Functional assay for rapidly determining immune status

Abstract

The present invention relates to a method for rapidly determining the immune status of an individual. The method includes the steps of collecting a certain amount of whole blood sample from the individual; stimulating the whole blood sample by incubating it with a certain amount of phytohemagglutinin (PHA) at a temperature between 35°C and 39°C for at least 3 to 3.5 hours; and evaluating the level of interferon-γ production induced by this incubation / stimulation, and the evaluated level then serves as an indicator of the immune status of the individual.

Description

Technical Field

[0001] The present invention relates to the evaluation and determination of an individual's immune state. More specifically, it relates to a method and tool for measuring the overall level of an individual's cell-mediated immunity, and its principle of operation is the same as that of a functional immunoassay.

[0002] By proposing a method and a clinical tool that enable a reliable and rapid evaluation of a patient's immune state and / or enable a diagnosis of a patient's immune response dysfunction or imbalance (immunodeficiency or immune hyperactivity), the present invention is advantageously positioned as a useful aid for a clinician's decision-making.

Background Art

[0003] An individual's immune state corresponds to the functional state of that individual's immune system, i.e., the ability to protect the body from potentially dangerous factors. These defense and protection mechanisms are mainly deployed against infectious and exogenous pathogens such as microorganisms such as viruses, bacteria, fungi, and protozoa. They may also be deployed against endogenous factors, especially cells that have changed as a result of physical and / or chemical damage (which may be the case for infected cells, cancer cells, or senescent cells, etc.).

[0004] Regardless of whether it is exogenous or endogenous, the immune system's response to an attack by a potentially dangerous factor is a dynamic phenomenon, which, if properly regulated, can maintain the integrity of the organism. Conversely, when the immune response weakens, becomes insufficient, or its balance is disrupted, the body's risk of developing a disease state increases. Therefore, when the immune response weakens or becomes insufficient, opportunistic infections, the onset of sepsis, and / or the reactivation of viruses are likely to occur, and when the immune response deteriorates, it may cause the onset of allergies, autoimmune diseases (e.g., multiple sclerosis, type 1 diabetes, lupus, autoimmune thyroiditis, rheumatoid arthritis, ankylosing spondylitis, Goujerot-Sjoegren syndrome, Crohn's disease, etc.).

Summary of the Invention

[0005] Therefore, enabling the determination of a patient's immune status and / or monitoring of its changes is a major clinical challenge. In this regard, numerous examples of clinical applications can be cited, and in particular, the following can be mentioned: - Identifying patients who may be immunocompromised (chronic or acute, acquired or induced), and where appropriate, - Providing appropriate care and medical support, and / or - Preventing intolerance to live attenuated vaccines or drugs that are contraindicated in cases of immunodeficiency to enable this, - Monitoring changes in the immune status of patients receiving immunosuppressive agents, such as candidates for solid organ transplantation, newly transplanted patients, etc., thereby optimally adjusting the dosage of the immunosuppressive agent used and establishing an immune level considered appropriate to prevent the risk of graft rejection while minimizing the risks of infection, reactivation of oncogenic viruses, and inhibition of the patient's anti-tumor immunity. Monitoring, - Monitoring the reconstitution of the immune system in patients after immunosuppressive therapy to ensure that the process proceeds smoothly, - Monitoring the impact of chemotherapy on the patient's immune status, thereby enabling optional readjustment or modification of the treatment, or - Managing and monitoring the treatment of patients receiving immunotherapy (especially cell therapy such as CAR-T (chimeric antigen receptor T) and treatment based on the injection of antibodies known as anti-checkpoint antibodies).

[0006] Enabling the determination of an individual's immune status and monitoring of its changes is also of great concern to the pharmaceutical industry and basic research related to human health. In this regard, numerous application examples can be cited, and in particular, - In the context of drug development, the need to evaluate the impact on the immune system, - In the context of vaccine development, for example, evaluating the potential for polarization of the immune response towards a Th1-type and / or Th2-type response, or - Evaluating the potential effects that pathological or environmental factors may have on an individual's immune system can be mentioned.

[0007] Currently, among the methods known to be able to determine and / or evaluate an individual's immune status, in particular, lymphocyte proliferation tests (LPT) and lymphoblast transformation tests (LTT) can be mentioned first. These are designed to quantify lymphocyte proliferation after stimulation with mitogens (such as lectins like phytohemagglutinin (PHA), concanavalin A (conA), and pokeweed mitogen (PWM)) or pathogen-specific antigens. These tests are particularly time-consuming. In particular, after isolation, the mononuclear cells need to be stimulated for 3 to 7 days. Then, the cells are harvested and DNA replication or cell division is measured by flow cytometry incorporating a tracer.

[0008] The HLA-DR assay by flow cytometry can be carried out much more rapidly and can measure the expression of HLA-DR ("human leukocyte antigen-D related") on the surface of monocytes; a low expression of this marker is a sign of immune system dysfunction. Similarly, in patients suffering from septic shock, a persistently low expression level of monocyte HLA-DR is generally a sign of low survival rate.

[0009] Currently, the method for assaying this HLA-DR can only be carried out using flow cytometry, and there are few healthcare centers or medical analysis laboratories equipped with appropriate equipment. As a result, the method for determining such an immune state is more suitable for observational and exploratory studies than for clinical diagnosis. Furthermore, this method is cumbersome and difficult to implement, and normalization / standardization is also very complex; temperature, the cell storage period before labeling, and erythrocyte lysis are all factors that strongly influence the variability of the measurement and thus need to be carefully controlled (Finck et al., 2003 - “Standardisation de la mesure de l’antigene HLA-DR monocytaire par cytometrie en flux : resultat preliminaire et application dans le suivi des chocs septiques [Standardization of monocyte HLA-DR antigen measurement by flow cytometry: preliminary results and application in the monitoring of septic shock]” - Ann. Biol. Clin. 2003, 61: 441-448).

[0010] A method for determining the immune state that requires an instrument that is easier to use and not so difficult to implement measures cell activities (including one or more types of immune cells such as lymphocytes, macrophages, monocytes, dendritic cells, granulocytes, etc.) in response to a specific stimulus and is known as IFA (Immune Functional Assay). Depending on the nature of the (one or more) stimulant(s) used for this purpose, the immune level being studied is either a specific immune level, i.e., a specific immune response developed specifically against a given target pathogen, or an overall immune level that reflects the general state of the individual's immune system. In either case, the measurement of cell activities consists of assaying one or more cytokines (such as IFNγ, TNFα, interleukin, etc.) whose expression is regulated by the stimulus.

[0011] To determine a specific immune level, the (one or more) stimulant(s) used generally reproduce epitope (protein and / or glycoside) units from the target pathogen (for example, in the case of the immune state against Mycobacterium tuberculosis, all or part of the protein sequences of markers such as ESAT-6, CFP-10, and TB7.7 are often used for the stimulus). For the determination of the overall immune level, one or more "non-specific" stimulants are used. Examples include protein kinase A (PKA), phorbol myristate acetate (phorbol-12-myristate-13-acetate or PMA), PHA, conA, staphylococcal enterotoxin B (SEB), or lipopolysaccharide (LPS), and cytokines such as interleukin IL-1, IL-2, and IL-12 can also be mentioned. Anti-CD3 (or rarely anti-CD2) monoclonal antibodies are also used, such as OKT-3, regardless of the presence or absence of anti-CD28.

[0012] The present invention particularly focuses on functional immunoassays specialized in determining the overall level of cellular immunity in an individual, such as in the case of the ImmuKnow® (Cylex Inc., USA) and QuantiFERON Monitor® (Qiagen GmbH, Germany) assays, both of which are commercially available.

[0013] The ImmuKnow® assay has been proposed for immunological monitoring of patients taking immunosuppressive agents after organ transplantation and is designed to identify situations of under - dosing and over - dosing. The principle of this assay is based on assaying intracellular ATP (adenosine triphosphate) synthesized by stimulated CD4 + T lymphocytes. The level of intracellular ATP measured in this way is thought to correlate with the overall lymphocyte activity of the patient. Thus, an activity level identified as low suggests over - dosing of immunosuppressive agents and the patient's risk of infection, while an activity level identified as high suggests under - dosing of immunosuppressive agents and the risk of graft rejection.

[0014] The ImmuKnow® assay is performed by stimulating a whole - blood sample with a mitogen (PHA in this case) for 15 - 18 hours. Next, CD4 + T lymphocytes are purified, lysed, and ATP is extracted. The latter is finally quantitatively measured by bioluminescence using the luciferin / luciferase system (Stewart, 2012 - “ImmuKnow as an immune monitoring tool following organ transplantation” - Le Courrier de la Transplantation, 2012, vol. VII No. 1).

[0015] Regarding the QuantiFERON Monitor® assay, its use in predicting the risk of infection in patients who have received allogeneic hematopoietic cell transplantation is described in Douglas et al., 2020 (“The QuantiFERON Monitor® assay is predictive of infection post allogeneic hematopoietic cell transplantation” - Transplant Infectious Disease, 2020, 22(3): 1-9). For this purpose, heparinized whole blood samples are stimulated at 37°C for 16 - 24 hours using an activator composition called QFM LyoSphere™. This composition contains an R848 reagent and a TLR7 receptor agonist to stimulate the patient's innate immunity, and an anti-CD3 antibody to stimulate the acquired immunity. After 16 - 24 hours of stimulation, plasma is collected and the gamma interferon (IFNγ) content is measured. The IFNγ content serves as an indicator of the patient's immune status (in this case, an indicator of the overall level of cell-mediated immunity). In this assay, both innate and acquired immune components are considered.

[0016] Similar to the ImmuKnow® assay, the QuantiFERON Monitor® assay is particularly time-consuming to perform, and its excessive duration is mainly due to the stimulation phase, which alone takes more than 15 - 16 hours.

[0017] Since immunocompromised patients frequently require special clinical and / or therapeutic management to address their high susceptibility to infection, immunocompromise screening may be urgent in many clinical situations, such as the following: - At the time of admission to a care center, - Before surgery, - Before prescribing drugs / treatments contraindicated for immunocompromised patients, - In cases of sepsis where particularly close monitoring is required, - Therefore, it is truly necessary for clinicians to be able to access, perform quickly, and provide results in the shortest possible time for the diagnosis / prognosis assay of the immunocompromised state.

[0018] Accordingly, an object of the present invention is to propose a functional immunoassay capable of determining and evaluating a patient's immune state in a significantly shortened time compared to currently commercially available functional immunoassays.

[0019] More generally, an object of the present invention is to propose a process for determining an individual's immune state in vitro or ex vivo, the implementation of which is intended to be simple and rapid using technical equipment available to healthcare centers and medical analysis laboratories.

[0020] The present invention meets all of the above objects. Before introducing its features and characteristics in more detail, the following definitions are given for better understanding.

[0021] In the context of this description, the term "determination / evaluation of the immune state" is used to indicate the body's ability of an individual to mount an immune response to protect itself from potentially dangerous factors. In a very similar way to the "immune state", the term "immune level" can also be used interchangeably.

[0022] The immune state determined / evaluated by the present invention may be a numerical value or a category, and may be reported by a value obtained directly or indirectly from the measurement of IFNγ generated in response to a stimulus generated according to the present invention. The result given in the form of a numerical value corresponds to a discrete variable or a continuous variable representing the immune level. The result given in the form of a categorical value can be combined with modifiers such as "normal", "low", or "high" for the individual's immune state, for example. Such a display is obtained from the interpretation / extrapolation based on the level of IFNγ production measured after stimulation and / or the comparison of this level with one or more reference IFNγ expression levels.

[0023] The term "whole blood sample" means a venous blood sample obtained from a sample taken from an individual / patient and consisting essentially of red blood cells, white blood cells, platelets, and plasma. Except for the possibility of adding an anticoagulant, optional dilution, and / or the possibility of storage between 2°C and 8°C, the whole blood sample subjected directly to the process of the present invention has not been subjected to any other treatment, and in particular, has not been subjected to any pretreatment that could significantly alter its composition (with respect to components and the ratio between components).

[0024] The term "evaluating the level of IFNγ production" (in this case, the production induced by the stimulation carried out according to the present invention) does not necessarily mean accurately measuring, more or less, the amount of IFNγ actually and specifically produced / secreted in response to the stimulation. Such an evaluation actually consists of reasonable estimations of the following indicators / parameters: - The total concentration / amount of IFNγ contained in the reaction mixture [whole blood stimulation solution], or a sub-fraction of this mixture, - The amount of mRNA transcribed in response to IFNγ stimulation and can be composed of, which, except for some factors and / or approximations, give a valid account of the IFNγ production thus studied.

[0025] Finally, the term "individual" refers to a human being, regardless of their state of health. A "healthy individual" for the purposes of the present invention is an individual who clearly does not have any disorders of the immune system. The term "patient" refers to an individual who is in contact with a medical professional such as a doctor (e.g., a general practitioner), and / or a medical facility (e.g., a hospital emergency room or intensive care unit), or a medical analysis laboratory.

[0026] Accordingly, the present invention is a method for determining the immune status of an individual, comprising: - providing a certain amount of whole blood sample from said individual; - stimulating said whole blood sample by incubating it with a certain amount of phytohemagglutinin (PHA) at a temperature between 35°C and 39°C for at least 3 hours, for example, between 3 hours and 8 hours; - A step of evaluating the level of induced IFNγ production, wherein the level thus evaluated provides an indicator of the immune state of the individual, the step of evaluating relates to a method comprising.

[0027] According to a particular embodiment, the stimulation time does not exceed 8 hours, preferably does not exceed 6 hours.

Brief Description of the Drawings

[0028]

Figure 1

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Modes for Carrying Out the Invention

[0029] The inventors have thus developed a highly reliable functional immunoassay that can give results particularly in a short time. Specifically, contrary to expectations, the inventors have succeeded in significantly shortening the duration of the stimulation step. All of this has basically been made possible by the following discoveries and demonstrations: 1) Stimulating whole blood with PHA elicits a cell-mediated immune response reflected in IFNγ production; a stimulation of 3 - 4 hours is sufficient to induce IFNγ production strong enough to be quantified by assay methods and equipment readily available at healthcare centers and medical analysis laboratories; 2) PHA stimulation induces IFNγ production that varies according to the functional state of the individual's immune system even when the duration is short (3 - 4 hours); 3) Since PHA-induced IFNγ production is highly sensitive to changes in the functional state of the immune system, the differences between the two specific functional states are reflected as differences in IFNγ production and can be easily measured by technical means commonly available in healthcare centers and medical analysis laboratories.

[0030] Therefore, IFNγ production in response to PHA stimulation of whole blood is considered a selection parameter for developing a system to stratify the overall state of an individual's immune system. The process for determining the immune state according to the present invention advantageously enables the distinction of the immune state of immunocompromised individuals from that of healthy patients. It is also possible to distinguish between different levels of immune competence in healthy individuals and different levels of immunodeficiency in immunosuppressed patients.

[0031] According to the present invention, the biological sample to be tested is a whole blood sample. Unlike other blood fractions, this blood fraction contains all white blood cells, red blood cells, platelets, and plasma. As a result, PHA-stimulating cells and cells that express IFNγ in response to PHA stimulation benefit from a relatively well-preserved cellular and biochemical environment, and the physiological interactions between various cell populations involved in the immune response remain intact. Therefore, the process for determining the immune state according to the present invention advantageously takes into account the full complexity of the intracellular and intercellular mechanisms of the cell-mediated immune response and can also be applied to individuals / patients under the influence of pharmaceuticals or environmental active agents having immunomodulatory effects.

[0032] According to the present invention, it is advantageous for the whole blood sample to be venous blood collected via the venous route. According to the present invention, prior to performing the process according to the present invention, the sample has not been subjected to any treatment other than the addition of an anticoagulant and / or dilution.

[0033] According to the present invention, the whole blood sample is subjected to a stimulation step (equally, also called an incubation step or a stimulation / incubation step) within 32 hours after collection. After collection, the whole blood sample is stored between 2°C and 8°C until the process of the present invention is carried out.

[0034] According to the present invention, it is advantageous that the whole blood sample is preferably treated with an anticoagulant immediately after collection.

[0035] According to the present invention, it is advantageous that the whole blood sample is heparinized (for example, treated with lithium heparin).

[0036] According to the present invention, the whole blood sample is subjected to a stimulation / incubation step with phytohemagglutinin (PHA), a lectin synthesized by plants and particularly known for its mitogenic effect on T lymphocytes.

[0037] According to the present invention, it is advantageous that the stimulation / incubation step (equally, also called an incubation step) is carried out using phytohemagglutinin P (PHA-P).

[0038] According to the present invention, it is advantageous that the stimulation / incubation step is carried out in an amount equal to at least 20 μg per 1 mL of whole blood using PHA, particularly PHA-P. According to a preferred embodiment, the amount of PHA, particularly PHA-P, is about 40 μg per 1 mL of whole blood.

[0039] Also, according to the present invention, the stimulation / incubation step is carried out at a temperature between 35°C and 39°C. According to the present invention, it is advantageous that this temperature is 37°C.

[0040] Regarding the duration of the stimulation / incubation step, it is equal to at least 3 hours and does not exceed 8 hours. Preferably, it is between 3 hours 30 minutes and 6 hours. Even more preferably, the minimum stimulation / incubation time is 3 hours 30 minutes.

[0041] According to certain preferred embodiments, the level of IFNγ production induced by PHA stimulation is evaluated by measuring the IFNγ present in the reaction mixture, which consists of a whole blood sample to which PHA has been added (e.g., in the form of a PHA solution).

[0042] According to a variation of one embodiment, the level of IFNγ production induced by PHA stimulation is evaluated by assaying the IFNγ present in the liquid fraction of the reaction mixture. For this purpose, upon completion of the stimulation / incubation step, the liquid fraction is recovered from the reaction mixture, optionally after a decantation or centrifugation step.

[0043] According to preferred embodiments, the level of stimulatory IFNγ production is evaluated by performing an IFNγ assay by immunoassay techniques.

[0044] Immunoassay methods are widely known to those skilled in the art. For example, the assay can be an enzyme immunoassay (EIA, enzyme immunoassay) type, i.e., an immunoassay in which the interaction between the binding partner and the target analyte is revealed by substrate hydrolysis (enzymatic catalyzed hydrolysis) and the release of a soluble product that is easily detectable and measurable. Detection and measurement of the soluble product reveals the presence and concentration of the target analyte in the test sample.

[0045] Depending on the nature of the enzyme substrate selected for the assay, colorimetric (ELISA, enzyme-linked immunosorbent assay), fluorescent (ELFA, enzyme-linked fluorescent assay), or chemiluminescent (CLIA, chemiluminescent immunoassay) soluble products with a detectable and easily measurable intensity are released by enzymatic hydrolysis.

[0046] According to the present invention, it is advantageous that IFNγ production is evaluated by assaying IFNγ using an ELFA test.

[0047] In this specific context and for the purposes of this specification, the term "immunoassay" shall be understood in a broad sense. Strictly speaking, it does not only refer to techniques for detecting and / or quantifying a target analyte having an operating principle based on antigen-antibody recognition and binding, but as a result, it requires the use of immunological properties or immunologically-derived tools such as antibodies or antibody fragments (Fab, Fab’, F(ab’)2, scFv (“single-chain fragment variable”) and dsFv (“double-stranded fragment variable”) types). More generally, this refers to techniques for detecting and / or quantifying a target analyte, in which an antibody or other functionally similar compound that is not necessarily immunological or of immunological origin is used as a binding partner in the process of recognizing and binding to the target analyte (or ligand).

[0048] In this regard, examples of binding partners for the purpose of performing an IFNγ immunoassay in the context of the present invention include the following: - Binding partners of immunological nature or origin, such as anti-IFNγ antibodies (monoclonal or polyclonal), or fragments of these antibodies (Fab, Fab’, F(ab’)2 fragments, scFv (“single-chain fragment variable”), and dsFv (“double-stranded fragment variable”)); - Non-immunological binding partners, such as the IFNγ receptor or a fragment of this receptor that can recognize and bind IFNγ, or oligonucleotides, nanobodies, aptamers, DARPins (Designed Ankyrin Repeat ProteINS) or other synthetic molecules that can recognize and bind IFNγ.

[0049] Therefore, according to the present invention, it is advantageous that stimulus-induced IFNγ production is evaluated by an immunoassay method. This can be quantitative or semi-quantitative.

[0050] Non-limiting examples of immunoassay devices suitable for the practice of the present invention include VIDAS® series (bioMerieux, France), Simoa® HD-1 (Quanterix, USA), Cobas® or Elecsys® (Roche Diagnostic, Switzerland), LIAISON® (DiaSorin, Italy), Architect® (Abbott, USA), Access 2 (Beckman Coulter, USA), Clarity™ (Singulex, USA), and Vitros® (Johnson & Johnson, USA).

[0051] According to certain embodiments of the objects of the present invention, this includes the measurement of basal IFNγ levels. This measurement is performed under the same conditions as the determination of stimulated IFNγ production according to the present invention, except that the whole blood sample is not exposed to any stimulation. In other words, prior to the IFNγ assay, the whole blood sample is incubated under the same conditions as the stimulated blood sample, especially with respect to temperature and incubation time, but in the absence of PHA and other stimulants. This specific IFNγ measurement value can be used as a control measurement value and can generate a value related to the basal IFNγ level specific to the whole blood sample analyzed.

[0052] Advantageously, the process for determining the immune status according to the present invention includes an additional result rendering step, according to which the results are - at least one discrete numerical value reflecting the immune level of the tested individual / patient, wherein the at least one numerical value is - the value of the assay of IFNγ produced in response to PHA stimulation, - the difference between the value of the assay of IFNγ produced in response to PHA stimulation and the measured basal IFNγ level, and / or - the ratio of the value of the assay of IFNγ produced in response to PHA stimulation to the measured basal IFNγ level At least one discrete numerical value corresponding thereto; - At least one category value estimated from a comparison of at least one of the previously listed numerical values with at least one reference value, - The at least one reference value has been previously determined from whole blood samples taken from the same individual / patient but at different times, and thus the process according to the invention provides an indication of the evolution over time of the immune state of said individual / patient and / or the effect of treatment on the immune system of said individual / patient; and / or - The at least one reference value has been previously determined from a set of whole blood samples collected from a population of individuals sharing the same particularity with respect to the immune system (e.g., a population of healthy individuals, a population of immunosuppressed individuals), and thus the process according to the invention provides an indication of whether said individual / patient belongs to the reference population and / or of the immune position of said individual / patient with respect to this reference population, At least one category value Provided in the form of an indicator selected from.

[0053] In addition to the identification of the immune state of an individual / patient, the process according to the invention has many important clinical applications. Thus, according to another aspect, the invention relates to the use of the method for determining the immune state of an individual / patient according to the invention for at least one of the following specific and particular applications: - Detecting immunodeficiency, - Monitoring the evolution of the immune state of patients undergoing immunosuppressive therapy, - Monitoring the reconstitution of the immune system of patients after immunosuppressive therapy, - Monitoring the effect of chemotherapy on the immune state of a patient and thereby enabling readjustment or modification of the treatment, - Investigating an active agent and its potential effect on the immune system, - Investigating environmental factors and their potential effect on the immune system, - Detecting and / or investigating infectious agents and their potential effect on the immune system, - Diagnosing and / or studying a disease and its potential impact on the immune system.

[0054] Other objects, features, and advantages of the present invention will become apparent from the following detailed description and the examples set forth hereinafter. These examples refer to the accompanying Figures 1 to 4 and show, in the form of box plots, the results of various implementations of the process according to the present invention.

Example

[0055] Example 1 : Stimulation of whole blood from healthy donors and patients undergoing chemotherapy. Origin of the blood sample to be analyzed Of the whole blood samples used in this example, the first batch was from 27 healthy adult individuals who, in principle, did not show symptoms of immunodeficiency. This first batch of samples was collected by the Etablissements Francais du Sang (EFS), a French blood institute.

[0056] Similarly, the second batch of blood samples was collected from 16 patients undergoing chemotherapy. These samples were collected at the hospital.

[0057] Each whole blood sample was collected in a sterile Vacutainer® tube (Becton-Dickinson) containing lithium heparin and stored upright at 2 - 8°C until the implementation of the process of the present invention.

[0058] Sample stimulation For each whole blood sample, after homogenization, 300 μL was collected and transferred to the wells of a VIDAS® strip (bioMerieux, located in France). Next, 300 μL of a 40 μg / mL PBS-diluted PHA-P solution (Medicago AB, located in Sweden) was added.

[0059] Similarly, to the second well, 300 μL of PBS buffer (without PHA-P) is added to 300 μL of whole blood to determine the basal IFNγ level.

[0060] Thereafter, while controlling evaporation, the reaction mixture is incubated at a temperature of 37 °C for 3 hours and 30 minutes. The stimulation / incubation step is performed herein using the VIDAS® 3 system, which is an immunoassay instrument.

[0061] Assay of INFγ produced after stimulation After 3 hours and 30 minutes of stimulation / incubation, 90 μL of the liquid fraction of the reaction mixture is collected and the IFNγ content is measured. For this purpose, the assay part of the VIDAS® TB-IGRA kit, which operates based on the principle of the ELFA test, is used. Similarly, the VIDAS® IFNγ RUO kit can also be used for the same purpose.

[0062] Results The results obtained are summarized in Table 1 below, and IFNγ production after stimulation with PHA-P (and without stimulation) is represented by the recorded fluorescence intensity (RFV, "relative fluorescence value") and the estimated IFNγ concentration. TIFF2025520850000001.tif250170

[0063] Figure 1 shows the same IFNγ assay results in graph and statistical formats.

[0064] Example 2 : Stimulation of whole blood from healthy donors and liver transplant patients. Origin of the blood sample to be analyzed The whole blood samples used in this example are from a cohort of volunteers registered in the EdMonHG clinical trial (ClinicalTrials.gov identifier: NCT03995537), - 11 healthy adult volunteers (i.e., without symptoms of immunodeficiency); and - 19 patients who have received a liver transplant and are undergoing immunosuppressive therapy It included. For each of these patients, a blood sample was collected before transplantation (the sample is denoted as Pre_TH), and then, weekly for one month after transplantation (the samples are denoted as D1-7, D8-14, D15-21, D22-31 in sequence).

[0065] Assay of sample stimulation and IFNγ secretion after stimulation The whole blood samples were stimulated with PHA according to the same protocol as the aforementioned stimulation protocol.

[0066] After the 3-hour and 30-minute stimulation ended, IFNγ present in the reaction medium was assayed according to the same protocol as the aforementioned stimulation protocol.

[0067] Results The results obtained are summarized in Table 2 below, and the INFγ production after stimulation (and without stimulation) is represented by the recorded fluorescence intensity (RFV, "relative fluorescence value") and the estimated IFNγ concentration. TIFF2025520850000002.tif255161TIFF2025520850000003.tif255160TIFF2025520850000004.tif50170

[0068] Figure 2 shows the results of these same IFNγ assays in graph and statistical formats.

[0069] Example 3 : Stimulation of whole blood from healthy donors and patients after septic shock Origin of the blood sample to be analyzed The whole blood samples used in this example were collected from 11 healthy volunteers and 22 patients who were admitted to the intensive care unit of Edouard Herriot Hospital in Lyon, France, after septic shock.

[0070] For each of these patients monitored after septic shock, the first blood sample was taken on the day of admission or the following day, and then, if possible, the second sample was taken on days 3 - 4 and finally on days 5 - 8 (the samples are designated as D1-2, D3-4, D5-8 in order). Four of these patients died during or shortly after this period.

[0071] Assay of sample stimulation and IFNγ secretion after stimulation Whole blood samples were stimulated with PHA according to the same protocol as the stimulation protocol described above.

[0072] After the end of the 3-hour and 30-minute stimulation, IFNγ present in the reaction medium was assayed according to the same protocol as the stimulation protocol described above.

[0073] Results The results obtained are summarized in Table 3 below, and INFγ production after stimulation (and without stimulation) is represented by the recorded fluorescence intensity (RFV, "relative fluorescence value") and the estimated IFNγ concentration. TIFF2025520850000005.tif255161TIFF2025520850000006.tif166170

[0074] Figure 3 shows all these IFNγ assay results in graphical and statistical form.

[0075] Figure 4 shows the results of the IFNγ assay after stimulation, distinguishing between data related to patients who survived (sp) during the follow-up period and data related to patients who died (dp) during or shortly after this follow-up period, in graphical and statistical form.

Claims

1. A method for determining the immune status of an individual, - A step of providing a certain amount of whole blood sample from the aforementioned individual, - A step of stimulating the whole blood sample by incubating it with a constant amount of phytohemagglutinin (PHA) at a temperature between 35°C and 39°C for a minimum of 3 hours, - A step of evaluating the level of IFNγ production induced by this incubation / stimulation, wherein the level thus evaluated provides an indicator of the individual's immune status. A method that includes this.

2. The method according to claim 1, wherein the shortest time is 3 hours and 30 minutes.

3. The method according to claim 1 or 2, wherein the duration of the stimulation / incubation step is between 3 hours and 8 hours.

4. The method according to claim 1 or 2, wherein the duration of the stimulation / incubation step is between 3 hours 30 minutes and 6 hours.

5. The method according to claim 1 or 2, wherein the stimulation / incubation step is performed using phytohemagglutinin P (PHA-P).

6. The method according to claim 1 or 2, wherein the stimulation / incubation step is performed using an amount of PHA equal to at least 20 μg per 1 mL of whole blood.

7. The method according to claim 1 or 2, wherein the stimulation / incubation step is performed using approximately 40 μg of PHA per 1 mL of whole blood.

8. The method according to claim 1 or 2, wherein the temperature of the stimulation / incubation step is approximately 37°C.

9. The method according to claim 1 or 2, wherein IFNγ production is evaluated by assaying IFNγ using an immunoassay technique.

10. The method according to claim 1 or 2, wherein IFNγ production is evaluated by assaying IFNγ using an ELFA assay.

11. The method according to claim 1 or 2, wherein a whole blood sample is heparinized.

12. Use of the method for determining the immune status of an individual according to claim 1 or 2 for the purpose of detecting immunodeficiency.

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