MXPA97006267A - Antigen of helicobacter pyl - Google Patents

Abstract

A novel antigen for H. pylori is provided. It also describes the use in the diagnosis of H. pylori infection including methods for said diagnosis, and equipment to use said method. In addition, novel antigenic fragments of the antigen are provided, as well as vaccines that comprise either the antigen or one or more antigenic fragments.

Description

ANTIGEN OF HELICOBACTER PYLORI DESCRIPTION OF THE INVENTION The present invention relates to a novel antigen of Helicobacter pylori, or antigenic fragments thereof, the use of the antigen of fragments thereof to detect Helicobacter pylori and kits comprising them, as well as vaccines comprising the antigen or fragments thereof and a method for isolating the antigen. Intestinal infections in mammals, and in particular humans, stimulate an immune response in mucous secretions, such as saliva, through the activation of the common mucosal immune system. This response usually initially forms an antibody response in serum although it is generally characterized by the presence of IgA antibodies. However, the immune response in the secretion, including saliva, rapidly decreases the subsequent elimination of the antigen (eg, bacteria or virus) from the body. Accordingly, the presence of the antibody in mucosal secretions reflects current, that is, contemporaneous infection. In the case of a microbial infection, for example, the antibodies in mucosal secretions, hereinafter referred to as secretory antibodies, reflect the current state of colonization of the microbe, such as in the intestine, and thus are a Useful monitor of contemporary infection. On the other hand, the antibody in the serum persists for some time after the microbe is removed from the body. A positive antibody test in the serum, therefore, reflects both past and present exposure to the antigen, which is less helpful to the physician. A positive secretion antibody test, on the other hand, indicates present or contemporaneous infection by the microbe. The diagnosis of H. pylori infection can be done through microscopy, microbiological culture, urea respiration test or by the presence of specific antibodies in ELI SAs in the serum. It can be predicted that H. pylori infection, being an infection of the gastric mucosa, could produce a response to IgA antibody in gastric secretion. However, it has been found that the specific H. pylori antibody in mucosal secretions is of the IgG class and not IgA, as expected. Very little IgA antibody is detected, if any. Accordingly, AU-A-9067676 is directed to the detection of IgG in secretion of the mucosa specific to the H. pylori antigen and thus provides means to verify the current infection, ie, contemporaneous, by that microorganism in mammals. The corresponding academic publication is Witt or others, Frontiers in Mucosal Immunology 1 693-696 (1991). The presence of IgG antibodies in the saliva of Helicobacter pylori positive patients has received some attention in the Procedures of the Annual Meetings of the American Gastroenterological Association (Annual Meeting of the American Gastroenterological Association). After the description, by Czinn and others, of the presence of such antibodies in the 1989 procedures, Larsen and others concluded in the May 1991 procedures that salivary IgG levels are a practical, noninvasive marker of the therapeutic response during the course of an antibiotic therapy. In the procedures of April 1992, Landes et al. Confirmed early observations and observed that the measurement of IgG in saliva for Helicobacter pylori is a simple, non-invasive test to detect H. pylori in positive patients, especially in disseminated or pediatric populations, where the other tests are not practical. WO-A-9322682 describes a convenient and reliable test, in vitro, for H. pylori. This test uses an antigen preparation in a reaction with the IgG antibody in a mucous secretion of a mammal being treated. Therefore, there is a need to identify, isolate and, in this way, provide novel antigens of H. pylori, that can be used in diagnostic tests. These antigens must be specific, reliably purifiable, and must be characterized by a good specific character and by the lack of false positive results, when used in such tests. In addition, they also form the basis of a vaccine, useful either for the treatment or prophylaxis of H. pylori infection. Thus, in a first aspect, the present invention provides a protein being an H. pylori antigen and having a molecular weight in the range of 55-65 kDa, as determined under denaturation and reduction conditions. Suitably, the antigenic protein has, at its amino terminal end, the following amino acid sequence: M V T L l N N E D D Met-Val-Thr-Leu-l le-Asn-Asn-Glu-Asp-Asp or a sequence substantially homologous to it. At the amino acid level, a protein sequence can be considered as substantially homologous to another protein sequence if a significant number of the constituent amino acids exhibit homology. At least 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids can be homologous. It has also been found that parts of the complete protein are themselves antigenic. Thus, in a second aspect, the present invention provides an antigenic fragment of the protein of the invention. In a preferred embodiment of this aspect of the invention, the antigenic fragment is a peptide having the sequence: M T L I N N Met-Val-Thr-Leu-lle-Asn-Asn-Glu Those skilled in the art will appreciate that some variation in the sequence of this fragment will be possible while still retaining its antigenic properties. Said variations also form part of the invention. The antigenic protein, or fragments thereof, of the present invention, may be provided alone, as a purified or isolated preparation, or as part of a mixture with other H. pylori antigenic proteins. In a third aspect, therefore, the invention provides an antigen composition comprising a protein of the invention and / or one or more antigenic fragments thereof. Said composition can be used for the detection and / or diagnosis of H. pylori. In one embodiment, the composition one or more additional H. pylori antigens. In a fourth aspect, the present invention provides a method for detecting and / or diagnosing H. pylori, which comprises: (a) contacting an antigenic protein, or antigenic fragment thereof, or an antigen composition of the invention with a sample that will be tested; and (b) detecting the presence of antibody for H. pylori. In particular, the protein, the antigenic fragment thereof or the antigen composition of the invention can be used to detect IgG antibodies. Suitably, the sample to be tested will be a biological sample, for example, a blood or saliva sample. An example of a method suitable for detecting H. pylori, using a sample of a mucosal secretion is that described in WO-A-9322682. In a fifth aspect, the invention provides the use of an antigenic protein, antigenic fragment thereof, or an antigenic composition of the present invention to detect and / or diagnose H. pylori. Preferably, the detection and / or diagnosis is performed in vi tro. In a sixth aspect, the present invention provides a method for isolating an antigenic protein of the invention, said method comprising the following steps: (a) preparing cultures of H. pylori, developing the cultures under appropriate conditions and harvesting them, followed by washing to produce a washed cell pellet; (b) resuspending the washed cells in an appropriate pH regulator, followed by the dissolution of the cells; (c) centrifuge to remove the cell waste and obtain the supernatant containing soluble cell proteins; (d) subjecting the obtained solution to ion-exchange chromatography with a gradient elution, and analyzing the fractions thus obtained for the presence of urease; (e) combining fractions containing urease and subjecting said combination to gel permeation chromatography; (f) selecting the appropriate peak; and (g) confirming the presence of the specific protein having a molecular weight in the range of 55-65 kDa in the isolate and isolating the protein. The antigenic protein, its antigenic fragment or the antigenic composition of the invention can be provided as part of a kit for use in the detection and / or in vitro diagnosis of H. pylori. Thus, in a seventh aspect, the present invention provides equipment for use in the detection and / or diagnosis of H. pylori, which comprises an antigenic protein, an antigenic fragment thereof or an antigenic composition of the invention. In addition, the antigenic protein or antigenic fragment thereof of the invention can be used to induce an immune response against H. pylori. Thus, in a further aspect, the present invention provides a composition capable of producing an immune response in a subject, which comprises the protein or one or more antigenic fragments thereof of the invention. Suitably, the composition will be a vaccine composition, which optionally comprises one or the other suitable adjuvant. Said vaccine composition can be a vaccine composition either prophylactic or therapeutic. The vaccine compositions of the invention may include one or more auxiliaries. Examples of auxiliaries well known in the art include inorganic gels, such as aluminum hydroxide or water-in-oil emulsions, such as incomplete Freund's adjuvant. Other useful auxiliaries will be well known to those skilled in the art. In still other aspects, the present invention provides: (a) the use of the protein or of one or more antigenic fragments thereof of the invention, for the preparation of an immunogenic composition, preferably a vaccine; (b) the use of said immunogenic composition to induce an immune response in a subject; and (c) a method for the treatment or prophylaxis of H. pylori infection in a subject, which comprises the step of administering to the subject an effective amount of the protein, at least one antigenic fragment or an antigenic composition of the invention, preferably as a vaccine. The preferred features of each aspect of the invention are, to each other, an aspect of mutatis mutandis. The invention will now be described with reference to the following examples, which should not be construed as limiting the invention in any way.

EXAMPLE 1 (a) H. pylori cultures were grown under appropriate conditions and the cells were harvested in pH regulated saline with phosphate. This was followed by repeated centrifugation to remove cell debris and other contaminants, e.g., agar, and fresh PBS was added, three times to produce a washed cell pellet; (b) The washed cells were resuspended in a pH regulator of 1 M TRIS-HCl, pH 7.2, for use in the step of ion exchange chromatography. The cell suspension was then subjected to sound application (6μ for 30 seconds, 60 seconds, repeated 25 times for a 10ml sample containing cells from 100 agar plates) of sufficient intensity and duration to ensure dissolution of the cells. cells; (c) The suspension was then centrifuged to remove the cell waste and the supernatant was obtained, containing soluble cell proteins; (d) The solution of step (c) was then subjected to fractionation through ion exchange chromatography, using a strong anion exchange resin such as Mono® or Q-Sepharose® (Pharmacia), using a gradient elution. based on the increase of the sodium chloride concentration of the pH regulator of the elution, from 0 to 1.0 M, in a predetermined manner. The fractions were then analyzed for the presence of urease; (e) Fractions containing urease were then combined and subjected to gel permeation chromatography, using a resin with a cut scale of 5 x 103-5 x 10 6 Da for lobular protein; (f) The appropriate peak was selected: (i) performing a urease test of all fractions and identifying the protein peak containing the urease activity; and (ii) analyzing all the fractions that showed to be positive to the urease and the protein containing peaks immediately adjacent to the urease peak, but of low molecular weight placing a microgram of the protein of these fractions on nitrocellulose or an equivalent, drying and then determining its ability to react with specific antibodies of H. pylori in serum or saliva samples of H. pylori-positive individuals; (g) The presence of the specific protein in the isolate was confirmed, subjecting it to polyacrylamide gel electrophoresis (PAGE) and Western staining, and analyzing staining using IgG from a prepared combination of human serum collected from H. pylori positive individuals. PAGE was carried out under denaturing conditions, and the identified protein was isolated by having a molecular weight on the scale of 55-65 kDa.

EXAMPLE 2 Rabbits were inoculated with the MVTLINNE peptide. Blood samples were analyzed, as described below, to test the antibody response.

Test Method To the blood samples were added 25 μl of a 1% solution of thiomersal per ml of blood (ie, a concentration of 25 μl / ml), the samples were then stored at -40 ° C before the proof. The analyzes were performed using plates, where each cavity was coated with 100 μl of 5 μg / ml of H. pylori antigen. (preparation of antigen as described in WO-A-93/22682), followed by 300 μl of a 1% solution of Byco A as a back coating. The blood samples were diluted using a CDL wash buffer to produce a scale of dilutions.

Regulated wash pH of CDL: Amounts for 1 dm 'Tris 12.11 g 5M HCl 15 ml Distilled water 400 ml NaCl 87.66 g Thiomersal 0.1 g Tween 80 50 g i) add tris to distilled water; ii) add 5M HCl until a pH of 7.80 is present at 20 ° C; iii) add Thiomersal; iv) add the NaCl; v) add Tween 80; vi) develop a final volume with distilled water. An anti-rabbit horseradish peroxidase conjugate Swine with ABTS (2,2-Azino-di- [3-ethyl-benzthiazoline-sulfonate]) diluted 1/25 with pH Regulator of Peroxidase Citrate as substrate was used, to detect a binding antibody, green in color, indicating a positive result.

Peroxidase pH regulator: amounts for dm 'Distilled water 700 ml Citric acid 23.0 g NaOH 100 ml 30% H202 0.5 ml Adjust the pH to 4.0 with 1M NaOH. The assay protocol was as follows: 1) add 100 μl of diluted sample to each well; 2) incubate for 30 minutes (with shaking); 3) wash 5x with CDL pH regulator and dry the plate; 4) add to each cavity 100 μl of HRP of anti-rabbit Swine; 5) incubate for 30 minutes (with shaking); 6) wash 5x and dry the plate; 7) add 100 μl of ABTS to each well; 8) incubate for 30 minutes; and 9) read the plate @ 414 nm.

Results The peak antibody response occurred around six months post-inoculation. Table 1, below, shows the results over time for blood samples from two rabbits inoculated with the peptide. The data is for samples diluted 1/100.

TABLE 1 These results are shown in Figure 1. Clearly, it can be seen that the peptide produces an anti-H. pylori antibody response.

Claims (18)

1. - A protein that is an antigen of H. pylori and that has a molecular weight in the range of 55-65 kDa, determined under conditions of denaturation and reduction conditions, which has, at its amino terminal end, the sequence: Met-Val-Thr-Leu-lle-Asn-Asn-Glu-Asp-Asp; or a sequence substantially homologous to it.

2. An antigenic fragment of a protein according to claim 1.

3. An antigenic fragment according to claim 2, having the sequence: M V T L I N N E Met-Val-Thr-Leu-lle-Asn-Asn-Glu or a sequence substantially homologous to it.

4. An antigen composition comprising a protein according to claim 1, or at least one antigenic fragment according to claim 2 or claim 3.

An antigen composition according to claim 4, which also includes one or more others

6. - A protein according to claim 1, an antigenic fragment according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5, for use for detection and / or diagnosis of H. pylori.

7 '.- A method for detecting and / or diagnosing H. pylori, which comprises: (a) contacting a protein according to claim 1, at least one antigenic fragment according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5 with a sample to be tested; and (b) detecting the presence of antibody for H. pylori.

8. A method according to claim 7, wherein the sample is a sample of saliva.

9. The use of a protein according to claim 1, at least one antigenic fragment according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5 for detecting and / or diagnose H. pylori.

10. The method according to claim 7 or claim 8, or the use according to claim 9, wherein the detection and / or diagnosis is performed in vitro.

11. A method for isolating a protein according to claim 1, which comprises: (a) preparing cultures of H. pylori, developing the cultures under appropriate conditions and harvesting them, followed by washing to produce a washed cell pellet; (b) resuspending the washed cells in an appropriate pH regulator, followed by the dissolution of the cells; (c) centrifuge to remove the cell waste and obtain the supernatant containing soluble cell proteins; (d) subjecting the obtained solution to ion-exchange chromatography with a gradient elution, and analyzing the fractions thus obtained for the presence of urease; (e) combining fractions containing urease and subjecting said combination to gel permeation chromatography; (f) selecting the appropriate peak; and (g) confirming the presence of the specific protein having a molecular weight in the range of 55-65 kDa in the isolate and isolating the protein.

12.- Equipment to be used in the detection and / or diagnosis of H. pylori, which comprises a protein according to claim 1, at least one antigenic fragment according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5.

13. - A composition capable of producing an immune response in a subject, which comprises a protein according to claim 1, at least one antigenic fragment according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5.

14. A composition according to claim 13, which is a vaccine composition, optionally comprising one or more auxiliaries.

15. The use of a protein according to claim 1, at least one antigenic fragment according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5 in the preparation of an immunogenic composition, preferably a vaccine.

16. The use of said immunogenic composition according to claim 15 for inducing an immune response in a subject.

17. A method for the treatment or prophylaxis of H. pylori infection in a subject, which comprises the step of administering to the subject an effective amount of a protein according to claim 1, at least one antigenic fragment. according to claim 2 or claim 3, or an antigen composition according to claim 4 or claim 5.

18. A method according to claim 17, wherein the protein, one or more fragments. or antigen composition is administered in the form of a vaccine.SUMMARY A novel antigen for H. pylori is provided. It also describes the use in the diagnosis of H. pylori infection including methods for said diagnosis, and equipment to use said method. In addition, novel antigenic fragments of the antigen are provided, as well as vaccines comprising either the antigen or one or more antigenic fragments.