T-cell responders

Abstract

The invention relates to methods for determining the presence or absence of a T-cell response to a disease (e.g. pathogen, a cancer, an autoimmune disease, an allergy or asthma) in a subject. Said methods allow quick and simple detection of at least one biomarker of early-stage T-cell activation.

Description

[0001] T-CELL RESPONDERS

[0002] FIELD OF INVENTION

[0003] The invention relates to methods for determining the presence or absence of an immune response, particularly a T-cell response to a disease (e.g. a response to a disease-related antigen such as a pathogen, a cancer antigen, an autoimmune disease target, asthma or allergy allergen) in a subject. Said methods allow quick and simple detection of at least one biomarker of early-stage T-cell activation.

[0004] BACKGROUND TO THE INVENTION

[0005] Clinical medicine and global public health surveillance make enormous use of assays that measure immune responses to assess a person’s immune status, whether in the context of diagnosing or monitoring infections such as COVID-19, TB or HIV, or assessing vaccine responses in individuals or populations at risk. Generally an immune response involves activation of humoral (antibodies) and cellular (T-cell) immunity. However, commercially available assays measuring the immune response have to date focused on antibodies. By contrast, the other extremely important arm of long-term protective immunity - the cellular (T-cell) response has been largely ignored. Assays for T-cell immunity, though critical for understanding a person’s long term and cross-protective immunity, have rarely been developed for widespread commercial use. This is largely due to the fact that assays measuring antibodies have been easily accessible, affordable, and relatively amenable to a range of scalable, high-throughput platforms, while assays measuring T-cell immunity have remained within the sphere of slow, costly, specialist tests done in a limited number of high-tech laboratories. As a result, these assays remain technically complex, slow, and costly. Indeed, assays measuring T-cell immunity tend to require specialist laboratories with access to specialised and expensive equipment with highly trained staff to perform, analyse and interpret the results of the assay. To date, T-cell specific assays have also been slow, generally taking several days to perform and requiring control samples to be run alongside the test sample to enable the analysis and interpretation of results. Additionally, T-cell specific assays usually require significant fresh samples (usually blood) which need to be taken by a trained individual. The sample must then be processed and analysed in a laboratory on the same day or stored in liquid nitrogen to be processed and analysed at a later date. For example, blood samples are processed by separating the white blood cells by density-gradient centrifugation, which are then cultured at least overnight under sterile tissue culture conditions with the antigen, cellular activation in response to the antigen is then analysed using specialist instruments such as a flow cytometer or an ELISPOT reader. Consequently, there are only asmall number of technologies for the assessment of T-cell immunity. These rely on cumbersome, specialist-lab-based approaches and equipment e.g. tissue culture hoods, centrifuges, incubators and analyser, and using either conventional immunology lab-based assays of T-cell effector pathways such as the interferon-gamma or interleukin-2 response, or expansion of T-cell receptor sequences.

[0006] It is an object of the present invention to address one or more of these deficiencies, and to provide a method to determine the presence or absence of a T-cell response to a disease (e.g. a response to a disease-related antigen such as a pathogen, a cancer antigen, an autoimmune disease target, asthma or allergy allergen) in a subject.

[0007] SUMMARY OF INVENTION

[0008] The inventors have demonstrated that CD28, ZAP70, LAT, LCK, and ARL13 are some of the earliest proteins to be released following T-cell activation. Notably, the inventors have shown that CD28, ZAP70, LAT, LCK, and ARL13 can be used as biomarkers for early-stage T-cell activation. Following identification of these early-stage T-cell activation biomarkers, the inventors have developed a method for determining the presence or absence of a T-cell response to a disease (e.g. a pathogen, a cancer, an autoimmune disease, asthma or an allergy) in the subject which uses the levels of at least one of these early-stage biomarkers, CD28, ZAP70, LAT, LCK, andARL13. By utilising biomarkers of early-stage T-cell activation, the inventors have developed a method which provides a quick (results can be obtained within 1-3 hours compared to current methods which can take a minimum of 24 hours before results are obtained) and simple test to measure the T-cell immune response to a disease (e.g. a disease-related antigen such as a pathogen, cancer antigen, an autoimmune disease target, asthma or allergy allergen).

[0009] Accordingly, the present invention provides a method for determining the presence or absence of a T-cell response to a disease in a subject comprising: (a) Contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; (b) Analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and (c) Determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0010] The present invention provides a method for determining the presence or absence of a T-cell response to a disease in a subject comprising: (a) Contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; (b) Analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a; and (c) Determining thepresence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0011] The present invention also provides a method for determining the presence or absence of a T-cell response to a disease in a subject comprising: (a) Contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; (b) Analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and (c) Determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0012] In the methods of the invention, the disease may be caused by a pathogen, or is a cancer, an autoimmune disease, asthma or an allergy.

[0013] In the methods of the invention, the at least one disease-related antigen may be comprised within a plurality of antigens or antigenic fragments from the disease.

[0014] In the methods of the invention, the at least one disease-related antigen may be an antigenic peptide or an antigenic carbohydrate.

[0015] In the methods of the invention, the at least one disease-related antigen may be immobilised on a support.

[0016] In the methods of the invention, the sample may be a blood sample.

[0017] In the methods of the invention, the sample may be incubated for at least 1 hour with at the least one disease-related antigen from the disease.

[0018] In the methods of the invention, incubation / contacting may occur at between 18 °C to 37 °C.

[0019] The methods of the invention may further comprise separating the test sample from the at least one disease-related antigen.

[0020] The methods of the invention may further comprise analysing the test sample for the presence of at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b.

[0021] The methods of the invention may further comprise analysing the test sample for the presence of all of CD28, ZAP70, LAT, LCK, and ARL13b.

[0022] The methods of the invention may further comprise determining the presence of a T-cell response if at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample.

[0023] The methods of the invention may further comprise determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample.

[0024] The methods of the invention may further comprise analysing the test sample for the presence of at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a.The methods of the invention may further comprise analysing the test sample for the presence of all of CD28, ZAP70, LAT, LCK, ARL13b and ARL13a.

[0025] The methods of the invention may further comprise determining the presence of a T-cell response if at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample.

[0026] The methods of the invention may further comprise determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample.

[0027] The methods of the invention may further comprise analysing the test sample for the presence of at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a.

[0028] The methods of the invention may further comprise analysing the test sample for the presence of all of CD28, ZAP70, LAT, LCK, and ARL13a.

[0029] The methods of the invention may further comprise determining the presence of a T-cell response if at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample.

[0030] The methods of the invention may further comprise determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample.

[0031] In the methods of the invention, the presence of a T-cell activation biomarker may be determined by mass spectrometry, ELISA or lateral flow device.

[0032] In the methods of the invention, the disease may be caused by a pathogen and the pathogen is selected from: (a) a virus or a bacteria; or (b) SARS-CoV-2, Cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, herpes simplex virus, Salmonella enterica, Samonella bongori, Yersinia pestis, Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Bacillus cereus biovar anthracis, Burkholderia cepacia complex, Burkholderia, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, zika virus, group A streptococcus, measles virus, mumps virus, rubella virus, Dengue virus, Chikungunya virus, Streptococcus pneumoniae, Neisseria meningitidis, Poliovirus, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae type b, rotavirus, Bordetella pertussis, human papillomavirus, human immunodeficiency virus, and influenza.

[0033] In the methods of the invention, the disease may be a cancer, and the cancer is selected from breast cancer, lung cancer, melanoma, hepatocellular and colon cancer.

[0034] In the methods of the invention, the disease may be an autoimmune disease, and the autoimmune disease is multiple sclerosis, rheumatoid arthritis or type 1 diabetes.

[0035] In the methods of the invention, the disease may be asthma or an allergy.Also provided is the use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b as a biomarker of a T-cell response to a disease.

[0036] Also provided is the use of at least one of CD28, ZAP70, LAT, LCK, ARL13b and ARL13a as a biomarker of a T-cell response to a disease.

[0037] Also provided is the use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to a disease.

[0038] In the use of the invention, the disease may be caused by a pathogen or is a cancer, an autoimmune disease, asthma or an allergy.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : A schematic of the steps taken of a method of the invention

[0041] Figure 2: Identification of T-cell activation biomarkers in blood samples from COVID immune individuals following activation of samples with SARS-CoV-2 spike peptide antigens. All five T-cell activation biomarkers (A) LCK, (B) ZAP70, (C) LAT, (D) ARL13a and (E) CD28, were found to have significantly higher levels following stimulation of samples with SARS-CoV-2 spike peptide antigens compared to DM SO control.

[0042] Figure 3: Correlation with T-cell responder status as assessed by conventional next-day ELISpot culture for T-cell activation biomarkers (A) LCK, (B) ZAP70, and (C) LAT.

[0043] Figure 4: Detection of T cell responses to SARS-CoV-2 spike peptide antigens using known ELISpot IFNy assay

[0044] Figure 5: Detection of T cell responses to Varicella Zoster virus

[0045] Figure 6: Detection of T cell responses to MAGE A1 cancer antigen

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with ageneral dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary.

[0049] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0050] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0051] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0052] As used herein, the term "capable of' when used with a verb, encompasses or means the action of the corresponding verb. For example, "capable of detecting" also means detecting, "capable of stimulating" also means stimulating and "capable of binding" also means binds.

[0053] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0054] As used herein, the articles "a" and “an” may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting.

[0055] “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term “about” shall be understood herein as plus or minus (±) 5%, preferably ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used.

[0056] Minor variations in the amino acid sequences of an antigen of the invention are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to the amino acid sequence of the invention or a fragment thereof as defined anywhere herein. The term homology is used herein to mean identity. As such, the sequence of a variant or analogue sequence of an amino acid sequence of the invention may differ on the basis of substitution (typically conservative substitution) deletion or insertion. Proteins comprising such variations are referred to herein as variants. Sequence identity is typically calculated over the full-length of an antigen.

[0057] The term “antigen” or “disease related antigen” refers to a molecule or substance associated with a disease which is capable of stimulating / activating an immune response. An antigen may be all, or a fragment of, a protein, peptide, carbohydrate or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal.

[0058] An antigen includes an “antigenic fragment”, which is a fragment of an antigen that is capable of stimulating / activating an immune response. The length of an antigenic fragment is not particularly limited, provided that said fragment is still immunogenic, i.e. it is capable of stimulating / activating an immune response. Thus, an antigenic fragment which is a peptide may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more of the original protein antigen. For example, antigenic fragment which is a peptidemay comprise at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400 or more amino acids of the protein antigen from which it is derived. Preferably a fragment may comprise no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 150, no more than 200, no more than 250 amino acids of the protein from which it is derived.

[0059] The term "peptide" as used herein also includes polypeptides and proteins unless the context clearly indicates otherwise.

[0060] The term "protein" also includes modified forms such as glycoproteins and phosphoproteins.

[0061] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the invention.

[0062] As used herein the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non-immunogenic ingredients).

[0063] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of” and / or “consisting essentially of” such features.

[0064] The term “contacting” as used herein refers to the process of allowing at least two distinct species to become sufficiently proximal to react, interact or physically touch. Contacting may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, an antigen as described herein and an immune cell, such as a T-cell within a sample.

[0065] The terms "decrease", "reduce", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. The terms "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" encompasses a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition (i.e. abrogation) as compared to a reference level.The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. The terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more compared with a reference level, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level.

[0066] As used herein, the term “amount” encompasses the mass, molar amount, the concentration, molarity, percentage, ratio or other appropriate parameter. An amount may be given in any appropriate units. For example, a concentration may be given in pg / ml, ng / ml or pg / ml.

[0067] As used herein, the term “sample” refers to any sample comprising immune cells capable of producing immune related molecules following stimulation with an appropriate antigen obtained from a subject. Any suitable sample may be used e.g. a body fluid sample comprising immune cells. A non-limiting list of samples are saliva, blood, and cerebrospinal fluid (CSF). Preferably, the sample is a blood sample, particularly a whole blood sample.

[0068] The terms "individual”, "subject”, and "patient”, are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimisation is desired. The mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In a preferred embodiment, the individual, subject, or patient is a human. An “individual” may be an adult, juvenile or infant. An “individual” may be male or female.

[0069] A "subject in need" of treatment for a particular disease can be an individual having that disease, diagnosed as having that disease, or at risk of developing that disease.

[0070] A subject can be one who has been previously diagnosed with or identified as suffering from or having a disease in need of treatment or one or more complications related to such a disease, and optionally, have already undergone treatment for a disease as defined herein or the one or more complications related to said disease. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease as defined herein or one or more complications related to said disease. For example, an individual can be one who exhibits one or more risk factors for a disease, or one or more complications related to said disease or a subject who does not exhibit risk factors.

[0071] As used herein, the term “healthy individual” refers to an individual or group of individuals who are in a healthy state, e.g. individuals who have not shown any symptoms of the disease, have not been diagnosed with the disease and / or are not likely to develop thedisease (e.g. infection with a pathogen described herein or a cancer as described herein or any other disease described herein). Preferably said healthy individual(s) has not been infected with a pathogen as described herein, or has not been diagnosed with a cancer, an autoimmune disease, asthma or an allergy as described herein. The one or more healthy individuals may have a similar sex, age, and / or body mass index (BMI) as compared with the test individual. Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.

[0072] The levels of a biomarker of the invention may be compared with a suitable control or reference population, such as a healthy individual, or an individual with an infection, a cancer, an autoimmune disease, asthma or an allergy of interest.

[0073] Herein the terms “control”, “threshold” and “reference population” are used interchangeably.

[0074] The term “T-cell activation” or “activation of T-cells” as used herein refers to the cellular process whereby T-cells are exposed to or stimulated by an antigen and results in a T-cell immune response.

[0075] The term “T-cell response” or “T-cell immune response” as used herein refers to the cellular events that follow activation of T-cells (also referred to as T lymphocytes) after, for example, infection with a pathogen, development of cancer, and / or development of an autoimmune disease, asthma or an allergy, and that may result in, for example, the secretion, or the down- or up-regulation of expression of surface or intracellular immune related molecules. The term “immune related molecules” as used herein refers to the molecules involved in an immune response following stimulation by an antigen, and hence encompasses immune effector molecules. For example, immune related molecules can include cytokines, chemokines, components of the complement system, enzymes, cell surface receptor and associated signalling molecules, cell surface ligand or other cell surface markers. A T-cell response may be in response to a disease-related antigen such as a pathogen, a cancer antigen, an autoimmune disease target, asthma or allergy allergen. A T-cell response may be associated with, or causal in, a disease. For example, a T-cell response may be associated with, or causal in, an autoimmune disease.

[0076] The term “T-cell activation biomarker” as used herein refers to immune related molecules which are released as part of a T-cell response which are indicative of T-cell activation. An “early-stage” T-cell activation biomarker as used herein refers to immune related molecules which are involved in the initial stages of activation of T-cells and not to the later, down-stream immune related molecules. These initial stages of T-cell activation typically occur within 5 hours of a T-cell being exposed to an antigen or antigenic fragment thereof, particularly within 3 hours or within 1 hour of exposure.Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

[0077] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0078] Disclosure related to the various methods of the invention are intended to be applied equally to other methods, therapeutic uses or methods, and vice versa.

[0079] Methods of determining a T-cell immune response

[0080] An important aspect of cellular immunity involves an antigen-specific response by T-cells. T-cells are capable of eliciting a strong immune response to antigens which they recognise. If T-cells have been previously exposed to one or more antigens or fragments thereof, T-cells with specific memory of that antigen are quickly re-stimulated, resulting in an antigen-specific T-cell response. These antigen-specific T-cells may secrete, down- or up-regulate the expression of a range of immune related molecules leading to an effector response in response to re-stimulation to the antigen. Immune related molecules released or upregulated in response to one or more disease related antigens can be used to test for the presence or absence of a T-cell response to a disease. The T-cell response may involve any type of T-cells. Typically, a T-cell response to a disease may involve memory T-cells, including memory CD8+ T-cells and / or memory CD4+ T-cells. Memory T-cells are T-cells which have previously been exposed to an antigen.

[0081] As exemplified herein, the present inventors have identified that CD28, ZAP70, LAT, LCK, and ARL13 can be used as early-stage biomarkers of T-cell activation and therefore can be used to determine the presence or absence of a T-cell response to a disease in a subject (e.g. a pathogen, a cancer, an autoimmune disease, asthma or an allergy). In particular, the present inventors have developed a method of determining the presence or absence of a T-cell response to a disease (e.g. a pathogen, a cancer, an autoimmune disease, asthma or an allergy) in a subject, which involves measuring at least one of these early-stage biomarkers of T-cell activation.

[0082] Accordingly, the invention provides a method for determining the presence or absence of a T-cell response to a disease in a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarkerselected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0083] Accordingly, the invention provides a method for determining the presence or absence of a T-cell response to a disease in a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a; and c) determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0084] Accordingly, the invention provides a method for determining the presence or absence of a T-cell response to a disease in a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0085] Any sample from a subject which comprises immune cells may be suitable for use in the methods of the invention. A non-limiting list of samples are saliva, blood, nasal swabs and cerebrospinal fluid (CSF). Typically, the sample is a blood sample, particularly whole blood. The blood sample may be a fresh blood sample or a dried blood sample. A blood sample may be described as fresh if it has been obtained from a subject within 24 hours, 12 hours, 8 hours, 4 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes or less before being used in a method of the invention. Where a fresh blood sample is used, the sample is typically tested in a method of the invention within 24 hours, 12 hours, 8 hours, 4 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes or less from the subject being obtained from a subject. A dried sample, such as a dried blood sample, may be reconstituted in a suitable buffer, particularly a saline buffer, prior to use in a method of the invention. A dried blood sample may be stored for a period of time prior to reconstitution and use in a method of the invention. For example, a dried blood sample may be stored for at least 2 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, 6 months, 12 months or more before being reconstituted and used in a method of the invention. A blood sample may be taken using capillary blood sample collection e.g. a finger-prick, ear-prick or heel-prick method. Such methods may involve collecting the blood sample in a capillary tube or blood spot collection on filter paper.

[0086] Advantageously, in the methods of the invention the sample may be any sample which reduces the complexity of the sample and removes the requirement for same day processingand / or expensive liquid nitrogen storage. Blood samples are particularly preferred in this regard.

[0087] The methods of the invention comprise contacting a sample from the subject with at least one disease-related antigen to generate a test sample. As used herein a “test sample” refers to a sample from a subject that has been contacted with at least one disease-related antigen (e.g. an antigen from a pathogen, a cancer, an autoimmune disease, asthma or an allergy) and which can be analysed for the presence of at least one T-cell activation biomarker. In other words, the sample has been contacted with at least one disease-related antigen for a sufficient amount of time to allow for re-stimulation of T-cells in the sample if they have previously been exposed to the at least one antigen, or a fragment thereof.

[0088] As used herein, “contacting” refers to the sample being placed in sufficient proximity to the at least one antigen so that the sample and antigen can react, interact or physically touch. Contacting may also be referred to as “incubating”. Contacting can occur through any suitable means as long as it allows the at least one antigen as described herein to react, interact or physically touch with an immune cell, such as a T-cell, comprised within the sample. Contacting or incubation allows re-stimulation of the immune cells such as T-cells within the sample by the at least one antigen. The sample may be added to the at least one antigen or the at least one antigen may be added to the sample. For example, the sample may be added to a tube or vessel containing the at least one antigen. The tube or vessel may be a collection tube or vessel which allows the sample to be collected and contacted with the at least one antigen at the same time.

[0089] The methods of the invention may include an initial mixing step to ensure an equal distribution of the at least one antigen and sample within the test sample. The remainder of the contacting or incubation step may occur without further mixing. Alternatively, the test sample may be mixed at least one further time during the contacting or incubation step.

[0090] The time required for the contacting or incubation step should be sufficient for the T-cells in the sample to produce immune related molecules in response to re-stimulation by the at least one antigen. Thus, the sample and the at least one antigen may be contacted for at least 1 hour. In other words, the sample and the at least one antigen may be incubated for at least 1 hour for example, the sample and the at least one antigen may be incubated for between 1 to 10 hours, 1 to 9 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, 2 to 3 hours, or 2 to 4 hours. Typically, the sample and the at least one antigen may be incubated for between 1 hour to 3 hours (e.g. about 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes or 180 minutes). In some preferred embodiments, the sample and the at least one antigen may be incubated for between 1 hour to 2 hours (e.g. about 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110minutes, 120 minutes). Preferably, the sample and the at least one antigen are incubated for 1 hour.

[0091] The temperature at which the contacting or incubation step occurs is not particularly limited. Typically, a method of the invention may comprise contacting or incubating the sample with at least one antigen at a temperature of about 16 °C to about 40 °C, such as about 18 °C to about 38 °C, about 18 °C to about 37 °C, about 18 °C to about 35 °C, about 18 °C to about 34 °C, about 18°C to about 33 °C, about 18°C to about 32 °C, about 18°C to about 31 °C, about 18 °C to about 30 °C, about 18 °C to about 28 °C, about 18 °C to about 27 °C, about 18 °C to about 25 °C, about 18 °C to about 24 °C, about 18 °C to about 23 °C, about 18 °C to about 22 °C, about 18 °C to about 21 °C. Preferably, a method of the invention comprises contacting or incubating the sample with at least one antigen at a temperature of about 18 °C to about 38 °C, more preferably 18 °C to about 21 °C.

[0092] A method of the invention may comprises separating the test sample from the at least one disease related antigen following the contacting / incubation step. Separation of the test sample from the at least one disease related antigen may occur using any suitable separation technique. For example, the test sample may be separated from the at least one disease related antigen through a washing step with a suitable buffer or wash.

[0093] T-cell Activation Biomarkers

[0094] T-cell activation biomarkers used in the methods of the invention are “early-stage T-cell activation biomarkers”. These biomarkers are immune-related molecules which are involved in the initial stages of T-cell activation, and therefore allow for a T-cell response to be measured without the long incubation times required in prior art methods which utilise downstream biomarkers. By taking a function-agnostic approach, the inventors identified a novel group of early-stage T-cell activation biomarkers, comprising CD28, ZAP70, LAT, LCK, and ARL13. The T-cell activation biomarkers of the invention are typically human. The methods of the invention, utilise at least one early-stage T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b, which advantageously allows for a rapid way of detecting a T-cell response to a disease related antigen. The methods of the invention, utilise at least one early-stage T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a, which advantageously allows for a rapid way of detecting a T-cell response to a disease related antigen. The methods of the invention, utilise at least one early-stage T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a, which advantageously allows for a rapid way of detecting a T-cell response to a disease related antigen.

[0095] CD28 is expressed on T-cells and provides a co-stimulatory signal required for T-cell activation and survival. A CD28 protein may have an amino acid sequence as set forth in SEQID NO: 1 or an amino acid sequence having at least 80% sequence identity thereto, as described herein.

[0096] ZAP70 is a protein normally expressed near the surface membrane, recruited upon antigen binding to the T cell receptor (TOR), and it plays a critical role in T cell signalling. A ZAP70 protein may have an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 80% sequence identity thereto, as described herein.

[0097] LAT (Linker for activation of T cells) is a transmembrane protein involved in the T-cell antigen receptor signal transduction pathway and which acts as a docking site for SH2 domain-containing proteins. Upon phosphorylation, LAT recruits multiple adaptor proteins and downstream signaling molecules into multimolecular signaling complexes located near the site of TCR engagement. A LAT protein may have an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least 80% sequence identity thereto, as described herein.

[0098] LCK (Tyrosin-protein kinase Lek) is responsible for the initiation of the TCR signaling cascade inside the cell by phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) within the TCR-associated chains. A LCK protein may have an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80% sequence identity thereto, as described herein.

[0099] ARL13b (ADP-ribosylation factor-like protein 13B) a small GTPase that contains both N-terminal and C-terminal guanine nucleotide-binding motifs. This protein is localized in the cilia and plays a role in cilia formation and in maintenance of cilia. AARL13b protein may have an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least 80% sequence identity thereto, as described herein.

[0100] ARL13a (ADP ribosylation factor like GTPase 13A) is a paralog of Arl13b and is also a small GTPase which regulates various cellular functions and plays an important role in cilia structure and signalling. AnARL13a protein may have an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence having at least 80% sequence identity thereto, as described herein.

[0101] In the methods of the invention, the test sample is analysed for the presence of at least one, at least two, at least three, at least four or at least five T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b.

[0102] The test sample may be analysed for the presence of at least one T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b. The at least one T-cell activation biomarker may be CD28. The at least one T-cell activation biomarker may be ZAP70. The at least one T-cell activation biomarker may be LAT. The at least one T-cell activation biomarker may be LCK. The at least one T-cell activation biomarker may be ARL13b.The test sample may be analysed for the presence of at least two T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b. The test sample may be analysed for the presence of at least CD28 and ZAP70. The test sample may be analysed for the presence of at least CD28 and LAT. The test sample may be analysed for the presence of at least CD28 and LCK. The test sample may be analysed for the presence of at least CD28 and ARL13b.The test sample may be analysed for the presence of at least ZAP70 and LAT. The test sample may be analysed for the presence of at least ZAP70 and LCK. The test sample may be analysed for the presence of at least ZAP70 and ARL13b. The test sample may be analysed for the presence of at least LAT and LCK. The test sample may be analysed for the presence of at least LAT and ARL13b. The test sample may be analysed for the presence of at least LCK and ARL13b.

[0103] The test sample may be analysed for the presence of at least three T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b. The test sample may be analysed for the presence of at least CD28, ZAP70, and LAT. The test sample may be analysed for the presence of at least CD28, ZAP70, and LCK. The test sample may be analysed for the presence of at least CD28, ZAP70, and ARL13b. The test sample may be analysed for the presence of at least CD28, LAT and LCK. The test sample may be analysed for the presence of at least CD28, LAT and ARL13b. The test sample may be analysed for the presence of at least CD28, LCK, and ARL13b. The test sample may be analysed for the presence of at least ZAP70, LAT and LCK. The test sample may be analysed for the presence of at least ZAP70, LAT and ARL13b. The test sample may be analysed for the presence of at least ZAP70, LCK and ARL13b. The test sample may be analysed for the presence of at least LAT, LCK and ARL13b.

[0104] The test sample may be analysed for the presence of at least four T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and LCK. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and ARL13b. The test sample may be analysed for the presence of at least CD28, ZAP70, LCK and ARL13b. The test sample may be analysed for the presence of at least CD28, LAT, LCK and ARL13b. The test sample may be analysed for the presence of at least ZAP70, LAT, LCK and ARL13b.

[0105] The test sample is analysed for the presence of all five of the T-cell activation biomarkers CD28, ZAP70, LAT, LCK, and ARL13b.

[0106] In the methods of the invention, the test sample is analysed for the presence of at least one, at least two, at least three, at least four or at least five T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a.

[0107] The test sample may be analysed for the presence of at least one T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a. The at least one T-cell activation biomarker may be CD28. The at least one T-cell activation biomarker may be ZAP70. The at least one T-cell activation biomarker may be LAT. The at least one T-cell activation biomarker may be LCK. The at least one T-cell activation biomarker may be ARL13a. The at least one T-cell activation biomarker may be ARL13b.

[0108] The test sample may be analysed for the presence of at least two T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28 and ZAP70. The test sample may be analysed for the presence of at least CD28 and LAT. The test sample may be analysed for the presence of at least CD28 and LCK. The test sample may be analysed for the presence of at least CD28 and ARL13b.The test sample may be analysed for the presence of at least CD28 and ARL13a.The test sample may be analysed for the presence of at least ZAP70 and LAT. The test sample may be analysed for the presence of at least ZAP70 and LCK. The test sample may be analysed for the presence of at least ZAP70 and ARL13b. The test sample may be analysed for the presence of at least ZAP70 and ARL13a. The test sample may be analysed for the presence of at least LAT and LCK. The test sample may be analysed for the presence of at least LAT and ARL13b. The test sample may be analysed for the presence of at least LAT and ARL13a. The test sample may be analysed for the presence of at least LCK and ARL13a. The test sample may be analysed for the presence of at least LCK and ARL13b. The test sample may be analysed for the presence of at least ARL13a and ARL13b.

[0109] The test sample may be analysed for the presence of at least three T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, and LAT. The test sample may be analysed for the presence of at least CD28, ZAP70, and LCK. The test sample may be analysed for the presence of at least CD28, ZAP70, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, and ARL13b. The test sample may be analysed for the presence of at least CD28, LAT and LCK. The test sample may be analysed for the presence of at least CD28, LAT and ARL13a. The test sample may be analysed for the presence of at least CD28, LAT and ARL13b. The test sample may be analysed for the presence of at least CD28, LCK, and ARL13a. The test sample may be analysed for the presence of at least CD28, LCK, and ARL13b.The test sample may be analysed for the presence of at least ZAP70, LAT and LCK. The test sample may be analysed for the presence of at least ZAP70, LAT and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LAT and ARL13b. The test sample may be analysed for the presence of at least ZAP70, LCK and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LCK and ARL13b. The test sample may be analysed for the presence of at least LAT, LCK and ARL13a. The test sample may be analysed for the presence of at least LAT, LCK and ARL13b.The test sample may be analysed for the presence of at least CD28, ARL13b,and ARL13a. The test sample may be analysed for the presence of at least ZAP70, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least LAT, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least LCK, ARL13b, and ARL13a.

[0110] The test sample may be analysed for the presence of at least four T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and LCK. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and ARL13b. The test sample may be analysed for the presence of at least CD28, ZAP70, LCK and ARL13a. The test sample may be analysed for the presence of at least CD28, LAT, LCK and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LAT, LCK and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LCK and ARL13b. The test sample may be analysed for the presence of at least CD28, LAT, LCK and ARL13b. The test sample may be analysed for the presence of at least ZAP70, LAT, LCK and ARL13b. The test sample may be analysed for the presence of at leastCD28, ZAP70, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, LAT, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LAT, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least LAT, LCK, ARL13b, andARL13a.

[0111] The test sample may be analysed for the presence of at least five T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, LAT, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LCK, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT, ARL13b, and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LAT, LCK, ARL13b, andARL13a.

[0112] The test sample is analysed for the presence of all of the T-cell activation biomarkers CD28, ZAP70, LAT, LCK, ARL13b, andARL13a.

[0113] In the methods of the invention, the test sample is analysed for the presence of at least one, at least two, at least three, at least four or at least five T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a.

[0114] The test sample may be analysed for the presence of at least one T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a. The at least one T-cell activation biomarker may be CD28. The at least one T-cell activation biomarker may beZAP70. The at least one T-cell activation biomarker may be LAT. The at least one T-cell activation biomarker may be LCK. The at least one T-cell activation biomarker may be ARL13a.

[0115] The test sample may be analysed for the presence of at least two T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a. The test sample may be analysed for the presence of at least CD28 and ZAP70. The test sample may be analysed for the presence of at least CD28 and LAT. The test sample may be analysed for the presence of at least CD28 and LCK. The test sample may be analysed for the presence of at least CD28 and ARL13a.The test sample may be analysed for the presence of at least ZAP70 and LAT. The test sample may be analysed for the presence of at least ZAP70 and LCK. The test sample may be analysed for the presence of at least ZAP70 and ARL13a. The test sample may be analysed for the presence of at least LAT and LCK. The test sample may be analysed for the presence of at least LAT and ARL13a. The test sample may be analysed for the presence of at least LCK and ARL13a.

[0116] The test sample may be analysed for the presence of at least three T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, and LAT. The test sample may be analysed for the presence of at least CD28, ZAP70, and LCK. The test sample may be analysed for the presence of at least CD28, ZAP70, and ARL13a. The test sample may be analysed for the presence of at least CD28, LAT and LCK. The test sample may be analysed for the presence of at least CD28, LAT and ARL13a. The test sample may be analysed for the presence of at least CD28, LCK, and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LAT and LCK. The test sample may be analysed for the presence of at least ZAP70, LAT and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LCK and ARL13a. The test sample may be analysed for the presence of at least LAT, LCK and ARL13a.

[0117] The test sample may be analysed for the presence of at least four T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and LCK. The test sample may be analysed for the presence of at least CD28, ZAP70, LAT and ARL13a. The test sample may be analysed for the presence of at least CD28, ZAP70, LCK and ARL13a. The test sample may be analysed for the presence of at least CD28, LAT, LCK and ARL13a. The test sample may be analysed for the presence of at least ZAP70, LAT, LCK and ARL13a.

[0118] The test sample is analysed for the presence of all five of the T-cell activation biomarkers CD28, ZAP70, LAT, LCK, and ARL13a.

[0119] The methods of the invention may comprise analysing the test sample for the presence of one or more further T-cell activation biomarker. The one or more further T-cell activationbiomarker may be selected from effector molecules produced by immune cells, particularly T cells, in response to antigen re-stimulation. The effector molecules may be selected from a cytokine, a component of the complement system, perforin, defensin, cathelicidin, granzyme, Fas ligand, CD40 ligand, exotaxin, a cytotoxin, a chemokine, or a monokine.

[0120] Detection of presence of T-cell response

[0121] The methods of the invention comprise the step of determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample. T-cell activation biomarkers may be detected by any suitable means known in the art. As used herein, the term “determining” may be used interchangeably with “detecting” and refers to the measurement, identification, and / or confirmation of the presence of a T-cell response or the presence of a T-cell activation biomarker. As used herein, the “presence of at least one of the T-cell activation biomarkers” refers to the T-cell activation biomarker being detected at a level known to be indicative of T-cell activation. Thus, references to determining the presence of at least one T-cell activation biomarker also encompass determining the level of the at least one T-cell activation biomarker.

[0122] References herein to "level of the at least one T-cell activation biomarker" or "level" refer to a measurement that is made using any analytical method that is suitable for detecting the T-cell activation biomarker in a test sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, a level, an expression level, a ratio of measured levels, or the like, of, for, or corresponding to the aggregates in the biological sample. The exact nature of the "level" depends on the specific design and components of the particular analytical method employed to detect the T-cell activation biomarker. The nature of the “level” for each of the at least one T-cell activation biomarker may be selected independently. By way of non-limiting example, the concentration a first T-cell activation biomarker may be determined and the absolute amount of a second T-cell activation biomarker may be determined. Typically, the nature of the “level” may be the same for each of the at least one T-cell activation biomarker. By way of non-limiting example, the concentration of each of the at least one T-cell activation biomarker may be determined.

[0123] The presence or level of at least one of the T-cell activation biomarkers in the test sample can be measured by any suitable method. The detection of at least one of the T-cell activation biomarkers in the test sample may occur at the peptide, protein, or nucleic acid level. Typically, the presence or level of at least one T-cell activation biomarker may be detected by mass spectrometry, ELISA, qPCR or lateral flow device. Preferably, the presence or level of at least one T-cell activation biomarker may be detected by mass spectrometry. The method used to measure the presence or level of each of the at least one T-cell activation biomarkers may be selected independently. By way of non-limiting example, the presence orlevel of a first T-cell activation marker may be measured using ELISA and the presence or level of a second T-cell activation marker may be measured using mass spectrometry. Typically, the same method is used to measure the presence or level of each of the at least one T-cell activation biomarkers. By way of non-limiting example, the presence or level of each of the at least one T-cell activation marker may be measured using mass spectrometry.

[0124] The presence of the at least one T-cell activation biomarker in the test sample is indicative of a T-cell response to the disease-related antigen, and can indicate that the subject has encountered or been exposed to the disease-related antigen previously. Thus, the methods of the invention have particular utility in determining whether the subject has previously encountered the disease-related antigens. This is useful in determining whether the subject has been exposed to a particular disease.

[0125] The presence of the at least on T-cell activation biomarker in the test sample may also indicate that the subject has the disease. Thus, determining the presence or absence of a T-cell response can be the equivalent to diagnosing whether a subject has a given disease.

[0126] The presence of a T-cell response to the disease may be determined by the presence of at least CD28. The presence of a T-cell response to the disease may be determined by the presence of at least ZAP70. The presence of a T-cell response to the disease may be determined by the presence of at least LAT. The presence of a T-cell response to the disease may be determined by the presence of at least LCK. The presence of a T-cell response to the disease may be determined by the presence of at least ARL13b. The presence of a T-cell response to the disease may be determined by the presence of at least ARL13a.

[0127] The methods of the invention may comprise determining the presence of a T-cell response if at least two (e.g. at least three, at least four, or at least five) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least two (e.g. at least three, at least four, or at least five) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least two (e.g. at least three, at least four, or at least five) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample.

[0128] The methods of the invention may comprise determining the presence of a T-cell response if at least two T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and ZAP70 are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and LAT are detected in the test sample. The methods of theinvention may comprise determining the presence of a T-cell response if at least CD28 and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LCK and ARL13b are detected in the test sample.

[0129] The methods of the invention may comprise determining the presence of a T-cell response if at least two T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and ZAP70 are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ARL13b and ARL13a are detected in the test sample.

[0130] The methods of the invention may comprise determining the presence of a T-cell response if at least two T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample. The methods of the invention may comprisedetermining the presence of a T-cell response if at least CD28 and ZAP70 are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28 and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70 and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LCK and ARL13a are detected in the test sample.

[0131] The methods of the invention may comprise determining the presence of a T-cell response if at least three T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LCK, and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LCK and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT, LCK and ARL13b are detected in the test sample.The methods of the invention may comprise determining the presence of a T-cell response if at least three T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LCK, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT, ARL13b, and ARL 13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LCK, ARL 13b, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, ARL13b, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ARL13b, and ARL13a are detected in the test sample.

[0132] The methods of the invention may comprise determining the presence of a T-cell response if at least three T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and LAT are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining thepresence of a T-cell response if at least CD28, LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LCK, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT, LCK and ARL13a are detected in the test sample.

[0133] The methods of the invention may comprise determining the presence of a T-cell response if at least four T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LCK and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT, LCK and ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT, LCK and ARL13b are detected in the test sample.

[0134] The methods of the invention may comprise determining the presence of a T-cell response if at least four T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13B and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT, ARL13b and ARL13a are detected in thetest sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least LAT, LCK, ARL13b and ARL13a are detected in the test sample.

[0135] The methods of the invention may comprise determining the presence of a T-cell response if at least four T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT and LCK are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT, LCK and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT, LCK and ARL13a are detected in the test sample.

[0136] The methods of the invention may comprise determining the presence of a T-cell response if at least five T-cell biomarkers selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT, LCK, ARL13b are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LAT, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, ZAP70, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least CD28, LAT, LCK, ARL13b and ARL13a are detected in the test sample. The methods of the invention may comprise determining the presence of a T-cell response if at least ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample.

[0137] The methods of the invention may comprise determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, and ARL13b are detected in the test sample.The methods of the invention may comprise determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, ARL13b and ARL13a are detected in the test sample.

[0138] The methods of the invention may comprise determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample.

[0139] The presence or level of a T-cell response may be determined by comparing the detected level of the at least one T-cell activation biomarker to a threshold level. Thus, the methods of the invention may comprise determining the level of the T-cell activation biomarker and comparison with the level to a threshold level. As used herein, the term “threshold level” refers to the level of a T-cell activation biomarker in a test sample which is indicative of the level of the biomarker known to be present following T-cell stimulation or re-stimulation by at least one disease-related antigen. The terms “threshold level” or “reference-level” may be used interchangeably. The threshold level is a level of a T-cell biomarker that may be used to differentiate between samples from subjects that have a T-cell response to the disease-related antigen and subjects that do not have a T-cell response to the disease-related antigen. In other words, the threshold level may be used to differentiate between samples from subjects that have been exposed to the disease-related antigen and subjects that have not been exposed to the disease related antigen. The threshold level may be the level at which the biomarkers are considered to be the background level. In other words, the level at which the biomarkers are present in a sample when no T-cell activation is present. Threshold levels can be determined using any suitable methods in the art. The threshold level for any one of the at least one T-cell activation markers may be independent of the threshold level for another of the at least one T-cell activation markers. Alternatively, threshold level for any two, three, four or five of the at least one T-cell activation markers may be interdependent.

[0140] The threshold level may refer to the level of a T-cell activation biomarker, or a score derived from the measurement of a T-cell activation biomarker, obtained from a “control”. In other words, the threshold level may be predetermined by analysing results from subjects and controls, and determining an appropriate value for detecting whether a subject as has a particular immune response state e.g. whether the subject has a T-cell response to a particular disease-related antigen (in other words, whether the subject has been exposed to a particular disease).

[0141] The threshold level may be indicative of the presence of a T-cell response to the disease. In other words, if the level of the at least one T-cell activation biomarker is equal to or above the threshold level, then the T-cell activation biomarker is determined to be present in the test sample and the presence of a T-cell response to the disease may be confirmed. However, if the level of the at least one biomarker is below the threshold level then the T-cell activation biomarker is determined to be absent and a T-cell response is considered to beabsent. Alternatively, the threshold level may be indicative of the absence of a T-cell response to the disease, if the level of the at least one T-cell activation biomarker is equal to or below the threshold level, then the T-cell activation biomarker is determined to be absent and a T-cell response is considered to be absent. The threshold level for the at least one T-cell activation marker may be dependent on the disease. Alternatively, the threshold level for the at least one T-cell activation marker may be disease-independent.

[0142] The threshold level may be determined using the level of the at least one T-cell activation biomarker in control subjects or group of control subjects. The method may comprise comparing the level of the at least one T-cell activation biomarker in the test sample to the level of the at least one T-cell activation biomarker in a control.

[0143] The control may be the level of the at least T-cell activation biomarker in a test sample from one or more healthy subjects (i.e. one or more subjects without the disease). Accordingly, (i) the same or a lower level of the at least one T-cell activation biomarker in the test sample compared to the level of the at least one T-cell activation biomarker in the control is indicative of the absence of a T-cell response to the disease (i.e. that the subject has not been exposed to the disease); and (ii) a higher level of the at least one T-cell activation biomarker in the test sample compared to the level of the at least one T-cell activation biomarker in the control is indicative of the presence of a T-cell response to the disease (i.e. that the subject has been exposed to the disease).

[0144] The control may be the level of the at least one T-cell activation biomarker in the test sample from one or more subjects with the presence of a T-cell response to the disease (i.e. subjects who have been exposed to the disease). Accordingly, (i) the same or a higher level of the at least one T-cell activation biomarker in the test sample compared to the level of the at least one T-cell activation biomarker in the control is indicative of the presence of a T-cell response to the disease (i.e. that the subject has been exposed to the disease).

[0145] It will be clear to those skilled in the art that the control subject(s) may be selected on a variety of factors which may include, for example, subjects known to have no T-cell response to the disease. Comparison with a control is well known in the field of diagnostics. The “control” may comprise healthy subjects without the disease and / or subjects with the disease.

[0146] It will be understood that it is not necessary to measure controls levels or determine a threshold level for comparative purposes on every occasion. For example, for healthy / non-diseased controls, once the ‘normal range’ is established it can be used as a benchmark for all subsequent tests. A normal range can be established by obtaining samples from multiple control subjects without disease / T-cell response and testing for the level of biomarker. Results (i.e. biomarker levels) for subjects suspected to have a T-cell response / disease can then be examined to see if they fall within, or outside of, the respective normal range. Use of a ‘normal range’ is standard practice for the detection of disease.The threshold level may be a predetermined threshold level which is indicative of the presence of or absence of a T-cell response to the disease.

[0147] The threshold level may be a level of the at least one T-cell activation biomarker that is known to be associated with the presence of a T-cell response to the disease. Accordingly, (i) the same or a higher level of the at least one T-cell activation biomarker in the test sample compared to the threshold level is indicative of the presence of a T-cell response to the disease (i.e. that the subject has been exposed to the disease); and (ii) a lower level of the at least one T-cell activation biomarker in the test sample compared to the threshold level is indicative of the absence of a T-cell response to the disease (i.e. that the subject has not been exposed to the disease).

[0148] The threshold level may be a level of the at least one T-cell activation biomarker that is known to be associated with the absence of a T-cell response to the disease. Accordingly, (i) the same or a lower level of the at least one T-cell activation biomarker in the test sample compared to the threshold level is indicative of the absence of a T-cell response to the disease (i.e. that the subject has been exposed to the disease).

[0149] Disease-related Antigens

[0150] As described herein, the methods of the invention utilise at least one disease-related antigen to re-stimulate antigen-specific T-cells in a sample obtained from a subject.

[0151] A “disease-related antigen” as described herein is a molecule or substance associated with a disease which is capable of stimulating / activating an immune response. An antigen may be all, or a fragment of, a protein, peptide, carbohydrate or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal. The antigen may be selected from the group consisting of peptides, proteins (including glycoproteins), carbohydrates, phospholipids, phosphoproteins, phospholipoproteins, and fragments of the foregoing. Preferably, the at least one disease-related antigen maybe an antigenic peptide or a fragment thereof, or an antigenic carbohydrate or a fragment thereof. Fragments of an antigen may be referred to as antigenic fragments. The disease-related antigen may be derived from screening for immunodominant targets and / or in silico analysis.

[0152] Typically, when the disease-related antigen is an antigenic peptide or fragment thereof, the antigenic peptides may each be between 5-30 amino acids, 5-25 amino acids, 5-20 amino acids, 10-30 amino acids, 10-25 amino acids, 10-20 amino acids 15-30 amino acids, 15-25 amino acids, 15-20 amino acids in length. Preferably, the antigen peptide is 20 amino acids in length (i.e. the peptide is a 20-mer).

[0153] The disease-related antigen used in the methods of the invention will depend on the specific disease. For example, if the method is determining the T-cell response to a virus, viral antigens such as the spike protein, capsid protein membrane, nucleoprotein from the virus inquestion may be used. The disease may be a virus and the at least one disease-related antigen may be selected from a spike protein, membrane, envelope, polymerase, nucleoprotein or whole viral lysate from the virus. The disease may be a bacteria and the at least one disease-related antigen may be selected based on characterization of immunoactive bacterial proteins such as a flagella antigen, a virulence antigen, or a fimbrial antigen.

[0154] The disease may be a cancer and the at least one disease-related antigen may be a tumor-specific antigen or a tumor-associated antigen.

[0155] The disease may be an autoimmune disease and the at least one antigen may be a self-antigen (e.g. myelin (or any component thereof, such as myelin basic protein, proteolipoprotein, myelin oligodendrocyte glycoprotein), collagen, or GAD65).

[0156] The disease may be an allergy or asthma and the at least one antigen may be an environmental antigen (e.g. pet dander, pollen, house dust mites, mould spores or food antigen).

[0157] The antigens used in the methods of the invention may be naturally occurring antigens, fragments of a naturally occurring antigen or synthetic antigens.

[0158] At least one disease-related antigen may be used in the methods of the invention. At least one, for example, at least two, at least three, at least four, at least five, at least six, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 disease-related antigens are contacted with the sample from the subject. The methods of the invention may comprise contacting a sample from the subject with 1-20, 2-20, 3-20, 4-20, 5-20, 1-18, 2-18, 3-18, 4-18, 5-18, 1-15, 2-15, 3-15, 4-15, 5-15, 1-12, 2-12, 4-12, 5-10, 1-5, 2-5 or 3-5 disease-related antigens. The methods of the invention may comprise contacting a sample from the subject with less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 disease-related antigens. The methods of the invention may comprise contacting a sample from the subject with 5, 4, 3, 2 or 1 disease-related antigens. Preferably, the methods of the invention may comprise contacting a sample from the subject with less than 5 disease-related antigens.

[0159] Reference herein to a disease-related antigen in the context of a pathogen, a cancer, an autoimmune disease, asthma or an allergy is synonymous with an antigen of a pathogen, a cancer antigen, an autoimmune disease target, or an asthma or allergy allergen. By way of example, a disease-related antigen in the context of a pathogen may be referred to as a pathogen. By way of further example, a disease-related antigen in the context of a cancer may be referred to as a cancer antigen. By way of further example, a disease-related antigen in the context of an autoimmune disease may be referred to as an autoimmune disease target. By way of further example, a disease-related antigen in the context of asthma or an allergy may be referred to as an asthma or allergy allergen.The disease-related antigen may be comprised within a plurality of antigens or antigenic fragments from the disease. The disease-related antigen may be comprised within a plurality of antigens or antigenic fragments from a pathogen. The disease-related antigen may be comprised within a plurality of antigens or antigenic fragments from the cancer. The disease-related antigen may be comprised within a plurality of antigens or antigenic fragments from self-antigens. The disease-related antigen may be comprised within a plurality of antigens or antigenic fragments from environmental antigens.

[0160] Where a plurality of antigens are used, the antigens may be added separately to the sample or the antigens may be added to the sample at the same time, such as in a single composition.

[0161] The at least one disease-related antigen may be immobilised on a support. Typically the support is an inert support. The support may be any suitable support known in the art. The support may be selected from agarose, cellulose, glass, paramagnetic beads, or polystyrene beads. For example, the at least one disease-related antigen may be immobilised on a support within a vessel such as a sample collection tube. The at least one disease-related antigen may be immobilised by coating the surface of a vessel or chamber of a vessel. The sample may be added to the vessel or the sample may be obtained directly into the vessel. For example, where the sample is a blood sample, the collection vessel may be a blood collection tube comprising a composition of antigens.

[0162] Pathogens

[0163] As described herein, the method of the invention may be used to determine the presence or absence of a T-cell response to a disease. The disease may be a disease caused by a pathogen. All disclosure herein in relation to disease or disease-related antigens applies equally and without reservation to embodiments relating to determining the presence or absence of a T-cell response to a disease mediated by a pathogen as described below.

[0164] Thus, the invention provides a method for determining the presence or absence of a T-cell response to a pathogen in a subject comprising: a) contacting a sample from the subject with at least one antigen from the pathogen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the presence of a T-cell response to the pathogen based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0165] The invention provides a method for determining the presence or absence of a T-cell response to a pathogen in a subject comprising: a) contacting a sample from the subject with at least one antigen from the pathogen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT,LCK, ARL13b and ARL13a; and c) determining the presence of a T-cell response to the pathogen based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0166] The invention provides a method for determining the presence or absence of a T-cell response to a pathogen in a subject comprising: a) contacting a sample from the subject with at least one antigen from the pathogen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the presence of a T-cell response to the pathogen based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0167] The antigen may be comprised within a plurality of antigens or antigenic fragments from a pathogen.

[0168] The pathogen may be selected from a virus or a bacteria. The pathogen may be selected from SARS-CoV-2, Cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, herpes simplex virus, Salmonella enterica, Samonella bongori, Yersinia pestis, Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Bacillus cereus biovar anthracis, Burkholderia cepacia complex, Burkholderia, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, zika virus, group A streptococcus, measles virus, mumps virus, rubella virus, Dengue virus, Chikungunya virus, Streptococcus pneumoniae, Neisseria meningitidis, Poliovirus, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae type b, rotavirus, Bordetella pertussis, human papillomavirus, human immunodeficiency virus, and influenza. The pathogen may be a virus. The pathogen may be SARS-CoV-2, Cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, herpes simplex virus, zika virus, measles virus, mumps virus, rubella virus, Dengue virus, Chikungunya virus, rotavirus, human papillomavirus, human immunodeficiency virus, and influenza. The pathogen may be a virus e.g. SARS-CoV-2.

[0169] Cancer

[0170] As described herein, the method of the invention may be used to determine the presence or absence of a T-cell response to a disease (e.g. a response to a disease-related antigen). The disease may be a cancer. All disclosure herein in relation to disease or disease-related antigens applies equally and without reservation to embodiments relating to determining the presence or absence of a T-cell response to a cancer (e.g. a response to a cancer antigen) as described below.

[0171] Thus, the invention provides a method for determining the presence or absence of a T-cell response to a cancer in a subject comprising: a) contacting a sample from the subject with at least one antigen from the cancer to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28,ZAP70, LAT, LCK, and ARL13b; and c) determining the presence of a T-cell response to the cancer based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0172] The invention provides a method for determining the presence or absence of a T-cell response to a cancer in a subject comprising: a) contacting a sample from the subject with at least one antigen from the cancer to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a; and c) determining the presence of a T-cell response to the cancer based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0173] The invention provides a method for determining the presence or absence of a T-cell response to a cancer in a subject comprising: a) contacting a sample from the subject with at least one antigen from the cancer to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the presence of a T-cell response to the cancer based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0174] The antigen may be comprised within a plurality of antigens or antigenic fragments from the cancer, based on tumour-specific antigens including those identified by neoantigen sequencing.

[0175] The cancer may be selected from breast cancer, lung cancer, melanoma, hepatocellular carcinoma and colon cancer.

[0176] The cancer may be selected from breast cancer, lung cancer, melanoma, hepatocellular carcinoma, colon cancer, bladder cancer, head and neck cancer, oesophageal cancer, ovarian cancer, thyroid cancer, prostate cancer, sarcoma, and neuroblastoma.

[0177] The subject may have a cancer and be undergoing immunotherapy treatment for the cancer. Thus, in such subjects the T-cell response may be indicative of the T-cell response to the cancer and / or the immunotherapy. Cancer may be associated with T-cell exhaustion which is characterised by loss of T-cell effector functions and self-renewal capacity. T-cell exhaustion may be a pathway for resistance to cancer treatments such as immunotherapy. Thus, for subjects who have a cancer and are undergoing immunotherapy treatment for the cancer, the presence of a T-cell response to the cancer may indicate that the immunotherapy is effective (e.g. the cancer has been reduced, eradicated or stabilized, or that T-cell exhaustion is not present), while the absence of a T-cell response to the cancer may indicate that the immunotherapy has not been effective (e.g. the cancer has progressed, or that T-cell exhaustion is present).Autoimmune diseases

[0178] As described herein, the method of the invention may be used to determine the presence or absence of a T-cell response to a disease (e.g. a response to a disease-related antigen). The disease may be an autoimmune disease. All disclosure herein in relation to disease or disease-related antigens applies equally and without reservation to embodiments relating to determining the presence or absence of a T-cell response to an autoimmune disease (e.g. a response to an autoimmune disease target) as describe below.

[0179] Thus, the invention provides method for determining the presence or absence of a T-cell response to an autoimmune disease (e.g. an autoimmune disease target) in a subject comprising: a) contacting a sample from the subject with at least one antigen from the autoimmune disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the presence of a T-cell response to the autoimmune disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0180] The invention provides method for determining the presence or absence of a T-cell response to an autoimmune disease (e.g. an autoimmune disease target) in a subject comprising: a) contacting a sample from the subject with at least one antigen from the autoimmune disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a; and c) determining the presence of a T-cell response to the autoimmune disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0181] The invention provides method for determining the presence or absence of a T-cell response to an autoimmune disease (e.g. an autoimmune disease target) in a subject comprising: a) contacting a sample from the subject with at least one antigen from the autoimmune disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the presence of a T-cell response to the autoimmune disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0182] The antigen may be comprised within a plurality of antigens or antigenic fragments from autoimmune self-antigens related to the autoimmune disease. For example, the autoimmune self-antigen may be myelin (or any component thereof, such as myelin basic protein, proteolipoprotein, myelin oligodendrocyte glycoprotein), collagen or GAD65.

[0183] The auto-immune disease may be selected from multiple sclerosis, rheumatoid arthritis and type 1 diabetes.

[0184] Asthma and allergiesAs described herein, the method of the invention may be used to determine the presence or absence of a T-cell response to a disease (e.g. a response to a disease-related antigen). The disease may be asthma or an allergy. All disclosure herein in relation to disease or disease-related antigens applies equally and without reservation to embodiments relating to determining the presence or absence of a T-cell response to asthma or an allergy (e.g. a response to an asthma or allergy allergen) as describe below.

[0185] Thus, the invention provides method for determining the presence or absence of a T-cell response to asthma or an allergy in a subject comprising: a) contacting a sample from the subject with at least one antigen from the asthma or the allergy to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the presence of a T-cell response to the asthma or the allergy based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0186] The invention provides method for determining the presence or absence of a T-cell response to asthma or an allergy in a subject comprising: a) contacting a sample from the subject with at least one antigen from the asthma or the allergy to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a; and c) determining the presence of a T-cell response to the asthma or the allergy based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0187] The invention provides method for determining the presence or absence of a T-cell response to asthma or an allergy in a subject comprising: a) contacting a sample from the subject with at least one antigen from the asthma or the allergy to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the presence of a T-cell response to the asthma or the allergy based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0188] The antigen may be comprised within a plurality of antigens or antigenic fragments from environmental antigens associated with asthma or allergies. For example, the environmental antigens associated with asthma or allergies may be pet dander, pollen, house dust mites, mould spores or food antigen.

[0189] Other methods or uses

[0190] The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b as a biomarker of a T-cell response to a disease. The disease may be a disease associated with a pathogen, a cancer, an autoimmune disease, asthma or an allergy asdescribed herein. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b as a biomarker of a T-cell response to a pathogen. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b as a biomarker of a T-cell response to a cancer. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b as a biomarker of a T-cell response to an autoimmune disease. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b as a biomarker of a T-cell response to an allergy or asthma.

[0191] The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13b, ARL13a as a biomarker of a T-cell response to a disease. The disease may be a disease associated with a pathogen, a cancer, an autoimmune disease, asthma or an allergy as described herein. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a as a biomarker of a T-cell response to a pathogen. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a as a biomarker of a T-cell response to a cancer. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a as a biomarker of a T-cell response to an autoimmune disease. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a as a biomarker of a T-cell response to an allergy or asthma.

[0192] The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to a disease. The disease may be a disease associated with a pathogen, a cancer, an autoimmune disease, asthma or an allergy as described herein. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to a pathogen. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to a cancer. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to an autoimmune disease. The invention also provides use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to an allergy or asthma.

[0193] The invention also provides a method of measuring a T-cell response to a disease in a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the level of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0194] The invention also provides a method of measuring a T-cell response to a disease in a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK,ARL13b, and ARL13a; and c) determining the level of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0195] The invention also provides a method of measuring a T-cell response to a disease in a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the level of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0196] The disease may be a disease associated with a pathogen, a cancer, an autoimmune disease, asthma or an allergy as described herein. The disease may be a disease associated with a pathogen or a cancer.

[0197] The invention also provides a method of determining T-cell function in a subject comprising a) contacting a sample from the subject with at least one disease-related antigen the subject has previously been exposed to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the T-cell function based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0198] The invention also provides a method of determining T-cell function in a subject comprising a) contacting a sample from the subject with at least one disease-related antigen the subject has previously been exposed to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a; and c) determining the T-cell function based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0199] The invention also provides a method of determining T-cell function in a subject comprising a) contacting a sample from the subject with at least one disease-related antigen the subject has previously been exposed to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the T-cell function based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0200] As used herein, the term “T-cell function” refers to the ability of T-cells to respond to a disease related antigen which the subject is known to have been exposed to previously. T-cell function can be indicative of whether a subject has a normal T-cell immune response to a disease or a reduce T-cell immune response to a disease. A “reduced T-cell function” may be indicative of the subject having a compromised immune system e.g. an immunodeficiency disorder.

[0201] T-cell function may be determined by comparison with a threshold level or control level. Where the threshold level is indicative of normal T-cell function, if the level of the at least oneT-cell activation biomarker is below the threshold level the subject will be considered to have a reduced T-cell function, and if the level of the at least one T-cell activation biomarker is the same or above the threshold level the subject will be consider to have a normal T-cell function.

[0202] The method may comprise contacting a second sample obtained from the subject at a subsequent timepoint with the at least one disease-related antigen to generate a second test sample and analysing the second test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and determining the T-cell function based on the presence of at least one of the T-cell activation biomarkers in the second test sample. The method may comprise contacting a second sample obtained from the subject at a subsequent timepoint with the at least one disease-related antigen to generate a second test sample and analysing the second test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a; and determining the T-cell function based on the presence of at least one of the T-cell activation biomarkers in the second test sample. The method may comprise contacting a second sample obtained from the subject at a subsequent timepoint with the at least one disease-related antigen to generate a second test sample and analysing the second test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and determining the T-cell function based on the presence of at least one of the T-cell activation biomarkers in the second test sample. The method may comprise monitoring the T-cell function of the subject by comparing (i) the level of the at least one T-cell activation biomarker in a test sample obtained from the subject at a first time point with (ii) the level of the at least one T-cell activation biomarker in a test sample obtained from the subject at a second time point; and determining whether the T-cell function of the subject has changed between the first and second time point based on the comparison step. The method may comprise contacting further samples taken from the subject at subsequent time points (e.g. at 3, 4, 5,6 or more time points) with the at least one disease-related antigen to generate a further test samples. Thus, the methods may involve monitoring the T-cell function at intervals, e.g. every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0203] The invention also provides a method for determining the cellular immune status of a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; and c) determining the T-cell immune status of the subject based on the presence of at least one of the T-cell activation biomarkers in the test sample. The method may comprise determining whether the subject is immunocompromised or vulnerable based on the level of the at least one biomarker.The invention also provides a method for determining the cellular immune status of a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, andARL13a; and c) determining the T-cell immune status of the subject based on the presence of at least one of the T-cell activation biomarkers in the test sample. The method may comprise determining whether the subject is immunocompromised or vulnerable based on the level of the at least one biomarker.

[0204] The invention also provides a method for determining the cellular immune status of a subject comprising: a) contacting a sample from the subject with at least one disease-related antigen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the T-cell immune status of the subject based on the presence of at least one of the T-cell activation biomarkers in the test sample. The method may comprise determining whether the subject is immunocompromised or vulnerable based on the level of the at least one biomarker.

[0205] The method may comprise monitoring the cellular immune status in the subject by comparing (i) the level of the at least one T-cell activation biomarker in a test sample obtained from the subject at a first time point with (ii) the level of the at least one T-cell activation biomarker in a test sample obtained from the subject at a second time point; and determining whether the cellular immune status of the subject has changed between the first and second time point based on the comparison step. The method may comprise contacting further samples taken from the subject at subsequent time points (e.g. at 3, 4, 5, 6 or more time points) with the at least one disease-related antigen to generate a further test samples. The methods may involve monitoring the cellular immune status at intervals, e.g. every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0206] The invention also provides a method of monitoring cellular immune status in a subject comprising: a) contacting a first sample taken from the subject at a first time point with at least one disease-related antigen to generate a first test sample; b) contacting a second sample taken from the subject at a second subsequent time point with the at least one disease-related antigen to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; d) comparing the level of the at least one biomarker in the first and second test samples; and (e) determining whether the cellular immune status of the subject has changed between the first and second time points.The invention also provides a method of monitoring cellular immune status in a subject comprising: a) contacting a first sample taken from the subject at a first time point with at least one disease-related antigen to generate a first test sample; b) contacting a second sample taken from the subject at a second subsequent time point with the at least one disease-related antigen to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a; d) comparing the level of the at least one biomarker in the first and second test samples; and (e) determining whether the cellular immune status of the subject has changed between the first and second time points.

[0207] The invention also provides a method of monitoring cellular immune status in a subject comprising: a) contacting a first sample taken from the subject at a first time point with at least one disease-related antigen to generate a first test sample; b) contacting a second sample taken from the subject at a second subsequent time point with the at least one disease-related antigen to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; d) comparing the level of the at least one biomarker in the first and second test samples; and (e) determining whether the cellular immune status of the subject has changed between the first and second time points.

[0208] The methods may comprise contacting further samples taken from the subject at subsequent time points (e.g. at 3, 4, 5, 6 or more time points) with the at least one disease-related antigen to generate a further test sample. The methods may involve monitoring the cellular immune status at intervals, e.g. every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months. The methods may comprise monitoring the cellular immune status of the population by comparing the cellular immune status of multiple members of a given population.

[0209] The invention also provides a method of monitoring the levels of T-cell immunity for a disease in a subject comprising: a) contacting a first sample taken from the subject at a first time point with at least one disease-related antigen to generate a first test sample; b) contacting a second sample taken from the subject at a second subsequent time point with the at least one disease-related antigen to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b; d) comparing the level of the at least one biomarker in the first and second test samples; and e) determining whether the level of T-cell immunity for the disease has changed between the first and second time points.

[0210] The invention also provides a method of monitoring the levels of T-cell immunity for a disease in a subject comprising: a) contacting a first sample taken from the subject at a first time point with at least one disease-related antigen to generate a first test sample; b)contacting a second sample taken from the subject at a second subsequent time point with the at least one disease-related antigen to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a; d) comparing the level of the at least one biomarker in the first and second test samples; and e) determining whether the level of T-cell immunity for the disease has changed between the first and second time points.

[0211] The invention also provides a method of monitoring the levels of T-cell immunity for a disease in a subject comprising: a) contacting a first sample taken from the subject at a first time point with at least one disease-related antigen to generate a first test sample; b) contacting a second sample taken from the subject at a second subsequent time point with the at least one disease-related antigen to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; d) comparing the level of the at least one biomarker in the first and second test samples; and e) determining whether the level of T-cell immunity for the disease has changed between the first and second time points.

[0212] The methods may comprise contacting further samples taken from the subject at subsequent time points (e.g. at 3, 4, 5, 6 or more time points) with the at least one disease-related antigen to generate a further test sample. The methods may involve monitoring the T-cell immunity at intervals, e.g. every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0213] The invention also provides a method of monitoring the immune response of a subject following vaccination comprising: a) contacting a first sample taken from the subject at a first timepoint before vaccination with at least one antigen from the vaccine to generate a first test sample; b) contacting a second sample taken from the subject after vaccination with the at least one antigen from the vaccine to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b to determine the level of vaccine immunity at the first and second timepoints; d) comparing the level of the at least one T-cell activation biomarker in the first and second test samples to determine if there is a change in the immune response of the subject following vaccination.

[0214] The invention also provides a method of monitoring the immune response of a subject following vaccination comprising: a) contacting a first sample taken from the subject at a first timepoint before vaccination with at least one antigen from the vaccine to generate a first test sample; b) contacting a second sample taken from the subject after vaccination with the at least one antigen from the vaccine to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a to determine the level of vaccine immunity atthe first and second timepoints; d) comparing the level of the at least one T-cell activation biomarker in the first and second test samples to determine if there is a change in the immune response of the subject following vaccination.

[0215] The invention also provides a method of monitoring the immune response of a subject following vaccination comprising: a) contacting a first sample taken from the subject at a first timepoint before vaccination with at least one antigen from the vaccine to generate a first test sample; b) contacting a second sample taken from the subject after vaccination with the at least one antigen from the vaccine to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a to determine the level of vaccine immunity at the first and second timepoints; d) comparing the level of the at least one T-cell activation biomarker in the first and second test samples to determine if there is a change in the immune response of the subject following vaccination.

[0216] The method may comprise monitoring the immune response of a subject following vaccination by comparing (i) the level of the at least one T-cell activation biomarker in a test sample obtained from the subject after vaccination with (ii) the level of the at least one T-cell activation biomarker in a test sample obtained from the subject at a subsequent time point; and determining whether the immune response of the subject has changed between the first and second time points based on the comparison step.

[0217] The methods may comprise contacting further samples taken from the subject at subsequent time points (e.g. at 3, 4, 5, 6 or more time points) with the at least one disease-related antigen to generate a further test sample. The methods may involve monitoring the immune response at intervals, e.g. every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0218] The invention also provides a method of determining vaccine immunity waning in a subject comprising: a) contacting a first sample taken from the subject after vaccination with at least one antigen from the vaccine to generate a first test sample; b) contacting a second sample taken from the subject at a subsequent timepoint with the at least one antigen from the vaccine to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b to determine the level of vaccine immunity at the first and second timepoints; d) comparing the level of the at least one T-cell activation biomarker in the first and second test samples; and e) determining whether the level of vaccine immunity has waned between the first and second time points.

[0219] The invention also provides a method of determining vaccine immunity waning in a subject comprising: a) contacting a first sample taken from the subject after vaccination withat least one antigen from the vaccine to generate a first test sample; b) contacting a second sample taken from the subject at a subsequent timepoint with the at least one antigen from the vaccine to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a to determine the level of vaccine immunity at the first and second timepoints; d) comparing the level of the at least one T-cell activation biomarker in the first and second test samples; and e) determining whether the level of vaccine immunity has waned between the first and second time points.

[0220] The invention also provides a method of determining vaccine immunity waning in a subject comprising: a) contacting a first sample taken from the subject after vaccination with at least one antigen from the vaccine to generate a first test sample; b) contacting a second sample taken from the subject at a subsequent timepoint with the at least one antigen from the vaccine to generate a second test sample; c) analysing the first and second test samples for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a to determine the level of vaccine immunity at the first and second timepoints; d) comparing the level of the at least one T-cell activation biomarker in the first and second test samples; and e) determining whether the level of vaccine immunity has waned between the first and second time points.

[0221] The invention also provides a method of determining the efficacy of a therapeutic intervention in a subject with a disease comprising: a) contacting a first sample taken from the subject prior to the therapeutic intervention with at least one disease-related antigen from the disease to generate a first test sample; b) contacting a second sample taken from the subject after the therapeutic intervention with the at least one disease-related antigen from the disease to generate a second test sample; c) comparing the level of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13b in the first test sample with the level of the at least one T-cell activation biomarker in the second test sample; and d) determining the efficacy of the therapeutic intervention based on the comparison performed in step c).

[0222] The invention also provides a method of determining the efficacy of a therapeutic intervention in a subject with a disease comprising: a) contacting a first sample taken from the subject prior to the therapeutic intervention with at least one disease-related antigen from the disease to generate a first test sample; b) contacting a second sample taken from the subject after the therapeutic intervention with the at least one disease-related antigen from the disease to generate a second test sample; c) comparing the level of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b and ARL13a in the first test sample with the level of the at least one T-cell activation biomarker in the second test sample; and d)determining the efficacy of the therapeutic intervention based on the comparison performed in step c).

[0223] The invention also provides a method of determining the efficacy of a therapeutic intervention in a subject with a disease comprising: a) contacting a first sample taken from the subject prior to the therapeutic intervention with at least one disease-related antigen from the disease to generate a first test sample; b) contacting a second sample taken from the subject after the therapeutic intervention with the at least one disease-related antigen from the disease to generate a second test sample; c) comparing the level of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a in the first test sample with the level of the at least one T-cell activation biomarker in the second test sample; and d) determining the efficacy of the therapeutic intervention based on the comparison performed in step c).

[0224] The disease may be caused by a pathogen or may be a cancer, an autoimmune disease, asthma or an allergy. The therapeutic intervention may be an immunotherapy, an antimicrobial treatment, or an antiviral treatment. Preferably the therapeutic intervention is an immunotherapy, such as an immune checkpoint inhibitor.

[0225] It will be understood that the efficacy of the therapeutic intervention will depend on the type of disease being treated and the type of therapeutic intervention used. The method may comprise a step of changing the therapeutic intervention if the efficacy of the therapeutic intervention is below a desired level. By way of non-limiting example, the method may be used to determine the efficacy of a chemotherapeutic for treating breast cancer. If the efficacy of the chemotherapeutic is determined to be below a desired level, the method may comprise a step of changing the chemotherapeutic for a second chemotherapeutic, or an immunotherapeutic. The method may be used to determine the efficacy of an immunotherapy for treating cancer. A higher level of at least one T-cell activation biomarker in the second test sample may be indicative of the presence of a T-cell response to the cancer and that the immunotherapy is effective (e.g. the cancer has been reduced, eradicated or stabilized, orT-cell exhaustion is not present). A lower level of at least one T-cell activation biomarker in the second test sample may be indicative of the absence of a T-cell response to the cancer and that the immunotherapy has not been effective (e.g. the cancer has progressed, or T-cell exhaustion is present). If the efficacy of the immunotherapy is determined to be below a desired level, the method may comprise a step of changing the immunotherapy for a second immunotherapeutic, or a chemotherapeutic.

[0226] The invention also provides a method for determining the status of the adaptive immune response to a disease comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28,ZAP70, LAT, LCK, and ARL13b; and c) determining the status of the adaptive immune response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0227] The invention also provides a method for determining the status of the adaptive immune response to a disease comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a; and c) determining the status of the adaptive immune response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0228] The invention also provides a method for determining the status of the adaptive immune response to a disease comprising: a) contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c) determining the status of the adaptive immune response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0229] The invention also provides a method of diagnosing a subject as having a disease comprising a) contacting a sample from the subject with at least one disease-related antigen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, andARL13b; and c) determining whether the subject has the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0230] The invention also provides a method of diagnosing a subject as having a disease comprising a) contacting a sample from the subject with at least one disease-related antigen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, ARL13b, and ARL13a; and c) determining whether the subject has the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

[0231] The invention also provides a method of diagnosing a subject as having a disease comprising a) contacting a sample from the subject with at least one disease-related antigen to generate a test sample; b) analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, andARL13a; and c) determining whether the subject has the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.The disease may be a disease associated with a pathogen, a cancer, an autoimmune disease, asthma or an allergy as described herein. The disease may be a disease associated with a pathogen or a cancer.

[0232] SEQUENCE INFORMATION

[0233] Sequence Key

[0234] SEQ ID NO: 1 - Amino acid sequence of Human CD28 (Uniprot: P10747-1, as accessed 20 March 2025)

[0235] M LRLLLALN LFPSIQVTGN KI LVKQSPM LVAYDN AVN LSCKYSYN LFSREFRASLH KGLDSAV EVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLD NEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0236] SEQ ID NO: 2 - Amino acid sequence of Human ZAP70 (Uniprot: P43403-1, as accessed 20 March 2025) MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIE RQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAM VRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQT DGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGL IYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPM PMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKV LKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKR EEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSY YTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAF IEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGST QKAEAACA

[0237] SEQ ID NO: 3 - Amino acid sequence of Human LAT (Uniprot: 043561-1, as accessed 20 March 2025) MEEAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQAG WGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLWLPDSTPATSTAAPSAPALSTP GIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEE VEEEGAPDYENLQELNSEQ ID NO: 4 - Amino acid sequence of Human LCK (Uniprot: P06239-1, as accessed 20 March 2025) MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPPASPL QDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSL EPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEWKHYKI RNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRE TLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRL YAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAA N I LVSDTLSCKI ADFGLARLI EDN EYTAREGAKFPI KWTAPEAI NYGTFTI KSDVWSFGI LLTEI V THGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVL EDFFTATEGQYQPQP

[0238] SEQ ID NO: 5 - Amino acid sequence of Human ARL13b (Uniprot: Q3SXY8-1, as accessed 20 March 2025) MFSLMASCCGWFKRWREPVRKVTLLMVGLDNAGKTATAKGIQGEYPEDVAPTVGFSKINLR QGKFEVTIFDLGGGIRIRGIWKNYYAESYGVIFWDSSDEERMEETKEAMSEMLRHPRISGK PILVLANKQDKEGALGEADVIECLSLEKLVNEHKCLCQIEPCSAISGYGKKIDKSIKKGLYWLL HVIARDFDALNERIQKETTEQRALEEQEKQERAERVRKLREERKQNEQEQAELDGTSGLAE LDPEPTNPFQPIASVIIENEGKLEREKKNQKMEKDSDGCHLKHKMEHEQIETQGQVNHNGQ KNNEFGLVENYKEALTQQLKNEDETDRPSLESANGKKKTKKLRMKRNHRVEPLNIDDCAPE SPTPPPPPPPVGWGTPKVTRLPKLEPLGETHHNDFYRKPLPPLAVPQRPNSDAHDVIS

[0239] SEQ ID NO: 6 -Amino acid sequence of Human ARL13a (Uniprot Q5H913, as accessed 16 March 2026) MFRLLSSCCSCLRTTEETRRNVTIPIIGLNNSGKTVLVEAFQKLLPSKTDHCMKSELTTLLLDE YELSIYDLNGDLKGREAWPNYYAQAHGLVFVLDSSDIRRMQEVKIILTHLLSDKRVAGKPILIL ANKQDKKKALMPCDIIDYLLLKKLVKENKCPCRVEPCSAIRNLERRNHQPIVEGLRWLLAVID TCQLPPTSSISISKNNTGSGERCSSHSFSTRTGMSKEKRQHLEQCSIEAKPLKSILQKEGTR LWSKKNMSVTFALDEPMKEGECSRRMRAQNTTKLCYN

[0240] EXAMPLES

[0241] The invention is now described with reference to the Examples below. These are not limiting on the scope of the invention, and a person skilled in the art would be appreciate that suitable equivalents could be used within the scope of the present invention. Thus, the Examples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination.Example 1

[0242] Materials and Methods

[0243] Antigens. For any given microbial pathogen, autoimmune self-antigen, or tumour, the amino acid sequence of the immunodominant antigen was used to synthesise a panel of short synthetic peptides. Synthetic peptide pools were prepared using conventional techniques, such as described in Reynolds et al. (1) (Science 2022 375,183-192) and Reynolds et al. (2) (Sci Immunol. 2020 Dec 23;5(54)). A preparation of a mixed solution of synthetic peptides was then stored ready for stimulation of blood cells. In the following examples, data is shown for COVID immune patient blood samples stimulated by peptides spanning the full-length amino acid sequence of SARS-CoV-2 spike. “Megapools” of peptides were prepared using conventional techniques, such as described in da Silva Antunes et al. (Curr Protoc. 2023 Nov;3(11):e934).

[0244] SARS-CoV-2 infection was determined by baseline and weekly nasal RNA stabilizing swabs and Roche cobas® SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) test and baseline and weekly antibody testing for S1 using the IgG EUROIMMUN enzyme-linked immunosorbent assay (ELISA) and nucleocapsid using the ROCHE Elecsys electrochemiluminescence immunoassay (ECLIA). Antibody ratios >1.1 were deemed positive for the EUROIMMUN SARS-CoV-2 ELISA and >1 was considered test positive for the ROCHE Elecsys anti-SARS-CoV-2 ECLIA, as evaluated by UK Health Security Agency (UKHSA)

[0245] Peptide panels are also prepared using the amino acid sequence of an alternate, diseasepertinent antigen such as MAGEA1 in the case of melanoma immunotherapy, varicella zoster virus glycoprotein E (gE) in the case of shingles, or haemagglutinin in the case of seasonal influenza or H5N1 avian flu. These peptide panels are then used to stimulate blood samples from patients to determine their immunity to the relevant disease.

[0246] Blood cell stimulation. Small blood samples (e.g. a sample obtained from capillary blood collection from a finger tip, earlobe or heel) were added to a tube containing sodium heparin (to give a final concentration of 171 U heparin / ml blood) and the synthetic SARS-CoV-2 peptide mixture / preparation. Alternatively, a clinical venepuncture blood sample was taken into a heparin blood-collection vacutainer, and then density gradient centrifugation was then used to purify a peripheral blood mononuclear cell (PBMC) preparation for assay. The blood samples (e.g. fingerprick whole-blood samples or purified PBMC samples) were thenincubated in the tube containing the synthetic peptide mixture / preparation for 1 hour at room temperature.

[0247] Activated test sample preparation. 400pL supernatant media from the peptide activated samples was added to a 1.5mL Eppendorf tube. 1.6mL of ice-cold acetone was added and samples incubated at -20°C for 2hours to precipitate protein. Following this, samples are centrifuged at 16,900g for 10 minutes at 4°C. The supernatant was discarded and pellets air dried for 1 hour before adding 70pl 0.5% sodium deoxycholate in 50mM ammonium bicarbonate buffer to each well, followed by the addition of 3pl of DTT solution (162mM DL-dithiothreitol in 0.5% sodium deoxycholate / 50mM ammonium bicarbonate buffer). The plates were capped and incubated at 85° for 15 minutes with shaking at 750rpm. After incubation, plates were allowed to cool to room temperature before adding 6pl of 162 mM iodoacetamide (IAA) in 0.5% sodium deoxycholate / 50 mM ammonium bicarbonate buffer. The plates were capped, briefly shaken, and incubated at room temperature for 30 minutes. Subsequently, 5pl trypsin (Sigma) (1mg / mL in 50 mM acetic acid) was added, and the mixture incubated at 45°C for 30 minutes. The digestion process was halted by adding 5pl of 6% TFA and mixing well. Plates were centrifuged at 4000g for 20 minutes at 10°C. Fifty microliters of supernatant was aliquoted into a fresh plate and analysed by LC-MS / MS.

[0248] Comparator ELISpot assay. ELISpot assays were carried out as previously described. Briefly, precoated ELISpot plates (Mabtech 3420-2APT) were washed x4 with PBS, blocked for 1h (room temperature) with supplemented RPMI1640 (GibcoBRL) [10% heat inactivated FCS; 1% 10Oxpenicillin, streptomycin and L-Glutamine solutions (GibcoBRL)]. 200,000 PBMC were seeded / well and stimulated 18-22h at 37 °C with SARS-CoV-2 recombinant protein [Wuhan Hu-1, B.1.617.2 (Delta) or B.1.1.529 (Omicron) SARS-CoV-2 S1 spike proteins (10 pg / ml)] or peptide pools (10 pg / ml / peptide). Negative and positive plate controls were medium or anti-CD3 (Mabtech mAb CD3-2). ELISpot plates were developed with 1 pg / ml biotinylated anti-human IFNy detection Ab conjugated to alk-phosphatase (7-B6-1-ALP, Mabtech), diluted in PBS / 0.5% FCS, adding 50 pl / well for 2h at room temperature followed by 50 pl / well BCIP / NBT-plus phosphatase substrate (Mabtech), 5 minutes (room temperature). Plates were washed and dried before analysis on an AID classic ELISpot plate reader (Autoimmun Diagnostika GMBH, Germany). ELISpot data was analyzed in Microsoft Excel. The average of two culture media alone wells was subtracted from all protein / peptide stimulated wells and any response that was lower in magnitude than 2 standard deviations of the sample specific control wells was not considered a stimulation-specific response. Results were expressed as difference in (delta) spot forming cells (SFC) / 106PBMC between negative control and protein / peptide stimulation conditions. Results were excluded if negative control wells showed>100 SFC / 106PBMC (n = 4) or cell viability was low with <1000 SFC / 106PBMC in anti-CD3 positive control wells (n = 5). Results were plotted using Prism 9.0 for Mac OS (GraphPad).

[0249] LC-MS / MS analysis. Digested samples were injected onto a Waters 50mm LIPLC Premier C18 1.7pm, 2.1 x 50mm column, operating at 45 °C for chromatographic separation, with a total method run time of 10 minutes. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in ACN, pumped at a flow rate of 0.3 mL / min. The initial conditions of 5% B were maintained for 0.1 minutes, followed by a linear gradient to 40% B over 7.7 minutes for peptide elution and separation. The gradient was then increased to 80% B over 0.2 minutes, held for 1 minute to wash the column, and returned to the initial conditions, with a 1-minute equilibration period before the next injection. The LC system was coupled to a Waters Xevo-TQ-S triple quadrupole mass spectrometer for multiple reaction monitoring (MRM) detection in positive electrospray ionisation mode. The capillary voltage was set to 2.5 kV, the source temperature to 150 °C, and the desolvation temperature to 600°C, with cone gas and desolvation gas flows at 150 and 600 L / hour, respectively. Nitrogen was used as the collision gas at a flow rate of 0.15 mL / min, and the nebuliser operated at 7 bar. The cone energy was set to 35 V, with collision energies optimized for each peptide.

[0250] Data analysis. Raw LC-MS / MS data was analysed using Skyline open-source software (https: / / skyline.ms / project / home / software / Skyline / begin.view). Analyte peptide identifications were determined from the analysis of digested PBMC / whole blood by a minimum of six transitions and matched to an in-silico spectral library (Prosit) for additional confirmation. Peptide abundance data were normalized to isotopically labelled lgG2 (QConCat), added to the whole blood samples. Exported data were then analysed using Microsoft Excel, GraphPad Prism v9, R Studio version 4.4.0 and SIMCA, version 17 (Umetrics Sartorius Stedim, Umea, Sweden).

[0251] Results

[0252] Values are presented as mean ± standard deviation (SD) or as otherwise stated. A paired Wilcoxon t-test is employed for individual comparisons. Results are considered statistically significant with a p-value < 0.05. Normality of the data is assessed using the Shapiro- Wilk test, and data considered to pass normality testing if p > 0.05

[0253] Five T cell immune-synapse related proteins (LCK, ZAP-70, CD28, LAT and ARL13) were identified which are detected within 1 hour of stimulation of T cells in blood samples of immune individuals by the SARS-CoV-2 spike peptide megapool (see Figure 2).The level of these T cell immune-synapse related proteins was found to correlate with T-cell responder status when assessed by conventional next-day ELISpot culture (see Figure 3).

[0254] Conclusion

[0255] The data demonstrate that LCK, ZAP-70, CD28, LAT and / or ARL13 may be used as T-cell activation biomarkers and that the present invention has utility for testing the T cell response to a disease of interest in a subject, particularly an infectious disease, a cancer, an autoimmune disease, asthma or an allergy.

[0256] Example 2

[0257] Materials and Methods

[0258] Study donors. Responses to by the SARS-CoV-2 spike peptide megapool were assayed in 6 healthy lab donors (2 males and 4 females) recruited using Imperial College Healthcare Tissue Bank donor consent in December 2025.

[0259] PBMC isolation. Blood samples were collected into lithium heparin vacutainers and PBMCs isolated using SepMate PBMC isolation tubes (StemCell Technologies).

[0260] IFN-y ELISpot. T cell responses to by the SARS-CoV-2 spike peptide megapool were measured by IFN-y ELISpot using a Mabtech alkaline phosphatase (APT) based IFN- y ELISpot kit (Mabtech 3420-2APT-10). Two hundred thousand PBMC cells were used per well and each assay condition was performed in duplicate. Recombinant proteins and peptide pools were used at a concentration of 2pg / ml and cells were cultured with antigen at 37°C and 5% CO2 for 20 hours before assay development. Data were collected using an AID ELIspot reader (Autoimmun diagnostika GMBH). Assay wells containing cells but no antigen were used as a negative control and responses were considered to be positive if after removal of the background values, the number of spot forming cells was greater than 2SD of the negative control wells. That is, all responses are indicated as difference in response (D), compared to a negative control without antigen.

[0261] Results

[0262] Figure 4 shows the T cell response to the SARS-CoV-2 spike peptide megapool measured in PBMCs from 6 healthy lab donors using the gold standard method - measuring IFN- y levels with ELISpot. Samples from all donors demonstrated a T-cell response measured as ASFCper 106PBMC (difference in response, A, compared to ‘no antigen’ negative control) ranging from <50 to >200 ASFC per 106PBMC.

[0263] As shown in Figure 3, the T cell response to the same SARS-CoV-2 spike peptide megapool was measured using early biomarkers of the invention with ELISpot. This resulted in a ASFC per 106PBMC of 100-1000.

[0264] Conclusion

[0265] Using the same SARS-CoV-2 spike peptide megapool the gold standard IFN-Y method and the method of the invention are both capable of measuring a T-cell response Moreover, the method of the invention appears to be capable of a higher ASFC per 106PBMC readout. Thus, the early-stage T cell response biomarkers of the invention may provide at least an equivalent, and potentially a more sensitive method of determining a T-cell response than the current gold standard.

[0266] Example 3

[0267] Materials and Methods

[0268] Study donors. Responses to Varicella Zoster virus (VZV) gE antigen were assayed in 6 healthy lab donors (2 males and 4 females), of whom only donor 1 had been immunized with the Shingrix VZ vaccine were tested using

[0269] PBMC isolation. Blood samples were collected into lithium heparin vacutainers and PBMCs isolated using SepMate PBMC isolation tubes (StemCell Technologies).

[0270] Recombinant proteins and peptide pools. VZV gE / gl heterodimeric recombinant protein was purchased from the Native Antigen Company (catalogue number REC31907). VZV gE peptide pool was purchased from Stem Cell Technologies (catalogue number 100-1408) and consists of 153 peptides (15 mers with 11 amino acid overlaps) that cover amino acids 1-623 of the VZV gE antigen.

[0271] IFN-y ELISpot T cell responses to VZV antigens were measured by IFN-y ELISpot using a Mabtech alkaline phosphatase (APT) based IFN- y ELISpot kit (Mabtech 3420-2APT-10). Two hundred thousand PBMC cells were used per well and each assay condition was performed in duplicate. Recombinant proteins and peptide pools were used at a concentration of 2pg / mland cells were cultured with antigen at 37°C and 5% CO2 for 20 hours before assay development. Data were collected using an AID ELIspot reader (Autoimmun diagnostika GMBH). Assay wells containing cells but no antigen were used as a negative control and responses were considered to be positive if after removal of the background values, the number of spot forming cells was greater than 2SD of the negative control wells. That is, all responses are indicated as difference in response (A), compared to a negative control without antigen.

[0272] Results

[0273] Figure 5 shows T cell responses to Varicella Zoster Virus (VZV) gE recombinant protein and peptide pool. With respect to a response to VZV gE, donor 1 shows a positive response to both the gE heterodimer and peptide pool. Donor 6 also has positive T cell immunity to VZV, presumably through natural infection, showing a positive response in the more sensitive of the two assays, measuring the response to the peptide pool.

[0274] Conclusion

[0275] Figure 5 demonstrates that VZV immunity can be detected using IFN- y ELISpot. Given the replication of results between the biomarkers of the invention and the known IFN- y ELISpot assay demonstrated in Examples 2-4 and that the biomarkers of the invention appear to provide a more sensitive assay, it is therefore credible that the same result will be seen using the biomarkers of the invention. Thus, the biomarkers of the invention are suitable for detecting a T-cell response to VZV and to detect VZV immunity.

[0276] Example 4

[0277] Materials and Methods

[0278] Study donors. Responses to the cancer antigen MAGE A1 were assayed 26 in an 84 year old male affected by unresectable hepatoceullar carcinoma (HCC) who had been undergoing immunotherapy treatment since May 2024.

[0279] Recombinant proteins and peptide pools. The MAGE A1 peptide pool is made up of 38 peptides (18 mers with 10 amino acid overlaps) that were purchased from GL Biochem, China.

[0280] IFN-y ELISpot. T cell responses to MAGE A1 antigens were measured by IFN-y ELI Spot using a Mabtech alkaline phosphatase (APT) based IFN- y ELISpot kit (Mabtech 3420-2APT-10). Two hundred thousand PBMC cells were used per well and each assay condition wasperformed in duplicate. Recombinant proteins and peptide pools were used at a concentration of 2pg / ml and cells were cultured with antigen at 37°C and 5% CO2 for 20 hours before assay development. Data were collected using an AID ELIspot reader (Autoimmun diagnostika GMBH). Assay wells containing cells but no antigen were used as a negative control and responses were considered to be positive if after removal of the background values, the number of spot forming cells was greater than 2SD of the negative control wells. That is, all responses are indicated as difference in response (A), compared to a negative control without antigen.

[0281] Results

[0282] Figure 6 shows T cell responses to the cancer antigen MAGE A1 (green bar) were measured by IFN- y ELISpot in PBMC from a cancer patient with hepatic cell carcinoma, and indicates a positive T cell response to MAGE A1 peptide pool (difference in response, A, compared to ‘no antigen’ negative control) in an HCC patient undergoing immunotherapy.

[0283] Conclusion

[0284] Figure 6 demonstrates that a T cell response to a cancer antigen can be detected using IFN-Y ELISpot. Given the replication of results between the biomarkers of the invention and the known IFN- y ELISpot assay demonstrated in Examples 2-4 and that the biomarkers of the invention appear to provide a more sensitive assay, it is therefore credible that the same result will be seen using the biomarkers of the invention. Thus, the biomarkers of the invention are suitable for detecting a T-cell response to a cancer.

Claims

CLAIMS1. A method for determining the presence or absence of a T-cell response to a disease in a subject comprising:a. Contacting a sample from the subject with at least one disease-related antigen from the disease to generate a test sample;b. Analysing the test sample for the presence of at least one T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a; and c. Determining the presence of a T-cell response to the disease based on the presence of at least one of the T-cell activation biomarkers in the test sample.

2. The method of claim 1, wherein the disease is caused by a pathogen, or is a cancer, an autoimmune disease, asthma or an allergy.

3. The method of claim 1 or claim 2, wherein the at least one disease-related antigen is comprised within a plurality of antigens or antigenic fragments from the disease.

4. The method of any one of the preceding claims, wherein the at least one disease- related antigen is an antigenic peptide or an antigenic carbohydrate.

5. The method of any one of the preceding claims, the at least one disease-related antigen is immobilised on a support.

6. The method of any one of the preceding claims, wherein the sample is a blood sample.

7. The method of any one of the preceding claims, wherein the sample is incubated for at least 1 hour with at the least one disease-related antigen from the disease.

8. The method according of any one of the preceding claims, wherein incubation / contacting occurs at between 18 °C to 37 °C.

9. The method of any one of the preceding claims, wherein the method further comprises separating the test sample from the at least one disease-related antigen.

10. The method according to any one of the preceding claims, wherein the method further comprises analysing the test sample for the presence of at least two (e.g. at least 3, at55least 4, or at least 5) T-cell activation biomarkers selected from CD28, ZAP70, LAT, LCK, andARL13a.

11. The method according to any one of the preceding claims, wherein the method further comprises analysing the test sample for the presence of all of CD28, ZAP70, LAT, LCK, and ARL13a.

12. The method according to any one of the preceding claims, wherein the method further comprises determining the presence of a T-cell response if at least two (e.g. at least 3, at least 4, or at least 5) T-cell activation biomarker selected from CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample.

13. The method according to any one of the preceding claims, wherein the method further comprises determining the presence of a T-cell response if all of CD28, ZAP70, LAT, LCK, and ARL13a are detected in the test sample.

14. The method according to any one of the preceding claims, wherein the presence of a T-cell activation biomarker is determined by mass spectrometry, ELISA or lateral flow device.

15. The method according to any one of the preceding claims, wherein the disease is a caused by a pathogen and the pathogen is selected from:a. a virus or a bacteria; orb. SARS-CoV-2, Cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, herpes simplex virus, Salmonella enterica, Samonella bongori, Yersinia pestis, Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Bacillus cereus biovar anthracis, Burkholderia cepacia complex, Burkholderia, Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, zika virus, group A streptococcus, measles virus, mumps virus, rubella virus, Dengue virus, Chikungunya virus, Streptococcus pneumoniae, Neisseria meningitidis, Poliovirus, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae type b, rotavirus, Bordetella pertussis, human papillomavirus, human immunodeficiency virus, and influenza.

16. The method according to any one of the preceding claims, wherein the disease is a cancer, and the cancer is selected from breast cancer, lung cancer, melanoma, hepatocellular carcinoma and colon cancer.

17. The method according to any one of the preceding claims, wherein the disease is autoimmune disease, and the autoimmune disease is multiple sclerosis, rheumatoid arthritis or type 1 diabetes.

18. The method according to any one of the preceding claims, wherein the disease is asthma or an allergy.

19. Use of at least one of CD28, ZAP70, LAT, LCK, and ARL13a as a biomarker of a T-cell response to a disease.

20. The use of claim 19, wherein the disease is caused by a pathogen or is a cancer, an autoimmune disease, asthma or an allergy.57

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