WO2011051402A1 - Nouveaux biomarqueurs pour déterminer un état d'allergie - Google Patents

Nouveaux biomarqueurs pour déterminer un état d'allergie Download PDF

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WO2011051402A1
WO2011051402A1 PCT/EP2010/066398 EP2010066398W WO2011051402A1 WO 2011051402 A1 WO2011051402 A1 WO 2011051402A1 EP 2010066398 W EP2010066398 W EP 2010066398W WO 2011051402 A1 WO2011051402 A1 WO 2011051402A1
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allergen
subject
allergic
biomarkers
sample
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PCT/EP2010/066398
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English (en)
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Haïfa HAMDI
Patrick Stordeur
Annick Ocmant
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Universite Libre De Bruxelles
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention lies in the field of medical diagnostics, more particularly in the diagnosis, prediction and prognosis of the allergic state of a subject.
  • the present invention particularly provides new tools and methods for assessing or predicting the allergic state and/or response of subjects to allergens or to immunotherapy. BACKGROUND OF THE INVENTION
  • the inventors identified new allergy-specific diagnostic and/or prognostic markers of peripheral blood basophils using a quantitative real-time PCR test performed and validated on PBMC's and on whole blood samples and evaluated the diagnostic power of said new markers.
  • the inventors investigated the differential expression of genes of Basophil cells, involved in allergy, before and after stimulation with real allergens. Several new biomarkers were identified and validated that can predict the allergic response of a subject to a specific allergen.
  • the present invention provides a method for determining whether a subject is allergic to or is prone to becoming allergic to a certain allergen comprising the steps of:
  • HAS1 Hyaluronan synthase 1
  • EGR1 Early Growth Factor 1
  • CD44 CD44
  • Rybp RING1 and YY1 binding protein
  • said expression level is assessed using PCR analysis, preferably quantitative PCR analysis.
  • the sample is selected from the group consisting of: blood, whole blood, peripheral blood mononuclear cells (PBMC) depleted from basophils or not, purified basophils, plasma or serum, preferably, a whole blood sample.
  • PBMC peripheral blood mononuclear cells
  • the allergen was added to the subject in vivo, i.e. by administering the allergen to the subject under investigation or under desensitisation treatment. A sample taken before said administration than acts as the reference sample.
  • the allergen is added to a sample of the subject in vitro, i.e. by adding the allergen to the sample after it was obtained from the subject.
  • the invention further provides a method for diagnosing or predicting the allergic response of a subject to a certain allergen comprising the steps of:
  • HAS1 Hyaluronan synthase 1
  • EGR1 Early Growth Factor 1
  • CD44 CD44
  • Rybp RING1 and YY1 binding protein
  • the invention further provides a method of defining immunotherapy in a subject allergic to a certain allergen comprising the steps of:
  • HAS1 Hyaluronan synthase 1
  • EGR1 Early Growth Factor 1
  • CD44 CD44
  • Rybp RING1 and YY1 binding protein
  • the invention provides a method for monitoring the response of a subject to an immunotherapy treatment comprising the steps of:
  • the addition of the allergen or allergen extract for immunotherapy treatment is, or has been done in vivo, i.e. by administering said allergen to the subject under investigation e.g. by sublingual, oral, or nasal administration, or by intraperitoneal, subcutaneous or intravenous injection.
  • the invention further provides a method for predicting the response to a vaccine or medicament comprising the steps of:
  • HAS1 Hyaluronan synthase 1
  • EGR1 Early Growth Factor 1
  • CD44 CD44
  • Rybp RING1 and YY1 binding protein
  • the subject is human. In a further preferred embodiment, the subject is an adult, an adolescent, a child, a toddler, a baby or a neonate.
  • the allergen is selected from the group consisting of foodstuffs such as cowmilk, gluten, wheat, peanuts, tree nuts, soy, egg, fish, shell fish, chocolate, strawberries etc.; insect venoms such as venom from bees, wasps, ants, mosquitos, flies etc.; venom from other animals such as snakes, spiders, etc; irritating secretions from plants, such as Bigsting (stinging nettle), Hogweed, thistle species etc.; tree, flower or grass-pollen; dust; small insects such as (house)mites etc.; other animal allergens such as hairs form e.g.
  • the invention thus provides methods for determining the response of a subject to any known substance or allergen (e.g. such as those exemplified herein) using the method according to the invention.
  • the stimulation with an allergen is done in vitro, by adding said allergen to the sample, after it was obtained from, or is taken from, the subject.
  • the mRNA or protein level of the biomarkers identified by the present invention is determined.
  • the sample is selected from the group consisting of: blood, whole blood, plasma or serum, purified Basophiles, or PBMCs.
  • said sample is a whole blood sample.
  • a single sample can be used, wherein the expression level of said one or more biomarkers is tested before and after addition of the allergen. An increase of the expression level of said one or more biomarkers after addition of the allergen indicates the subject is allergic to or prone to become allergic to said allergen.
  • the expression level of all 4 biomarkers listed therein is measured and optionally also that of the following biomarkers: ENPP3, CISH and/or Dcun1 D3.
  • the invention further provides a kit for diagnosing allergy in a subject comprising or consisting of:
  • (iii) means and/or instructions for performing the diagnosis according to the method of the present invention.
  • kit of the invention comprises:
  • HAS1 Hyaluronan synthase 1
  • EGR1 Early Growth Factor 1
  • CD44 CD44
  • Rybp RING1 and YY1 binding protein
  • RNA stabilizing agent optionally an RNA stabilizing agent
  • said vessel comprises: i) a vessel capable of accepting a blood sample, and optionally: ii) a container in which a stabilizing agent is present, iii) a connection between the inside of said vessel (i) and the inside of said container (ii), (iv) a physical barrier that temporarily blocks said connection; and
  • a container in which the allergen is present optionally: (v) a container in which the allergen is present, vi) a connection between the inside of said vessel (i) and the inside of said container (v), and (vii) a physical barrier that temporarily blocks said connection between (i) and (v).
  • the allergen is already present in said vessel (i).
  • the invention further provides for the use of a kit according to the invention, for diagnosing, predicting or prognosticating the allergic active state in a subject, preferably by performing the method according to the invention.
  • the means for determining either biomarker level is a means for determining the mRNA or protein level, such as end-point-PCR, real-time-PCR, quantitative-PCR, digital-PCR, or northern blot, capable of determining the mRNA level of the biomarkers, or ELISA and other EIA, ELISPOT, Luminex's xMAP technology, flow cytometry, nephelometry, turbidimetry, immunoprecipitation, capable of determining the concentration of the specific biomarker protein(s).
  • means for determining the expression level of said one or more biomarker(s) is a specific binding assay, immunodetection assay, Mass-spectrometry assay, capable of determining the protein level of the biomarkers.
  • kits comprise primer pairs suitable for detecting the expression level of all 4 biomarkers listed therein and optionally also for the following biomarkers: ENPP3, CISH and/or Dcun1 D3.
  • the kits comprise primer pairs suitable for detecting the expression level of all 4 biomarkers listed therein and optionally also for the IL-6 biomarker.
  • the invention provides for the use of the methods and kits of the invention for determining the treatment needed for an allergic patient comprising performing the method steps or using the kits as defined herein at different time points during the treatment, wherein increased expression levels of said one or more biomarkers of the invention point to an active allergic phase in the subject under observation, indicating the need for anti allergic treatment.
  • Figure 4 Histogram with means +/- SEMs per group for marker EGR1 .
  • p 0.288
  • the inventors used basophil cells or PBMCs or basophils-depleted PBMCs, stimulated or not with an allergen in order to identify genes that are specifically upregulated upon stimulation with allergens.
  • a real cat allergen such as Fel d1 (formerly called "cat allergen 1 ") as a test case
  • the inventors analysed changes in the expression level genes in the transcriptome of PBMCs, basophils and basophils-depleted PBMCs, isolated from peripheral blood of subjects allergic to the cat. This study was approved by the local ethics committee and patients and controls gave informed consent.
  • Table 1 List of up-regulated genes after allergen stimulation
  • the present method thus comprises obtaining or providing two blood samples from a subject, one sample before the subject is treated with the allergen and one sample after the subject is treated with the allergen.
  • the blood samples are not further treated and are analyzed as such.
  • the subject is submitted to a treatment with the allergen during a suitable period of time which may vary from 1 to 24 hours, depending on the allergen that is administrated to the patient and which for instance may be about 2, 3, 4, 5, 6, 7 or 8 hours.
  • Other allergens may be administrated for months, like for desensitization therapy.
  • the allergen is not administered to the subject under treatment, but is added in vitro to the sample or a portion thereof obtained from the subject.
  • a single sample of the subject can be divided in two portions: one portion to which the allergen is added in vitro and one portion to which no allergen is added.
  • the increased expression level of the one or more biomarkers of the invention can then be calculated based on the differential mRNA levels of said biomarkers between both samples, i.e. with and without allergen added.
  • all blood samples i.e. the blood samples obtained from a subject before and after treatment with the allergen or the blood samples incubated or not with an allergen in vitro
  • the stabilizing agent is an inhibitor of cellular RNA degradation and/or gene induction.
  • said inhibitor of cellular RNA degradation and/or gene induction is that as found in a PAXgeneTM Blood RNA Tube or alternatively in a TempusTM Blood RNA Tube.
  • a quaternary amine surfactant may be used as a stabilizing agent. Suitable quaternary amine surfactants, able to stabilize RNA from biological samples, are described in US Pat. No.
  • quaternary amine which can be used in the method of the present invention is tetradecyltrimethyl-ammonium oxalate. (US Pat. No 5,985,572).
  • said cationic detergent may be Catrimox-14TM (US Pat. No 5,010,183).
  • the method of the invention can comprise the steps of:
  • said increase in expression level is 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5 fold.
  • all 4 biomarkers are used in parallel, i.e. the expression levels of all 4 genes is determined in the sample of the subject, in order to obtain an expression profile encompassing data of all 4 biomarkers.
  • the method of the present invention may use a blood collecting vessel and a container in which a stabilizing agent is present.
  • a blood collecting vessel and a container Preferably, the inside of said blood collecting vessel and the inside of said container are connected, and a physical barrier temporarily blocks said connection.
  • the method of the invention can comprise the steps of:
  • step e) comparing the mRNA levels of said biomarkers determined in step c) and d), and evaluating the allergy status of the individual, wherein an increase in the expression levels of the one or more biomarkers after addition of the allergen indicates the patient is allergic to the allergen.
  • the method of the present invention may use a blood collecting vessel and a container in which a stabilizing agent is present as described above, wherein either an amount of allergen is present in the vessel (i) used for collecting the second blood sample, or wherein said blood collecting vessel additionally comprises a container (v) comprising an amount of allergen, connected with vessel (i) by a connection (vi), said connection being temporarily blocked by a physical barrier (vii).
  • the method of the invention comprises the steps of:
  • the first and second (portions of) blood samples are preferably stabilized with stabilizing agent, as fast as possible after blood collection or after the ex vivo incubation.
  • the allergen can also be added in vitro, i.e. after the sample of e.g. blood has been taken from the subject under investigation.
  • One part of the blood sample is then contacted with the allergen and the resulting RNA expression pattern resulting there from is determined subsequently by fixing the RNA with an RNA stabilizing agent, followed by RNA analysis as indicated for the other embodiments.
  • the remaining part of the blood sample is also analysed for its RNA expression pattern in the same manner, safe from the addition of the allergen.
  • the two expression patterns can then be compared to give an indication of the allergic state of the subject.
  • the method involves incubating a blood sample in the presence of an allergen in an incubator, preferably at a temperature of about 37°C.
  • the method is performed in the absence of controlling the air composition during incubation.
  • an oven can be used that is working under ambient atmospheric conditions, i.e. without any regulation of the amounts of C0 2 and H 2 0 present in the oven.
  • the sample is preferably maintained at 37°C.
  • a cell culture incubator wherein atmospheric conditions and the concentration of e.g. C0 2 are controlled, is not required for carrying out the incubation step of the present method.
  • the method of the invention can comprise the steps of:
  • a vessel comprising: (i) a suitable amount of allergen present inside said vessel, (ii) a container in which a stabilizing agent is present, (iii) a connection between the inside of said vessel and the inside of said container, and (iv) a physical barrier that temporarily blocks said connection.
  • step g) comparing the expression levels in e) and f) and evaluating the allergic state of the subject, wherein an increased expression of said one or more biomarkers in step e) as compared to step f) indicates the subject is allergic to the tested allergen.
  • said first blood sample (or first blood portion) is also incubated, similarly to said second sample (or blood portion), in a vessel as described above but free of allergen.
  • This first blood sample is also stabilized with stabilizing agent, as fast as possible after blood collection, or alternatively, after incubation as done for the tube containing the allergen. This can again be done by perforating or removing the temporary physical barrier (iv) between the sample vessel (i) and the container (ii) comprising the stabilizing agent.
  • the allergen is present in the sample vessel prior to the addition of the second (portion of the) sample.
  • said allergen is present in an additional container (v), in connection with said sample vessel, wherein the connection (vi) is temporary blocked by a physical barrier (vii).
  • a physical barrier (vii)
  • two identical vessels can be used for the first and second (portion of) the blood sample, wherein for the first (portion of the) sample, the physical barrier between the allergen container and the sample vessel is left intact, and wherein for the second (portion of the) sample, said barrier is perforated, thereby only bringing the second (portion of the) sample in contact with the allergen.
  • the method comprises the determination of mRNA levels of the one or more biomarkers of the invention in a first blood sample of an individual and in a second blood sample that has been incubated with the allergen.
  • the mRNA level of the one or more biomarkers is compared in both samples and based on the results thereof, the allergy status of the individual is evaluated.
  • the present invention therefore also provides a method for monitoring the in vivo response of an individual to a treatment with an anti-allergic agent, comprising incubating a first blood sample of said individual in vitro with a suitable amount of the allergen and a second blood sample of said individual in vitro with a suitable amount of the allergen and the anti-allergic agent for a suitable period of time, and comparing mRNA levels of the one or more biomarkers of the invention in both blood sample. If the mRNA levels are reduced or modified in any way, upon addition of the anti allergic agent, it indicates that the symptoms of the patient can likely be reduced or alleviated when the anti-allergic agent is administered to the patient.
  • the method can be applied using the steps, conditions, amounts, examples of incubation times and conditions as described above.
  • the invention also provides a method for identifying an individual as a responder or non responder to a treatment with an anti allergic agent, comprising incubating a blood sample of said individual in vitro with a suitable amount of said allergen for a suitable period of time, and determining mRNA levels of the one or more biomarkers in said blood sample.
  • the method can be applied using the steps, conditions, amounts, examples and incubation times and conditions as described above.
  • the invention further provides a method for adjusting an anti-allergy therapy in a patient, comprising the step of identifying an individual as a responder or non responder to a treatment using the method of the present invention, and adjusting said therapy when the patient is a non-responder.
  • said adjusting step comprises discontinuing the therapy.
  • said adjusting step comprises using a different antiallergic agent.
  • the identification step is performed at least twice before adjusting the therapy.
  • the at least two successive identification steps are separated by 3 to 6 months.
  • the method can be applied using the steps, conditions, amounts, examples and conditions or as described above.
  • blood sample applied in the present method generally refers to a "whole blood sample”.
  • whole blood refers to blood as it is collected by venous sampling, i.e. containing white and red cells, platelets, and plasma.
  • the sample can be purified basophils or PBMCs, or depleted-PBMCs, all obtained from blood, e.g. peripheral blood.
  • mRNA levels of the biomarkers of the invention can be determined using any known method in the art. Examples are: Polymerase Chain Reaction (PCR), Real-Time quantitative PCR (RT-qPCR), End-Point PCR, digital PCR (dPCR), RNA or cDNA hybridization techniques, microarrays, RNA-in-situ hybridization (RISH), Northern-Blotting, digital analysis of gene expression (DAGE), sequence-analysis based expression analysis, Supported oligonucleotide detection, Pyrosequencing, Polony Cyclic Sequencing by Synthesis, Simultaneous Bi-directional Sequencing, Single-molecule sequencing, Single molecule real time sequencing, True Single Molecule Sequencing, Hybridization- Assisted Nanopore Sequencing or Sequencing by synthesis.
  • PCR Polymerase Chain Reaction
  • RT-qPCR Real-Time quantitative PCR
  • dPCR digital PCR
  • RNA or cDNA hybridization techniques microarrays, RNA-in-situ hybrid
  • the biomarker mRNA levels are determined by real-time quantitative polymerase chain reaction (qPCR, qc-PCR, RT-PCR).
  • qPCR real-time quantitative polymerase chain reaction
  • qc-PCR real-time quantitative polymerase chain reaction
  • kits for use in practicing the subject methods.
  • kit refers to any combination of reagents or apparatus that can be used to perform a method of the invention.
  • a kit for diagnosing allergic response to an allergen in a subject according to the invention typically comprises:
  • said vessel comprises: i) a vessel capable of accepting a blood sample, and optionally ii) a container in which a stabilizing agent is present, iii) a connection between the inside of said vessel (i) and the inside of said container (ii), and iv) a physical barrier that temporarily blocks said connection.
  • the kit according to the invention optionally additionally comprises:
  • control primer pair specific to the mRNA of a control gene which is suitable for the transcription of mRNA of said control gene into cDNA and the amplification of the latter, and a control probe designed to anneal to an internal region of the produced control cDNA.
  • the allergen can either be added in vitro, i.e. after the sample of e.g. blood has been taken from the subject under investigation.
  • One part of the blood sample is then contacted with the allergen and the resulting RNA expression pattern resulting from the stimulation is determined subsequently by fixing the RNA with an RNA stabilizing agent, followed by RNA analysis as indicated for the other embodiments.
  • the remaining part of the blood sample is also analysed for its RNA expression pattern in the same manner, safe from the addition of the allergen.
  • the two expression patterns can then be compared to give an indication of the active state of the MS in the subject.
  • a typical kit according to the invention can thus comprise:
  • said vessel (i) comprises: a) an allergen present inside said vessel, optionally present in a container (v) separated from said vessel by a physical barrier (vii) temporarily blocking the connection (vi), b) a container (ii) in which an RNA-stabilizing agent is present, c) a connection between the inside of said vessel (i) and the inside of said container (ii), d) a physical barrier (iv) that temporarily blocks said connection (iii).
  • any one of the physical barrier(s) in the kits or methods of the invention may be opened by the application of physical force to said vessel.
  • Said force may transmit an opening means to said physical barrier.
  • Such physical barriers include rotary valve, aperture valve, slit valve, diaphragm valve, ball valve, flap valve.
  • said force may irreversibly open said physical barrier.
  • Other examples of such physical barriers include a plug which is forced out of position, a barrier which shatters upon the application of force.
  • the inside of said container and the inside of said vessel are connected, and the flow of stabilizing agent from the container to the vessel is prevented by the surface tension of the stabilizing agent in combination with the aperture size of the connection.
  • an application of force which transmits to the stabilizing agent forces the stabilizing agent from the container into the vessel.
  • the force may be applied, for example, by squeezing, continually inverting, and agitating.
  • the allergen can be provided in said vessel in a liquid or lyophilized form, not immobilized.
  • the allergen can also be immobilized on part or all of the inside surface of said vessel.
  • the inside wall of the vessel may be lined with a suitable coating enabling the allergen to be attached.
  • said allergen is immobilized on a solid support.
  • the solid support may be attached to the inside of the vessel. Alternatively, the solid support may be free of the inside of the vessel. Examples of solid supports include, but are not limited to, chromatography matrix, magnetic beads.
  • the vessel may be sealed with resealing means such as a screw-cap, push-on cap, and a flip-cap.
  • Said vessel may comprise one or more openings.
  • the vessel as described above comprises one or more areas suitable for puncture by a syringe needle, such as a re-sealable septum.
  • the vessel may comprise a fitting suitable for receiving a syringe or a syringe needle and transmitting the contents therein to the interior of said vessel.
  • Suitable vessel may further comprise cannular suitable for withdrawing bodily fluids.
  • Suitable vessel may further comprise a valve which is capable of minimizing the flow of gas/liquid from vessel, and allowing the flow of biological sample into the vessel.
  • Suitable vessel may further comprise a means through which displaced gas may be expelled.
  • Said means are known the art and include valves, non-drip holes, vents, clothed-vents, expandable vessel walls, use of negative pressure within said vessel.
  • Said vessel may further be held under negative pressure.
  • the negative pressure may be utilized to relieve the pressure build-up upon introduction of whole blood into said sealed vessel. Alternatively, or in addition, the negative pressure may be at a predetermined level and may be utilized so as to allow the introduction of a fixed volume of whole blood.
  • Suitable vessel may comprise an indication for dispensing a known volume of stabilizing agent therein.
  • the qPCR control gene used in some of the kits or methods according to the invention is selected from the group comprising mRNAs for certain ribosomal proteins such as RPLP0 (ribosomal protein, large, P0), glyceraldehyde-3-phosphate dehydrogenase mRNA, beta actin mRNA, MHC I (major histocompatibility complex I) mRNA, cyclophilin mRNA, 28S or 18S rRNAs (ribosomal RNAs).
  • RPLP0 ribosomal protein, large, P0
  • glyceraldehyde-3-phosphate dehydrogenase mRNA beta actin mRNA
  • MHC I major histocompatibility complex I
  • cyclophilin mRNA 28S or 18S rRNAs
  • the kit can further comprise additional components for carrying out the method of the invention, such as RNA extraction solutions, purification column and buffers and the like.
  • the kit of the invention can further include any additional reagents, reporter molecules, buffers, excipients, containers and/or devices as required described herein or known in the art, to practice a method of the invention.
  • the various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container, as desired.
  • the kits may further include instructions for practicing the present invention. These instructions may be present in the kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.
  • kits g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e. g., diskette, CD, etc., on which the information has been recorded. Any convenient means may be present in the kits.
  • the allergens are the allergens.
  • the allergy of the subject can be to any product, e.g. foodstuffs such as cowmilk, gluten, wheat, peanuts tree nuts, hazelnuts, soy, egg, fish, shell fish, chocolate, strawberries, apple etc.; insect venoms such as from bees, wasps, ants, etc.; venom from other animals such as snakes, spiders, etc; irritating secretions and other allergens from plants, such as Bigsting (stinging nettle), timothy grass etc.; pollen from trees such as birch; dust or small insects such as (house)mites etc.; animal allergens such as hairs form e.g.
  • foodstuffs such as cowmilk, gluten, wheat, peanuts tree nuts, hazelnuts, soy, egg, fish, shell fish, chocolate, strawberries, apple etc.
  • insect venoms such as from bees, wasps, ants, etc.
  • venom from other animals such as snakes, spiders, etc
  • the general treatment of an allergic reaction is nowadays mainly focussed on alleviating the symptoms of the allergic reaction, which is of course highly desired for the subject, but does in no way prevent the recurrence of the allergic reaction upon new exposure to the allergen in question.
  • Immunosuppressants such as e.g. antihistamines or corticosteroids can therefore help the patient to feel better, but can never cure him.
  • One way of actually curing the subject is using allergy immunotherapy. A three-to-five-year individually tailored regimen of injections may result in long-term benefits.
  • Allergy immunotherapy (also called hyposensitization therapy, immunologic desensitization or allergen-specific immunotherapy) involves a series of injections with an allergy extract given regularly for several years (usually 3 to 5 years). The first injections only contain very small amounts of the allergen or antigen to which the subject is allergic and this dose is progressively increased over time. The immune system of the subject is thus gradually exposed to higher dosages of the allergen and becomes less sensitive to it. This process is called desensitization.
  • a good example of such an immunotherapy is the sublingual (under the tongue) tablet Grazax, containing a grass pollen extract which has been shown effective with few side effects and can even be self-administered at home.
  • Allergen specific immunotherapy is the only treatment strategy which treats the underlying cause of the allergic disorder and has been shown to produce long term remission of allergic symptoms, reduce severity of associated asthma as well as reduce the chances of new sensitizations to allergens developing.
  • the allergy extract is usually administered sublingually (under the tongue), by nasal or oral administration or by injections under the skin (subcutaneous).
  • Subcutaneous injection immunotherapy has been shown to be highly efficient treatment for allergic disease, but can lead to anaphylaxis and is therefore restricted to specialized clinical settings.
  • Sublingual therapy which can be safely administered at home is currently preferred.
  • Another possibility is the use of DNA vaccines that incorporate a gene encoding the entire or partial allergen sequence and containing at least one T-cell epitope sequence.
  • the expression levels of the biomarkers are often depicted as “ratios” or “stimulation index” or “stimulation ratio” herein. Said ratios are calculated by dividing the expression level of a certain biomarker of allergen-treated blood samples by the expression level of said biomarker in the non-allergen-treated blood sample. If the ratio is close to 1 , there is no or little response of the biomarker expression level to the allergen. If on the other hand the ratio is significantly higher than 1 , there is an increase in biomarker expression upon stimulation with the allergen. The ratio represents the "fold induction" of the expression level of the biomarker.
  • the goal of the diagnostic methods according to the invention is to provide a more accurate indication whether or not a patient can be classified as allergic to a certain allergen.
  • a significant increase in expression level for classifying or diagnosing a subject of being allergic to said tested allergen or allergen extract is e.g. 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, or more than 20, wherein a ratio of 1.5 indicates that the expression level of said biomarker after stimulation with the allergen is 1 .5 times higher than the expression level without stimulation with the allergen.
  • Example 1 Identification of new allergy specific genes in activated basophils with DNA Microarrays
  • Example 2 Expression of Basophil LDA cards qPCRs
  • Betvl Bet allergen
  • HAS1 27 8.3 14.8 18.1 0.4 28.3 18.7 16.7
  • CD44 2.4 1 .4 1 .8 3.5 1 .8 1 .7 1 .57 1 .9
  • Patient 5 was left out of the test, since a further analysis revealed unresponsiveness to anti-Fee receptor antibody, which is used as a positive control. Unresponsiveness to the positive control is considered as unresponsiveness of the basophils, and is one of the criteria of exclusion. This also explains the absence of response to the 4 studied genes.
  • the cat allergen Fel d1 leads to an increased stimulation ratio in previously diagnosed cat allergic patients, while the unrelated Betvl allergen does not. This implies that the response of said 5 identified biomarkers is specific to the concerned allergen and thus related to the clinics, i.e. in cat allergic patients, a cat allergen induces their expression, while a non-related allergen does not.
  • the ratios are calculated by dividing the expression level of a certain biomarker of allergen-treated blood samples by the expression level of said biomarker in the non- allergen-treated blood sample. If the ratio is close to 1 , there is no or little response of the biomarker expression level to the allergen. If on the other hand the ratio is significantly higher than 1 , there is an increase in biomarker expression upon stimulation with the allergen.
  • Example 3 Analysis of markers in pediatric patients tested for food allergy
  • the expression level of the 4 genes of the present invention will be tested on mRNA isolated form said samples.
  • Peripheral blood cells were purified from Buffy coats using HetasepTM solution. Basophils were purified using the Basophil purification kit from Stemcell Technologies, Grenoble, France, and subsequently activated for 2 hours with an anti-FceRI antibody, (0.2 ug/ml, obtained from Bijhlmann Laboratories AG, Switzerland). Peripheral blood mononuclear cells (PBMCs) from blood of cat allergic patients were isolated through a ficoll gradient. A fraction of blood was depleted in Basophils using the Basophil depletion kit from Stemcell Technologies, Grenoble, France.
  • PBMCs Peripheral blood mononuclear cells
  • PBMC and Basophil depleted PBMCs were stimulated in parallel with the allergens for 2 hours.
  • Whole blood was stimulated for 2 hours with allergens (12,5 ug/ml Fel d1 or Betvl , obtained from Indoor Biotechnologies Wiltshire, UK) and Paxgene (1 .5 ml for 500 ⁇ of whole blood) was added (Becton Dickinson, Erembodegem, Belgium) at this end of the incubation in order to stabilize total RNA.
  • allergens (12,5 ug/ml Fel d1 or Betvl , obtained from Indoor Biotechnologies Wiltshire, UK) and Paxgene (1 .5 ml for 500 ⁇ of whole blood) was added (Becton Dickinson, Erembodegem, Belgium) at this end of the incubation in order to stabilize total RNA.
  • Basophils were isolated from Buffy coats using HetasepTM (Stemcell Technologies, Grenoble, France) density gradient centrifugation followed by a negative selection kit: EasySepTM Human Basophil Enrichment Kit (Stemcell Technologies, Grenoble, France) as described previously (B. F. Gibbs et al., 2008, Clin Exp Allergy, 2008 Mar; 38(3):480- 5). Basophils were then activated for 2 hours with an anti-FceRI antibody (0.2 ug/ml, obtained from Bijhlmann Laboratories AG, Switzerland). PBMCs were separated from the heparinized peripheral blood of two cat allergic patients by LymphoprepTM (NYCOMED PHARMA, Switzerland) density gradient centrifugation.
  • LymphoprepTM NYCOMED PHARMA, Switzerland
  • RNA from Basophils or PBMCs and the corresponding Basophil depleted PBMCs were extracted with the micro-RNAeasyTM kit (Qiagen). RNA quality was controlled using the Agilent 2100 Bioanalyser.
  • Double strand cDNA was synthesized from 30 ng total RNA to which Affymetrix GenechipTM Poly- A Controls were added. The cDNA was transcribed in vitro in order to produce biotin labeled aRNA. 10 ⁇ g of the cleaned up aRNA was then fragmented using the GenechipTM Sample Cleanup module (Affymetrix) in order to produce fragments of 35 to 200 bases. 300 ⁇ of hybridization mix was produced including the 10 ⁇ g of fragmented aRNA, the oligonucleotide B2 control, the Affymetrix hybridization controls, herring sperm DNA, acetylated BSA, hybridization buffer and DMSO.
  • Quantitative real-time PCR (TaqmanTM) assays were performed using a 96-genes format TaqmanTM Low Density Arrays (TLDA) on an ABI PRISM 7900 thermocycler.
  • TaqManTM Universal PCR Master Mix (ABI) was used for PCRs.
  • the oligonucleotide primers and fluorogenic probes were prepared and spotted in the TLDA by ABI.
  • the 96-gene format low-density array allowed simultaneous measurement of 92 selected target genes and 3 housekeeping genes (Rplpl , RplpO, and ⁇ -actin).
  • delta-delta-Ct method was used to calculate the gene expression values. Genes where no amplified product could be detected were given an arbitrary Ct of 36 cycles, the maximum number of cycles carried out by the thermocycler.

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Abstract

La présente invention porte sur de nouveaux procédés pour le diagnostic, la prédiction et/ou le pronostic de la réponse allergique d'un sujet à un certain allergène, sur base des niveaux d'expression de 4 biomarqueurs identifiés dans des Basophiles, après une stimulation avec un allergène réel, et sur des ensembles pour mettre en œuvre lesdits procédés.
PCT/EP2010/066398 2009-11-02 2010-10-28 Nouveaux biomarqueurs pour déterminer un état d'allergie WO2011051402A1 (fr)

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CN102353771A (zh) * 2011-06-23 2012-02-15 湖南中医药大学 一种中药注射剂致敏原的综合检测方法
CN103439508A (zh) * 2013-08-25 2013-12-11 河南科技学院 用于检测铬离子的间接竞争酶联免疫试剂盒及其组建和检测方法
ITMI20131094A1 (it) * 2013-06-28 2014-12-29 Ornella Cazzalini Metodo per predire e determinare allergie
EP2753711B1 (fr) * 2011-09-07 2019-07-24 Stallergenes Procédés pour identifier des sous-ensembles de cellules dendritiques pour déterminer si un patient développe une réponse immunitaire régulatrice ou effectrice et pour déterminer la réponse à une immunothérapie
EP3599286A1 (fr) * 2018-07-27 2020-01-29 Administración General De La Communidad Autónoma De Euskadi Biomarqueurs pour le diagnostic et le pronostic de la fragilité
US20210018490A1 (en) * 2019-07-19 2021-01-21 The Regents Of The University Of Michigan Compositions and methods for individualized characterization of non-ige mediated food allergies
WO2023250419A1 (fr) * 2022-06-23 2023-12-28 Regeneron Pharmaceuticals, Inc. Procédés d'identification et d'évaluation des signatures génétiques de l'allergie au chat chez un sujet en déterminant un score stratifié fondé sur l'expression des gènes.

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102353771A (zh) * 2011-06-23 2012-02-15 湖南中医药大学 一种中药注射剂致敏原的综合检测方法
EP2753711B1 (fr) * 2011-09-07 2019-07-24 Stallergenes Procédés pour identifier des sous-ensembles de cellules dendritiques pour déterminer si un patient développe une réponse immunitaire régulatrice ou effectrice et pour déterminer la réponse à une immunothérapie
ITMI20131094A1 (it) * 2013-06-28 2014-12-29 Ornella Cazzalini Metodo per predire e determinare allergie
CN103439508A (zh) * 2013-08-25 2013-12-11 河南科技学院 用于检测铬离子的间接竞争酶联免疫试剂盒及其组建和检测方法
EP3599286A1 (fr) * 2018-07-27 2020-01-29 Administración General De La Communidad Autónoma De Euskadi Biomarqueurs pour le diagnostic et le pronostic de la fragilité
WO2020021028A1 (fr) * 2018-07-27 2020-01-30 Administración General De La Comunidad Autónoma De Euskadi Biomarqueurs de diagnostic et/ou de pronostic de fragilité
US20210018490A1 (en) * 2019-07-19 2021-01-21 The Regents Of The University Of Michigan Compositions and methods for individualized characterization of non-ige mediated food allergies
WO2021016090A1 (fr) * 2019-07-19 2021-01-28 The Regents Of The University Of Michigan Compositions et procédés pour la caractérisation individualisée d'allergies alimentaires non induites par les ige
WO2023250419A1 (fr) * 2022-06-23 2023-12-28 Regeneron Pharmaceuticals, Inc. Procédés d'identification et d'évaluation des signatures génétiques de l'allergie au chat chez un sujet en déterminant un score stratifié fondé sur l'expression des gènes.

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