Classifications

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  • 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
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6818—Sequencing of polypeptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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  • 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
  • C12Q1/6813—Hybridisation assays
• • 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
  • C12Q1/6844—Nucleic acid amplification reactions
• • 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

Definitions

• the present invention relates to an in vitro or ex vivo method for determining the ability of an individual to respond to a stimulus, based on the measurement of the expression of at least two different biomarkers, chosen from different lists among three lists of. biomarkers, from a blood sample of said individual, incubated with said stimulus, as well as tools allowing the implementation of this method and the use of these tools.
• the immune system is the body's defense system against what is recognized as non-self, such as pathogens.
• the immune response requires very fine regulation and can sometimes be impaired, especially in the case of inflammatory, allergic or autoimmune diseases (in which the immune system is more active than normal), or diseases characterized by immunosuppression (in which the immune system is less active than normal).
• This immunosuppression can have different origins, take many forms, and affect innate immunity and / or adaptive immunity.
• sepsis was recognized as a health priority by the WHO in 2017, and represents a global problem in terms of morbidity, mortality, as well as costs. It is estimated that 31.5 million people develop sepsis each year around the world, of which 6 million will die of the pathology and 3 million will suffer from disorders that may lead to readmission to hospital. In a patient with sepsis (also known as sepsis), the immune response is deregulated, following infection, leading to multiple organ failure and dysfunction and potentially fatal. This immune response is complex and evolves over time, with excessive pro-inflammatory and anti-inflammatory phenomena which may be concomitant. All of these disorders of the immune system lead to organ failure, paralysis immune system and secondary infections.
• Septic shock is a subtype of sepsis, in which hypotension persists despite adequate blood supply. At the initial stage of sepsis, it is an inflammatory response, even hyper-inflammatory (including cytokine shock), which seems to predominate, and which is the cause of tissue damage and organ failure, particularly in the kidney. This is why clinical trials in the field of sepsis have long focused on anti-inflammatory treatments, but with inconclusive results. More recent studies on the pathophysiology of sepsis have shown that an anti-inflammatory or immunosuppressive response occurs in patients with sepsis, either concomitantly with the initial inflammation, or later, in an attempt to compensate for the hyper-inflammatory response.
• the patient can then find himself in a state of immunosuppression, potentially severe, depending on the respective degrees of pro-inflammatory and anti-inflammatory responses.
• These immunocompromised patients present a high risk of developing nosocomial infections (or HAI, Hospital-Acquired Infections or Healthcare-Associated Infections) and of being subject to viral reactivation, and could advantageously benefit from immunostimulatory treatments.
• HAI Hospital-Acquired Infections or Healthcare-Associated Infections
• early studies in patients with septic shock showed no benefit with such treatments. This may be due to the complexity of the pathophysiology of sepsis (including the inter-individual variability of the immune response), but also to the dynamics of the host response.
• HLA-DR human leucocyte antigen - D related
• CD88 CD88 in neutrophils
• Functional tests directly measure, ex vivo, the ability of one or more cell population (s) to respond to a stimulus with which cells are placed in contact, and have for example been used in research to study the monocyte anergy, most often by measuring TNF ⁇ at the protein level after ex vivo stimulation with lipopolysaccharide (LPS), as well as clinically, in the case of tuberculosis, by measuring interferon g at the protein level after a stimulation with Mycobacteria tuberculosis antigen.
• LPS lipopolysaccharide
• the functional test according to the invention makes it possible in particular to demonstrate three categories of individuals: individuals exhibiting an unaltered to slightly altered immune profile (cluster SI), individuals exhibiting a strongly altered immune profile (cluster S2) and individuals with an intermediate immune profile (cluster S3).
• cluster SI an unaltered to slightly altered immune profile
• cluster S2 individuals exhibiting a strongly altered immune profile
• cluster S3 individuals with an intermediate immune profile
• the individuals of cluster S2 whose immunity appears to be strongly impaired and with a greater probability of mortality, could advantageously benefit from more "aggressive" and / or earlier therapeutic interventions, while the standard of care would be sufficient for individuals.
• the SI cluster whose immunity is little altered; in individuals of cluster S3, whose immunity appears to be restorable, personalized treatments (eg IL-7, interferon g) could advantageously be tested.
• the present invention relates to an in vitro or ex vivo method for determining the capacity of an individual to respond to a stimulus, preferably for determining the capacity of the immune system of an individual to respond to a stimulus, comprising: a) a step of incubating a blood sample of said individual with said stimulus, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least two different biomarkers, chosen respectively from at least two different lists, from the following lists:
• the term “individual” designates a human being, whoever he is (and in particular whatever his state of health, whether it is a healthy individual or a sick individual).
• the term “patient” designates an individual who has come into contact with a health professional, such as a doctor (for example, a general practitioner) or a medical structure (for example, a hospital, and more particularly the department of emergency, 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 (such as, for example, an elderly person coming to be vaccinated);
• the “stimulus” corresponds to one or more molecules capable of inducing an immune response and making it possible to qualitatively and / or quantitatively assess the immune response of the individual; in particular, it may be immunogen (s) (or “challenge (s)”) or molecule (s) for therapeutic purposes;
• Determining the "capacity of an individual to respond to a stimulus” can have several uses, both diagnostic (eg identifying the immune status status, which may be normal status, inflammation status, or immunosuppression status) than prognostic (eg identify individuals whose immune status may change - for example, from normal to immune status). inflammation or vice versa, or individuals who will go from an immunosuppression status to an inflammation status), for example in order to adapt the therapeutic management, or even to predict and / or monitor the 'effectiveness of response to treatment;
• diagnostic eg identifying the immune status status, which may be normal status, inflammation status, or immunosuppression status
• prognostic eg identify individuals whose immune status may change - for example, from normal to immune status. inflammation or vice versa, or individuals who will go from an immunosuppression status to an inflammation status
• a "blood sample” means a whole blood sample or a cellular sample derived from blood (ie a sample obtained from blood and containing at least one type of cells, such as a sample of peripheral blood mononuclear cells or PBMCs) ;
• 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 biomarker, 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);
• the at least two different biomarkers are chosen respectively from at least two different lists, from the following lists:
• the at least two different biomarkers are chosen respectively from at least two different lists, from the following lists:
• the at least two different biomarkers are chosen respectively from at least two different lists, from the following lists:
• the method as described above is an in vitro or ex vivo method for determining the ability of an individual to respond to a stimulus, preferably for determining the ability of the immune system of an individual to respond to a stimulus, comprising: a) a step of incubating a blood sample of said individual with said stimulus, and b) a step of measuring the expression, from the stimulated blood sample resulting from step a), at least three different biomarkers, chosen respectively from:
• step b) the expression is measured, from the stimulated blood sample resulting from step a):
• At least 4 at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 , at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40 , at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 different biomarkers chosen from each of Lists S1, S2 and S3;
• At least 4 at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 , at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40 , at least 41, at least 42, at least 43, at least 44, at least 45 different biomarkers chosen from each of Lists S1-1, S2-1 and S3-1;
• the method as described above in all of its embodiments, is applied to a blood sample from a patient, preferably a patient in the hospital, more preferably a patient in the emergency department, an intensive care unit, intensive care unit or continuing care unit, even more preferably a patient with trauma (preferably severe trauma), burns (preferably severe burn), having received surgery (in particular, major surgery) or in a septic state, and very particularly preferably a patient in septic shock.
• septic patient or patient with sepsis
• septic shock is meant a subtype of sepsis, in which hypotension persists despite adequate vascular filling.
• the method as described above in all of 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 PBMCs, Peripheral Blood Mononudear 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.
• a sample of whole blood that is to say containing all the leukocytes, erythrocytes, platelets and the plasma
• a sample of whole blood such as collected by the venous route (for example by using tubes containing an anticoagulant)
• a sample of whole blood that is to say containing all the leukocytes, erythrocytes, platelets and the plasma
• collected by the venous route for example by using tubes containing an anticoagulant
• PBMCs only contain mononuclear cells
• whole blood also contains granulocytes (or polymorphs).
• semi-closed culture systems eg tubes
• these tubes can also allow the collection of the blood sample (which allows the cells to be stimulated at the time of collection), and more preferably, they allow the collection of a precise volume of blood.
• TruCulture ® tubes mention may be made of TruCulture ® tubes.
• the blood sample may have been taken at the request of the doctor, for example to know whether an individual will respond to a vaccine injection.
• the sample may also have been taken on admission or at the end of the patient's evolution; 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 D3 to D7, and in particular on D3 / 4) after the assault (ie sepsis or trauma) or after septic shock (in particularly, when the patient needs vasopressors and his lactate exceeds 2 mmol / L).
• the step of incubating the blood sample of the individual with the stimulus can be carried out at different temperatures (preferably at 37 ° C) and at different times. incubation (preferably between 1 hour and 48 hours of incubation; for example with an incubation of ih 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 when performing the test in the clinic.
• the stimulus used in the method as described above in all its embodiments, can be of different types.
• the stimulus can comprise one (or more) molecule (s) of immunogenic type.
• the method is particularly useful for determining a diagnosis (in particular concerning the immune status of the individual), a prognosis (in particular concerning the evolution of the immune status of the individual), and / or adapting the therapeutic management of said individual.
• the immunogenic-type stimulus may for example comprise one (s) molecule (s) capable of binding: at least one type of antigen-presenting cell (APC), said APC possibly being in particular a type of cell of the 'innate immunity (eg a monocyte, a macrophage, or a dendritic cell) or a type of cell of adaptive immunity (eg a B lymphocyte), on the one hand, and at least one type of adaptive immunity cell (such as a T lymphocyte), on the other hand.
• APC antigen-presenting cell
• this stimulus comprises a molecule of superantigen type or a molecule analogous to a superantigen.
• Superantigens are toxins of a protein nature, capable of stimulating a large number of T lymphocytes, through their simultaneous binding to the b chain of the variable domain (Vp) of a T cell receptor via the hypervariable region CDR4, and an MHC II molecule (major class II histocompatibility complex), present on the surface of an antigen presenting cell (APC).
• Vp variable domain
• APC antigen presenting cell
• the blood sample used in the method according to the invention contains T lymphocytes and antigen-presenting cells.
• the superantigens more particularly of interest, mention may in particular be made of the superantigens produced by the staphylococcal species and the superantigens produced by the streptococcal species.
• the stimulus comprises at least one molecule chosen from SEB (Staphylococcal Enterotoxin B) and SEA (Staphylococcal Enterotoxin A).
• bispecific antibodies capable of binding on the one hand to a T lymphocyte, and on the other hand to an antigen-presenting cell (such as, for example, antibodies capable of binding, on the one hand, to V on T lymphocytes, and, on the other hand, to an MHCII molecule or to a TLR-type receptor, on antigen-presenting cells).
• an antigen-presenting cell such as, for example, antibodies capable of binding, on the one hand, to V on T lymphocytes, and, on the other hand, to an MHCII molecule or to a TLR-type receptor, on antigen-presenting cells.
• MHCII MHCII molecule
• TLR-type receptor on antigen-presenting cells
• these antibodies being associated physically and / or chemically with one another, more preferably by coupling to polymers, by coupling on balls or by coupling between them.
• They may for example be anti-CD3 antibodies (such as Muromonab-CD3, marketed under the name Orthoclone OKT3), preferably combined with anti-CD28, anti-CD2 and / or anti-CD137 / TNFRSF9 antibodies.
• It may also be a stimulus of the imidazoquinolines type, structural analogues of a nucleoside, comprising a ring in their structure, of low molecular weight. This type of stimulus produces antiviral and antitumor effects in vivo.
• Resiquimod As an example of imidazoquinoline-type stimulus, mention may be made of Resiquimod (R848), which binds to human TLR7 and TLR8 on dendritic cells, or more generally on antigen-presenting cells, or APC (NF response -KB dependent). Direct effects on T lymphocytes have also been described (Smits et al (2008), Oncologist 13 (8): 859-875).
• the stimulus may comprise, preferably consist essentially of, more preferably still consist of, a molecule for therapeutic purposes (in particular, a drug or a drug candidate), and more preferably a molecule having an immunomodulatory effect. (in particular, a molecule having an immunostimulating or anti-inflammatory effect). Mention may be made, by way of examples, of IL-7 or interferon ⁇ .
• the method is particularly useful for predicting and / or monitoring the efficiency of response to said molecule for therapeutic purposes.
• Measuring the expression (or the level of expression) of a biomarker consists in quantifying at least one product of expression of this biomarker.
• the product of expression of a biomarker within the meaning of the invention is any biological molecule resulting from the expression of this biomarker. More particularly, the product of expression of a biomarker can be an RNA transcript.
• the term “transcribed” is understood to mean the RNAs, and in particular the messenger RNAs (mRNAs), obtained 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 is measured at the RNA or mRNA transcribed 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 carried out 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.
• RNA probes for example Kricka et al., Clinical Chemistry, 1999, No. 45 (4), p.453-458; Relier GH et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p.173-249.
• Expression of the biomarkers may especially be measured by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably RT-PCR or quantitative RT-qPCR (e.g. 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 for multiplexing (like 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 derived value therefrom.
• a value derived from the quantity may 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 integrates the values of a reference sample, a calibrator and one or more housekeeping genes (also called reference genes).
• reference genes mention may be made of the PPIB, PPIA, GLYR1, RANBP3, HPRT1, 18S, GAPDH, RPLPO and ACTB genes.
• the expression of the biomarkers is normalized relative to the expression of one or more of the following reference genes: HPRT1, DECRI and TBP; in particular, the geometric mean of the 3 genes HPRT1, DECRI and TBP can be used for normalization.
• 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 stimuli), the same biomarkers as those measured from the stimulated blood sample.
• 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 optionally steps of transforming into reduced centered variables.
• a subject of the invention is also a kit comprising means for amplifying and / or detecting (preferably primers and / or probes) of at least two different biomarkers, chosen respectively from at least two different lists from: - Lists SI, S2 and S3;
• At least 4 at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 , at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40 , at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 different biomarkers chosen from each of Lists S1, S2 and S3;
• At least 4 at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 , at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40 , at least 41, at least 42, at least 43, at least 44, at least 45 different biomarkers chosen from each of Lists S1-1, S2-1 and S3-1; - at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least
• kits being characterized in that all of the amplification and / or detection means of said kit allow the detection and / or amplification of at most 100 (preferably at most 90, preferably at most 80, preferably at most 70, preferably at most 60, preferably at most 50, preferably at most 40, preferably at most 30, preferably at most 20, preferably at most 10, preferably at most 5) biomarkers , in total.
• biomarkers preferably at most 100 (preferably at most 90, preferably at most 80, preferably at most 70, preferably at most 60, preferably at most 50, preferably at most 40, preferably at most 30, preferably at most 20, preferably at most 10, 5) biomarkers , in total.
• said kit can for example also comprise means for amplifying and / or detecting one or more housekeeping genes.
• the kit can also comprise 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.
• primer or “amplification primer” is understood to mean a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably 15 to 30 nucleotides, and having 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 are used, consisting of two primers.
• probe or “hybridization probe” is understood to mean a nucleotide fragment typically consisting of 5 to 100 nucleotides, preferably from 15 to 90 nucleotides, even more preferably from 15 to 35 nucleotides, possessing a hybridization specificity. under conditions determined 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 may 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 obtained by reverse transcription of said mRNA.
• biomarkers e.g. genes
• several different probes are preferably used, each preferably having an ability to specifically hybridize 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 stringency of the operating conditions. Hybridization is all the more specific the more it is carried out at higher stringency.
• Stringency is defined in particular as a function of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids. Stringency can also be depending on the reaction parameters, such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of denaturing agents and / or the hybridization temperature. The stringency of the conditions under which a hybridization reaction is to be carried out will depend primarily 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.
• the temperature for the hybridization reaction is between about 20 and 70 ° C, in particular between 35 and 65 ° C in 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 patent US-A-5,399,491, and LAMP (Loop mediated isothermal amplification) with the patent US6410278.
• RT-PCR reverse transcription
• MRNA messenger RNA reverse-transcription step
• cDNA complementary DNA
• amplification and / or detection means preferably primers and / or probes
• amplification and / or detection means preferably primers and / or probes of at least two different biomarkers, chosen respectively from at least two different lists from lists SI to S3 (or S1-1 to S3-1, or S1-2 to S3-2, or S1-3 to S3-3), more preferably means of amplification and / or detection of at least three different biomarkers, chosen respectively from each of the three lists SI to S3 (or S1-1 to S3-1, or S1-2 to S3-2, or S1-3 to S3- 3)), or
• kits comprising such amplification and / or detection means, preferably all of the amplification and / or detection means of said kit allow the detection and / or amplification of at most 100 ( preferably at most 90, preferably at most 80, preferably at most 70, preferably at most 60, preferably at most 50, preferably at most 40, preferably at most 30, preferably at most 20, preferably at most 10, preferably at most 5) biomarkers, in total, and optionally said kit comprises means for amplifying and / or detecting one or more household genes and / or positive control means making it possible to qualify the quality of RNA extraction, the quality of any amplification and / or hybridization process, to determine the ability of an individual to respond to a stimulus, preferably the ability of an individual's immune system to respond to a stimulus.
• FIG 1 The biomarkers contributing the most to the variance for the response of healthy individuals and patients with septic shock, following stimulation with SEB.
• PCA Principal Component Analysis
• Each individual (“donor”, D) is labeled by his number.
• the percentage of variance explained by each axis of the Principal Component (PC) is indicated, as well as the total variance.
• the position of the vector for each individual was plotted.
• the most important variables are represented graphically in (B) (representing 20% of the total weight of the variables for PCI and PC2).
• Figure 2 Multivariate dustering analysis, following stimulation with SEB.
• the dendrogram is based on the distance between individuals from the medoid of each cluster found by the PAM method.
• a higher intensity of the gray level (approaching black) on the heatmap (or heatmap) indicates a higher value of the expression ratio or fold change (stimulated sample / control sample) of the biomarkers and a lower intensity of the biomarkers.
• gray level indicates a lower value of the expression ratio or fold change (stimulated sample / control sample) of the biomarkers.
• the 10,000 Ab / c value was used as a cutoff for high and low mHLA-DR levels.
• HLA-DR human leukocyte antigen DR.
• FIG. 3 Distribution of protein TNF ⁇ secretion post-stimulation with LPS and mHLA-DR by defined clusters following stimulation with SEB.
• A the secretion of protein TNF ⁇ was measured ex vivo 24 hours post-stimulation with LPS in healthy individuals (circles) and patients with septic shock (squares)
• B mHLA-DR was measured by flow cytometry, only in patients with septic shock (squares).
• Mortality non-surviving individuals
• the clusters defined post-stimulation with SEB were obtained using the PAM method with correlation distance. ** p ⁇ 0.001; *** p ⁇ 0.0001.
• SEB staphylococcal enterotoxin B
• LPS lipopolysaccharide
• mHLA-DR monocyte human leukocyte antigen DR.
• Septic shock was defined according to the Sepsis-3 consensus of the Society for Critical Care Medicine and the European Society for Critical Care Medicine (Singer et al (2016), JAMA 315: 801-10): patients requiring administration of vasopressor and having a measurement of the serum lactate concentration greater than 2 mmol / L in the absence of hypovolemia in a patient having an infection, or suspected of having an infection (ie criteria which define the onset of shock septic in a patient with sepsis). The exclusion criteria were an age below 18 years and the presence of aplasia or a known immunosuppressive disease.
• data collected included demographic characteristics (age, sex) and site of primary infection; the initial severity was assessed by the simplified severity index (IGS II; range of values: 0-163) on admission. Information regarding death during ICU stay was collected, and the severity 24 hours after admission was assessed by the sequential failure assessment score. organs (SOFA) (range of values: 0-24). Laboratory data during follow-up was also collected, including monocyte HLA-DR values (mHLA-DR), as well as measurement of TNF ⁇ protein secretion after stimulation with LPS. At the same time, blood samples from healthy individuals (or healthy volunteers) were obtained from the national blood service (Étableau für du Sang) and used immediately.
• IGS II simplified severity index
• SOFA range of values: 0-24
• Nanostring technology was used for the detection of mRNA from a panel of 46 biomarkers (Table 3) - this is a hybridization-based multiplex assay characterized by the absence of a step of amplification; 300 ng of RNA was hybridized to the probes at 67 ° C for 18 hours using a thermal cycler (Biometra, Professional TRIO, Analytik Jena AG, Jena, Germany).
• samples were loaded into nCounter Prep Station (NanoString Technologies, Seattle, WA, USA) for purification and immobilization on the inner surface of a sample cartridge for 2-3 hours.
• the sample cartridge was then transferred and imaged on the nCounter Digital Analyzer (NanoString Technologies) where the color codes were counted and tabulated for the 46 biomarkers.
• a first standardization step using internal positive controls made it possible to correct the source of potential variation associated with the technical platform. To do this, we calculated for all samples the mean background noise level as the median +3 standard deviations of all six negative probes. Each sample below the background noise level was set at this value.
• a scale factor for a sample was a ratio of the geometric mean of the sample and the mean of all geometric means. For each sample, we divided all the gene values by the corresponding scale factor.
• HLA-DR HLA-DR on the surface of circulating monocytes (mHLA-DR) of patients was assessed on days 3-4 after the onset of septic shock, in peripheral whole blood collected in EDTA tubes, by flow cytometry. (NAVIOS; Beckman-Coulter, Brea, CA, United States). The results are expressed as the number of antibodies bound per cell (Ab / C). - Protein detection
• the TNF ⁇ protein in the supernatant of the TruCulture tubes was quantified, for patients with septic shock and healthy individuals, using the ELLA nanofluidic system (Biotechne, Minneapolis, M1, USA), according to the manufacturer's instructions. The results are expressed in pg / ml.
• Cluster S2 has the lowest median level of TNF ⁇ protein among the 3 clusters. Comparing the SI cluster to S3, there is a significant difference for the two parameters (p ⁇ 0.001), the S3 cluster exhibits an intermediate median level of TNFa protein concentration after LPS stimulation between the 3 clusters, while the levels medians of mHLA-DR are the lowest ( Figure 3).
• the immune functional test developed has therefore made it possible to demonstrate that, if the immune response of healthy individuals is homogeneous, the immune response of patients with septic shock is heterogeneous, and the heterogeneity of the response lies in the adaptive arm. of immunity.
• the patients grouped together in the SI cluster, along with healthy individuals, have a more "normal” / "healthy” immune profile, unlike the other patients. A priori these patients would not require particular vigilance and a standard ofcare would be sufficient.
• the patients in the S2 cluster correspond to “severe” patients, characterized by a high mortality rate. These patients, whose immunity appears to be strongly impaired and with a greater probability of mortality, could advantageously benefit from more “aggressive” and / or earlier therapeutic interventions.
• the third group corresponds to patients of intermediate to severe phenotype, which may present a degree of immune recovery.
• these patients whose immunity appears to be restorable could be the subject of personalized treatments (e.g. IL-7, interferon y).
• personalized treatments e.g. IL-7, interferon y.

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