Functional assay for quickly determining immune status

Abstract

The present invention relates to a method for quickly determining the immune status of an individual. The method comprises the following steps: taking a volume of a whole blood sample from the individual; stimulating the whole blood sample by incubating the latter with a quantity of phytohemagglutinin (PHA) at a temperature between 35°C and 39°C for a minimum of 3 to 3.5 hours; evaluating the level of interferon-gamma production induced by this incubation / stimulation, the evaluated level subsequently providing an indication of the individual's immune status.

Description

[0001] FUNCTIONAL TEST FOR RAPID DETERMINATION

[0002] IMMUNE STATUS

[0003] The present invention relates to the assessment and determination of the immune status of an individual. More particularly, it relates to methods and tools dedicated to measuring the overall level of cell-mediated immunity of an individual, and the operating principle of which is that of a functional immune test.

[0004] By proposing a method and clinical tools capable of enabling a reliable and rapid assessment of a patient's immune status and / or the diagnosis of a possible dysfunction or imbalance in their immune response (immune deficiency or hyperactivity), the invention positions itself advantageously as a valuable aid offered to clinicians in their decision-making.

[0005] An individual's immune status corresponds to the functional state of their immune system, that is, their body's ability to defend itself against potentially dangerous agents. These defense and protection mechanisms are deployed primarily against pathogens of infectious origin and exogenous to the body, such as microorganisms - such as viruses, bacteria, fungi and protozoa -. They can also be deployed against endogenous agents, particularly against cells transformed following physical and / or chemical damage (as may be the case for infected cells, cancer cells or senescent cells).

[0006] The immune system's response to an attack by a potentially dangerous agent, whether exogenous or endogenous, is a dynamic phenomenon which, when adapted, helps maintain the body's integrity. Conversely, a weakened, insufficient or unbalanced immune response exposes the body to a high risk of developing pathologies. A weak or ineffective immune response thus promotes opportunistic infections, the occurrence of sepsis and / or viral reactivations, while an exacerbated immune response can explain the occurrence of allergies, autoimmune diseases (e.g., multiple sclerosis, type 1 diabetes, lupus, autoimmune thyroiditis, rheumatoid arthritis, ankylosing spondylitis, Goujerot-Sjögren syndrome, Crohn's disease).

[0007] Being able to determine a patient's immune status and / or monitor the evolution of their immune status therefore represents a major clinical challenge. In this respect, numerous examples of clinical utility can be cited, in particular: - the identification of patients with a possible immune deficiency (chronic or acute, acquired or induced) and, where appropriate, being able to:

[0008] - provide appropriate medical care and supervision; and / or

[0009] - prevent intolerance to live attenuated vaccines, to drugs contraindicated in cases of immunodeficiency;

[0010] - monitoring the evolution of the immune status of patients receiving immunosuppressive therapy - for example, candidates for solid organ transplants, newly transplanted patients -; this would make it possible to best adapt the dosage of immunosuppressants used in order to establish a level of immunity deemed appropriate to prevent the risk of transplant rejection, while minimizing the risk of infection, reactivation of oncogenic viruses and inhibiting the antitumor immunity of patients;

[0011] - monitoring the reconstitution of the immune system of patients after immunosuppressive therapy, with a view to ensuring that it is progressing properly;

[0012] - monitoring the impact of chemotherapy on a patient's immune status, and allowing for possible readjustment or change in therapy; or

[0013] - the management and monitoring of patients receiving immunotherapy treatment (in particular treatment with CAR-T cells (for “Chimeric Antigenic Receptor-T”) and treatment based on an injection of antibodies known as anti-checkpoint antibodies).

[0014] Being able to determine an individual's immune status and monitor its evolution is also of great interest to the pharmaceutical industry and basic research into human health. In this regard, numerous examples of applications can be cited, in particular:

[0015] - in the context of drug development where its impact on the immune system must be assessed;

[0016] - in the context of the development of a vaccine, for example to assess its effects on a possible polarization of the immune response, towards a Thl and / or Th2 type response; or

[0017] - to assess the possible impact of a pathology or an environmental factor on an individual's immune system.

[0018] Among the methods known to date as being able to determine and / or evaluate the immune status of an individual, we can firstly cite the lymphocyte proliferation test (LPT) and the lymphoblastic transformation test (LTT), which aim to quantify the proliferation of lymphocytes following stimulation by mitogens (for example lectins such as phytohemagglutinin (PHA), concanavalin A (conA) and mitogenic Pokeweed (PWM)) or by pathogen-specific antigens. The implementation of these tests is particularly long. In particular, after being isolated, the mononuclear cells must be placed under stimulation for 3 to 7 days. The cells are then recovered and DNA replication or cell division is measured by flow cytometry, thanks to the incorporation of tracers.

[0019] Much faster to implement, HLA-DR assay by flow cytometry allows the measurement of HLA-DR (for "human leukocyte antigen - D related") expression on the surface of monocytes; low expression of this marker is a sign of immune system failure. Also, in patients with septic shock, persistent low levels of monocyte HLA-DR expression generally indicate poor survival rates.

[0020] Because for the time being, this method of HLA-DR dosage can only be done by flow cytometry and few care centers and medical analysis laboratories have appropriate equipment, such a method of determining immune status is more suited to observational studies, exploratory research work, than to use in diagnosis for clinical purposes. What is more, it is cumbersome and delicate to implement, and remains very complicated to standardize / normalize; the temperature and duration of storage of the cells before their labeling, as well as the lysis of the red blood cells, are all factors having a strong impact on the variability of the measurements and which must therefore be finely controlled (Fink et al., 2003 - "Standardization of the measurement of the monocyte HLA-DR antigen by flow cytometry: preliminary result and application in the monitoring of septic shock" - Ann. Biol. Clin., 2003, 61 : 441-448).

[0021] Requiring more accessible equipment and benefiting from less restrictive implementation, the methods for determining immune status, which are qualified as immune functional assays (or IFA, an English acronym for "Immune Functional Assays"), are based on the measurement of cellular activity (involving one or more types of immune cells - lymphocytes, macrophages, monocytes, dendritic cells, granulocytes -) in response to a particular stimulation. Depending on the nature of the stimulant(s) used for this purpose, the level of immunity studied is either a level of specific immunity, that is to say an immune response specifically deployed and directed against a given target pathogen, or an overall level of immunity which reflects the general state of the immune system of this individual.In both cases, the measurement of said cellular activity consists of a dosage of one or more cytokines whose expression is modulated by stimulation (for example IFNy, TNFa, interleukins, etc.).

[0022] To determine a specific immunity level, the stimulant(s) used generally reproduce epitopic motifs (protein and / or glycosidic) from the targeted pathogen (e.g., for an immune status possibly directed against Mycobacterium tuberculosis, all or part of the protein sequence of markers such as ESAT-6, CFP-10 and TB7.7 is frequently used for stimulation). To determine an overall immunity level, one or more so-called 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) and lipopolysaccharide (LPS), as well as cytokines such as interleukins IL-1, IL-2 and IL-12. Anti-CD3 (or more rarely anti-CD2) monoclonal antibodies are also used, such as OKT-3 with or without anti-CD28.

[0023] The present invention is more specifically concerned with functional immune tests dedicated to determining the overall level of cellular immunity of an individual, as is the case for the ImmuKnow® (Cylex Inc., USA) and QuantiFERON Monitor® (Qiagen GmbH, Germany) tests, both commercially available.

[0024] The ImmuKnow® test, proposed for the immunological monitoring of patients placed on immunosuppressants after an organ transplant, aims to identify situations of under- and overdose. The principle of this test is based on a measurement of intracellular ATP (adenosine triphosphate) synthesized by CD4 T lymphocytes +stimulated. This level of intracellular ATP thus measured is supposed to be correlated with the overall lymphocyte activity of the patient. An activity level identified as low thus reveals an overdose of immunosuppressant and a risk of infection for the patient, while an activity level identified as high is a sign of an underdose of immunosuppressant and a risk of graft rejection.

[0025] The ImmuKnow® test involves stimulating a whole blood sample for 15 to 18 hours with a mitogen, in this case PHA. CD4 T lymphocytes + are then purified and lysed to extract F ATP. The latter is finally measured quantitatively by bioluminescence using a luciferin / luciferase system (Stewart, 2012 - “ImmuKnow as an immune monitoring tool following organ transplantation” - Le courrier de la transplantation, 2012, vol. VII n°1).

[0026] Regarding the QuantiFERON Monitor® test, 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) describes its use for prognostic purposes of the risk of infection in patients who have undergone allogeneic hematopoietic stem cell transplantation. To do this, a heparinized whole blood sample is stimulated at 37°C for 16 to 24 hours, using a composition of actives, called QFM LyoSphere™. This composition contains an R848 reagent and a TLR7 receptor agonist, to stimulate the patient's innate immunity, as well as an anti-CD3 antibody to stimulate their adaptive immunity. After these 16 to 24 hours of stimulation, the plasma is collected and the interferon gamma (IFNY) content is measured.The latter provides an indication of the patient's immune status, in this case an indication of their overall level of cell-mediated immunity. This test cumulatively takes into account the components of innate immunity and adaptive immunity.

[0027] Like the ImmuKnow® test, the QuantiFERON Monitor® test suffers from a particularly long implementation time; an excessive duration mainly due to the stimulation phase which, on its own, requires more than 15-16 hours.

[0028] Because immunodeficient patients often require specific clinical and / or therapeutic management to address their high susceptibility to infections, screening for immunodeficiency may be urgent in many clinical situations, for example:

[0029] - upon admission to a care center,

[0030] - before surgery,

[0031] - before prescribing drugs / treatments contraindicated for immunocompromised patients,

[0032] - in the case of a septic condition, where particularly sustained and close monitoring is required,

[0033] - there is therefore a real need for clinicians to have a diagnostic / prognostic test for an immunodeficiency state, which can be implemented quickly and is capable of providing a result in the shortest possible time.

[0034] The present invention thus aims to propose a functional immune test capable of enabling a determination and evaluation of the immune status of a patient in a significantly reduced time compared to functional immune tests currently available commercially.

[0035] More generally, the present invention aims to propose a method for determining in vitro or ex vivo the immune status of an individual, the implementation of which is intended to be simple, rapid and feasible with technical equipment accessible to care centers and medical analysis laboratories.

[0036] The present invention meets all of the above-mentioned objectives. Before presenting its characteristics and particularities in more detail, the following definitions are given to allow a better understanding.

[0037] In the context of this description, the term "determining / evaluating immune status" means giving an indication of the capacity of an individual's body to be able to implement an immune response in order to defend itself, to protect itself against potentially dangerous agents. In a very similar way to "immune status", the expression "level of immunity" can also be used interchangeably.

[0038] The immune status determined / evaluated with the present invention may be reported by means of a value, which may be numerical or categorical, and which results directly or indirectly from a measurement of IFNγ produced in response to stimulation carried out in accordance with the present invention. A result given in the form of a numerical value then corresponds to a discrete or continuous variable, representative of the level of immunity. A result given in the form of a categorical value may for example associate the immune status of an individual with qualifiers such as "normal", "low" or "high". Such an indication results from an interpretation / extrapolation based on the level of IFNγ production measured after stimulation and / or from a comparison of this level with one or more reference IFNγ expression levels.

[0039] By "whole blood sample" is meant a venous blood sample, obtained from a sample taken from an individual / patient and essentially consisting of erythrocytes, leukocytes, platelets and plasma. Apart from the possible addition of an anticoagulant, an optional dilution and / or possible storage between 2°C and 8°C, the whole blood sample directly subjected to the method of the invention has not undergone any other treatment, in particular no prior treatment likely to significantly modify its composition (in terms of constituents and proportions between the constituents).

[0040] By "evaluating the level of IFNy production", in this case the production induced by a stimulation carried out in accordance with the present invention, is not necessarily meant the fact of measuring with more or less precision the quantity of IFNy which is actually and specifically produced / secreted in response to the stimulation. Such an evaluation may in fact consist of a reasoned estimation of indicators / parameters such as: - the total concentration / quantity of IFNy found in the reaction mixture [whole blood - stimulation solution], or in a sub-fraction of this mixture;

[0041] - the quantity of mRNA transcribed in response to IFNy stimulation, and which, apart from a few factors and / or approximations, validly account for the production of IFNy thus studied.

[0042] Finally, the term "individual" refers to a human being, regardless of their state of health. A "healthy individual" within the meaning of the present invention is an individual who does not apparently have a deregulation of the immune system. The term "patient" refers to an individual in contact with a health professional - such as a doctor (for example, a general practitioner) - and / or a medical facility (for example, the emergency or intensive care department of a hospital, or an intensive care unit) or a medical analysis laboratory.

[0043] The present invention therefore relates to a method for determining the immune status of an individual; which comprises the following steps:

[0044] - have a volume of whole blood sample from said individual;

[0045] - stimulating said whole blood sample by incubating it with a quantity of phytohemagglutinin (PHA), at a temperature between 35°C and 39°C, for a minimum period of 3 hours, for example for a period of 3 hours to 8 hours;

[0046] - assess the level of induced IFNy production; said level thus assessed gives an indication of the immune status of said individual.

[0047] According to a particular embodiment, the stimulation duration does not exceed 8 hours and, preferably, it does not exceed 6 hours.

[0048] The inventors have thus developed a reliable functional immune test capable of producing results in a particularly short time. Indeed, and against all expectations, they have managed to significantly reduce the duration of the stimulation stage. This was made possible mainly thanks to the following observations and demonstrations:

[0049] 1) stimulation of whole blood with PHA causes a cell-mediated immune response resulting in IFNy production; 3 to 4 hours of stimulation are sufficient to induce IFNy production sufficiently intense to be quantified, including by methods and assay equipment readily available to healthcare centers and medical analysis laboratories; 2) PHA stimulation, even of short duration, of 3 to 4 hours, induces IFNy production that varies according to the functional state of the individual's immune system;

[0050] 3) PHA-induced IFNy production is sufficiently sensitive to variations in the functional state of the immune system so that the difference between two particular functional states translates into a difference in IFNy production, easily measurable including by the technical means commonly available to care centers and medical analysis laboratories.

[0051] The production of IFNy in response to whole blood stimulation by PHA thus appears to be a parameter of choice for the development of a system for stratifying the overall state of the immune system of individuals. The method for determining immune status according to the invention advantageously makes it possible to distinguish the immune status of immunodeficient individuals from that of healthy patients. It also makes it possible to distinguish different levels of immunocompetence in healthy individuals, and different levels of immunodeficiency in immunocompromised patients.

[0052] According to the invention, the biological sample tested is a whole blood sample. Unlike other blood fractions, this contains all leukocytes, erythrocytes, platelets and plasma. As a result, the cells stimulable by PHA and the cells expressing IFNy in response to stimulation by PHA benefit from a relatively well-preserved cellular and biochemical environment, in which physiological interactions between the different cell populations involved in the immune response remain possible. The method for determining the immune status according to the invention thus advantageously takes into account the entire complexity of the intra- and intercellular mechanisms of the cell-mediated immune response, and also applies to individuals / patients under the influence of a medicinal or environmental active ingredient with immunomodulatory effects.

[0053] Advantageously and according to the invention, the whole blood sample is venous blood, collected intravenously. According to the invention, prior to implementing the method according to the invention, it has not undergone any treatment other than the possible addition of an anticoagulant and / or dilution.

[0054] According to the invention, the whole blood sample is subjected to the stimulation step (equivalently, it may also be referred to as an incubation step or a stimulation / incubation step) within 32 hours of collection. After collection and until the implementation of the method of the invention, the whole blood sample is stored between 2°C and 8°C.

[0055] Advantageously and according to the invention, the whole blood sample was treated with an anticoagulant, preferably immediately after its collection.

[0056] Advantageously and according to the invention, the whole blood sample has been heparinized (treated for example with lithium heparin).

[0057] According to the 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 action on T lymphocytes.

[0058] Advantageously and according to the invention, the stimulation / incubation step (equivalently, one can also speak of an incubation step) is carried out with phytohemagglutinin P (PHA-P).

[0059] Advantageously and according to the invention, the stimulation / incubation step is carried out with PHA, in particular with PHA-P, in an amount at least equal to 20 pg per mL of whole blood. According to a preferred embodiment, the amount of PHA, in particular PHA-P, is of the order of 40 pg per mL of whole blood.

[0060] Also and according to the invention, the stimulation / incubation step is carried out at a temperature between 35°C and 39°C. Advantageously and according to the invention, this temperature is 37°C.

[0061] Regarding the duration of the stimulation / incubation step, this is at least 3 hours and does not exceed 8 hours. Preferably, it is between 3.5 and 6 hours. Even more preferably, the minimum stimulation / incubation duration is 3.5 hours.

[0062] According to a particularly preferred embodiment, the level of IFNy production induced by PHA stimulation is evaluated by measuring the IFNy present in the reaction mixture, the latter being composed of the whole blood sample to which PHA has been added - for example in the form of a PHA solution.

[0063] According to an alternative embodiment, the level of IFNy production induced by PHA stimulation is evaluated by measuring the IFNy present in the liquid fraction of the reaction mixture. To do this, once the stimulation / incubation step is completed, the liquid fraction is recovered from the reaction mixture, possibly after a decantation or centrifugation step.

[0064] According to a preferred embodiment, the level of FNγ production induced by the stimulation is evaluated by performing an IFNγ assay using an immunoassay technique (or immunoassay).

[0065] Immunoassay methods are widely known to those skilled in the art. For example, this may involve an enzyme immunoassay (EIA), i.e., an immunoassay test in which the interaction between the binding partner and the target analyte is revealed by the hydrolysis of a substrate (an enzyme-catalyzed hydrolysis) and the release of an easily detectable and measurable lysis product. The detection and measurement of the lysis product thus provide an indication of the presence and concentration of the target analyte in the sample being tested.

[0066] Depending on the nature of the enzymatic substrate chosen to implement the assay, enzymatic hydrolysis releases a colorimetric lysis product (this is then referred to as an ELISA test, for “Enzyme-Linked Immunosorbent Assay”), fluorescent (ELF A test, for “Enzyme Linked Fluorescent Assay”) or chemiluminescent (CLIA test, for “Chemiluminescence Immuno Assays”), which is detectable and has an intensity that is easy to measure.

[0067] Advantageously and according to the invention, the production of IFNy is evaluated by measuring the IFNy using an ELFA test.

[0068] In this particular context and within the meaning of this description, the terms "immunoassay" and "immunotest" are to be understood in the broad sense. They do not refer strictly speaking and only to techniques for detecting and / or quantifying a target analyte, the operating principle of which is based on antigen-antibody recognition and coupling which, therefore, requires the use of tools of an immune nature or origin, such as antibodies or antibody fragments (fragments of the Fab, Fab', F(ab')2, scFv ("Single chain fragment variable") and dsFv ("Double-stranded fragment variable") type).They more generally refer to techniques for detecting and / or quantifying a target analyte, in which antibodies or any other functionally analogous compound, not necessarily of immune nature or origin, can be used as binding partners in a process of recognition and coupling to the target analyte (or ligand). In this regard, as examples of binding partners for carrying out an IFNy immunoassay within the meaning of the present invention, mention may be made of:

[0069] - binding partners of immunological nature or origin, such as anti-IFNy antibodies (monoclonal or polyclonal), or fragments of these antibodies (such as Fab, Fab', F(ab')2 fragments, scFv chains ("Single chain fragment variable") and dsFv ("Doublestranded fragment variable"));

[0070] - binding partners without immunological origin such as the F IFNy receptor or a fragment of this receptor capable of recognizing and binding F IFNy, or oligonucleotides, nanofitins, aptamers, DARPins (for “Designed Ankyrin Repeat ProteINS”) or any other synthetic molecule which could recognize and bind to F IFNy.

[0071] Thus, advantageously and according to the invention, the production of IFNy induced by the stimulation is evaluated by means of an immunoassay method. This can be quantitative or semi-quantitative.

[0072] Non-limiting examples of immodosing instruments suitable for implementing the present invention include instruments from the VIDAS® range (bioMérieux, 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).

[0073] According to a particular embodiment of the method according to the invention, the latter further comprises a measurement of the basal level of IFNy. This measurement is carried out under the same conditions as for a determination of the production of IFNy induced by a stimulation carried out in accordance with the present invention, but with the difference that the whole blood sample is not subjected to any stimulation. In other words, prior to the F IFNy assay, the whole blood sample is incubated under the same conditions as a stimulated blood sample, in particular in terms of temperature and incubation time, but in the absence of PHA and any other stimulant. This particular measurement of IFNy, which can be used as a control measurement, makes it possible to obtain a value related to the basal level of IFNy, specific to the whole blood sample analyzed.Advantageously, the method for determining immune status according to the invention comprises an additional step of rendering a result, by which said result is delivered in the form of an indication chosen from:

[0074] - at least one discrete numerical value reflecting the level of immunity of the individual / patient tested, said at least one numerical value corresponding to:

[0075] - the value of the IFNy dosage produced in response to stimulation by PHA;

[0076] - the difference between the value of the IFNy dosage produced in response to PHA stimulation and the basal level of IFNy measured; and / or

[0077] - a ratio between the value of the IFNy dosage produced in response to stimulation by PHA and the basal level of IFNy measured;

[0078] - at least one category value deduced from the comparison between at least one of the previously listed numerical values ​​and at least one reference value:

[0079] - said at least one reference value having been previously determined from a whole blood sample from the same individual / patient but taken at a different time; the method according to the invention thus provides an indication as to the evolution over time of the immune status of said individual / patient, and / or as to the impact of a possible treatment on his / her immune system; and / or

[0080] - said at least one reference value having been previously determined from a set of whole blood samples collected from a population of individuals sharing the same particularity in their immune system (for example a population of healthy individuals, a population of immunocompromised individuals); the method according to the invention thus provides an indication as to the possible membership of said individual / patient in the reference population and / or their immune positioning in relation to this reference population.

[0081] Beyond the identification of the immune status of an individual / patient, the method according to the invention finds numerous important clinical applications. Also, according to another aspect, the present invention relates to the use of a method for determining the immune status of an individual / patient according to the invention, for at least one of the following particular and specific applications:

[0082] - the detection of a possible immune deficiency; - monitoring the evolution of the immune status of a patient placed on immunosuppressive therapy;

[0083] - monitoring the reconstitution of a patient's immune system after immunosuppressive therapy;

[0084] - monitoring the impact of chemotherapy on a patient's immune status, and allowing for possible readjustment or change in therapy;

[0085] - the study of a medicinal active ingredient and its possible impact on the immune system;

[0086] - the study of an environmental factor and its possible impact on the immune system;

[0087] - the detection and / or study of an infectious agent and its possible impact on the immune system;

[0088] - the diagnosis and / or study of a disease and its possible impact on the immune system.

[0089] Other aims, characteristics and advantages of the invention will appear in view of the detailed description which follows and the examples developed below. These examples refer to the appended figures 1 to 4, which present, in the form of a box diagram, the results of different implementations of a method according to the invention.

[0090] EXAMPLES

[0091] EXAMPLE 1: Stimulation of whole blood from healthy donors and patients undergoing chemotherapy.

[0092] Origins of the analyzed samples

[0093] Among the whole blood samples used in this example, a first batch comes from 27 healthy, adult individuals who do not appear to have any symptoms of immunodeficiency. The samples from this first batch were collected by the French Blood Establishments (EFS).

[0094] Similarly, a second batch of blood samples came from 16 patients undergoing chemotherapy. These samples were collected in a hospital setting. Each of the whole blood samples was collected in sterile Vacutainer® tubes (Becton-Dickinson) containing lithium heparin, then stored upright and at 2-8°C pending implementation of the method of the invention.

[0095] Sample stimulation

[0096] For each whole blood sample, after homogenization, 300 pL are collected and transferred into a well of a VIDAS® strip (bioMérieux, France). 300 pL of a 40 pg / mL PHA-P solution (Medicago AB, Sweden) diluted with PB S is then added.

[0097] Similarly, in a second well, to determine the basal level of ZFNy, 300 pL of PBS buffer (without PHA-P) is added to 300 pL of whole blood.

[0098] The reaction mixtures are then incubated for 3.5 hours at a temperature of 37°C, with controlled evaporation. The stimulation / incubation step is carried out using an immunoassay instrument, the VIDAS® 3.

[0099] Dosage of INF produced after stimulation

[0100] After 3.5 hours of stimulation / incubation, 90 pL of the liquid fraction of the reaction mixture is collected and the IFNY content is measured. To do this, the assay part of a VIDAS® TB-IGRA kit, which works according to the principle of an ELFA test, is used. Similarly, the VIDAS® IFNY RUO kit can be used for this same purpose.

[0101] Results

[0102] The results obtained are compiled in Table 1 below, in which the production of INFy after stimulation with PHA-P (and without stimulation), is expressed in recorded fluorescence intensity (RFV, for “Relative Fluorescence Value”) and in estimated IFNy concentration.

[0103] Table 1: IFNy assay without and after PHA-P stimulation, on whole blood from healthy donors and patients undergoing chemotherapy.

[0104] Figure 1 presents these same results of IFNy assays, in graphical and statistical forms. EXAMPLE 2: Stimulation of whole blood from healthy donors and patients with liver transplants.

[0105] Origin of the blood samples analyzed

[0106] The whole blood samples used in this example are from a cohort of volunteers enrolled in the EdMonHG clinical study (ClinicalTrials.gov Identifier: NCT03995537), which included:

[0107] - 11 healthy adult volunteers (i.e. who do not show any symptoms of immunodeficiency); and

[0108] - 19 patients followed for a liver transplant and under immunosuppression. For each of these patients, a blood sample was taken before the transplant (samples noted Pre TH), then every week following the transplant, for one month (samples noted successively Dl-7, D8-14, D15-21 and D22-31).

[0109] Stimulation of samples and dosage of ITFNy secreted after stimulation

[0110] Whole blood samples were stimulated with PHA following the same stimulation protocol as previously described.

[0111] At the end of 3.5 hours of stimulation, the IFNy present in the reaction medium is measured following the same assay protocol as previously described.

[0112] Results

[0113] The results obtained are compiled in Table 2 below, in which the production of INFy after stimulation (and without stimulation) is expressed in recorded fluorescence intensity (RFV, for “Relative Fluorescence Value”) and in estimated IFNy concentration.

[0114] Table 2: IFNy T assay without and after PHA-P stimulation, in whole blood from healthy donors and liver transplant patients. Figure 2 shows these same IFNy assay results, in graphical and statistical forms.

[0115] EXAMPLE 3: Stimulation of whole blood from healthy donors and patients after septic shock

[0116] Origin of the analyzed samples

[0117] The whole blood samples used in this example come from 11 healthy volunteers and 22 patients admitted to intensive care at the Edouard Herriot Hospital in Lyon (France), after septic shock.

[0118] For each of these patients followed after septic shock, a first blood sample was taken on the day of their admission or the following day, then if possible, a second sample on the 3-4 eme , and finally at 5-8 eme days (samples noted successively Dl-2, D3-4, D5-8). 4 of these patients died during this period or shortly after.

[0119] Stimulation of samples and dosage of IFNy secreted after stimulation

[0120] Whole blood samples were stimulated with PHA following the same stimulation protocol as previously described.

[0121] At the end of 3.5 hours of stimulation, the IFNy present in the reaction medium is measured following the same assay protocol as previously described.

[0122] Results

[0123] The results obtained are compiled in Table 3 below, in which the production of INFy after stimulation (and without stimulation) is expressed in recorded fluorescence intensity (RFV, for “Relative Fluorescence Value”) and in estimated IFNy concentration.

[0124] Table 3: Dosage of T IFNy without and after PHA-P stimulation, on whole blood from healthy donors and patients followed after septic shock.

[0125] Figure 3 shows all of these IFNy assay results in graphical and statistical form.

[0126] Figure 4 shows these results of IFNy dosages after stimulation, in graphical and statistical forms, differentiating the data associated with patients alive (sp) during the follow-up week, from those associated with patients who died (dp) during this follow-up period or shortly after.

Claims

CLAIMS 1. Method for determining the immune status of an individual, comprising the following steps: - have a volume of whole blood sample from said individual; - stimulate said whole blood sample by incubating it with a quantity of phytohemagglutinin (PHA), at a temperature between 35°C and 39°C, for a minimum period of 3 hours; - assess the level of IFNy production induced by this incubation / stimulation; said level thus assessed gives an indication of the immune status of said individual.

2. Method according to claim 1, wherein said minimum duration is 3h30.

3. Method according to claim 1 or 2, in which the duration of the stimulation / incubation step is between 3 hours and 8 hours.

4. Method according to claim 1 or 2, in which the duration of the stimulation / incubation step is between 3h30 and 6 hours.

5. A method according to any one of the preceding claims, wherein the stimulation / incubation step is carried out with phytohemagglutinin P (PHA-P).

6. Method according to any one of the preceding claims, wherein the stimulation / incubation step is carried out with an amount of PHA at least equal to 20 pg per mL of whole blood.

7. Method according to any one of the preceding claims, in which the stimulation / incubation step is carried out with of the order of 40 pg of PHA per mL of whole blood.

8. Method according to any one of the preceding claims, in which the temperature of the stimulation / incubation step is of the order of 37°C.

9. A method according to any preceding claim, wherein IFNγ production is assessed by assaying IFNγ using an immunoassay technique.

10. A method according to any preceding claim, wherein IFNγ production is assessed by assaying IFNγ using an ELFA assay.

11. Method according to any one of the preceding claims, wherein the whole blood sample is heparinized.

12. Use of a method for determining the immune status of an individual according to any one of claims 1 to 11, for the purpose of detecting a possible immune deficiency.

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