ES2813623T3 - An assay of cell-mediated immune response - Google Patents

Abstract

A method for measuring a cell-mediated immune response activity, said method comprising: (a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon stimulation with a peptide antigen, with at least one peptide antigen having a selected length of 5 to 100 amino acids, and with at least one non-reducing sugar capable of increasing the interferon-gamma response of immune cells responding to the antigen; and (b) detecting the presence or level of at least one immunological effector molecule, wherein detecting the presence or level of at least one immunological effector molecule comprises detecting the presence or level of the interferon-gamma (INF- gamma) and / or an immune effector molecule whose production is induced or enhanced by interferon-gamma.

Description

DESCRIPTION

An assay of cell-mediated immune response

Field of the invention

This description relates generally to the field of immunologically based assays and describes methods for measuring cell-mediated immune responsiveness. The present disclosure discloses methods, compositions, and kits for measuring cell-mediated immune response activity with higher sensitivity and better shelf-life stability.

Background of the invention

Immuno-based diagnostic assays have wide application in the medical field. In particular, they are important tools to aid in the detection and management of a variety of disease disorders. The efficacy of these types of assays is based, in part, on the specificity of components within the immune system, such as T lymphocytes. Despite this specificity, immunologically based diagnoses are not necessarily sensitive enough to detect low-level infections, the presence of a persistent low-level infection, detect infections in subjects with latent or active infectious disease states or in subjects showing immunodeficiency or any form of immunosuppression. Desirable performance characteristics of a cell-mediated immune response assay, in particular for detecting an antigen-specific T-cell response, include adequate sensitivity, specificity, reliability and reproducibility and, furthermore, must be capable of being performed easily and quickly. .

An established form of an immunologically based diagnostic assay involves the stimulation of T cells or other cells of the immune system with antigens, followed by the detection of immune effector molecules, such as IFN-gamma or other cytokines produced in response to stimulation with the antigen. Immunological effector molecules are detected using well-known techniques, such as enzyme immunoassays, multiplex sphere analysis, ELISA, ELISpot, and flow cytometry. The presence or increase in the level of immunological effector molecules can also be determined based on the level of RNA. These assays are useful, for example, to detect disease-specific immune responses, in particular pathogen-specific immune responses. The respective assays are commercially available under the trademark QuantiFERON (Registered Trade Mark; Cellestis Limited) and can be used, for example, to diagnose a pathogen infection or to monitor cell-mediated immunity against disease.

Other applications of the respective cell-mediated immune response assays include the analysis or monitoring of immune responses to vaccines or immunotherapy, such as, for example, cancer immunotherapy.

There is a great demand for these assays with increased sensitivity. Previously, methods for measuring cell-mediated immune responses have been improved by incubating the sample, such as a whole blood sample, with the antigen in the presence of a simple sugar, such as dextrose (see, for example, WO 2004 / 042396 A1). Simple sugars, such as dextrose and glucose, have been found to increase IFN-gamma production by immune cells and thus increase the sensitivity of the assay. The use of simple sugars, such as glucose and dextrose, is believed to be essential for cells to be able to utilize this energy source and thus benefit from the addition of sugar during incubation with the antigen. Significant increases in the IF- ^y level have been observed when simple sugar is added directly to the sample in addition to the antigen. However, to simplify the performance of the method it is preferable to provide ready-to-use reagent compositions and avoid manual handling steps. Therefore, it would be desirable to provide a single composition that includes the antigen and the simple sugar. Said composition can be provided in a sample collection tube, in which the sample is in direct contact with the antigen and the simple sugar in the appropriate concentration, thereby avoiding handling errors. It has been found herein that the activity of the assay decreases over time when the respective sample collection tubes comprising the antigen and the simple sugar are stored at room temperature or elevated temperatures. This has been discovered with certain antigens. Therefore, the shelf life of these kit components is limited and, after a certain storage time, the assay provides only low sensitivity or even the assay activity is completely lost. Therefore, the respective test kits / materials in which the antigen is conveniently provided together with a simple sugar in a composition are not shelf stable and therefore pose a risk that the sensitivity of the assay will be affected. reduce or even be lost over time. This is not observed when the simple sugar is not included together with the antigen in the sample collection tube, but is added separately to the sample for incubation with the antigen.

EP 1321 670 A1 relates to methods for inducing, maintaining and expanding cytotoxic T cells having antigen-specific activity by using, as an effective ingredient, at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides , acid monosaccharides and their salts.

Xu Zhi-Jun et al. (Archives of Pathology and Laboratory Medicine, 1993, 117 (11): 1174-1177) refers to the field of immunocytological assays and immunohistochemistry, and indicates that a sucrose-magnesium chloride-glycerol solution can be used to preserve cytological preparations. .

Fujita et al. (Microbiology and Immunology, 1990, 34 (6): 523-532) refers to the in vivo immunomodifying activity of a glycolipid mycoloil, trehalose 2,3,6'-trimicholate (GaGM) from R. auranticus.

Roser (Biopharm., 1991, 4 (8): 47-53) refers to a method called "trehalose drying" as a new substitute for lyophilizing macromolecules.

DATABASE WPI Week 200103 Thompson Scientific, London, UK, AN 2001-016743 and CN 1262131 A (CHANGSHENG IND CO LTD CHANGC HUN CITY) indicates that trehalose can be used as a protector of biological preparations.

US 4,891,319 A relates to the protection of proteins and other biological macromolecules with tertiary structures against denaturation during drying in the presence of trehalose.

WO 98/58259 A1 is directed to improve immunoassays that are immunoadsorbent assays and in which antibodies are captured, such as ELISA assays.

EP 1529536 A1 refers to an immunogenic composition having a greater capacity for immunostimulation of the immune system of a host to which it is administered, and the composition comprises the compound sucrose acetate isobutyrate (SAIB).

WO 00/56365 A1 refers to a liquid vaccine composition comprising at least one antigen consisting of a polysaccharide bound to a carrier protein and trehalose.

The object of the present invention is to overcome at least one of the drawbacks of the prior art methods. In particular, the object of the present invention is to provide sensitive and reliable methods for measuring cell-mediated immune responsiveness, as well as kits and kit components that have good shelf-life stability.

Summary of the invention

The invention can be defined by the appended claims.

Accordingly, the present invention provides a method for measuring cell-mediated immune response activity, said method comprising:

(a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon stimulation with a peptide antigen, with at least one peptide antigen having a selected length of 5 to 100 amino acids, and with at least one non-reducing sugar capable of increasing the interferon-gamma response of immune cells that respond to the antigen; Y

(b) detecting the presence or level of at least one immunological effector molecule, wherein detecting the presence or level of at least one immunological effector molecule comprises detecting the presence or level of the interferon-gamma (INF-gamma ) and / or an immune effector molecule whose production is induced or enhanced by interferon-gamma.

The present invention also provides a composition according to claim 14, a sample collection container according to claim 16, a kit according to claim 17, a use according to claim 19, a use according to claim 20 and an incubation composition according to claim 21.

The inventors have found that the addition of a non-reducing sugar during the incubation of the sample with the antigen surprisingly increases the response levels and thus the sensitivity of the method in a manner similar to that observed when a simple sugar, such as dextrose and glucose, during incubation. This is unexpected because it was previously believed that an increase in sensitivity can only be achieved with simple sugars and thus monosaccharides, which represent reduced sugars. Furthermore, it has surprisingly been found that the method can maintain its highest sensitivity even over long storage periods of the test materials, even if the non-reducing sugar and antigen are provided in the form of a single composition. . Thus, the inventors have surprisingly discovered that the use of a non-reducing sugar, rather than a simple sugar, significantly increases the shelf-life of the assay components, while increasing the response levels. and thus can improve the sensitivity of the assay. Although no limitation is intended by theory, it is believed that non-reducing sugar increases the amount of immune effector molecules released in the event of a positive cell-mediated immune response, thereby improving the sensitivity of the assay. The production of immune effector molecules appears to be enhanced. Thus, the use of a non-reducing sugar, as described herein, may also allow earlier detection of cell stimulation. immunological than with other possible forms. The ability to increase the sensitivity of a cell-mediated immune response assay may also allow the use of less sensitive means of detecting effector molecules and / or the use of smaller sample sizes. Furthermore, these beneficial effects are also achieved after prolonged storage of the test materials, thereby improving the reliability and reproducibility of the test.

The following aspects are also described:

According to a first aspect, a method is provided for measuring a cell-mediated immune response activity, said method comprising:

(a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon challenge with an antigen, with at least one antigen, and with at least one non-reducing sugar, and

(b) detecting the presence or level of an immunological effector molecule.

Such a method can be used, for example, to measure cell-mediated immune response activity in a subject, such as a patient. The presence (or absence) or level of the detected immunological effector molecule is indicative of the level or ability of a subject to mount a cell-mediated immune response against the antigen tested.

According to a second aspect, there is provided a composition for inducing a cell-mediated immune response, said composition comprising:

a) at least one antigen;

b) at least one non-reducing sugar; Y

c) optionally at least one anticoagulant.

A respective composition may be conveniently employed in the method according to the first aspect of the invention to prepare the incubation composition by contacting the sample comprising the immune cells with said composition.

According to a third aspect, there is provided a sample collection container comprising the composition according to the second aspect. The sample collection container can be conveniently used in the method according to the first aspect. Preferably, the sample collection container is a vacuum blood collection tube.

According to a fourth aspect, there is provided a kit for measuring cell-mediated immune response activity in a subject, comprising at least one antigen, at least one non-reducing sugar, at least one sample collection container which is preferably a tube blood collection system, and at least one detection means for at least one immunological effector molecule. A respective kit can be used to carry out the method according to the first aspect.

According to a fifth aspect, the present description refers to the use of a non-reducing sugar in an immunological assay to measure the activity of the cell-mediated response, in which the addition of the non-reducing sugar increases the release of at least one immunological effector molecule. , preferably IFN-gamma, from immune cells that respond to the antigen tested in said assay.

Other objects, features, advantages, and aspects of the present application will be apparent to those skilled in the art from the following description and the appended claims.

Brief description of the figures

Figure 1: Effect of trehalose addition on IFN-gamma response in a QFN-CMV assay (** p <0.01; *** p <0.001).

Figure 2: Effect of trehalose addition on IFN-gamma response in a QFN-TB assay.

Figure 3: Four different non-reducing sugars increase the result of a quantitative QFN-CMV.

Detailed description of the invention

The invention can be defined by means of the appended claims.

Throughout this specification, unless the context indicates otherwise, the term "comprises" or variations such as "comprising", imply the inclusion of an element or integer or method step or group of elements or numbers. integers or steps of the method mentioned, but not the exclusion of any another element or integer or method step or groups of elements or integers or method steps.

As used herein, the singular forms "a" and "the" include the plural aspects, unless the context clearly indicates otherwise. Thus, for example, reference to "a T cell" includes a single T cell, as well as two or more T cells, and reference to "an antigen" includes a single antigen, as well as two or more antigens. Similarly, reference to an "agent", "reagent", "molecule" and "compound" includes individual entities and combinations of two or more of such entities. Reference to "the description" includes individual or multiple aspects disclosed herein, and the like.

According to a first aspect, a method is provided for measuring a cell-mediated immune response activity, said method comprising:

(a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon challenge with an antigen, with at least one antigen, and with at least one non-reducing sugar, and

(b) detecting the presence or level of an immunological effector molecule.

Advantageous embodiments and applications of such a method are described herein. According to an embodiment of the first aspect, there is provided a method for measuring a cell-mediated immune response activity in a subject, said method comprising:

(a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon challenge with an antigen obtained from a subject, with at least one antigen, and with at least one non-reducing sugar, and

(b) detecting the presence or an increase in the level of an immunological effector molecule.

The presence (or absence) or an elevated level of the immunological effector molecule is indicative of the level or capacity of the subject's cell-mediated immune response. In particular, said method makes it possible to determine whether said subject has previously encountered the antigen or with an antigen of which the antigen tested is representative. This can determine whether the subject is capable of eliciting a cell-mediated immune response against said antigen. In certain embodiments, the quantitative level of cell-mediated immune responsiveness can also be determined. The magnitude of the cell-mediated immune response detected in the assay described herein can be correlated, in certain embodiments, with a disease state, with the progression and / or severity of a disease. Thus, the present disclosure provides a means of determining cell-mediated immune responsiveness in a subject. The described method allows and / or supports, among others, the diagnosis of diseases, in particular infectious diseases, pathological conditions, allows to determine the level of immunocompetence and allows the evaluation of the response capacity of immune cells to endogenous or exogenous agents, as well like protein poisons. The assay also makes it possible to select or monitor subjects previously exposed to a particular antigen, such as an antigen associated with a disease, an infection, or a contaminant. Other important applications and utilities of this method will be described below.

The individual method steps and preferred embodiments of the method according to the first aspect of the invention will now be explained in detail.

Stage (a)

In step (a), a sample comprising immune cells capable of producing immune effector molecules after stimulation with an antigen is contacted with at least one antigen and with at least one non-reducing sugar. The immune cells and / or the whole sample can be obtained, for example, from a subject whose cell-mediated immune responsiveness is to be determined.

Reference to a "subject" includes, for example, a human or a non-human species, including primates, cattle (for example, sheep, cows, pigs, horses, donkeys, goats), laboratory test animals (for eg, mice, rats, rabbits, guinea pigs, hamsters), companion animals (eg, dogs, cats), avian species (eg, poultry, aviary birds), reptiles, and amphibians. The method described herein can be applied to research, human medicine, as well as livestock, veterinary and wildlife applications. Preferably, the subject is human and the cell-mediated immune response method described herein is employed to screen for responsiveness to pathogenic microorganisms, viruses and parasites, disease conditions, potential for development or control of autoimmune disorders, monitoring a subject's response to an oncological challenge or immunotherapy, monitoring cell-mediated immunity against disease, and for determining the presence of any immunodeficiency or immunosuppression. The latter can occur, for example, due to certain drugs, including various chemotherapeutic agents. Alternatively, exposure to environmental protein poisons and contaminants can be determined using the method according to the present invention.

The sample comprises immune cells capable of producing immune effector molecules upon challenge with an appropriate antigen. "Immune cells" include, but are not limited to, lymphocytes, which include natural killer cells ("natural killers", NKs), T cells, B cells, macrophages and monocytes, dendritic cells, or any other immune cell that is capable of producing one or more immunological effector molecules in response to direct or indirect stimulation with an antigen. Preferably, the sample comprises lymphocytes, more preferably T lymphocytes. The terms "T cell" and "T lymphocytes" are used interchangeably herein. T cells are capable of eliciting a strong immune response if they recognize the offered antigen. If the T cells have previously been exposed to the tested antigen or to an antigen of which the tested antigen is representative, rapid restimulation of the memory T cells specific to that antigen occurs. These antigen-specific T cells respond by secreting immune effector molecules, such as, in particular, interferon gamma. Interferon gamma, or an immunological effector molecule released in response to interferon gamma released, can then be measured as a specific marker of immune responsiveness against the antigen tested. Thus, according to one embodiment, the sample comprises T lymphocytes, preferably CD4 + helper T cells and / or CD8 + cytotoxic T cells. Preferably, the sample also comprises stimulator cells, in particular antigen presenting cells that are capable of presenting the tested antigen to T cells. However, suitable antigen presenting cells can also be added separately to the incubation composition. The respective added antigen presenting cells (APCs) include natural and artificial antigen presenting cells or particles. For example, optionally stimulator cells, such as HLA-matched antigen-presenting cells or irradiated autologous, can be added separately to the incubation composition, which then presents the antigen to the T cells. This embodiment is possible, for example, if the sample does not comprise the respective stimulator cells necessary to induce a T cell response. Artificial antigen presenting embodiments include, but are not limited to, lipid particles or vesicles with recombinant MHC peptides or molecules and associated recombinant costimulatory molecules.

Preferably, the sample is obtained from a subject. According to one embodiment, the sample is a body fluid comprising immune cells or it is an immune cell containing a portion derived from a respective body fluid. According to a preferred embodiment, the sample is whole blood. "Whole blood" means blood from a subject that has not been substantially diluted or fractionated. According to one embodiment, the whole blood sample is peripheral blood. Although whole blood is the preferred and most convenient sample for determining cell-mediated immune response activity, other samples containing immune cells can also be used. Examples include, but are not limited to, lymphatic fluid, brain fluid, tissue fluid (such as bone marrow or thymus fluid), and respiratory fluid, including nasal and pulmonary fluid and bronchioalveolar lavage. Portions or derivatives of the above-mentioned samples may also be sampled, eg, unnecessary cell depleted samples to measure cell-mediated immune response, and this can be achieved by processing the sample. For example, whole blood can be treated to remove components unnecessary for the MIC response, such as erythrocytes and / or platelets, by methods known in the art, or it can be processed to enrich for leukocytes. Buffy coat cells or peripheral blood mononuclear cells (PBMC) can also be obtained by methods known in the art and can be used as a sample. According to one embodiment, cultured immune cells are used as a sample. Furthermore, cryopreserved cells, eg cryopreserved PBMC cells, can also be used as a source of immune cells from the subject and thus as a sample. For example, thawed PBMC cells can be contacted with a culture medium to provide the sample comprising immune cells, which is then contacted and incubated with an antigen and the non-reducing sugar, which are preferably added in the form of a unique composition. According to one embodiment, the sample comprises all the immune cells necessary to mediate a cellular immune response. However, as described above, stimulator cells, in particular antigen presenting cells, can also be added separately within the scope of the present invention. According to one embodiment, the sample comprises at least T cells (T lymphocytes) and NK cells (NK lymphocytes). According to one embodiment, the sample is not diluted more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 3% before contacting the sample with the antigen and / or non-reducing sugar.

The sample to be analyzed is contacted with at least one antigen and with at least one non-reducing sugar to provide an incubation composition.

The term "antigen" as used herein refers, in particular, to any molecule or agent that is capable of stimulating or restimulating an immune response and, in particular, that is capable of stimulating or restimulating an immune response. mobile. Therefore, the term "antigen" is used in a broad sense. The term refers, in particular, to any molecule or agent that can bind to a major histocompatibility complex (MHC) and that can be presented to a T-cell receptor or can be bound by an antibody. The term can also refer to any molecule or agent that can bind to non-classical MHC proteins, such as, for example, CD1d or other members of the CD1 family. According to one embodiment, the antigen is an immunogen. According to one embodiment, the antigen is not an immunogen. Antigens include, but are not limited to, peptides, proteins, haptens, allergens, or toxins, or any natural or synthetic molecule or one of its parts. According to one embodiment, the antigen is an inactive pathogen or a portion thereof or its lysates. According to one embodiment, the antigen is selected from the group consisting of peptides, proteins, including glycoproteins, carbohydrates, phospholipids, phosphoproteins, phospholipoproteins, and their fragments. The term "peptide" as used herein also includes polypeptides and proteins, unless the context indicates otherwise. The term "protein" also includes modified forms, such as glycoproteins and phosphoproteins. According to one embodiment, the antigen comprises one or more full-length or part-length peptides. According to one embodiment, the antigen is provided by a peptide. According to one embodiment, said one or more peptides used as antigen have a selected length of 5 to 100 amino acids, preferably 7 to 50 amino acids. According to one embodiment, the antigen is provided by a set of peptides of one or more different full-length or part-length peptides. A set of peptides comprises at least two peptides and includes, in one embodiment, a series of overlapping or non-overlapping peptides. A respective set of peptides can cover the full length or a part of the natural protein antigen. However, the peptides need not be overlapping or may overlap by a single amino acid or multiple amino acids. According to one embodiment, a set of peptides is employed that includes 80-100% of a natural protein or peptide antigen.

According to one embodiment, the antigen is provided by at least one peptide that is recognized by a CD8 + cytotoxic T cell. For this embodiment, the antigen is preferably provided by at least one peptide that is less than 15 amino acids in length, preferably 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, or 10 amino acids or less. Suitable size ranges for the respective peptide that is recognized by a CD8 + cytotoxic T cell include 7-14 amino acid residues, 7-13 amino acid residues, 8 to 12 amino acid residues, 8-11 amino acid residues, and 8 to 10 residues. amino acids. A set of peptides comprising or consisting of peptides that are recognized by CD8 + cytotoxic T cells can also be employed. Such peptides can include all or part of a protein antigen, such as a natural protein antigen. Assays incorporating the respective short peptides as antigens have been found to show a significant decrease in assay activity during storage in the presence of simple sugars, such as glucose or dextrose. This is not observed when non-reducing sugars are used, as disclosed by the present invention. At present, the sensitivity of the assay remains high due to the incorporation of the non-reducing sugar, even after a long shelf-life of the assay components. This is an important advantage when using a composition comprising a peptide antigen that is recognized by a CD8 + cytotoxic T cell and a non-reducing sugar, for example, as a kit compound, respectively component.

According to one embodiment, the antigen is provided by at least two sets of peptides, the first set comprising at least one peptide with a length of about 7 to 14 amino acids, and a second set comprising at least one peptide of 15 amino acid residues or more. , wherein said peptides include all or part of a protein antigen. Each respective set comprises from at least one peptide to a series of overlapping or non-overlapping peptides. Co-incubation with peptides of 7 to 14 amino acids and peptides of> 15 amino acids derived from or corresponding to a protein antigen representative of the disease or disorder to be tested with the immune cells comprised in the sample produces a more sensitive assay. , thereby allowing earlier detection of stimulation of immune cells and, in particular, lymphocytes, than with other possible ways. The ability to increase the sensitivity of a cell-mediated immune response assay advantageously lowers the detection limit and / or allows the use of less sensitive means to detect effector molecules. Therefore, the use of at least two sets of the respective peptides in combination with a non-reducing sugar is beneficial. Although no limitation is intended by theory or mode of action, it is believed that the two sets of peptides, peptides 7 to 14-mer and peptides> 15-mer, allow detection by CD4 + and CD8 + T cells. CD4 + T cells recognize> 15-mer peptides and CD8 + T cells recognize 7 to 14-mer peptides. These peptides may be referred to herein as "CD4 + peptides"(> 15-mer peptides) or "CD8 + peptides" (7 to 14-mer peptides). Each set comprises at least one peptide and includes, in one embodiment, a series of overlapping peptides. Thus, a first set may contain a series of overlapping peptides 7 to 14 amino acid residues in length. These peptides are recognized by CD8 + T cells (CD8 + peptides). A second set may contain a series of overlapping peptides more than 15 amino acid residues in length. These peptides are recognized by cytotoxic CD4 + T cells (CD4 + peptides). Both sets of peptides can cover the full length or a portion of a protein antigen, eg, a natural protein antigen representative of the disease or disorder being tested. The peptides need not be overlapping or may overlap by a single amino acid or multiple amino acids. Peptides include sets of peptides that include and thus cover 80-100% of a protein antigen. Del "80-100%" means 80, 81.82, 83, 84, 85, 86, 87, 88, 89, 90, 91.92, 93, 94, 95, 96, 97, 98, 99 or 100% . Reference to a series of overlapping peptides with a length of about 7 to 14 amino acid residues that include all or part of a protein antigen according to one embodiment means a peptide with a length of about 7 amino acid residues up to a maximum of 14 amino acid residues that in total, they range from each amino acid residue which, in total, includes all amino acid residues up to 6 amino acid residues of a protein antigen from its N-terminal end to its C-terminal end or one of its parts. Thus, if the length of a particular peptide is x amino acids, where x is approximately 7 to 14, then the degree of overlap between two consecutive peptides is x-1 to x-6. In one embodiment, the overlap of each consecutive peptide is x-1. A series of overlapping peptides> 15 amino acid residues in length also span all or part of a protein antigen, where each peptide is at least 15 amino acids long or up to the length of the entire protein antigen. In one embodiment, a peptide> 15 amino acid residues in length is 15 to 50 amino acids, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

The present disclosure includes the case where each peptide in the series or set of peptides has the same length (ie, x). However, the peptide array or peptide pool may comprise a mixture of x ¹ , x ² , xj ... xi peptides in which, according to one embodiment, each of the xi peptides is about 7 to 14 amino acid residues or a length of> 15 amino acid residues. CD4 + and / or CD8 + peptides can be divided into different peptide groupings. They can be added separately to the sample or they can be included in a composition, preferably together with the non-reducing sugar. This composition can then be contacted with the sample to prepare the incubation composition.

In some embodiments, one or more antigens are employed that mimic one or more of the effects of the antigens presented to the immune system in vivo. According to one embodiment, the antigen is selected from an autoantigen, an antigen derived from an antigen or cross-reactive with an antigen derived from a pathogenic organism, a metal or inorganic molecule that stimulates an immune response, or a tumor-associated antigen. According to one embodiment, the antigen is derived from and therefore cross-reactive with an antigen, derived from a pathogen associated with a disease disorder, or is a tumor associated antigen associated with cancer, or is or is derived of a poison. According to one embodiment, the sample is contacted with an antigen that is specific for the disease or disorder for which the cell-mediated immune response is to be tested, for example, an antigen associated with or that is representative of a disease or disorder to be tested. According to one embodiment, the antigen is a disease-specific antigen, in particular a pathogen-specific antigen. In some embodiments, the pathogen is a bacterium, virus, parasite, or fungus. In an illustrative embodiment, the antigen is an antigen from a mycobacterium, in particular Mycobacterium tuberculosis. Thus, in some embodiments, the antigen is a tuberculosis (TB) specific antigen. For example, the antigen can be a protein derivative purified from Mycobacterium tuberculosis or M. avium. In some embodiments, the antigen mimics mycobacterial proteins, such as ESAT-6, CFP-10, and TB7, respectively TB7.7. In another illustrative embodiment, the antigen is derived from or specific to a virus, such as cytomegalovirus (CMV).

Other examples of antigens, in particular disease-specific antigens, are also described below.

Furthermore, the sample is contacted in step a) with a non-reducing sugar. A "non-reducing sugar" refers, in particular, to a sugar that does not react with a detection reagent for reducing sugars, such as Fehling's solution, Benedict's reagent, or Tollens reagent. A non-reducing sugar does not comprise a free reducing end and consequently does not comprise a free aldehyde or free ketone group. The non-reducing sugar can be of any length and can be linear or branched. In certain embodiments, the non-reducing sugar comprises at least two monosaccharide units. According to one embodiment, in any and all monosaccharide units of the non-reducing sugar, the carbon atoms next to the oxygen atom in the ring structure do not comprise a hydroxyl group and therefore do not comprise an anomeric hydroxyl group. According to one embodiment, the ring structures of the monosaccharide units of the non-reducing oligosaccharide do not comprise a hemiacetal or hemiketal group. According to one embodiment, the non-reducing sugar is an oligosaccharide comprising 10 monosaccharide units or less, more preferably 8 monosaccharide units or less, 6 monosaccharide units or less, 5 monosaccharide units or less, 4 monosaccharide units or less, 3 monosaccharide units or less, or 2 monosaccharide units. Preferably, the non-reducing sugar is a disaccharide. According to one embodiment, glycosidic linkages are formed between monosaccharide units by joining the reducing end of one monosaccharide unit with the reducing end of another monosaccharide unit. Preferred examples of the non-reducing sugar are sucrose and trehalose. Furthermore, as shown in the examples, mannitol and raffinose can also be used as the non-reducing sugar. Thus, according to one embodiment, the non-reducing sugar is selected from trehalose, mannitol, sucrose, and raffinose. As demonstrated in the examples, these examples of non-reducing sugars increase the magnitude of the response. Trehalose is particularly preferred because experiments have shown that trehalose increases the magnitude of the response and thus can increase the sensitivity of the assay and, furthermore, a composition comprising trehalose and an antigen shows excellent shelf stability. As demonstrated in the examples, among the non-reducing sugars tested, the most potent effect-enhancing response was obtained with trehalose. However, the non-reducing sugar can also be a monosaccharide in which, for example, the reducing end is coupled to another chemical entity and thus blocked. Consequently, the non-reducing sugar can be derivatized. Examples of derivatives of sugars are amino sugars, in which one or more hydroxyl groups are substituted by an amino group or an acetylamino group. In preferred embodiments, the non-reducing sugar is unsubstituted and, in particular, it is not derivatized. According to one embodiment, the non-reducing sugar is not a polysaccharide. In certain embodiments, the non-reducing sugar is not bound to a protein, peptide or lipid, or other macromolecule. According to one embodiment, the non-reducing sugar is not comprised in a cell culture medium or other medium. According to one embodiment, the non-reducing sugar is not comprised in a liquid. Non-reducing sugar can be metabolized by the immune cells included in the sample. According to one embodiment, sugar does not "reducing" is a non-reducing sugar which, when present in an appropriate concentration in the incubation composition comprising the sample and the antigen, is capable of enhancing the release of interferon gamma by restimulated T cells.

Contacting the sample with the antigen, non-reducing sugar, and optionally other additives provides an incubation composition. Preferably, said incubation composition is incubated above room temperature and, therefore, at elevated temperatures. Preferably, the incubation temperature is greater than 30 ° C, preferably greater than 35 ° C. Suitable ranges for the incubation temperature include 30 ° C to 40 ° C, preferably 35 ° C to 40 ° C. Conveniently, the incubation composition is incubated at 37 ° C / -1 ° C. Preferably, the incubation composition is incubated for at least 2 hours at said elevated temperatures to allow the stimulation of the immune cells by the antigen and the production of immune effector molecules. The incubation stage can last from 2 to 50 hours, such as 2 to 40 hours, 5 to 30 hours, 8 to 24 hours, 16 to 24 hours, or a period of time in between these, which includes 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 hours. In some embodiments, after an optional initial mixing step to distribute the antigen (s), non-reducing sugar, and sample through the incubation composition, incubation is performed without remixing.

In the incubation composition, the non-reducing sugar is present in a concentration where it is effective to enhance the stimulation, respectively the restimulation of the cells of the immune system by the antigen. Therefore, the non-reducing sugar is added at a concentration where, in a positive sample, that is, a sample that is immunoreresponsive to the antigen, the addition of the non-reducing sugar to the incubation composition increases the level of immunological effector molecules produced. , preferably gamma interferon, compared to the case where the non-reducing sugar is not added. Thus, the non-reducing sugar is added at a concentration where it enhances the magnitude of the response of immune effector molecules, since more immune effector molecules are produced in a sample exhibiting a cell-mediated immune response. Preferably, the non-reducing sugar is employed at a concentration in which it enhances the response of immune effector molecules by at least 1.1 times, preferably by at least 1.2 times, more preferably by at least 1.3 times. According to one embodiment, the non-reducing sugar maintains the ability of the immune cells comprised in the sample to mediate a respective response over extended periods of time. According to one embodiment, the concentration of the non-reducing sugar in the incubating composition is at least 1 mg / ml, at least 1.5 mg / ml, preferably at least 1.75 mg / ml, more preferably at least 2 mg / ml. ml. Examples of ranges include, but are not limited to, 1 mg / ml to 20 mg / ml, 1.5 mg / ml to 17.5 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 15 mg / ml, 4 mg / ml to 12.5 mg / ml, and 5 mg / ml to 10 mg / ml. As the examples show, these ranges are suitable, for example, for trehalose. They are also suitable for other non-reducing sugars, such as sucrose, mannitol and raffinose, as demonstrated in the examples. According to one embodiment, the concentration of the non-reducing sugar in the incubation composition is in a range from 1.5 mg / ml to 10 mg / ml, for example, from 1.75 mg / ml to 7.5 mg / ml. , or from 2 mg / ml to 5 mg / ml. Those skilled in the art can also determine suitable concentrations by following the guidelines described herein.

One or more additional additives can be added and are therefore included in the incubation composition. For example, one or more additives that are necessary or advantageous for sample preparation and / or sample preservation may be added, such as, for example, a suitable anticoagulant if the sample is a blood sample. Preferably the anticoagulant is heparin. Additives should not be at a concentration where they can interfere with the cell-mediated immune response. According to one embodiment, no simple sugar is added to the incubation composition in addition to the non-reducing sugar. According to one embodiment, no reducing sugar, in particular a reducing monosaccharide, is added to the incubation composition in addition to the non-reducing sugar.

According to one embodiment, the sample is contacted with a composition comprising the antigen and the non-reducing sugar. This embodiment is particularly advantageous in that the user does not have to add the non-reducing sugar separately to the incubation composition. Providing these ready-to-use compositions avoids handling errors and saves personnel. Optionally, a diluent or a solvent is comprised in the composition comprising the antigen and the non-reducing sugar. Furthermore, one or more additives may be included in said composition if they are to be included in the incubation composition. Additives should not interfere with the cell-mediated response. According to one embodiment, the composition further comprises an anticoagulant, preferably heparin. According to one embodiment, the composition does not comprise a simple sugar. According to one embodiment, the composition does not comprise a reducing sugar, in particular it does not comprise a reducing monosaccharide.

To prepare the incubation composition, the composition comprising the antigen, the non-reducing sugar, and optionally one or more additional additives, such as an anticoagulant in the case of a blood sample, is contacted with the sample. The sample can be added to the composition or vice versa. To prepare the incubation composition, the sample, the antigen, the non-reducing sugar and the additional additive (if present) are preferably mixed.

Examples of suitable composition forms include liquid compositions, semi-liquid compositions, gel-type compositions and solid compositions, in particular dried compositions. According to one embodiment, the composition comprising the antigen, the non-reducing sugar, and optionally an additional additive, is comprised in a sample collection container, preferably a sample collection tube, such as a blood collection tube. This is particularly convenient since the sample is directly contacted with the composition after collection. According to one embodiment, the composition comprising the antigen, the non-reducing sugar and optionally an additional additive is spray dried into the sample collection container. Spray drying methods are well known in the prior art and, therefore, a detailed description is not necessary here.

According to one embodiment, the sample is obtained from a subject and is not diluted, such as, for example, with tissue cultures, media, excipients, or other liquid agents prior to contacting the non-reducing sugar and / or antigen. According to one embodiment, the incubation composition comprises at least 10% by volume of the sample. The expression "at least 10% by volume" includes sample volumes of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100% by volume of the total volume of the incubation composition.

As shown in the examples, the addition of a non-reducing sugar in step a) surprisingly increases the release of immunological effector molecules, such as, in particular, interferon gamma, thereby increasing the sensitivity of the assay. This effect is very surprising, since it was believed that this effect can only be achieved with simple sugars and therefore reducing monosaccharides. Furthermore, it has been found that compositions comprising the antigen and a non-reducing sugar show better shelf stability even at elevated temperatures. The effect observed with reducing sugars that the sensitivity and quality of the assay may decrease over time is not observed with non-reducing sugars. Thus, the present invention makes an important contribution to the prior art by providing a shelf-stable and sensitive assay that also maintains assay performance over extended shelf-lives. The indications of the present invention conveniently allow the antigen and the non-reducing sugar to be provided in the form of a shelf-stable composition. In the present invention, the non-reducing sugar is not used as a stabilizer for the antigen. Through experiments, antigens, in particular peptide antigens, have been shown to be very stable during storage. Therefore, no decrease in assay performance is observed if the antigen is preserved in the absence of a sugar. Therefore, non-reducing sugar is used to enhance the sensitivity of the assay, in particular by enhancing the production and / or release of immunological effector molecules, in particular cytokines, such as gamma interferon.

Stage (b)

In step (b) the presence or an increase in the level of an immunological effector molecule is detected. As described above, the presence (including the absence) or the level of an immunological effector molecule is indicative of the level or capacity of the subject's cell-mediated immune response to the antigen tested. In particular, said method makes it possible to determine whether said subject has previously encountered the antigen tested or with an antigen that shows cross-reactivity with the antigen tested, such as the pathogen to be detected. Hereby it can be determined whether the subject is capable of eliciting a cell-mediated immune response against said antigen, respectively the antigen, pathogen or disease for which the antigen tested is representative.

Detection of the immunological effector molecule can occur at the peptide or protein level or at the nucleic acid level, in particular at the level of mRNA expression of the immunological effector molecule. Consequently, the reference to detecting "the presence or level" of the immunological effector molecule includes direct and indirect data. For example, the presence or quantity of immunological effector molecules can be determined directly using appropriate detection methods, such as ELISA or ELISpot. However, in one embodiment, the presence or level of the immunological effector molecule is measured based on the level of expression of its RNA. Elevated levels of the mRNA of the immune effector molecule are indirect data showing an increase in the levels of the immune effector molecule. Suitable methods for determining the expression level of an mRNA of a target gene are well known in the prior art and, therefore, a detailed description is not necessary. Accordingly, in some embodiments, the immunological effector molecule can be detected using ligands or binding molecules, such as antibodies specific for the effector molecule, or by measuring the level of expression of genes encoding the immunological effector molecule.

The immunological effector molecules to be detected can be any of a range of molecules that are produced in response to activation, stimulation, or restimulation of cells by an antigen. Furthermore, in step (b) more than one immunological effector molecule or a pattern of immunological effector molecules released after contact of the sample with the tested antigen can be detected. The immune effector molecule to be measured can be produced by immune cells, in particular it can be produced by lymphocytes, such as T cells, in particular CD4 + helper T cells and / or CD8 + cytotoxic T cells. Thus, in some embodiments, the method is based on measuring the production of one or more immunological effector molecules by the cells of the immune system, particularly T cells, in response to antigenic stimulation. However, also non-immune cells can release immune effector molecules in response to a stimulation, respectively a restimulation, of the immune cells by the antigen, since they are stimulated by the immune effector molecules that are released by the immune cells, in particular by immunological effector molecules, such as IFN-gamma, released by restimulated T cells. These immune effector molecules can also be an important source of information. Thus, according to one embodiment, the immunological effector molecule to be detected may be the immediate effector molecule produced by the effector T cells in response to restimulation with an antigen. In other embodiments, a downstream immune effector molecule is measured. For example, IFN-gamma or other immediate immune effector molecules produced by immune cells, particularly T cells that are (re) stimulated by the antigen tested, can be measured in step (b). However, as described above, these molecules often induce or enhance the production of other immune effector molecules by other cells. The production of these other immunological effector molecules (downstream) can also be measured in step (b). The present invention also includes detecting more than one type of immunological effector molecule in step (b). According to one embodiment, the presence or level of a pattern of immunological effector molecules is detected in step (b), both individually and in addition to the immediate immunological effector molecules, such as IFN-gamma. A respective standard comprises more than two, preferably more than three different immunological effector molecules. Analysis of a respective pattern can provide valuable information on the immune status of the subject. For example, specific immunological effector molecules or patterns of specific immunological effector molecules can be characteristic of specific diseases.

According to one embodiment, the immunological effector molecule to be measured in step (b) is a cytokine, such as a lymphokine, an interleukin, or a chemokine. An interferon (IFN), such as IFN-gamma, is particularly useful as an immunological effector molecule to be determined. Other examples of immunological effector molecules include, but are not limited to, a range of cytokines, such as interleukins (IL), eg IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10 , IL-12, IL-13, IL-16 (LCF) or IL-17, IL-1a (IL-1 F1), IL-1 p (IL-1F2), IL-1 ra (IL-1F3), tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-p), colony stimulating factor (CSF), such as granulocyte CSF ((G) -CSF) or granulocyte macrophage CSF ((GM ) -CSF), complement component 5a (C5a), Groa (CXCL1), slCAM-1 (CD54), IP-10 (CXCL10), l-TAC (CXCL11), MCP-1 (CCL2), MIF (GIF) , MIP-1a (CCL3), MIP-1 p (CCL4), Serpin E1 (PAI-1), RANTES (CCL5) or MIG (CXCL9). In some embodiments, the present invention provides methods in which the immunological effector molecule to be detected in step (b) is a cytokine, a component of the complement system, perforin, defensin, cathelicidin, granzyme, Fas ligand, CD-40 ligand, exotaxin, a cytotoxin, a chemokine, or a monokine. In preferred embodiments, the immunological effector molecule detected in step (b) is IFN-gamma. Thus, according to a preferred embodiment, the present disclosure provides a method for measuring a cell-mediated immune response in a subject, said method comprising collecting a sample from said subject into a collection container, wherein said sample comprises cells of the immune system. that are capable of producing IFN-gamma after being stimulated with an antigen, incubating said sample with an antigen and a non-reducing sugar, and then measuring the presence or an elevation in the level of an IFN-gamma, in which the presence or the level of IFN-gamma is indicative of the ability of said subject to mount a cell-mediated immune response.

Furthermore, in step (b) a combination of immunological effector molecules can be detected.Thus, step (b) comprises, in particular, detecting an immunological effector molecule or a combination of immunological effector molecules, in particular cytokines, released in response to the stimulation with the antigen and which are characteristic of the disease or disorder to be tested. Furthermore, the level of said one or more immunological effector molecules can be selected individually or in combination with other biomarkers or disease indicators.

According to one embodiment, the immunological effector molecule is detected using a ligand that specifically binds to the immunological effector molecule. Immunological effector ligands are particularly useful for detecting and / or quantifying these molecules. Cells comprised in the incubation composition can be removed prior to detecting the immune effector molecule. Techniques for detection assays that can be used in step (b) are known in the art and include, for example, radioimmunoassays, "sandwich" assays, ELISA, and ELISpot. Antibodies against immunological effectors are particularly useful as ligands. Reference to "antibodies" includes portions of antibodies that specifically bind to the immunological effector molecule, such as Fab fragments, mammalianized (eg, humanized) antibodies, deimmunized antibodies, recombinant or synthetic antibodies, and hybrid and single chain antibodies. Polyclonal and monoclonal antibodies can be obtained by immunization with the immunological effector molecules or their antigenic fragments, and either type can be used in immunoassays. Methods for obtaining both types of antibodies are well known in the art. Polyclonal antibodies are less preferred, but are relatively easily prepared by injecting into a suitable laboratory animal an effective amount of the immune effector, or an antigenic portion thereof, collecting serum from the animal, and isolating the specific serum by any of the known immunoadsorbent techniques. . Although the antibodies produced by this method can be used in almost any type of immunoassay, are generally less appreciated due to the potential heterogeneity of the product. The use of monoclonal antibodies in an immunoassay is particularly useful due to the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for the production of monoclonal antibodies by fusing an immortal cell line and sensitized lymphocytes against the immunogenic preparation can be accomplished by techniques well known to those of skill in the art. Antibodies against specific immune effector molecules are also commercially available.

According to one embodiment, step (b) comprises contacting the incubation composition, or one of its portions, such as, for example, a cell-depleted portion thereof, with an antibody, or one of its fragments, specific for the immunological effector molecule to be detected, for a time and under conditions sufficient for an antibody-effector complex to be formed, and then to detect said complex. As described above, cells comprised in the incubation composition can be removed, for example, by centrifugation, prior to detection. For example, when blood is used as a sample, cells can be separated from the incubation composition after incubation, and thus the production and release of immunological effector molecules occurs prior to detection, thereby basically providing a plasma sample.

A wide range of immunoassay techniques are available, as can be seen by referring to US Patent Nos. 4,016,043, 4,424,279, and 4,018,653. The respective assays that can be used in conjunction with the cell-mediated immune response assays described herein to detect the immune effector molecules produced are also described in WO2004 / 042396, WO2008 / 113119, WO2010 / 009494 and WO2011 / 075773. Also in Clay et al., Assays for monitoring cellular immune responses to active immunotherapy of cancer, Clinical Cancer Research, 2001, 7: 1127-1135, various methods are described to monitor cellular immune responses, therefore suitable assays are also described to detect immunological effector molecules produced in response to (re) stimulation with an antigen. This article describes, for example, ELISA-based assays, ELISpot assays, and nucleic acid-based assays, such as measurement of cytokine mRNA levels by quantitative real-time RT-PCR. Optionally, when the level of an immunological effector molecule is determined based on the level of expression of its RNA, the data obtained can be normalized to the expression of a control gene, such as, for example, CD8. The respective methods can also be used in conjunction with the present invention to detect the immune effector molecules produced. According to one embodiment, a nucleic acid-based assay is employed to detect the presence or level of an immunological effector molecule. Nucleic acids, in particular RNA, can be isolated from the incubation composition, or its cellular portion, using standard methods well known in the prior art. Preferably, the presence or increased expression of the immunological effector molecule is detected in this embodiment using amplification-based assays, preferably PCR-based assays. The isolated RNA can first be reverse transcribed to produce cDNA prior to amplification using primers and / or probes specific for the immunological effector molecule to be detected. Preferably, the detection is quantitative. A suitable method is real-time reverse transcription PCR (RT-PCR).

Preferably, the detection of the immunological effector molecule in step (b) is a quantitative detection.

Specific realizations

Specific and preferred embodiments of the method according to the present invention and description and the components used therein will be described below.

As described above, in step (a) one or more additional additives may be included in the incubation composition. According to one embodiment, an agent is added to the incubation composition to modulate the activity of regulatory T cells (T-reg cells). This includes inhibiting the suppressor function of T-reg cells. Agents that modulate T-reg cells included herein include, but are not limited to, a CD25 ligand, a sense or antisense oligonucleotide with respect to a genetic material encoding JAK1 or TYK2, a neutralizing antibody, an oligonucleotide containing CpG, an oligonucleotide that acts as a toll-like receptor (TLR) modulating agent, and other TLR modulating agents. In a particular embodiment, the T-reg cells are immune response suppressor cells whose activity is inhibited by the modulating agent. A "CpG molecule" means an oligonucleotide comprising a CpG sequence or motif. Examples of inhibitors or modulators of T-reg function include CD25 ligands including, but not limited to, a polyclonal or monoclonal antibody against CD25, or one of its antigen-binding fragments, humanized or deimmunized polyclonal or monoclonal antibodies. against CD25, or a recombinant or synthetic form of the polyclonal or monoclonal antibodies. Other examples of agents include sense or antisense nucleic acid molecules targeted to mRNA or DNA encoding Janus tyrosine kinase 1 (JAK1) or tyrosine kinase 2 (TYK2) or small molecule inhibitors of the JAK1 or TYK2 proteins. Reference to "small molecules" includes novel immunoglobulin antigen receptors (IgNARs), as described in International Patent Publication No. WO 2005/118629. Other examples of suitable agents are stimulating agents, such as CpG molecules that act through Toll-like receptors (TLRs) and / or other mechanisms. Thus, oligonucleotides containing CpG and an oligonucleotide acting as an agent TLR modulator are also part of the present description. A single type of agent can be used or two or more types of agents can be used to modulate T-reg cells. For example, the assay can be performed with a CD25 ligand and a JAK1 / TYK2 sense or antisense oligonucleotide; a CD25 ligand and a TLR modulating agent; a JAK1 / TYK2 sense or antisense oligonucleotide and a TLR modulating agent; or a CD25 ligand, a JAK1 / TYK2 sense or antisense oligonucleotide, and a TLR modulating agent. Alternatively, only one type of agent is used. In another alternative, an oligonucleotide comprising CpG and a TLR modulating agent is employed. The respective T-reg modulating agents can be added separately to the sample and / or the incubation composition or they can be included in the composition comprising the antigen and / or the non-reducing sugar.

According to one embodiment, the method according to the present invention comprises:

(a) preparing an incubation composition by contacting a whole blood sample obtained from a human subject with a composition comprising at least one peptide antigen and at least one non-reducing disaccharide, preferably trehalose or sucrose, and incubating the composition of incubation for at least 2 hours above room temperature; Y

(b) detecting the presence or level of IFN-gamma;

wherein the presence or level of IFN-gamma is indicative of the level of cell-mediated immune responsiveness of the human subject.

Preferably, the peptide antigen is a disease or pathogen specific antigen. Non-limiting examples of pathogens are described herein. According to one embodiment, the pathogen is a virus and the method makes it possible to determine whether the human subject has the ability to mount an antiviral cell-mediated immune response.

According to one embodiment, the method further comprises:

(c) comparing the level of the detected immunological effector molecule, or a value derived therefrom, with a reference level.

Step (c) comprises comparing the level of the determined immunological effector molecule, or a value derived therefrom, with a reference level. This embodiment is particularly useful for diagnosing an infection with a pathogen for which the antigen is representative. If said subject is infected by the pathogen, the level of the immunological effector molecule determined, or a value derived from it, is above the reference level, and if said subject is not infected by the pathogen, the level of the effector molecule immunological test, or a value derived from it, is below the reference level. The same principle applies to determining whether said subject is capable of mounting a cell-mediated immune response against a pathogen for which the antigen is representative.

According to one embodiment, the sample is divided into at least two fractions, and in step (a), the first fraction of the sample is contacted with an antigen and a non-reducing sugar to generate a response sample, and in the that the second fraction of the sample is contacted with an inactive solution to generate a negative (null) control sample. In step (b) of this embodiment, the presence or level of the immunological effector molecule in the two fractions is determined. In step (c), the antigen-dependent immune effector molecule response is determined by subtracting the level of the immunological effector molecule determined in the negative control sample from the level of the immunological effector molecule determined in the response sample. The antigen-dependent immune effector molecule response, or a value derived therefrom, can then be compared to a reference level, thereby providing an aid in determining whether the subject has previously encountered the antigen. With this, it can be determined, for example, whether the subject has generated an immunological reactivity against the antigen, whether he is at risk of developing a disease and / or whether he is susceptible to infection with a pathogen.

Optionally, the method may further comprise dividing the sample into at least three fractions and incubating the third fraction of the sample with a T cell activator, such as a mitogen, to generate a positive control sample. In this case, the immune cells can be incubated, for example, in three different populations: a negative control sample (for example, saline), an antigen-stimulated response sample, and a positive control sample (using, for example , a T-cell activator, eg, a mitogen, such as phytohemagglutinin). Thus, according to one embodiment, the sample is divided into at least three fractions. In step (a), the first fraction of the sample is contacted with an antigen and a non-reducing sugar to generate a response sample, the second fraction of the sample is contacted with an inactive solution to generate a sample. negative control, and the third sample fraction is contacted with a stimulation solution (comprising, for example, a mitogen) to generate a positive control sample. In step (b) of this embodiment, the presence or level of the immunological effector molecule in the three fractions is determined. In step (c), the antigen-dependent immune effector molecule response of the response sample is determined by subtracting the level of the immunological effector molecule determined in the negative control sample from the level of the immunological effector molecule determined in the sample. response, and comparing the antigen-dependent immune effector molecule response, or a value derived therefrom, with a reference level; The immune effector molecule response of the positive control sample is determined by subtracting the level of the immunological effector molecule determined in the negative control sample from the level of the immunological effector molecule determined in the positive control sample, and comparing the response of the resulting immunological effector molecule with a reference level, or a value derived therefrom. This can determine whether the subject has previously encountered the tested antigen or an antigen that shows cross-reactivity with the tested antigen, and thus has generated immunological reactivity against the antigen. This provides a valuable aid in determining, for example, whether the subject has an active, recent, or latent infection, or whether the subject is responding to treatment, is going to develop an infection or disease, or is immunosuppressed. For example, if the response of the antigen-dependent immunological effector molecule is above the reference level, the result can be evaluated as positive, that is, the subject is capable of producing a cell-mediated response against said antigen. If the response of the antigen-dependent immune effector molecule is below the reference level and the response of the positive control-dependent immune effector is above the reference level, the result can be evaluated as negative, that is, the subject is not capable of producing a cell-mediated response against said antigen.

Mitogens that can be used in the present invention as a positive control include all mitogens known to those of skill in the art and include, but are not limited to, phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), and phytolac mitogen. (PWM). Other examples of immunostimulants, in addition to mitogens, that can be used to provide a positive control include, but are not limited to, chemical compounds, such as R848.

As described above, in some embodiments, the container in which the sample, antigen, and non-reducing sugar are co-incubated is also the collection container used to collect the sample from the subject. Any of a large number of different containers available may be employed, provided they provide adequate dimensions for the sample. In some embodiments, the container is a tube that comprises a vacuum to facilitate collection of the sample, such as blood, from a subject. The respective vacuum blood collection tubes are well known in the prior art and, therefore, a detailed description is not necessary here. In other embodiments, the container is a capillary tube. In some embodiments, a capillary tube is used to collect blood from the surface of the skin by capillary action. In some embodiments, the sample is collected from a subject into a collection container that contains at least one antigen, at least one non-reducing sugar, and at least one anticoagulant, preferably heparin, or to which an antigen, a non-reducing sugar, is subsequently added. reducing agent and an anticoagulant, preferably heparin. In some embodiments, blood samples are taken using a capillary sampling device, such as a lancing device, and the blood is filled into a heparinized collection container and then transferred to an appropriate container for co-incubation with the antigen. and non-reducing sugar. Preferably, the antigen, the non-reducing sugar, and optionally the anticoagulant, are provided in the form of a single composition, as described above. In some embodiments, whole blood from a subject is collected into a container containing the antigen, the non-reducing sugar, and optionally the anticoagulant. In other embodiments, the antigen, non-reducing sugar, and / or anticoagulant are added to the whole blood sample after collection.

The present invention is particularly useful in screening for exposure to pathogens, in particular mycobacteria, such as M. tuberculosis. Therefore, the present description discloses a method for measuring the activity of cell-mediated immune response in a subject, said method comprising contacting a sample comprising immune cells capable of producing immunological effector molecules after stimulation with an antigen, with a tuberculosis-specific antigen and with a non-reducing sugar. The peptide or peptides used as antigen may include all or part of a M. tuberculosis protein antigen. After incubation, the level of the immune effector molecule, preferably gamma interferon, produced by the immune cells is determined, wherein the level of the immune effector molecule is indicative of the level of cell-mediated immune responsiveness to M tuberculosis.

ESAT-6 is a 6 kDa early secreted antigenic target of M. tuberculosis. The ESAT-6 protein ("early secreted antigenic target 6") is an important secreted antigen that has been purified from filtrates of short-term cultures of M. tuberculosis. As indicated in the present ESAT-6, CFP-10 ("culture filtrate protein 10", culture filtrate protein 10) and 85B can be obtained from the lysate and purification of cells, by means of recombinant techniques or they can be produced as peptides. synthetics. For example, ESAT6 can be obtained as a recombinant protein from Statens Serum Institute (SSI, Copenhagen, Denmark). Other suitable target protein antigens for M. tuberculosis include TB7.7 and TB37.6. CFP10 is also known as the ESAT-6-like protein eesxB and secreted antigenic protein MTSA-10.

Tuberculin or PPD ("purified protein derivative") differs from ESAT-6 (early secreted antigenic target 66), CFP-10 (culture filtrate protein 10), and TB7.7, which are encoded by genes (in the RD-I region) located only within the genome of M. tuberculosis and are not contained in BCG (Bacillus Calmette-Guerin). It differs from PPD in that PPD also contains other antigens that are shared, for example, with substrains of BCG and with several non-tuberculous mycobacterial species with low or no pathogenicity. According to one embodiment, PDD is used as the antigen. Specifically, as used herein, the term "Purified Protein Derivative" (PPD or tuberculin) is a precipitate of non-species specific molecules. PPD or tuberculin is obtained by extracting proteins from a mixture of M. tuberculosis or other mycobacteria, such as M. avium. PPD is commonly used to test for the presence of cellular immunity or the Th1 response generated against BCG or against M. tuberculosis. For example, it can be obtained from Connaught Laboratories Limited's TubersolB prepared from a larger master batch, Master Batch, Connaught Tuberculin (CT68), or as RT23 obtained from Statens Serum Institute (SSI, Copenhagen, Denmark).

Preferably, the antigen is selected from CFP10, ESAT-6, TB7.7 and TB37.6 of Mycobacterium tuberculosis. According to one embodiment, the antigen is provided by peptides that correspond to these protein antigens and / or that show cross-reactivity with them.

In one embodiment, the sample is contacted with a combination of CD4 + and CD8 + peptides, which have been described in detail above. It refers to the previous description.

Cells of the immune system lose the ability to mount a cell-mediated immune response in whole blood after long periods after blood is drawn from the subject, and responses without intervention are often severely reduced or absent by 24 hours after drawing blood. The reduction of work and the need for specialized equipment in the present invention allows for a stimulation of the immune response mediated by cells with antigens in the healthcare setting, such as doctor's offices, clinics, outpatient facilities and veterinary or farm clinics. . After antigen stimulation has been completed, fresh, active cells are no longer required. IFN-gamma and other cytokines or immunological effector molecules released due to stimulation with antigen are stable in cell-depleted or cell-depleted fluids, such as plasma, and thus the sample can be stored or transferred without special conditions or requirements for quickly in a manner similar to conventional plasma or serum samples used for other diagnoses of infectious or other diseases. Therefore, detection of the actual released immune effector molecules is preferred. However, the cells comprised in the incubation composition or the entire incubation composition may, after incubation, be contacted with a nucleic acid stabilizing composition comprising reagents that stabilize the RNA expression pattern, if the presence or level of the immunological effector molecule based on the level of RNA expression of the immunological effector molecule. Various stabilizer compositions are available on the market. For example, PreAnalytiX provides compositions containing reagents for immediate stabilization of the RNA gene expression profile in blood. The respective compositions can also be used to stabilize the RNA gene expression profile of the cells comprised in the incubation composition. The respective stabilizing composition allows transport and storage at room temperature without the risk of changes in the RNA profile by inducing genes and degradation of transcripts (see, for example, US 6,617,170, US 7,270,953, Kruhoffer et al., 2007). The respective compositions are marketed under the name PAXgene Blood RNA Tubes.

Applications, diseases and disorders

In the following, non-limiting applications will be described, including uses, diseases and disorders. As will be apparent from this, the method according to the invention, as well as the compositions and kits described herein, can be widely used in the medical and diagnostic field and can be used, for example, in in vitro assays suitable for analyzing the cell-mediated responsiveness of patients. Furthermore, the method according to the invention, as well as the compositions and kits described herein, are valuable analytical tools for testing the ability of agents, such as, for example, cancer immunotherapeutics, to stimulate and thus enhance the cell-mediated immunity.

The method disclosed herein allows, for example, the detection of the presence or absence, or the level or stage of a disease or disorder in a subject, such as an infection by a pathogen, an autoimmune disease, cancer, exposure to an inflammatory agent, exposure to a drug, exposure to a toxic protein agent, and immunodeficiency or immunosuppression disorders, such as those induced by a disease disorder or induced by pharmaceutical agents. The ability to reliably and sensitively measure cell-mediated immunity is important, for example, to assess a subject's ability to respond to infection by a pathogen, such as a microorganism, virus, or parasite, to mount an autoimmune response, to respond to a vaccine or immunotherapeutic product, to protect against cancers or other oncological disorders, to detect an inflammatory disorder, or to detect a subject's exposure or sensitivity to a toxic agent, such as beryllium or environmental agents. The assay described herein allows for early and / or more sensitive detection of immune responsiveness. The assay described herein also enables and aids in the detection of disease disorders leading to immunosuppression or to detect drug-induced immunosuppression. Accordingly, "measuring a cell-mediated immune response in a subject" as disclosed herein has many useful applications in the medical field and will be described below. non-limiting applications and uses.

For example, the method described herein can be used for the immunodiagnosis of infectious and autoimmune diseases, as a marker for immunocompetence, as well as a marker for inflammatory diseases, allergens, cancer, the effect of immunotherapeutics, and as a marker for toxic agents. Furthermore, the method is useful, in general, for the detection of T cell responses to endogenous and / or exogenous antigens (including measuring the efficacy of a vaccine).

Cell-based functional immune response assays are also accepted as surrogate markers of efficacy in the development of vaccines, immunotherapeutics, and biologics that impact immune responses. The assays can be used in academic settings, in pharmaceutical settings, and in the research and development of vaccines and biologics. Assessing the functional capacity of immune cells is critical to understanding various disease disorders and the efficacy of therapeutic strategies targeting them. Immune cells are responsible for prevention or therapy, eg, in infectious diseases, such as HIV, or are responsible for the disease disorder itself, eg, autoimmune disease disorders. The specific reactivity of autoantigens can be measured in patients affected by an autoimmune disease, such as multiple sclerosis, using the method according to the present invention. The present invention provides assays that are useful for these purposes.

Many cancer immunotherapy strategies are currently being tested in clinical trials. Although clinical efficacy would be the final test of these strategies, the long and complicated path of the development of these articles necessitates the evaluation of the immune responses as intermediate markers of the most probable candidates for their success. This emphasizes the need for assays that accurately detect and quantify antigen-specific immune responses mediated by T cells. For the respective immunotherapeutic agents that, for example, are not necessarily expected to cause tumor regression, but that continue to have a beneficial effect on the disease, a biological marker should be chosen based on the presumed mode of activity. For immunotherapy, this marker is the stimulation of the tumor antigen-specific immune response that can be detected by one or more immunological assays. Herein, assays evaluating the function of CD8 + cytotoxic T cells that directly recognize tumor peptides presented by MHC molecules on the surface of a tumor cell are preferably employed as an activator for direct cytolysis and T cell responses. CD4 + helpers, in particular type 1 helper T cells, which lead to the generation of cytotoxic T cells. The present invention provides assays that are useful for this purpose because of their sensitivity and reliability. Thus, the method, kits, and compositions described herein can be used to test whether a pharmaceutical agent, such as, for example, a cancer immunotherapeutic, is capable of enhancing cell-mediated immunity. This can be tested, for example, on a general level using available, for example, standardized immune cells (the examples of which have been described above) or it can also be tested in an individual patient to analyze whether the specific immunotherapeutic product, such as, for example, a cancer immunotherapeutic product, is capable of enhancing cell-mediated immunity in said patient. This assay is valuable for clinical development, as well as in the subsequent therapeutic setting, to analyze whether a patient would benefit from therapy with an immunotherapeutic. Examples of immunotherapeutic products include, but are not limited to, biological products, such as therapeutic antibodies that enhance the activity of T cells, particularly cytotoxic T cells. The mode of action is not important, as long as it produces or is supposed to produce a stimulation / enhancement of cell-mediated immunity. For example, activity can be enhanced by direct stimulation of immune cells or by reducing or turning off inhibitory mechanisms that negatively affect the activity of such T cells, thereby indirectly stimulating / increasing cell-mediated immunity. An example is the cancer immunotherapeutic antibody ipilimumab.

Furthermore, the method according to the present invention can be used to detect the presence, absence, level or stage of a disease or disorder in a subject, wherein the presence or level of the immunological effector molecule detected using the method according to the present invention is indicative of the disease or disorder.

Furthermore, the method according to the present invention can be used to determine whether an agent or disease disorder induces or is associated with immunosuppression in a subject, wherein the presence or level of the immunological effector molecule detected using the method according to The present invention is indicative of the degree of immunosuppression induced by the agent or induced or associated with the disease disorder.

One aspect of the present application includes methods that demonstrate the cell-mediated immune responsiveness of a subject by measuring responsiveness to a particular antigen using the method according to the present invention. In one embodiment, a sample, such as whole blood, a leukocyte-enriched fraction, or a bronchioalveolar lavage may be obtained from a subject suffering from or suspected of developing a particular disease (eg, an autoimmune disease, a disease caused by by an infection with a pathogen, or exposure to a toxic agent) and the immune response is measured using the method of the present invention, for example, by detecting immunological effector molecules released from effector T cells (eg, CD4 + T cells and / or CD8 + cytotoxic T cells) in response to stimulation with antigen. The method of the present invention is particularly useful for detecting and / or monitoring a disease or disorder, including the level or stage of the disease or disorder in a subject, such as an infection with a pathogen, an autoimmune disease, cancer or an inflammatory disorder. Other disorders include exposure to toxic agents, such as beryllium. The assay of the present invention is also useful for monitoring therapeutic protocols.

The presence of the level of the immunological effector molecule produced in response to the antigen tested is indicative of the level of the subject's cell-mediated immune responsiveness. In particular, the level of responsiveness is indicative of the presence or absence or the level or stage of a disease or disorder selected from the list comprising an infection by a pathogen, an autoimmune disease, a cancer, a inflammatory disorder, and exposure to a toxic agent. In particular, the presence or level of the detected immunological effector molecule is indicative of the presence, absence, level or stage of a disease or disorder for which the antigen tested is representative.

The method can also be used to monitor a response to a therapeutic protocol for a disease or disorder in a subject. The presence, level, or pattern of the immunological effector molecule produced may be indicative of the efficacy of the therapeutic protocol.

The method of the present invention can also be referred to as a "test". The method is an ex vivo method. The assay described herein is useful, among other things, for assessing the general immune responsiveness of a subject or is useful for detecting responsiveness to specific disease disorders, such as autoimmune disease, celiac disease, cancer or infection by a pathogenic organism or agent, exposure to a toxic agent or drug, and immunodeficiency or immunosuppression disorders, such as those induced by a disease disorder or by a therapeutic agent.

In one embodiment, the subject is a human and the cell-mediated immune response assay is used to screen for responsiveness to pathogenic microorganisms, viruses, and parasites, the potential to develop an autoimmune disorder, or to monitor an autoimmune disorder, disease. celiac disease, monitor a subject's response to an oncological challenge and to determine the presence of any immunodeficiency or immunosuppression. The latter can occur, for example, due to certain drugs, including various chemotherapeutic agents. Alternatively, exposure to environmental poisons and pollutants can be tested. In one embodiment, disease disorders that lead to immunosuppression include chronic infection and cancer. Other disease disorders that can lead to immunosuppression include inflammatory disease disorders. Medications that can lead to immunosuppression include those used to treat rheumatoid arthritis, cancer, and inflammatory bowel disease or those given in conjunction with an organ transplant.

In one embodiment, the method described herein can be used for the diagnosis or monitoring, or as an aid thereto, of subjects suspected of having tuberculosis (eg, an active, latent or recent TB infection) and, in particular, in patients with an increased risk of progression from latent tuberculosis to active tuberculosis, for example, in patients receiving immunosuppressive medication (specifically, a treatment with monoclonal antibodies (anti-CD20 antibody (for example, Rituximab ©) or a blocking treatment TNF-alpha (for example, Remicade ©, Enbrel ©, Humira ©))) or steroids or cancer chemotherapy, or patients suffering from immunosuppressive disorders (for example, HIV infection, cancer, IDDM or non-insulin dependent diabetes mellitus (NIDDM), autoimmune disorders, malnutrition, aging, intravenous drug use (IVDU), or inherited immune disorders), and in individuals who have recently been infected two.

In one embodiment, the method described herein can be used to monitor subjects diagnosed with infections or other disease disorders. This can help, for example, to evaluate the effectiveness of treatment during and after the completion of a treatment, for example, by monitoring and predicting the possible recurrence of infection.

According to one embodiment, the method is to determine whether a subject is infected and / or capable of mounting a cell-mediated immune response against a pathogen for which the antigen is representative.

Pathogens or infections include bacteria, parasites, and viruses. Examples of bacteria include Gram positive and Gram negative microorganisms, such as Mycobacterium species, Staphylococcus species, Streptococcus species, Escherichia coli, Salmonella species, Clostridium species, Shigella species, Proteus species, Bacillus species, of Hemophilus, Borrelia species, among others. Mycobacterium tuberculosis is a particularly useful target, as well as disorders arising from M. tuberculosis infection, such as tuberculosis (TB). Examples of viruses include the hepatitis virus (hepatitis B virus and hepatitis C virus), herpes virus and CMV virus, human immunodeficiency virus (HIV), as well as the diseases that result from these. Parasites include Plasmodium species, ringworm, liver parasites, and the like. Other pathogens include eukaryotic cells, such as yeast and fungi. In general, it is important to assess the potential or actual cell-mediated responsiveness in subjects. exposed to these infectious entities. The method of the present disclosure can also be used to detect the presence or absence of these infections, as well as the level or stage of the disease process.

The method can also be used to monitor the level of cell-mediated immunity against certain diseases and / or infectious agents in people who are at risk of developing the respective disease. An example is a CMV infection in immunosuppressed patients, such as organ transplant patients. CMV infections frequently appear as a complication of immunosuppression, particularly after transplantation, and contribute significantly to morbidity and mortality in transplant recipients. Current immunosuppressive therapies used to prevent rejection of a transplanted organ have detrimental effects on T lymphocytes and, therefore, on cell-mediated immune responses, producing a greater susceptibility to viral infections after transplantation. The importance of T cell function and the suppression of CMV replication is also emphasized by the fact that CMV-specific CD8 + cytotoxic T lymphocytes (CTL) can protect against virus-associated pathogenesis. Another example of immunosuppressed patients is HIV-infected patients. The method of the present invention can be used to serially monitor the level of disease immunity, such as, for example, anti-CMV immunity, in people at risk of developing a respective disease, since the loss of immune function it can be associated with the development of a disease, such as CMV disease. Preferably, interferon gamma is determined as the effector molecule in a respective assay. The immune status of the transplant recipient can influence the (re) activation of CMV in transplant recipients. For example, a robust gamma-interferon response induced by a CMV-specific antigen indicates a lower risk of CMV disease, since the patient exhibits a strong cell-mediated response and thus is protected against the virus. A minimal gamma interferon response indicates an increased risk of CMV disease, since there is little or no cell-mediated immunity. The data demonstrate that patients with a positive CMV test remain free from CMV disease significantly more often and for longer than patients with a negative test after cessation of antiviral prophylaxis. Thus, patients who present a cellular immune response to CMV at the end of prophylaxis have a significantly lower risk of developing CMV disease than those who do not present a detectable immune response. Thus, the method according to the present invention can predict the development of late-onset CMV disease in transplant recipients and is therefore very useful in treating patients.

Alternatively, the subject may present or may be tested for a disease disorder selected from celiac disease, autoimmune diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, multiple sclerosis, autoimmune disease of the adrenal gland, hemolytic anemia. autoimmune, autoimmune hepatitis, autoimmune oophoritis and orchitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome (CFIDS), demyelinating chronic inflammatory polyneuropathy, Churg-Straus syndrome, scar pemphigoid, CREST syndrome cold agglutinin disease, Crohn's disease, dermatitis herpetiformis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura openia purpura ”, ITP), IgA nephropathy, insulin-dependent diabetes (type I), lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, personae syndrome , systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and inflammatory bowel disease.

Alternatively, the subject can have or can be tested for cancer. Cancer therapy is also dependent to some extent on cell-mediated immunity, and the cancer itself or the drugs used to treat cancer can lead to immunosuppression. Cancers contemplated herein include a group of diseases and disorders that are characterized by uncontrolled cell growth (eg, tumor formation) without differentiation of these cells into specialized and different cells. These diseases and disorders include ABL1 proto-oncogene, AIDS-related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft tissue sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brainstem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small and round cell tumor, ductal carcinoma , endocrine cancers, endometrial cancer, ependymoma, cancer is Ophagic, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal carcinoid tumor, cancers genitourinary, germ cell tumors, gestational trophoblastic disease, glioma, gynecological cancers, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer male breast, malignant rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplasia syndromes, myeloma, myeloproliferative diseases, nasal cancer, nasopharyngeal cancer , nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen rupture syndrome, cancer of a type other than melanoma, breast cancer Non-small cell lung cancer (NSCLC), eye cancers, esophageal cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, paranasal cancer, parathyroid cancer, cancer of the parotid gland, cancer of the penis, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, rare cancers and associated disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, cancer of the glands salivary, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), cancer of the small intestine, soft tissue sarcoma, spinal cord tumors, squamous cell carcinoma (skin ), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional cell cancer (bladder), transitional cell cancer (renal-pelvic / ureter), cancer r trophoblastic, urethral cancer, urinary system cancer, uroplakines, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms tumor.

Alternatively, the subject is exposed or can be tested for exposure to a protein venom.

Autoimmune diseases contemplated herein for detection and / or control include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, multiple sclerosis, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, Autoimmune oophoritis and orchitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue syndrome (CFIDS), chronic demyelinating inflammatory polyneuropathy, Churg-Straus syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatitis herpetiformis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy (insulin-dependent diabetes type I), Lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, primary polymyositis and dermatomyositis, primary agammaglobulinemia , psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff person syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis and vitiligo.

Other contemplated disease disorders include inflammatory disease disorders, since they can lead to immunosuppression. Examples of inflammatory disease disorders contemplated by the present disclosure include, but are not limited to, diseases and disorders that arise in response to redness, swelling, pain, and a sensation of heat in certain areas that are intended to protect the tissues affected by injury or disease. Inflammatory diseases that can be treated using the methods of the present disclosure include, but are not limited to, acne, angina, arthritis, aspiration pneumonia disease, empyema, gastroenteritis, inflammation, intestinal flu, NEC, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, PID, pleurisy, strep throat, flushing, flushing, sore throat, stomach flu and urinary tract infections, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy. In terms of non-human applications, the present disclosure extends to detecting EIPH in horses and various disorders in animals, such as facial tumor disease in Tasmanian devil.

In the above aspects, the antigen may be derived from a pathogen, it may be associated with the disease disorder or cancer, or it may be a poison. Alternatively, infection, disease disorder, cancer, or poison can suppress cell-mediated immunity, in which case any antigen to which the subject has been previously exposed can be employed.

The particularly advantageous embodiments described in the description and in the claims of the method according to the first aspect are described again below. According to the first aspect, the present description provides a method for measuring a cell-mediated immune response activity, said method comprising:

(a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon challenge with an antigen, with at least one antigen, and with at least one non-reducing sugar, and

(b) detecting the presence or level of at least one immunological effector molecule.

According to one embodiment, the non-reducing sugar is a non-reducing disaccharide, preferably selected from trehalose and sucrose. The concentration of the non-reducing sugar in the incubation composition is, according to one embodiment, at least 1.5 mg / ml, preferably at least 2 mg / ml. Suitable ranges for the concentration of the non-reducing sugar in the incubation composition were also described above and indicated in the above description.

According to one embodiment, the non-reducing sugar is selected from trehalose, mannitol, sucrose, and raffinose. As demonstrated in the examples, these non-reducing sugars increase response levels. Trehalose is particularly preferred because the greatest increase in response level was observed here. Furthermore, the enhancing effect can be maintained during storage.

According to one embodiment, in step (a), the sample is contacted with a composition comprising the antigen and the non-reducing sugar. According to one embodiment, when the sample is a whole blood sample, the composition further comprises an anticoagulant, preferably heparin. According to one embodiment, the composition comprising the antigen, the non-reducing sugar, and optionally an anticoagulant, is comprised in a sample collection container. As described herein, such a sample collection container may be, for example, a vacuum blood collection container. According to one embodiment, the composition is a spray-dried composition. Suitable embodiments are described herein. According to one embodiment, the composition has one or more of the following characteristics:

i) the sample is obtained from a human subject;

ii) the sample is obtained from a human subject who is immunosuppressed or immunodeficient;

iii) the sample comprises immune cells selected from the group consisting of NK cells, T cells, B cells, dendritic cells, macrophages and monocytes; me

iv) the sample is whole blood.

According to one embodiment, the antigen is selected from the group consisting of peptides, proteins, including glycoproteins, phosphoproteins, and phospholipoproteins, carbohydrates, phospholipids, and fragments of the foregoing, and is preferably provided by one or more peptides.

According to one embodiment, two or more different antigens are used in step (a) and / or two or more different effector molecules are detected in step (b).

According to one embodiment, one or more peptides are employed as antigens having a selected length of 5 to 100 amino acids, or 7 to 50 amino acids. According to an advantageous embodiment, the antigen is provided by one or more peptides that are recognized by a CD8 + cytotoxic T cell. Preferably, in this embodiment, the antigen is provided by one or more peptides that are less than 15 amino acids in length, preferably that are selected 7-14 amino acids in length. The details of the respective peptides are described above and are indicated in the previous description.

According to one embodiment, the antigen is provided by at least two sets of peptides, the first set comprising at least one peptide with a length of about 7 to 14 amino acids, and a second set comprising at least one peptide of 15 amino acid residues or more. , wherein said peptides include all or part of a protein antigen. The details of the respective sets of peptides are described above and are indicated in the description above.

According to an advantageous embodiment, the antigen is provided by one or more synthetic peptides. The details of the peptide (s) are described above and are indicated in the description above.

According to one embodiment, the sample is contacted with an antigen associated with or representative of a disease or disorder for which the cell-mediated immune response is to be tested. According to an advantageous embodiment, the antigen is a disease-specific antigen, in particular a pathogen-specific antigen. For example, the antigen can be derived and thus can be cross-reactive with an antigen from a pathogen associated with a disease disorder or is a tumor associated antigen associated with a cancer. As described above, the pathogen can be a bacterium, a virus, a parasite, a yeast, or a fungus. Next, non-limiting examples will be described. For example, the bacterium can be selected from Gram positive and Gram negative microorganisms, in particular Mycobacterium species, such as Mycobacterium tuberculosis, Staphylococcus species, Streptococcus species, Escherichia coli, Salmonella species, Clostridium species, Shigella species, Proteus, Bacillus species, Hemophilus species, and Borrelia species. The virus can be selected from hepatitis viruses, such as hepatitis B virus and hepatitis C virus, herpes virus, CMV virus, and human immunodeficiency virus (HIV). The parasite can be selected from species of Plasmodium, ringworm and parasites liver

According to one embodiment, the antigen is derived from or specific to a virus, preferably cytomegalovirus (CMV). Preferably, said antigen is provided by one or more peptides having a length of 7 to 14 amino acid residues, 7 to 13 amino acid residues, or 8 to 12 amino acid residues. As described above, said one or more peptides can be synthetic peptides.

According to one embodiment, the immunological effector molecule detected in step (b) has one or more of the following characteristics:

i) is a cytokine;

ii) is a chemokine;

iii) occurs in response to activation, stimulation or restimulation of cells by antigen;

iv) is selected from the group consisting of interferons, interleukins (IL), tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-p), colony stimulating factor (CSF), such as CSF from granulocytes ((G) -CSF) or granulocyte macrophage CSF ((GM) -CSF), complement component 5a (C5a), Groa (CXCL1), slCAM-1 (CD54), IP-10 (CXCL10), l- TAC (CXCL11), MCP-1 (CCL2), MIF (GIF), MIP-1a (CCL3), MIP-1P (CCL4), Serpin E1 (PAI-1), RANTES (CCL5) or MIG (CXCL9); me

v) the immunological effector molecule is IFN-gamma.

According to one embodiment of the method, the presence or the level of an immunological effector molecule is determined based on the immunological effector molecule or is determined based on the expression level of RNA of the immunological effector molecule. Suitable embodiments were described above.

According to one embodiment, the method according to any of the above embodiments is to monitor or determine the presence, absence, level or stage of a disease or disorder selected from the group consisting of an infection by a pathogen, an autoimmune disease , a cancer, an inflammatory disorder, exposure to a toxic agent, response to a therapeutic agent, an immunodeficiency, and an immunosuppression. Preferably, the magnitude of the cell-mediated immune response correlates with the status, progression, and / or severity of a disease disorder. According to one embodiment, the method according to any of the above embodiments is for detecting or controlling a disease, an infection and / or the response to a therapy, in particular an immunotherapy or a therapy with immunosuppressive agents.

According to one embodiment, the method according to any of the previous embodiments comprises:

(a) providing an incubation composition by contacting a whole blood sample obtained from a human subject with a composition comprising at least one peptide antigen and at least one non-reducing disaccharide, preferably trehalose or sucrose, and incubating the composition of incubation for at least 2 hours; Y

(b) measuring the presence or level of IFN-gamma released due to stimulation with the antigen;

wherein the presence or amount of IFN-gamma detected is indicative of the level of cell-mediated immune responsiveness of the human subject.

According to one embodiment, the method according to any of the previous embodiments comprises:

(c) comparing the level of the determined immunological effector molecule, or a value derived therefrom, with a reference level.

The present description further provides a method of treating a subject suffering from a pathogenic infection, an autoimmune disorder or a cancer or a propensity to develop said disorder or disease, said method comprising performing the method for measuring cell-mediated response activity according to the first aspect, wherein the presence or the determined level of the immunological effector molecule is indicative of the level of the subject's cell-mediated responsiveness, which is indicative of the presence, absence, level or status of the disorder or condition, and then treating the disorder or condition. The details of the method according to the first aspect were described above and indicated in the above description.

Compositions, kits and uses

Claim 14 defines a composition for inducing a cell-mediated immune response, comprising:

a) at least one isolated peptide antigen having a selected length of 5 to 100 amino acids; b) at least one non-reducing sugar capable of increasing the interferon-gamma response of antigen-responsive immune cells; Y

c) at least one anticoagulant.

Also described is a composition for inducing a cell-mediated immune response in a sample, comprising:

a) at least one isolated antigen;

b) at least one non-reducing sugar;

c) optionally at least one anticoagulant.

Details regarding the composition, the form of the composition, antigens, non-reducing sugars and suitable anticoagulants were described above in conjunction with the method according to the present invention and are indicated in the above description. Said composition can be used, for example, in the method according to the first aspect. Suitable applications, including uses / targets, diseases and disorders that can be analyzed using said composition and, consequently, suitable uses of said composition are described in detail above and indicated in the description above. The composition is particularly suitable for use in the methods, applications and uses described above. According to one embodiment, the composition does not comprise a simple sugar. According to one embodiment, the composition does not comprise a reducing sugar. Preferably, the composition is a semi-liquid, is gel-like, or is a solid composition. Preferably it is a dried composition. According to one embodiment, the composition is a spray-dried composition.

Advantageous non-limiting embodiments are again described.

The non-reducing sugar may have the characteristics described above together with the method according to the first aspect and indicated in the above description, which also apply at this point. As described above, the non-reducing sugar is not used as a stabilizer for the antigen, but is used to increase the level of response. As shown in the examples, non-reducing sugars, such as trehalose, mannitol, sucrose, and raffinose, increase response levels. According to one embodiment, the non-reducing sugar is a non-reducing disaccharide. The non-reducing disaccharide can be selected from trehalose and sucrose. The concentration of the non-reducing sugar in the composition according to the second aspect is, according to one embodiment, such that when said composition is contacted with the intended amount of sample, the resulting incubation composition comprises the non-reducing sugar in a concentration which is at least 1.5 mg / ml, preferably at least 2 mg / ml. Suitable ranges for the concentration of the non-reducing sugar in the incubation composition were also described above in conjunction with the method according to the first aspect and are indicated in the above description. Suitable and preferred sample materials are also described above in conjunction with the method according to the first aspect and are indicated in the above description. According to one embodiment, the composition has one or more of the following characteristics:

i) the sample is obtained from a human subject;

ii) the sample is obtained from a human subject who is immunosuppressed or immunodeficient;

iii) the sample comprises immune cells selected from the group consisting of NK cells, T cells, B cells, dendritic cells, macrophages and monocytes; me

iv) the sample is whole blood.

According to one embodiment, the antigen comprised in the composition is selected from the group consisting of peptides, proteins, including glycoproteins, phosphoproteins and phospholipoproteins, carbohydrates, phospholipids, and fragments of the foregoing, and is preferably provided by one or more peptides.

According to one embodiment, the composition comprises two or more different antigens.

According to one embodiment, the composition comprises one or more peptides as antigens, wherein said one or more peptides have a length selected from 5 to 100 amino acids, or from 7 to 50 amino acids. According to an advantageous embodiment, the composition comprises as antigen one or more peptides that are recognized by a CD8 + cytotoxic T cell. Preferably, in this embodiment, the antigen is provided by one or more peptides that are less than 15 amino acids in length, preferably that are selected 7-14 amino acids in length. The details of the respective peptides were described above together with the method according to the first aspect and are indicated in the above description.

According to one embodiment, the composition comprises an antigen that is provided by at least two sets of peptides, the first set comprising at least one peptide with a length of about 7 to 14 amino acids, and a second set comprising at least one peptide of 15 amino acid residues or more, in the that said peptides include all or part of a protein antigen. The details of the respective sets of peptides were described above together with the method according to the first aspect and are indicated in the above description.

According to an advantageous embodiment, the composition comprises an antigen that is provided by one or more synthetic peptides. The details of the peptide (s) are described above and are indicated in the description above.

According to one embodiment, the composition comprises an antigen associated with or representative of a disease or disorder for which the cell-mediated immune response is to be tested.

According to an advantageous embodiment, the antigen comprised in the composition is a disease-specific antigen, in particular a pathogen-specific antigen. For example, the antigen can be derived and thus cross-reactive with an antigen from a pathogen associated with a disease disorder or is a tumor associated antigen associated with a cancer. As described above, the pathogen can be a bacterium, a virus, a parasite, a yeast, or a fungus. Next, non-limiting examples will be described. For example, the bacterium can be selected from Gram positive and Gram negative microorganisms, in particular Mycobacterium species, such as Mycobacterium tuberculosis, Staphylococcus species, Streptococcus species, Escherichia coli, Salmonella species, Clostridium species, Shigella species, Proteus, Bacillus species, Hemophilus species, and Borrelia species. The virus can be selected from hepatitis viruses, such as hepatitis B virus and hepatitis C virus, herpes virus, CMV virus, and human immunodeficiency virus (HIV). The parasite can be selected from Plasmodium species, ringworm, and liver parasites.

According to one embodiment, the composition comprises an antigen that is derived from or specific to a virus, such as cytomegalovirus (CMV). Preferably, said antigen is provided by one or more peptides having a length of 7 to 14 amino acid residues, 7 to 13 amino acid residues, or 8 to 12 amino acid residues. As described above, said one or more peptides can be synthetic peptides.

The composition may be comprised in a sample collection container and suitable and advantageous embodiments of such a container, such as a vacuum blood collection container, are described herein and indicated in the respective description.

Claim 21 defines an incubation composition comprised in a sample collection container, the incubation composition comprising:

a) an undiluted sample obtained from a subject, the sample comprising immune cells capable of producing immune effector molecules after stimulation by an antigenic peptide, preferably T lymphocytes; b) at least one isolated peptide antigen having a selected length of 5 to 100 amino acids;

c) at least one non-reducing sugar capable of increasing the interferon-gamma response of immune cells that respond to the antigen; Y

d) optionally an anticoagulant.

The present invention also discloses an incubation composition, comprising at least one isolated antigen, at least one non-reducing sugar, immune cells capable of producing immune effector molecules after stimulation with an antigen, preferably T lymphocytes, and optionally at least one anticoagulant. . Preferably, the incubation composition is prepared from and therefore comprises a whole blood sample. According to one embodiment, the incubation composition is prepared by contacting a composition according to the second aspect with a sample. Suitable and preferred sample materials were described above in conjunction with the method according to the first aspect and are indicated in the above description. According to one embodiment, the sample has one or more of the following characteristics:

i) the sample is obtained from a human subject;

ii) the sample is obtained from a human subject who is immunosuppressed or immunodeficient;

iii) the sample comprises immune cells selected from the group consisting of NK cells, T cells, B cells, dendritic cells, macrophages and monocytes; me

iv) the sample is whole blood.

Preferably, the incubation composition is obtained by contacting a composition according to the second aspect with a whole blood sample obtained from a subject, preferably obtained from a human. The details regarding the composition according to the second aspect, the incubation composition, its preparation, the antigens, non-reducing sugars and suitable anticoagulants were described above together with the method according to the present invention and the composition according to the second aspect and are indicated on the previous description. The anticoagulant is preferably heparin. According to one embodiment, the incubation composition comprises the non-reducing sugar in a concentration of at least 1.5 mg / ml, preferably at least 2 mg / ml. Suitable ranges for the concentration of the non-reducing sugar in the incubation composition were also described above in conjunction with the method according to the first aspect and are indicated in the above description. According to one embodiment, the non-reducing sugar is selected from trehalose, mannitol, sucrose, and raffinose. As described above, according to an advantageous embodiment, the non-reducing sugar is a non-reducing disaccharide, preferably selected from trehalose and sucrose.

Claim 16 defines a sample collection container, comprising the composition according to claim 14.

Also described is a sample collection container, in particular a sample collection tube, comprising a respective composition comprising:

a) at least one isolated antigen;

b) at least one non-reducing sugar;

c) optionally at least one anticoagulant.

The composition comprised in the sample collection container is preferably the composition according to the second aspect. Details regarding composition, antigens, non-reducing sugars, and suitable anticoagulants are described above and indicated in the description above. Such a sample collection container can be used, for example, in the method according to the first aspect. Suitable applications, including uses / targets, diseases and disorders that can be analyzed using such a sample collection container are described in detail above and are indicated in the description above. The sample collection container according to the third aspect is suitable, in particular, for use in the methods, applications and uses described above. Preferably, the composition is sprayed onto the interior of the container which is preferably a vacuum blood collection tube. By using such ready-to-use collection containers in the method of the present invention, method conditions are optimized and standardized and handling is simplified.

The method according to the invention is preferably carried out using a kit that provides the materials necessary to carry out the steps of the method. This kit preferably comprises a standardized material that ensures that the method is performed under optimized conditions, thereby ensuring that the results obtained from different samples or patients or by different physicians can be compared with each other. Thus, in a fourth aspect, the present disclosure also provides a kit for measuring cell-mediated immune response activity in a subject, comprising at least one antigen, at least one non-reducing sugar, at least one collection container for samples and at least one detection means to detect at least one immunological effector molecule.

Claim 17 defines a kit for measuring cell-mediated immune response activity in a subject, comprising at least one peptide antigen having a selected length of 5 to 100 amino acids, preferably 7 to 50 amino acids, at least one sugar non-reducing agent capable of increasing the interferon-gamma response of antigen-responsive immune cells, at least one sample collection container and at least one detection means for interferon-gamma and / or an immune effector molecule whose production is induced or enhanced by interferon-gamma ..

Preferably, the antigen and the non-reducing sugar are provided as a single composition. For this purpose, the composition according to the second aspect can be used and the details of said composition are indicated in the above description. Details regarding the composition, antigen and non-reducing sugar were also described above together with the method and composition according to the present invention and are indicated in the above description. According to one embodiment, the kit comprises a sample collection container, such as a blood collection tube, comprising the composition comprising the antigen and the non-reducing sugar. Preferably, the composition further comprises an anticoagulant, such as heparin. As described, the composition comprised in the sample collection container can be the composition according to the second aspect. Preferably, the detection means is an immunodetection reagent, such as a labeled antibody. However, for assays that are based on the detection of the expression level of the mRNA, the means of detection can be provided by primers and / or probes specific for the immunological effector molecule to be detected. Suitable assays and means of detection are known to those of skill in the art and are also described above.

According to another aspect, the present invention relates to the use of a non-reducing sugar in an immunological assay to measure the activity of the cell-mediated response, in which the addition of the non-reducing sugar during the incubation of the sample with the antigen increases the release of an immunological effector molecule from immune cells that respond to the antigen tested in said assay.

Claim 19 defines the use of a non-reducing sugar in an immunological assay to measure the activity of cell-mediated response against a peptide antigen having a selected length of 5 to 100 amino acids, preferably 7 to 50 amino acids, wherein the addition of the non-reducing sugar increases the release of interferon-gamma from immune cells that respond to the peptide antigen tested in said assay, and wherein preferably the non-reducing sugar has one or more of the following characteristics: i) the non-reducing sugar is a disaccharide;

ii) the non-reducing sugar is selected from trehalose and sucrose;

iii) the non-reducing sugar is selected from trehalose, mannitol, sucrose and raffinose;

iv) the non-reducing sugar is trehalose;

v) the non-reducing sugar is used in a concentration of at least 1 mg / ml, preferably 2 mg / ml in the incubation composition comprising the sample to be tested and the antigen;

vi) the non-reducing sugar is provided in the form of a composition further comprising the antigen to be tested and, optionally, an anticoagulant;

vii) the addition of the non-reducing sugar during the incubation of the sample with the antigen increases the release of interferon-gamma from immune cells that respond to the peptide antigen tested in said assay.

Claim 20 defines the use of a non-reducing sugar in an immunological assay to measure the activity of cell-mediated response to increase the release of interferon-gamma from immune cells that respond to an antigen tested and said assay, optionally wherein the antigen and / or the non-reducing sugar have one or more of the characteristics defined in claim 15.

The details regarding the non-reducing sugar, the preferred concentrations, the immunological assay and its applications in the medical field, the suitable and preferred antigens, the immunological effector molecules and the immune cells were described above in conjunction with the method according to the present invention and are indicated in the description above. Non-reducing sugar can have one or more of the following characteristics:

i) the non-reducing sugar is a disaccharide;

ii) the non-reducing sugar is selected from trehalose and sucrose;

iii) the non-reducing sugar is used in a concentration of at least 1 mg / ml, preferably 2 mg / ml in the incubation composition comprising the sample to be tested and the antigen; me

iv) the non-reducing sugar is provided in the form of a composition further comprising the antigen to be tested and, optionally, an anticoagulant.

According to one embodiment, the non-reducing sugar is selected from trehalose, mannitol, sucrose, and raffinose.

The use of a non-reducing sugar, as described herein, for example trehalose, in an assay to determine or monitor cell-mediated immunity has significant advantages. As described above, preferably the non-reducing sugar is provided together with the antigen in the form of a single composition that is contacted with the sample. According to one embodiment, a reducing sugar is not added for incubation and / or is not comprised in the composition comprising the non-reducing sugar and the antigen. As described above, the non-reducing sugar is not used as a stabilizer for the antigen. According to an advantageous embodiment, the antigen is provided by one or more peptides, preferably synthetic peptides, which are recognized by a CD8 + cytotoxic T cell. Preferably, in this embodiment, the antigen is provided by one or more peptides, preferably synthetic peptides, which are less than 15 amino acids in length, preferably having a selected length of 7-14 amino acids.

According to another aspect, the present description refers to the use of the composition according to the second aspect, to a sample collection container according to the third aspect and / or to a kit according to the fourth aspect in an assay for measuring response activity mediated by cells. Details regarding suitable and preferred uses and applications are described above and indicated in the above description. According to one embodiment, the assay is to monitor or determine the presence, absence, level, or stage of a disease or disorder selected from the group consisting of a pathogen infection, an autoimmune disease, a cancer, a disorder. inflammatory, exposure to a toxic agent, response to a therapeutic agent, an immunodeficiency and an immunosuppression. According to one embodiment, the test is to detect or monitor a disease, an infection and / or the response to a therapy, in particular an immunotherapy or a therapy with immunosuppressive agents. According to one embodiment, the composition according to the second aspect, a sample collection container according to the third aspect and / or a kit according to the fourth aspect is used in the method according to the first aspect which is described in detail above and in the claims.

The numerical ranges described herein include the numbers that define the range. Headers provided herein are not limitations on the various aspects or embodiments of this invention, and may be read by reference to the specification as a whole. According to one embodiment, the matter described herein, comprising certain steps in the case of methods or comprising certain ingredients in the case of, for example, compositions, solutions and / or mixtures, refers to matter consisting of the respective steps or ingredients. It is preferred to select and combine the preferred embodiments described herein, and the specific matter arising from a respective combination of the preferred embodiments also pertains to the present disclosure.

The invention will be illustrated in more detail below by the following non-limiting examples.

Examples

Example 1: Effects of trehalose on the sensitivity of the Epstein-Barr virus assay

Investigations were conducted using QuantiFERON technology to measure responses to the Epstein-Barr virus EBNA-1 antigen model to assess increases in assay sensitivity induced by the addition of non-reducing sugars to the incubation mix. Briefly, EBNA-1 peptides were added to a blood collection tube plus / minus 3 concentrations of a trehalose solution (0.5, 1.0 or 2.0 mg / ml trehalose). The QuantiFERON assay measuring anti-EBV cellular immune responses was run according to the manufacturer's instructions. IFN-gamma response was determined and compared depending on trehalose concentration. An increase in IFN-gamma response was observed as the trehalose concentration increased. A concentration of 2 mg / ml of trehalose provided the best results in this test.

These studies demonstrate that the addition of a non-reducing sugar, such as trehalose, to the whole blood assay produced a significant increase in the IFN-gamma response in a cell-mediated immune response assay. Due to the increase in IFN-gamma response that is induced by the addition of the non-reducing sugar, the sensitivity of the assay is enhanced.

Example 2: Storage stability of a prior art assay

In this example, a cell-mediated immune response assay against CMV was performed and the shelf-life stability of the assay components was tested. Synthetic peptides 8-12 amino acids long from antigens associated with cytomegalovirus were used as antigens. The CMV-associated synthetic peptides were formulated into a solution containing a simple sugar (glucose) and sprayed onto the inside of a sample collection tube which was then dried. Therefore, the antigen and the simple sugar were comprised in a single composition. The stability test of said composition contained in the blood collection tube indicated that the activity of the test decreased throughout the storage time at room temperature. Furthermore, the assay activity of the CMV device is lost when the blood collection device comprising the spray-dried composition comprising the antigen and glucose is stored at 55 ° C for a period greater than 3 weeks (21 days). Therefore, the respective compositions and consequently the collection tubes comprising the respective compositions are not suitable for use as components of a ready-to-use kit due to lack of shelf stability. Removal of glucose from the composition restored the stability and function of the assay. Therefore, it is not feasible to include the simple sugar together with the antigen in a composition. Instead, to increase the sensitivity of the assay, simple sugar should be added separately to the sample during preparation of the incubation composition.

In contrast, compositions comprising the antigen and a non-reducing sugar as disclosed herein can maintain the highest assay sensitivity over long storage periods as well. These results were also confirmed by other experiments. CMV tubes containing or not glucose were stored for three weeks at 4 ° C, 22 ° C, 37 ° C and 55 ° C and then tested with blood samples from 4 reactive donors and one non-reactive donor. Table 1 shows the average data obtained with the reactive donors. As indicated by the temperatures, CMV tubes containing glucose are less reactive than tubes stored at 4 ° C or 22 ° C and show a marked decrease in responses relative to the liquid formulation (reference standard) used to prepare the tests. CMV tubes. In contrast, the tubes lacking glucose did not show a respective loss in reactivity.

Table 1 - Effect of glucose during storage The difference in number of times with respect to retention is shown (average IU / ml)

Example 3: Effect of Trehalose Addition on IFN-gamma Response in a QFN-CMV Tube QuantiFERON (QFN) CMV is an EC-registered assay that monitors T-cell immunological memory directed against Cytomegalovirus-derived antigens. QFN-CMV blood tubes contain peptides designed to specifically activate CD8 + T cells to produce interferon gamma (IFN-gamma). These tubes contain only peptide and heparin, without the addition of any sugar molecules.

This experiment is aimed at investigating the effect on IFN-gamma response when the non-reducing sugar trehalose is added to QFN-CMV tubes. 0, 1, 5 or 10 mg / ml of a trehalose solution (in water) was added to QFN-CMV blood collection tubes. Then 1 ml of blood from 17 healthy donors with a known anti-CMV T cell response was added to each of the four tubes, and furthermore, one null tube and one mitogen tube were tested as controls. The QFN test was then performed according to the insert of the container. All values were subtracted the null value. IFN-gamma levels in lU / ml were normalized against the respective QFN-CMV 0 mg / ml trehalose (untreated control) to generate a unit of "number of fold change" to control the interdonating variant in response magnitudes. . Healthy donors with no detectable anti-CMV immune response were included as controls against induction of nonspecific IFN-gamma by trehalose.

The addition of trehalose produced a concentration-dependent increase in the average level of IFN-gamma (expressed as IFN-gamma fold change versus the corresponding untreated control). A significant increase in effect was observed with the addition of 5 and 0 mg / ml trehalose (Friedman's test with Dunn's multiple comparison test). The results are shown in Figure 1. No significant increase in the background level of IFN-gamma was observed in the CMV negative control donors (data not shown).

The addition of trehalose to a QFN assay significantly increases the level of IFN-gamma produced in response to the specific antigen (s) contained in the blood collection tube without non-specifically increasing the background level of IFN-gamma.

Example 4: Effect of the addition of trehalose on the IFN-gamma response in a QFN-TB tube To confirm the observation that the addition of the non-reducing sugar trehalose to an assay evaluating peptide-specific cellular immunity, such as The QuantiFERON (QFN) assay, enhances the quantitative result of the assay, QFN tubes were made with the addition of a non-reducing sugar. The QFN blood collection tubes were manufactured to contain synthetic peptides of the Mycobacterium tuberculosis (MTB) antigens ESAT-6 and CFP-10 without the addition of any sugar or with the addition of the non-reducing sugar trehalose.

Whole blood was assayed from 20 subjects with evidence of MTB infection, confirmed using the QuantiFERON Gold In Tube Assay, using the established QuantiFERON platform technology, but using the modified QFN TB antigen tubes described above that were manufactured without a non-reducing sugar (no sugar) or with a non-reducing sugar (trehalose). The test was performed according to the QFT Gold package insert which is an established test. In this paired clinical analysis, the inclusion of the non-reducing sugar trehalose in the TB antigen tube significantly enhanced the quantitative response of the assay (P = 0.0005), as shown in Figure 2. The tube values ( ordinate axis) are shown in transformed logarithmic values of IFN-gamma lU / ml (having subtracted the values of the null tubes). A paired one-tailed t-test was performed to show statistical significance between the two cohorts. The results clearly demonstrate that the addition of trehalose increases the gamma interferon response, as demonstrated in subjects with TB infection.

Example 5: The addition of different non-reducing sugars increases the quantitative response of INF-gamma. The potentiation effect observed was also demonstrated with other non-reducing sugars. For this experiment, four different non-reducing sugars were added to the QFN CMV antigen tubes and to a corresponding null tube. To reach a final blood concentration of 2 mg / ml, a corresponding solution of sucrose, trehalose, mannitol and raffinose in PBS was added to whole blood obtained from 5 donors. Therefore, a QFN assay was performed with blood from 5 donors using QFN tubes without (N / S) or with the addition of a non-reducing sugar (sucrose, trehalose, mannitol or raffinose). The test was performed according to the QFN package insert which is an established test. The results are shown in Figure 3. The abscissa axis (see Figure 3) indicates the conditions tested. The background (value of the null tubes with / without the addition of the respective sugar) was subtracted from the corresponding CMV tube and the percentage increase in the IFN-gamma value (lU / ml) was calculated compared to the tube without additive (axis ordinate). The results demonstrate that an increased response to CMV antigens, as measured by the quantitative value of IFN-gamma (IU / ml), is observed with all four non-reducing sugars. Thus, all non-reducing sugars tested had a beneficial effect on the INF-gamma response. The greatest increase was observed with trehalose, followed by mannitol, sucrose and raffinose.

Claims (22)

1 A method for measuring a cell-mediated immune response activity, said method comprising: (a) providing an incubation composition by contacting a sample comprising immune cells capable of producing immune effector molecules upon stimulation with a peptide antigen, with at least one peptide antigen having a selected length of 5 to 100 amino acids, and with at least one non-reducing sugar capable of increasing the interferon-gamma response of immune cells that respond to the antigen; Y (b) detecting the presence or level of at least one immunological effector molecule, wherein detecting the presence or level of at least one immunological effector molecule comprises detecting the presence or level of the interferon-gamma (INF-gamma ) and / or an immune effector molecule whose production is induced or enhanced by interferon-gamma. 2. - The method according to claim 1, wherein the non-reducing sugar is a non-reducing disaccharide, preferably selected from trehalose and sucrose. 3. - The method according to claim 1 or 2, wherein the concentration of the non-reducing sugar in the incubation composition is at least 1.5 mg / ml, preferably at least 2 mg / ml. 4. - The method according to one or more of claims 1 to 3, wherein, in step (a), the sample is contacted with a composition comprising the antigen and the non-reducing sugar, and wherein preferably, said composition is comprised in a sample collection container. 5. - The method according to claim 4, wherein the composition further comprises an anticoagulant, preferably heparin, and wherein the sample is a whole blood sample. 6. - The method according to any one of claims 1 to 5, wherein the sample has one or more of the following characteristics: i) the sample is obtained from a human subject; ii) the sample is obtained from a human subject who is immunosuppressed or immunodeficient; iii) the sample comprises immune cells selected from the group consisting of NK cells, T cells, B cells, dendritic cells, macrophages and monocytes; me iv) the sample is whole blood. 7. - The method according to any one of claims 1 to 6, wherein the antigen has one or more of the following characteristics: i) one or more peptides are used as antigen, wherein said one or more peptides have a length selected from 7 to 50 amino acids; ii) the antigen is provided by one or more synthetic peptides; iii) the antigen is provided by at least two sets of peptides, the first set comprising at least one peptide with a length of about 7 to 14 amino acids, and a second set comprising at least one peptide of 15 amino acid residues or more, in that said peptides include all or part of a protein antigen; iv) the antigen is provided by one or more peptides that are recognized by a CD8 + cytotoxic T cell; and / or v) the antigen is provided by one or more peptides that are recognized by a CD8 + cytotoxic T cell, and wherein said one or more peptides are less than 15 amino acids in length, preferably have a selected length of 7-14 amino acids . 8. - The method according to one or more of claims 1 to 7, in which, in step (a), two or more different antigens are used and / or in which, in step (b), they are detected two or more different effector molecules. 9. - The method according to one or more of claims 1 to 8, wherein the antigen is a disease-specific antigen, in particular a pathogen-specific antigen. 10. - The method according to one or more of claims 1 to 9, wherein the antigen has one or more of the following characteristics: i) the antigen is associated with or representative of a disease or disorder for which the antigen is to be tested cell-mediated immune response; ii) the antigen is derived and thus cross-reactive with an antigen from a pathogen associated with a disease disorder or is a tumor associated antigen associated with a cancer; iii) the antigen is a pathogen-specific antigen, and wherein the pathogen is a bacterium, virus, parasite, yeast, or fungus; iv) the antigen is a pathogen-specific antigen, specifically a bacterial-specific antigen, and wherein the bacterium is selected from Gram-positive and Gram-negative microorganisms, in particular Mycobacterium species, such as Mycobacterium tuberculosis, Staphylococcus species, Staphylococcus species. from Streptococcus, Escherichia coli, Salmonella species, Clostridium species, Shigella species, Proteus species, Bacillus species, Hemophilus species and Borrelia species; v) the antigen is a pathogen-specific antigen, specifically a virus-specific antigen, and wherein the virus is selected from the hepatitis virus, such as the hepatitis B virus and the hepatitis C virus, herpes virus , CMV virus and human immunodeficiency virus (HIV); me vi) the antigen is derived from or specific to a virus, preferably cytomegalovirus (CMV), and is provided by one or more peptides that are 7 to 14 amino acid residues, 7 to 13 amino acid residues, or 8 to 12 residues in length amino acids. 11. - The method according to any one of claims 1 to 10 to control or determine the presence, absence, level or stage of a disease or a disorder selected from the group consisting of an infection by a pathogen, a disease autoimmune, cancer, inflammatory disorder, exposure to a toxic agent, response to a therapeutic agent, immunodeficiency, and immunosuppression. 12. - The method according to any one of claims 1 to 11, comprising: (a) providing an incubation composition by contacting a whole blood sample obtained from a human subject with a composition comprising at least one peptide antigen having a selected length of 5 to 100 amino acids, and at least one non-reducing disaccharide , preferably trehalose or sucrose, and incubating the incubation composition for at least 2 hours; Y (b) measuring the presence or level of IFN-gamma released due to stimulation with the antigen; wherein the presence or amount of IFN-gamma detected is indicative of the level of cell-mediated immune responsiveness of the human subject. 13. - The method according to any one of claims 1 to 12, in which the non-reducing sugar is selected from trehalose, mannitol, sucrose and raffinose, and in which, preferably, the non-reducing sugar is trehalose. 14. - A composition to induce a cell-mediated immune response in a sample, comprising: a) at least one isolated peptide antigen having a selected length of 5 to 100 amino acids; b) at least one non-reducing sugar capable of increasing the interferon-gamma response of antigen-responsive immune cells; Y c) at least one anticoagulant. 15. - The composition according to claim 14, which has one or more of the following characteristics: i) the non-reducing sugar is a non-reducing disaccharide, preferably selected from trehalose and sucrose; ii) the non-reducing sugar is selected from trehalose, mannitol, sucrose and raffinose; iii) the non-reducing sugar is trehalose; iv) the antigen has one or more of the characteristics defined in one or more of claims 7 to 10; v) the antigen is provided by one or more peptides, preferably synthetic peptides, having a length less than 15 amino acids, preferably having a selected length of 7-14 amino acids; vi) the antigen is a disease-specific or disease-associated antigen vii) the anticoagulant is heparin; me viii) The composition is a dried composition, optionally spray dried. 16. - A sample collection container comprising the composition according to claim 14 or 15. 17. - A kit for measuring cell-mediated immune response activity in a subject, comprising at least one peptide antigen having a selected length of 5 to 100 amino acids, preferably 7 to 50 amino acids, at least one non-sugar reducer capable of increasing the interferon-gamma response of antigen-responsive immune cells, at least one sample collection container and at least one detection medium for interferon-gamma and / or an immune effector molecule whose production is induced or enhanced by interferon-gamma. 18. - The kit according to claim 17, wherein the sample collection container has the characteristics of the sample collection container according to claim 16 and / or wherein the kit comprises an anticoagulant, preferably heparin. 19. - Use of a non-reducing sugar in an immunological assay to measure the cell-mediated response activity against a peptide antigen having a selected length of 5 to 100 amino acids, preferably 7 to 50 amino acids, in which addition Non-reducing sugar increases the release of interferon-gamma from immune cells that respond to the peptide antigen tested in said assay, and in which, preferably, the non-reducing sugar has one or more of the following characteristics: i) the non-reducing sugar is a disaccharide; ii) the non-reducing sugar is selected from trehalose and sucrose; iii) the non-reducing sugar is selected from trehalose, mannitol, sucrose and raffinose; iv) the non-reducing sugar is trehalose; v) the non-reducing sugar is used in a concentration of at least 1 mg / ml, preferably 2 mg / ml in the incubation composition comprising the sample to be tested and the antigen; vi) the non-reducing sugar is provided in the form of a composition further comprising the antigen to be tested and, optionally, an anticoagulant; 20. Use of a non-reducing sugar in an immunological assay to measure cell-mediated response activity to increase the release of interferon-gamma from immune cells that respond to an antigen tested in said assay, optionally wherein the antigen and / or The non-reducing sugar has one or more of the characteristics defined in claim 15. 21. An incubation composition comprised in a sample collection container, the incubation composition comprising: a) an undiluted sample obtained from a subject, the sample comprising immune cells capable of producing immune effector molecules after stimulation by an antigen of peptide, preferably T lymphocytes; b) at least one isolated peptide antigen having a selected length of 5 to 100 amino acids; c) at least one non-reducing sugar capable of increasing the interferon-gamma response of immune cells that respond to the antigen; and d) optionally an anticoagulant. 22. The incubation composition according to claim 21, having one or more of the following characteristics: i) the sample is a body fluid; ii) the sample is whole blood and the incubation composition comprises an anticoagulant, preferably heparin; and / or iii) the antigen and / or the non-reducing sugar have one or more of the characteristics defined in claim 15.