ITTO950545A1 - VACCINE / THERAPAUTICAL COMPOSITIONS OF HELYCOBACTER PYLORI, THEIR PREPARATION AND USE. - Google Patents

Description

Description of the industrial invention entitled:

"Method for the diagnosis of Helvcobacter nylori infections and related diagnostic kit"

HELYCOBACTER PYLORI CARBON ANHYDRASE-TYPE PROTEIN The present invention refers to the carbonic anhydrase (CAL) protein of Helycobacter nylori (H. pylori) and, in particular, to the use of this protein in the diagnosis of H. pylori infection.

H. oylori (formerly Camnylobacter pyloridis or C. oyloridis) is a spiral-shaped Gram negative human gastric pathogen. H. pylori is well documented in its association with gastric and duodenal ulcer, as well as with gastric cancer. The organism has been described as the most common chronic infectious agent in humans. Despite its spread, most of the pathogenic mechanism affecting H. is not known. nylori in gastroduodenal pathology.

The action of autoantibodies directed against specific target tissues is known to have etiological ramifications in organ-specific autoimmune diseases. known examples include: the acetylcholine receptor in myasthenia gravis; streptococcal antigens in rheumatic heart disease; glutamate-decarboxylase in insulin-dependent diabetes mellitus (IDDM); Lyme disease; and Goodpasture syndrome.

The production of cross-reacting antibodies with gastric autoantigens has been proposed as a pathological mechanism linking H. pylori and gastritis (Negrini et al, Gastroenterolocrv. 1991, 101.-437-445), although to date such gastric autoantigens do not have been identified. However, the opposite has also been suggested, since H. pylori-associated gastritis is rare in AIDS patients, possibly as a result of immunodeficiency (Marano et al. Am. J. Gastroenterol., 1993, 8B, 887-890 ).

Carbonic anhydrase (CA) is a ubiquitous gastric metalloenzyme that has three isoforms in human tissue, CAI and CAII which are present in most tissues including the stomach and CAIII which is only present in skeletal muscle. The tertiary structures of all three CAs and the amino acid sequences involved in zinc binding are conserved. Carbonic anhydrase has also been implicated in the formation and secretion of gastric and pancreatic juices.

We have now surprisingly found that H. nylori possesses carbonic anhydrase-like proteins. We showed that patients with high antibody titers against H. oylori exhibited an antibody response against carbonic anhydrase (CA).

Prior art in diagnosing H. pylori-associated gastric disease includes Campylobacter DNA probes capable of hybridizing with H. oylori rRNA (EP-A-0350205); H. pylori oligonucleotides specific for sequences of the H. pylori urease gene (WO 91/09049); detection and serological diagnosis of H. oylori infection by serumimmunoassays; and detection of H. pylori antigens and antigenic fragments (WO 89/08843; WO 89/9497 and EP-A-0329570).

It would, however, be highly desirable to have reliable and rapid means of diagnosing H, pylori infection from a patient clinical specimen, as a means of early diagnosis of gastric or peptic ulcer or gastric cancer.

Similarly, the present invention provides a method for diagnosing H. oylori infection by detecting the presence of antibodies against the protein carbonic anhydrase or a fragment thereof, comprising contacting a sample to be tested with the protein carbonic anhydrase or a fragment thereof and detecting the presence of the antibody antigen complex.

The present invention also provides a method for diagnosing H. pylori infection by detecting the presence of antibodies against carbonic anhydrase or a fragment thereof, comprising contacting a sample to be tested with the protein carbonic anhydrase or a fragment thereof, then furthermore by contacting the sample that has reacted with the carbonic anhydrase protein or a fragment thereof with a second anti-human antibody and detecting both the presence of the antigen-secondary antibody complex and detecting the secondary antibody bound by reaction with a third labeled antibody .

It will be understood that the term "protein carbonic anhydrase" refers to the protein carbonic anhydrase or the protein type carbonic anhydrase. The term "fragment" will be understood to include any carbonic anhydrase or protein carbonic anhydrase that is capable of developing an immune response in the host.

The detection of the antigen-antibody complex can take place by any method known in the art. Such methods include identification with a marker attached to the carbonic anhydrase protein or fragment thereof or by immunoprecipitation. Markers can include radioactive, fluorescent, chemiluminescent, dye molecules or enzyme markers. Detection of the antigen-antibody complex may include an amplification system such as those using biotin or avidin.

The second anti-human antibody may be a rabbit anti-human antibody mixture or a goat anti-human antibody mixture. Alternatively, the secondary anti-human antibody can be anti-human IgA, anti-human IgG, or anti-human IgM.

The third antibody that can be used to detect the bound secondary antibody is selected to react with the secondary antibody. For example, if the secondary antibody is a rabbit anti-human antibody, the third antibody may be a horse anti-rabbit antibody or a goat anti-rabbit antibody.

The sample to be tested may include gastric mucosa, dental plaque, saliva, gastric juice, a gastric biopsy sample, or stool. Preferably the carbonic anhydrase protein or a fragment thereof is H. pylori carbonic anhydrase. although it may be human CAI or CAII, or other carbonic anhydrase proteins or their fragments.

The present invention also provides a kit for diagnosing H. oylori infection by detecting antibodies against carbonic anhydrase, comprising the protein carbonic anhydrase or a fragment thereof. Kits according to the present invention may include appropriately labeled reagents and additional substances, such as buffer solutions, other IL proteins.

pylori, such as ureases, H. oylori cytoxins and flagella, means for detecting the results of the diagnostic procedure and instructions for the assay. Kit components can be packaged in an appropriate kit container.

The present invention will now be described in greater detail with reference to the following examples.

Example l

Growth of an H. oylori strain and protein extraction.

The H. pylori strain NCTC 11683 was grown in liquid culture by adding a culture plate collected to 100 ml of Brucella (Difco) broth containing 2% beta-cyclodextrin (Sigma) and 0.2 ml of selective supplement for H. oylori reconstituted (Oxoid) in a 500 ml conical flask. The flask was incubated with gentle agitation (100 r.p.m.) for three days under microaerophilic conditions (Campypak, BBL) at 37 C. H. pylori cells were harvested by centrifugation and the supernatant proteins precipitated as previously described by Milton D.L. , Norqvist A. and Wolf Watz H., (1992), J. Bacteriol .. 174: 7235-7244. Cellular proteins were extracted by resuspending two growth plates in 1.5 mL of protein extraction buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.15 mM EDTA, 1 mM DTT, 1 mM PMSF, 2 mg / mL pepstatin A (Sigma) 0.5 mg / ml leupeptin (Sigma), adding 200 ugml of 10% SDS and boiling for 5 minutes. The suspension was then cooled on ice for 5 minutes and the supernatant collected by centrifugation at 12500 r.p.m. for 5 The protein concentration was determined according to Bradford, M :, (1976), Anal. Biochem .. 72: 248-252 (1976).

Identification of carbonic anhydrase-like proteins in H. pylori protein extracts.

40 ug of H. pylori total soluble (HpT) and extracellular (HpE) protein extracts were analyzed on vertical minigels (Hoefer Scientific), including a 5% acrylamide packing gel and a 13% resolution gel, according to the process of Laemli U.K., 1970, Nature.

227, 680-685.

Electrophoresis was performed at 20 mA. The proteins were transferred onto a nitrocellulose membrane by semi-dry transfer, using transfer buffer (48 mM Tris, pH 8, 39 mM glyein, 20% methanol, 1.3 mM SDS) and an ATTO AE-6675 Horizoblot transfer unit (Genetic Research International), according to the manufacturer's instructions. The nitrocellulose membrane is. it was then dried and placed in blocking solution (1% bovine serum albumin, 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature with constant stirring. The primary antibody (both rabbit human CAI and rabbit human CAII prepared as described below) was added to the blocking buffer at a concentration of 1: 1000 and the incubation was continued for 1 hour. The membrane was then briefly washed twice with wash buffer, once for 15 minutes and once for 5 minutes with agitation. The antibody labeled with HRP (goat anti-rabbit) was added to the membrane at a concentration of 1: 1000 in wash buffer and incubated for one hour as above. The membrane was then washed once for 15 minutes and four times for 5 minutes with wash buffer as above. The membrane was then developed using ECL substrate reagents (Amersham) and exposed to a Fuji X-ray film, according to the manufacturer's instructions. The rehybridization of the membranes was carried out after the detachment of the antibodies bound to the transferred proteins, incubating it in 20 mM gliein pH 2.5, 0.055% Tween 20, overnight at room temperature with constant stirring.

The Western blot result of the two SDS-PAGE separations of total (HpT) and extracellular (HpE) proteins of H. pylori is shown in Fig. 1. Bovine carbonic anhydrase was clearly detectable in the molecular weight marker trace ( MWM).

H. nylori Total Protein (Hpt) extracts contain three major proteins that reacted with both anti-CAI and anti-CAII antibodies. The relative molecular weight of these H. oylori CAL proteins was 57 (the main), 42 and 34 kDa (indicated with an arrow with the molecular weights in Figure 1). Only the 57 protein was the major one in H. pylori extracellular protein extracts (HpE). Total cellular proteins of H, pylori were fractionated on an affinity column of Sepharose AH conjugated with beta-aminomethylbenzene sulfonamide hydrogen chloride. When subjected to SDS-PAGE and Western blot analysis, only the potassium thiocyanate and urea fractions reacted with anti-CAI antibodies (not shown), indicating that these proteins bind strongly to sulfonamide with an active site for CA.

H. pylori CAL proteins were not shown to be essential for growth, as nine culture collections and seven clinical isolates were resistant to three CA sulfonamide inhibitors, as well as antimicrobial sulfanylamide (MIC> 64 mg / 1). .

Preparations of rabbit anti-human CAI and rabbit anti-human CAII primary antibodies.

500 ml of human red blood cells were extracted with chloroform at 4 C and separated by centrifugation. The red blood cells were then purified on a Sepharose AH affinity column conjugated with beta-aminomethyl benzene sulfonamide hydrogen chloride. The potassium iodide fraction contained the CAI protein and the azide fraction contained the CAII protein. The fractions were dialyzed against 10 mM Tris, pH 8.3 and purified on a MonoQ FPLC column (Pharmacia) according to the manufacturer's instructions. The CAI protein was eluted from the potassium iodide fraction with 0.1M NaCl and the CAII protein was eluted from the azide fraction with 0.1M NaCl. The extracts were freeze-dried and the protein content was evaluated with Pierce's BCA Protein Analytical Reagent according to the manufacturer's instructions (Pierce (UK), Ltd.).

Preparations comprising 300 µg of CAI or CAII proteins dissolved in 1.5 ml of water and 1.5 ml of Freund's complete adjuvant were prepared and 1 ml was injected subcutaneously into rabbits. After one month the rabbits were injected intramuscularly with 300 ug of CAI or CAII protein absorbed on alum and the procedure was repeated after 6 months.

The rabbits were then bled and the antibodies recovered according to ordinary procedures known in the art.

Example 2

Correlation of CAL proteins in H. oylori protein extracts with serum from infected patients.

To correlate H. pylori CAL proteins with antibody proteins in patients infected with this microorganism, the protein separations of Example 1 were dehybridized overnight at room temperature and rehybridized with the pooled sera from seven patients (absorbed with Escherichia coli), serum positive or serum negative (in ELISA) for H. oylori (as shown in Fig. 2). Sera from patients infected with H, pylori (detected with an antibody conjugated with HRP anti-human Ig at 1: 1000 dilution) strongly identified proteins of 57 and 34 kDa, CAL and HAP respectively, among others of H. oylori. as well as the molecular weight marker carbonic anhydrase (30 kDa). Sera from serum-negative patients did not react with the CA marker or other proteins in the lanes of H. pylori (not shown). This suggests that H, oylori infected patients produce a significant antibody response to both H. pylori metalloenzymes (CAL and HAP) and since CAL is a predominant native gastric enzyme, this would result in antibody-mediated gastroduodenal inflammation.

Example 3

Blockade of rabbit antibodies to CAI by serum from patients infected with H. oylori.

To confirm that the CA epitopes were similar in rabbit and human polyclonal antibodies, a H. oylori total protein Western blot as described in Example 2 was first blocked with sera from patients with high or low antibody titers against H. oylori. .

Three identical cell protein separations of H. pylori (HpT) were pre-blocked with either sera from patients with high or low antibody titers against H. pylori in blocking solutions (dilution 1: 250), or in blocking solution from alone. The molecular weight marker CA (MWM) served as a positive control.

After washing in the manner described for the CAI antibody in Example 1, the separation was then hybridized and detected using rabbit anti-CAI as in Figure 1 (except that the sera were pre-absorbed with E. coli and normal human serum). Figure 4 shows an overall signal reduction in the blocked ward with serum from infected patients versus the control ward (without blocking) or with sera from patients with low H. pylori antibody titers. In particular, the signal from the predominant protein of 57 kDa CAL of H. pylori (arrow) is significantly reduced in the serum-positive trace, but not in the control or serum-negative traces. This confirms that H. pylori infected patients produce antibodies against the same epitopes as the rabbit anti-human CAI polyclonal antibody.

Claims (9)

CLAIMS 1. Procedure for the diagnosis of H, pylori infection by detecting the presence of antibodies against the protein carbonic anhydrase or its fragment comprising the contact of a sample to be tested with the protein carbonic anhydrase or its fragment and by detecting the presence of antigen-antibody complex . 2. Procedure for diagnosing H. pylori infection by detecting the presence of antibodies against carbonic anhydrase or its fragment, including contacting a sample to be tested with protein carbonic anhydrase or its fragment and then placing the reacted sample further in contact with the protein carbonic anhydrase or its fragment with a second anti-human antibody and detecting both the presence of the antigen-secondary antibody complex, and detecting the secondary antibody bound by reaction with a third antibody. 3. Process as claimed in Claim 2, wherein the secondary anti-human antibody is labeled. 4. A process as claimed in Claim 2 or Claim 3 wherein the secondary antibody is a mixture of rabbit anti-human antibodies. 5. A process as claimed in claim 2 or claim 3 wherein the secondary anti-human antibody is anti-human IgA, anti-human IgG, or anti-human IgM. The process as claimed in any one of Claims 1 to 5, wherein the sample to be tested comprises gastric mucosa, dental plaque, saliva, gastric juice, gastric biopsy sample or faeces. A process as claimed in any one of Claims 1 to 6, wherein the carbonic anhydrase protein or a fragment thereof is derived from H. nylori. 8. Kit for diagnosing H. pylori infection, comprising the protein carbonic anhydrase or a fragment thereof. 9. A kit as claimed in Claim 8, further comprising methods for detecting an antigen-antibody complex.