FR2791356A1 - Isolated, established culture of Tropheryma whippelii, useful for producing diagnostic and therapeutic agents for Whipple disease - Google Patents

Abstract

The bacterium Tropheryma whippelii, the causative agent of Whipple disease, in isolated form and established in culture, is new. Independent claims are also included for the following: (1) antigens (Ag) from T. whippelii; (2) antibody (Ab) specific for Ag or T. whippelii; (3) serological in vitro diagnosis of Whipple disease; (4) kits for carrying out the method of (3); (5) total or partial sequences (I) of the rpoB gene of T. whippelii; and (6) a method for detecting presence or absence of T. whippelii in a sample by formation of nucleic acid complex with a specific probe.

Description

l 2791356 The present invention relates to the field of diagnostics. More

  specifically, the invention relates to a method for the in vitro serological diagnosis of Whipple's disease as well as to a device for implementing this method. The invention also relates to a kit for the in vitro detection of the bacteria responsible for WVVhipple disease. Whipple's disease is a disease that comes in many forms. The most classic form is that of a fever with chronic diarrhea which leads to weight loss, but it is also a disease which is capable of giving chronic joint damage, brain damage with dementia and also cardiac damage in particular a endocarditis

negative blood culture.

  As early as his original description in 1907, Whipple evoked the existence of a

  bacteria associated with "intestinal lipodystrophy" after observation of numerous microorganisms after silver staining of a mesenteric ganglion (Whipple, Bull. John Hopkins Hosp. 1907; 18: 328-391). The demonstration of the positive (non-specific "periodic acid Schiff") PAS character of this bacterium, then the observations by electron microscopy confirm the presence of an intracellular bacterial species of Gram positive structure (Chears et al., Gastroenterology 1961; 41: 129-138). The universal molecular tool 16S rRNA made it possible to confirm this hypothesis by specifying the phylogenic taxonomy of this new bacterial species, and by assigning it the provisional name of Tropheryma whipelii to evoke the notion of intestinal malabsorption and to honor the discoverer of the affection ( Relman et al., N. Engl. J. Med. 1992; 327: 293-301). Direct sequencing of 721 bases of an amplified fragment from a biopsy of the small intestine of a patient (Wilson et al., Lancet 1991; 338: 474-475), then from a lymph node d 'Another patient (Wilson et al., ASM News 1992; 58: 318-321) confirms the originality of the bacterial species associated with WVhipple's disease. Sequencing by Relman et al. (op. cited) of 1,321 bases representing 90% of the gene in a sample, and a fragment of 284 bases in four other patients made it possible to confirm that the bacterial species associated with Whipple's disease represented a new species, to specify its taxonomic position in the phylum of actinomycetes, that is to say bacteria of Gram positive structure with high guanosine content

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  plus cytosine, representing a new branch relatively close to two known species in human pathology, Actinomyces pyogenes and Rothia dentocariosa. The diagnosis of the disease is currently made by observation after staining of a microscopic smear obtained from a biopsy or by amplification and

  sequencing of the universal gene tool 16S rRNA (Relman et al., op. cit.).

  To date, the bacterium responsible for Whipple's disease has not

  could be isolated and cultured in such a way that serology could be envisaged.

  Against all expectations, the Applicant has developed a method for

  culture of the bacteria responsible for Whipple's disease.

  This method, which is detailed in sections 1 and 2 below, includes the inoculation of a ground valve heart on human fibroblasts

  of the HEL line in MEM medium.

  The bacteria responsible for Whipple's disease was isolated and established in culture after a minimum of two months of incubation, the culture medium being replaced regularly. By "established in culture" is meant that the bacteria is

  obtained in a reproducible manner and multiplies over time.

  The present invention therefore relates to the bacteria thus isolated and established as a source of antigen. This bacterium has been deposited in the collection of the Rickettsia Unit - WHO Collaborating Center (CNRS unit UPRES A 6020 from

  the University of the Mediterranean Aix-Marseille II) under the code name "TWIST".

  The present invention also relates to a method for the in vitro serological diagnosis of Whipple's disease, which comprises bringing the serum or any other biological fluid of a patient into contact with the bacterium such as

  defined above, and detection of the immunological reaction.

  Advantageously, the diagnostic method of the invention implements

  an ELISA type immunoenzymatic assay or an immunofluorescent assay.

  More particularly, the method according to the invention comprises: - the deposition in or on a solid support of a solution of bacteria isolated and established as indicated above; The introduction into or onto said support of the diluted serum or of the biological fluid to be tested;

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  - the introduction into or onto the support of an anti-immunoglobulin solution

  marked human; - observation of an incubation period; - possible rinsing of the solid support; - the actual detection of the immunological reaction. As a solid support, any device suitable for handling cellular and bacterial suspensions can be used, and in particular tubes, glass slides, jewelry tubes or rigid microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose comprising

  microwells, glass slides being preferred.

  The antibody detected is an immunoglobulin, in particular of the G, M or A type, specific for the bacteria responsible for Whipple's disease. As a type of labeling of anti-human immunoglobubin, an enzymatic labeling is used,

  radioactive or fluorescent, the latter type of labeling being preferred.

  The expression "fluorescent labeling" means that the antibody has been made fluorescent by an appropriate fluorescent agent such as fluorescein iso (thio) cyanate combined with an animal immunoglobin recognizing the human antibody. The expression "radioactive labeling" means that the antibody carries, either on an element of its structure, for example the constituent tyrosine residues, or on an appropriate radical which has been attached to it, a radioactive isotope making it possible to assay it by

  counting the radioactivity associated with it.

  The expression "enzymatic labeling" means that the antibody is coupled to an enzyme which, combined with the use of appropriate reagents, allows a measurement

  quantitative of this specific antibody.

  The substrate and the reagents are chosen so that the final product of the reaction or of the reaction sequence caused by the enzyme and using these substances is: - or a colored or fluorescent substance which diffuses into the surrounding liquid medium the sample tested and which is the subject either of the final spectrophotometric or fluorimetric measurement, respectively, or of an evaluation by eye, possibly with respect to a range of calibrated shades,

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  - or an insoluble colored substance which is deposited on the test sample and which can be the subject either of a photometric measurement by reflection, or of an evaluation by eye, possibly in relation to a range of

calibrated shades.

  When using a fluorescent antibody, the associated fluorescence

  to the test sample is read directly on a suitable device.

  When using a radioactive probe, such as for example iodine 125, the radioactivity associated with the test sample is counted in a gamma counter according to any appropriate method and for example after solubilization of the cells with an alkaline solution (for example a soda solution) and recovery of

  solution containing radioactivity using an absorbent pad.

  When an enzyme is used on the specific antibody, the appearance of a colored or fluorescent product is obtained by adding a solution containing the substrate of the enzyme and one or more auxiliary reagents making it possible finally to obtain as a product of reaction, either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as explained above. The light signal coming from the samples thus treated is then measured, using the apparatus adapted to each case: transmission, or reflection or fluorometer photometer respectively. Alternatively, it is also possible to evaluate the coloration obtained by eye, possibly with the aid of a

  range of calibrated colored solutions.

  By using alkaline phosphatase as the enzyme, the coupling of this enzyme with the specific antibody is carried out according to the method proposed by Boehringer Mannheim-Biochemica. The preferred substrates for this enzyme are paranitrophenylphosphate for a spectrophotometric final reading or methyl-4-umbelliferyl phosphate for a fluorometric reading or 5-chloro-4-chloro-indolyl-3 phosphate to obtain an insoluble colored reaction product. The enzyme 13-galactosidase can likewise be used, the preferred substrates of which are orthonitrophenyl J3-D-galactopyranoside or methyl-4

umbelliferyl, -D-galactopyranoside.

  Preferably, the specific antibodies can be coupled to the

  peroxidase. In this case, the coupling process is derived from that described by M.B.

2791356

  WILSON and P.K. NAKANE in Immunofluorescence and Related Staining

  Techniques, W. Knapp, K. Kolubar, G. Wicks ed. Elsevier / North Holland.

Amsterdam 1978, p. 215-224.

  Reagents used to reveal peroxidase conjugated to specific antibodies contain hydrogen peroxide, the enzyme substrate, and an appropriate chromogen, for example orthophenylenediamine or azino-2,2 'bis (3-ethyl-thiazoline) acid sulfonic-6) or ABTS to obtain a final reaction product which is colored and soluble in the medium or alternatively 3,3-diamino benzidine or 3-amino-9-ethyl carbazole or 4-chloro a-naphthol to obtain a insoluble final reaction product, o or parahydroxyphenyl propionic acid to obtain a reaction product

fluorescent soluble in the medium.

  Another embodiment of the invention is the use of antibodies

  specific coupled to acetylcholinesterase.

  Acetylcholinesterase is coupled to the antibody, preferably using a method derived from that described in French Patent No. 2,550,799 or a method which schematically comprises the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate heterobifunctional agent and finally the coupling of the products thus obtained. Other known methods of constructing conjugates

  immunoenzymatics can also be used in this case.

  The revelation of the enzymatic activity specifically linked to the antigen recognized by the conjugate with acetylcholinesterase is preferably carried out according to the well-known technique which employs acetylthiocholine as substrate of the enzyme and the reagent of Ellman, or acid dithio-5,5 '2-nitro-2 benzoic as chromogen, according to any variant adapted to the case examined, for example that described by

  Pradelles et al. in Anal. Chem. 1985, 57: 1170-1173.

  The chromogens mentioned are used as such or in the form of salts

soluble in water.

  The serological diagnostic method of the invention is suitable for use in biology and / or anatomopathology laboratories. To do this, we propose a device for implementing this method, which

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  includes a solid support on or in which a solution containing

  the bacteria as defined above.

  According to another aspect, the invention also relates to a kit for the in vitro detection of the bacteria responsible for Whipple's disease. This kit comprises as components: - a solution containing the bacteria responsible for Whipple's disease, isolated and established as described above, as a positive control; - a solution containing a specific labeled antibody;

  - optionally, a washing solution.

  The specific antibody used in the kit of the invention is advantageously labeled with a radioactive probe, an enzyme or a fluorescent agent. When the specific antibody is labeled with an enzyme, the kit further includes the enzyme substrate and one or more reagents to visualize the activity of the enzyme. When the specific antibody is labeled with a fluorescent agent,

  preferably fluorescein iso (thio) cyanate.

  According to a preferred embodiment of the invention, an immunoglobulin, in particular an immunoglobulin of

mouse.

  The invention will be better understood using the following description, divided into sections, which relate to experiments carried out with the aim of carrying out

  the invention, and which are given purely by way of illustration.

  Section I: Priming The priming was carried out by the centrifugation technique on jewelry tubes inoculated with a line of human HEL fibroblasts available from ATCC. HEL cells are cultured on MEM medium (Gibco) supplemented with% fetal calf serum (Gibco) and 2 mM L-glutamine (Gibco). The jewelry tubes (Sterilin-Felthan-England, 3.7 ml) comprising a support lamella 12 mm in diameter are inoculated with 1 ml of culture medium containing approximately 50,000 cells and incubated at 37 ° C. for 3 days under 5% of CO2 so as to obtain a mat of confluent cells. The studied heart valve was ground in

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  MEM medium, and the suspension was used to inoculate the 3 jewelry tubes. These tubes were then centrifuged at 700 g for 1 hour at 220C. The supernatant was then removed, the carpets were washed twice with sterile PBS buffer, then incubated with 1 ml of medium at 37 ° C. under 5% CO 2. The cultures were monitored by cytocentrifugation of 100 μl of the supernatant from the Gimenez jewelry and staining tubes. This procedure was repeated at 10, 20 and 30 days. After 30 days, the supernatant and the cell carpet of the jewelry tubes were collected and subcultured on a confluent cell carpet in a 25 cm 2 culture dish (box 1) with 15 ml of culture medium and incubated at 370C under 5 % of CO2. Each week every 10 for the following 6 weeks (D72), the cell carpet was examined using an inverted microscope for a cytopathogenic effect and the culture medium was replaced by fresh medium. Before changing the medium, 200 μl of supernatant was used to carry out a cytocentrifugation colored by a Gimenez stain. No cytopathic effect was detected before the 65th day. On the 72nd day, the examination of the cell mat in reverse microscopy made it possible to detect small dark and irregular inclusions in the HEL cells. On the Gimenez stain of the cytocentrifugation of the supernatant from box I several fine bacilli were detected, most of them in intracellular location where they appear smaller than the extracellulars. However, most of them were poorly colored or not colored by the Gimenez coloring and appeared pale blue. Numerous bacilli were also detected during Gram staining. Most appeared Gram positive but several were only partially purple or appeared Gram negative. When stained with Ziehl, these bacilli are not acid-alcohol resistant. On PAS staining, the PAS positive bacilli appeared more numerous than on previous stains. Most long, fine bacilli are observable in extracellular localization. HEL cells appear filled with conglomerates

  NOT positive and short, fine bacilli NOT positive.

  Section 2: Propagation of the isolate The entire propagation procedure was carried out on HEL cells cultured on MEM medium supplemented with 10% fetal calf serum and 2 mM L-glutamine and incubated at 37 ° C. under 5% of CO2. On day 75, 3 ml of x8 2791356 supernatant from box I was used to inoculate 10 jewelry tubes by the method described above and 2 ml of supernatant was used to inoculate a confluent cell mat in a 25 cm2 culture dish (dish A) with 15 ml of medium. The cells of box I as well as the rest of the supernatant were harvested, making it possible to obtain 10 ml of suspension. This suspension was then divided into 2 ml aliquots. One of the aliquots was frozen in liquid nitrogen. An aliquot was inoculated on a confluent cell carpet in a 25 cm 2 culture dish (box B) with 15 ml of medium. The cells of an aliquot were lysed by 4 freeze-thaw cycles using liquid nitrogen and hot water (55 ° C.) and then inoculated on a cell mat confluent in a 25 cm 2 culture dish (dish C) with 15 ml of medium. An aliquot was inoculated into a 25 cm 2 cell culture dish (cell D) with 15 ml of medium. On the 85th day, the medium of all the boxes and jewelry tubes was replaced by fresh medium. The cells were harvested and inoculated in a 75 cm2 culture dish (D2 dish) with 30 ml of medium. Before changing the medium, 200 µl of supernatant was used to perform stained cytocentrifugation by PAS staining and the rest of the supernatant was frozen for use as an antigen for serology. On the 95th and 105th days, the center of all the jewelry boxes and tubes was changed as described above. Small portions of the cell carpet were scraped in order to make cell smears for PAS staining. The effectiveness of the propagation of the strain was achieved by a semi-quantitative evaluation. The presence of PAS positive bacilli was evaluated microscopically using X1000 magnification as follows: 0, absence; +, present but difficult to find; ++, easy to find but not present in all fields; +++, present in all fields. These

  assessments were performed blind.

  All propagation methods have been shown to be effective since

  all found the isolate after 30 days of subculture (Table 1).

  The semi-quantitative evaluation made it possible to observe that the most effective procedures are subculture in jewelry tubes, the supernatant subculture (box A),

  and the subculturing of cells (boxes D, D2).

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Table 1

  Tube Box A Box B Box C Box D Box D2 jewelry Day 10 Sumrnageant + - + - + NF

Day 20 Supernatant + - + + NF -

NF + + + NF cell smear -

  Day 30 Supernatant +++ +++ ++ + NF ++ Cell smear NF ++ + + NF ++ NF: not done Section 3: Detection by immunofluorescence The detection of intracellular bacteria was carried out on the 105th day directly in a tube immunofluorescence jewelry. After fixation with acetone, the tube was rinsed twice with PBS. 100 μl of the patient's serum diluted 1:50 with PBS with 3% skimmed milk powder was added and the tube was incubated in a humid chamber at 37 ° C. for 30 min. After 3 rinses with PBS, the tube was incubated for 30 min at 37 ° C. with 100 μl of anti-human goat immunoglobulin labeled with fluorescein isothiocyanate (Fluoline H, Biomerieux, Marcy I'Etoile, France) diluted to 1 : 200 with PBS added with 0.2% Evans blue. After 3 rinses with PBS the coverslip was mounted (cells down) in buffered glycerin (pH 8) and examined with X400 magnification using a Zeiss fluorescence microscope and a confocal microscope (LEICA DMIRBE)

  equipped with an immersion X100 (NA. 1.4) objective.

  The immunofluorescence examination shows that the PAS staining and the other stains underestimate the cellular infection. On the coverslip after 30

  days of subculture all cells are filled with the bacterial antigen.

  The confocal microscopy study confirms the intracellular localization of the bacteria. Several bacteria are detected in isolation in the form of fine bacilli resembling those observed by PAS staining. However, most of the immunopositive material corresponds to larger inclusions where it is impossible to individualize bacteria. No immunopositive material is detected in the

nucleus of cells.

  Section 4: Electron Microscopy On the 105th day, 300 μl of a solution containing the cells harvested in the D2 dish were prepared for the study by electron microscopy. The cells were fixed in a 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer containing 0.1 M of sucrose for 1 h at 4 C. The cells were rinsed overnight in the same buffer and then fixed 1 h at room temperature in Io osmium tetraoxide in 0.1 M cacodylate tamon Dehydration was carried out

  by successive rinses in ethanol solutions of increasing concentrations.

  The cells were then included in blocks of Epon 812. Thin sections were then cut from the blocks using an LKB Ultratome III microtome and then stained with a saturated solution of uranyl acetate in methanol. and an aqueous solution of lead citrate before examination on an electron microscope

Jeol JEM 1200 EX.

  The electron microscopy study confirms that the PAS positive inclusions and the immunopositive material correspond to intact or degrading bacteria. The cytoplasmic membrane of these bacteria is made up of two dense layers of electrons. The thin bacterial wall is sometimes covered with an external pseudo-membrane which gives a trilamellar appearance. Bacteria in

division courses are observed.

  Section 5: Production of polyclonal antibodies by the mouse against the bacteria responsible for Whipple disease A mouse of the Balb C strain was injected intraperitoneally with 0.5 ml of supernatant containing 104 bacteria responsible for Whipple disease. The mouse was then reinjected 1, 2 and 3 weeks later with 0.5 ml of the same suspension. The mouse was bled 1 week after this last inoculation. The serum was tested on the one hand against the bacteria responsible for Whipple's disease in culture and on the other hand on the valve of a patient suffering from Whipple's disease, once by immunofluorescence and once by immunoperoxidase. The revealing antibodies were anti-mouse antibodies I 2791356 labeled with fluorescein or labeled with immunoperoxidase (supplied by the company Immunotech). The bacteria could be visualized inside the cells. The present request therefore also relates to the direct detection of the bacteria responsible for Whipple's disease in biopsies and different samples, for example: heart valve, digestive biopsy, or biopsy of any other organ suspected of being

  infected with the bacteria responsible for Whipple's disease.

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Claims (15)

  1. The bacteria responsible for Whipple's disease isolated and established in culture. 2. Bacterium according to claim 1, which is obtained after at least two months   incubation in a MEM-based culture medium.   3. A method for the in vitro serological diagnosis of Whipple's disease, which comprises the following steps: - the deposition in or on a solid support of a solution containing the bacteria as defined in claim 1 or 2; The introduction into or onto said support of the serum or of the biological fluid to be tested diluted;   - the introduction into or onto the support of an anti-immunoglobulin solution   marked human; - observation of an incubation period; - possible rinsing of the solid support; and   - the actual detection of the immunological reaction.   4. The method of claim 3, wherein the immunoglobulin is   labeled with a radioactive probe, an enzyme or a fluorescent agent.   5. Method according to claim 4, in which the fluorescent agent is   fluorescein iso (thio) cyanate.   6. Method according to one of claims 3 to 5, wherein one uses   about 0.5 to about 5 HlI, preferably about lpl, of said solution containing the bacterium. 13 2791356   7. Device for implementing the method according to one of the   claims 3 to 6 which comprises a solid support in or on which a   solution containing the bacteria was disposed.   8. Device according to claim 7, wherein the solid support is a glass coverslip.   9. Kit for the in vitro detection of the bacterium responsible for Whipple's disease, comprising as components: 1o - a solution containing the bacterium as defined in claim 1 or 2; a solution containing a specific labeled antibody;   - optionally, a washing solution.   10. The kit of claim 9 wherein the specific antibody is   labeled with a radioactive probe, an enzyme or a fluorescent agent.   11 The kit of claim 10, wherein the specific antibody is labeled with an enzyme.   12. The kit of claim 11, which further comprises the substrate of   the enzyme and one or more reagents to visualize the activity of the enzyme.   13. The kit of claim 10, wherein the specific antibody is marked with a fluorescent agent.   14. The kit of claim 13, wherein the fluorescent agent is   fluorescein iso (thio) cyanate.   15. Kit according to one of claims 9 to 14, in which the antibody   specific is an immunoglobulin, preferably a mouse immunoglobulin.