Detection of antigen-specific t cell response in whole blood and PBMC samples

Abstract

This application relates to a method for detecting an antigen-specific T cell response in a whole blood or PBMC sample comprising collecting a sample, incubating the sample simultaneously with an antigen and a reporter cell line expressing at least one reporter gene that detects the presence of an antigen-specific T cell response product. It also relates to a vector for detecting an antigen-specific T cell response comprising a transcriptional response element and at least one reporter gene and the kit thereof.

Description

DETECTION OF ANTIGEN-SPECIFIC T CELL RESPONSE IN WHOLE BLOOD AND PBMC SAMPLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore Application No.10202403596X, filed 18 November 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The invention relates generally to detection of cell responses in biological samples. Particularly, the invention relates to methods, compositions and kits for detecting an antigenspecific T cell response in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs).BACKGROUND OF THE INVENTION

[0003] Assessing humoral and cellular response is important for public health strategy in the context of disease management and vaccination effectiveness. Moreover, previous studies have demonstrated the persistence of cellular response despite waning antibodies response. Humoral response is usually assessed from routine immunogenicity monitoring, but cellular response is not included in the monitoring due to the complexity and tediousness of assessing cellular response. Detection of human Interferon gamma (IFN-y) secreted by antigen specific T cells can be evaluated using flow cytometry or ELISPOT. However, these assays typically have long turnaround times of 4 to 6 hours after stimulation and require large amounts of whole blood (3-5 ml) and cells (100,000-300,000) to detect an antigen specific response. Conventional methods such as those based on enzyme-linked immunosorbent assay (ELISA) detection of interferon require multiple processing steps to obtain the optimal amount and types of biological samples to provide reliable results. These processing steps are detrimental to small volume samples and may not provide reliable results. Hence, the conventional methods are not amendable to small volume samples.

[0004] Accordingly, although assessment of cellular response is critical to determine long-term protection in individuals, it is typically not included in immune monitoring due to its complexity and tediousness. As such, there is a need for improved methods for assessing cellular response, such as for detecting antigen specific T cell response.SUMMARYrooos] In one aspect, provided herein is a method for detecting an antigen-specific T cell response in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs), comprising the steps of:(1) collecting a sample;(2) incubating the sample simultaneously with the following:(a) an antigen to induce production of an antigen- specific T cell response product, and(b) a reporter cell line capable of expressing at least one reporter gene, wherein presence of the antigen-specific T cell response product induces expression of the at least one reporter gene by the reporter cell line; and(3) detecting at least one reporter gene product as an indicator of the antigen- specific T cell response, thereby detecting the antigen- specific T cell response in the sample. rooo6] In another aspect, provided herein is a vector for expressing a reporter gene in the presence of an antigen- specific T cell response product, comprising at least one reporter gene operably linked to at least one inducible promoter comprising at least one transcriptional response element (TRE), wherein the at least one inducible promoter is induced in the presence of the antigen-specific T cell response product, thereby expressing the reporter gene.

[0007] In another aspect, provided herein is a kit for detecting antigen- specific T cell responses in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs) using the method as described herein, comprising:(a) reporter cell line capable of expressing at least one reporter gene in the presence of an antigen-specific T cell response product;(b) an antigen for stimulating PBMCs or whole blood to induce production of the antigen-specific T cell response product; and(c) reagents for detecting at least one reporter gene product;wherein the kit is configured to process a sample volume of 50 -100 pL collected via fingerprick or venipuncture and complete the detection process of the antigen- specific T cell responses in 12.5-24.5 hours.DEFINITIONS

[0008] As used herein, the term “antigen- specific T cell” refers to a T cell capable of recognizing and binding to a specific antigen. Examples of antigen-specific T cells include an antigen- specific CD4+T cell or an antigen- specific CD8+T cell.

[0009] As used herein, the term “antigen-specific T ceil response” refers to an immune response that occurs where an antigen- specific T cell is activated or induced by an antigen. Unless otherwise specified, the terms “activate” and “induce” in the context of this disclosure are interchangeable and refer to activating an antigen-specific T cell to generate an antigenspecific T cell response. Such antigen- specific T cell responses include the production and / or secretion of one or more gene products, i.e., an “antigen- specific T cell response product”, as a result of induction by an antigen.

[0010] As used herein, “cell line” refers to a cell or a population of cells derived from a single source that can be cultured and propagated indefinitely in-vitro.

[0011] As used herein, “reporter cell line” refers to a cell line that responds to the presence of an antigen-specific T cell response, typically by expressing one or more reporter genes which are under inducible control of an antigen-specific T cell response product. The expression of a reporter gene can be detected by a detection means, and a positive detection of a reporter gene indicates the presence of an antigen- specific T cell response product, which in turn indicates the presence of an antigen-specific T cell response.

[0012] As used herein, the term "about", in the context of time period, typically means + / - 10% of the stated value.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0014] FIG. 1 is a schematic of the process of detection of cytokines produced by antigenspecific T cells as part of an antigen-specific T cell response, using reporter cell lines and detection using a luminometer or flow cytometry machine.

[0015] FIG. 2 presents data from various experiments for detection of human IFN-y using an exemplary reporter cell line, HEK-Dual™ ZFN-y cells, in human whole blood and PBMC samples. FIG. 2A is a schematic of an existing procedure: a sample e.g., a whole blood sample or a PBMC sample (depicted as a microccntrifugc tube) is first incubated with antigenic peptides (depicted as curly lines) for 20 - 22 hours. Next, the supernatant containing cytokines produced as part of an antigen-specific T cell response is incubated with a reporter cell line (depicted as a culture vessel containing the supernatant and a reporter cell) for ~19 hours. Finally, the luciferase produced by the reporter cell is detected via luminescence assay using a luminometer (depicted as a plate reader). The total duration from sample collection to dataacquisition in this existing procedure takes between upwards of 39 - 41 hours. FIG. 2B is a chart showing detection of Luciferase activity via luciferase assay in the supernatant of a sample of stimulated human PBMCs incubated with a HEK-Dual IFN-y reporter cell line, as an example of the existing procedure. The horizontal dotted line represents the threshold that determines a positive antigen-specific T cell response in the sample. FIG.2C is a schematic of the method of the present invention, where the reporter cell line is simultaneously incubated with the sample (e.g., whole blood or PBMCs) in addition to stimulant (e.g., a non-specific chemical stimulant or antigenic peptides). FIG.2D is a chart showing the detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen- specific T cell response via Luciferase assay in PBMCs and whole blood samples (obtained via venipuncture or fingerprick) using the method of the present invention. The results are represented as relative change in luciferase activity in stimulated compared to unstimulated samples. The horizontal dotted line represents the threshold that determines a positive antigen- specific T cell response in the sample. FIG. 2E is a chart showing the enhancement of luciferase activity in a SARS-CoV-2 specific T cell response detected using the method of the present invention when the sample is additionally incubated with monoclonal antibodies (mAb) against an antigen-specific T cell costimulatory molecule. The horizontal dotted line represents the positive antigen- specific T cell response for the sample incubated without mAb i.e., the “antigen only” sample. A PBMC sample was used in this experiment.

[0016] FIG. 3 depicts the plasmid maps of the plasmids used in establishing various reporter cell lines. FIG. 3A is a commercially available plasmid, the pGreenFirel-GAS (EFla-puro) vector. FIG. 3B is a plasmid in accordance with an embodiment of the present invention, referred to as “ST-8”. FIG. 3C is another plasmid in accordance with an embodiment of the present invention, and lacks the optional hPEST feature. GAS TRE - gamma interferon activation site transcriptional response element; mCMV - minimal cytomegalovirus (CMV) promoter. Luciferase -luciferase gene; dscGFP - destabilized copepod green fluorescent protein gene; EGFP - enhanced green fluorescent protein gene; hPEST - human proline, glutamate, serine and threonine (PEST) sequence; DsRed2 - Discosoma sp. red fluorescent protein; T2A- Thosea asigna virus 2A peptide; HygR - hygromycin resistance gene; AmpR -ampicillin resistance gene; ori - origin of replication.

[0017] FIG. 4 is data from detection of human IFN-y using an in-house cell line in stimulated human PBMC samples. FIG. 4A is a chart showing the Luciferase activity in supernatant of stimulated human PBMCs after incubation with the in-house cell line. The horizontal dotted line represents the threshold that determines a positive antigen-specific T cell response in thesample. FIG. 4B is a chart showing the relative change in luciferase activity of the in-house cell line incubated with supernatant of stimulated human PBMCs against unstimulated PBMCs.FIG. 4C is a dot plot of GFP+ expression of the in-house cell line incubated with supernatant of un stimulated PBMCs and PMA stimulated human PBMCs. FIG. 4D is a chart showing the relative change in GFP expression of the in-house cell line incubated with supernatant of stimulated human PBMCs against unstimulated PBMCs. The leftmost bar represents incubation with unstimulated PBMCs, the middle bar represents incubation with PMA-stimulatcd PBMCs, and the rightmost bar is a control where the reporter cells were incubated with recombinant IFN-y (no PBMCs). The vertical axis represents the relative change in the number of GFP positive cells, i.e. change in cell count, compared to the unstimulated sample.

[0018] FIG.5 is a chart comparing the effect of co-incubating the sample with antigen (S ARS-CoV-2) and monoclonal antibodies that bind to co-stimulatory molecules compared to incubation with antigen alone, and detecting the antigen- specific T cell response using the HEK-Dual™ IFN-y cells.

[0019] FIG. 6 shows the comparative results of detection of antigen-specific T cell using the method of the present invention compared to other conventional approaches such as flow cytometry detection and ELISA-based (ELISPOT) detection. The detection threshold for the respective methods is indicated on each chart. FIG. 6A is a chart showing detection using the method of the present invention compared to detection using a luminometer compared to detection using a conventional flow cytometer method. The vertical axis represents the luminometer reading (relative light units or RLU) and the horizontal axis represents the flow cytometer reading (% cells double positive for IFN-y (reporter gene) and CD3 (T cell marker).FIG. 6B is a chart showing detection using the method of the present invention compared to detection using a conventional ELISPOT method. The vertical axis represents the luminometer reading (relative light units or RLU) and the horizontal axis represents the ELISPOT reader reading (number of spot-forming units (SFU) per unit cell, in this example, SFU per 200,000 PBMCs).

[0020] FIG. 7 are charts showing validation data for a reporter cell line expressing a vector in accordance with the present invention. FIG.7A presents luciferase assay data of three variants, ST-8.1 A, ST-8. IB and ST-8.1C tested using supernatant from PBMCs stimulated with PMA, interferon gamma (IFN-y), or interferon beta (fFN-|3). DMSO was included as a vehicle control ;FIG. 7B presents luciferase assay data of the ST-8.1C line incubated simultaneously with PBMCs and antigen (either SARS-CoV-2 peptide pool or PMA). DMSO was included as a vehicle control. FIG. 7C presents flow cytometry data of the ST-8.1C line incubatedsimultaneously with PBMCs and antigen (either SARS-CoV-2 peptide pool or PMA). DMSO was included as a vehicle control. Expression of the GFP was detected via flow cylomclry (FITC filter).DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides improved methods for detecting an antigen specific T cell response.

[0022] In one aspect, provided herein is a method for detecting an antigen-specific T cell response in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs), comprising the steps of:(1) collecting a sample;(2) incubating the sample simultaneously with the following:(a) an antigen to induce production of an antigen- specific T cell response product, and(b) a reporter cell line capable of expressing at least one reporter gene, wherein presence of the antigen-specific T cell response product induces expression of the at least one reporter gene by the reporter cell line; and(3) detecting the expression of the at least one reporter gene product as an indicator of the antigen- specific T cell response, thereby detecting the antigen-specific T cell response in the sample.

[0023] An antigen- specific T cell response is generated by an antigen-specific T cell upon activation or induction by an antigen. An antigen-specific T cell response can be induced when an antigen- specific T cell binds to an antigen, for example, via binding of its antigen-binding receptor to an antigen. Thus, an antigen-specific T cell in a sample can be induced by contacting the antigen-specific T cell with an antigen in the sample or incubating an antigen-specific T cell with an antigen in the sample. Examples of antigen-specific T cells include but are not limited to antigen- specific CD4+cells and antigen-specific CD8+cells.

[0024] In one example, the antigen- specific T cell response product comprises a cytokine, an enzyme, or combinations thereof.

[0025] In some examples, the cytokine or enzyme is selected from the group consisting of interferon gamma (IFN-y), interleukin 2 (IL-2), Tumour Necrosis Factor Alpha (TNF-a), Granzyme B (GzB), and combinations thereof.

[0026] It will be understood by those skilled in the art that an antigen- specific T cell response product is any compound produced and / or secreted by an antigen- specific T cell upon activation or induction by an antigen.

[0027] An example of an antigen-specific T cell response product is a cytokine. The cytokine can be an antimicrobial cytokine (e.g., an antibacterial, antiviral, antifungal cytokine), an antiparasitic cytokine, a proliferative cytokine, an anti-proliferative cytokine, an inflammatory cytokine, an anti-inflammatory cytokine, an immunomodulatory cytokine, or combinations thereof. The cytokine can be an autocrine or paracrine cytokine. For example, an antigenspecific T cell response product can be an interferon cytokine or an interleukin cytokine.

[0028] Examples of interferons produced and / or secreted by antigen- specific T cells upon activation or induction by an antigen include but are not limited to Type T interferons e.g., interferon alpha (IFN-a) and interferon beta (IFN-0), Type II interferons e.g., interferon gamma (IFN-y), and Type 111 interferons e.g., interferon lambda (1FN- ).

[0029] Examples of interleukins produced and / or secreted by antigen-specific T cells upon activation or induction by an antigen include but arc not limited to interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 14 (IL-14), interleukin 17 (IL-17), interleukin 21 (IL-21) and interleukin 22 (IL-22).

[0030] Other examples of compounds produced and / or secreted by antigen-specific T cells upon activation or induction by an antigen include but are not limited to Tumour Necrosis Factor Alpha (TNF-a) and Granzyme B (GzB).

[0031] Thus, in accordance with what is described herein, a detection of a reporter gene product in step (3) indicates the presence of an antigen-specific T cell response product that was produced in step (2), thereby indicating an antigen- specific T cell response in the sample.

[0032] As described herein, the method for detecting an antigen-specific T cell response provides an improvement in the duration (i.e., reduction in total duration) from collection of the sample to detecting the antigen- specific T cell response. Detecting the antigen-specific T cell response is achieved at the point of time of acquisition of data of the expression of the reporter gene, e.g., at the point of time that a reporter gene product, such as a polynucleotide or polypeptide encoded by the reporter gene, is detected.

[0033] A total duration from incubation of sample to detecting an antigen-specific T cell response refers to a duration that includes the step (2) of incubating the sample with both of an antigen to induce an antigen- specific T cell response and a reporter cell line capable of expressing a reporter gene , and the duration for the step (3) of detecting the reporter geneproduct. The step (2) of incubating the sampic simultaneously with both of an antigen to induce an antigen- specific T cell response and a reporter cell line capable of responding to an antigenspecific T cell response by expressing a reporter gene, reduces the total duration from incubating the sample to detecting an antigen-specific T cell response.

[0034] In one example, a total duration from incubating the sample to detecting the at least one reporter gene product is 12.5-24.5 hours, and the duration for incubation in step (2) is about 12-24 hours.

[0035] ft will be understood by those skilled in the art that the duration for incubation in step (2) may be any duration suitable for inducing an antigen-specific T cell response (e.g., induction of production of interferon, such as IFN-y) and for the presence of the antigenspecific T cell response (e g., presence of IFN-y) to induce expression of a reporter gene by the reporter cell line.

[0036] For example, the duration for incubation in step (2) may be about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0037] The duration for incubation in step (2) may also be any duration from about 12 hours to about 30 hours, for example, about 12-14 hours, about 12-16 hours, about 12-20 hours, about 14-22 hours, about 16-24 hours, about 19-24 hours, about 19-27 hours, or about 19-30 hours.

[0038] In one example, the duration for incubation in step (2) is about 12-24 hours.

[0039] Together with the step (3) of detecting a reporter gene product, a total duration from collection of the sampic to acquisition of data of the expression of the reporter gene may be any duration from about 12.5 hours to about 36 hours, for example, about 12.5 hours to about 20 hours, about 12.5 hours to about 24 hours, about 12.5 hours to about 36 hours, about 20 hours to about 24 hours, or about 24 hours to about 36 hours.

[0040] For example, a total duration from incubating the sample to detecting the reporter gene product is 12.5-36 hours, and the duration for incubation in step (2) is about 12-30 hours.

[0041] In one example, the sample comprises whole blood.

[0042] The sampic may comprise a sampic collected from a subject. Some examples of samples collected from a subject include but are not limited to a whole blood sample, a peripheral blood sample, and combinations thereof.

[0043] The sample may also comprise a sample derived from a subject. The term “sample derived from” in this context refers generally to samples collected from a subject that require additional processing steps from the point of sample collection, such as fractionation, filtration,mechanical cell dissociation, cell sorting, etc. Such processing steps will be well understood by those skilled in the art. Some examples of samples derived from a subject include but are not limited to whole blood samples, PBMC samples derived from fractionated blood samples, and combinations thereof.

[0044] It will be understood by those skilled in the art that the methods described herein allow for the determination of whether a subject is capable of an antigen- specific T cell response against an antigen, i.e., the antigen that is incubated with the sample in step (2)(a). For example, a positive detection of an antigen-specific T cell response in a sample collected from or derived from a subject, indicates that the subject is capable of an antigen-specific T cell response against an antigen, wherein the antigen is the antigen incubated with the sample in step (2)(a). On the other hand, a negative detection of an antigen-specific T cell response (i.e., no response) in a sample collected from or derived from a subject, indicates that the subject is not capable of an antigen- specific T cell response against an antigen, wherein the antigen is the antigen incubated with the sample in step (2)(a).

[0045] In one example, the sample comprising whole blood comprises a volume of at least 50 L.

[0046] In some examples, the sample comprising whole blood comprises a volume of 50 -100 pL.

[0047] For example, the sample comprising whole blood may comprise a volume of at least 50 pL, at least 60 pL, at least 70 pL, at least 80 pL, at least 90 pL or at least 100 pL.

[0048] The sample comprising whole blood may comprise a volume of whole blood that is suitable for detecting an antigen-specific T cell response. Unless otherwise specified, the term “volume” refers to the volume of sample collected from a subject and excludes any volume of additives or materials added post-obtention / collection.

[0049] In one example, the sample comprising whole blood is collected from a subject via finger-prick or venipuncture.

[0050] The sample comprising whole blood may also be collected from a subject via any suitable means for obtaining a whole blood sample, which are well understood by those skilled in the art.

[0051] In one example, the sample comprising whole blood comprises a volume of 50 -100 pL collected from a subject via finger-prick or venipuncture.

[0052] In another example, the sample comprises peripheral blood or comprises peripheral blood mononuclear cells (PBMCs).

[0053] The sample comprising peripheral blood or comprising PBMCs may comprise a number of PBMCs that is suitable for detecting an antigen- specific T cell response. It is to be understood in the context of this disclosure that a sample comprising peripheral blood also refers to a sample comprising PBMCs.

[0054] For example, a sample comprising PBMCs may comprise at least 1 x 105PBMCs, at least 1.1 x 105PBMCs, at least 1.2 x 105PBMCs, at least 1.3 x 105PBMCs, at least 1.4 x 105PBMCs, at least 1.5 x 105PBMCs or at least 2 x 10 PBMCs.

[0055] In one example, the sample comprising PBMCs comprises at least 1 x 105PBMCs.

[0056] The sample comprising peripheral blood or comprising PBMCs may be collected from a subject via any suitable means for collecting a peripheral blood sample or a sample comprising PBMCs, which are well understood by those skilled in the art. For example, a peripheral blood sample or a sample comprising PBMCs may be collected from a subject by density gradient centrifugation of a whole blood sample collected from a subject or collected from a subject by leukapheresis or venipuncture.

[0057] In one example, the sample comprising whole blood or PBMCs is collected from a human or an animal.

[0058] It will be understood by those skilled in the art that the sample comprising whole blood or PBMCs may be collected from any subject potentially possessing antigen-specific T cells or potentially capable of an antigen- specific T cell response. It will be understood by those skilled in the art that such subjects generally refer to subjects capable of an adaptive immune response. For example, the subject may be a human subject or an animal subject. Animal subjects capable of an adaptive immune response may include mammals, birds, reptiles and fish.

[0059] The reporter cell line used in the method of the invention may be any cell line capable of expressing one or more reporter gene(s) in the presence of an antigen specific T cell response product.

[0060] It will be understood by those skilled in the art that the presence of the antigen specific T cell response product may directly or indirectly induce expression of a reporter gene.

[0061] In the context of this disclosure, a “reporter gene” refers to a gene whose expression is inducible by an antigen-specific T cell response product, such as an interferon gamma (IFN-y) cytokine. A reporter cell line may express a reporter gene because it endogenously expresses the reporter gene upon induction, or it may be a cell engineered to express a reporter gene upon induction. Methods of engineering a cell to express a gene, e.g., a reporter gene, are well understood by those skilled in the art.

[0062] The reporter cell line may express one or more reporter genes upon direct or indirect induction by an antigen- specific T cell response product. For example, the presence of an interferon may directly or indirectly induce expression of one reporter gene, two reporter genes, three reporter genes, or more than three reporter genes, by the reporter cell line. The term “directly induce” refers to when the antigen- specific T cell response product itself induces the expression of a reporter gene. The term “indirectly induce” refers to when the antigen-specific T cell response product induces expression of a reporter gene via secondary mediators, such as when said antigen- specific T cell response product initiates a signalling cascade that results in induction of a reporter gene.

[0063] A reporter gene can be any gene which gene product is detectable by a suitably corresponding detection means. A gene product can be any product produced when the gene encoding for the gene product is expressed. For example, a gene product can be an RNA polynucleotide (e.g., a mRNA) or a polypeptide (e.g., a protein or an enzyme).

[0064] Suitably corresponding means for detecting expressed gene products are well understood in the art. For example, expression of an mRNA gene product may be detected via quantitative reverse-transcriptase PCR (qRT-PCR), and expression of a polypeptide gene product may be detected by enzyme-linked immunosorbent assay (ELISA). Expression of a polypeptide gene product such as a protein or an enzyme may also be detected by any means suitable for detecting the protein or enzyme that depends on its specific properties. For example, expression of a fluorescent protein can be detected by flow cytometry, fluorometer, or fluorescence microscopy. Expression of an enzyme can be detected by assays designed to detect an enzyme product. For example, expression of a luciferase enzyme can be detected by a luminescence assay, using a luminometer.

[0065] It will be understood by those skilled in the art that any combination of detection means can also be used to detect expression of a reporter gene, depending on the expressed gene product. For example, a combination of flow cytometry and luminometry can be used to detect expression of a fluorescent protein and a luciferase enzyme in a single reporter cell line.

[0066] It will be understood by those skilled in the art that since expression of a reporter gene by a reporter cell line in a sample is directly or indirectly induced by an antigen- specific T cell response product in the sample, such expression of a reporter gene indicates the presence of an antigen- specific T cell response in the sample. For example, presence of an antigen in a sample induces production and / or secretion of interferon by antigen- specific T cells present in the sample, the interferon produced and / or secreted by the antigen- specific T cells in the sample in turn induces expression of a reporter gene by a reporter cell line in the sample, and the reportergene product can be detected by a means described above for detecting gene products, thereby detecting the antigen-specific T cell response in the sample.

[0067] Thus, a reporter cell line may be any cell line that expresses a polynucleotide or polypeptide in the presence of an antigen-specific T cell response product.

[0068] For example, the reporter cell line may be a cell line that expresses or has been engineered to express an mRNA, a polypeptide, or a combination thereof in the presence of an antigen-specific T cell response product (e.g., IFN-y, IL-2, TNF-a, GzB etc). The reporter cell line may also be a cell line that expresses or has been engineered to express more than one mRNA and / or more than one polypeptide in the presence of an antigen-specific T cell response product (e.g., IFN-y, IL-2, TNF-a, GzB etc). The reporter cell line may be a cell line that expresses or has been engineered to express an enzyme, e.g., a luciferase enzyme, and a fluorescent protein in the presence of an antigen-specific T cell response product (e.g., IFN-y, IL -2, TNF-a, GzB etc).

[0069] The expression of a reporter gene may be under operable control of one or more inducible promoters comprising a transcriptional response clement (TRE). It will be understood by those skilled in the art that the expression of a gene that is operably linked to an inducible promoter is considered to be “under operable control of’ that inducible promoter. In the context of this disclosure, a “transcriptional response element” refers to a polynucleotide sequence within a promoter that interacts with (e.g., by binding to) an inducer, enabling the inducer to induce expression of one or more genes under inducible control of the promoter. For example, a minimal CMV (mCMV) promoter comprising a gamma-activated sequence (GAS) TRE will be induced when a Stall homodimcr or hctcrodimcr binds to the GAS TRE, resulting in induction of one or more genes downstream of the mCMV promoter.

[0070] Examples of suitable TREs include but are not limited to a gamma-activated sequence TRE (GAS TRE), an interferon-stimulated response element (ISRE), and an interleukin response element (IRE).

[0071] Where the reporter cell line expresses more than one reporter gene, the expression of each reporter gene may be induced by a single inducible promoter or the expression of each gene may be induced by a different inducible promoter. It will be understood by those skilled in the art that the expression of any reporter gene under operable control of an inducible promoter comprising a specific TRE will be induced by the same inducer. For example, any reporter gene under operable control of an inducible promoter comprising a GAS TRE will be induced by IFN-y, and any reporter gene under operable control of an inducible promotercomprising an interleukin response element (IRE) will be induced by an interleukin (e.g., IL-21-

[0072] It will be appreciated by those skilled in the art that the expression of different reporter genes in a reporter cell line may also be under operable control of separate inducible promoters comprising different TREs, and thus, be induced by different antigen-specific T cell products. For example, the expression of a luciferase gene may be under operable control of an inducible promoter comprising a GAS TRE while the expression of a fluorescent protein (e.g. GFP) gene may be under operable control of an inducible promoter comprising an interleukin response element (IRE). Such a reporter cell line would express a luciferase enzyme in the presence of IFN-y and a GFP in the presence of IL -2. If both IFN-y and IL-2 are present in the sample at the same time, then such a reporter cell line would express both a luciferase enzyme and a GFP.

[0073] In some examples, the reporter cell line expresses or is engineered to express one reporter gene in the presence of an antigen- specific T cell response product. In other examples, the reporter cell line expresses or is engineered to express more than one reporter gene in the presence of an antigen-specific T cell response product.

[0074] It will be understood by those skilled in the art that any cell line that expresses a reporter gene, e.g., a fluorescent protein or a luciferase enzyme, in the presence of an antigen- specific T cell response product may be suitable as a reporter cell line.

[0075] A reporter cell line may also be any cell line engineered to express a reporter gene in the presence of an antigen- specific T cell response product. Some examples of suitable cell lines for engineering include but are not limited to a human embryonic kidney (HEK) cell line and an A549 lung adenocarcinoma cell line. Some examples of suitable HEK cell lines include but are not limited to a HEK293 cell line, and a HEK293T cell line. It will generally be understood any other cell lines that can express the reporter gene can be included.

[0076] Tn one example, the at least one reporter gene product comprises a fluorescent protein, a luciferase enzyme, or combinations thereof.

[0077] In one example, the reporter gene product is a fluorescent protein.

[0078] Some examples of suitable fluorescent proteins include but are not limited to green fluorescent protein (GFP), Enhanced Green Fluorescent Protein (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mRFP-1, mGrape2, mGrape3 and mPlum, and all derived fluorescent products thereof.

[0079] A “derived fluorescent product” in this context refers to a fluorescent protein product that has been obtained via modification of any one of the above fluorescent proteins to improve its properties for use in fluorescent cell biology techniques, for example to improve its fluorescent properties and / or its stability in cells.

[0080] In some examples, the reporter gene product is a green fluorescent protein (GFP), such as Enhanced Green Fluorescent Protein (EGFP).

[0081] In one example, reporter gene product is a luciferase enzyme.

[0082] Some examples of suitable luciferase enzymes include but arc not limited to Firefly luciferase (FLuc), Renilla luciferase (RLuc), Gaussia luciferase (GLuc), Oplophorus luciferase (OpLuc), Metridia luciferase (MetLuc), Lucia® luciferase, and all derived products thereof. In some examples, the luciferase is a non-secreted luciferase. In some examples, the luciferase is a secreted luciferase, i.e., expressed and secreted by the engineered cell line, for example a Lucia® luciferase.

[0083] In one example, the reporter cell line is an engineered cell line capable of expressing a fluorescent protein and / or a luciferase enzyme in the presence of IFN-y.

[0084] In one example, the expression of the fluorescent protein and / or luciferase enzyme is detected using flow cytometry, a fluorescence microscope, and / or a luminometer.

[0085] It will be understood by those skilled in the art that any means suitable for detecting a fluorescent protein expressed by a cell may be used to detect expression of the fluorescent protein. Some examples include a flow cytometer, fluorescence microscope, fluorometer etc.

[0086] It will also be understood by those skilled in the ail that any means suitable for detecting a luciferase enzyme expressed by a cell may be used to detect expression of the luciferase enzyme. An example of such means is a luciferase assay, which involves detecting a luciferase enzyme via measuring luciferase activity using a luminometer.

[0087] In one example, the antigen is selected from the group consisting of a non-specific chemical stimulant, one or more microbe-derived antigen, one or more neoantigen, one or more immunogenic peptide, a cytokine and transcription factor activator or inhibitor, and combinations thereof.

[0088] A “microbe-derived antigen” in this context refers to antigen (c.g., a peptide) whose origin can be traced to a specific microbe.

[0089] It will be understood by those skilled in the art that the antigen can be any antigen that induces an antigen- specific T cell response in a sample. Some examples of suitable antigens (types of antigens) include but are not limited to a non-specific chemical stimulant, a microbe-derived antigen, a pathogen-derived antigen, a neoantigen, an immunogenic peptide, a cytokine, a transcription factor activator or inhibitor, and combinations thereof.

[0090] In some examples, one type of antigen is incubated with the sample. In other examples, more than one type of antigen (e.g., a non-specific chemical stimulant and a microbe-derived antigen) is incubated with the sample. In still other examples, a pool of one type of antigen (e.g., a pool of pathogen-derived peptides) is incubated with the sample.

[0091] In one example, the non-specific chemical stimulant is phorbol myristate (PMA) and / or lonomycin.

[0092] In another example, the microbe-derived antigen comprises one or more pathogen derived peptide selected from the group consisting of a viral peptide, a bacterial peptide, a fungal peptide, a protozoan peptide, and a parasitic peptide.

[0093] A “pathogen-derived peptide” in this context refers to a peptide whose origin can be traced to a specific pathogen.

[0094] In another example, the viral peptide is a peptide derived from SARS-CoV-2.

[0095] A “peptide derived from SARS-CoV-2” in this context refers to a peptide whose origin can be traced to a SARS-CoV-2 virus. Those skilled in the art will understand that such peptides may include recombinant SARS-CoV-2 peptides or peptides obtained from SARS-CoV-2 virus particles, as well as how to obtain such peptides.

[0096] In one example, the neoantigen is an antigen derived from a cancer cell selected from the group consisting of bladder cancer, breast cancer, brain cancer, spinal cord cancer, colorectal cancer, rectal cancer, kidney cancer, lung cancer, liver cancer, bowel cancer, bone cancer, bile duct cancer, eye cancer, gallbladder cancer, stomach cancer, head and neck cancer, laryngeal cancer, nasopharyngeal cancer, oesophageal cancer, tongue cancer, tonsil cancer, lymphoma, leukaemia, myeloma, pancreatic cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, thyroid cancer, skin cancer, and uterine cancer.

[0097] An “antigen derived from a cancer cell” in this context refers to antigen (e.g., a peptide) whose origin can be traced to a specific cancer. Such antigens may also be referred to as “cancer-derived antigens” or “cancer neoantigens”.

[0098] An antigen- specific T cell response may be enhanced by co- stimulation to fully activate the T cell immune response in the presence of an antigen. This may be achieved by incubating the sample in the presence of an antigen and a monoclonal antibody that binds to a T cell costimulatory molecule.

[0099] In one example, a monoclonal antibody that binds specifically to a co -stimulatory molecule is incubated with the sample together with the antigen in step (2) to enhance the antigen- specific T cell responses.

[0100] In some examples, the co-stimulatory molecule is selected from the group consisting of CD28, CTLA-4, PD1, iCOS and LAG3.

[0101] It will be understood by those skilled in the art that any T cell co-stimulatory molecule that is involved in the production and / or secretion of an antigen-specific T cell response product may be included. Examples of T cell co-stimulatory molecules include but arc not limited to CD28, CTLA-4, PD1, iCOS, LAG3, and combinations thereof.

[0102] In one example, the monoclonal antibody is incubated with the sample for about 12-16 hours.

[0103] It will be understood by those skilled in the art that the monoclonal antibody can be incubated with the sample in step (2) for part of the duration of incubation of the sample with the antigen, or the entire duration of incubation of the sample with the antigen. For example, the monoclonal antibody can be incubated with the sample for about 12-14 hours, about 12-16 hours, about 12-20 hours, about 12-24 hours, about 16-20 hours, about 16-24 hours, or about 20-24 hours.

[0104] Also provided herein, in another aspect, is a vector for expressing a reporter gene in the presence of an antigen- specific T cell response product, comprising at least one reporter gene operably linked to at least one inducible promoter comprising at least one transcriptional response element (TRE), wherein the at least one inducible promoter is induced in the presence of the antigen- specific T cell response product, thereby expressing the reporter gene.

[0105] In one example, the TRE comprises a gamma-interferon activated sequence TRE (GAS TRE), an interleukin response element (IRE), an interferon- stimulated response element (ISRE), or combinations thereof.

[0106] It will be understood by those skilled in the art that the TRE may be any transcriptional response element (TRE) that binds to an antigen- specific T cell response product.

[0107] In one example, the GAS TRE is encoded for by the sequence set forth in SEQ ID NO: 3.

[0108] It will be understood by those skilled in the art that a GAS TRE may also be referred to as a Signal Transducer and Activator of Transcription 1 TRE (STAT1 TRE). Those skilled in the art will also understand that such a GAS TRE comprises the consensus nucleotide sequence, TTC(N)2-4GAA, where “N” refers to any nucleotide and there are 2-4 nucleotides separating the core palindromic sequences “TTC” and “GAA”.

[0109] In one example, the inducible promoter comprises a minimal cytomegalovirus (mCMV) promoter, a SV40 minimal promoter, a P-globin minimal promoter, or combinations thereof

[0110] It will be understood by those skilled in the art that the inducible may be any promoter that is inactive or has negligible activity in the absence of an inducer. The effect of using such promoters is that in the absence of the antigen- specific T cell response product, no reporter gene product is detectable i.e., the amount of gene product present is below the minimum detection limit of the means used to detect the reporter gene product. For example, if the reporter gene is a luciferase gene and the antigen-specific T cell product is IFN-y, then in the absence of IFN-y, the promoter is not induced and the amount of luciferase enzyme expressed by a cell line comprising the vector is below the detection limit of a typical luminometer.

[0111] Examples of such inducible promoters include but is not limited to a minimal cytomegalovirus (mCMV) promoter, a SV40 minimal promoter, and a P-globin minimal promoter.

[0112] In one example, the inducible promoter is a mCMV promoter encoded by the sequence set forth in SEQ ID NO: 4.

[0113] In one example, the at least one reporter gene comprises a fluorescent protein gene, a luciferase gene, or combinations thereof. In some examples, the fluorescent protein gene and luciferase gene comprise the sequence set forth in SEQ ID NOs: 5 and 7, respectively.

[0114] In one example, the vector comprises at least two reporter genes.

[0115] In some examples, the vector comprises three reporter genes.

[0116] In one example, at least two of the at least two reporter genes are separated by a polynucleotide sequence encoding a linker. In some examples, the linker is encoded for by the sequence set forth in SEQ ID NO: 6.

[0117] It will be understood by those skilled in the art that when a continuous polypeptide comprising two or more functional polypeptides is desired, a polypeptide linker, such as a flexible polypeptide linker, can be included between each functional polypeptide. A “functional polypeptide” in this context refers to a gene product. The functional polypeptides on a continuous polypeptide will generally be expressed at a comparative level (i.e., approximately 1 : 1 ratio). For example, a fluorescent protein and a luciferase enzyme expressed as a continuous polypeptide will be expressed in an approximately 1:1 ratio, thus, when expression of the fluorescent protein and the luciferase enzyme is detected (e.g., using flow cytometry and luciferase assay respectively), the expression levels of each reporter gene will be generally similar with each other. In some examples, flexible polypeptide linkers that reduce or avoid steric hindrance between functional polypeptides may be utilized. An example of such a linkeris “GGGSSRSGGGSSRSGGGSS” (SEQ ID NO: 6), which consists of two repeats of the sequence “GGGSSRS” and a third portion “GGGSS”. The length of the linker be extended or reduced by either changing the number of glycine residues, and / or number of repeats.

[0118] In one example, the antigen-specific T cell response product comprises a cytokine, an enzyme, or combinations thereof.

[0119] In some examples, the cytokine or enzyme is selected from the group consisting of interferon gamma (IFN-y), interleukin 2 (IL-2), Tumour Necrosis Factor Alpha (TNF-a), Granzymc B (GzB), and combinations thereof.

[0120] In one example, the vector further comprises a polynucleotide sequence encoding for a polypeptide rich in proline, glutamic acid, serine and threonine (PEST sequence). In some examples, the PEST sequence is encoded by the sequence set forth in SEQ ID NO: 8.

[0121] It will be understood by those skilled in the art that a PEST sequence functions as a proteolytic signal in cells and promotes the degradation of a polypeptide that it is conjugated to. Thus, linking a PEST sequence to a reporter gene product such as a fluorescent protein and / or a luciferase enzyme may promote its degradation in the reporter cell. The effect achieved by including a PEST sequence is that the reporter gene product will be present in the sample only if presence of the antigen-specific T cell response product is maintained at sufficiently high levels. In other words, expression of the reporter gene product will be detected only if a robust antigen-specific T cell response is present, which reduces false positives in detecting an antigen- specific T cell response.

[0122] In one example, the vector further comprises at least one gene that encodes for a selection marker.

[0123] It will be understood by those skilled in the art that a “selection marker” refers to a gene product that aids in identifying, selecting, and / or isolating cells that express a vector that comprises a gene encoding for the selection marker. Some examples of selection markers include but are not limited to a fluorescent protein and an antibiotic resistance gene product, i.e., a gene product that confers resistance to an antibiotic.

[0124] In one example, the gene encoding a selection marker comprises a fluorescent protein gene, an antibiotic selection gene, or combinations thereof.

[0125] In one example, the fluorescent protein gene encodes for a Discosoma sp. red fluorescent protein (DsRed2). In some examples, the fluorescent protein gene comprises the sequence set forth in SEQ ID NO: 11.

[0126] In one example, the antibiotic selection gene is a hygromycin resistance gene. In some examples, the hygromycin resistance gene comprises the sequence set forth in SEQ ID NO: 13.

[0127] In one example, expression of the at least one gene encoding for the selection marker is operably linked to a constitutive promoter.

[0128] In some examples, the constitutive promoter is a SV40 promoter. In some examples, the SV40 promoter is encoded by the polynucleotide sequence set forth in SEQ ID NO: 10.

[0129] It will be understood by those skilled in the art that any constitutive promoter that maintains expression of the selection marker at a level sufficient to aid in identifying, selecting, and / or isolating cells that comprise the vector can be included.

[0130] In one example, the vector comprises at least two genes that each encode for a selection marker.

[0131] In one example, the at least two genes that encode for a selection marker are separated by a polynucleotide sequence that encodes for a self-cleaving peptide.

[0132] It will be understood by those skilled in the art that a “self-cleaving peptide” refers to a peptide sequence that promotes ribosomal “skipping” during translation of the RNA transcript. Generally, the self-cleaving peptide sequence triggers the skipping of peptide bond formation by the ribosome after translation of the self-cleaving peptide, causing the ribosome to fall off and resulting in termination of translation. A self-cleaving peptide is typically utilized when it is desired to express more than one physically separated polypeptide under the same promoter, (i.e., to avoid or reduce expression of a fusion protein).

[0133] In some examples, the self-cleaving peptide is a T2A peptide. In some examples, the polynucleotide sequence that encodes for a self-cleaving peptide comprises the sequence set forth in SEQ ID NO: 12.

[0134] In another aspect, provided herein is a vector for expressing an EGFP gene and a luciferase gene in the presence of interferon gamma (IFN-y), wherein the EGFP gene and luciferase gene are separated by a linker sequence, wherein the EGFP gene and luciferase gene are operably linked to a minimal cytomegalovirus (mCMV) promoter comprising a gammaactivated sequence transcriptional response element (GAS TRE), and wherein the mCMV promoter is induced by IFN-y, thereby expressing the reporter gene. In some examples, the EGFP gene, luciferase gene, linker sequence, mCMV promoter and GAS TRE comprise the sequence set forth in SEQ ID NOs: 5, 7, 6, 4 and 3, respectively.

[0135] In one example, the vector / engineered vector further comprises a PEST sequence linked to the luciferase gene, wherein the PEST sequence comprises the sequence set forth in SEQ ID NO: 8.

[0136] In one example, the vector / engineered vector further comprises a DsRed gene and a hygromycin resistance gene, wherein the DsRed gene and hygromycin resistance gene areseparated by a polynucleotide sequence encoding for a T2A peptide, wherein the DsRcd gene, hygromycin resistance gene and polynucleotide sequence encoding for a T2 A peptide comprise the sequence set forth in SEQ ID NOs: 11, 13 and 12, respectively.

[0137] In another aspect, provided herein is a vector / engineered vector comprising the sequence set forth in SEQ ID NOs: 1 or 2.

[0138] Also provided herein is a kit of parts for use in the methods as described herein.

[0139] Also provided herein, in another aspect, is a kit for detecting antigen- specific T cell responses in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs) using the method as described herein, comprising:(a) reporter cell line capable of expressing at least one reporter gene in the presence of an antigen-specific T cell response product;(b) an antigen for stimulating PBMCs or whole blood to induce production of the antigen-specific T cell response product; and(c) reagents for detecting at least one reporter gene product;wherein the kit is configured to process a sample volume of 50 -100 pL collected via fingerprick or venipuncture and complete the detection process of the antigen- specific T cell responses in 12.5-24.5 hours.

[0140] In one example, the reporter cell line is the reporter cell line as described herein.

[0141] In one example, the reporter cell line is an engineered cell line capable of expressing a fluorescent protein and / or a luciferase enzyme in the presence of the antigen-specific T cell response product. In some examples, the antigen- specific T cell response product is interferon gamma (IFN-y).

[0142] In one example, the reagents for detecting at least one reporter gene product are reagents for detecting a fluorescent protein and / or a luciferase using flow cytometry and / or a luminometer.

[0143] The kit may further comprise a tool for obtaining a sample from a subject. For example, the kit may comprise a tool for collecting a blood sample, a peripheral blood sample, or combinations thereof. It will be understood by those skilled in the art that the kit as described herein may comprise one or more of any tool suitable for obtaining a sample described herein.

[0144] In one example, the kit further comprises a sample collection tool selected from the group consisting of a finger-prick tool and a venipuncture tool.

[0145] The kit may further comprise a data acquisition module that provides quantitative measurements of fluorescent and / or luminescent signals in a sample comprising one or more cell(s) expressing a fluorescent protein and / or luciferase. The data acquisition module maycomprise a means for detecting the expression of the fluorescent protein and / or luciferase, and a means to display the fluorescent protein and / or luciferase expression data quantitatively to a user.

[0146] Examples of means for detecting the expression of the fluorescent protein and / or luciferase include but are not limited to flow cytometry, a fluorescence microscope, and / or a luminometer.

[0147] Examples of fluorescent protein and / or luciferase expression data include but are not limited to relative fluorescence units (RFU) and relative luminescence units / rclativc light units (RLU).

[0148] It will be understood by those skilled in the art that the kit as described herein may comprise one or more of a data acquisition module comprising one or more of any means suitable for detecting expression of a fluorescent protein and / or luciferase as described herein, together with a means to display the fluorescent protein and / or luciferase expression data quantitatively to a user

[0149] In one example, the kit further comprises a data acquisition module that provides quantitative measurements of fluorescent and / or luminescent signals.

[0150] In another example, the kit further comprises a sample collection tool selected from the group consisting of a finger-prick tool or a venipuncture tool, and a data acquisition module that provides quantitative measurements of fluorescent and / or luminescent signals.

[0151] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0152] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0153] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTION

[0154] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0155] Human T cells produce human interferon gamma (IFN-y) upon response after nonspecific chemical stimulation using phorbol myristate (PMA) / lonomycin) or after stimulation with their cognate antigens. To detect secretion of IFN-gamma released by T cells, reporter cells expressing fluorescent protein or / and luciferase in the presence of human interferon gamma are used. This approach was validated using commercially available and inhouse developed cell lines.

[0156] Example 1: Commercial cell lines and SARS-CoV-2 application

[0157] Supernatants of chemically or peptides stimulated PBMCs or whole blood samples were collected after 20-24 hours incubation. Using SARS-CoV-2 infection / vaccination as a proof of concept to detect antigen specific T cells response, supernatants of PBMCs stimulated with various stimulants (c.g. antigens or PMA) were incubated with HEK-Dual™ IFN-y cells for another 19 hours. After 19 hours, supernatants were obtained to detect luciferase activity in unstimulated, PMA / Ionomycin or SARS-CoV-2 peptides stimulated PBMCs using a luminometer (Figure 2B). This experiment was based on the conventional approach for detecting an antigen- specific T cell response (Figure 2A).

[0158] The presently improved approach of combining antigenic peptides, HEK-Dual™ IFN-y cells and, PBMCs and whole blood in a single incubation of 19 hours (Figure 2C) allowed detection of SARS-CoV-2 specific T cells response in PBMCs, whole blood (100 pl via venipuncture & 50 pl via finger-prick collections) (Figure 2D).

[0159] Co-incubation of samples with antigen and commercially available monoclonal antibodies (mAb) against co-stimulatory molecules (CD28 or CTLA-4) was determined to improve detection of antigen specific T cell response (Figure 2E). Without intending to bebound by theory, it is believed that incubation with such monoclonal antibodies can elevate IFN-y secreted by an individual T cell and / or elevate the number of IFN-y producing T cells.

[0160] Further tests validated the approach of co-incubating the sample with a mAb that binds to co-stimulatory molecules. Human PBMC samples were either co-incubated with an antigen (SARS-CoV-2) and an mAb against PD1, CTLA4 or LAG3 in the presence of a reporter cell (HEK-Dual™ IFN-y), or with antigen only in the presence of a reporter cell. The antigenspecific T cell response of the antigen-stimulated samples was compared to respective unstimulatcd (i.c., antigen absent) samples. The unstimulatcd samples (controls) were cither Unstimulated samples consisted of only PBMCs only or PBMCs only with mAb. No antigen was present in the unstimulated samples. There was a statistically significant difference in the relative change in the response compared to unstimulated between the 12-hour antigen-only and 19-hour antigen-only incubated samples (Figure 5). However, there was no statistically significant difference in relative change in the response (measured as RLU compared to unstimulated) between the 12-hour antigen and mAb co-incubated samples compared to the 19-hour antigen-only incubated sample (Figure 5). This indicated that co-incubating the sample with antigen and mAb for 12 hours produced an antigen- specific T cell response that was comparable to incubating with antigen alone for 19 hours.

[0161] The above approaches have allowed the reduction of use of multiple different kits for data acquisition, and reduction of manhours needed to incubate the sample simultaneously with (a) an antigen to induce production of interferon gamma (IFN-y), and (b) a reporter cell line capable of expressing a fluorescent protein and / or luciferase, wherein presence of IFN-y induces expression of the fluorescent protein and / or luciferase by the reporter cell line, as well as to detect the expression of the fluorescent protein and / or luciferase as an indicator of the antigen- specific T cell response, thereby detecting the antigen- specific T cell response in the sample, to 2 hours or less through the whole process after sample collection to data acquisition.

[0162] Example 2: In-house developed reporter cell lines and SARS-CoV-2 application

[0163] Reporter cell lines were established using a commercially purchased lentiviral transfer plasmid (pGreenFirel-GAS) or in-house designed plasmids (ST-8) expressing both luciferase and fluorescent protein (Figure 3). The complete sequence of the ST-8 plasmids used arc presented in Tables 1 and 2.

[0164] HEK-293T cells were either transduced with the pGreenFirel-GAS plasmid according to the manufacturer’s protocol or transfected with the ST-8 plasmid using standard transfection methods (Lipofectamine 2000 per the manufacturer’s protocol). Subsequently, transduced / transfected cells were selected for stable expression of the reporter gene (i.e., theluciferase and GFP genes linked to the mCMV promoter linked to the GAS TRE) by utilizing DMEM medium containing respective antibiotics, ensuring the acquisition of drug-resistant clones. In addition, fluorescent-activated cell sorter (FACS) was used to select for the fluorescent protein-positive cells. These enabled the establishment of a stable cell line expressing the transfected GAS TRE to detect human interferon gamma.

[0165] For subsequent experiments, the cell line stably expressing the pGreenFirel-GAS plasmid was named “ST-1”, and the cell line stably expressing the ST-8 plasmid was named “ST-8”, taking the name of the ST-8 plasmid.

[0166] Validation of an in-house developed reporter cell lines was conducted using a similar approach as used with the commercial cell line in Example 1.

[0167] Using the ST-1 reporter cell line as an example, supernatants of chemically or antigenic peptides stimulated PBMCs or whole blood samples were incubated with the in-house cell line for 19 hours. After 19 hours, supernatants were obtained to detect luciferase activity using a luminometer. It was confirmed that the in-house developed reporter cell line could detect human interferon gamma produced in PBMC samples stimulated with PMA / Ionomycin or SARS-CoV-2, both by assaying luciferase activity (Figure 4A & B) as well as by fluorescent protein expression (Figure 4C & D).

[0168] Example 3: Validation of the ST-8 reporter cell line

[0169] Cell lines were established using three variants of ST-8. ST-8.1 A and ST8.1B each included only one reporter gene, i.e., luciferase and EGFP, respectively. ST-8.1C included both luciferase and EGFP. Figure 7A shows the results of a luciferase assay performed with the three cell lines, ST-8.1 A, ST-8. IB and ST-8.1C. The cells were incubated with cither supernatant of PMA-stimulated PBMCs, with IFN-y, or with IFN- . “DMSO” was included as a vehicle control sample as DMSO was used to resuspend PMA and peptide. The luciferase data was plotted after normalizing the luminescence signal against an unstimulated sample (unstimulated sample not shown). This showed that the ST-8.1C dual reporter system is able to perform at least as well as the single (luciferase) reporter system, with the added benefit of being able to report the presence of IFN-y via flow cytometry as well by detecting GFP if desired.

[0170] Next, performance of the ST-8.1C cell line was validated using a SARS-CoV-2 peptide pool. ST-8.1C cells were incubated with PBMCs together with either a SARS-CoV2 peptide pool or with PMA. As above, DMSO was included as a vehicle control. Figure 7B shows that the dual reporter cell line ST-8.1C can indeed detect SARS-CoV-2-specific T cell response via luciferase assay.

[0171] Finally, the ability of the ST-8.1C cell line to report the presence of ZFN-y via fluorescence (flow cytometry) was validated. ST-8.1C cells were incubated with PBMCs together with either a SARS-CoV2 peptide pool or with PMA, and DMSO was included as a vehicle control. Figure 7C shows that the dual reporter cell line ST-8.1 C can also detect S ARS-CoV-2-specific T cell response via fluorescence-based techniques, i.e., flow cytometry in this case.

[0172] Table 1: ST-8 plasmid

[0173] Example 4: Comparison of the present approach with conventional approaches

[0174] The method of the present invention was compared to other alternative approaches in this experiment. PBMC samples were either unstimulated or stimulated with PMA or SARS-CoV-2, and the antigen-specific T cell response was detected using a) the method of the present invention, using a reporter cell line expressing luciferase reporter gene, b) a conventional flowcytometry based detection method, i.e., based on detection of cells co-expressing a T cell marker (CD3) and an antigen-specific T cell response marker (IFN-y), or c) a conventional ELISA-bascd detection method (ELISPOT), i.e., antibody-based detection of cells expressing IFN-y. As shown in Figure 6, a greater number of stimulated samples were above the luminometer threshold than the FC threshold or ELISPOT reader threshold, indicating that the method of the present invention using luciferase (luminometer) detection was more sensitive than both the conventional flow cytometer-based detection and ELIS A-based (ELISPOT reader) detection. Furthermore, the present invention allowed for determining of the presence of an antigen- specific T cell response with a total duration of about 12.5 - 19.5 hours from the beginning of incubation to data acquisition, while the conventional flow cytometer and ELISPOT methods take at least 3.5 - 4 hours longer.

[0175] Equivalents

[0176] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.

[0177] The present invention provides improved methods for detecting an antigen specific T cell response. Each process in the present invention has been specifically designed to integrate in a 'ay that allows the use of smaller sample volumes while still producing results comparable to existing methods. This integration creates a compounded effect that cannot be achieved by combining existing methods. The present invention does not require multiple sample processing steps, and the minimal sample processing steps allow the present invention to detect antigen specific response using a small sample volume as low as 50 pL. This allows for the use of finger-prick blood samples, unlike the conventional methods which require a much larger blood sample obtained for example by venipuncture.

Claims

ClaimsWhat is claimed is:

1. A method for detecting an antigen- specific T cell response in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs), comprising the steps of:(1) collecting a sample;(2) incubating the sample simultaneously with the following:(a) an antigen to induce production of an antigen- specific T cell response product, and(b) a reporter cell line capable of expressing at least one reporter gene, wherein presence of the antigen-specific T cell response product induces expression of the at least one reporter gene by the reporter cell line; and(3) detecting at least one reporter gene product as an indicator of the antigen- specific T cell response, thereby detecting the antigen- specific T cell response in the sample.

2. The method of claim 1, wherein the antigen- specific T cell response product comprises a cytokine, an enzyme, or combinations thereof; optionally wherein the cytokine or enzyme is selected from the group consisting of interferon gamma (IFN-y), interleukin 2 (IL-2), Tumour Necrosis Factor Alpha (TNF-a), Granzyme B (GzB), and combinations thereof.

3. The method of claim 1 , wherein a total duration from incubating the sample to detecting the at least one reporter gene product is 12.5-24.5 hours, and wherein the duration for incubation in step (2) is about 12-24 hours.

4. The method of claim 1, wherein the sample comprising whole blood comprises a volume of at least 50 pL; optionally wherein the sample comprising whole blood comprises a volume of 50 -100 pL.

5. The method of claim 4, wherein the sample comprising whole blood comprises a volume of 50 -100 pL collected from a subject via finger-prick or venipuncture.

6. The method of claim 1, wherein the sample comprising PBMCs comprises at least 1 x 105PBMCs.

7. The method of any one of claims 1-6, wherein the sample comprising whole blood or PBMCs is collected from a human or an animal.

8. The method of any one of claims 1-7, wherein the at least one reporter gene product comprises a fluorescent protein, a luciferase enzyme, or combinations thereof.

9. The method of any one of claims 1-8, wherein the reporter cell line is an engineered cell line capable of expressing a fluorescent protein and / or a luciferase enzyme in the presence ofIFN-y.

10. The method of claim 8 or claim 9, wherein the expression of the fluorescent protein and / or luciferase enzyme is detected using flow cytometry, a fluorescence microscope, and / or a luminometer.

11. The method of claim 1, wherein the antigen is selected from the group consisting of a nonspecific chemical stimulant, one or more microbe-derived antigen, one or more neoantigen, one or more immunogenic peptide, a cytokine and transcription factor activator or inhibitor, and combinations thereof.

12. The method of claim 11, wherein the non-specific chemical stimulant is phorbol myristate (PMA) and / or lonomycin.

13. The method of claim 11, wherein the microbe-derived antigen comprises one or more pathogen derived peptide selected from the group consisting of a viral peptide, a bacterial peptide, a fungal peptide, a protozoan peptide, and a parasitic peptide.

14. The method of claim 13, wherein the viral peptide is a peptide derived from SARS-CoV-2.

15. The method of claim 11, wherein the neoantigen is an antigen derived from a cancer cell selected from the group consisting of bladder cancer, breast cancer, brain cancer, spinal cord cancer, colorectal cancer, rectal cancer, kidney cancer, lung cancer, liver cancer, bowel cancer, bone cancer, bile duct cancer, eye cancer, gallbladder cancer, stomach cancer, head and neck cancer, laryngeal cancer, nasopharyngeal cancer, oesophageal cancer, tongue cancer, tonsil cancer, lymphoma, leukaemia, myeloma, pancreatic cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, thyroid cancer, skin cancer, and uterine cancer.

16. The method of claim 1, wherein a monoclonal antibody that binds specifically to a costimulatory molecule is incubated with the sample together with the antigen in step (2) toenhance the antigen-specific T cell responses; optionally wherein the co- stimulatory molecule is selected from the group consisting of CD28, CTLA-4, PD1, iCOS and LAG3.

17. The method of claim 16, wherein the monoclonal antibody is incubated with the sample for about 12-16 hours.

18. A vector for expressing a reporter gene in the presence of an antigen- specific T cell response product, comprising at least one reporter gene operably linked to at least one inducible promoter comprising at least one transcriptional response element (TRE), wherein the at least one inducible promoter is induced in the presence of the antigen- specific T cell response product, thereby expressing the reporter gene.

19. The vector of claim 18, wherein the TRE comprises a gamma-interferon activated sequence TRE (GAS TRE), an interleukin response element (IRE), an interferon-stimulated response element (ISRE), or combinations thereof; optionally wherein the GAS TRE is encoded for by the sequence set forth in SEQ ID NO: 3.

20. The vector / engineered vector of claim 18, wherein the inducible promoter comprises a minimal cytomegalovirus (mCMV) promoter, a SV40 minimal promoter, a P-globin minimal promoter, or combinations thereof; optionally wherein the inducible promoter is a mCMV promoter encoded by the sequence set forth in SEQ ID NO: 4.

21. The vector / engineered vector of claim 18, wherein the at least one reporter gene comprises a fluorescent protein gene, a luciferase gene, or combinations thereof; optionally wherein the fluorescent protein gene and luciferase gene comprise the sequence set forth in SEQ ID NOs: 5 and 7, respectively.

22. The vector / engineered vector of any one of claims 18 to 21 , wherein the vector comprises at least two reporter genes.

23. The vector / engineered vector of claim 22, wherein the at least two reporter genes are separated by a polynucleotide sequence encoding a linker; optionally wherein the linker is encoded for by the sequence set forth in SEQ ID NO: 6.

24. The vector / engineered vector of claim 18, wherein the antigen- specific T cell response product comprises a cytokine, an enzyme, or combinations thereof; optionally wherein the cytokine or enzyme is selected from the group consisting of interferon gamma (IFN-y),interleukin 2 (IL-2), Tumour Necrosis Factor Alpha (TNF-a), Granzyme B (GzB), and combinations thereof.

25. The vector / engineered vector of claim 18, wherein the vector further comprises a polynucleotide sequence encoding for a polypeptide rich in proline, glutamic acid, serine and threonine (PEST sequence); optionally wherein the PEST sequence is encoded by the sequence set forth in SEQ ID NO: 8.

26. The vector / engineered vector of claim 18, wherein the vector further comprises at least one gene that encodes for a selection marker.

27. The vector / engineered vector of claim 26, wherein the gene encoding a selection marker comprises a fluorescent protein gene, an antibiotic selection gene, or combinations thereof.

28. The vector / engineered vector of claim 27, wherein the fluorescent protein gene encodes for a Discosoma sp. red fluorescent protein (DsRed2); optionally wherein the fluorescent protein gene comprises the sequence set forth in SEQ ID NO: 11.

29. The vector / engineered vector of claim 27, wherein the antibiotic selection gene is a hygromycin resistance gene; optionally wherein the hygromycin resistance gene comprises the sequence set forth in SEQ ID NO: 13.

30. The vector / engineered vector of claim 26, wherein the at least one gene encoding for the selection marker is operably linked to a constitutive promoter; optionally wherein the constitutive promoter is a SV40 promoter, further optionally wherein the S V40 promoter is encoded by the polynucleotide sequence set forth in SEQ ID NO: 10.

31. The vector / engineered vector of any one of claims 26 to 30, wherein the vector comprises at least two genes that each encode for a selection marker.

32. The vector / engineered vector of claim 31, wherein the at least two genes that encode for a selection marker are separated by a polynucleotide sequence that encodes for a selfcleaving peptide; optionally wherein the self-cleaving peptide is a T2A peptide, further optionally wherein the polynucleotide sequence that encodes for a self-cleaving peptide comprises the sequence set forth in SEQ ID NO: 12.

33. A kit for detecting antigen-specific T cell responses in a sample comprising whole blood or peripheral blood mononuclear cells (PBMCs) using the method of any one of claims 1-17, comprising:(a) a reporter cell line capable of expressing at least one reporter gene in the presence of an antigen- specific T cell response product;(b) an antigen for stimulating PBMCs or whole blood to induce production of the antigen-specific T cell response product; and(c) reagents for detecting at least one reporter gene product;wherein the kit is configured to process a sample volume of 50 -100 pL collected via fingerprick or venipuncture and complete the detection process of the antigen- specific T cell responses in 12.5-24.5 hours.

34. The kit of claim 33, wherein the reporter cell line is an engineered cell line capable of expressing a fluorescent protein and / or a luciferase enzyme in the presence of the antigenspecific T cell response product.

35. The kit of claim 33 or 34, wherein the antigen- specific T cell response product is interferon gamma (IFN-y).

36. The kit of any one of claims 33 to 35, further comprising a sample collection tool selected from the group consisting of a finger-prick tool or a venipuncture tool, and a data acquisition module that provides quantitative measurements of fluorescent and / or luminescent signals.

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