type-specific H. pylori Type I antigen" is meant a polypeptide, as defined below, derived from H. pylori Type I which reacts predominantly with antibodies against H. pylori Type I, but not with antibodies against H. pylori Type II. Representative type-specific H. pylori Type I antigens include: H. pylori VacA, also known as CT; and H. pylori CagA, also known as CAI antigen and tagA; and epitopes from these antigens which are capable of reacting with antibodies against H. pylori Type I but not H. pylori Type II. Both VacA and CagA are discussed further below. It is to be understood that other type-specific H. pylori Type I antigens, not specifically described herein, are also captured by this definition.

The term "polypeptide" when used with reference to a type-common or type-specific H. pylori antigen, such as VacA, CagA or any of the other type-specific or type common antigens described above, refers to a VacA, CagA etc., whether native, recombinant or synthetic, which is derived from any of the various H. pylori strains. In the case of type-specific H. pylori Type I antigens, the polypeptide will be derived from an H. pylori Type I strain. In the case of a type-common antigen, the polypeptide may be derived from either of H. pylori Type I or Type II. The polypeptide need not include the full-length amino acid sequence of the reference molecule but can include only so much of the molecule as necessary in order for the polypeptide to react with the appropriate H. pylori antibodies. Thus, only one or few epitopes of the reference molecule need be present. Furthermore, the polypeptide may comprise a fusion protein between the full-length reference molecule or a fragment of the reference molecule, and another protein that does not disrupt the reactivity of the H. pylori polypeptide. It is readily apparent that the polypeptide may therefore comprise the full-length sequence, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. The term also intends deletions, additions and substitutions to the reference sequence, so long as the polypeptide retains the ability to react with H. pylori antibodies.

In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that do not substantially affect the antibody binding capabilities of the protein, are therefore within the definition of the reference polypeptide.

By "VacA polypeptide" is meant a polypeptide as defined above which is derived from the antigen known as the H. pylori Type I Cytotoxin and which reacts predominantly with antibodies against H. pylori Type I, but not H. pylori Type II. The VacA protein induces vacuolization in epithelial cells in tissue culture and causes extensive tissue damage and ulceration when administered orally to mice. The DNA and corresponding amino acid sequences for VacA are known and reported in, e.g., International Publication No. WO 93/18150, published 16 Sep. 1993. The gene for the VacA polypeptide encodes a precursor of about 140 kDa that is processed to an active molecule of about 90-100 kDa. This molecule, in turn, is slowly proteolytically cleaved to generate two fragments that copurify with the intact 90 kDa molecule. See, Telford et al., Trends in Biotech. (1994) 12:420-426. Thus, the definition of "VacA polypeptide" as used herein includes the precursor protein, as well as the processed active molecule, proteolytic fragments thereof or portions or muteins thereof, which retain specific reactivity with antibodies present in a biological sample from an individual with H. pylori Type I infection. For example, the VacA polypeptide depicted in FIGS. 3A-3B and used in assays described herein includes a VacA fragment from Gly-311 to Ile-819, inclusive, of the full-length molecule, fused by a linker sequence of five amino acids to 154 amino acids of human SOD to facilitate recombinant expression.

By "CagA polypeptide" is meant a polypeptide as defined above which is derived from the H. pylori Type I cytotoxin associated immunodominant antigen and which reacts predominantly with antibodies against H. pylori Type I, but not H. pylori Type II. CagA is expressed on the bacterial surface. The DNA and corresponding amino acid sequences for CagA are known. See, e.g., International Publication No. WO 93/18150, published 16 Sep. 1993. The full-length CagA antigen described therein includes about 1147 amino acids with a predicted molecular weight of about 128 kDa. The native protein shows interstrain size variability due to the presence of a variable number of repeats of a 102 bp DNA segment that encodes repeats of a proline-rich amino acid sequence. See, Covacci et al., Proc. Natl. Acad. Sci. USA (1993) 90:5791-5795. Accordingly, the reported molecular weight of CagA ranges from about 120-135 kDa. Hence, the definition of "CagA polypeptide" as used herein includes any of the various CagA variants, fragments thereof and muteins thereof, which retain the ability to react with antibodies in a biological sample from an individual with H. pylori Type I infection but does not substantially react with antibodies generated against H. pylori Type II. For example, the CagA polypeptide depicted in FIG. 4 and used in assays described herein is a truncated protein of 268 amino acids and includes Glu-748 to Glu-1015, inclusive, of the full-length molecule.

By "epitope" is meant a site on an antigen to which specific B cells and T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." An epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids. Methods of determining spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Furthermore, the identification of epitopes in a given protein is readily accomplished using techniques well known in the art, such as by the use of hydrophobicity studies and by site-directed serology. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA (1984) 81:3998-4002 (general method of rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); U.S. Pat. No. 4,708,871 (procedures for identifying and chemically synthesizing epitopes of antigens); and Geysen et al., Molecular Immunology (1986) 23:709-715 (technique for identifying peptides with high affinity for a given antibody). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

A "purified" protein or polypeptide is a protein which is recombinantly or synthetically produced, or isolated from its natural host, such that the amount of protein present in a composition is substantially higher than that present in a crude preparation. In general, a purified protein will be at least about 50% homogeneous and more preferably at least about 80% to 90% homogeneous.

As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, samples derived from the gastric epithelium and gastric mucosa, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.

As used herein, the terms "label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used under the invention include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, NADPH and α-β-galactosidase.

II. Modes of Carrying Out the Invention

The present invention is based on the discovery of novel diagnostic methods for accurately detecting H. pylori infection and for discriminating between H. pylori Type I and H. pylori Type II infection. The methods utilize one or more H. pylori type-common antigens, either purified or present in lysates derived from the bacterium, as well as purified type-specific H. pylori antigens. The use of both type-common and type-specific antigens reduces the incidence of false positive results. The methods can be practiced in a simple one-step assay format which allows for both detection of infection, as well as identification of the type of infection present, in a single assay. The method can also be practiced in two steps wherein the sample is first reacted with the H. pylori type-common antigens and if positive, reacted with one or more type-specific Type I antigens. The methods can also employ type-specific Type II antigens.

More particularly, the use of the H. pylori type-common antigens allows the diagnosis of H. pylori infection in general. The presence of one or more type-specific antigens allows determination of the bacterial type, i.e., whether the infection is caused by H. pylori Type I and/or H. pylori Type II. Due to the presence of the H. pylori type-common antigens, positive results will occur even in untypable samples. Hence, the incidence of false negatives is reduced. Furthermore, if H. pylori Type I infection is present, the individual can be administered antibiotics to treat or prevent type B gastritis, peptic ulcers, and gastric tumors.

Furthermore, the assays described herein are useful for monitoring the course of treatment in a subject to determine whether antibiotic therapy is effective.

The antigens for use in the subject diagnostic techniques can be produced using a variety of techniques. For example, the type-common antigens can be provided in a lysate that can be obtained using methods well known in the art. Generally, such methods entail extracting type-common proteins from either H. pylori Type I or Type II bacteria using sonication, pressure disintegration, detergent extraction, fractionation, and the like. Type-common antigens present in such lysates can be further purified if desired, using standard purification techniques. H. pylori strains for use in such methods are readily available from several sources including the American Type Culture Collection (ATCC, Rockville, Md.). For example, ATCC strain designations NCTC 11637, 11639 and 11916, will find use as a source of the lysate. Other useful strains are known in the art.

The type-specific H. pylori Type I antigens can also be obtained using standard purification techniques. In this regard, particular antigens can be isolated from Type I H. pylori ulcer-producing strains using standard purification techniques such as column chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography and immunoprecipitation. See, e.g., International Publication No. WO 96/12965, published 2 May 1996, for a description of the purification of several antigens from H. pylori. For example, ATCC strain designation NCTC 11916 is a Type I ulcer-producing strain of H. pylori and can therefore be used as a source for one or more type-specific antigens for use in the subject invention.

The H. pylori antigens can also be generated using recombinant methods, well known in the art. In this regard, oligonucleotide probes can be devised based on the known sequences of the H. pylori genome and used to probe genomic or cDNA libraries for H. pylori genes encoding for the antigens useful in the present invention. The genes can then be further isolated using standard techniques and, if desired, restriction enzymes employed to mutate the gene at desired portions of the full-length sequence.

Similarly, H. pylori genes can be isolated directly from bacterial cells using known techniques, such as phenol extraction, and the sequence can be further manipulated to produce any desired alterations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA. Finally, the genes encoding the H. pylori antigens can be produced synthetically, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311.

Once coding sequences for the desired polypeptides have been isolated or synthesized, they can be cloned into any suitable vector or replicon for expression in a variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all well known in the art. In particular, host cells are transformed with expression vectors which include control sequences operably linked to the desired coding sequence.

The control sequences will be compatible with the particular host cell used. For example, typical promoters for mammalian cell expression include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter, among others. Other non-viral promoters, such as a promoter derived from the murine metallothionein gene, will also find use in mammalian constructs. Mammalian expression may be either constitutive or regulated (inducible), depending on the promoter. Typically, transcription termination and polyadenylation sequences will also be present, located 3′ to the translation stop codon. Examples of transcription terminator/polyadenylation signals include those derived from SV40 (Sambrook et al., supra). Introns, containing splice donor and acceptor sites, may also be designed into the constructs of the present invention.

Enhancer elements can also be used in the mammalian constructs to increase expression levels. Examples include the SV40 early gene enhancer (Dijkema et al., EMBO J. (1985) 4:761) and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777) and human cytomegalovirus (Boshart et al., Cell (1985) 41:521). A leader sequence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the gene of interest such that the leader sequence can be cleaved either in vivo or in vitro. The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Once complete, the mammalian expression vectors can be used to transform any of several mammalian cells. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are also known and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), as well as others.

The constructs of the present invention can also be expressed in yeast. Control sequences for yeast vectors are known in the art and include promoters such as alcohol dehydrogenase (ADH) (EP Publication No. 284,044)., enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EP Publication No. 329,203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al., Proc. Natl. Acad. Sci. USA (1983) 80:1). In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, or PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP Publication No. 164,556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.

Other control elements which may be included in the yeast expression vectors are terminators (e.g., from GAPDH and from the enolase gene (Holland, J. Biol. Chem. (1981) 256:1385), and leader sequences which encode signal sequences for secretion. DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP Publication No. 012,873; JPO Publication No. 62,096,086) and the α-factor gene (U.S. Pat. Nos. 4,588,684, 4,546,083 and 4,870,008; EP Publication No. 324,274; PCT Publication No. WO 89/02463). Alternatively, leaders of non-yeast origin, such as an interferon leader, also provide for secretion in yeast (EP Publication No. 060,057).

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Saccharomyces cerevisiae (Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163); Saccharomyces carlsbergeneis; Candida albicans (Kurtz et al., Mol. Cell. Biol. (1986) 6:142); Candida maltosa (Kunze et al., J. Basic Microbiol. (1985) 25:141); Hansenula polymorpha (Gleeson et al., J. Gen. Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302); Kluyveromyces fragilis (Das et al., J. Bacteriol. (1984) 158:1165); Kluyveromyces lactis (De Louvencourt et al., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology (1990) 8:135); Pichia guillerimondii (Kunze et al., J. Basic Microbiol. (1985) 25:141); Pichia pastoris (Cregg et al., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555); Schizosaccharomyces pombe (Beach and Nurse, Nature (1981) 300:706); and Yarrowia lipolytica (Davidow et al., Curr. Genet. (1985) 10:380471; Gaillardin et al., Curr. Genet. (1985) 10:49).

Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.

Bacterial expression systems can also be used with the present constructs. Control elements for use in bacteria include promoters, optionally containing operator sequences, and ribosome binding sites. Useful promoters include sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the b-lactamase (bla) promoter system, bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), which do not occur in nature also function as in bacterial host cells.

The foregoing systems are particularly compatible with E. coli. However, numerous other systems for use in bacterial hosts such as Bacillus spp., Streptococcus spp., and Streptomyces spp., among others, are also known. Methods for introducing exogenous DNA into these hosts typically include the use of CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation.

Other systems for expression of the desired antigens include insect cells and vectors suitable for use in these cells. The systems most commonly used are derived from the baculovirus Autographa californica polyhedrosis virus (AcNPV). Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene into the baculovirus genome); and appropriate insect host cells and growth media.

Promoters for use in the vectors are typically derived from structural genes, abundantly transcribed at late times in a viral infection cycle. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression" in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EP Publication Nos. 127,839 and 155,476; and the gene encoding the p10 protein Vlak et al., J. Gen. Virol. (1988) 69:765. The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al., Ann. Rev. Microbiol. (1988) 42:177) and a procaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli. DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al., Gene (1988) 73:409), as well as mammalian signal sequences such as those derived from genes encoding human α-interferon, Maeda et al., Nature (1985) 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., Molec. Cell. Biol. (1988) 8:3129; human IL-2, Smith et al., Proc. Natl. Acad. Sci. USA (1985) 82:8404; mouse IL-3, (Miyajima et al., Gene (1987) 58:273; and human glucocerebrosidase, Martin et al., DNA (1988) 7:99.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 bps downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31).

The desired DNA sequence is inserted into the transfer vector, using known techniques (see, Summers and Smith, supra; Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989) and an insect cell host is cotransformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by cotransfection. The vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, for example, Invitrogen, San Diego Calif. ("MaxBac" kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter "Summers and Smith").

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.