EP0859788A1 - Helicobacter pylori protein - Google Patents

Helicobacter pylori protein

Info

Publication number
EP0859788A1
EP0859788A1 EP96932692A EP96932692A EP0859788A1 EP 0859788 A1 EP0859788 A1 EP 0859788A1 EP 96932692 A EP96932692 A EP 96932692A EP 96932692 A EP96932692 A EP 96932692A EP 0859788 A1 EP0859788 A1 EP 0859788A1
Authority
EP
European Patent Office
Prior art keywords
pylori
coccoid
antigenic protein
spiral
coccoids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96932692A
Other languages
German (de)
English (en)
French (fr)
Inventor
Bow National University of Singapore Faculty Ho
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cortecs International Ltd
Chapman Paul William
Original Assignee
Cortecs International Ltd
Chapman Paul William
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cortecs International Ltd, Chapman Paul William filed Critical Cortecs International Ltd
Publication of EP0859788A1 publication Critical patent/EP0859788A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K2039/106—Vibrio; Campylobacter; Not used, see subgroups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a novel antigen derived from the coccoid form of H. pylori , its general use in medicine, its use in the preparation of vaccines, as well as its use in the detection of the coccoid form and the diagnosis of H. pylori infection, as well as determination of the disease prognosis of a subject.
  • H. pylori is a Gram negative bacteria that has been strongly implicated in chronic active gastritis and peptic ulcer disease (Marshall et al , Medical Journal of Australia , 142:439-444 (1985); Buck, G.E., Journal of clinical Microbiology, 3:1-12 (1990)) . In in vi tro culture, H.
  • pylori exists in two distinct morphological forms, the culturable spiral form and the non-culturable coccoid form (Marshall e ⁇ al , Microbios letters, 25:83-88 (1984); Kung, J.S.L., and HO, B., Workshop on Gastroduodenal Pathology and Campylobacter pylori (abstract P9) , edited by F. Megraud and H. Lamouliatte, Bordeaux, France (1988)) .
  • the spiral form of the bacterium does not survive beyond about 2 hrs when exposed to air. Under unfavourable conditions, the spiral form undergoes differentiation into the coccoid form (Vijayakumari and Ho, Acta Gastro-enterologica Belgica, 56:101 (1993) ) .
  • H. pylori spiral form various antigens of the H. pylori spiral form have been identified (see, for example, WO 93/22682) and are used, for example, in diagnostic kits for the detection of H. pylori , e.g. the HELISALTM test marketed by CORTECS LTD.
  • the results described herein indicate that it is essential to be able to detect the coccoid form of the bacterium to ensure accurate and complete (i.e. coccoid only infection) diagnosis.
  • no specific antigens for the coccoid form have been identified.
  • the present invention provides an antigenic protein having a molecular weight of 60 kDa, as determined by native PAGE, obtainable from the coccoid form of H. pylori .
  • the protein can furhter be characterised in that it has the following N- termmal amino acid sequence:
  • the invention provides the use of the antigen of the invention in the detection of antibodies against H. pylori , and specifically detecting the coccoid form.
  • the novel antigen of the invention can be used in combination with other antigens, particularly those obtainable from the spiral form of H.pylori to provide more sensitive methods of detecting H.pylori .
  • the coccoid antigen of the invention can be used to raise antibodies, which antibodies can then be used to detect the antigen, including the antigen when present as part of the intact coccoid.
  • the antibodies could be labelled and used as a means of detecting coccoids in tissue samples and the like. Methods of raising antibodies using the antigen are well known to the skilled man, as are means of labelling such antibodies for use in such methods.
  • the present invention provides the use of the coccoid antigen of the invention in the preparation of antibodies, e..g polyclonal antibodies.
  • the invention provides the use of such antibodies in the detection of the coccoid form of H. pylori which comprises antibodies raised against the coccoid antigen of the invention.
  • the antigen of the invention also finds use as part of an antigen composition, which may contain antigens against both che spiral and coccoid forms of H. pylori .
  • the invention provides a composition comprising the antigenic protein of the invention, optionally together with one or more other H. pylori antigens, these one or more other antigens being obtainable from either the spiral or coccoid form of H. pyl ori .
  • the invention provides a method of detecting the coccoid form of H. pylori , e.g. by detecting antibodies, which includes the step of contacting the antigen of the invention, or the antigen composition of the invention, with a sample.
  • a sample will be a biological sample, eg a blood sample, a urine sample or a saliva sample.
  • the antigen or antigen composition can be brought_ into direct contact with the biological sample.
  • the biological sample can first be treated to render it more suitable, eg by filtration, pH adjustment etc. Examples of suitable methods are those described in UK patent application no. 9422991.1.
  • the present invention provides a method for the diagnosis of H.pylori infection which includes the step of contacting the antigenic protein of the invention with a biological sample obtained from a subject.
  • the antigen can be provided in the form of an antigenic composition as described herein.
  • the diagnostic method of the invention will also include the step of detection of other antigens obtainable from either the spiral or coccoid form of H.pylori . In this way a more sensitive method of diagnosis for H.pylori infection is provided.
  • the diagnostic method of the invention is carried out on a sample of blood, a sample of urine or a samoie of saliva.
  • the diagnostic method of the invention will be be carried using a test device or test kit, e.g. that used in the HELISALTM test.
  • the present invention provides a kit for use in the diagnosis of H.pylori infection which comprises the antigenic protein of the invention.
  • the kit of the invention will also include one or more other antigens obtainable from either the spiral or coccoid form of H.pylori .
  • the identification of the unique antigen of the invention also opens up the possibility of providing a vaccine against H.pylori which will be active against both the spiral and coccoid forms of the bacterium.
  • the present invention provides a vaccine for the prophylaxis or treatment of H.pylori infections which comprises the antigen of the invention together with one or more adjuvants and/or carriers.
  • the vaccine includes one or more antigens derived from the spiral form of H.pylori .
  • these additional antigens will include at least one which is unique to the spiral form.
  • the vaccine can comprise the coccoid form (either killed or "live") of H. pylori itself since the cells could be taken up in the GI tract and then induce an immune response.
  • forms of H. pylori which exist between the true coccoid and true spiral forms could be used on the basis that they are expressing the novel antigen.
  • the coccoid, or intermediate, form(s) of H. pylori are used as a vehicle for delivery of the novel antigen to achieve an immune response.
  • the novel antigen of the invention can be used to detect IgM antibodies produced in children in response to H. pylori infection.
  • the present invention provides a method of detecting IgM antibodies against the coccoid form of H. pylori in children, which comprises the step of bringing the antigen of the invention into contact with a biological sample obtained from a child.
  • the biological sample will be a blood sample, a urine sample or a saliva sample.
  • the present invention provides a method of culturing the coccoid form of H. pylori which comprises the step of regularly adding carbon dioxide to a culture medium comprising the spiral form of H. pylori such that conversion to the coccoid form occurs and wherein the coccoid form obtained is viable.
  • C0 2 is added at least twice a day and the culture is allowed to run for nine weeks to ensure conversion.
  • FIGURE 1 shows a typical growth curve for H. pylori grown in a chemostat with concurrent pH and urease measurements
  • FIGURE 2 shows wet preparations of (a) spirals and (b) coccoids seen under phase contrast microscope, magnification xlOOO. Spiral cells were uniformly dense while coccoids were of two types : (A) compact with dense cytoplasm and (B) those with loose cytoplasm like "ghost" cells;
  • FIGURE 3 shows a transmission electron micrograph of a coccoid, magnification x 80,000;
  • FIGURE 4 shows a transmission electron micrograph of a coccoid with flagella, magnification x 80,000;
  • FIGURE 5 shows a silver stained SDS-PAGE protein profile.
  • FIGURE 6 shows modified periodic acid Schiff stained smears of (a) spirals and (b) coccoids, magnification x 1000;
  • FIGURE 7 shows modified gram staining of coccoids, wherein in (a) they have been treated with the salivary enzyme ot amylase and in (b) they have not;
  • FIGURE 8 shows DNA of H.pylori . Lanes: 1 Hindlll cut ⁇ DNA: 2 spiral NCTC 11637: 3 coccoid NCTC 11637: 4 spiral V 2 : 5 coccoid V-,;
  • FIGURE 9 shows the results of modified Albert's stain, magnification x 1000
  • FIGURE 10 shows detection of urease enzyme activity on PAGE. Lanes: 1 high molecular weight marker (Pharmacia) : 2 spiral NCTC 11637:
  • FIGURE 11 shows silver stained native PAGE protein profile.
  • FIGURE 12 shows a western immunoblot under non-denaturing conditions with the coccoid antigen. Lanes: 1 molecular weight marker
  • a preimmune anti-spiral serum B anti-spiral serum: C preimmune anti-coccoid serum: D anti-coccoid serum;
  • FIGURE 13 shows indirect fluorescent antibody test of coccoids, magnification X1000, wherein it can be seen that, like the spirals, the coccoids fluoresce under ultra violet light, indicating that their surface antigens are similar.
  • a local H. pylori strain V- isolated from a patient with non-ulcer dyspepsia was used. This strain was initially grown on chocolate blood agar (CBA) to check for purity. The plate culture was then used as inoculum for a 250ml Schott flat-bottomed round bottle containing 30ml BHIH (brain heart infusion supplemented with 10% horse serum and 0.4% yeast extract) , and incubated at 37°C for 72h. This in turn serves as the inoculum for chemostat or batch cultures.
  • CBA chocolate blood agar
  • a 1.5L fermenter containing 540ml BHIH was set up as described in Ho and Vijayakumari ( ⁇ icrojbios, 76:59-66 U993) .
  • the medium was inoculated with 2x30ml of 3 day old H.pylori cul ture, giving a ratio of 1:10
  • the culture was maintained under these conditions for up to 3 months during which daily monitoring of the cells was continued.
  • the cells were harvested by centrifugation at 10,000g for 40min. and washed once. The pellet was then used to prepare coccoid antigen using the modified glycine method (Ho, B., and Jiang, B., European Journal of Gastroenterology and Hepatology, 7:121-124 (1995) .
  • a IL Schott round-bottomed bottle or IL Erlenmeyer flask with a side-arm and fitted with a tight fitting rubber bung, containing 270ml BHIH was used.
  • a "'mm diameter hole was bored so as to accomodate the fitting of a disposable filter unit containing a 0.22 ⁇ m filter having a diameter of 50mm (e.g. Gelman) .
  • Each 270ml of BHIH was inoculated with 30ml of 3 day old H.pylori culture. Carbon dioxide was supplied twice daily via the 0.22 ⁇ m filter.
  • the culture was incubated in a 37°C shaker incubator (New Brunswick) maintained at 90rpm for up to nine weeks and the cells were subsequently harvested by centrifugation at 10,000g for 4Omin. The cell pellet was washed once and antigen prepared as described above.
  • Figure 1 shows viability, pH and urease specific activity for a typical culture. From this it can be seen that at 9 weeks the culture had become a coccoid culture. This was confirmed by microscopic examination failure of spiral for growth on CBA. The time taken for the spiral form to differentiate into the coccoid form is dependent on the constant supply of Carbon Dioxide. It is also clear that there are two forms of coccoid. One has a dense cytoplasm while the other has a "ghost" -like appearance . This latter form is considered to be non- viable . In contrast to other reports (Nilius et al , infra) , the chemostat culture showed mostly dense coccoids. These coccoids were also harvested and suspended in BHIH supplemented with 20% glycerol. The suspended coccoids were then stored at -80°C until needed.
  • H. pylori Two strains of H. pylori were used, the local H. pylori strain V 2 referred to in example 1 above isolated from a patient with non-ulcer dyspepsia and the standard strain NCTC 11637.
  • Coccoid and spiral sultures were prepared as described in example 1.
  • coccoids a batch culture was grown as described earlier (Ho and Vijayakumari, infra) . A small aliquot was aseptically removed at time intervals to assess culturability on chocolate blood agar, and the viable count enumerated using the Miles and Misra technique (Miles and Misra, Journal of Hygiene, 38:732- 738 (1938) . The percentage of coccoids was estimated by counting in triplicate the number of spirals to coccoids using a Neubauer bacterial cell counting chamber under a phase contrast microscope. Urease specific activity was measured using the phenol spectrophotometric method of Hamilton-Miller and Gergan, infra , while pH of the culture medium was monitored.
  • ATP was quantitated using the bioluminescence assay kit (Bio-Orbit, Finland) and the polysaccharide content measured by the L-cysteine sulphuric acid assay as described by Chaplin and Kennedy
  • the D ⁇ A of both forms were extracted according to the procedure of Clayton et al ( Infection and Immuni ty, 57:623-629 (1988)) and electrophoresed on a 1% agarose gel . Total D ⁇ A content per cell was assayed according to the method by Kapuscinski and Skoczylas (Analytical Biochemistry, 83:252-257 (1977)) .
  • Protein profiles were elucidated by polyacrylamide gel electrophoresis (PAGE) according to the method of Laemmli ( Nature , 227:680-685 (1970)) .
  • PAGE polyacrylamide gel electrophoresis
  • 30 ⁇ g total protein of whole cell preparations were electropnoresed on a 6% separating gel and 5% stacking gel.
  • SDS sodium dodecyl sulphate
  • the same amount of protein was electrophoresed on a 10% separating gel and 5% stacking gel. Relative molecular weight was determined with reference proteins run under the respective electrophoretic conditions.
  • Antibodies raised in rabbits against either the spiral or the coccoid were used as probes to identify the specific and immunogenic proteins in both forms.
  • Haemagrg-lutina ion and -haemagg-luti ⁇ ation inhibi tion assay A slight modification of the microtitration plate assay of Morgan et al ( Journal of Clinical Microbiology 29: 395-397 (1991)) was carried out with 20 ⁇ l of 2% v/v red blood cells (human or rabbit) which were added to 25 ⁇ l of bacterial culture containing a range of 10 7 -10 12 cells/ml in individual microtitration wells. Each mixture was incubated in quadruplicates at 4°C overnight before the haemagglutination patterns were read.
  • the haemagglutination inhibition assay was performed with bacteria pretreated with 1 mg ml "1 protease (pronase E, Sigma) at 37°C for 60 minutes or heated at 60°C for 10 minutes. Similarly, the red blood cells were pretreated with 4.0 ⁇ g ml "1 Neuraminidase (Sigma) or 1 mg ml "1 protease at 37°C for 60 minutes before haemagglutination assay.
  • FIG. 1 shows a typical growth curve for H. pylori in a chemostat. Growth in the first two weeks was similar to that described by Ho and Vijayakumari ( infra) . The late stationary phase showed a gradual decrease in viable counts to 10 s CFU/ml within the next two weeks. Subsequently, the declining death phase continued linearly for the following five weeks. Throughout the approximately nine week culturing period, the percentage of coccoids was shown to be inversely proportional to spirals.
  • the pH of culture medium decreased from neutral to 6.58 in the first 3 days and remained at 6.53 +/- 0.13 in the stationary phase for the next four weeks. It then increased to a maximum of 6.98 on the 7th week, after which the pH was stable at 6.84 +/- 0.02 in the following two weeks.
  • Catrenich & Makin ⁇ Scandinavian Journal of Gastroenterology, 6(suppl. 181) : 58-64 (1991)) reported a similar inversely proportional relationship between pH and viable counts and postulated that the loss of viability and conversion to coccoids was due to basic pH endogenously produced by deaminase activity. However, in our study, as the pH of the culture medium remained within the tolerable range for the growth of H.
  • the increase in USA in the stationary phase may be an adaptive response of the spirals to the increasing acid pH due to metabolism. Concomitantly, as the pH increases in the declining phase, the USA is decreased.
  • cytoplasmic density fig 2a
  • the coccoids on the other hand were circular and consisted of two types: one type was shown to be compact with dense cytoplasm, while the other type was with loose cytoplasm and had the appearance of "ghost" cells (Fig 2b) . It was not possible to separate the two types of coccoids using sucrose density gradient centrifugation.
  • the coccoids were non-motile when observed under the phase contrast microscope. Transmission electron microscopy, on the other hand, showed the presence of flagella in some coccoids (Fig 4) . This could either mean that the flagella is a remnant of the spirals (Marshall et al , 1984 infra) or that the coccoids actually possess flagella but they are inactive due to the dormant state or the lack of energy to drive it.
  • the coccoids could possibly survive outside the human body with protection offered by the thick polysaccharide layer from atmospheric oxygen tension as well as the unfavourable environment. Similar observations were rendered for Campylobacter jejuni by Rollins and Colwell (Applied and Environmental Microbiology, 52:531-538 (1985)) where an increase in viscosity of the culture suspension was noted as it underwent transition from the spirals to coccoids. They suggested that the production of an extracelluar viscous polysaccharide as an adaption to ensure extended survival for C. j ejuni .
  • the coccoids contained ATP but 100 x less than in the spirals (Table 2) . This signifies that the coccoids are a viable but dormant form. Similarly, a 99% reduction in endogenous respiration was exhibited by the marine vibrio ANT 300 as part of its survival strategy under long term nutrient starvation (Novitsky & Morita, Applied and Environmental Microbiology, 32:617- 622 (1976) ) .
  • the mean urease specific activity of coccoids was twenty times less than that of the spirals having an activity of 0.18 +/- 0.03 U mg "1 protein as compared to 3.61 +/- 0.52 U mg 'l protein in the spirals.
  • the low urease actvity in the coccoids could either be due to the preformed enzymes left in the coccoids or that the dormant coccoids do not require as much urease enzyme activity as the actively reproducing spirals.
  • haemagglutinin in the coccoid similar to that observed by Huang et ai (FEMS Microbiology Letters, 56: 109-112 (1988)) in the spirals, is a protein while the receptor is not a protein but sialic acid.
  • the haemagglutinating property of the spirals is retained in the coccoid as was also observed by Wadstrom et ai (European Journal of Gastroenterology and Hepatology, 5( ⁇ uppl.2) :S12-S15 (19931).
  • the coccoid form can exist in a viable form, contrary to what was believed previously. It has intact DNA, ATP enzyme activities, presence of novel and conserved protein and the presence of a thick polysaccharide coat to protect it under adverse conditions in the environment.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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EP96932692A 1995-09-29 1996-09-27 Helicobacter pylori protein Withdrawn EP0859788A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9519865.1A GB9519865D0 (en) 1995-09-29 1995-09-29 Protein
GB9519865 1995-09-29
PCT/GB1996/002404 WO1997012910A1 (en) 1995-09-29 1996-09-27 Helicobacter pylori protein

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EP0859788A1 true EP0859788A1 (en) 1998-08-26

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EP96932692A Withdrawn EP0859788A1 (en) 1995-09-29 1996-09-27 Helicobacter pylori protein

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EP (1) EP0859788A1 (xx)
JP (1) JP2000500123A (xx)
KR (1) KR19990063833A (xx)
CN (1) CN1201464A (xx)
AU (1) AU7137896A (xx)
BR (1) BR9610953A (xx)
CA (1) CA2233328A1 (xx)
GB (1) GB9519865D0 (xx)
NO (1) NO981407L (xx)
WO (1) WO1997012910A1 (xx)
ZA (1) ZA968185B (xx)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6599509B2 (en) * 1997-09-02 2003-07-29 Massachusetts Institute Of Technology Compositions and methods comprising helicobacter antigens for treatment and prevention of inflammatory bowel disease
MY120554A (en) * 1997-10-29 2005-11-30 Honda Motor Co Ltd Valve operating system in internal combustion engine
IT1299312B1 (it) * 1998-02-13 2000-03-16 Consortia Lab Srl Dosaggio nei liquidi biologici di anticorpi diretti contro uno o piu' antigeni del helicobacter pylori mediante metodo immunologico
GB9806039D0 (en) * 1998-03-20 1998-05-20 Cortecs Ltd Therapy
KR101600867B1 (ko) * 2008-06-03 2016-03-10 삼성전자주식회사 냉장고

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9712910A1 *

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AU7137896A (en) 1997-04-28
MX9802486A (es) 1998-10-31
CA2233328A1 (en) 1997-04-10
KR19990063833A (ko) 1999-07-26
CN1201464A (zh) 1998-12-09
BR9610953A (pt) 1999-12-21
NO981407L (no) 1998-05-28
GB9519865D0 (en) 1995-11-29
ZA968185B (en) 1998-03-27
NO981407D0 (no) 1998-03-27
WO1997012910A1 (en) 1997-04-10
JP2000500123A (ja) 2000-01-11

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