WO2022106795A1 - Method for classifying an individual - Google Patents

Method for classifying an individual Download PDF

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Publication number
WO2022106795A1
WO2022106795A1 PCT/FR2021/052060 FR2021052060W WO2022106795A1 WO 2022106795 A1 WO2022106795 A1 WO 2022106795A1 FR 2021052060 W FR2021052060 W FR 2021052060W WO 2022106795 A1 WO2022106795 A1 WO 2022106795A1
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Prior art keywords
expression
hla
group
mpla
biomarker
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PCT/FR2021/052060
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French (fr)
Inventor
Chloé ALBERT VEGA
Sophie ASSANT-TROUILLET
François Mallet
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bioMérieux
Hospices Civils De Lyon
Universite Claude Bernard Lyon 1
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Publication of WO2022106795A1 publication Critical patent/WO2022106795A1/en

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the present invention relates to an in vitro or ex vivo method for classifying an individual, preferably a patient, into a group associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment, based on a functional test in wherein a blood sample of said individual is incubated with monophosphoryl lipid A (MPLA) or a derivative of MPLA, and then the expression of at least one biomarker is measured in said blood sample.
  • MPLA monophosphoryl lipid A
  • Functional tests or immune functional tests (IFA, Immune Functional Assays), measure directly, in vitro or ex vivo, the capacity of one or more cell population(s) of a sample from an individual, to respond to a stimulus with which these cells are brought into contact, and can allow such classifications.
  • IFA Immune Functional Assays
  • a functional test is used clinically, in which interferon y is measured at the protein level after stimulation with an antigen of Mycobacteria tuberculosis, for indirect detection of the pathogen.
  • Functional tests were also used as part of a study aimed at defining the limits of a normal immune response (/.e.
  • LPS lipopolysaccharide
  • LPS have certain advantages, particularly in terms of ease of preparation. However, the use of LPS in functional tests leads to variable responses depending on the studies, in particular due to differences in terms of source and purification of LPS (Segre and Fullerton (2016), Shock 45:490-494; Kiers et al (2019), Innate Immun. 25:34-45).
  • MPLA Monophosphoryl lipid A
  • LPS Long fatty acid
  • MPLA and LPS are both TLR4 agonists and they induce similar transcriptional profiles in healthy individuals, but differences should be noted, especially in terms of coreceptors, pro-inflammatory effect and ability to induce the production cytokines (Chentouh et al (2016), Sci. Rep. 8:7096).
  • MPLA induces the production of pro-inflammatory cytokines more weakly than LPS.
  • MPLA is mainly used as an adjuvant in vaccines, due to its effects on the innate immune system, while being less toxic than LPS (Luan et al (2017), Sci. Rep. 7:40050; Ito el al ( 2017), PLoS One 12: e0188934).
  • MPLA and MPLA derivatives can be used as stimuli in functional immune tests, for the classification of an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or responsiveness to treatment.
  • MPLA is, unlike LPS, a product which can be chemically well defined and homogeneous, which reduces inter-test variability.
  • stimulation by MPLA in functional tests allows a finer classification of individuals, in particular of patients, at early times (within 48 hours from admission) , than stimulation by LPS. Improved classification or stratification of patients has many advantages. Indeed, the finer the classification of individuals into groups or subgroups exhibiting specific characteristics, the more it is possible to provide a suitable treatment for said individual who thus benefits from the most optimal immunomodulation strategy.
  • MPLA or one of its derivatives comprising in particular a single phosphate group on a glucosamine unit, makes it possible to achieve this objective when it is used as a stimulus in the immune functional tests known to those skilled in the art and used Routine.
  • the method according to the invention thus advantageously makes it possible to define the capacity of the immune system of an individual to react to a stimulus, thus determining the activity of said immune system. This determination finds a very particular utility when it comes to defining the most suitable treatment for the patient, in particular the immunomodulatory treatment.
  • the subject of the present invention is an in vitro or ex vivo method for classifying an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment, comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives, in particular MPLA derivatives comprising a single phosphate group on one glucosamine unit, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least one, 2, 3, 4, 5, 6, 7 , 8, 9, 10,
  • MPLA Monophosphoryl lipid A
  • MPLA derivatives in particular MPLA derivatives comprising a single phosphate group on one glucosamine unit
  • the invention can also be defined as an in vitro or ex vivo method for determining the activity of the immune system of an individual, comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives, in particular MPLA derivatives comprising a single phosphate group on a glucosamine unit, and b) a step for measuring the expression, from of the stimulated blood sample resulting from step a), of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
  • MPLA Monophosphoryl lipid A
  • MPLA derivatives in particular MPLA derivatives comprising a single phosphate group on a glucosamine unit
  • the term "individual” designates a human being, whoever he is (and in particular whatever his state of health, whether he is a healthy individual or a sick individual).
  • the term “patient” refers to an individual who has come into contact with a health care professional, such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital, and especially the emergency department, intensive care unit, intensive care unit or continuing care unit).
  • a patient is generally a sick individual, but it can also be a healthy individual (for example, an elderly person coming to be vaccinated);
  • the “stimulus” corresponds to one or more molecule(s), capable of inducing an immune response and making it possible to qualitatively and/or quantitatively evaluate the immune response of the individual;
  • MPLA stands for Monophosphoryl lipid A.
  • MPLA is a derivative of LPS lipid A, which has only one phosphate group on one of the glucosamines.
  • MPLA consists of two glucosamine units (only one of which bears a phosphate group) linked by a P(1->6) type bond, and having fatty acid chains attached to them.
  • Natural MPLAs generally comprise 5, 6 or 7 chains of fatty acids (possibly mixed), of which 4 are attached directly to the glucosamine units. These 4 fatty acid chains are beta hydroxy acyl chains, generally comprising between 10 and 16 carbon atoms. Additional fatty acid chains (usually two) are attached to the beta hydroxy group.
  • the fatty acid chains can be of identical or different lengths.
  • MPLA from Salmonella minnesota R595 has up to 7 fatty acid chains with 12 to 16 carbon atoms.
  • Synthetic MPLAs have also been proposed, which may comprise a variable number of fatty acid chains, which may have variable lengths (commonly, between 10 and 16 carbon atoms, ie 10, 11, 12, 13, 14, 15 or 16 carbon atoms).
  • the structure of a commonly used synthetic MPLA (consisting of 6 fatty acid chains, comprising 14 carbon atoms) is shown in Figure 1.
  • MPLA derivatives refer to molecules derived from the chemical structure of MPLA, comprising a single phosphate group on a glucosamine unit and retaining TLR4 agonist properties.
  • MPLA derivatives mention may be made of the molecules of the family of aminoalkyl glucosaminide 4-phosphates (AGPs), as described in Stover et al (2004), J. Biol. Chem 279(6): 4440-9, such as CRX-512, CRX-526, CRX-527, CRX-555, CRX-560, CRX-565, CRX-566, CRX-567, CRX-568, CRX-569, CRX-570 and in particular CRX-527, which is a compound that contains only the glucosamine unit carrying the phosphate group (and no second gluocosamine unit), with 3 chains of fatty acids with 14 carbon atoms and 3 chains of fatty acids with 10 carbon atoms;
  • AGPs aminoalkyl glucosaminide 4-phosphates
  • a “blood sample” means a sample of whole blood or a cell sample derived from blood (i.e. a sample obtained from blood and containing at least one type of cells, such as a sample of peripheral blood mononuclear cells or PBMC) ;
  • a “biomarker” or “marker” is an objectively measurable biological characteristic that represents an indicator of normal or pathological biological processes or of pharmacological response to a therapeutic intervention. It may in particular be a molecular or protein biomarker. When it is a molecular biomarker, it is preferably detectable at the mRNA level. More particularly, the biomarker can be an endogenous biomarker or loci (such as a gene or a HERV/Human Endogenous RetroVirus, which are found in the chromosomal material of an individual) or an exogenous biomarker (such as a virus). When it is a protein biomarker, it is preferably a secreted protein, more particularly a cytokine.
  • the classification obtained using the process described above can therefore be useful for diagnosis (eg classification of healthy individuals versus sick individuals), prognostic (eg severity of the disease, occurrence of an infection associated with treatment, septic shock, mortality, etc. ) and/or prediction of response to a treatment (in particular, to an immunomodulating agent, and more particularly, an immunostimulating agent).
  • a treatment in particular, to an immunomodulating agent, and more particularly, an immunostimulating agent.
  • individuals or patients classified as having a less severe disease would have an at least partially functional immunity, on which it could be possible to act by means of certain treatments, such as immunomodulatory treatments (eg IFNgamma, GM-CSF, IL7).
  • immunomodulatory treatments eg IFNgamma, GM-CSF, IL7
  • Classification of an individual therefore means the possibility of classifying or stratifying said individual in a group or subgroup of individuals sharing a common pathology and/or common biological characteristics, in particular common immune characteristics. For example, it may involve classifying or stratifying an individual into a healthy group or a sick group, or even, within a group of sick individuals, stratifying said individuals according to the activity of the immune system. .
  • the individual is a patient, more preferably a patient within a health establishment, in particular in a hospital, more preferably a patient within the emergency department , an intensive care unit, an intensive care unit or a continuing care unit, even more preferably a patient suffering from trauma (preferably severe trauma), burns (preferably severe burns), having undergone surgery (in particular, major surgery) or in a septic state.
  • trauma preferably severe trauma
  • burns preferably severe burns
  • it may be a patient in a septic state, and more particularly in septic shock.
  • a sepsis patient is defined as a patient with at least one life-threatening organ failure caused by an inappropriate host response to an infection.
  • septic shock is meant a subtype of sepsis, in which hypotension persists, despite adequate vascular filling. It may also be an individual presenting, or likely to present, pathological immune alterations, due to an autoimmune disease (e.g. multiple sclerosis), inflammatory disease (e.g. rheumatoid arthritis), HIV, cancer ...
  • autoimmune disease e.g. multiple sclerosis
  • inflammatory disease e.g. rheumatoid arthritis
  • HIV cancer ...
  • step b in the method as described above, in all its embodiments, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
  • biomarker(s) chosen from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, 03, CCL2 , CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A,
  • step b) the expression of at least one, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 selected biomarker(s)( s) in the group consisting of: ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/ RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18
  • step b) the expression of at least one, 2, 3, 4, 5, 6, 7 chosen biomarker(s) is measured. ) in the group consisting of: CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2. More preferably, also, in the method as described above, in step b), the expression of at least one, 2, 3, 4, 5 biomarker(s) chosen from the group consisting of: ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA.
  • the individual is a patient in a septic state, more particularly in septic shock.
  • the use of MPLA as a stimulus in blood samples makes it possible to obtain a stratification of the patients, in particular according to the activity of their immune system.
  • This aspect is demonstrated in particular in the embodiments of the invention.
  • the level of stratification obtained is more precise than that obtained for the same type of sample using LPS as a stimulus in patients in a septic state, particularly in septic shock.
  • more precise stratification or “finer stratification”, we mean the possibility of identifying new subgroups of patients in known groups. For example, within a group of sick patients, to identify subgroups of patients according to the activity of their immune system, in particular the functional capacity of said immune system.
  • the method is therefore an in vitro or ex vivo method for determining the activity of the immune system of an individual, preferably an individual in a septic state, especially in septic shock, said method comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives comprising a single phosphate group on a glucosamine unit, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least a biomarker as defined previously.
  • MPLA Monophosphoryl lipid A
  • MPLA derivatives comprising a single phosphate group on a glucosamine unit
  • step b) the expression of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • biomarker(s) chosen from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10 /IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, I
  • step b) the expression of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 biomarker(s) chosen from the group consisting of: ADGRE3 , AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1 , IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B,
  • step b) the expression of at least one, 2, 3, 4, 5, 6, 7 biomarker(s) chosen in the group consisting of: CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, and POU2F2.
  • step b) the expression of at least one, 2, 3, 4, 5 biomarker(s) chosen from the group consisting of: ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA.
  • the individual is a patient in a septic state, more particularly in septic shock.
  • the method as described previously is applied to a blood sample containing leukocytes.
  • the blood sample can for example be a sample of peripheral blood mononuclear cells (or PBMC, Peripheral Blood Mononuclear Cells), which consists of lymphocytes (B, T and NK cells), dendritic cells and monocytes, and which is generally obtained by the Ficoll method, well known to those skilled in the art.
  • PBMC peripheral blood mononuclear cells
  • B lymphocytes
  • T and NK cells dendritic cells and monocytes
  • a whole blood sample directly (that is to say containing all the leukocytes, erythrocytes, platelets and plasma), as collected by the venous route (for example in using tubes containing an anticoagulant), in order to minimize manipulations of the sample and to preserve the physiological cellular interactions between the different cell populations involved in the immune response, and to better reflect the complexity of the innate and adaptive immune responses in the individual.
  • PBMCs only contain mononuclear cells
  • whole blood also contains granulocytes (or polymorphonuclear cells).
  • the blood sample may have been taken at the doctor's request.
  • the sample may also have been taken on admission or during the evolution of the patient; in particular, for patients suffering from sepsis or patients suffering from trauma, the sample may in particular have been taken during the first week (eg from D1 to D7, and in a particularly advantageous manner in the first 48 hours) after the aggression (ie sepsis or trauma) or after septic shock (in particular, when the patient needs vasopressors and his lactate exceeds 2 mmol/L).
  • the first week eg from D1 to D7, and in a particularly advantageous manner in the first 48 hours
  • the aggression ie sepsis or trauma
  • septic shock in particular, when the patient needs vasopressors and his lactate exceeds 2 mmol/L.
  • the stimulus is MPLA or an MPLA derivative, in particular an MPLA derivative comprising a single phosphate group on a glucosamine unit; preferably it is MPLA.
  • Doses comprised between 100 ⁇ g/mL and 100 ⁇ g/mL can in particular be used, preferably between 10 ng/mL and 1 ⁇ g/mL (in particular for MPLA).
  • the step of incubating the blood sample of the individual with the stimulus can be carried out at different temperatures (in particular, at 37° C.) and at different incubation time (preferably between 1 hour and 48 hours of incubation; for example with an incubation of 1 hour or less, 2 hours or less, 4 hours or less, 12 hours or less, 24 hours or less, or 48 hours or less ). Short incubation times are particularly advantageous for carrying out the test in the clinic.
  • the measurement of the expression (or of the level of expression) of a biomarker consists in quantifying at least one expression product of this biomarker.
  • the expression product of a biomarker within the meaning of the invention is any biological molecule resulting from the expression of this biomarker. More particularly, the expression product of a biomarker can be an RNA transcript.
  • RNA transcript we mean the RNAs, and in particular the messenger RNAs (mRNAs), resulting from the transcription of the biomarker. More precisely, the transcripts are the RNAs produced by the transcription of a gene followed by the post-transcriptional modifications of the pre-RNA forms.
  • the expression of the biomarkers can be measured at the RNA or mRNA transcript level.
  • the measurement of the level of expression of one or more RNA transcripts of the same biomarker can be carried out.
  • the determination of the quantity of several transcripts can be implemented sequentially or simultaneously, according to methods well known to those skilled in the art.
  • the detection of an mRNA transcript can be carried out by a direct method, by any method known to those skilled in the art making it possible to determine the presence of said transcript in the sample, or by indirect detection of the transcript after transformation of the latter into DNA. , or after amplification of said transcript or after amplification of the DNA obtained after transformation of said transcript into DNA.
  • the expression of the biomarkers can in particular be measured by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably by quantitative RT-PCR or RT-qPCR (for example using FilmArray® technology), by sequencing (preferably by sequencing high throughput) or by hybridization techniques (for example with hybridization microchips or by techniques of the NanoString® nCounter® type). Techniques allowing multiplexing (such as FilmArray® or NanoString® nCounter®) are preferred.
  • the measurement of the level of expression makes it possible to determine the quantity of one or more transcripts present in the sample tested or to give a value derived therefrom.
  • a value derived from the quantity can for example be the absolute concentration, calculated using a calibration curve obtained from successive dilutions of a solution of amplicons of known concentration. It can also correspond to the value of the standardized and calibrated quantity, such as the CNRQ. (Calibrated Normalized Relative Quantity, (Hellemans et al (2007), Genome biology 8(2):R19)), which incorporates the values of a reference sample, a calibrator and one or more housekeeping genes (also called reference genes).
  • housekeeping gene mention may be made of the ACTB, DECRI, GAPDH, GLYR1, HPRT1, PPIB, PPIA, RANBP3, RPLPO, TBP and 18S genes.
  • the expression of the biomarker(s) is normalized with respect to the expression of one or more of the following housekeeping genes: DECRI, HPRT1 and TBP; in particular, the geometric mean of the 3 genes DECRI, HPRT1 and TBP can be used for normalization.
  • the expression of the biomarkers can also be measured at the protein level.
  • the techniques allowing such measurement of protein expression are well known to those skilled in the art.
  • assays by immunoassays such as ELISA (Enzyme Linked ImmunoSorbent Assay) and ELFA (Enzyme Linked Fluorescent Assay), by ECL (electrochemiluminescence) and assays by mass spectrometry.
  • the method as described above can also comprise a step of measuring the expression, from a control blood sample without stimulation (that is to say the sample blood incubated under the same conditions as the stimulated blood sample, but in the absence of stimulus), of the same biomarker(s) as that (those) measured from the blood sample stimulated.
  • the method comprises a step of calculating the ratios of the expression (preferably, the normalized expression) of each biomarker in the stimulated blood sample, relative to the expression (preferably, the normalized expression), of the same biomarker in the control blood sample.
  • the method comprises a step of transforming the ratios obtained by a base 10 logarithmic transformation, and possibly steps of transforming into reduced centered variables.
  • a subject of the invention is also a kit comprising: at least one stimulus chosen from the group consisting of MPLA and MPLA derivatives, in particular MPLA derivatives comprising a single phosphate group on a glucosamine unit, and one or more (s) amplification (preferably primers) and / or one or more detection means (preferably probes, DNA or PNA probes, antibodies or antibody analogs such as aptamers ) of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • biomarker(s) (and preferably no more than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 99, 98, 97, 96, 95, 94, 93, 92 , 91, 90, 89, 88, 87, 86,
  • biomarker(s) possibly being selected from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, 03, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA- DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA
  • primer or “amplification primer” is understood to mean a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably of 15 to 30 nucleotides, and possessing a specificity of hybridization with a target nucleotide sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence.
  • primers consisting of two primers.
  • probe or “hybridization probe” is understood to mean a nucleotide fragment typically consisting of 5 to 100 nucleotides, preferably of 15 to 90 nucleotides, even more preferably of 15 to 35 nucleotides, possessing a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence.
  • the probe also comprises a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence.
  • the target nucleotide sequence can be a nucleotide sequence included in a messenger RNA (mRNA) or a nucleotide sequence included in a complementary DNA (cDNA) obtained by reverse transcription of said mRNA.
  • mRNA messenger RNA
  • cDNA complementary DNA
  • probes are preferably used, each preferably having a capacity to hybridize specifically with a different biomarker.
  • hybridization is meant the process during which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, having sufficiently complementary sequences, are capable of forming a double strand with stable and specific hydrogen bonds.
  • a nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner.
  • the hybridization conditions are determined by the stringency, that is to say the rigor of the operating conditions.
  • the hybridization is all the more specific as it is carried out at higher stringency.
  • Stringency is defined in particular according to the base composition of a probe/target duplex, as well as by the degree of mismatch between two nucleic acids.
  • the stringency can also be a function of the reaction parameters, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and/or the hybridization temperature.
  • the stringency of the conditions under which a hybridization reaction must be carried out will mainly depend on the hybridization probes used. All of these data are well known and the appropriate conditions can be determined by those skilled in the art. In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70°C, in particular between 35 and 65°C in a saline solution at a concentration of about 0 .5 to 1 M. A step of detecting the hybridization reaction is then carried out.
  • enzymatic amplification reaction is meant a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme.
  • amplification reactions are well known to those skilled in the art and the following techniques may be mentioned in particular: PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), RCR (Repair Chain Reaction), 3SR (Self Sustained Sequence Replication) with patent application WO-A-90/06995, NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification) with the patent US-A-5,399,491, and LAMP (Loop mediated isothermal amplification) with patent US6410278.
  • RT-PCR reverse transcription
  • mRNA messenger RNA
  • cDNA complementary DNA
  • the invention also relates to the use of a kit as described previously, in all its embodiments, to classify an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or a ability to respond to treatment, or to determine the activity of an individual's immune system.
  • a subject of the invention is also the use of MPLA or one of its derivatives as defined previously, in an immune functional test, to determine the activity of the immune system of an individual from a blood sample of said individual.
  • a subject of the invention is also the use of MPLA or one of its derivatives as defined above in one or more blood samples, preferably within an immune functional test, as a stimulus of the immune system to stratify a group sick individuals, in particular individuals in a septic state or septic shock, depending on the activity of said immune system.
  • FIG. 1 Chemical structure of a synthetic MPLA, comprising 6 chains of fatty acids with 14 carbon atoms.
  • FIG. 2 Heat map (heat-map) after modulation by stimulation with LPS (thin line) or MPLA (thick line), based on the expression of 93 biomarkers, in 10 healthy volunteers (on the ordinate, L for LPS and M for MPLA).
  • FIG. 3 Heat-map after modulation by stimulation with LPS (thin line) or MPLA (thick line), based on the expression of 93 biomarkers, in 20 patients with sepsis (on the ordinate, L for LPS and M for MPLA).
  • FIG. 4 TNF ⁇ concentration (pg/mL) measured after MPLA stimulation in healthy volunteers and patients with sepsis (* p ⁇ 0.05).
  • FIG. 5 Pooling analysis performed on healthy volunteers and patients with sepsis, after LPS stimulation (top) or MPLA stimulation (bottom).
  • HLA-DR circulating monocytes
  • TruCulture Stimulation Heparinized whole blood (1 ml) from patients, collected on the day of study inclusion, and from healthy volunteers (VS) was dispensed into pre-warmed TruCulture tubes (Myriad Rbm, Austin, TX, USA) containing the medium alone (NUL), medium with LPS (100 ng/mL; Invivogen, San Diego, CA, USA) or medium with MPLA (100 ng/mL; Invivogen, San Diego, CA, USA - see Figure 1 ). These tubes were then inserted into a dry block incubator and maintained at 37°C for 24 hours.
  • the cell pellet was resuspended in 2 mL of TRI Reagent® LS (Sigma-Aldrich, Deisenhofen, Germany), vortexed for 2 minutes, and allowed to stand for 10 minutes at room temperature before storage at - 80°C.
  • TRI Reagent® LS Sigma-Aldrich, Deisenhofen, Germany
  • Table 1 Molecular panel of 93 biomarkers and 3 housekeeping genes, used to evaluate the response to LPS and MPLA stimulation on whole blood from healthy volunteers and patients with sepsis with Nanostring technology. . Generation of normalized data
  • Each sample was analyzed in a separate multiplexed reaction each comprising 8 negative probes and 6 serial concentrations of positive control probes. Negative control analysis was performed to determine background for each sample.
  • a scale factor for a sample was a ratio of the geometric mean of the sample and the mean of all the geometric means. For each sample, we divided all biomarker values by the corresponding scale factor.
  • the results are expressed in counts (analysis within a population) and in ratio (ratio between the stimulation condition (LPS or MPLA) with the control condition (NUL), in order to compare competent and immunosuppressed populations.
  • TNF ⁇ protein in healthy volunteers (VS) and in patients with sepsis was quantified in the TruCulture supernatant using the ELLA nanofluidic system (Biotechne, Minneapolis, ML, USA), according to the manufacturer's instructions. The results are expressed in pg/ml.
  • Counts and frequency were used for qualitative data, and medians and IQ.R (interquartile range: [Q.1-Q.3]) for quantitative data.
  • Qualitative variables were compared using the Chi-square test (or Fisher's exact test for expected small numbers).
  • the distribution of quantitative data was compared using Student's t-test (or Mann-Whitney t-test when the distribution was not normal or Welch's test when homoscedasticity was rejected) if 2 groups were compared. If more than 2 groups, the distribution of quantitative data was compared using the Anova test (or the Kruskal-Wallis test when the distribution was not normal or when homoscedasticity was rejected). Spearman's test was used for correlation analysis.
  • biomarkers were differentially expressed after LPS stimulation (53 were over-expressed and 11 were under-expressed) and 55/93 (59%) were differentially expressed after stimulation.
  • MPLA 51 were over-expressed and 4 under-expressed
  • 46 biomarkers were common to both stimulations (44 over-expressed and 2 under-expressed).
  • biomarkers were differentially expressed after LPS stimulation (all over-expressed) and 22/93 (24%) were differentially expressed after MPLA stimulation (20 over-expressed and 2 underexpressed) compared to the control condition.
  • 12 differentially regulated biomarkers were common to both stimulations, all over-expressed (see Table 2).
  • Biomarkers differentially expressed following stimulation with LPS or MPLA in healthy volunteers and patients with sepsis are differentially expressed following stimulation with LPS or MPLA in healthy volunteers and patients with sepsis (the number of over-expressed biomarkers is specified in parentheses).
  • biomarkers 38 of the 93 biomarkers (i.e. 41%) were found to be differentially expressed between the two stimulations in healthy volunteers. Among these 38 biomarkers, 29 biomarkers are found to be more strongly expressed following stimulation by LPS than following stimulation by MPLA, and 9 biomarkers are found to be more strongly expressed following stimulation by MPLA than following stimulation by LPS. In patients with sepsis, 10 biomarkers out of 93 (i.e. 11%) are differently modulated between LPS and MPLA.
  • biomarkers have a higher expression differential following stimulation by LPS than following stimulation by MPLA (indicated LPS in the LPS/MPLA column of Table 3 below), and 5 biomarkers (ADGRE3 , CD74, HLA.DPA1, HLA.DMB and HLA.DRA) have a higher expression differential following MPLA stimulation than following LPS stimulation (indicated MPLA).
  • septic patients have a higher loss of differentially expressed biomarkers during LPS stimulation (72%, p-value ⁇ 0.001) compared to LPS stimulation.
  • MPLA (60%, p-value ⁇ 0.01) (see Table 4). The fact that the loss of biomarkers involved in the post MPLA signaling pathway is less important than in that of LPS, indicates that the response to MPLA in septic patients is less altered (compared to healthy volunteers) and more informative than the response at LPS.
  • MPLA stimulation is able to reveal immune alterations in patients with sepsis.
  • the TNF ⁇ secreted after ex vivo stimulation of whole blood by MPLA reveals a lower protein induction in patients with sepsis, compared to healthy volunteers (p value ⁇ 0.05) ( Figure 4).
  • the L1 cluster was composed of only healthy volunteers and the L3 cluster consisted of only 2 individuals, excluding statistical analysis of clinical parameters. It seems that following LPS stimulation, profound and common alterations are observed in the innate arm of patients' immunity. septic, which did not allow further segregation of patients on the basis of these.
  • the M1 cluster consisted of only healthy volunteers and the M2 cluster consisted of 3 low mHLA-DR patients (1864, 2560, and 5427 Ab/C, respectively), with varying levels of post-stimulation TNF ⁇ protein.
  • LPS (229, 73 and 1010 pg/mL, respectively).
  • the individuals (patients) of the M2 group presented at admission a SOFA score of 13, 14 and 7 and a lactate of 2.5, 3.6 and 1 mmol/L, respectively. All were non-survivors at D28 and only patient G3 had a nosocomial infection before dying.
  • SOFA sequential organ failure assessment
  • CCI Charlson Comorbidity Index
  • H LA-DR human leukocyte antigen DR
  • TNF ⁇ tumor necrosis factor alpha
  • LPS lipopolysaccharide
  • IQR Interguartile range
  • Table 7 below shows the biomarkers differentially expressed between the M3 and M4 clusters.

Abstract

The present invention relates to an in vitro or ex vivo method for classifying an individual, preferably a patient, into a group associated with a diagnosis, prognosis and/or ability to respond to a treatment, based on a functional assay in which a blood sample of said individual is incubated with monophosphoryl lipid A (MPLA) or a derivative of MPLA, and then the expression of at least one biomarker is measured in said blood sample.

Description

DESCRIPTION DESCRIPTION
Titre : Procédé de classification d'un individu Title: Process for classifying an individual
La présente invention concerne un procédé in vitro ou ex vivo de classification d'un individu, de préférence un patient, dans un groupe associé à un diagnostic, un pronostic et/ou une capacité de réponse à un traitement, basé sur un test fonctionnel dans lequel un échantillon sanguin dudit individu est incubé avec du monophosphoryl lipid A (MPLA) ou un dérivé de MPLA, puis l'expression d'au moins un biomarqueur est mesurée dans ledit échantillon sanguin. The present invention relates to an in vitro or ex vivo method for classifying an individual, preferably a patient, into a group associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment, based on a functional test in wherein a blood sample of said individual is incubated with monophosphoryl lipid A (MPLA) or a derivative of MPLA, and then the expression of at least one biomarker is measured in said blood sample.
La classification d'individus, en particulier de patients, dans des groupes associés à un diagnostic, à un pronostic et/ou à une capacité de réponse à un traitement, revêt une grande importance pour une meilleure prise en charge de ces individus. The classification of individuals, in particular of patients, into groups associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment, is of great importance for better management of these individuals.
Les tests fonctionnels, ou tests fonctionnels immunitaires (IFA, Immune Functional Assays), mesurent directement, in vitro ou ex vivo, la capacité d'une ou plusieurs population(s) cel lulaire(s) d'un échantillon d'un individu, à répondre à un stimulus avec lequel ces cellules sont mises en contact, et peuvent permettre de telles classifications. Dans le cas de la tuberculose, par exemple, on utilise en clinique un test fonctionnel, dans lequel on mesure l'interféron y au niveau protéique après une stimulation avec un antigène de Mycobacteria tuberculosis, pour une détection indirecte du pathogène. Des tests fonctionnels ont également été utilisés dans le cadre d'une étude visant à définir les limites d'une réponse immunitaire normale (/.e. en contexte « sain ») en réponse à différents challenges infectieux (Urrutia et al (2016), Cell Reports 16 : 2777-2791). Un autre test fonctionnel est utilisé en recherche pour étudier l'anergie des monocytes, qui est un trait caractéristique de l'immunosuppression induite par le sepsis ; dans ce test, on mesure le plus souvent le TNFa au niveau protéique après une stimulation ex vivo avec du lipopolysaccharide (LPS) (Antonakos et al (2017), Crit Care 21 :48). Les LPS sont des molécules se trouvant dans la membrane externe des bactéries à Gram négatif, qui se lient au récepteur toll-like 4 (TLR4) et induisent la libération de cytokines pro-inflammatoires. Ils sont notamment utilisés pour établir des modèles in vitro ou in vivo de pathologies inflammatoires. Les LPS présentent certains avantages, notamment en terme de facilité de préparation. Cependant, l'utilisation de LPS dans des tests fonctionnels conduit à des réponses variables selon les études, notamment du fait de différences en termes de source et de purification du LPS (Segre and Fullerton (2016), Shock 45:490-494 ; Kiers et al (2019), Innate Immun. 25 :34-45). Functional tests, or immune functional tests (IFA, Immune Functional Assays), measure directly, in vitro or ex vivo, the capacity of one or more cell population(s) of a sample from an individual, to respond to a stimulus with which these cells are brought into contact, and can allow such classifications. In the case of tuberculosis, for example, a functional test is used clinically, in which interferon y is measured at the protein level after stimulation with an antigen of Mycobacteria tuberculosis, for indirect detection of the pathogen. Functional tests were also used as part of a study aimed at defining the limits of a normal immune response (/.e. in a "healthy" context) in response to different infectious challenges (Urrutia et al (2016), Cell Reports 16:2777-2791). Another functional test is used in research to study monocyte anergy, which is a characteristic feature of sepsis-induced immunosuppression; in this test, TNFα is most often measured at the protein level after ex vivo stimulation with lipopolysaccharide (LPS) (Antonakos et al (2017), Crit Care 21:48). LPS are molecules found in the outer membrane of Gram-negative bacteria, which bind to the toll-like receptor 4 (TLR4) and induce the release of pro-inflammatory cytokines. They are used in particular to establish in vitro or in vivo models of inflammatory pathologies. LPS have certain advantages, particularly in terms of ease of preparation. However, the use of LPS in functional tests leads to variable responses depending on the studies, in particular due to differences in terms of source and purification of LPS (Segre and Fullerton (2016), Shock 45:490-494; Kiers et al (2019), Innate Immun. 25:34-45).
Le monophosphoryl lipid A (MPLA) est un dérivé du lipide A du LPS, qui ne comporte qu'un seul groupe phosphate sur une des glucosamines. Le MPLA et le LPS sont tous deux des agonistes du TLR4 et ils induisent des profils transcriptionnels similaires chez des individus sains, mais des différences sont à noter, notamment en termes de corécepteurs, d'effet pro-inflammatoire et de capacité à induire la production de cytokines (Chentouh et al (2018), Sci. Rep. 8:7096). En particulier, le MPLA induit plus faiblement la production de cytokines pro-inflammatoires, que les LPS. Le MPLA est principalement utilisé comme adjuvant dans des vaccins, du fait de ses effets sur le système immunitaire inné, tout en étant moins toxique que le LPS (Luan et al (2017), Sci. Rep. 7:40050 ; Ito el al (2017), PLoS One 12 : e0188934). Monophosphoryl lipid A (MPLA) is a derivative of LPS lipid A, which has only one phosphate group on one of the glucosamines. MPLA and LPS are both TLR4 agonists and they induce similar transcriptional profiles in healthy individuals, but differences should be noted, especially in terms of coreceptors, pro-inflammatory effect and ability to induce the production cytokines (Chentouh et al (2018), Sci. Rep. 8:7096). In particular, MPLA induces the production of pro-inflammatory cytokines more weakly than LPS. MPLA is mainly used as an adjuvant in vaccines, due to its effects on the innate immune system, while being less toxic than LPS (Luan et al (2017), Sci. Rep. 7:40050; Ito el al ( 2017), PLoS One 12: e0188934).
Or, il a à présent été découvert que le MPLA et les dérivés de MPLA pouvaient être utilisés comme stimuli dans des tests fonctionnels immunitaires, pour la classification d'un individu, de préférence un patient, dans un groupe associé à un diagnostic, un pronostic et/ou une capacité de réponse à un traitement. De manière avantageuse, le MPLA est, contrairement au LPS, un produit qui peut être bien défini chimiquement et homogène, ce qui réduit les variabilités inter-tests. Par ailleurs, de manière surprenante, il a été montré que la stimulation par MPLA dans des tests fonctionnels, permettait une classification plus fine des individus, en particulier des patients, à des temps précoces (dans les 48 heures à partir de l'admission), qu'une stimulation par LPS. L'amélioration de classification ou de la stratification des patients présente de nombreux avantages. En effet, plus la classification des individus dans des groupes ou sous-groupes présentant des caractéristiques propres est fine, et plus il est possible de fournir un traitement adapté pour ledit individu qui bénéficie ainsi de la stratégie d'immunomodulation la plus optimale. However, it has now been discovered that MPLA and MPLA derivatives can be used as stimuli in functional immune tests, for the classification of an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or responsiveness to treatment. Advantageously, MPLA is, unlike LPS, a product which can be chemically well defined and homogeneous, which reduces inter-test variability. Furthermore, surprisingly, it has been shown that stimulation by MPLA in functional tests, allows a finer classification of individuals, in particular of patients, at early times (within 48 hours from admission) , than stimulation by LPS. Improved classification or stratification of patients has many advantages. Indeed, the finer the classification of individuals into groups or subgroups exhibiting specific characteristics, the more it is possible to provide a suitable treatment for said individual who thus benefits from the most optimal immunomodulation strategy.
Le MPLA, ou l'un de ses dérivés comprenant notamment un seul groupe phosphate sur une unité glucosamine, permet d'atteindre cet objectif lorsqu'il est utilisé en tant que stimulus dans les tests fonctionnels immunitaires connus de l'homme du métier et utilisés en routine. MPLA, or one of its derivatives comprising in particular a single phosphate group on a glucosamine unit, makes it possible to achieve this objective when it is used as a stimulus in the immune functional tests known to those skilled in the art and used Routine.
Le procédé selon l'invention permet ainsi, avantageusement, de définir la capacité du système immunitaire d'un individu à réagir à un stimulus, déterminant ainsi l'activité dudit système immunitaire. Cette détermination trouve une utilité toute particulière lorsqu'il s'agit de définir le traitement le plus adapté pour le patient, notamment le traitement immunomodulateur. The method according to the invention thus advantageously makes it possible to define the capacity of the immune system of an individual to react to a stimulus, thus determining the activity of said immune system. This determination finds a very particular utility when it comes to defining the most suitable treatment for the patient, in particular the immunomodulatory treatment.
Ainsi, la présente invention a pour objet un procédé in vitro ou ex vivo de classification d'un individu, de préférence un patient, dans un groupe associé à un diagnostic, un pronostic et/ou une capacité de réponse à un traitement, comprenant : a) une étape d'incubation d'un échantillon sanguin dudit individu avec un stimulus, ledit stimulus étant choisi dans le groupe constitué par le Monophosphoryl lipid A (MPLA) et les dérivés de MPLA, notamment les dérivés de MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et b) une étape de mesure de l'expression, à partir de l'échantillon sanguin stimulé résultant de l'étape a), d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10,Thus, the subject of the present invention is an in vitro or ex vivo method for classifying an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment, comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives, in particular MPLA derivatives comprising a single phosphate group on one glucosamine unit, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least one, 2, 3, 4, 5, 6, 7 , 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 biomarqueur(s). 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 biomarker(s).
L'invention peut également être définie comme un procédé in vitro ou ex vivo de détermination de l'activité du système immunitaire d'un individu, comprenant : a) une étape d'incubation d'un échantillon sanguin dudit individu avec un stimulus, ledit stimulus étant choisi dans le groupe constitué par le Monophosphoryl lipid A (MPLA) et les dérivés de MPLA, notamment les dérivés de MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et b) une étape de mesure de l'expression, à partir de l'échantillon sanguin stimulé résultant de l'étape a), d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,The invention can also be defined as an in vitro or ex vivo method for determining the activity of the immune system of an individual, comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives, in particular MPLA derivatives comprising a single phosphate group on a glucosamine unit, and b) a step for measuring the expression, from of the stimulated blood sample resulting from step a), of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 biomarqueur(s). 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 biomarker(s).
Dans le cadre de la présente invention : In the context of the present invention:
Le terme « individu » désigne un être humain, quel qu'il soit (et notamment quel que soit son état de santé, qu'il s'agisse d'un individu sain ou d'un individu malade). Le terme « patient » désigne un individu qui est entré en contact avec un professionnel de la santé, tel qu'un médecin (par exemple, un médecin généraliste) ou une structure médicale ou un établissement de santé (par exemple, un hôpital, et plus particulièrement le service des urgences, le service de réanimation, une unité de soins intensifs ou une unité de soins continus). Un patient est généralement un individu malade, mais il peut également s'agir d'un individu sain (comme par exemple, une personne âgée venant se faire vacciner) ; Le « stimulus » correspond à une ou plusieurs molécule(s), capable(s) d'induire une réponse immune et permettant d'évaluer qualitativement et/ou quantitativement la réponse immune de l'individu ; The term "individual" designates a human being, whoever he is (and in particular whatever his state of health, whether he is a healthy individual or a sick individual). The term "patient" refers to an individual who has come into contact with a health care professional, such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital, and especially the emergency department, intensive care unit, intensive care unit or continuing care unit). A patient is generally a sick individual, but it can also be a healthy individual (for example, an elderly person coming to be vaccinated); The “stimulus” corresponds to one or more molecule(s), capable of inducing an immune response and making it possible to qualitatively and/or quantitatively evaluate the immune response of the individual;
Le MPLA désigne le Monophosphoryl lipid A. Le MPLA est un dérivé du lipide A du LPS, qui ne comporte qu'un seul groupe phosphate sur l'une des glucosamines. Ainsi, le MPLA consiste en deux unités glucosamines (dont une seule porte un groupe phosphate) reliées par une liaison de type P(l— >6), et comportant des chaînes d'acides gras qui s'y rattachent. Les MPLA naturels comprennent généralement 5, 6 ou 7 chaînes d'acides gras (possiblement en mélange), dont 4 sont attachées directement aux unités glucosamines. Ces 4 chaînes d'acides gras sont des chaînes beta hydroxy acyl, comportant généralement entre 10 et 16 atomes de carbone. Des chaînes d'acide gras supplémentaires (généralement deux) sont attachées au groupe beta hydroxy. Les chaînes d'acides gras peuvent être de longueurs identiques entre elles ou différentes. Par exemple, le MPLA de Salmonella minnesota R595 présente jusqu'à 7 chaînes d'acides gras comportant de 12 à 16 atomes de carbone. Des MPLA synthétiques ont également été proposés, pouvant comporter un nombre variable de chaînes d'acides gras, pouvant présenter des longueurs variables (couramment, entre 10 et 16 atomes de carbone, i.e. 10, 11, 12, 13, 14, 15 ou 16 atomes de carbone). La structure d'un MPLA synthétique couramment utilisé (comportant 6 chaînes d'acides gras, comportant 14 atomes de carbone) est représentée sur la Figure 1. Les « dérivés de MPLA » désignent des molécules dérivées de la structure chimique du MPLA, comportant un seul groupe phosphate sur une unité glucosamine et conservant les propriétés d'agoniste du TLR4. A titre d'exemples de dérivés du MPLA, on peut citer les molécules de la famille des aminoalkyl glucosaminide 4-phosphates (AGPs), telles que décrites dans Stôver et al (2004), J. Biol. Chem 279(6) : 4440-9, comme par exemple le CRX- 512, CRX-526, CRX-527, CRX-555, CRX-560, CRX-565, CRX-566, CRX-567, CRX- 568, CRX-569, CRX-570 et en particulier le CRX-527, qui est un composé qui ne comporte que l'unité glucosamine portant le groupe phosphate (et pas de deuxième unité gluocosamine), avec 3 chaînes d'acides gras à 14 atomes de carbone et 3 chaînes d'acides gras à 10 atomes de carbone ; MPLA stands for Monophosphoryl lipid A. MPLA is a derivative of LPS lipid A, which has only one phosphate group on one of the glucosamines. Thus, MPLA consists of two glucosamine units (only one of which bears a phosphate group) linked by a P(1->6) type bond, and having fatty acid chains attached to them. Natural MPLAs generally comprise 5, 6 or 7 chains of fatty acids (possibly mixed), of which 4 are attached directly to the glucosamine units. These 4 fatty acid chains are beta hydroxy acyl chains, generally comprising between 10 and 16 carbon atoms. Additional fatty acid chains (usually two) are attached to the beta hydroxy group. The fatty acid chains can be of identical or different lengths. For example, MPLA from Salmonella minnesota R595 has up to 7 fatty acid chains with 12 to 16 carbon atoms. Synthetic MPLAs have also been proposed, which may comprise a variable number of fatty acid chains, which may have variable lengths (commonly, between 10 and 16 carbon atoms, ie 10, 11, 12, 13, 14, 15 or 16 carbon atoms). The structure of a commonly used synthetic MPLA (consisting of 6 fatty acid chains, comprising 14 carbon atoms) is shown in Figure 1. "MPLA derivatives" refer to molecules derived from the chemical structure of MPLA, comprising a single phosphate group on a glucosamine unit and retaining TLR4 agonist properties. As examples of MPLA derivatives, mention may be made of the molecules of the family of aminoalkyl glucosaminide 4-phosphates (AGPs), as described in Stover et al (2004), J. Biol. Chem 279(6): 4440-9, such as CRX-512, CRX-526, CRX-527, CRX-555, CRX-560, CRX-565, CRX-566, CRX-567, CRX-568, CRX-569, CRX-570 and in particular CRX-527, which is a compound that contains only the glucosamine unit carrying the phosphate group (and no second gluocosamine unit), with 3 chains of fatty acids with 14 carbon atoms and 3 chains of fatty acids with 10 carbon atoms;
Un « échantillon sanguin » désigne un échantillon de sang total ou un échantillon cellulaire dérivé du sang (i.e. un échantillon obtenu à partir du sang et contenant au moins un type de cellules, tel qu'un échantillon de cellules mononuclées du sang périphérique ou PBMC) ; A “blood sample” means a sample of whole blood or a cell sample derived from blood (i.e. a sample obtained from blood and containing at least one type of cells, such as a sample of peripheral blood mononuclear cells or PBMC) ;
Un « biomarqueur » ou « marqueur » est une caractéristique biologique mesurable objectivement qui représente un indicateur des processus biologiques normaux ou pathologiques ou de réponse pharmacologique à une intervention thérapeutique. Il peut s'agir en particulier d'un biomarqueur moléculaire ou protéique. Lorsqu'il s'agit d'un biomarqueur moléculaire, il est de préférence détectable au niveau ARNm. Plus particulièrement, le biomarqueur peut être un biomarqueur endogène ou loci (tel qu'un gène ou un HERV / Human Endogenous RetroVirus, qui se retrouvent dans le matériel chromosomique d'un individu) ou un biomarqueur exogène (tel qu'un virus). Lorsqu'il s'agit d'un biomarqueur protéique, il s'agit de préférence d'une protéine sécrétée, plus particulièrement une cytokine. A "biomarker" or "marker" is an objectively measurable biological characteristic that represents an indicator of normal or pathological biological processes or of pharmacological response to a therapeutic intervention. It may in particular be a molecular or protein biomarker. When it is a molecular biomarker, it is preferably detectable at the mRNA level. More particularly, the biomarker can be an endogenous biomarker or loci (such as a gene or a HERV/Human Endogenous RetroVirus, which are found in the chromosomal material of an individual) or an exogenous biomarker (such as a virus). When it is a protein biomarker, it is preferably a secreted protein, more particularly a cytokine.
La classification obtenue grâce au procédé décrit ci-dessus peut donc avoir une utilité diagnostique (e.g. classification individus sains versus individus malades), pronostique (e.g. sévérité de la maladie, occurrence d'une infection associée aux soins, choc septique, mortalité...) et/ou de prédiction de réponse à un traitement (en particulier, à un agent immunomodulateur, et plus particulièrement, un agent immunostimulant). En particulier, les individus ou patients classifiés comme ayant une maladie moins sévère auraient une immunité au moins partiellement fonctionnelle, sur laquelle il pourrait être possible d'agir au moyen de certains traitements, tels que des traitements immunomodulateurs (e.g. IFNgamma, GM-CSF, IL7). Au sens de la présente invention, les termes « classification » et « stratification » sont considérés comme des synonymes. The classification obtained using the process described above can therefore be useful for diagnosis (eg classification of healthy individuals versus sick individuals), prognostic (eg severity of the disease, occurrence of an infection associated with treatment, septic shock, mortality, etc. ) and/or prediction of response to a treatment (in particular, to an immunomodulating agent, and more particularly, an immunostimulating agent). In particular, individuals or patients classified as having a less severe disease would have an at least partially functional immunity, on which it could be possible to act by means of certain treatments, such as immunomodulatory treatments (eg IFNgamma, GM-CSF, IL7). Within the meaning of the present invention, the terms “classification” and “stratification” are considered to be synonyms.
Par « classification d'un individu », on entend donc la possibilité de classer ou stratifier ledit individu dans un groupe ou sous-groupe d'individus partageant une pathologie commune et/ou des caractéristiques biologiques communes, notamment des caractéristiques immunitaires communes. Par exemple, il peut s'agir de classer ou stratifier un individu dans un groupe sain ou un groupe malade, ou encore, au sein d'un groupe d'invididus malades, de stratifier lesdits individus en fonction de l'activité du système immunitaire. “Classification of an individual” therefore means the possibility of classifying or stratifying said individual in a group or subgroup of individuals sharing a common pathology and/or common biological characteristics, in particular common immune characteristics. For example, it may involve classifying or stratifying an individual into a healthy group or a sick group, or even, within a group of sick individuals, stratifying said individuals according to the activity of the immune system. .
De préférence, dans le procédé tel que décrit précédemment, l'individu est un patient, de préférence encore un patient au sein d'un établissement de santé, en particulier à l'hôpital, de préférence encore un patient au sein du service des urgences, d'un service de réanimation, en unité de soins intensifs ou en unité de soins continus, de manière encore plus préférée un patient atteint de traumatismes (de préférence, de traumatismes graves), de brûlures (de préférence, de brûlure graves), ayant reçu une chirurgie (notamment, une chirurgie lourde) ou en état septique. De manière particulièrement préférée, il peut s'agir d'un patient en état septique, et plus particulièrement en choc septique. Par patient en état septique (ou patient atteint de sepsis), on entend un patient présentant au moins une défaillance d'organe menaçant le pronostic vital et causée par une réponse inappropriée de l'hôte à une infection. Par choc septique, on entend un sous-type de sepsis, dans lequel une hypotension persiste, malgré un remplissage vasculaire adéquat. Il peut encore s'agir d'un individu présentant, ou susceptible de présenter, des altérations immunitaires pathologiques, du fait d'une maladie auto-immune (e.g. sclérose en plaques), inflammatoire (e.g. polyarthrite rheumatoide), un VIH, un cancer... Preferably, in the method as described above, the individual is a patient, more preferably a patient within a health establishment, in particular in a hospital, more preferably a patient within the emergency department , an intensive care unit, an intensive care unit or a continuing care unit, even more preferably a patient suffering from trauma (preferably severe trauma), burns (preferably severe burns), having undergone surgery (in particular, major surgery) or in a septic state. In a particularly preferred manner, it may be a patient in a septic state, and more particularly in septic shock. A sepsis patient (or sepsis patient) is defined as a patient with at least one life-threatening organ failure caused by an inappropriate host response to an infection. By septic shock is meant a subtype of sepsis, in which hypotension persists, despite adequate vascular filling. It may also be an individual presenting, or likely to present, pathological immune alterations, due to an autoimmune disease (e.g. multiple sclerosis), inflammatory disease (e.g. rheumatoid arthritis), HIV, cancer ...
De préférence, dans le procédé tel que décrit précédemment, dans tous ses modes de réalisation, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,Preferably, in the method as described above, in all its embodiments, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 biomarqueur(s) choisi(s) dans le groupe constitué par : ADGRE3, AHNAK, ARL14EP, BPGM, BST2, 03, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 biomarker(s) chosen from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, 03, CCL2 , CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A,
CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA- DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, I L10, IL18, IL18R1, ILIA, I LIB, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, P0LR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020. CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, I L10, IL18, IL18R1, ILIA, I LIB, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, P0LR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020.
De préférence encore, dans le procédé tel que décrit précédemment, dans tous ses modes de réalisation, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 biomarqueur(s) choisi(s) dans le groupe constitué par : ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116. De manière encore plus préférée, dans le procédé tel que décrit précédemment, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5, 6, 7 biomarqueur(s) choisi(s) dans le groupe constitué par : CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2. De préférence encore, également, dans le procédé tel que décrit précédemment, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5 biomarqueur(s) choisi(s) dans le groupe constitué par : ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA. Preferably again, in the method as described previously, in all its embodiments, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 selected biomarker(s)( s) in the group consisting of: ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/ RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116. Even more preferably, in the method as described above, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7 chosen biomarker(s) is measured. ) in the group consisting of: CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2. More preferably, also, in the method as described above, in step b), the expression of at least one, 2, 3, 4, 5 biomarker(s) chosen from the group consisting of: ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA.
Selon un mode de réalisation préférée, l'individu est un patient en état septique, plus particulièrement en choc septique. According to a preferred embodiment, the individual is a patient in a septic state, more particularly in septic shock.
Selon ce mode de réalisation préféré, la mise en oeuvre du MPLA en tant que stimulus dans des échantillons sanguin, permet d'obtenir une stratification des patients, notamment en fonction de l'activité de leur système immunitaire. Cet aspect est notamment démontré dans les exemples de réalisation de l'invention. De manière particulièrement avantageuse, le niveau de stratification obtenu est plus précis que celui obtenu pour un même type d'échantillon en utilisant le LPS comme stimulus chez des patients en état septique, particulièrement en choc septique. According to this preferred embodiment, the use of MPLA as a stimulus in blood samples makes it possible to obtain a stratification of the patients, in particular according to the activity of their immune system. This aspect is demonstrated in particular in the embodiments of the invention. In a particularly advantageous manner, the level of stratification obtained is more precise than that obtained for the same type of sample using LPS as a stimulus in patients in a septic state, particularly in septic shock.
Par « stratification plus précise » ou « stratification plus fine », on entend la possibilité d'identifier de nouveaux sous-groupes de patients dans des groupes connus. Par exemple, au sein d'un groupe de patients malades, d'identifier des sous-groupes de patients en fonction de l'activité de leur système immunitaire, notamment de la capacité de fonctionnalité dudit système immunitaire. By “more precise stratification” or “finer stratification”, we mean the possibility of identifying new subgroups of patients in known groups. For example, within a group of sick patients, to identify subgroups of patients according to the activity of their immune system, in particular the functional capacity of said immune system.
Le professionnel de santé peut ainsi adapter plus finement le traitement et mettre en place la stratégie immunomodulatrice la plus optimale pour le patient. Selon ce mode de réalisation préféré, le procédé est donc un procédé in vitro ou ex vivo de détermination de l'activité du système immunitaire d'un individu, de préférence un individu en état septique, tout particulièrement en choc septique, ledit procédé comprenant : a) une étape d'incubation d'un échantillon sanguin dudit individu avec un stimulus, ledit stimulus étant choisi dans le groupe constitué par le Monophosphoryl lipid A (MPLA) et les dérivés du MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et b) une étape de mesure de l'expression, à partir de l'échantillon sanguin stimulé résultant de l'étape a), d'au moins un biomarqueur tel que défini précédemment. The healthcare professional can thus adapt the treatment more finely and implement the most optimal immunomodulatory strategy for the patient. According to this preferred embodiment, the method is therefore an in vitro or ex vivo method for determining the activity of the immune system of an individual, preferably an individual in a septic state, especially in septic shock, said method comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives comprising a single phosphate group on a glucosamine unit, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least a biomarker as defined previously.
Ainsi, selon ce mode de réalisation préféré, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,Thus, according to this preferred embodiment, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 biomarqueur(s) choisi(s) dans le groupe constitué par : ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, POLR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116 et 043166701-MALR1020. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 biomarker(s) chosen from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10 /IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1 , IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2 , PCGF5, PDCD10, POLR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10 , TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116 and 043166701-MALR1020.
De préférence, selon ce mode de réalisation préféré, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 biomarqueur(s) choisi(s) dans le groupe constitué par : ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B et 121601901-HERV0116. Preferably, according to this preferred embodiment, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 biomarker(s) chosen from the group consisting of: ADGRE3 , AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1 , IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B and 121601901-HERV0116.
De préférence encore, selon ce mode de réalisation préféré, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5, 6, 7 biomarqueur(s) choisi(s) dans le groupe constitué par : CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, et POU2F2. More preferably, according to this preferred embodiment, in step b), the expression of at least one, 2, 3, 4, 5, 6, 7 biomarker(s) chosen in the group consisting of: CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, and POU2F2.
De préférence encore, selon ce mode de réalisation préféré, dans l'étape b), on mesure l'expression d'au moins un, 2, 3, 4, 5 biomarqueur(s) choisi(s) dans le groupe constitué par : ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA. More preferably, according to this preferred embodiment, in step b), the expression of at least one, 2, 3, 4, 5 biomarker(s) chosen from the group consisting of: ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA.
Selon un mode de réalisation préférée, l'individu est un patient en état septique, plus particulièrement en choc septique. According to a preferred embodiment, the individual is a patient in a septic state, more particularly in septic shock.
De préférence, le procédé tel que décrit précédemment, dans tous ses modes de réalisation, est appliqué à un échantillon sanguin contenant des leucocytes. L'échantillon sanguin peut par exemple être un échantillon de cellules mononuclées du sang périphérique (ou PBMC, Peripheral Blood Mononuclear Cells), qui est constitué des lymphocytes (B, T et cellules NK), des cellules dendritiques et des monocytes, et qui est généralement obtenu par la méthode Ficoll, bien connue de l'homme du métier. Cependant, de manière particulièrement avantageuse, on préférera utiliser directement un échantillon de sang total (c'est-à-dire contenant l'ensemble des leucocytes, érythrocytes, plaquettes et le plasma), tel que collecté par la voie veineuse (par exemple en utilisant des tubes contenant un anticoagulant), afin de minimiser les manipulations de l'échantillon et de préserver les interactions cellulaires physiologiques entre les différentes populations cellulaires impliquées dans la réponse immunitaire, et de mieux refléter la complexité des réponses immunitaires innées et adaptatives chez l'individu. En particulier, alors que les PBMC ne contiennent que les cellules mononuclées, le sang total contient également des granulocytes (ou polynucléaires). Le prélèvement de l'échantillon sanguin peut avoir été réalisé à la demande du médecin. Le prélèvement peut également avoir été réalisé à l'admission ou au décours de l'évolution du patient ; notamment, pour des patients atteints de sepsis ou des patients atteints de traumatismes, le prélèvement peut en particulier avoir été réalisé lors de la première semaine (e.g. de J1 à J7, et de manière particulièrement avantageuse dans les 48 premières heures) après l'agression (i.e. le sepsis ou le traumatisme) ou après le choc septique (en particulier, lorsque le patient a besoin de vasopresseurs et que son lactate dépasse les 2 mmol/L). Preferably, the method as described previously, in all its embodiments, is applied to a blood sample containing leukocytes. The blood sample can for example be a sample of peripheral blood mononuclear cells (or PBMC, Peripheral Blood Mononuclear Cells), which consists of lymphocytes (B, T and NK cells), dendritic cells and monocytes, and which is generally obtained by the Ficoll method, well known to those skilled in the art. However, in a particularly advantageous manner, it will be preferred to use a whole blood sample directly (that is to say containing all the leukocytes, erythrocytes, platelets and plasma), as collected by the venous route (for example in using tubes containing an anticoagulant), in order to minimize manipulations of the sample and to preserve the physiological cellular interactions between the different cell populations involved in the immune response, and to better reflect the complexity of the innate and adaptive immune responses in the individual. In particular, while PBMCs only contain mononuclear cells, whole blood also contains granulocytes (or polymorphonuclear cells). The blood sample may have been taken at the doctor's request. The sample may also have been taken on admission or during the evolution of the patient; in particular, for patients suffering from sepsis or patients suffering from trauma, the sample may in particular have been taken during the first week (eg from D1 to D7, and in a particularly advantageous manner in the first 48 hours) after the aggression (ie sepsis or trauma) or after septic shock (in particular, when the patient needs vasopressors and his lactate exceeds 2 mmol/L).
Dans le procédé tel que décrit précédemment, dans tous ses modes de réalisation, le stimulus est le MPLA ou un dérivé de MPLA, notamment un dérivé de MPLA comprenant un seul groupe phosphate sur une unité glucosamine ; de préférence, il s'agit du MPLA. Des doses comprises entre 100 pg/mL et 100 pg/mL peuvent notamment être utilisées, de préférence entre 10 ng/mL et 1 pg/mL (en particulier pour le MPLA). In the method as described previously, in all its embodiments, the stimulus is MPLA or an MPLA derivative, in particular an MPLA derivative comprising a single phosphate group on a glucosamine unit; preferably it is MPLA. Doses comprised between 100 μg/mL and 100 μg/mL can in particular be used, preferably between 10 ng/mL and 1 μg/mL (in particular for MPLA).
Il est particulièrement avantageux d'utiliser, dans le cadre du procédé selon l'invention, des systèmes permettant une standardisation des procédures ; en particulier, on pourra utiliser des systèmes de culture semi-fermés (e.g. des tubes) pré-remplis avec le milieu de culture et le stimulus d'intérêt, qui sont standardisés, e.g. qui contiennent un stimulus bien défini (i.e. sans variabilité inter-lots au niveau de la production du stimulus, quant à sa nature/sa composition) et/ou chargés en « batch », de manière à contrôler la quantité de stimulus dans le tube et avoir une reproductibilité de tube à tube. De préférence, cestubes peuvent également permettre la collecte de l'échantillon de sang (ce qui permet de stimuler les cellules au moment de la collecte), et de préférence encore, ils permettent la collecte d'un volume précis de sang. On peut citer à titre d'exemple de systèmes standardisés les tubes TruCulture®. Dans le procédé tel que décrit précédemment, dans tous ses modes de réalisation, l'étape d'incubation de l'échantillon sanguin de l'individu avec le stimulus peut être réalisée à différentes températures (notamment, à 37°C) et à différents temps d'incubation (préférentiellement entre lh et 48h d'incubation ; par exemple avec une incubation d'ih ou moins, de 2h ou moins, de 4h ou moins, de 12h ou moins, de 24h ou moins, ou de 48h ou moins). Des temps d'incubation courts sont particulièrement avantageux pour la mise en œuvre du test en clinique. It is particularly advantageous to use, within the framework of the process according to the invention, systems allowing a standardization of the procedures; in particular, it is possible to use semi-closed culture systems (eg tubes) pre-filled with the culture medium and the stimulus of interest, which are standardized, eg which contain a well-defined stimulus (ie without inter- batches at the level of the production of the stimulus, as to its nature/its composition) and/or loaded in "batch", so as to control the quantity of stimulus in the tube and to have tube-to-tube reproducibility. Preferably, these tubes can also allow the collection of the blood sample (which makes it possible to stimulate the cells at the time of collection), and more preferably, they allow the collection of a precise volume of blood. One example of standardized systems is the TruCulture® tubes. In the method as described previously, in all its embodiments, the step of incubating the blood sample of the individual with the stimulus can be carried out at different temperatures (in particular, at 37° C.) and at different incubation time (preferably between 1 hour and 48 hours of incubation; for example with an incubation of 1 hour or less, 2 hours or less, 4 hours or less, 12 hours or less, 24 hours or less, or 48 hours or less ). Short incubation times are particularly advantageous for carrying out the test in the clinic.
La mesure de l'expression (ou du niveau d'expression) d'un biomarqueur consiste à quantifier au moins un produit d'expression de ce biomarqueur. Le produit d'expression d'un biomarqueur au sens de l'invention est toute molécule biologique issue de l'expression de ce biomarqueur. Plus particulièrement, le produit d'expression d'un biomarqueur peut être un transcrit ARN. Par « transcrit », on entend les ARN, et en particulier les ARN messagers (ARNm), issus de la transcription du biomarqueur. Plus précisément, les transcrits sont les ARN produits par la transcription d'un gène suivi des modifications post-transcriptionnelles des formes pré-ARN. The measurement of the expression (or of the level of expression) of a biomarker consists in quantifying at least one expression product of this biomarker. The expression product of a biomarker within the meaning of the invention is any biological molecule resulting from the expression of this biomarker. More particularly, the expression product of a biomarker can be an RNA transcript. By “transcript”, we mean the RNAs, and in particular the messenger RNAs (mRNAs), resulting from the transcription of the biomarker. More precisely, the transcripts are the RNAs produced by the transcription of a gene followed by the post-transcriptional modifications of the pre-RNA forms.
Ainsi, dans le procédé tel que décrit précédemment, dans tous ses modes de réalisation, l'expression des biomarqueurs peut être mesurée au niveau transcrit ARN ou ARNm. Dans le cadre de la présente invention, la mesure du niveau d'expression d'un ou plusieurs transcrits ARN du même biomarqueur peut être effectuée. La détermination de la quantité de plusieurs transcrits peut être mise en œuvre séquentiellement ou simultanément, selon des procédés bien connus de l'homme du métier. La détection d'un transcrit ARNm peut être réalisée par une méthode directe, par tout procédé connu de l'homme du métier permettant de déterminer la présence dudit transcrit dans l'échantillon, ou par détection indirecte du transcrit après transformation de ce dernier en ADN, ou après amplification dudit transcrit ou après amplification de l'ADN obtenu après transformation dudit transcrit en ADN. De nombreuses méthodes existent pour la détection des acides nucléiques (voir par exemple Kricka et al., Clinical Chemistry, 1999, n° 45(4), p.453-458 ; Relier G. H. et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 et 6, p.173-249). L'expression des biomarqueurs peut notamment être mesurée par Reverse Transcription-Polymerase Chain Reaction ou RT-PCR, de préférence par RT-PCR quantitative ou RT-qPCR (par exemple en utilisant la technologie FilmArray®), par séquençage (de préférence par séquençage haut débit) ou par des techniques d'hybridation (par exemple avec des micropuces d'hybridation ou par des techniques du type NanoString® nCounter®). Les techniques permettant le multiplexage (comme le FilmArray® ou le NanoString® nCounter®) sont préférées. Thus, in the method as described previously, in all its embodiments, the expression of the biomarkers can be measured at the RNA or mRNA transcript level. In the context of the present invention, the measurement of the level of expression of one or more RNA transcripts of the same biomarker can be carried out. The determination of the quantity of several transcripts can be implemented sequentially or simultaneously, according to methods well known to those skilled in the art. The detection of an mRNA transcript can be carried out by a direct method, by any method known to those skilled in the art making it possible to determine the presence of said transcript in the sample, or by indirect detection of the transcript after transformation of the latter into DNA. , or after amplification of said transcript or after amplification of the DNA obtained after transformation of said transcript into DNA. Many methods exist for the detection of nucleic acids (see for example Kricka et al., Clinical Chemistry, 1999, n° 45(4), p.453-458; Relier GH et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p.173-249). The expression of the biomarkers can in particular be measured by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably by quantitative RT-PCR or RT-qPCR (for example using FilmArray® technology), by sequencing (preferably by sequencing high throughput) or by hybridization techniques (for example with hybridization microchips or by techniques of the NanoString® nCounter® type). Techniques allowing multiplexing (such as FilmArray® or NanoString® nCounter®) are preferred.
Dans un mode de réalisation de la présente invention, la mesure du niveau d'expression permet de déterminer la quantité d'un ou plusieurs transcrits présents dans l'échantillon testé ou d'en donner une valeur dérivée. Une valeur dérivée de la quantité peut par exemple être la concentration absolue, calculée grâce à une courbe de calibration obtenue à partir de dilutions successives d'une solution d'amplicons de concentration connue. Elle peut également correspondre à la valeur de la quantité normalisée et calibrée, comme le CNRQ. (Calibrated Normalized Relative Quantity, (Hellemans et al (2007), Genome biology 8(2):R19)), qui intègre les valeurs d'un échantillon de référence, d'un calibrateur et d'un ou plusieurs gènes de ménage (appelés également gènes de référence). A titre d'exemples de gène de ménage, on peut citer les gènes ACTB, DECRI, GAPDH, GLYR1, HPRT1, PPIB, PPIA, RANBP3, RPLPO, TBP et 18S. In one embodiment of the present invention, the measurement of the level of expression makes it possible to determine the quantity of one or more transcripts present in the sample tested or to give a value derived therefrom. A value derived from the quantity can for example be the absolute concentration, calculated using a calibration curve obtained from successive dilutions of a solution of amplicons of known concentration. It can also correspond to the value of the standardized and calibrated quantity, such as the CNRQ. (Calibrated Normalized Relative Quantity, (Hellemans et al (2007), Genome biology 8(2):R19)), which incorporates the values of a reference sample, a calibrator and one or more housekeeping genes ( also called reference genes). As examples of a housekeeping gene, mention may be made of the ACTB, DECRI, GAPDH, GLYR1, HPRT1, PPIB, PPIA, RANBP3, RPLPO, TBP and 18S genes.
De préférence, dans le procédé tel que décrit précédemment, dans tous ses modes de réalisation, l'expression du(des) biomarqueur(s) est normalisée par rapport à l'expression d'un ou plusieurs des gènes de ménage suivants : DECRI, HPRT1 et TBP ; en particulier, on peut utiliser pour la normalisation la moyenne géométrique des 3 gènes DECRI, HPRT1 et TBP. Preferably, in the method as described previously, in all its embodiments, the expression of the biomarker(s) is normalized with respect to the expression of one or more of the following housekeeping genes: DECRI, HPRT1 and TBP; in particular, the geometric mean of the 3 genes DECRI, HPRT1 and TBP can be used for normalization.
Dans le procédé tel que décrit précédemment, l'expression des biomarqueurs peut également mesurée au niveau protéique. Les techniques permettant un telle mesure de l'expression protéique sont bien connues de l'homme du métier. A titre d'exemples, on peut citer les dosages par immunoessais, tels qu'ELISA (Enzyme Linked ImmunoSorbent Assay) et ELFA (Enzyme Linked Fluorescent Assay), par ECL (Electrochimiluminescence) et les dosages par spectrométrie de masse. In the method as described previously, the expression of the biomarkers can also be measured at the protein level. The techniques allowing such measurement of protein expression are well known to those skilled in the art. As examples, mention may be made of assays by immunoassays, such as ELISA (Enzyme Linked ImmunoSorbent Assay) and ELFA (Enzyme Linked Fluorescent Assay), by ECL (electrochemiluminescence) and assays by mass spectrometry.
De préférence, le procédé tel que décrit précédemment, dans tous ses modes de réalisation, peut comprendre également une étape de mesure de l'expression, à partir d'un échantillon sanguin contrôle sans stimulation (c'est-à-dire l'échantillon sanguin incubé dans les mêmes conditions que l'échantillon sanguin stimulé, mais en l'absence de stimulus), du(des) même(s) biomarqueur(s) que celui(ceux) mesuré(s) à partir de l'échantillon sanguin stimulé. De préférence encore, le procédé comprend une étape de calcul des ratios de l'expression (préférentiellement, l'expression normalisée) de chaque biomarqueur dans l'échantillon sanguin stimulé, par rapport à l'expression (préférentiellement, l'expression normalisée), du même biomarqueur dans l'échantillon sanguin contrôle. De manière encore plus préférée, le procédé comprend une étape de transformation des ratios obtenus par une transformation logarithmique de base 10, et éventuellement des étapes de transformation en variables centrées réduites. Preferably, the method as described above, in all its embodiments, can also comprise a step of measuring the expression, from a control blood sample without stimulation (that is to say the sample blood incubated under the same conditions as the stimulated blood sample, but in the absence of stimulus), of the same biomarker(s) as that (those) measured from the blood sample stimulated. Preferably again, the method comprises a step of calculating the ratios of the expression (preferably, the normalized expression) of each biomarker in the stimulated blood sample, relative to the expression (preferably, the normalized expression), of the same biomarker in the control blood sample. Even more preferably, the method comprises a step of transforming the ratios obtained by a base 10 logarithmic transformation, and possibly steps of transforming into reduced centered variables.
L'invention a également pour objet un kit comprenant : au moins un stimulus choisi dans le groupe constitué par le MPLA et les dérivés de MPLA, en particulier les dérivés de MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et un ou plusieurs moyen(s) d'amplification (de préférence, des amorces) et/ou un ou plusieurs moyen(s) de détection (de préférence, des sondes, des sondes DNA ou PNA, des anticorps ou des analogues d'anticorps tels que des aptamères) d'au moins un, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,A subject of the invention is also a kit comprising: at least one stimulus chosen from the group consisting of MPLA and MPLA derivatives, in particular MPLA derivatives comprising a single phosphate group on a glucosamine unit, and one or more (s) amplification (preferably primers) and / or one or more detection means (preferably probes, DNA or PNA probes, antibodies or antibody analogs such as aptamers ) of at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 biomarqueur(s) (et de préférence, d'au plus 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 biomarker(s) (and preferably no more than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 99, 98, 97, 96, 95, 94, 93, 92 , 91, 90, 89, 88, 87, 86,
85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65,85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65,
64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44,64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44,
43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,
22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 biomarqueurs) ; le(s)dit(s) biomarqueur(s) pouvant en particulier être choisi(s) dans le groupe constitué par : ADGRE3, AHNAK, ARL14EP, BPGM, BST2, 03, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, POLR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020 ; de préférence dans le groupe constitué par: ADGRE3, AHNAK, 03, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116; de préférence encore dans le groupe constitué par : CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2 ; de préférence également dans le groupe constitué par : ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA. Ledit kit peut comprendre également des moyens d'amplification et/ou de détection d'un ou plusieurs gènes de ménage, et/ou des moyens de contrôle positif permettant de qualifier la qualité de l'extraction de l'ARN, la qualité de tout procédé d'amplification et /ou d'hybridation. 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 biomarkers); the said biomarker(s) possibly being selected from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, 03, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA- DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, POLR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020; preferably from the group consisting of: ADGRE3, AHNAK, 03, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/ RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116; more preferably from the group consisting of: CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2; preferably also from the group consisting of: ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA. Said kit can also comprise means for amplifying and/or detecting one or more housekeeping genes, and/or positive control means making it possible to qualify the quality of the extraction of the RNA, the quality of any amplification and/or hybridization process.
On entend par « amorce » ou « amorce d'amplification », un fragment nucléotidique pouvant être constitué de 5 à 100 nucléotides, de préférence de 15 à 30 nucléotides, et possédant une spécificité d'hybridation avec une séquence nucléotidique cible, dans des conditions déterminées pour l'initiation d'une polymérisation enzymatique, par exemple dans une réaction d'amplification enzymatique de la séquence nucléotidique cible. Généralement, on utilise des « couples d'amorces », constitués de deux amorces. Lorsque l'on souhaite réaliser l'amplification de plusieurs biomarqueurs (e.g des gènes) différents, plusieurs couples d'amorces différents sont de préférence utilisés, ayant préférentiellement chacun une capacité à s'hybrider spécifiquement avec un biomarqueur différent. The term “primer” or “amplification primer” is understood to mean a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably of 15 to 30 nucleotides, and possessing a specificity of hybridization with a target nucleotide sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence. Generally, “pairs of primers” are used, consisting of two primers. When it is desired to carry out the amplification of several different biomarkers (e.g. genes), several different pairs of primers are preferably used, each preferably having a capacity to hybridize specifically with a different biomarker.
On entend par « sonde » ou « sonde d'hybridation », un fragment nucléotidique constitué typiquement de 5 à 100 nucléotides, de préférence de 15 à 90 nucléotides, de manière encore plus préférée de 15 à 35 nucléotides, possédant une spécificité d'hybridation dans des conditions déterminées pour former un complexe d'hybridation avec une séquence nucléotidique cible. La sonde comporte également un rapporteur (tel qu'un fluorophore, une enzyme ou tout autre système de détection), qui va permettre la détection de la séquence nucléotidique cible. Dans la présente invention, la séquence nucléotidique cible peut être une séquence nucléotidique comprise dans un ARN messager (ARNm) ou une séquence nucléotidique comprise dans un ADN complémentaire (ADNc) obtenu par transcription inverse dudit ARNm. Lorsque l'on souhaite cibler plusieurs biomarqueurs (e.g. des gènes) différents, plusieurs sondes différentes sont de préférence utilisées, ayant préférentiellement chacune une capacité à s'hybrider spécifiquement avec un biomarqueur différent. Par « hybridation », on entend le processus au cours duquel, dans des conditions appropriées, deux fragments nucléotidiques, tels que par exemple une sonde d'hybridation et un fragment nucléotidique cible, ayant des séquences suffisamment complémentaires, sont susceptibles de former un double brin avec des liaisons hydrogènes stables et spécifiques. Un fragment nucléotidique "capable de s'hybrider" avec un polynucléotide est un fragment pouvant s'hybrider avec ledit polynucléotide dans des conditions d'hybridation, qui peuvent être déterminées dans chaque cas de façon connue. Les conditions d'hybridation sont déterminées par la stringence, c'est-à- dire la rigueur des conditions opératoires. L'hybridation est d'autant plus spécifique qu'elle est effectuée à plus forte stringence. La stringence est définie notamment en fonction de la composition en bases d'un duplex sonde/cible, ainsi que par le degré de mésappariement entre deux acides nucléiques. La stringence peut également être fonction des paramètres de la réaction, tels que la concentration et le type d'espèces ioniques présentes dans la solution d'hybridation, la nature et la concentration d'agents dénaturants et/ou la température d'hybridation. La stringence des conditions dans lesquelles une réaction d'hybridation doit être réalisée dépendra principalement des sondes d'hybridation utilisées. Toutes ces données sont bien connues et les conditions appropriées peuvent être déterminées par l'homme du métier. En général, selon la longueur des sondes d'hybridation utilisées, la température pour la réaction d'hybridation est comprise entre environ 20 et 70°C, en particulier entre 35 et 65°C dans une solution saline à une concentration d'environ 0,5 à 1 M. On réalise ensuite une étape de détection de la réaction d'hybridation. The term “probe” or “hybridization probe” is understood to mean a nucleotide fragment typically consisting of 5 to 100 nucleotides, preferably of 15 to 90 nucleotides, even more preferably of 15 to 35 nucleotides, possessing a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence. The probe also comprises a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence. In the present invention, the target nucleotide sequence can be a nucleotide sequence included in a messenger RNA (mRNA) or a nucleotide sequence included in a complementary DNA (cDNA) obtained by reverse transcription of said mRNA. When it is desired to target several different biomarkers (eg genes), several different probes are preferably used, each preferably having a capacity to hybridize specifically with a different biomarker. By "hybridization" is meant the process during which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, having sufficiently complementary sequences, are capable of forming a double strand with stable and specific hydrogen bonds. A nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner. The hybridization conditions are determined by the stringency, that is to say the rigor of the operating conditions. The hybridization is all the more specific as it is carried out at higher stringency. Stringency is defined in particular according to the base composition of a probe/target duplex, as well as by the degree of mismatch between two nucleic acids. The stringency can also be a function of the reaction parameters, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and/or the hybridization temperature. The stringency of the conditions under which a hybridization reaction must be carried out will mainly depend on the hybridization probes used. All of these data are well known and the appropriate conditions can be determined by those skilled in the art. In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70°C, in particular between 35 and 65°C in a saline solution at a concentration of about 0 .5 to 1 M. A step of detecting the hybridization reaction is then carried out.
Par « réaction d'amplification enzymatique », on entend un processus générant de multiples copies d'un fragment nucléotidique cible, par l'action d'au moins une enzyme. De telles réactions d'amplification sont bien connues de l'homme du métier et on peut citer notamment les techniques suivantes : PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), RCR (Repair Chain Reaction), 3SR (Self Sustained Séquence Replication) avec la demande de brevet WO-A-90/06995, NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification) avec le brevet US-A-5,399,491, et LAMP (Loop mediated isothermal amplification) avec le brevet US6410278. Lorsque la réaction d'amplification enzymatique est une PCR, on parlera plus particulièrement de RT-PCR (RT pour « reverse transcription »), lorsque l'étape d'amplification est précédée d'une étape de réverse-transcription d'ARN messager (ARNm) en ADN complémentaire (ADNc), et de qPCR ou RT-qPCR lorsque la PCR est quantitative. By “enzymatic amplification reaction”, is meant a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme. Such amplification reactions are well known to those skilled in the art and the following techniques may be mentioned in particular: PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), RCR (Repair Chain Reaction), 3SR (Self Sustained Sequence Replication) with patent application WO-A-90/06995, NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification) with the patent US-A-5,399,491, and LAMP (Loop mediated isothermal amplification) with patent US6410278. When the enzymatic amplification reaction is a PCR, we will speak more particularly of RT-PCR (RT for "reverse transcription"), when the amplification step is preceded by a step of reverse-transcription of messenger RNA ( mRNA) to complementary DNA (cDNA), and qPCR or RT-qPCR when the PCR is quantitative.
L'invention a également pour objet l'utilisation d'un kit tel que décrit précédemment, dans tous ses modes de réalisation, pour classifier un individu, de préférence un patient, dans un groupe associé à un diagnostic, un pronostic et/ou une capacité de réponse à un traitement, ou encore pour déterminer l'activité du système immunitaire d'un individu. The invention also relates to the use of a kit as described previously, in all its embodiments, to classify an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or a ability to respond to treatment, or to determine the activity of an individual's immune system.
L'invention a également pour objet l'utilisation du MPLA ou un de ses dérivés tels que définis précédemment, dans un test fonctionnel immunitaire, pour déterminer l'activité du système immunitaire d'un individu à partir d'un échantillon sanguin dudit individu. A subject of the invention is also the use of MPLA or one of its derivatives as defined previously, in an immune functional test, to determine the activity of the immune system of an individual from a blood sample of said individual.
L'invention a également pour objet l'utilisation du MPLA ou un de ses dérivés tels que définis précédemment dans un ou plusieurs échantillons sanguins, de préférence au sein d'un test fonctionnel immunitaire, en tant que stimulus du système immunitaire pour stratifier un groupe d'individus malades, notamment des individus en état septique ou choc septique, selon l'activité dudit système immunitaire. A subject of the invention is also the use of MPLA or one of its derivatives as defined above in one or more blood samples, preferably within an immune functional test, as a stimulus of the immune system to stratify a group sick individuals, in particular individuals in a septic state or septic shock, depending on the activity of said immune system.
Figures tricks
[Fig. 1] : Structure chimique d'un MPLA synthétique, comportant 6 chaînes d'acides gras à 14 atomes de carbone. [Fig. 1]: Chemical structure of a synthetic MPLA, comprising 6 chains of fatty acids with 14 carbon atoms.
[Fig. 2] : Carte thermique (heat-map) après modulation par stimulation au LPS (trait fin) ou au MPLA (trait épais), basée sur l'expression de 93 biomarqueurs, chez 10 volontaires sains (en ordonnée, L pour LPS et M pour MPLA). [Fig. 3] : Carte thermique (heat-map) après modulation par stimulation au LPS (trait fin) ou au MPLA (trait épais), basée sur l'expression de 93 biomarqueurs, chez 20 patients ayant un sepsis (en ordonnée, L pour LPS et M pour MPLA). [Fig. 2]: Heat map (heat-map) after modulation by stimulation with LPS (thin line) or MPLA (thick line), based on the expression of 93 biomarkers, in 10 healthy volunteers (on the ordinate, L for LPS and M for MPLA). [Fig. 3]: Heat-map after modulation by stimulation with LPS (thin line) or MPLA (thick line), based on the expression of 93 biomarkers, in 20 patients with sepsis (on the ordinate, L for LPS and M for MPLA).
[Fig. 4] : Concentration de TNFa (pg/mL) mesurée après stimulation par MPLA chez des volontaires sains et des patients ayant un sepsis (* p <0.05). [Fig. 4]: TNFα concentration (pg/mL) measured after MPLA stimulation in healthy volunteers and patients with sepsis (* p <0.05).
[Fig. 5] : Analyse de regroupement réalisée sur des volontaires sains et des patients ayant un sepsis, après stimulation par LPS (haut) ou stimulation par MPLA (bas). [Fig. 5]: Pooling analysis performed on healthy volunteers and patients with sepsis, after LPS stimulation (top) or MPLA stimulation (bottom).
La présente invention est illustrée de manière non limitative par les exemples suivants. The present invention is illustrated without limitation by the following examples.
Exemples Examples
Matériels et Méthodes Materials and methods
Individus (patients et volontaires sains) testés Individuals (patients and healthy volunteers) tested
Entre octobre 2018 et janvier 2019, 20 patients ayant un sepsis (selon la définition Sepsis-3) et un dysfonctionnement respiratoire (défini par un ratio PaO2/FiO2 inférieur à 200), ainsi que 10 volontaires sains (VS ou HV, « healthy volunteers »), ont été recrutés de manière prospective. Between October 2018 and January 2019, 20 patients with sepsis (according to the Sepsis-3 definition) and respiratory dysfunction (defined by a PaO2/FiO2 ratio of less than 200), as well as 10 healthy volunteers (VS or HV, “healthy volunteers ”), were recruited prospectively.
Mesure de mHLA-DR par cytométrie en flux Measurement of mHLA-DR by flow cytometry
L'expression monocytaire de HLA-DR des monocytes circulants (mHLA-DR) des patients a été évaluée le jour de l'inclusion à l'étude, sur du sang total périphérique prélevé dans des tubes anticoagulants EDTA, par cytométrie en flux (NAVIOS; Beckman-Coulter, Brea, CA, USA). Les résultats sont exprimés en nombre d'anticorps liés par cellule (Ab/C), le seuil d'immunocompétence étant à 15000 Ab/C (Dôcke et al. (2005), Clin. Chem. 51(12):2341-7). The monocyte expression of HLA-DR of circulating monocytes (mHLA-DR) of the patients was evaluated on the day of inclusion in the study, on peripheral whole blood collected in EDTA anticoagulant tubes, by flow cytometry (NAVIOS Beckman-Coulter, Brea, CA, USA). The results are expressed as the number of antibodies bound per cell (Ab/C), the immunocompetence threshold being at 15,000 Ab/C (Dôcke et al. (2005), Clin. Chem. 51(12):2341-7 ).
Test fonctionnel immunitaire Immune functional test
Stimulation TruCulture Du sang total hépariné (1 ml) de patients, prélevé le jour de l'inclusion dans l'étude, et de volontaires sains (VS) a été distribué dans des tubes TruCulture préchauffés (Myriad Rbm, Austin, TX, USA) contenant le milieu seul (NUL), le milieu avec du LPS (100 ng/mL; Invivogen, San Diego, CA, USA) ou le milieu avec du MPLA (100 ng/mL; Invivogen, San Diego, CA, USA - voir Figure 1). Ces tubes ont ensuite été insérés dans un incubateur à blocs secs et maintenus à 37°C pendant 24 heures. Après l'incubation, le culot cellulaire a été remis en suspension dans 2 ml de TRI Reagent® LS (Sigma-Aldrich, Deisenhofen, Allemagne), agité au vortex pendant 2 minutes et reposé pendant 10 minutes à température ambiante avant un stockage à -80°C. TruCulture Stimulation Heparinized whole blood (1 ml) from patients, collected on the day of study inclusion, and from healthy volunteers (VS) was dispensed into pre-warmed TruCulture tubes (Myriad Rbm, Austin, TX, USA) containing the medium alone (NUL), medium with LPS (100 ng/mL; Invivogen, San Diego, CA, USA) or medium with MPLA (100 ng/mL; Invivogen, San Diego, CA, USA - see Figure 1 ). These tubes were then inserted into a dry block incubator and maintained at 37°C for 24 hours. After incubation, the cell pellet was resuspended in 2 mL of TRI Reagent® LS (Sigma-Aldrich, Deisenhofen, Germany), vortexed for 2 minutes, and allowed to stand for 10 minutes at room temperature before storage at - 80°C.
Analyse de l'expression des biomarqueurs Analysis of biomarker expression
Après extraction de l'ARNm sur colonne (kit Macherey-Nagel), l'expression a été évaluée à l'aide d'un panel de 93 biomarqueurs, et 3 gènes de ménage (voir Tableau 1) à l'aide la technologie Nanostring. Le traitement et la normalisation des données ont été réalisés avec le logiciel d'analyse nSolver (version 4.0, technologies NanoString) comme décrit précédemment (Mouton et al. (2020), Clin. Immunol. 210, 108312). After extraction of the mRNA on a column (Macherey-Nagel kit), the expression was evaluated using a panel of 93 biomarkers, and 3 housekeeping genes (see Table 1) using Nanostring technology . Data processing and normalization was performed with nSolver analysis software (version 4.0, NanoString technologies) as previously described (Mouton et al. (2020), Clin. Immunol. 210, 108312).
[Tableau 1]
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
[Table 1]
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Tableau 1. Panel moléculaire de 93 biomarqueurs et 3 gènes de ménage, utilisé pour évaluer la réponse aux stimulations LPS et MPLA sur du sang total de volontaire sain et de patient ayant un sepsis avec la technologie Nanostring. . Génération de données normalisées Table 1. Molecular panel of 93 biomarkers and 3 housekeeping genes, used to evaluate the response to LPS and MPLA stimulation on whole blood from healthy volunteers and patients with sepsis with Nanostring technology. . Generation of normalized data
Chaque échantillon a été analysé dans une réaction multiplexée distincte comportant chacune, 8 sondes négatives et 6 concentrations en série de sondes témoins positives. Une analyse de témoins négatifs a été réalisée pour déterminer le bruit de fond pour chaque échantillon. Each sample was analyzed in a separate multiplexed reaction each comprising 8 negative probes and 6 serial concentrations of positive control probes. Negative control analysis was performed to determine background for each sample.
Les données ont été importées dans le logiciel d'analyse nSolver (version 4.0, NanoString technologies) pour le contrôle de qualité et la normalisation des données. Une première étape de normalisation utilisant des témoins positifs internes a permis de corriger la source de variation potentielle associée à la plate-forme technique. Pour ce faire, nous avons calculé pour tous les échantillons le niveau du bruit de fond moyen comme étant la médiane +3 écarts-types de l'ensemble des six sondes négatives. Chaque échantillon sous le niveau de bruit de fond a été fixé à cette valeur. Data were imported into nSolver analysis software (version 4.0, NanoString technologies) for quality control and data normalization. A first standardization step using internal positive controls corrected for the potential source of variation associated with the technical platform. To do this, we calculated for all the samples the level of the average background noise as being the median +3 standard deviations of all the six negative probes. Each sample below the background noise level was set to this value.
Ensuite, nous avons calculé pour chaque échantillon la moyenne géométrique des sondes positives. Un facteur d'échelle pour un échantillon était un ratio de la moyenne géométrique de l'échantillon et de la moyenne de toutes les moyennes géométriques. Pour chaque échantillon, nous avons divisé toutes les valeurs des biomarqueurs par le facteur d'échelle correspondant. Then, we calculated for each sample the geometric mean of the positive probes. A scale factor for a sample was a ratio of the geometric mean of the sample and the mean of all the geometric means. For each sample, we divided all biomarker values by the corresponding scale factor.
Enfin, pour normaliser les différences de quantité d'ARN introduit, nous avons utilisé le même procédé que celui dans la normalisation par des témoins positifs, sauf que les moyennes géométriques ont été calculées pour les trois gènes de ménage DECRI, HPRT1 et TBP. Finally, to normalize for differences in the amount of RNA introduced, we used the same method as in normalization by positive controls, except that geometric means were calculated for the three housekeeping genes DECRI, HPRT1 and TBP.
Afin de comparer l'effet de la stimulation, les résultats sont exprimés en dénombrements (analyse au sein d'une population) et en ratio (rapport entre la condition de stimulation (LPS ou MPLA) avec la condition témoin (NUL), afin de comparer les populations compétentes et immunosupprimées. In order to compare the effect of the stimulation, the results are expressed in counts (analysis within a population) and in ratio (ratio between the stimulation condition (LPS or MPLA) with the control condition (NUL), in order to compare competent and immunosuppressed populations.
Détection de protéines Protein detection
La protéine TNFa chez les volontaires sains (VS) et chez les patients ayant un sepsis a été quantifiée dans le surnageant TruCulture en utilisant le système nanofluidique ELLA (Biotechne, Minneapolis, Ml, USA), selon les instructions du fabricant. Les résultats sont exprimés en pg/ml. TNFα protein in healthy volunteers (VS) and in patients with sepsis was quantified in the TruCulture supernatant using the ELLA nanofluidic system (Biotechne, Minneapolis, ML, USA), according to the manufacturer's instructions. The results are expressed in pg/ml.
Analyses statistiques Les nombres et la fréquence ont été utilisés pour les données qualitatives, et les médianes et l'IQ.R (intervalle interquartile : [Q.1-Q.3]) pour les données quantitatives. Les variables qualitatives ont été comparées en utilisant le test du Chi carré (ou le test exact de Fisher pour les petits nombres attendus). La distribution des données quantitatives a été comparée en utilisant le test t de Student (ou le test t de Mann-Whitney lorsque la distribution n'était pas normale ou le test de Welch lorsque l'homoscédasticité était rejetée) si 2 groupes étaient comparés. Si plus de 2 groupes, la distribution des données quantitatives a été comparée en utilisant le test d'Anova (ou le test de Kruskal-Wallis lorsque la distribution n'était pas normale ou lorsque l'homoscédasticité était rejetée). Le test de Spearman a été utilisé pour l'analyse de corrélation. Des analyses statistiques ont été réalisées à l'aide du logiciel GraphPad Prism® (version 6; logiciel GraphPad, La Jolla, CA, USA) et R (version 3.5.1). Une valeur de p<0,05 a été considérée comme statistiquement significative. L'analyse en composantes principales (ACP) a été réalisée à l'aide de Genomics Suite 7 (Partek, St. Louis, MO, USA). Statistical analyzes Counts and frequency were used for qualitative data, and medians and IQ.R (interquartile range: [Q.1-Q.3]) for quantitative data. Qualitative variables were compared using the Chi-square test (or Fisher's exact test for expected small numbers). The distribution of quantitative data was compared using Student's t-test (or Mann-Whitney t-test when the distribution was not normal or Welch's test when homoscedasticity was rejected) if 2 groups were compared. If more than 2 groups, the distribution of quantitative data was compared using the Anova test (or the Kruskal-Wallis test when the distribution was not normal or when homoscedasticity was rejected). Spearman's test was used for correlation analysis. Statistical analyzes were performed using GraphPad Prism® software (version 6; GraphPad software, La Jolla, CA, USA) and R (version 3.5.1). A value of p<0.05 was considered statistically significant. Principal component analysis (PCA) was performed using Genomics Suite 7 (Partek, St. Louis, MO, USA).
Construction d'un cluster Building a cluster
Les données ont été transformées en loglO, centrées et mises à l'échelle. Deux matrices de distances et une matrice de corrélation ont été construites sur les données et 10 méthodes de regroupement ont été lancées ("hiérarchique", "k-means", "diana", "fanny", "som", "model", "sota", " pam "," clara "et" agnes "). Pour chaque méthode, k=2 à k=6 clusters ont été testés. Les meilleures méthodes de clusters ont été sélectionnées à l'aide de 7 indices combinant des mesures internes (connectivité, largeur de silhouette et indice Dunn) et de stabilité (la proportion moyenne de non- chevauchement (APN), la distance moyenne (AD), la distance moyenne entre les moyennes (ADM ) et la figure de mérite (FOM)). Data were log10 transformed, centered and scaled. Two distance matrices and a correlation matrix were built on the data and 10 clustering methods were launched ("hierarchical", "k-means", "diana", "fanny", "som", "model", "sota", "pam", "clara" and "agnes"). For each method, k=2 to k=6 clusters were tested. The best clustering methods were selected using 7 indices combining internal measures (connectivity, silhouette width and Dunn's index) and stability (the average proportion of non-overlapping (APN), the average distance (AD) , the average distance between the means (ADM ) and the figure of merit (FOM)).
Résultats Results
Comparaison des réponses aux stimulations par LPS et MPLA chez des volontaires sains et des patients ayant un sepsis Nous avons observé que le MPLA et le LPS induisaient une réponse immunitaire quantitativement distincte chez des volontaires sains (Figure 2), mettant en évidence que la nature du stimulus représente le principal facteur de variabilité dans la réponse observée et que pour chacune des stimulations, le groupe des volontaires sains a une réponse homogène. Au contraire, la réponse des patients ayant un sepsis, après stimulation par LPS et MPLA, s'organise de façon différente (Figure 3). En effet, la réponse à chacune des deux stimulations est regroupée pour chaque patient, soulignant que la physiopathologie représenterait ici le principal facteur de variabilité, et non la nature du stimulus. Comparison of responses to LPS and MPLA stimulation in healthy volunteers and patients with sepsis We observed that MPLA and LPS induced a quantitatively distinct immune response in healthy volunteers (Figure 2), highlighting that the nature of the stimulus represents the main factor of variability in the response observed and that for each of the stimulations, the group of healthy volunteers has a homogeneous response. On the contrary, the response of patients with sepsis, after stimulation by LPS and MPLA, is organized differently (Figure 3). Indeed, the response to each of the two stimuli is grouped for each patient, emphasizing that the physiopathology would represent here the main factor of variability, and not the nature of the stimulus.
Dans la population de volontaires sains, 64/93 (69%) biomarqueurs étaient différentiellement exprimés après une stimulation par LPS (53 étaient sur-exprimés et 11 étaient sous-exprimés) et 55/93 (59%) étaient différentiellement exprimés après une stimulation par MPLA (51 étaient sur-exprimés et 4 sous-exprimés) par rapport à la condition de contrôle. Parmi ces biomarqueurs différentiellement exprimés, 46 biomarqueurs étaient communs aux deux stimulations (44 sur-exprimés et 2 sous- exprimés). In the population of healthy volunteers, 64/93 (69%) biomarkers were differentially expressed after LPS stimulation (53 were over-expressed and 11 were under-expressed) and 55/93 (59%) were differentially expressed after stimulation. by MPLA (51 were over-expressed and 4 under-expressed) compared to the control condition. Among these differentially expressed biomarkers, 46 biomarkers were common to both stimulations (44 over-expressed and 2 under-expressed).
Dans la population de patients ayant un sepsis, 18/93 (19%) biomarqueurs étaient différentiellement exprimés après une stimulation par LPS (tous sur-exprimés) et 22/93 (24%) étaient différentiellement exprimés après une stimulation par MPLA (20 surexprimés et 2 sous-exprimés) par rapport à la condition de contrôle. Parmi ces biomarqueurs, 12 biomarqueurs régulés différentiellement étaient communs aux deux stimulations, tous sur-exprimés (cf. Tableau 2). In the population of patients with sepsis, 18/93 (19%) biomarkers were differentially expressed after LPS stimulation (all over-expressed) and 22/93 (24%) were differentially expressed after MPLA stimulation (20 over-expressed and 2 underexpressed) compared to the control condition. Among these biomarkers, 12 differentially regulated biomarkers were common to both stimulations, all over-expressed (see Table 2).
[Tableau 2]
Figure imgf000028_0001
Figure imgf000029_0001
[Table 2]
Figure imgf000028_0001
Figure imgf000029_0001
Tableau 2. Biomarqueurs différentiellement exprimés suite aux stimulations par le LPS ou le MPLA chez les volontaires sains et les patients ayant un sepsis (le nombre de biomarqueurs sur-exprimés est précisé entre parenthèses). Table 2. Biomarkers differentially expressed following stimulation with LPS or MPLA in healthy volunteers and patients with sepsis (the number of over-expressed biomarkers is specified in parentheses).
Au niveau de la modulation des biomarqueurs, 38 des 93 biomarqueurs (soit 41%) ont été trouvés différentiellement exprimés entre les deux stimulations chez les volontaires sains. Parmi ces 38 biomarqueurs, 29 biomarqueurs se retrouvent plus fortement exprimés suite à une stimulation par LPS que suite à une stimulation par MPLA, et 9 biomarqueurs se retrouvent plus fortement exprimés suite à une stimulation par MPLA que suite à une stimulation par LPS. Chez les patients ayant un sepsis, 10 biomarqueurs sur 93 (soit 11%) sont différemment modulés entre le LPS et le MPLA. Parmi ces 10 biomarqueurs, 5 biomarqueurs ont un différentiel d'expression plus élevé suite à une stimulation par LPS que suite à une stimulation par MPLA (indiqué LPS dans la colonne LPS/MPLA du Tableau 3 ci-dessous), et 5 biomarqueurs (ADGRE3, CD74, HLA.DPA1, HLA.DMB et HLA.DRA) ont un différentiel d'expression plus élevé suite à une stimulation par MPLA que suite à une stimulation par LPS (indiqué MPLA). At the level of biomarker modulation, 38 of the 93 biomarkers (i.e. 41%) were found to be differentially expressed between the two stimulations in healthy volunteers. Among these 38 biomarkers, 29 biomarkers are found to be more strongly expressed following stimulation by LPS than following stimulation by MPLA, and 9 biomarkers are found to be more strongly expressed following stimulation by MPLA than following stimulation by LPS. In patients with sepsis, 10 biomarkers out of 93 (i.e. 11%) are differently modulated between LPS and MPLA. Among these 10 biomarkers, 5 biomarkers have a higher expression differential following stimulation by LPS than following stimulation by MPLA (indicated LPS in the LPS/MPLA column of Table 3 below), and 5 biomarkers (ADGRE3 , CD74, HLA.DPA1, HLA.DMB and HLA.DRA) have a higher expression differential following MPLA stimulation than following LPS stimulation (indicated MPLA).
Ainsi, par rapport au total des biomarqueurs différentiellement exprimés pour la population de volontaires sains, les patients septiques ont une perte plus élevée de biomarqueurs différentiellement exprimés lors de la stimulation par LPS (72%, valeur p <0,001) par rapport à la stimulation par MPLA (60%, valeur p <0,01) (cf. Tableau 4). Le fait que la perte des biomarqueurs impliqués dans la voie de signalisation post MPLA soit moins importante que dans celle du LPS, indique que la réponse au MPLA chez les patients septiques est moins altérée (par rapport aux volontaires sains) et plus informative que la réponse au LPS. Thus, compared to the total differentially expressed biomarkers for the healthy volunteer population, septic patients have a higher loss of differentially expressed biomarkers during LPS stimulation (72%, p-value <0.001) compared to LPS stimulation. MPLA (60%, p-value <0.01) (see Table 4). The fact that the loss of biomarkers involved in the post MPLA signaling pathway is less important than in that of LPS, indicates that the response to MPLA in septic patients is less altered (compared to healthy volunteers) and more informative than the response at LPS.
[Tableau 3]
Figure imgf000029_0002
Figure imgf000030_0001
Figure imgf000031_0001
[Table 3]
Figure imgf000029_0002
Figure imgf000030_0001
Figure imgf000031_0001
Tableau 3. Biomarqueurs différentiellement exprimés post-stimulation par LPS ou MPLA, chez des volontaires sains et des patients ayant un sepsis. Table 3. Biomarkers differentially expressed post-stimulation by LPS or MPLA, in healthy volunteers and patients with sepsis.
[Tableau 4]
Figure imgf000031_0002
[Table 4]
Figure imgf000031_0002
Tableau 4. Comparaison entre l'expression plus fortement induite suite à une stimulation par LPS ou par MPLA. Table 4. Comparison between more strongly induced expression following LPS or MPLA stimulation.
Au niveau protéique, la stimulation par MPLA est capable de révéler des altérations immunitaires chez les patients ayant un sepsis. Le TNFa sécrété post stimulation ex vivo du sang total par MPLA révèle une induction de la protéine plus faible chez les patients ayant un sepsis, par rapport aux volontaires sains (valeur p <0,05) (Figure 4). At the protein level, MPLA stimulation is able to reveal immune alterations in patients with sepsis. The TNFα secreted after ex vivo stimulation of whole blood by MPLA reveals a lower protein induction in patients with sepsis, compared to healthy volunteers (p value <0.05) (Figure 4).
Potentiel de stratification du LPS et du MPLA Stratification potential of LPS and MPLA
En prenant en considération les deux populations, nous avons effectué un regroupement non supervisé avec l'ensemble du panel moléculaire afin d'identifier des profils de biomarqueurs, en profitant de la réponse hétérogène obtenue suite aux deux stimulations chez les patients ayant un sepsis. La méthode la plus stable pour le LPS était la méthode hiérarchique utilisant la corrélation et le regroupement à liaison unique (indice de score = 46), et la méthode la plus stable pour MPLA était la méthode k-moyennes (k-means) ou partitionnement en k-moyennes utilisant la corrélation et la distance de Ward (indice de score = 24). Considering the two populations, we performed an unsupervised grouping with the entire molecular panel in order to identify biomarker profiles, taking advantage of the heterogeneous response obtained following the two stimulations in patients with sepsis. The most stable method for LPS was the hierarchical method using correlation and single-link clustering (score index = 46), and the most stable method for MPLA was the k-means (k-means) or partitioning method. in k-means using correlation and Ward's distance (score index = 24).
Les volontaires sains ont été regroupés en un seul groupe après stimulation par LPS et MPLA, montrant une grande homogénéité dans la réponse immunitaire de cette population et une réponse clairement différente par rapport aux patients ayant un sepsis (Figure 5). Après stimulation par LPS, les patients ayant un sepsis constituaient principalement un cluster (n=18, cluster L2) à l'exception de deux patients (n=2, cluster L3). Après stimulation par MPLA, trois groupes de patients ont été identifiés, un groupe ne contenant que 3 patients (n=3, cluster M2) et le reste des patients ont été séparés en deux groupes de nombre presque égal (n=8, cluster M3 et n=9, cluster M4). La composition de chaque cluster est présentée dans le Tableau 5. Healthy volunteers were grouped into a single group after stimulation with LPS and MPLA, showing great homogeneity in the immune response of this population and a clearly different response compared to patients with a sepsis (Figure 5). After LPS stimulation, patients with sepsis mainly formed a cluster (n=18, L2 cluster) except for two patients (n=2, L3 cluster). After stimulation with MPLA, three groups of patients were identified, one group containing only 3 patients (n=3, cluster M2) and the rest of the patients were separated into two groups of almost equal number (n=8, cluster M3 and n=9, cluster M4). The composition of each cluster is shown in Table 5.
[Tableau 5]
Figure imgf000032_0001
[Table 5]
Figure imgf000032_0001
Tableau 5. Composition des clusters obtenus après stimulation par LPS (lignes) et MPLA (colonnes). Les patients non-survivants sont barrés et les patients ayant eu une infection nosocomiale sont soulignées. VS : Volontaires sains, G : Patients ayant sepsis. Table 5. Composition of the clusters obtained after stimulation with LPS (rows) and MPLA (columns). Non-surviving patients are crossed out and patients with nosocomial infection are underlined. VS: Healthy volunteers, G: Patients with sepsis.
Une analyse bivariée a ensuite été réalisée entre les clusters et les paramètres biologiques ou cliniques. A bivariate analysis was then carried out between the clusters and the biological or clinical parameters.
Pour la stimulation par LPS, le cluster L1 était composé uniquement de volontaires sains et le cluster L3 ne comprenait que 2 individus, excluant l'analyse statistique des paramètres cliniques. Il semble que suite à la stimulation par LPS, des altérations profondes et communes soient observées dans le bras innée de l'immunité des patients septiques, ce qui n'a pas permis de séparer davantage les patients sur la base de ces derniers. For LPS stimulation, the L1 cluster was composed of only healthy volunteers and the L3 cluster consisted of only 2 individuals, excluding statistical analysis of clinical parameters. It seems that following LPS stimulation, profound and common alterations are observed in the innate arm of patients' immunity. septic, which did not allow further segregation of patients on the basis of these.
Pour la stimulation par MPLA, le cluster Ml n'était composé que de volontaires sains et le cluster M2 regroupait 3 patients de faible mHLA-DR (1864, 2560 et 5427 Ab/C, respectivement), avec divers niveaux de protéine TNFa post stimulation LPS (229, 73 et 1010 pg/mL, respectivement). En ce qui concerne les paramètres cliniques, les individus (patients) du groupe M2 présentaient à l'admission un score SOFA de 13, 14 et 7 et un lactate de 2,5, 3,6 et 1 mmol/L, respectivement. Tous étaient non-survivants à J28 et seul le patient G3 avait une infection nosocomiale avant de mourir. Enfin, les deux groupes restants d'un nombre presque égal de patients (n = 8, groupe M3 et n = 9, groupe M4) présentaient des niveaux similaires de mHLA-DR (M3; médiane 3938 Ab/C [3114-5062] et M4; médiane 3370 Ab/C [2297-4908]) mais supérieurs à ceux des patients du cluster M2. For MPLA stimulation, the M1 cluster consisted of only healthy volunteers and the M2 cluster consisted of 3 low mHLA-DR patients (1864, 2560, and 5427 Ab/C, respectively), with varying levels of post-stimulation TNFα protein. LPS (229, 73 and 1010 pg/mL, respectively). Regarding the clinical parameters, the individuals (patients) of the M2 group presented at admission a SOFA score of 13, 14 and 7 and a lactate of 2.5, 3.6 and 1 mmol/L, respectively. All were non-survivors at D28 and only patient G3 had a nosocomial infection before dying. Finally, the remaining two groups of nearly equal number of patients (n=8, M3 group and n=9, M4 group) had similar mHLA-DR levels (M3; median 3938 Ab/C [3114-5062] and M4; median 3370 Ab/C [2297-4908]) but higher than those of patients in the M2 cluster.
Le cluster M4 présentait le taux le plus élevé de non-survivants à J28 (n = 7), par rapport au cluster M3 (n = 3) et au cluster M2 (n = 3). Les patients du cluster M4 avaient un lactate statistiquement plus élevé à l'admission (médiane 2,3 mmol/L, IQ.R: [2, 3-6,4], Tableau 6) par rapport à ceux du groupe M3 (médiane 1,6 mmol/L, I Q.R: [1,2-1, 8]; valeur de p = 0,05) ainsi qu'un indice de comorbidité Charlson plus élevé (médiane: 8 et IQ.R: [7-9] vs médiane: 5,5 et IQ.R: [5-6]; valeur p <0,05), indiquant un phénotype plus sévère pour les patients regroupés dans le cluster M4. Il convient de noter que les marqueurs conventionnels comme la CRP ou la PCT n'étaient pas discriminants entre les deux groupes (valeur p> 0,05). The M4 cluster had the highest rate of non-survivors at D28 (n = 7), compared to the M3 cluster (n = 3) and the M2 cluster (n = 3). Patients in the M4 cluster had a statistically higher lactate on admission (median 2.3 mmol/L, IQ.R: [2.3-6.4], Table 6) compared to those in the M3 group (median 1.6 mmol/L, I Q.R: [1.2-1.8]; p-value = 0.05) as well as a higher Charlson comorbidity index (median: 8 and IQ.R: [7- 9] vs median: 5.5 and IQ.R: [5-6]; p value <0.05), indicating a more severe phenotype for patients grouped in the M4 cluster. It should be noted that conventional markers like CRP or PCT were not discriminating between the two groups (p value > 0.05).
[Tableau 6]
Figure imgf000033_0001
Figure imgf000034_0001
[Table 6]
Figure imgf000033_0001
Figure imgf000034_0001
SOFA: sequential organ failure assessment, CCI: Charlson Comorbidity Index ; H LA-DR: human leukocyte antigen DR ;SOFA: sequential organ failure assessment, CCI: Charlson Comorbidity Index; H LA-DR: human leukocyte antigen DR;
TNFa: tumor necrosis factor alpha; LPS: lipopolysaccharide ; IQR: Interguartile range TNFα: tumor necrosis factor alpha; LPS: lipopolysaccharide; IQR: Interguartile range
Tableau 6. Analyses bivariées entre les clusters M3 et M4 lors de la stimulation par MPLA et des paramètres cliniques et biologiques. Une analyse statistique a été effectuée entre les clusters M3 (n = 8) et M4 (n = 9) définis à l'aide de la méthode k- moyennes utilisant la corrélation et la distance de Ward. Table 6. Bivariate analyzes between M3 and M4 clusters during MPLA stimulation and clinical and biological parameters. A statistical analysis was performed between the M3 (n = 8) and M4 (n = 9) clusters defined using the k-means method using correlation and Ward's distance.
Dans le Tableau 7 ci-dessous, sont représentés les biomarqueurs différentiellement exprimés entre les clusters M3 et M4. Table 7 below shows the biomarkers differentially expressed between the M3 and M4 clusters.
[Tableau 7]
Figure imgf000034_0002
[Table 7]
Figure imgf000034_0002
Tableau 7. Biomarqueurs différentiellement exprimés entre les clusters M3 et M4Table 7. Differentially expressed biomarkers between M3 and M4 clusters
Stratification par MPLA Dans le Tableau 8 ci-dessous, figurent les biomarqueurs différentiellement exprimés entre les volontaires sains et les patients ayant un sepsis suite à une stimulation MPLA. Stratification by MPLA Table 8 below shows the biomarkers differentially expressed between healthy volunteers and patients with sepsis following MPLA stimulation.
[Tableau 8]
Figure imgf000035_0001
Figure imgf000036_0001
[Table 8]
Figure imgf000035_0001
Figure imgf000036_0001
Tableau 8. Biomarqueurs différentiellement exprimés entre les volontaires sains et les patients ayant un sepsis suite à une stimulation MPLA. Table 8. Biomarkers differentially expressed between healthy volunteers and patients with sepsis following MPLA stimulation.

Claims

36 REVENDICATIONS 36 CLAIMS
1. Procédé in vitro ou ex vivo de classification d'un individu, de préférence un patient, dans un groupe associé à un diagnostic, un pronostic et/ou une capacité de réponse à un traitement, comprenant : a) une étape d'incubation d'un échantillon sanguin dudit individu avec un stimulus, ledit stimulus étant choisi dans le groupe constitué par le Monophosphoryl lipid A (MPLA) et les dérivés de MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et b) une étape de mesure de l'expression, à partir de l'échantillon sanguin stimulé résultant de l'étape a), d'au moins un biomarqueur. 1. In vitro or ex vivo method for classifying an individual, preferably a patient, into a group associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment, comprising: a) an incubation step of a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives comprising a single phosphate group on a glucosamine unit, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least one biomarker.
2. Procédé selon la revendication 1, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, POLR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, SOCS1, SOCS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020. 2. Method according to claim 1, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, PCGF5, PDCD10, POLR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, SOCS1, SOCS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV01166, 7043-6 MALR1020.
3. Procédé selon la revendication 1 ou 2, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, SOCS1, SOCS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116. 37 3. Method according to claim 1 or 2, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, SOCS1, SOCS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116. 37
4. Procédé selon l'une des revendications 1 à 3, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2. 4. Method according to one of claims 1 to 3, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: CD83, CXCL2/MIP2A, ILIA is measured , IRAK2, NFKB1, NFKBIA, POU2F2.
5. Procédé selon la revendication 1 ou 2, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA. 5. Method according to claim 1 or 2, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: ADGRE3, CD74, HLA-DMB, HLA- DPA1, HLA-DRA.
6. Procédé selon l'une des revendications précédentes, caractérisé en ce que l'échantillon sanguin est un échantillon de sang total. 6. Method according to one of the preceding claims, characterized in that the blood sample is a whole blood sample.
7. Procédé selon l'une des revendications précédentes, caractérisé en ce que le stimulus est le MPLA ou un de ses dérivés comprenant un seul groupe phosphate sur une unité glucosamine. 7. Method according to one of the preceding claims, characterized in that the stimulus is MPLA or one of its derivatives comprising a single phosphate group on a glucosamine unit.
8. Procédé selon l'une des revendications précédentes, caractérisé en ce que, dans l'étape b), l'expression est mesurée au niveau ARN messager (ARNm). 8. Method according to one of the preceding claims, characterized in that, in step b), the expression is measured at the messenger RNA (mRNA) level.
9. Procédé selon l'une des revendications précédentes, caractérisé en ce que, dans l'étape b), l'expression est mesurée par RT-PCR, de préférence par RT-qPCR, par séquençage ou par hybridation. 9. Method according to one of the preceding claims, characterized in that, in step b), the expression is measured by RT-PCR, preferably by RT-qPCR, by sequencing or by hybridization.
10. Procédé selon l'une des revendications précédentes, caractérisé en ce que l'expression mesurée à l'étape b) est normalisée par rapport à l'expression d'un ou plusieurs gènes de ménage. 10. Method according to one of the preceding claims, characterized in that the expression measured in step b) is normalized with respect to the expression of one or more housekeeping genes.
11. Procédé selon l'une des revendications 1 à 7, caractérisé en ce que, dans l'étape b), l'expression est mesurée au niveau protéique. 11. Method according to one of claims 1 to 7, characterized in that, in step b), the expression is measured at the protein level.
12. Procédé selon l'une des revendications 1 à 7 et 11, caractérisé en ce que, dans l'étape b), l'expression est mesurée par ELISA, ELFA, ECL ou spectrométrie de masse. 12. Method according to one of claims 1 to 7 and 11, characterized in that, in step b), the expression is measured by ELISA, ELFA, ECL or mass spectrometry.
13. Procédé selon l'une des revendications précédentes, caractérisé en ce qu'il comprend en outre une étape de mesure de l'expression, à partir d'un échantillon sanguin contrôle sans stimulation, du(des) même(s) biomarqueur(s) que celui(ceux) mesuré(s) à l'étape b), à partir de l'échantillon sanguin stimulé. 13. Method according to one of the preceding claims, characterized in that it further comprises a step of measuring the expression, from a control blood sample without stimulation, of the same biomarker(s) ( s) than that (those) measured in step b), from the stimulated blood sample.
14. Procédé selon la revendication 13, caractérisé en ce qu'il comprend une étape de calcul des ratios de l'expression, de préférence l'expression normalisée, de chaque biomarqueur dans l'échantillon sanguin stimulé, par rapport à l'expression, de préférence l'expression normalisée, du même biomarqueur dans l'échantillon sanguin contrôle. 14. Method according to claim 13, characterized in that it comprises a step of calculating the ratios of the expression, preferably the normalized expression, of each biomarker in the stimulated blood sample, relative to the expression, preferably the normalized expression of the same biomarker in the control blood sample.
15. Kit comprenant : un stimulus choisi dans le groupe constitué par le Monophosphoryl lipid A (MPLA) et les dérivés de MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et un ou plusieurs moyen(s) d'amplification et/ou un ou plusieurs moyen(s) de détection d'au moins un biomarqueur. 15. Kit comprising: a stimulus chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives comprising a single phosphate group on a glucosamine unit, and one or more amplification means and/or a or more means(s) for detecting at least one biomarker.
16. Kit selon la revendication 15, caractérisé en ce que ledit au moins un biomarqueur est choisi parmi les biomarqueurs listés dans la revendication 2, de préférence parmi ceux de la revendication 3, de préférence encore parmi ceux de la revendication 4, ou de préférence également parmi ceux de la revendication 5. 16. Kit according to claim 15, characterized in that said at least one biomarker is chosen from the biomarkers listed in claim 2, preferably from those of claim 3, more preferably from those of claim 4, or preferably also among those of claim 5.
17. Utilisation d'un kit selon la revendication 15 ou 16, pour classifier un individu, de préférence un patient, dans un groupe associé à un diagnostic, un pronostic et/ou une capacité de réponse à un traitement. 17. Use of a kit according to claim 15 or 16, to classify an individual, preferably a patient, in a group associated with a diagnosis, a prognosis and/or a capacity to respond to a treatment.
18. Procédé in vitro ou ex vivo de détermination de l'activité du système immunitaire d'un individu, ledit procédé comprenant : a) une étape d'incubation d'un échantillon sanguin dudit individu avec un stimulus, ledit stimulus étant choisi dans le groupe constitué par le Monophosphoryl lipid A (MPLA) et les dérivés de MPLA comprenant un seul groupe phosphate sur une unité glucosamine, et b) une étape de mesure de l'expression, à partir de l'échantillon sanguin stimulé résultant de l'étape a), d'au moins un biomarqueur choisi dans le groupe constitué par : ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA- DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, 0AS1, 0AS2, PCGF5, PDCD10, P0LR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020. 18. In vitro or ex vivo method for determining the activity of the immune system of an individual, said method comprising: a) a step of incubating a blood sample of said individual with a stimulus, said stimulus being chosen from the group consisting of Monophosphoryl lipid A (MPLA) and MPLA derivatives comprising a single phosphate group on a glucosamine unit, and b) a step for measuring the expression, from the stimulated blood sample resulting from step a), at least one biomarker chosen from the group consisting of: ADGRE3, AHNAK, ARL14EP, BPGM, BST2, C3, CCL2, CCL20, CCL4, CCL8, CCNB1IP1, CCR1, CD209/DC-SIGN, CD3D, CD44, CD74, CD83, CDKN1A, CLEC7A/DECTIN1, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, DYRK2, EIF2AK4, FAM89A, GADD45A, HAVCR2/TIM3, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DRA, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFITM1, IFNG, IL10, IL18, IL18R1, ILIA, IL1B, IL1R2, IL2, IL6, IL7R, IRAK2, IRF3, IRF7, JAK2, LILRB1, MDC1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, 0AS1, 0AS2, PCGF5, PDCD10, P0LR2A, POU2F2, PTGS2, PTX3, RARRES3, RELB, RPL19, RPLPO, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TBX21, TDRD9, TGFB1, TMEM173/STING, TNFA, TNFAIP3, TNFSF10, TNFSF13B, ZAP70, ZBP1, ZBTB16, 121601901-HERV0116, 043166701-MALR1020.
19. Procédé selon la revendication 18, caractérisé en ce que l'individu est un patient en état septique, de préférence en choc septique. 19. Method according to claim 18, characterized in that the individual is a patient in a septic state, preferably in septic shock.
20. Procédé selon la revendication 18 ou 19, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116. 20. Method according to claim 18 or 19, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: ADGRE3, AHNAK, C3, CCL2, CCL20, CCL4, CCL8, CCR1, CD209/DC-SIGN, CD44, CD83, CDKN1A, CX3CR1, CXCL10/IP10, CXCL2/MIP2A, CXCL9, DDX58/RIG1, HAVCR2/TIM3, HLA-DPA1, IDO1, IFI27, IFI35, IFI44L, IFIH1, IFNG, IL10, IL18, ILIA, IL1B, IL6, IRAK2, IRF7, LILRB1, MERTK, MX1, NFKB1, NFKB2, NFKBIA, NFKBIZ, OAS1, OAS2, POU2F2, PTX3, RELB, S100A9, SLAMF7, S0CS1, S0CS3, SRC, STAT2, TAP2, TNFA, TNFSF10, TNFSF13B, 121601901-HERV0116.
21. Procédé selon l'une des revendications 18 à 20, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : CD83, CXCL2/MIP2A, ILIA, IRAK2, NFKB1, NFKBIA, POU2F2. 21. Method according to one of claims 18 to 20, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: CD83, CXCL2/MIP2A, ILIA is measured , IRAK2, NFKB1, NFKBIA, POU2F2.
22. Procédé selon l'une des revendications 18 à 20, caractérisé en ce que, dans l'étape b), on mesure l'expression d'au moins un biomarqueur choisi dans le groupe constitué par : ADGRE3, CD74, HLA-DMB, HLA-DPA1, HLA-DRA. 22. Method according to one of claims 18 to 20, characterized in that, in step b), the expression of at least one biomarker chosen from the group consisting of: ADGRE3, CD74, HLA-DMB is measured , HLA-DPA1, HLA-DRA.
23. Utilisation du MPLA ou un de ses dérivés comprenant un seul groupe phosphate sur une unité glucosamine dans un ou plusieurs échantillons sanguins, de préférence au sein d'un test fonctionnel immunitaire, en tant que stimulus du système immunitaire pour stratifier un groupe d'individus malades, de préférence des individus en état septique ou choc septique, selon l'activité dudit système immunitaire. 23. Use of MPLA or a derivative thereof comprising a single phosphate group on a glucosamine unit in one or more blood samples, preferably within an immune functional test, as an immune system stimulus to stratify a group of sick individuals, preferably individuals in a septic state or septic shock, depending on the activity of said immune system.
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