Method for in vitro or ex vivo detection of an immunocompromised status in a subject

Abstract

The present invention relates to a method for in vitro or ex vivo detection of an immunocompromised status in a subject, comprising quantifying, in a biological sample of said subject, the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1R2, and identifying whether or not a variation in their expression is present as compared to a reference value. The invention also relates to tools for implementing said method, and to uses thereof.

Description

[0001] TITLE: METHOD FOR IN VITRO OR EX VIVO DETECTION OF AN IMMUNODEPRESSED STATUS IN A SUBJECT

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for in vitro or ex vivo detection of the presence or absence of an immunocompromised status in a subject comprising a step of quantifying, in a biological sample of said subject, the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2, and a step of comparing the expression of each of the genes respectively to a reference value so as to identify the presence or absence of a variation in the expression.

[0004] STATE OF THE ART

[0005] Sepsis affects nearly 50 million people worldwide each year, more than 40% of whom are children under the age of 5. It is a leading cause of death worldwide, with more than 11 million deaths each year. However, the majority of these deaths are preventable. In Europe, sepsis is estimated to be responsible for nearly 700,000 deaths each year, including nearly 57,000 deaths in France, with an average cost of approximately €16,000 per hospitalization.

[0006] Sepsis is a systemic inflammatory response to an infection (bacterial, viral, fungal, or parasitic) that is defined as an acute state of dysregulation of the body's response to this infection, resulting in loss of organ function and a risk to the patient's life. The most severe form of this condition is septic shock. Sepsis is accompanied by an exacerbated production of inflammatory mediators. For example, we speak of a "cytokine storm" to refer to the massive production of cytokines (chemical mediators that allow communication between our cells), which acutely affect the functioning of vital organs and can lead to longer-term functional sequelae. Patients with sepsis are generally admitted to intensive care, where they receive antibiotics and the necessary supports to maintain vital functions.However, for more than twenty years, despite considerable progress made in understanding the pathophysiology associated with sepsis, no new sepsis-specific therapy has emerged.

[0007] The high heterogeneity of patients has been identified as the main reason for the failure of decades of treatment strategies, including anti-inflammatory strategies, in sepsis. This underscores the crucial need for patient stratification and a more individualized approach.

[0008] Regarding the pathophysiology of sepsis, after the initial inflammation, persistent immunosuppression has been repeatedly described and found to have adverse consequences for patients (increased nosocomial infection risk, mortality risk, and length of stay) and healthcare costs. Therefore, adjuvant immunostimulation is now being considered to counterbalance this immunosuppressive response and restore patients' immune functions. However, this new approach relies on the ability to identify or enrich patient groups with the most immunosuppressed functions. Since there is no clinical sign of immunosuppression, patient stratification must be based on immune biomarkers.

[0009] Monocytes are by nature extremely "plastic" cells. Thus, throughout the progression of a disease, monocytes can alternate between being pro- and anti-inflammatory cells. The outcome of these pro- and anti-inflammatory forces acting on monocytes at a given time can be broadly reflected by the level of mHLA-DR expression on their surface.

[0010] Therefore, one of the reference tests to monitor immune alterations in patients in intensive care units (e.g. patients with sepsis, trauma, major surgery, burns, or patients with pancreatitis) is the decrease in the expression of a surface receptor belonging to the major histocompatibility complex (MHC) class II, namely HLA-DR (human leukocyte antigen-D related), on the surface of monocytes (mHLA-DR), measured by flow cytometry. Indeed, this marker provides valuable information in terms of mortality prediction or the assessment of the risk of secondary infections in these patients (Venet et al. (2018) Nat Rev Nephrol 2018;14:121-137).

[0011] Measuring mHLA-DR expression is a well-known method in the literature to identify whether a patient, for example with sepsis, is immunocompromised or not (Monneret and Venet (2016) Cytometry Part B (Clinical Cytometry) 90B:376-386). Using a standardized measurement, a clinical decision threshold has been proposed, namely an mHLA-DR of at least 5000 antibodies bound per cell (AB / C), such as 8000 AB / C, and used to identify immunocompromised patients in intensive care.

[0012] To date, mHLA-DR measurement is based on flow cytometry, a technology that cannot be implemented in all hospital sites for clinical routine due to numerous limitations. Indeed, flow cytometry has preanalytical constraints and requires skilled technicians to exploit and interpret the results. In addition, the deficiencies in terms of flow cytometer implementation in hospitals are not compensated by 24 / 7 accessibility when they are present (Monneret and Venet (2014), Grit Care 18:102). Finally, it is a time-consuming technology that is rarely used when the patient is in the intensive care unit or intensive care unit.

[0013] To overcome these drawbacks, other biomarkers, using molecular biology tools, have been proposed. For example, we can cite a biomarker based on the ratio of the expression level, at the messenger RNA (mRNA) level, of CD74 on day 3 (following the patient's admission to a medical facility) to the expression level of CD74 on day 1. CD74 represents the invariant chain y of HLA-DR. It has been shown that the CD74 expression ratio D3 / D1 is associated with the occurrence of secondary infections acquired in intensive care (Peronnet et al. (2017) Intensive Care Medicine;43(7): 1013-20).

[0014] Also proposed in document WO2013 / 156627 is a method for determining immunocompromised or non-immunocompromised status, based on the determination of the anellovirus load in a biological sample.

[0015] In parallel, a prototype multiplex molecular tool to evaluate on an automated platform the expression of a set of 16 biomarkers, including mRNAs, was implemented to identify the immune profile of patients. (Tawfik et al. (2020) JID 2020:222 (suppl 2)). However, the biomarkers are not derived from a dedicated cohort study and are therefore not sufficiently reliable to discriminate and stratify patients. In addition, the performances obtained need to be improved and no correlation is made between the expressions of the different biomarkers and an mHLA-DR value characteristic of immunocompetence.

[0016] To date, several genes have been identified as potentially linked to immune status in a subject. For example,

[0017] - the TAP2 (Antigen peptide transporter 2) gene encodes a membrane-associated protein of the ABC (ATP-binding cassette) transporter superfamily, which is involved in the transport of peptides from the cytoplasm to the endoplasmic reticulum as part of the antigen presentation process of class I molecules. The expression of the transcript of this gene has also been studied in septic patients, in whom it has been shown to identify patients with a particular endotype associated with the occurrence of death at 28 days (Scicluna et al. (2017), Lancet Respir Med 5(10): 816-826);

[0018] - the C3AR1 gene (complement component 3a receptor 1) codes for the complement receptor C3a, a protein released during complement activation. Binding of the anaphylatoxin C3a to its receptor activates chemotaxis and opsonization of bacteria. In the literature, it has been shown that the transcript of the C3AR1 gene was more highly expressed in septic patients than in healthy volunteers (Napier et al. (2016) J. Exp. Med. 213(11):2365-2382);

[0019] - the CD177 gene codes for the neutrophil membrane glycoprotein CD177, discovered in 1971 in a case of neonatal neutropenia. A study has shown an increase in the level of expression of CD177, at the mRNA and protein levels, in circulating neutrophils from patients who have had septic shock (Demaret et al. (2016), Immunol. Letters 178: 122-130);

[0020] - the IL1 R2 gene encodes an interleukin-1 receptor, specifically interleukin-1a (IL1A) and interleukin-1p (IL1B), which transmit intracellular signals via pathways shared by other receptors such as the interleukin-18 receptor (IL18), leading to the secretion of inflammatory mediating factors. The IL1 R2 receptor is described as a "decoy" receptor, which binds IL1A and IL1B and therefore prevents them from binding to their regular receptors, resulting in an inhibition of signal translation normally mediated by these interleukins. The IL1 R2 transcript was identified as differentially expressed in neutrophils from septic patients in a bioinformatics study (He et al. (2019) 35(6):481-490). IL1 R2 has been proposed as a diagnostic marker for sepsis, based on results of measuring serum IL1 R2 concentration in healthy volunteers and septic patients (Lang et al.(2017) Shock 47(1): 119-124). Chromatin immunoprecipitation analyses showed differences in chromatin (histone methylation and acetylation) of the IL1 R2 gene promoter between septic patients and healthy volunteers (Weiterer et al. (2015) PLoS One 10(3):e0121748).

[0021] Although these markers are described individually, to date, no study has demonstrated a sufficiently robust link between their combined expressions and patient immunosuppression, nor has any correlation been demonstrated between these markers and commonly used immunosuppression markers, such as HLA-DR expression on monocytes, the percentage of regulatory T cells, or the number of CD4+ T cells (Demaret et al. (2016) Immunology Letters 178:122-130). In other words, there is no alternative to flow cytometry techniques.

[0022] There therefore remains a clear need to identify a reliable (i.e., presenting acceptable sensitivity and / or specificity) and rapid means, which is also easy to read / interpret, simple to use and can be integrated into clinical routine, in order to determine the patient's immune status, in particular to identify immunosuppression.

[0023] SUMMARY OF THE INVENTION A first subject relates to a method for in vitro or ex vivo detection of an immunocompromised status in a subject, comprising the following steps: a. Quantification, in a biological sample of said subject, of the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1R2; b. Comparison of the expression of each of the genes respectively to a reference value so as to identify the presence or absence of a variation in the expression, the reference value corresponding to the expression of said target gene in a subject having a normal immune status defined by an mHLA-DR greater than 5000 antibodies bound per cell (AB / C), preferably greater than 8000 antibodies bound per cell (ABC), the immunocompromised status being detected on the basis of at least two variations in the expression, said variations being chosen from:

[0024] - underexpression of TAP2,

[0025] - overexpression of C3AR1,

[0026] - overexpression of CD177, and

[0027] - overexpression of IL1 R2.

[0028] According to a particular embodiment, step a) comprises the quantification of the expression of at least 3 target genes chosen from the group consisting of TAP2, C3AR1, CD177 and IL1 R2.

[0029] According to another particular embodiment, step a) comprises the quantification of the expression of the target gene TAP2 and of at least one other target gene chosen from C3AR1, CD177 and IL1 R2.

[0030] According to another particular embodiment, step a) comprises the quantification of the expression of the 4 target genes TAP2, C3AR1, CD177 and IL1 R2.

[0031] Advantageously, the method according to the invention further comprises a step of quantifying the expression of at least one other target gene selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9, the immunocompromised status being detected by taking into account at least one additional variation in the expression, said additional variation being chosen from

[0032] - an underexpression of CD3D,

[0033] - a subexpression of CIITA,

[0034] - an underexpression of CD74,

[0035] - underexpression of CTLA4, - underexpression of CX3CR1,

[0036] - overexpression of IFNy, and

[0037] - overexpression of S100A9,

[0038] According to another particular embodiment, the biological sample is a blood sample, preferably a whole blood sample.

[0039] According to another particular embodiment, the biological sample comes from a patient in the emergency department, the intensive care unit, in the intensive care unit (ICU) or in the continuing care unit, preferably from a patient in the ICU.

[0040] According to another particular embodiment, the expression is measured at the RNA or messenger RNA level, preferably by RT-PCR, preferably RT-qPCR, by sequencing or by hybridization.

[0041] According to a particular embodiment, the expression is normalized relative to the expression of one or more housekeeping genes selected from the group consisting of DECR1, HPRT 1, PPIB, GAPDH, and ACTB.

[0042] Another subject of the invention relates to a kit for its use in the detection of an immunocompromised status in a subject, said kit comprising means for amplification and / or means for detection of the expression of at least 2 target genes, preferably at least 3 target genes, selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2. Advantageously, the kit comprises means for amplification and / or means for detection of the expression of the 4 target genes TAP2, C3AR1, CD177 and IL1 R2.

[0043] According to a particular embodiment, the kit further comprises means for amplifying and / or means for detecting the expression of at least one other gene target selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9.

[0044] Advantageously, the amplification and / or detection means are reagents specific to the expression products of said target genes chosen from amplification primers or hybridization probes.

[0045] DETAILED DESCRIPTION OF THE INVENTION After extensive research, it is to the credit of the inventors that they were able to identify, in an unexpected and surprising manner, a signature of several genes whose deregulated expression is characteristic of an immunocompromised status.

[0046] An objective of the present invention is to identify the same immunocompromised patients, particularly in intensive care units, as those characterized by mHLA-DR expression <8,000 bound antibodies / cell (AB / C).

[0047] Thus, a first subject of the present invention relates to a method for in vitro or ex vivo detection of an immunocompromised status in a subject, comprising the following steps: a. Quantification, in a biological sample from said subject, of the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2; b. Comparison of the expression of each of the genes respectively to a reference value so as to identify the presence or absence of a variation in the expression.

[0048] The present invention therefore has various advantages, in particular the fact of allowing a generalization of the immunomonitoring of subjects, such as that of patients in intensive care, in a simple and rapid manner by means of a technology (tools and method) which can be implemented on all hospital sites, unlike flow cytometry. The present invention also makes it possible to identify immunocompromised patients, for whom it would be relevant to administer immunostimulating treatments in the context of clinical trials, and thus to demonstrate the effectiveness of such treatments. In addition, the invention opens the way to personalized medicine in patients, in particular those in intensive care.

[0049] To the inventors' knowledge, it has never been described or suggested that the demonstration of a variation in the expression of at least 2 target genes selected from the group consisting of the TAP2, C3AR1, CD177 and IL1 R2 genes makes it possible to detect, in vitro or ex vivo, an immunocompromised status in a subject. In addition, it has never been described or suggested that the result of this detection is correlated with a quantification of the expression of HLA-DR on the surface of monocytes (mHLA-DR) less than 8000 AB / C, advantageously less than 7500 AB / C, less than 7500 AB / C, preferably less than 7000 AB / C, less than 6500 AB / C, or even less than 6000 AB / C, less than 5500 AB / C or even less than 5000 AB / C, measured by flow cytometry.

[0050] The target genes whose expression is quantified in the method which is the subject of the present invention are known to those skilled in the art and the chromosomal locations are given in particular in Table 1 below.

[0051] [Table 1]

[0052] In the context of the invention, and as opposed to a normal immune status, described as immunocompetent, the expressions “immunocompromised”, “immunosuppressed”,

[0053] “Immunodeficient,” “hypoactive immune status,” or “immune paralysis” are used interchangeably and refer to a less active than normal immune system / immune response in response to an infection or inflammatory condition. Specifically, it is an immunosuppression characterized by impaired innate and adaptive immune responses, including increased apoptosis and lymphocyte dysfunction, impaired phagocytic functions, monocyte deactivation with decreased HLA class II surface expression, and impaired cytokine production.

[0054] In the context of the invention, the term "subject" designates a human being and preferably, the subject is a patient. The patient is defined as a person who has come into contact with a healthcare professional, in particular a doctor, a medical facility or a healthcare establishment.

[0055] According to a particular embodiment, the subject is a patient within a healthcare establishment, preferably within a hospital, more preferably within the emergency department, the intensive care department, in an intensive care unit (ICU) or in a continuing care unit, and in particular, the subject is a patient in an ICU.

[0056] In the context of the invention, the expressions "biomarker" and "marker" are used interchangeably and designate an objectively measurable biological characteristic which represents an indicator of normal or pathological biological processes. It may in particular be a molecular biomarker, preferably detectable at the mRNA level. More particularly, the biomarker may be an endogenous biomarker or loci, such as a HERV or a gene.

[0057] “Sepsis” is a pathology in which the immune response is deregulated in an individual, following an infection, leading to multiple and potentially fatal organ failure and dysfunction. “Septic shock” is a subtype of sepsis, in which hypotension persists, despite adequate vascular filling. In the context of the invention, the expression “biological sample” designates any sample originating from a subject, and which may be of different natures, such as blood or its derivatives, sputum, urine, stool, skin, cerebrospinal fluid, bronchoalveolar lavage fluid, abdominal cavity puncture fluid, saliva, gastric secretions, semen, seminal fluid, tears, spinal cord, trigeminal nerve ganglion, adipose tissue, lymphoid tissue, placental tissue, gastrointestinal tract tissue, genital tract tissue, central nervous system tissue.In particular, this sample may be a biological fluid, such as a blood sample or a blood-derived sample, which may in particular be chosen from whole blood (as collected from the venous route, i.e. containing white and red cells, platelets and plasma), plasma, serum, as well as any type(s) of cells extracted from blood, such as peripheral blood mononuclear cells (or PBMC, containing lymphocytes (B, T and NK cells), dendritic cells and monocytes), B cell subpopulations, purified monocytes, or neutrophils.

[0058] In the context of the invention, the expressions “reference value”, “normal value”, “norm”, “usual value” or “reference biological sample” are used interchangeably and designate a value or an average of the values ​​of a parameter (e.g. gene expression, in particular at the mRNA level, etc.) from one or more healthy subjects, presenting a normal or immunocompetent immune status, that is to say a subject whose immune status is known not to be immunocompromised with in particular an mHLA-DR value greater than 5000 AB / C, in particular greater than 5500 AB / C, greater than 6000 AB / C, greater than 6500 AB / C, or even greater than 7000 AB / C, greater than 7500 AB / C, and very particularly, greater than 8000 AB / C, that the subject does not present an altered immune response following inflammation or sepsis, as opposed to a “tested value”, “test value” or “test biological sample”.The reference value and the test value are obtained by implementing the same detection, quantification and identification process.

[0059] In the context of the invention, the expression “variation of expression” designates an overexpression or an underexpression of the gene. In particular:

[0060] - the term "overexpression" means that the expression of a gene is increased compared to a reference value, i.e. obtained in an immunocompetent subject defined by an mHLA-DR value greater than 5000 AB / C, in particular greater than 5500 AB / C, advantageously greater than 6000 AB / C, in particular greater than 6500 AB / C, preferably greater than 7000 AB / C, in particular greater than 7500 AB / C, and very particularly by an mHLA-DR value greater than 8000 AB / C;

[0061] - the term "under-expression" means that the expression of a gene is reduced compared to a reference value, i.e. obtained in an immunocompetent subject defined by an mHLA-DR value greater than 5000 AB / C, in particular greater than 5500 AB / C, advantageously greater than 6000 AB / C, in particular greater than 6500 AB / C, preferably greater than 7000 AB / C, in particular greater than 7500 AB / C, and very particularly by an mHLA-DR value greater than 8000 AB / C.

[0062] The terms "coding" or "coding for," "code" or "codes for" are used interchangeably and refer to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers having a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is generally described in sequence listings and databases, and the non-coding strand, used as a template for transcription of a gene or cDNA, may be designated as coding for the protein or other product of that gene or cDNA.

[0063] In the context of the invention, the expression "amplification primer" designates a nucleotide fragment which may comprise from 5 to 100 nucleotides, preferably from 15 to 30 nucleotides, and which has 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, "primer pairs" are used, consisting of two primers. When it is desired to carry out the amplification of several different genes, several different primer pairs are preferably used, each preferably having a capacity to hybridize specifically with a different gene.

[0064] In the context of the invention, the expression "hybridization probe" designates a nucleotide fragment typically comprising from 5 to 100 nucleotides, preferably from 15 to 90 nucleotides, even more preferably from 15 to 35 nucleotides, having 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 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.When it is desired to target several different genes, several different probes are preferably used, each preferably having the ability to hybridize specifically with a different gene. In the context of the invention, "hybridization" means 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, i.e. the rigor of the operating conditions.The higher the stringency, the more specific the hybridization is. Stringency is defined in particular as a function of the base composition of a probe / target duplex, as well as the degree of mismatch between two nucleic acids. Stringency may also be a function of 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 must be carried out will depend mainly 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.

[0065] In the context of the invention, the expression "enzymatic amplification reaction" designates 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 cited in particular: Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Repair Chain Reaction (RCR), Self Sustained Sequence Replication (3SR) with patent application WO-A-90 / 06995, Nucleic Acid Sequence-Based Amplification (NASBA), Transcription Mediated Amplification (TMA) with patent US-A-5,399,491, and Loop mediated isothermal amplification (LAMP) with patent US6410278.When the enzymatic amplification reaction is a PCR, we will speak more specifically of RT-PCR (RT for "reverse transcription"), when the amplification step is preceded by a step of reverse transcription of messenger RNA (mRNA) into complementary DNA (cDNA), and of qPCR or RT-qPCR when the PCR is quantitative.

[0066] Preferably, the subject of the present invention is a method for in vitro or ex vivo detection of a depressed immune status in a subject, as defined above and having the following technical characteristics, taken alone or in combination:

[0067] - step a) includes the quantification of the expression of the target genes TAP2 and IL1 R2;

[0068] - step a) includes the quantification of the expression of the target genes TAP2 and C3AR1;

[0069] - step a) includes the quantification of the expression of the target genes TAP2 and CD177;

[0070] - step a) includes the quantification of the expression of the target genes C3AR1 and CD 177;

[0071] - step a) includes the quantification of the expression of the target genes C3AR1 and IL1 R2;

[0072] - step a) includes the quantification of the expression of the target genes CD177 and IL1 R2;

[0073] - step a) comprises the quantification of the expression of at least 3 target genes chosen from the group consisting of TAP2, C3AR1, CD177 and IL1 R2;

[0074] - step a) comprises quantifying the expression of the target gene TAP2 and at least one other target gene chosen from C3AR1, CD177 and IL1 R2;

[0075] - step a) comprises the quantification of the expression of the target gene TAP2 and at least two other target genes chosen from C3AR1, CD177 and IL1 R2;

[0076] - step a) includes the quantification of the expression of the 3 target genes TAP2, C3AR1 and IL1 R2;

[0077] - step a) includes the quantification of the expression of the 3 target genes TAP2, C3AR1 and CD177;

[0078] - step a) includes the quantification of the expression of the 3 target genes TAP2, CD177 and IL1 R2;

[0079] - step a) includes the quantification of the expression of the 4 target genes TAP2, C3AR1, CD177 and IL1 R2;

[0080] - the reference value corresponds to the expression of said target gene in a subject with normal immune status or a subject whose immune status is known not to be immunocompromised;

[0081] - the immunocompromised status is detected on the basis of at least two variations of the expression, said variations being chosen from an underexpression of TAP2, an overexpression of C3AR1, an overexpression of CD177, and an overexpression of IL1 R2;

[0082] - the immunocompromised status is detected on the basis of two variations in expression, said variations being chosen from underexpression of TAP2, overexpression of C3AR1, overexpression of CD177, and overexpression of IL1 R2;

[0083] - the immunocompromised status is detected on the basis of three variations in expression, said variations being chosen from underexpression of TAP2, overexpression of C3AR1, overexpression of CD177, and overexpression of IL1 R2;

[0084] - immunocompromised status is detected based on TAP2 underexpression, C3AR1 overexpression, CD177 overexpression, and IL1 R2 overexpression;

[0085] - the method of the invention further comprises a step of quantifying the expression of at least one additional target gene selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9; the immunocompromised status is then further detected by taking into account at least one variation of at least one additional target gene, said variations being chosen from an under-expression of CD3D, an under-expression of CIITA, an under-expression of CD74, an under-expression of CTLA4, an under-expression of CX3CR1, an over-expression of IFNy, and an over-expression of S100A9;

[0086] - the method of the invention comprises a step of quantifying the expression of all of the following target genes: TAP2, C3AR1, CD177, IL1 R2, CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9;

[0087] - the subject is a patient, in particular a seriously ill patient in a septic state, more particularly, in septic shock; suffering from burns, more particularly, serious burns; suffering from trauma, more particularly, serious trauma; or operated on by surgery, more particularly, major surgery;

[0088] - the biological sample is a blood sample;

[0089] - the biological sample is a whole blood sample;

[0090] - the biological sample is a sample from a patient in the emergency department, the intensive care unit, the intensive care unit (ICU) or the continuing care unit, and preferably from a patient in the ICU;

[0091] - the test biological sample and / or the reference biological sample according to the invention is a blood sample, preferably a whole blood, plasma or serum sample, or a sample of peripheral blood mononuclear cells, extracted from a blood sample;

[0092] - the reference value of a target gene corresponds to the expression of said target gene in a subject having a normal immune status defined by an mHLA-DR value greater than 5000 AB / C, in particular greater than 5500 AB / C, advantageously greater than 6000 AB / C, in particular greater than 6500 AB / C, preferably greater than 7000 AB / C, in particular greater than 7500 AB / C, and very particularly by an mHLA-DR value greater than 8000 AB / C;

[0093] - the expression of target genes is quantified at the RNA or messenger RNA (mRNA) transcript level;

[0094] - the expression of target genes is measured at the DNA or complementary DNA (cDNA) level;

[0095] - the expression of the target genes is measured by implementing a molecular detection method and / or direct quantification by any method known to those skilled in the art making it possible to determine the presence of an mRNA transcript in a sample such as hybridization methods, preferably with a hybridization chip, by in situ hybridization or by Northern blot; amplification methods, preferably by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), more preferably by quantitative RT-PCR (RT-qPCR), in particular nested PCR (or nested PCR), the PCR reactions can also be multiplexed; sequencing methods, preferably by high-throughput sequencing;

[0096] - quantification of the expression of the target genes is carried out by RT-PCR, by sequencing or by hybridization. Preferably, the quantification is carried out by RT-PCR, and in particular by RT-qPCR;

[0097] - the expression of the target genes is measured by implementing a method of indirect detection and / or quantification of the mRNA 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 mRNA transcript into DNA;

[0098] - the expression of the target genes is normalized relative to the expression of one or more housekeeping genes, advantageously relative to the expression of one of the housekeeping genes selected from the group consisting of DECR1, HPRT1, PPIB, GAPDH, ACTB and their combinations;

[0099] - the expression of target genes is normalized relative to the combination of the expression of the housekeeping genes DECR1, HPRT1 and PPIB;

[0100] - the method according to the invention makes it possible to identify the immunocompromised status in a subject with a sensitivity of at least 70%, and a specificity of at least 80%;

[0101] - the method of the invention further comprises a step of administering a treatment, preferably an immunomodulatory treatment and / or an anti-inflammatory treatment, adapted to the immune status of the individual;

[0102] - the immunostimulating treatment is chosen from the group consisting of interleukins, in particular IL-7, IL-15 or IL-3; growth factors, in particular GM-CSF; interferons, in particular IFNy, Toll agonists; antibodies, in particular anti-PD1, anti-PDL1, anti-LAG3, anti-TIM3, anti-IL-10 or anti-CTLA4 antibodies; transferrins and apoptosis inhibitory molecules; FLT3L; Thymosin a1; adrenergic antagonists;

[0103] - anti-inflammatory treatment is chosen from the group consisting of glucocorticoids, cytostatic agents, molecules acting on immunophilins and cytokines, molecules blocking the IL-1 receptor and anti-TNF treatments.

[0104] According to the invention:

[0105] - the CD3D gene (CD3 Delta Subunit Of T-Cell Receptor Complex) encodes a protein that is a constituent of the T-cell receptor / CD3 complex (TCR / CD3 complex) and is involved in T-cell development and signal transduction. The protein encoded by this gene is the delta subunit of the CD3 complex and, together with four other CD3 subunits, binds to either the alpha / beta TCR or the gamma / delta TCR to form the TCR / CD3 complex on the surface of T cells. The chromosomal location of CD3D is: chr11:118,338,954-118,342,744;

[0106] - the CIITA (Class II Major Histocompatibility Complex Transactivator) gene encodes a transactivator of the class II major histocompatibility complex. The protein, once in the cell nucleus, is thought to act as a positive co-regulator for the transcription of genes of the class II major histocompatibility complex, a major component of the immune defense. The chromosomal location of CIITA is: chr16:10,866,212- 10,943,021; - the CD74 (Invariant Polypeptide Of Major Histocompatibility Complex, Class II Antigen-Associated or HLA class II histocompatibility antigen gamma chain) gene encodes a protein that associates with the class II major histocompatibility complex (MHC) and is an important chaperone protein that regulates antigen presentation for the immune response.It also serves as a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), which, when bound to the protein encoded by the CD74 gene, initiates cell survival pathways and proliferation. This protein also interacts with amyloid precursor protein (APP) and suppresses the production of amyloid beta or p-amyloid. The chromosomal location of CD74 is: chr5:150, 400, 041-150, 412, 936;.

[0107] - the CTLA4 gene (or CTLA-4; Cytotoxic T-Lymphocyte Associated Protein 4) is part of the immunoglobulin superfamily and codes for a protein that transmits an inhibitory signal to T lymphocytes.; The chromosomal location of CTLA4 is as follows: chr2:203, 867, 771-203, 873, 965;

[0108] - the CX3CR1 gene (C-X3-C Motif Chemokine Receptor 1) encodes the fractalkine receptor (or CX3CL1). Fractalkine is a transmembrane protein and a chemokine notably involved in the adhesion and migration of leukocytes. The chromosomal location of CX3CR1 is as follows: chr12:8,058,302-8,066,471.

[0109] - the IFNy (Interferon y) gene encodes a soluble cytokine belonging to the type II interferon class. The encoded protein is secreted by cells of the innate and adaptive immune systems. The active protein is a homodimer that binds to the interferon gamma receptor, which triggers a cellular response to viral and microbial infections. The chromosomal location of IFNG is: chr12:68, 154, 768-68, 159, 741; and

[0110] - the S100A9 gene (S100 Calcium Binding Protein A9 or MRP14 for migration inhibitory factor-related protein 14) codes for a protein belonging to the S100 family of proteins containing 2 calcium-binding motifs in the EF hand (i.e. a structural domain or a helix-loop-helix motif). The chromosomal location of S100A9 is as follows: chr12:68,154,768- 68,159,741.

[0111] Another subject of the present invention relates to the use for detecting in vitro or ex vivo, an immunocompromised status in a subject, of the quantification, in a biological sample of said subject, of the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2; and the comparison of the expression of each of the genes respectively to a reference value so as to identify the presence or absence of a variation in the expression, as described previously. The use according to the invention further comprises a step of quantifying the expression of at least one other target gene selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9.Another subject of the present invention also relates to a kit for implementing the method for in vitro or ex vivo detection of a depressed immune status in a subject, as defined above, comprising means for amplification and / or means for detecting the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2.

[0112] Preferably, the subject of the present invention is a kit for implementing the method for in vitro or ex vivo detection of a depressed immune status in a subject, as defined, having the following technical characteristics, taken alone or in combination:

[0113] - said kit comprises the means of amplification and / or means of detection of the expression of at least 3 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2;

[0114] - the kit includes the means of amplification and / or means of detection of the expression of the 4 target genes TAP2, C3AR1, CD177 and IL1 R2;

[0115] - the kit further comprises means for amplifying and / or means for detecting the expression of at least one other target gene selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9 and combinations thereof;

[0116] - the kit comprises the means of amplification and / or means of detection of the expression of the following 11 target genes: TAP2, C3AR1, CD177, IL1 R2, CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9;

[0117] - the amplification and / or detection means are reagents specific to the expression products of said target genes chosen from amplification primers or hybridization probes;

[0118] - the kit further comprises a test biological sample (positive control sample) calibrated to contain the quantities of mRNA of the target genes TAP2, C3AR1, CD177 and IL1 R2 which correspond to the quantity or concentration representative of the level of expression measured in a pool of samples from a subject having an immunocompetent immune status with an mHLA-DR value greater than 5000 AB / C, in particular greater than 5500 AB / C, advantageously greater than 6000 AB / C, in particular greater than 6500 AB / C, preferably greater than 7000 AB / C, in particular greater than 7500 AB / C, and very particularly by an mHLA-DR value greater than 8000 AB / C.

[0119] The present invention also relates to the use of a kit as described above, for detecting in vitro or ex vivo a depressed immune status in a subject, preferably a patient, in particular a seriously ill patient in a septic state (more particularly, in septic shock), suffering from burns (more particularly, severe burns), suffering from trauma (more particularly, severe trauma), or operated on by surgery (more particularly, major surgery). FIGURES

[0120] [Fig. 1]: Distribution of expression levels of different markers in sample groups defined according to the immune status of the patients. The normalized expression values ​​of all genes were significantly different between immune status groups (Mann-Whitney test, p<0.001). n=1183 samples in the group with mHLA-DR >8,000 AB / C (immunocompetent) and n=658 samples in the group with mHLA-DR <8,000 AB / C (immunocompromised).

[0121] [Fig. 2]: Concordance between mHLA-DR categories and stratification by target genes of the pocket prototype across all subjects (patients and healthy; n=1300). Sample groups based on mHLA-DR value (<8000 AB / C for immunocompromised state and or 8000 AB / C for immunocompetent state) are shown on the left and stratification using the target gene set is shown on the right.

[0122] [Fig. 3]: Concordance between mHLA-DR categories and stratification by target genes of the pouch prototype across all patients (n= 541). Sample groups based on mHLA-DR value (<8,000 AB / C for immunocompromised state and or 8,000 AB / C for immunocompetent state) are shown on the left and the prediction using the target gene set is shown on the right.

[0123] EXAMPLES OF ACHIEVEMENT

[0124] Example 1: Ex vivo detection of an immunocompromised status in a subject by implementing the method of the invention

[0125] 1. Materials and methods

[0126] 1.1. Patients and samples

[0127] Patients and samples were obtained from the REALISM study, which enrolled 377 critically ill patients with various etiologies (sepsis, trauma, elective surgery, burns) and 175 healthy volunteers. Peripheral whole blood was collected in EDTA tubes and PAXgene Blood RNA tubes at various time points between study enrollment and day 60 (D60), with up to 7 samples per patient. Healthy volunteers were sampled once.

[0128] 1.2. Measurement of mHLA-DR

[0129] Determination of the number of HLA-DR molecules per monocyte was performed using the standardized BD Quantibrite method (HLA-DR:340827; Quantibrite: 340495; Becton Dickenson, New Jersey, USA) on fresh EDTA blood samples, within 3 h of collection, as previously described.

[0130] 1.3. Quantification of target gene expression

[0131] PAXgene samples were stabilized for at least 2 hours after collection at room temperature and frozen at -80°C according to the manufacturer's recommendations. RNA was then isolated using the Maxwell® HT simplyRNA Kit (AX2420, Promega Corporation, Madison, WI, USA). RNA concentration was determined using the QuantiFluor RNA System (E3310, Promega) on the GloMax® Discover Microplate Reader (Promega). RNA integrity was assessed using the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA) on the Agilent 2100 Bioanalyzer (Agilent Technologies).

[0132] Samples were tested using a prototype FilmArray® pouch optimized to detect by nested PCR the target genes TAP2, C3AR1, CD177 and IL1 R2, and the additional target genes CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9.

[0133] The determination of the mRNA expression level of the target genes was carried out with the pocket prototype on the FilmArray® Torch instrument (BioFire®, USA), following the manufacturer's recommendations, by injecting 200 ng of RNA into the said pocket prototype. The results of the expression levels are obtained automatically, in less than one hour, before being compiled for analysis. The normalized expression values ​​of the target markers (relative to the reference genes DECR1, HPRT1 and PPIB) were calculated and used for the analyses. All samples for which mHLA-DR measurements were available were used in the analysis. Samples were considered immunocompromised when mHLA-DR quantification was less than 8,000 AB / C; otherwise, these samples were considered immunocompetent. The data were divided into a training set containing 1,300 samples (70%) and a test set containing 541 samples (30%). The distribution between the training set and the test set was balanced based on the distribution of mHLA-DR values, sampling time, and patient etiology. A Partial Least Square (PLS) regression model combining the expression level of the prototype pocket markers was trained by repeated 20X-5-fold cross-validation for the prediction of mHLA-DR less than 8,000 AB / C.Synthetic Minority Over-Sampling Technique (SMOTE) was used to balance the training set across categories. The number of components was manually scaled from 1 to the maximum possible. Model performance was evaluated on the test set by calculating the area under the ROC curve (AUC), accuracy, sensitivity, specificity, positive predictive value, and negative predictive value.

[0134] Statistical analyses were performed using R software, version 3.6.2.

[0135] 2. Results

[0136] Both mHLA-DR and target gene expression data were available for 1841 samples distributed as follows: 163 samples from healthy volunteers and 1678 patient samples corresponding to 106 sepsis, 136 trauma, 109 surgeries, and 24 burns.

[0137] Overall, mHLA-DR values ​​ranged from 439 to 80,066 AB / C, with 36% of values ​​below 8,000 AB / C.

[0138] The detection of variations in the expression of the markers of the pocket prototype (target and additional genes) according to the invention was carried out.

[0139] The data are represented in Figure 1.

[0140] In the immunocompromised group, downregulation of the expression of: CD3D, CD74, CIITA, CTLA4, CX3CR1, IFNy and TAP2 was observed; while the following markers had their expression upregulated: C3AR1, CD177, IL1 R2, S100A9.

[0141] Individual normalized expression levels of the mRNA markers TAP2, C3AR1, CD177, and IL1 R2, as well as those of different combinations of the markers TAP2, C3AR1, CD177, IL1 R2, CD3D, CIITA, CD74, CTLA4, CX3AR1, IFNy, and S100A9 were combined in a model trained for the identification of immunocompromised patients (mHLA-DR <8,000 AB / C).

[0142] The performance data of the different combinations of target genes according to the invention are shown in Table 2.

[0143] [Table 2] The model performed very well in identifying patients with immunocompromised status, with an AUC greater than 0.82 for all gene sets tested.

[0144] For comparison, the performance obtained on the test set with CD74 (which represents the invariant y chain of HLA-DR) as the sole substitute for mHLA-DR in a generalized linear model was lower, with an AUC of 0.77 (95% CI [0.73-0.81]), thus demonstrating the full interest of the combinations of target genes according to the invention.

[0145] Concordance was performed between the mHLA-DR results and the identification with the target genes on the training set (n=1300 samples; Figure 2) and on the test set (n=541; Figure 3). The sample groups based on the mHLA-DR value (<8,000 AB / C for immunosuppression and or 8,000 AB / C for immunocompetence) are represented on the left and the prediction using the target gene set according to the invention is represented on the right. It appears that the concordance obtained in all samples between the mHLA-DR model and the target genes is 80%, whether on the training set or the test set.

[0146] 3. Conclusion

[0147] It clearly appears that the method of the invention for in vitro or ex vivo detection of an immunocompromised status in a subject is effective and appears to be an excellent alternative to mHLA-DR quantification, since the correlation of the data from the prototype pocket and the mHLA-DR shows a similarity in the identification of immunocompromised or immunocompetent patients.

[0148] More importantly, the method of the invention has several other advantages. For example, the pre-analytical process is facilitated by the stabilization of whole blood RNA in the PAXgene tube, requiring no sample preparation. In addition, results are obtained within one hour, without manipulation, thanks to its implementation on the fully automated platform such as the F / 7mArray® (BioFire®), already widely used in clinics for microbiological diagnosis. At a minimum, the method according to the invention thus allows early identification of a patient's immunocompromised status, for example until it is possible to perform the mHLA-DR measurement.

[0149] Despite good agreement between the data from mHLA-DR quantification and those from the method of the invention, some samples are misclassified. This could be due to the intrinsic variability of the two techniques. Another explanation could be the differences in regulation between a protein (mHLA-DR) and the expression of several mRNA markers. Furthermore, while HLA-DR is measured at the cellular level only in monocytes, the method of the invention allows quantification at the mRNA level in whole blood, including monocytes but also other cells expressing HLA-DR (e.g. B lymphocytes), and thus captures other immune alterations, not necessarily related to mHLA-DR regulation. However, this does not detract from the advantages of the method according to the invention for the identification of an immunocompromised status in a subject.

Claims

CLAIMS 1. Method for in vitro or ex vivo detection of an immunocompromised status in a subject, comprising the following steps: a. Quantification, in a biological sample of said subject, of the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1R2; b. Comparison of the expression of each of the genes respectively to a reference value so as to identify the presence or absence of a variation in the expression, the reference value corresponding to the expression of said target gene in a subject having a normal immune status defined by an mHLA-DR greater than 5000 antibodies bound per cell (AB / C), preferably greater than 8000 antibodies bound per cell (ABC), the immunocompromised status being detected on the basis of at least two variations in the expression, said variations being chosen from: - underexpression of TAP2, - overexpression of C3AR1, - overexpression of CD177, and - overexpression of IL1 R2.

2. Method according to claim 1, characterized in that step a) comprises the quantification of the expression of at least 3 target genes chosen from the group consisting of TAP2, C3AR1, CD177 and IL1 R2.

3. Method according to claim 1 or 2, characterized in that step a) comprises the quantification of the expression of the target gene TAP2 and of at least one other target gene chosen from C3AR1, CD177 and IL1 R2.

4. Method according to claim 1, characterized in that step a) comprises the quantification of the expression of the 4 target genes TAP2, C3AR1, CD177 and IL1 R2.

5. Method according to any one of claims 1 to 4, characterized in that it further comprises a step of quantifying the expression of at least one other target gene selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNy and S100A9, the immunocompromised status being detected by taking into account at least one additional variation in the expression, said additional variation being chosen from - an underexpression of CD3D, - a subexpression of CIITA, - an underexpression of CD74, - underexpression of CTLA4, - an underexpression of CX3CR1, - overexpression of IFNy, and - overexpression of S100A9, 6. Method according to any one of claims 1 to 5, characterized in that the biological sample is a blood sample, preferably a whole blood sample.

7. Method according to any one of claims 1 to 6, characterized in that the biological sample comes from a patient in the emergency department, the intensive care unit, in the intensive care unit (ICU) or in the continuing care unit, preferably from a patient in the ICU.

8. Method according to any one of claims 1 to 7, characterized in that the expression is measured at the RNA or messenger RNA level.

9. Method according to any one of claims 1 to 8, characterized in that the expression is measured by RT-PCR, preferably RT-qPCR, by sequencing or by hybridization.

10. Method according to any one of the preceding claims, characterized in that the expression is normalized relative to the expression of one or more housekeeping genes selected from the group consisting of DECR1, HPRT1, PPIB, GAPDH, and ACTB.

11. Kit for implementing the method according to any one of claims 1 to 10 comprising means for amplification and / or means for detection of the expression of at least 2 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2.

12. Kit according to claim 11, characterized in that it comprises means for amplification and / or means for detection of the expression of at least 3 target genes selected from the group consisting of TAP2, C3AR1, CD177 and IL1 R2, preferably, the means for amplification and / or means for detection of the expression target genes TAP2, C3AR1, CD177 and IL1 R2. Kit according to claim 11 or 12, characterized in that it further comprises means for amplification and / or means for detection of the expression of at least one other target gene selected from the group consisting of CD3D, CIITA, CD74, CTLA4, CX3CR1, IFNγ and S100A9. Kit according to any one of claims 11 to 13, characterized in that the amplification and / or detection means are reagents specific for the expression products of said target genes chosen from amplification primers or hybridization probes.

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