IE940538A1 - Vaccine - Google Patents
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- IE940538A1 IE940538A1 IE940538A IE940538A IE940538A1 IE 940538 A1 IE940538 A1 IE 940538A1 IE 940538 A IE940538 A IE 940538A IE 940538 A IE940538 A IE 940538A IE 940538 A1 IE940538 A1 IE 940538A1
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Abstract
This invention relates to a protein or derivative or fragment thereof obtained from Heliocobacter pylori, methods to use this protein as a vaccine to provide immunological protection against H. pylori infection, and methods to use this protein in diagnostic assays relating to H. pylori.
Description
Field of the Invention
This invention relates to a protein or derivative or fragment thereof obtained from Helicobacter pylori, methods to use this protein as a vaccine to provide immunological protection against H. pylori infection, and methods to use this protein in diagnostic assays relating to H. pylori.
Background '“•ί'**» 15
Helicobacter pylori is a widely prevalent organism found on gastric biopsy in approximately 30% of the population less than 40 years old with increasing incidence thereafter. The organism is a causative agent of chronic gastritis in humans (e.g. Marshall & Warren 1984*; Blaser, 1990*). Epidemiological studies have shown that H. pylori is most commonly found in association with gastritis. Serological investigations have demonstrated that evidence of a current or prior infection can be found in 30 - 50% of a randomly chosen population of blood donors. No direct causal relationship has been conclusively proven for duodenal ulcer disease. However, the organism is found in 95% of patients with duodenal ulcer. Furthermore, eradication of the organism results in rapid ulcer healing (e.g. Rauws & Tytgat, 1990*). These data provide strong evidence that H. pylori is a dominant factor in the development of duodenal ulcer. Additional evidence for the pathogenic involvement of H. pylori in these conditions has been provided by studies with gnotobiotic piglets (Lambert et al., 1987*) and the fulfilment of Koch's postulates with human volunteers (Marshall et al., 1985*; Morris & Nicholson, 1987*).
In addition, there is now strong circumstantial evidence implicating H. pylori in the pathogenesis of gastric carcinoma (e.g. Jiang et al., 1987*; Lambert et al., 1986*; Crabtree et al. , 1992; 1993; Forman et al. , 1990*, 1991; Nomura et al. , 1991; Parsonnet et al. ,
- 2 t- 9 4 0 5 ο «
1991; Correa et al., 1990). Most recently, the Eurogast Study Group, led by Forman (1993), demonstrated a significant relationship between H. pylori seropositivity and gastric cancer mortality and incidence. Indeed, there is now a convincing body of literature implying infection with H. pylori in a considerable proportion of upper gastrointestinal morbidity. A number of hypotheses have been suggested for the pathogenic mechanisms of H. pylori induced gastroduodenal disease, including the production of cytotoxins and mechanical disruption of the epithelium (e.g. Blaser, 1992*). Interestingly, however, many infected persons remain asymptomatic despite the persistent presence of the pathogen (Taylor & Blaser, 1991*).
Diagnosis of infections with H. pylori is based mainly on histology and culture from gastric biopsy specimens or on indirect methods based on urease activity. Various serological assays have been developed for the detection of anti-ff.
pylori antibodies in epidemiological studies in addition to more molecular orientated approaches such as the cloning of H. pylori species-specific antigens for use in, for example, PCRbased serological investigations (e.g. Clayton et al., 1989). However, the use of recombinant species-specific antigens has not yet received widespread use and, consequently, the majority of immunosorbent-based assays currently in use employ various subcellular fractions of H. pylori as a source of antigen. The fractions of proteins used in these assays are frequently heterogenous in composition preparation. Interestingly, compared the inter-assay sensitivity and specificity of several commercially available ELISA kits manufactured specifically for serological studies. Not surprisingly, considerable inter-assay variation was observed. Caution, therefore, must be exercised before employing a particular preparation of protein for use in such immunosorbent assays, particularly in view of the significant genetic heterogeneity of different strains as are their methods of a number of groups have of H. pylori (e.g. Xia et al. , 1994; Owen et al. , 1991) .
We have studied the prevalence of immuno-reactivity to H. pylori in both infected and un-infected individuals and found that un-infected individuals have a high response to H. pylori both in their B-cell and T-cell systems. Specifically, the T-cell immune response to H. pylori seems to be stronger in individuals who are negative for the organism. In this regard we have examined the secretion of the cytokine γ-interferon which is extremely important for the killing of microorganism by macrophages. Secretion of y-interferon by T-cells of patients infected with H. pylori was considerably less than secretion by un-infected individuals when their T-cells were exposed to the organism (Fan et al., 1993*). Hence, these data suggest that individuals who are H. pylori negative have been exposed to the organism and may potentially have cleared the organism. Furthermore, the response to the organism is considerably more potent in this group of individuals than it is in the H. pylori positive patients.
Further studies revealed that patients who are H. pylori negative do indeed have low blood detectable levels of circulating antibodies to H. pylori. Finally a cytokine produced by macrophages called interleukin 12 may significantly enhance -interferon production in response to antigen. As stated previously, antigenspecific interferon production is reduced with H. pylori positive individuals. The addition of IL-12 to immunisation schedules with the 25 kDa protein would be expected to boost host immunity to H. pylori by augmenting the g-interferon response.
An inherent constraint in the design of ELISA based detection systems is that of establishing a cut off point such that all samples below this threshold are considered negative. Clearly, many seropositive cases will remain undetected in this situation and a true estimate of the incident of prior contact with the organism will thereby be underestimated. In this . lM 4 V 5 J a
- 4 approach we use Western blotting to investigate antigen specificity of systemic responses to H. pylori in both healthy and H. pylori-infected individuals and shown that the incidence of seropositivity in H. pylori negative individuals is much greater than has previously been demonstrated. Furthermore, we have demonstrated that antibodies to a 25 kDa protein are detectable in the majority of H. pylori negative individuals. These were detected using a technique which we have modified called Enchansed Chemiluminescence. Enhanced
Chemiluminescence on Western blot analysis reveals that the majority of uninfected individuals have antibodies which are specific for H. pylori and recognise antigens which are not present on other micro organisms. Of these antigens the most common one recognised in a 25 kDa protein which appears to be specific to H. pylori. Hence, these data suggest that immunisation with the 25 kDa protein or sub-unit thereof could have the potential to confer protective immunity on individuals who are either un-infected with the organism or individuals in whom the organism has been cleared by anti-bacterial treatment. A second protein was also identified at 18 kDa in a large subgroup of H. pylori negative individuals .
According to one aspect the invention provides a H. pylori protein or derivative or fragment thereof which generates an antibody response in H. pylori negative individuals .
In another aspect the invention provides a vaccine against H. pylori including at least one of a H. pylori protein or derivative or fragment thereof.
The vaccine may be in combination with a suitable adjuvant.
In a further aspect the invention provides a method of inducing protective antibodies against H. pylori in animals including humans, the method comprising the step of administering a H. pylori protein or derivative or t-84 0 i.
- 5 fragment thereof to generate specific autosensor. The H. pylori protein is administrered in combination with at least one other pharmaceutical agent.
The invention further provides the use of 25 kDa or 18 kDa H. pylori protein or derivative or fragment thereof in immunoassays.
In addition, the invention provides the use of H. pylori proteins to which immunoreactivity is detected in H. pylori negative individuals as vaccines or immunogens.
The invention also provides the use of H. pylori protein from which 25 kDa and/or 18 kDa H. pylori protein or derivative or fragment thereof has been removed in immunoassays .
According to a further aspect the invention provides the use of proteins below 30 kDa as the basis for a vaccine or immunogen.
The invention also provides the use of proteins above 30 kDa as the basis for immunoassay for H. pylori.
It is an object of the present invention to develop antibodies to the 25 kDa protein which might be useful as passive immunotherapy for established H. pylori infection.
It is an object of the present invention to provide a purified 25 kDa protein which might be used in combination with other H. pylori sub-units as a combined vaccine against H. pylori.
It is an object of the present invention to increase the discriminatory power of ELISA testing for H. pylori by generating H. pylori protein preparations for use in ELISA tests from which the 25 kDa protein has been removed. Removal of a strongly immunogenic antigen to which antibodies are present in H. pylori negative individuals should increase the discriminatory
- 6 capabilities of ELISA in identifying people with active infection.
It is also an object of the present invention to increase the discriminatory power of ELISA testing for H. pylori by generating H. pylori protein preparations for use in ELISA tests from which the 25 kDa protein — and other antigens to which immunoreactivity is detected in H. pylori negative individuals- has been removed. Removal of a strongly immunogenic antigen to which antibodies are present in H. pylori negative individuals should increase the discriminatory capabilities of ELISA in identifying people with active infection.
It is also an object of the invention to provide other purified proteins of H. pylori to antibodies are detected in H. individuals .
which constitutive pylori negative
The invention includes the use of interleukin 12 in combination with the 25 kDa protein or any other H. pylori subunit as an adjuvant therapy.
A further object of the present invention is to identify a 18 kDa protein associated with H. pylori which induces as strong immune response in individuals who are negative for the organism.
It is also an object of the present invention to provide a purified 18 kDa protein for use as a vaccine against H. pylori.
It is a further object of the present invention to provide a purified 18 kDa protein for use as a therapeutic immunogen for eradication of H. pylori infection.
It is also an object of the present invention to develop antibodies to the 18 kDa protein which might be useful as passive immunotherapy for established H. pylori infection.
i-94 0 538
The invention also provides a purified 18 kDa protein which might be used in combination with other H. pylori sub-units as a combined vaccine against H. pylori.
It is a further object of the present invention to increase the discriminatory power of ELISA testing for H. pylori by generating H. pylori protein preparations for use in ELISA tests from which the 18 kDa protein has been removed. Removal of a strongly immunogenic antigen to which antibodies are present in H. pylori negative individuals should increase the discriminatory capabilities of ELISA in identifying people with active infection.
It is also an object of the present invention to increase the discriminatory power of ELISA testing for H. pylori by generating H. pylori protein preparations for use in ELISA tests from which the 18 kDa protein — and other antigens to which immunoreactivity is detected in H. pylori negative individuals- has been removed. Removal of a strongly immunogenic antigen to which antibodies are present in H. pylori negative individuals should increase the discriminatory capabilities of ELISA in identifying people with active infection.
It is an object of the current invention other purified proteins of H. pylori constitutive antibodies are detected in negative individuals .
to provide to which
H. pylori
It is also an object of the present patent to provide a H. pylori protein preparation from which proteins below 30 kDa have been removed on the basis for immunoassay for H. pylori.
It is an object of the present patent to provide a H. pylori protein preparation of below 30 kDa molecular weight for use as an immunogen.
The invention also provides a H. pylori protein preparation of below 30 kDa molecular weight for use as a vaccine.
- 8 B-9405381
The invention also includes the use of interleukin 12 in combination with the 18 kDa protein or any other H. pylori subunit as an adjuvant therapy.
We have developed a novel assay for detection of antibodies to H. pylori. This assay uses Western blotting and Enhanced Chemiluminescence (ECL). Using this assay we have demonstrated that approximately 75% of individuals who are negative for H. pylori by routine testing such as the rapid urease test have in fact got detectable antibodies to H. pylori (Fig. 1).
Furthermore, these antibodies are not absorbed by C. jejuni or by E. coli suggesting that his is a specific antibody response (Fig.2 ). Of particular note we have performed characterisation of the antigens recognised by these antibodies by molecular weight, using ECL Western blotting. Sera from un-infected individuals recognize a range of antigens on H. pylori. The most common antigen recognised is a 25 kDa protein which is recognised in over 70% of individuals who are negative for the organism on Rapid urease testing. Hence this suggests that the 25 kDa protein may be an immunodominant antigen which evokes a powerful immune response in individuals who are negative for the organism. A second protein was identified at 18 kDa which elicited significant antibody responses in H. pylori-negative children.
Detailed Description of the Invention
Methods used in the identification and partial purification of two novel antigens from Helicobacter pylori
Methods
Western Blotting. Proteins from SDA-PAGE gels (30% T/2.67% C) were electroblotted (0.8 mA/cm2 for 1 h) to PVDF membrane using a semi-dry blotting apparatus (LKB/Pharmacia). Primary antibodies (human serum; 1/50 - 1/100 dilution) were detected using a 1/5,000 dilution
^.-94 0 5 of anti-human IgG (horseradish peroxidase-conjugated) in combination with enhanced chemiluminescence (see below). Blots were washed in phosphate buffered saline (pH 7.5) containing fat-free dried skimmed milk (5%, w/v) and Tween-20 (0.05%, v/v). Blots were exposed to Kodak XOMAT S film for 1-10 s. Exposed films were developed in Kodak LX-24 developer and fixed in Kodak dental X-ray fixer.
Enhanced Chemiluminescence (ECL)
The use of chemiluminescence to detect antibodies in Western blotting in preference to the conventional procedures of employing chromogenic substrates as detection reagents was adopted primarily because of the reporting gain in the sensitivity of detection (approximately 10-fold) over that found when chromogens are used. Oxidized luminol emits visible light and the intensity of this light emission is increased 1000-fold in the presence of chemical enhancers (e.g. iodophenol). The method is described blow:
Substrate
Concentration/Amount
Luminol
4-Iodophenol
1.2 mM (in 0.1 Μ-Tris (50ml), pH 0.4 mM (dissolved in DMSO before
Hydrogen Peroxide 17 μΐ of a 30% (v/v) solution
8.8) use)
Blots were incubated in the above mixture for one minute and then exposed to X-ray film as described above.
Partial Purification of 18 and 25 kDa Proteins
Both proteins were partially purified from whole Helicobacter pylori on the basis of molecular weight (Fig. ) using preparative continuous-elution sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on a Model 491 Prep-Cell (Bio-Rad). This method enables us to quantitively purify preparative amounts of proteins in a soluble form.
Purification Method
-»405 mg H. pylori were precipitated with ice-cold acetone, washed once in acetone and the precipitate then solubilised in 3.8 ml SDS-PAGE sample buffer (62 mM Tris, pH 6.8; glycerol (10%, v/v) ; SDS (2%, v/v) ; 2mercaptoethanol (5%, v/v); bromophenol blue (0.002%, v/v). Published electrophoretic procedures, with very minor modifications, were followed throughout sample preparation.
Loading: The protein mixture, in sample buffer, was loaded onto a 12.5% polyacrylamide tube gel (30% T/2.67% C) . The dimensions of the tube gel were: 28 mm internal diameter; upper surface 3.6 cm2; stacking gel 2 cm; resolving gel 10 cm.
Running Conditions: Electrophoresis was performed at 40 mA (constant current) overnight at room temperature. Fractions (1 ml) were collected at 0.1 ml/min. Samples of each fraction (5 μΐ) were subjected to analytical SDS-PAGE to assess the purity and antigenicity of each protein. Every fraction within the molecular mass region of interest was screened by both SDS-PAGE (to assess purity) and Western blotting (to assess antigenicity) in an attempt to isolate and characterise the individual immunogenic proteins. The resolution of this technique is such that pure preparations of single proteins may be achieved once optimal electrophoretic conditions have been established. Preliminary optimization protocols entailed electrophoresing mixtures of H. pylori proteins under conditions designed to favour high resolution of low molecular weight proteins. The final electrophoretic conditions used to achieve partial purification of the selected proteins are detailed in the Methods section. Using these exact conditions the 18 kDa proteins eluted between 11-14 ml and the 25 kDa protein eluted within 16-20 ml. The molecular weights of the proteins were determined by analytical SDS-PAGE using a range of low molecular weight marker proteins (range: 14.5 kDa - 66 kDa; code: Sigma SDS-7) and Western blotting confirmed that these proteins were the immunogens of interest.
840538
Figure 1 shows Western blot analysis of antibody responses to H. pylori in individuals negative for H. pylori on Rapid urease testing. Western blotting was performed as previously described using an enhanced chemiluminescence detection system. Antibodies to a large range of H. pylori proteins were seen in individuals who are H. pylori negative on Rapid urease testing. The most common antigen to which an antibody was detected with the 25 kDa protein. Figure 3 shows a preparative SDS gel elution profile of the 25 kDa and 18 kDa proteins .
Detailed Description
Materials & Methods
Materials. All antibodies were obtained from Dako Ltd., High Wycombe, Bucks., U.K. All other chemicals and solvents were obtained from either the Sigma Chemical Company Ltd., Poole, Dorset, United Kingdom or BDH Chemicals Ltd., Poole, Dorset, United Kingdom.
SDS-PAGE. Discontinuous SDS-PAGE was performed essentially as described by Laemmli (1970). A total of 5 mg of acetone-precipitated H. pylori protein were located into each well. Gels were either stained with Coomassie Blue R-250 or processed for immunoblotting. Broad range molecular weight markers were purchased from Bio-Rad Laboratories, 3300 Regatta Blvd., Richmond, CA 94804. The molecular masses are expressed as kDa.
Western Blotting. Proteins from SDS-PAGE gels (30% T/2.67% C) were electroblotted (0.8 mA/cm2 for 1 h) to PVDF membrane using a semi-dry blotting apparatus (LKB/Pharmacia), essentially as described by Towbin et al, (1979). Primary antibodies (human serum; 1/50 1/100 dilution) were detected using a 1/5,000 dilution of anti-human IgG (horseradish peroxidase-conjugated) in combination with enhanced chemiluminescence. Blots were washed in PBS containing fat-free dried skimmed milk (5%, w/v) and Tween-20 (0.05%, v/v). Blots were exposed to Kodak X-OMAT S film for 1-10 s. Exposed films were developed in Kodak LX-24 developer and fixed in Kodak dental X-ray fixer.
Sera. Serum samples were obtained from the Research Centre, Our Ladies Hospital for Sick Children, Crumlin, Dublin. All subjects were attended for medical conditions other than gastroenterological disorders. In addition, blood samples were obtained from a randomly selected cohort of children (Harcourt Street Childrens Hospital, Dublin) or from adults attending the gastenterology unit at St. James's Hospital, Dublin. All patients had a rapid urease (CLOtest) performed. Patients were defined as H. pylori positive or negative on the basis of positive or negative responses on rapid urease test.
Anti-tf. pylori antiserum. Anti-tf. pylori antiserum was a kind gift from Prof. B. Drumm and Dr. M. Clyne. The antiserum was raised in New Zealand white rabbits against whole H. pylori using conventional immunizing and boosting procedures .
Protein Measurements. Protein was measured by the method of Markwell et al. (1978) with bovine serum albumin as the protein standard.
Absorption of sera. Antisera were absorbed with either E. coli or C. jejuni by incubating a suspension of the bacteria with patient sera for 2 h at room temperature with gentle mixing. The bacteria were removed from suspension by centrifugation (12,000 x g, 3 min).
Bacterial strains and growth conditions. The clinical isolates H. pylori used in this study were isolated from antral biopsies obtained from patients attending the gastroenterology clinic at St. James's Hospital, Dublin. H. pylori was grown under microaerophilic conditions for 4 days on 7% lysed horse blood agar at 37°C. Cells were harvested into ice-cold phosphate buffered saline (pH 7.5) containing PMSF (1 mM), EDTA (1 mM) , and leupeptin (50 μg/ml). The cells were washed twice by centrifugation ( 10,000 x g, 5 min, 4°C) in this buffer fc'r- 9 4 0 5 3 8 $ before use. C. jejuni was a clinical isolate from stool in a patient with C. _jejuni enteritis and was grown for two days exactly as described above with the exception that the incubation temperature was 42°C. The strain of E. coli used in this study is commercially available (Gibco) and was kindly provided by Dr. Ciaran Cronin, Dpt. Pharmacology, University College Dublin.
EXAMPLE 1
CLP negative adults
Similarly, a cohort of 19 adult sera was screened for anti-#, pylori IgG antibodies. Each of these subjects was CLO negative, yet 83% had detectable antibodies (IgG) to H. pylori (Fig. 4a). Taken together, these data suggest extensive prior contact with H. pylori. The most common antigen to which an antibody was detected was a 25 kDa species.
CLO negative children
The systemic humoral immune response (IgG) to H. pylori was studied in two groups of children also. None of these subjects had received any form of anti-H. pylori therapy. However, in almost all cases the children had a specific antibody response to H. pylori. The first cohort studies consisted of twenty children (age range: 4-15 years), negative for H. pylori on CLO test. Of these, 75% had detectable IgG antibodies to H. pylori (Fig. 4b).
The second cohort of children (n = 20) were asymptomatic and presented in hospital with conditions other than gastrointestinal disorders. Yet (note only 18/20 screened so far) 13/18 (72%) had detectable IgG antibodies to several H. pylori specific antigens (Fig. 1). However, from the intensity of the response the data suggest that the antibody response is most likely due to prior contact with the bacterium, when compared to the considerably stronger response observed with H. pylori positive individuals.
^»94 0 5 3 8 4 ί
EXAMPLE 2
Cross Reactivity with other Bacteria
As many bacteria share common antigenic determinants, we examined the extent of cross-reactivity between H. pylori and the closely related C. jejuni, in addition to E. coli, using two complimentary approaches. Firstly, the ability of the anti-tf. pylori polyclonal antiserum to recognise antigens on both C. jejuni and E. coli was examined by Western blotting (Fig. 2).
Anti-tf. pylori antiserum recognized a number of antigenic determinants on both E. coli and C. jejuni. Specifically, the antiserum recognises proteins of molecular mass 72, 50, 40, 36, and 25 kDa on C. jejuni and proteins of molecular mass 200, 116, 45, and 38 kDa on E. coli (Fig. 3). Of these, only 3 proteins (70, 25 kDa from C. jejuni and 200 kDa from E. coli) show pronounced cross-reactivity with anti-tf. pylori antiserum. Therefore, the observed cross reactivity is clearly not extensive. Secondly, absorption experiments demonstrated that this cross reactive antigen recognition was of minor significance. Serum samples absorbed with clinical isolates of H. pylori and C. jejuni in addition to a commercially available strain of E. coli demonstrated that seroreactivity could be eliminated by absorbing with H. pylori but not with C. jejuni or E. coli (Fig. 5). Figure 5 is a representative experiment. Absorption studies were performed on approximately half of the serum samples screened in this study with similar results to those shown.
Having partially purified the 26-26 kDa protein by preparation continuous-elution electrophoresis as shown in Fig. 3, we confirmed the antigenicity of the 24-26 kDa protein by incubating a Western blot (Fig. 6) which was run in parallel with the analytical SDS-PAGE gel shown in Fig. 3a. The example shown in Fig. 6 is a representative experiment where the blot was incubated with the serum from an H. pylori un-infected individual.
^-940538
Clearly, this serum sample contains antibodies that specifically recognise the 24-26 kDa protein and furthermore, the results of this experiment demonstrate that the antigen preparation is highly enriched for this protein and that no other immunogenic proteins are present in this preparation. We have obtained similar results with the 16-20 kDa protein.
It will be appreciated that while we have referred to a molecular mass of 25 kDa and 18 kDa the molecular mass may lie in the 24-26 kDa and 17-19 kDa range.
Many variations on the specific embodiments described will be readily apparent and accordingly the invention is not limited to the embodiments hereinbefore described which may be varied in detail.
Claims (32)
1.
2. .
3.
4. .
5.
6.
7. .
8.
9.
10.
11. A H. pylori protein or derivative or fragment thereof which generates an antibody response in H. pylori negative individuals. A H. pylori protein as claimed in calim 1 which has a molecular weight of less than 30 kDa. A H. pylori protein as claimed in claim 1 or 2 which is a 25 kDa H. pylori protein or derivative or fragment thereof. A H. pylori protein as claimed in claim 1 or 2 which is an 18 kDa H. pylori protein or derivative or fragment thereof. A vaccine against H. pylori including at least one of a H. pylori protein or derivative or fragment thereof as claimed in any preceding claim. A vaccine as claimed in claim 4 in combination with a suitable adjuvant. A vaccine as claimed in claim 4 or 5 including at least one other pharmaceutical product. A vaccine as claimed in claim 6 wherein the pharmaceutical product is an antibiotic. A vaccine as claimed in claim 7 wherein the antibiotic is selected from one or more of metranidazole, amoxycillin, tetracycline or erythromyc in. A vaccine as claimed in any of claims 6 to 8 wherein the pharmaceutical product is an antibacterial agent such as bismuth salts. A vaccine as claimed in any of claims 4 to 9 in a form for oral administration.
12.
13.
14. .
15.
16. .
17. .
18.
19.
20.
21.
22. . A vaccine as claimed in any of claims 4 to 9 in a form for intranasal administration. A vaccine as claimed in any of claims 4 to 12 including a peptide delivery system. A process for generating a H. pylori protein or derivative or fragment thereof by preparative SDS-PAGE electrophoresis. A process as claimed in claim 11 wherein the H. pylori protein is a 25 kDa H. pylori protein. A process as claimed in claim 11 wherein the H. pylori protein is an 18 kDa H. pylori protein. A method of inducing protective antibodies against H. pylori in animals including humans, the method comprising the step of administering a H. pylori protein or derivative or fragment thereof to generate specific antibodies. A method as claimed in claim 17 wherein the H. pylori protein has a molecular weight of less than 30 kDa. A method as claimed in claim 17 or 18 wherein the H. pylori protein is a 25 kDa H. pylori protein. A method as claimed in claim 17 or 18 wherein the H. pylori protein is a 18 kDa H. pylori protein. A method as claimed in any of claims 17 to 20 wherein the H. pylori protein is administered in combination with at least one other pharmaceutical agent. A method as claimed in claim 21 wherein the pharmaceutical agent is an antibiotic. *-»4
23. A method as claimed in claim 22 wherein the antibiotic is selected from one or more of metranidazole, amoxycillin, tetracycline or erythromycin
24. A method as claimed in claim 23 wherein the pharmaceutical agent is an antibacterial agent such as bismuth salts.
25. Use of 25 kDa H. pylori protein or derivative or fragment thereof in immunoassays.
26. Use of 18 kDa H. pylori protein or derivative or fragment thereof in immunoassays.
27. Use of H. pylori proteins to which immunoreactivity is detected in H. pylori negative individuals as vaccines or immunogens.
28. Use of H. pylori protein preparation, from which H. pylori proteins to which immunoreactivity is detected in H. pylori negative individuals has been removed, in immunoassays.
29. Use of H. pylori protein preparation from which 25 kDa H. pylori protein or derivative or fragment thereof has been removed in immunoassays .
30. Use of H. pylori protein preparation from which 18 kDa H. pylori protein or derivative or fragment thereof has been removed in immunoassays .
31. The use of proteins below 30 kDa as the basis for a vaccine or immunogen.
32. The use of proteins above 30 kDa as the basis for immunoassay for H. pylori.
Priority Applications (22)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IE940538A IE940538A1 (en) | 1994-07-01 | 1994-07-01 | Vaccine |
IE950496A IE80851B1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
CA002194236A CA2194236C (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
PCT/IE1995/000037 WO1996001273A1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
AU27513/95A AU695769B2 (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
GB9626483A GB2303854B (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
IE950497A IE81023B1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
GB9626484A GB2303855B (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
JP8503771A JPH10502366A (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigen protein preparation and immunoassay |
ZA955500A ZA955500B (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
JP8503770A JPH10502365A (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
ZA955499A ZA955499B (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
EP95922709A EP0769019A1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
AT95922708T ATE229978T1 (en) | 1994-07-01 | 1995-07-03 | HELICOBACTER PROTEINS AND VACCINES |
CA002194237A CA2194237A1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
PCT/IE1995/000036 WO1996001272A1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
AU27514/95A AU696941B2 (en) | 1994-07-01 | 1995-07-03 | Helicobacter pylori antigenic protein preparation and immunoassays |
EP95922708A EP0769018B1 (en) | 1994-07-01 | 1995-07-03 | Helicobacter proteins and vaccines |
DE69529219T DE69529219T2 (en) | 1994-07-01 | 1995-07-03 | HELICOBACTER PROTEINS AND VACCINES |
US08/775,765 US20010010821A1 (en) | 1994-07-01 | 1996-12-31 | Helicobacter proteins and vaccines |
JP2006190939A JP2006348036A (en) | 1994-07-01 | 2006-06-13 | Helicobacter protein and vaccine |
US11/518,467 US20070104731A1 (en) | 1994-07-01 | 2006-09-08 | Helicobacter proteins and vaccines |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IE940538A IE940538A1 (en) | 1994-07-01 | 1994-07-01 | Vaccine |
Publications (1)
Publication Number | Publication Date |
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IE940538A1 true IE940538A1 (en) | 1997-08-27 |
Family
ID=11040445
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
IE940538A IE940538A1 (en) | 1994-07-01 | 1994-07-01 | Vaccine |
Country Status (2)
Country | Link |
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IE (1) | IE940538A1 (en) |
ZA (2) | ZA955500B (en) |
-
1994
- 1994-07-01 IE IE940538A patent/IE940538A1/en unknown
-
1995
- 1995-07-03 ZA ZA955500A patent/ZA955500B/en unknown
- 1995-07-03 ZA ZA955499A patent/ZA955499B/en unknown
Also Published As
Publication number | Publication date |
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ZA955500B (en) | 1996-01-02 |
ZA955499B (en) | 1996-01-02 |
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