Method for detecting an immune response

Abstract

The present invention relates to the field of medical diagnostics. In particular, the present invention relates to compositions, methods, and kits for detecting immune cells for diagnosing allergies, monitoring vaccination responses, and determining immune responses to pathogens and the therapeutic effectiveness of allergen immunotherapy. For example, the present invention provides a method for determining allergic reactivity in a subject, the method comprising providing a sample from the subject, contacting the sample with a recombinant or synthetic allergen linked to a detectable label under conditions that allow binding of the allergen to IgE molecules present in the sample, and determining binding of the allergen to the IgE molecules in the sample by detecting the label, wherein detection of the label indicates that the subject has allergic reactivity. For example, the present invention provides a method for detecting antigen-specific B cells in a subject, the method comprising providing a sample from the subject, contacting the sample with an antigen linked to a detectable label under conditions that allow binding of the antigen to Ig molecules on the surface of B cells present in the sample, and determining binding of the antigen to the Ig molecules in the sample by detecting the label, wherein detection of the label indicates that the subject has antigen-specific B cells.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis. In particular, the present invention relates to compositions, methods and kits for detecting immune cells for the diagnosis of allergies, determining the immune response to pathogens, and monitoring the therapeutic efficacy of vaccination responses and allergen immunotherapy.

[0002] Cross-reference to prior applications This application claims priority from Australian Provisional Patent Applications Nos. 2019903919 and 2020901811, the entire contents of each application being incorporated herein by reference in their entirety.

Background Art

[0003] The immune system can be activated in response to the presence of pathogens such as viruses, protozoa and bacteria, in response to the presence of vaccines, and also in response to the presence of allergens. One aspect of the immune response is the production of immunoglobulins expressed or bound by specific cells. For example, detecting the presence of B cells expressing immunoglobulins or basophils that bind to immunoglobulins can inform an individual's immune status, identify immune-mediated disorders, and provide information about treatment options and responses.

[0004] Allergic diseases are one of the most common chronic immune-mediated disorders and can present with a vast diversity in clinical severity and range of symptoms. As a result, there are significant challenges in diagnosis, predicting disease progression / development, and treatment, which continue to this day.

[0005] The allergic reaction in a subject is characterized by the induction of an immune response to a harmless antigen or allergen. Allergens cause the activation of IgE-binding cells, leading to a series of responses characteristic of allergy. The rapid and efficient detection of allergic patients remains a challenge in current tests based on serum IgE levels, local allergic responses in the skin (skin prick test), and / or the presence of reactive IgE-bearing basophils in peripheral blood (basophil activation test (BAT)). Indeed, tests of patients via exposure to allergens, such as skin prick tests or food challenges, can lead to adverse events. In many cases, the tests require close monitoring of subjects within a clinic. In contrast, tests based on the measurement of allergen-specific IgE, such as the RAST test, can be performed using a patient's blood sample and are minimally invasive to the patient. However, a potential pitfall of these tests is that they detect "free" serum IgE rather than "functional" IgE that is bound to effector cells such as mast cells and basophils. The measurement of functional allergen-specific IgE can be performed by measuring basophil activation (BAT test). In the BAT test, a patient's basophils are isolated and incubated for a short period (20 minutes to 1 hour) with various potential allergens. The activation of basophils is then measured by histamine release or cell surface expression of CD63 or upregulation of surface CD203c. The BAT test is very specific, but there are pitfalls because allergic patients may show a mild response or no response at all. Furthermore, this test is very laborious as a cytometry test and does not allow component breakdown diagnosis in a single assay. Therefore, there is a need for new or improved methods for detecting patients at risk of an allergic response.

[0006] Patients diagnosed with an allergy to a particular allergen may seek treatment to reduce the symptoms and reactions associated with the allergy. Conventionally, patients have been treated with one of two approaches: (1) controlling symptoms through pharmacological neutralization of effector molecules such as antihistamines; and (2) altering the immune response to the allergen via allergen immunotherapy (AIT). In AIT patients, the allergen is repeatedly exposed over a long period, thereby altering the immune response. Since AIT is often effective only if the patient undergoes treatment for several years, this can lead to difficulties with compliance and completion of treatment.

[0007] Over the past 20 years, the treatment of allergic diseases has changed dramatically with the introduction of therapeutic agents that target different pathways in allergy. These include monoclonal antibodies against IgE and type 2 cytokines and their receptors, small molecule inhibitors of signaling pathways, and others. The therapeutic agents have shown some promise in the regulation of severe allergic responses, but are expensive, not effective for all patients, and may require lifelong administration. The effectiveness of these therapies is conditional upon patients undertaking long-term treatment at high cost.

[0008] In the current COVID-19 pandemic, the world is facing an extreme situation with a highly infectious coronavirus (SARS-CoV-2, also known as 2019-nCoV) to which the global population is immunologically naive. The number of COVID-19 infections has exceeded 36 million worldwide, and more than 1 million people have died to date. The available PCR-based tests for detecting SARS-CoV-2 RNA are the gold standard for confirming current infections. The severity of the disease ranges from mild to life-threatening, and the mortality rate is also quite high. To manage the current situation and accelerate vaccine development, there is an urgent need to identify sensitive and specific immunological markers that demonstrate the generation of protective immune responses by the host. The presence or absence of these immune markers early after infection can be used to stratify patients with mild disease from those at risk of severe complications. The latter group can then be treated at the early stages of infection to prevent the disease or shorten the hospital stay. Furthermore, evidence of immunity in previously infected healthcare workers can expedite their return to the frontline. Finally, comparison of immune markers between recovered patients and immunized individuals in vaccine trials can shed light on the functionality of the vaccine. This will be necessary to focus resources on the most promising candidates and prevent delays in large-scale production for administration to those at risk of severe disease.

[0009] Accordingly, there is a need for diagnostic and prognostic methods for (1) diagnosing allergies, (2) predicting the progression of allergic diseases in patients, (3) defining patients who would benefit from specific immunotherapy treatments, (4) determining responders to allergy treatments, (5) detecting immune responses and memory to pathogens, and / or (6) monitoring vaccine inoculation responses.

[0010] References to prior art in this specification are not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would reasonably be expected to be understood, regarded as relevant, and / or combined by persons skilled in the art with other prior art.

Summary of the Invention

Means for Solving the Problems

[0011] In one aspect, the present invention provides a method for determining the allergenicity in a subject, the method comprising: providing a sample from the subject; contacting the sample with a natural, recombinant or synthetic allergen linked to a detectable label under conditions that allow binding of the allergen to IgE molecules present in the sample; determining the binding of the allergen to IgE molecules in the sample by detecting the label, wherein detection of the label indicates that the subject is allergenic.

[0012] In any aspect of the present invention, the allergen is recombinant or synthetic.

[0013] In any aspect of the present invention, the allergen is selected from food-based allergens, airborne or environmental allergens, drug allergens, peptide allergens, goat milk allergens, plant allergens, animal allergens or arthropod allergens, preferably, the arthropod is an insect, myriapod, arachnid or crustacean (e.g., insect, mite, crustacean). Preferably, the allergen is a protein and often an enzyme. When the allergen is an enzyme, preferably, the enzyme is modified to reduce its activity, e.g., the modification is a point mutation or cleavage.

[0014] Suitable allergens include food-based allergens such as nuts, sesame, buckwheat, peanuts, milk proteins, and egg white. Other interesting allergens include various airborne antigens such as grass pollen, animal dander, and house dust mite feces, as well as insect venom and mold allergens. Typical food allergens include milk allergens (Bos d 4, 5, and 8), peanut allergens (Ara h 1, 2, 3, 6, 8, and 9), hazelnut (Cor a 9 and 14), cashew nut (Ana o 3), walnut (Jug r 1), Brazil nut (Ber e 1), sesame (Ses i 1), buckwheat (Fag e 3), almond (Pru du 6), peach (Pru p 1 and Pru p 3), shrimp (Pen m 1), and wheat (Tri a 19; omega-5-gliadin). Common aeroallergens include the house dust mite (Dermatophagoides pteryonyssinus) (Der p 1 and 2); rye grass (Lol p 1, 5), timothy grass (Phl p 1, 5), bahiagrass (Pas n 1), bermudagrass (Cyn d 1), ragweed (Amb a 1), Parietaria species (Par o 1; Par j 1, 2), pollen allergens from birch (Bet v 1), and other airborne pollens including olive (Olea europaea), Artemisia species, and Gramineae; as well as animal dander from, for example, cats (Fel d 1) and dogs (Can f 1). Other allergens include venom allergens from honeybees (Api m 1, 3, 10); phospholipases from the yellow jacket Vespula maculifrons and the North American yellow jacket Dolichovespula maculata, and venom from the jumper ant Myrmecia pilosula).Other interesting allergens are those that cause mold allergies (especially those derived from Alternaria, Aspergillus, and Cladosporium species), as well as blood-sucking arthropods such as mosquitoes (Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies (Phlebotomus sp., Culicoides sp.), especially Diptera including midges, black flies, and sand flies; mites (Dermacenter sp., Ornithodoros sp., Otobius sp.); fleas, for example, those that cause allergic dermatitis caused by the order Siphonaptera including Xenopsylla, Pulex, and Ctenocephalides. Specific allergens can be polysaccharides, fatty acid moieties, proteins, etc. In many cases, allergen epitopes are polypeptides. Recombinant allergens may be produced by expression from recombinant DNA, may be obtained commercially, or may be obtained by other techniques well known in the art.

[0015] Typically, a recombinant allergen or antigen is linked to a tag that facilitates binding to a detectable label. For example, the tag can bind non-covalently to a detectable label or can form a covalent interaction with the detectable label. Suitable tags are known in the art and include streptavidin and its derivatives, avidin and its derivatives, biotin, immunoglobulins, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments and their derivatives, the leucine zipper domain of AP-1, jun, fos, hexa his, hexa hat glutathione S-transferase, glutathione affinity, calmodulin binding peptide, Strep-tag, cellulose binding domain, maltose binding protein, S-peptide-tag, chitin binding tag, immunoreactive epitope, epitope tag, E2 tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope, carbohydrates, lipids and lectins that mediate binding to diverse compounds including proteins, Con A or WGA and tetranectin or protein A and protein G. Most preferably, the tag is Bir-A. Even more preferably, the Bir-A tag is biotinylated.

[0016] In any aspect of the present invention, a detectable label is any moiety that enables detection. For example, the label may enable colorimetric or fluorescence detection. Suitable fluorescent labels are known in the art and include fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor 488, Alexa 647, Alexa 710, Alexa fluor 405, cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), Pacific Blue (PacB), Horizon Violet 450 (HV450), Pacific Orange (PacO), Horizon-V500 (HV500), Krome Orange, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 711 (BV711), Brilliant Violet 785 (BV785), Brilliant Ultraviolet 395 (BV395), Brilliant Ultraviolet 496 (BV496), Brilliant Ultraviolet 737 (BV737), Orange Cytognos (OC)515, quantum dots, and conjugates thereof conjugated to PE, APC, or PerCP (e.g., PE / Cy5, PE / Cy5.5, PE / Cy7, PerCP / Cy5.5, APC / Cy7, APC-H7, APC-Alex750, PE-Texas Red, PE-Dazzle, PE-CF594), or any additional compatible fluorescent dye or fluorescent dye tandem, etc. Suitable labels can be directly or indirectly linked to a recombinant allergen or antigen by use of a suitable tag containing any tag described herein. In a preferred embodiment, the detectable label is linked to streptavidin. When two or more allergens or antigens are tested, each allergen or antigen can be linked to a different detectable label, i.e., each label is uniquely identifiable.

[0017] In any aspect of the present invention, the recombinant allergen or antigen is linked to a biotinylated Bir-A tag that can bind to streptavidin linked to a detectable label. In this embodiment, the recombinant allergen or antigen is linked to the detectable label via the interaction between streptavidin and the biotinylated Bir-A.

[0018] In any aspect of the present invention, the allergen or antigen is linked to a tag or a detectable label at the N-terminus of the allergen or antigen. Alternatively, the allergen or antigen is linked to a tag or a detectable label at the C-terminus of the allergen or antigen.

[0019] In any aspect of the present invention, the sample is contacted with two or more, three or more, four or more, five or more, six or more, or seven or more allergens or antigens, each linked to a different detectable label.

[0020] In any aspect, the sample is contacted with the same allergen or antigen linked to two or more different labels (e.g., an antigen linked to a first label and the same antigen linked to a separately detectable label) (e.g., as shown as dual discrimination in the examples).

[0021] In any aspect of the present invention, the sample can be a body fluid, such as a blood sample. Alternatively, the sample may be a tissue sample. In one embodiment, the sample can include a body fluid and a tissue sample. The blood sample can be whole blood, cerebrospinal fluid, peripheral blood mononuclear cells (PBMC), cord blood, a purified or sorted cell population, or a body fluid. Body fluids include lymph, semen, nasal secretions, bronchial secretions, alveolar fluid, cerebrospinal fluid, endolymph, synovial fluid, pleural effusion, pericardial fluid (pericardial membrane fluid), menstrual fluid, or a combination thereof. The tissue sample may be selected from tonsils, lymph nodes, bronchi, nose, or intestine, or a skin biopsy.

[0022] In a preferred embodiment, the sample is a whole blood sample.

[0023] In any aspect, the method includes determining the binding of an allergen to IgE molecules present on the surface of cells in a sample.

[0024] In an alternative embodiment, the sample is contacted with beads, microparticles or a chemical substrate to enable capture of cell-free IgE molecules. Alternatively, the sample is contacted with an array tissue culture plate or chip to enable capture of cell-free IgE molecules. Cell-free IgE can be captured via any molecule known in the art or described herein that binds to IgE.

[0025] IgE molecules can be present on the surface of immune cells. Preferably, on the surface of immune cells expressing the Fc epsilon receptor, FcεRI or FcεRII (CD23), such as basophils, eosinophils, mast cells, monocytes, macrophages, B cells, activated T cells, platelets, follicular dendritic cells or thymic epithelial cells. Typically, IgE molecules are present on the surface of basophils. IgE molecules are present on the surface of basophils via binding to high-affinity FcεRI.

[0026] In any aspect of the invention, the method further includes removing cells present in the sample that do not have IgE molecules bound to an allergen linked to a detectable label on their surface. Preferably, the IgE molecules on the surface of the cells are bound to FcεRI or FcεRII (CD23). For example, cell removal can be performed by electronic gating via sorting or by subsequent flow cytometry software analysis to quantify the number of allergen-binding cells.

[0027] In any aspect of the invention, the method further includes removing an allergen linked to a detectable label that is not bound to IgE. Preferably, the allergen is removed by washing the sample.

[0028] In any aspect of the present invention, the method further comprises contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types. Preferably, this molecule binds to an immune cell marker and enables detection. Typically, the molecule is linked to a detectable label or is the detectable label itself. For example, the molecule can be a fluorescent dye, an antibody, a nucleotide probe, or an enzyme that results in the production of a substrate. Alternatively, the molecule is linked to a tag that facilitates binding to a detectable label. For example, the tag can bind non-covalently to the detectable label or can form a covalent interaction with the detectable label. Suitable tags are known in the art and are described herein.

[0029] In a preferred embodiment, the molecule is an antibody that detects the marker of interest and the detectable label is a fluorescent dye. Suitable fluorescent dyes are known in the art and are described herein.

[0030] In a further preferred embodiment, the marker of interest is a phenotypic marker used to identify the cell of interest, for example, a marker that enables the identification of basophils. Preferably, this molecule distinguishes basophils from other cell types. For example, basophils can be identified using a pan-basophil marker. Suitable basophil markers are CD63, IgE, 2D7 antigen, CD117, CD124, CD203c, CD200R3, or FcεRIα.

[0031] In any aspect of the present invention, a recombinant allergen or antigen linked to a detectable label may be contacted with the sample before contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types. Alternatively, a recombinant allergen or antigen linked to a detectable label may be contacted with the sample after contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types.

[0032] In any aspect of the present invention, a sample may be contacted with a molecule that enables the identification of one or more, preferably two or more, immune cell types, and simultaneously with a recombinant allergen or antigen linked to a detectable label.

[0033] In any aspect of the present invention, detection of a label linked to a recombinant or synthetic allergen, antigen or molecule is performed by any method known in the art or described herein, including flow cytometry.

[0034] In any aspect of the present invention, if an individual is determined by the method of the present invention to be allergic reactive, the method further comprises administering to the subject an allergy immunotherapy, preferably an allergen-specific immunotherapy.

[0035] In another aspect, the present invention provides a method for detecting sensitization to an allergen in a subject, the method comprising: providing a sample from the subject; contacting the sample with a recombinant allergen linked to a detectable label under conditions that allow binding of the allergen to IgE molecules present in the sample; determining binding of the allergen to IgE molecules in the sample by detecting the label; detection of the label indicates that the subject is sensitized to the allergen.

[0036] In another aspect, the present invention provides a method for treating a subject identified as having allergic reactivity, the method comprising determining whether the subject has allergic reactivity by performing or having performed a method as described herein; if the subject has allergic reactivity, administering to the subject an immunotherapy specific for the allergen to which the subject is reactive.

[0037] In another aspect, the present invention provides a method for treating a subject identified as having an allergic reactivity, the method comprising: providing or having provided a sample from the subject, contacting or having contacted the sample with a recombinant or synthetic allergen linked to a detectable label under conditions that permit binding of the allergen to IgE molecules present in the sample, determining or having determined binding of the allergen to IgE molecules in the sample by detecting the label, and determining thereby whether the subject has allergic reactivity, wherein if the subject has allergic reactivity, administering to the subject a specific immunotherapy for the allergen to which the subject is reactive.

[0038] In another aspect, the present invention provides a method for determining the efficacy of allergy immunotherapy in a subject, the method comprising: providing a first sample obtained from the subject prior to receiving allergy immunotherapy; providing a second sample obtained from the subject during or after receiving allergy immunotherapy; contacting the first and second samples from the subject with a recombinant allergen linked to a detectable label under conditions that permit binding of the allergen to IgE molecules on the surface of cells present in the samples; determining binding of the allergen to IgE molecules on the surface of cells present in the first and second samples by detecting the label; and a decrease in the total number or percentage of IgE-binding cells in the second sample relative to the first sample indicates the efficacy of allergy immunotherapy in the subject.

[0039] In any aspect, the allergy immunotherapy is allergen-specific immunotherapy.

[0040] In another aspect, the present invention provides a method for determining the efficacy of allergy immunotherapy in a subject, the method comprising: providing a first sample obtained from a subject before receiving allergy immunotherapy; providing a second sample obtained from the subject during or after receiving allergy immunotherapy; contacting the first and second samples derived from the subject with a recombinant allergen linked to a detectable label under conditions that allow binding of the allergen to Ig molecules on the surface of B cells present in the samples; determining the binding of the allergen to Ig molecules on the surface of B cells present in the first and second samples by detecting the label; and an increase in the total number or proportion of IgG-expressing B cells in the second sample relative to the first sample indicates the effectiveness of allergy immunotherapy in the subject; an increase in the ratio of IgG:IgE-expressing B cells in the second sample relative to the first sample indicates the effectiveness of allergy immunotherapy in the subject; or an increase in the total number or proportion of IgG2 and / or IgG4-expressing B cells in the second sample relative to the first sample indicates the effectiveness of allergy immunotherapy in the subject.

[0041] In one embodiment, the method further comprises contacting the first and second blood samples with a molecule that enables identification of B cells expressing IgG.

[0042] In another embodiment, the method further comprises contacting the first and second blood samples with a molecule that enables identification of B cells expressing IgM, IgA, IgG, IgD, and IgE.

[0043] In this aspect, the method of the invention can be used in a wide variety of biological samples known or suspected to contain B cells. The sample can be blood, bone marrow or lymphoid tissue. The tissue may be selected from tonsil, lymph node, bronchus, nasal or intestinal biopsies. Alternatively, the blood sample can be a whole blood sample, a cerebrospinal fluid sample, a peripheral blood mononuclear cell (PBMC) sample, cord blood, a purified or sorted cell population or a body fluid. Body fluids include samples from the group consisting of lymph amniotic fluid, nasal secretions, bronchial secretions, alveolar fluid, endolymph, pericardial fluid (pericardial fluid), peritoneal fluid, breast milk, or combinations thereof. Alternatively, the tissue may be selected from tonsil, lymph node, bronchus, nasal or intestinal biopsies.

[0044] In a preferred embodiment, the sample is a whole blood sample. In this aspect of the invention, the method may further comprise removing cells present in the sample that do not have surface Ig receptors bound to an allergen or antigen conjugated to a detectable label. For example, cell removal can be performed by electronic gating by sorting, or by subsequent analysis with flow cytometry software, to quantify the number of allergens or antigens bound per cell.

[0045] In one embodiment, a recombinant allergen or antigen conjugated to a detectable label that does not bind to surface immunoglobulin (Ig) on B cells is removed. Preferably, the recombinant allergen or antigen is removed by washing the sample.

[0046] In this aspect of the invention, the method further comprises contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types. Preferably, the molecule binds to an immune cell marker and enables visual detection. Typically, the molecule is either conjugated to a detectable label or is the detectable label itself. For example, the molecule may be a fluorescent dye, an antibody, a nucleotide probe, or an enzyme that results in the production of a substrate. Alternatively, the molecule is linked to a tag that facilitates binding to a detectable label. For example, the tag can bind non-covalently to the detectable label or can form a covalent interaction with the detectable label. Suitable tags are known in the art and are described herein.

[0047] In a preferred embodiment, the molecule is an antibody that detects the marker of interest and the detectable label is a fluorescent dye. Suitable fluorescent dyes are known in the art and are described herein.

[0048] In a more preferred embodiment, the marker of interest is a phenotypic marker used to identify the cell of interest. Preferably, the molecule distinguishes B cells from other cells. For example, B cells can be identified using a pan-B cell marker. Suitable B cell markers are CD19, CD20, CD79a, or CD22. Preferably, it is the CD19 antigen. Fluorescent dye-labeled antibodies for use in the recombinant or synthetic allergens or antigens, methods, or kits of the present invention can be prepared according to routine techniques or they can be commercially obtained from various sources. Most preferably, the method comprises contacting the sample with a labeled fluorescent dye-conjugated antibody against any one or more of the markers: CD123, CD27, IgM, IgA, IgG, IgD, CD19, CD21, CD38, and IgE; preferably, any one or more of CD27, IgM, IgA, IgE, IgG, IgD, CD19, CD21, and CD38. The antibody is provided with a detectable label, for example, any detectable label described herein. Typically, the detectable label enables separate detection and quantification by flow cytometry.

[0049] In one aspect, the present invention provides a method for detecting antigen-specific B cells in a subject, the method comprising: providing a sample from the subject; contacting the sample with an antigen linked to a detectable label under conditions that allow binding of the antigen to Ig molecules on the surface of B cells present in the sample; determining binding of the antigen to Ig molecules in the sample by detecting the label, wherein detection of the label indicates that the subject has antigen-specific B cells.

[0050] In one embodiment, the antigen is derived from a vaccine. In another embodiment, the antigen is not derived from a vaccine and may be derived from an infectious agent or pathogen such as a virus, bacterium, fungus, protozoan, or parasite.

[0051] Exemplary viruses include those associated with or causing a respiratory condition or disease. The antigen-specific B cells can be specific for a particular viral protein, such as a nucleocapsid protein (NCP) or spike protein, or a domain within a nucleocapsid protein (NCP) or spike protein (e.g., S1B). Exemplary viral proteins and domains include those defined herein, including in the examples.

[0052] The virus can be selected from coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus (RSV), adenovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), dengue virus, rhinovirus, herpes simplex virus and enterovirus. More preferably, the virus is coronavirus or influenza virus. Even more preferably, the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and most preferably SARS-CoV-2.

[0053] Representative bacteria are Clostridium tetani and Corynebacterium diphtheria, or bacteria that cause tetanus and diphtheria.

[0054] Exemplary parasites are those that cause malaria in humans. For example, parasites belonging to the genus Plasmodium (phylum Apicomplexa), particularly Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and Plasmodium knowlesi.

[0055] In one embodiment, the antigen is a self-antigen.

[0056] In another aspect, the present invention provides a nucleic acid comprising a first nucleotide sequence encoding an allergen or antigen and a second nucleotide sequence encoding a tag. Preferably, the allergen or antigen is any one described herein, including those listed in Table 1, for example, SEQ ID NOs: 7-51, 56-61, or 76-86. Exemplary nucleotide sequences encoding allergens or antigens include any one described herein, including those listed in Table 1 shown in, for example, SEQ ID NOs: 52-55, 62-71, or 87-90. Preferably, the tag is any one described herein, including those listed in Table 1, for example, SEQ ID NO: 4, 5, 6, or 72.

[0057] In another aspect, the present invention provides a recombinant or synthetic polypeptide comprising an allergen or antigen and a tag, preferably, the tag is any one described herein. Preferably, the allergen or antigen is any one described herein, including those listed in Table 1, for example, SEQ ID NOs: 7-51, 56-61, or 76-86. Preferably, the tag is any one described herein, including those listed in Table 1, for example, SEQ ID NO: 4, 5, or 6. Preferably, the recombinant or synthetic polypeptide is obtained or can be obtained by the expression of the nucleic acid of the present invention described herein.

[0058] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention described herein.

[0059] In another aspect, the present invention provides a cell comprising the vector or nucleic acid of the present invention described herein.

[0060] The present invention provides a diagnostic or prognostic kit comprising one or more recombinant allergens or antigens, a detectable label, and instructions for use, buffer, and / or control samples. For example, a diagnostic kit for use in any method described herein is provided. Preferably, the recombinant allergen or antigen conjugated to the detectable label is suitable for use in flow cytometry immunophenotyping.

[0061] In any aspect, one or more or all of the steps of any method of the present invention are performed in vitro or ex vivo. In one embodiment, in any method of the present invention, the method does not include obtaining a sample from a subject.

[0062] As used herein, unless the context otherwise requires, the term "comprise" and variations of the term such as "comprising," "comprises," and "comprised" do not purport to exclude further additions, components, integers, or steps.

[0063] Further aspects of the present invention, and further embodiments of the aspects described in the previous paragraph, will become apparent from the following description given by way of example, with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0064]

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Mode for Carrying Out the Invention

[0065] It will be understood that the invention disclosed and defined herein extends to all alternatives, combinations of two or more of the features mentioned in the text or drawings, or obvious from them. All of these different combinations constitute various alternative aspects of the invention.

[0066] Further aspects of the invention, and further embodiments of the aspects described in the previous paragraph, will become apparent from the following description given by way of example, with reference to the accompanying drawings.

[0067] Here, specific embodiments of the invention will be referred to in detail. While the invention is described in relation to embodiments, it is understood that the invention is not intended to be limited to these embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may fall within the scope of the invention as defined by the claims.

[0068] As outlined previously, current diagnosis of allergy testing requires either invasive intradermal challenge, in vitro culturing of cells with allergens that may or may not damage the cells, or concentration of cells from blood before obtaining sufficient cells to detect the necessary immune cells. These limit the number of allergens that can be tested at a given time and / or require much larger amounts of blood to be drawn.

[0069] The present invention provides a means for diagnosing allergy in a subject. This diagnosis utilizes a patient sample, such as blood, without the need for concentration of cells by magnetic means or culturing of cells with allergens to induce activation. The diagnosis relies on differences in the amount of allergen that binds to cells via expressed or cell-bound immunoglobulin in allergic and non-allergic subjects. In the blood of a subject not sensitized to a particular allergen, cells expressing IgE or cells bearing cell-bound IgE do not bind to this allergen. In blood from a sensitized subject, IgE-expressing and IgE-bearing cells (particularly basophils) can be labeled by the allergen. The presence of labeled basophils indicates a hypersensitive IgE response to that particular allergen. In addition, the present invention can further be used to determine the effectiveness of allergen immunotherapy (AIT). The effectiveness depends on changes in the frequency or fraction of allergen-binding B cells of either IgE or IgG in a subject undergoing AIT therapy. In particular, an increase in the fraction of allergen-specific IgG-B cells with respect to the frequency of IgM-expressing and / or IgE-expressing B cells (either memory cells or plasma cells) indicates the effectiveness of the treatment, similar to an increase in the fraction of IgG2 and / or IgG4 in total IgG-expressing B cells.

[0070] The inventors have developed a method using recombinant allergens that are modified to reduce their toxicity while maintaining their conformation. These recombinant allergens are linked to a detectable label, for example in the form of a fluorescent molecule.

[0071] The present invention provides compositions and methods that enable the diagnosis of allergic hypersensitivity in a subject or the determination of the effectiveness of allergen immunotherapy. An advantage of aspects of the present invention is that the diagnosis of allergic hypersensitivity does not require the use of invasive intradermal challenges. Further, multiple allergens can be tested, enabling the determination of a subject's hypersensitivity to multiple allergens in a single assay (e.g., multiplexing in flow cytometry). Another advantage of aspects of the methods of the present invention is that whole blood can be used, avoiding the need for steps to process or fractionate the blood. Further, for example, for certain types of immune cells, there is no need to concentrate the sample. Finally, since this method enables ex vivo analysis of blood samples, there is no risk of systemic reactions with positive results as compared to food challenges, skin prick tests, or intradermal skin tests.

[0072] The present invention also provides methods and compositions for monitoring vaccine responses, such as influenza, tetanus, and diphtheria vaccines.

[0073] The present invention also provides methods and compositions for determining whether an individual has an immune response to an infectious agent, such as a virus, parasite, or bacterium, including any of those described herein. An advantage of the present invention is that it enables the identification of vaccinated or naturally infected subjects. For example, in the context of SARS-CoV-2, natural infection in a subject will result in separate B cell populations specific for the nucleocapsid protein and specific for the spike protein. However, many vaccine approaches do not include the nucleocapsid protein, and thus, subjects who have had natural infection will exhibit a population of B cells specific for the nucleocapsid protein. This can be applied to patient stratification for clinical trials.

[0074] General Throughout this specification, unless otherwise specified or the context otherwise requires, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be construed to include one and more (i.e., one or more) of these steps, compositions of matter, groups of steps, or groups of compositions of matter. Accordingly, as used herein, the singular forms "a", "an", and "the" include plural aspects unless the context clearly dictates otherwise, and vice versa. For example, a reference to "a" includes one and two or more, a reference to "an" includes one and two or more, a reference to "the" includes one and two or more, and so on.

[0075] Those skilled in the art will appreciate that the present invention is capable of receiving variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions, and compounds individually or collectively recited or shown herein, as well as any and all combinations or any two or more of the foregoing steps or features.

[0076] Those skilled in the art will recognize numerous methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0077] All patents and publications referred to herein are hereby incorporated by reference in their entirety.

[0078] The present invention should not be limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0079] Any example or embodiment of the present invention herein shall, unless otherwise specified, be applicable to any other example or embodiment of the present invention with the necessary modifications.

[0080] Unless otherwise defined, all technical and scientific terms used in this specification shall be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0081] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those of ordinary skill in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0082] The term "and / or", e.g., "X and / or Y", should be understood to mean either "X and Y" or "X or Y", and should be construed as providing explicit support for both meanings or either meaning.

[0083] As used herein, the term "derived from" should be construed to indicate that a particular integer can be obtained from a particular source, although not necessarily directly from that source.

[0084] Selected definitions As used herein, the term "allergen" refers to any naturally occurring protein or mixture of proteins or chemicals / drugs that has been reported to induce an allergic (i.e., IgE-mediated) reaction upon repeated exposure to them in an individual.

[0085] "Allergy", also referred to herein as "allergy reactivity", is any condition in which an unwanted (e.g., type 1 hypersensitivity) immune response (i.e., allergic response or reaction) to a substance exists. Such a substance is referred to herein as an allergen. Allergies or allergic conditions include, but are not limited to, allergic asthma, hay fever, hives, eczema, plant allergies, bee sting allergies, pet allergies, latex allergies, mold allergies, cosmetic allergies, food allergies, allergic rhinitis or hay fever, local allergic reactions, anaphylaxis, atopic dermatitis, hypersensitivity reactions, and other allergic conditions. An allergic reaction can be the result of an immune reaction to any allergen. In some embodiments, the allergy is a food allergy. Food allergies include, but are not limited to, milk allergy, egg allergy, nut allergy, fish allergy, shellfish allergy, soybean allergy, or wheat allergy.

[0086] As used herein, the term "hypersensitivity" refers to unwanted reactions produced by normal immune responses, including allergies and autoimmunity. These overreactions of the immune system can be damaging, uncomfortable, or even fatal. Hypersensitivity reactions require prior sensitization of the host.

[0087] As used herein, the term "allergen sensitization" or "sensitization to an allergen" refers to the production of IgE antibodies following the first exposure to an allergen or antigen that later results in an allergic reaction or allergic reactivity.

[0088] As used herein, the terms "antibody," "immunoglobulin," or "Ig" refer to proteins that can specifically bind to one or a few closely related antigens by antigen-binding domains contained within the Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, bispecific antibodies). Antibodies generally include constant domains that can be arranged in a constant region or a constant fragment or crystallizable fragment (Fc). Exemplary forms of antibodies include a four-chain structure as their basic unit. Full-length antibodies include two covalently linked heavy chains (about 50-70 kDa) and two light chains (each about 23 kDa). Light chains generally include a variable region (if present) and a constant domain, and in mammals are either κ light chains or λ light chains. Heavy chains generally include a variable region and one or two constant domains linked by a hinge region to additional constant domains. Mammalian heavy chains are one of the α, δ, ε, γ, or μ types. Each light chain is also covalently bound to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each about 110 amino acids in length) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL, about 110 amino acids in length) aligns with and is disulfide-bonded to the first constant domain of the heavy chain (CH1, 330-440 amino acids in length). The light chain variable region aligns with the variable region of the heavy chain. The antibody heavy chain can include two or more additional CH domains (e.g., CH2, CH3, etc.) and can include a hinge region between the CH1 constant domain and the CH2 constant domain. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as human) antibody.In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.

[0089] As used herein, the term "immune cell" refers to any cell involved in an immune response. Such cells include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, dendritic cells, natural killer cells, NKT cells, NK-like cells, T cells, B cells, and plasma cells.

[0090] As used herein, the term "FcεRI+ immune cell" is intended to refer to a cell that expresses the FcεRI high-affinity receptor and can release pharmacological mediators upon IgE-induced sensitization and exposure to an antigen of interest (e.g., mast cells and basophils).

[0091] The term "isolated protein" or "isolated polypeptide" is not associated with the naturally associated components that accompany it in its native state by virtue of its origin or source of derivation; it is a protein or polypeptide that is substantially free of other proteins from the same source. A protein can be made substantially free of naturally associated components using protein purification techniques known in the art or can be substantially purified by isolation. "Substantially purified" means that the protein is substantially free of contaminants, e.g., contains less than at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% contaminants.

[0092] The term "recombinant" shall be understood to mean a product of artificial gene recombination. Thus, in the context of a recombinant allergen or antigen, this term does not include allergens or antigens that occur naturally. However, if such allergens or antigens are isolated, they are considered isolated allergens or antigens. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant allergen or antigen. Recombinant proteins also include proteins expressed by artificial recombinant means, for example, if they are within the cells, tissues or subjects in which they are expressed.

[0093] The term "protein" should be interpreted to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other by covalent or non-covalent bonds (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using appropriate chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0094] The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraphs to mean a series of consecutive amino acids linked by peptide bonds.

[0095] As used herein, the term "binds" in relation to the interaction between an allergen or antigen and an antibody means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the allergen or antigen. For example, an antibody generally recognizes and binds to a specific protein structure rather than a protein. If an antibody binds to epitope "A", in a reaction containing labeled "A" and the protein, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0096] As used herein, the term "epitope" (synonymous with "antigenic determinant") is understood to mean the region of a protein (such as an allergen or antigen) to which the antigen-binding domain of an antibody binds.

[0097] As used herein, the term "condition" refers to the disruption or interference of normal function and should not be limited to any particular condition, and may include diseases or disorders.

[0098] As used herein, the term "diagnosis" refers to the act of identifying a disease or condition using its signs or symptoms.

[0099] As used herein, the term "subject" should be construed to mean any animal, including a human, such as a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0100] Allergy and Sensitization It is understood that the methods or compositions of the present invention have several important uses, particularly in the field of human medical diagnosis.

[0101] Hypersensitivity reactions due to an immunological response can be classified into four broad classes. In particular, type I hypersensitivity reactions are immediate allergic reactions mediated by IgE antibodies. In most allergies, such as food, pollen, and house dust mites, the reaction is caused by the production of IgE antibodies against harmless antigens (allergens), resulting in sensitization to harmless antigens. Subsequent exposure to the allergen induces the activation of IgE-binding cells, including mast cells and basophils in tissues or blood, triggering a series of responses characteristic of this type of reaction, including degranulation of effector cells and the release of histamine, heparin, eosinophil and neutrophil chemotactic factors, leukotrienes, and thromboxane. Allergic immune responses are characterized by the production of high concentrations of IgE antibodies detectable in the blood and the production of IgE-specific B cells.

[0102] Conventional allergy tests include the skin prick test, where an allergen is injected intracutaneously, or sometimes intradermally. An allergic or allergic response causes the rapid production of wheals and erythema within 30 minutes. Other tests for allergies are known to those skilled in the art and include immunoassay tests such as enzyme-linked immunosorbent assay (ELISA, or EIA) and radioallergosorbent test (RAST). In the ELISA test, the amount of allergen-specific antibodies in the blood is measured, and in the RAST test, specific allergen-related antibodies are sought to identify the cause of the allergy.

[0103] The test of the present invention can be used to diagnose allergic reactivity or allergen sensitivity in situations where a skin prick test cannot be guaranteed, for example, (i) when the patient is using a medicament known to interfere with the skin prick test, such as an antihistamine, a steroid, or a specific antidepressant; (ii) when the subject cannot tolerate the scratching by the many needles required for the skin test; (iii) when the subject has an unstable heart condition; (iv) when the subject has poorly controlled asthma, severe eczema, dermatitis, psoriasis, or other severe skin conditions; and / or (v) when the subject has had an extreme reaction during the skin test or has a history of life-threatening allergic reactions, such as anaphylaxis.

[0104] The types of allergies include, but are not limited to, food allergies, skin allergies, dust or pollen allergies, insect sting allergies, pet allergies, eye allergies, drug allergies, allergic rhinitis, particularly latex allergy in type I IgE-mediated allergic reactions, mold allergies, allergy-related sinus infections, and cockroach allergies. Food allergies include, but are not limited to, allergies to milk, eggs, peanuts, tree nuts, soybeans, wheat, fish, and shellfish. Drug allergies include IgE mediated by reacting to substances. The most common drug allergies include penicillin and other related antibiotics, antibiotics containing sulfonamides, anticonvulsants, aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs (NSAIDs), and chemotherapy drugs. The most common skin allergies include eczema (atopic dermatitis), hives (urticaria), and contact dermatitis. The most common form of eye allergy is induced by outdoor allergens such as pollen from grass, trees, and weeds, indoor allergens such as pet dander, dust mites, and mold, and irritants such as tobacco smoke, fragrances, and diesel exhaust. The most common dust or pollen allergies include dust mites, cockroaches, mold, pollen, pet hair, fur, or feathers. The types of symptoms of allergic reactions include, but are not limited to, mucus production, loss of smell or taste, sore throat and / or cough, fatigue, fever or tremors, facial congestion, headache, toothache, postnasal drip, wheezing, shortness of breath, difficulty breathing, swelling of the throat and mouth, nausea, vomiting, bloating, diarrhea, stomach pain, cramping abdominal pain, skin rash, itching (especially of the nose, eyes, ears, and mouth), eye redness and watering, swelling around the eyes, hives, swelling of the lips, tongue, or throat, high blood pressure, dizziness and / or fainting, severe asthma episodes (asthma attacks), chronic asthma, and anaphylaxis

[0105] Allergen Suitable allergens include food-based allergens such as nuts, sesame, buckwheat, peanuts, milk protein, egg white, and shrimp. Other interesting allergens include various airborne antigens such as grass pollen, animal dander, and house dust mite feces, as well as insect venom and mold allergens. Typical food allergens include milk allergens (Bos d 4, 5, and 8), peanut allergens (Ara h 1, 2, 3, 6, and 8), hazelnut (Cor a 9 and 14), cashew nut (Ana o 3), walnut (Jug r 1), Brazil nut (Ber e 1), sesame (Ses i 1), buckwheat (Fag e 3), almond (Pru du 6), black tiger shrimp (Pen m 1), and wheat (Tri a 19). Common aeroallergens include Dermatophagoides pteryonyssinus (Der p 1 and 2); rye grass (Lol p 1, 5), timothy grass (Phl p 1, 5), bahiagrass (Pas n 1), bermudagrass (Cyn d 1), ragweed (Amb a 1), Parietaria species (Par o 1; Par j 1, 2), pollen allergens from birch (Bet v 1), and other airborne pollens including olive (Olea europaea), Artemisia sp., and gramineae; as well as animal dander from, for example, cats (Fel d 1) and dogs (Can f 1). Other allergens include venom allergens from honeybees (Api m 1, 3, 10); phospholipase from Vespula maculifrons and Dolichovespula maculata (North American yellowjackets), and venom from the jumper ant Myrmecia pilosula.Other interesting allergens are those that cause mold allergies (especially those derived from Alternaria, Aspergillus, and Cladosporium species), as well as hematophagous arthropods, such as mosquitoes (Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies (Phlebotomus sp., Culicoides sp.), especially Diptera including midges, black flies, and sand flies; mites (Dermacenter sp., Ornithodoros sp., Otobius sp.); fleas, such as those of the order Siphonaptera including the genera Xenopsylla, Pulex, and Ctenocephalides, which cause allergic dermatitis. The allergen may be derived from bacteria, for example, the protein MGL_1304 secreted by the bacterium Malassezia (M.) globosa, which is the main allergen in sweat allergy.

[0106] Certain allergens can be polysaccharides, fatty acid moieties, proteins, etc. In many cases, allergen epitopes are polypeptides. Recombinant allergens may be produced by expression from recombinant DNA, may be commercially obtained, or may be obtained by other techniques well known in the art.

[0107] The subject can be tested with one or a panel of different suspected allergens. Determination of the specific allergen to which a patient is hypersensitive enables the affected individual to seek treatment, e.g., desensitization, and avoid activities that increase the risk, e.g., exposure to the allergen. The panel can include many different pollens, groups of suspected food allergens, animal allergens, etc. To facilitate detection, the allergens can be multiplexed by including different labels with different allergens. In one embodiment, the allergens and labels are those described in the examples including Example 6.

[0108] Typically, recombinant or synthetic allergens or antigens are linked to a tag that facilitates binding to a detectable label. For example, the tag can bind non-covalently to the detectable label or form a covalent interaction with the detectable label. Suitable tags are known in the art and include streptavidin and its derivatives, avidin and its derivatives, biotin, immunoglobulins, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments and their derivatives, the leucine zipper domain of AP-1, jun, fos, hexa his, hexa hat glutathione S-transferase, glutathione affinity, calmodulin binding peptide, Strep-tag, cellulose binding domain, maltose binding protein, S-peptide-tag, chitin binding tag, immunoreactive epitope, epitope tag, E2 tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope, carbohydrates, lipids and proteins, lectins that mediate binding to diverse compounds including Con A or WGA and tenascin or protein A and protein G.

[0109] Most preferably, the tag is a Bir-A moiety that is later biotinylated upon contact with Bir-A enzyme. The biotinylated recombinant allergen or antigen can be used in combination with any detectable label that can be linked by this moiety. Suitable detectable labels for each tag are known in the art.

[0110] The following are examples of a set of criteria for the design and manufacture of recombinant or synthetic allergens or antigens: 1. The allergen or antigen must be non-pathogenic, non-toxic, and without enzymatic activity in order to minimize risk to the researcher and prevent cell death in the target assay; 2. The allergen or antigen should fold naturally and, if applicable, should include post-translational modifications to ensure the presence of conformational epitopes; 3. The allergen or antigen must be able to be purified without affecting its protein structure; 4. The allergen or antigen may include a tag to facilitate binding in order to enable multimerization and / or detection.

[0111] The recombinant or synthetic allergen may be generated based on any of the sequences from the WHO / IUIS allergen nomenclature database at http: / / www.allergen.org. The sequence may be further modified by any of one or more amino acid substitutions, one or more amino acid deletions, or one or more amino acid additions. This can be achieved by techniques known to those skilled in the art and described herein. In particular, any mutations that preserve the structural conformation of the Ig-binding motif are tolerated and intended within the scope of the present invention.

[0112] For example, allergy sensitization to honey bee (Apis mellifera) venom and venom from ryegrass (Lolium perenne) and their major allergens (Api m 1 and Lol p 1, respectively) were recombinantly generated based on sequences from the WHO / IUIS Allergen Nomenclature Database (http: / / www.allergen.org / ). Both constructs contained an Api m 1 N-terminal leader sequence for extracellular production, as well as C-terminal AviTag and 6-His sequences. Enzymatic activity was abrogated by introduction of the point mutations H34Q for Api m 1 (described by Forster E et al. (1995) J Allergy Clin Immunol. 95(6):1229-35) and H104V for Lol p 1 (described by Grobe K et al. (2002), Eur J Biochem 269(8):2083-92). Both constructs were codon optimized for Spodoptera frugiperda (fall armyworm) and cloned into the pFastBac vector (Thermo Fisher Scientific) prior to their incorporation into Bacmid for baculovirus expression.

[0113] Many allergens have enzymatic activity. Examples of enzymes are cysteine proteases: mite group I allergens (Der p 1) and grass pollen group I allergens (Phl p 1, Lol p 1, etc.); other proteases: bumblebee group IV (Bom p 4); trypsin: mite group III (Der p 3); chymotrypsin: mite group VI (Der p 6); amylase: mite group IV (Der p 4); nuclease: grass pollen group V (Phl p 5) and tree pollen group I (Bet v 1), phospholipase: insect group I (Api m 1), hyaluronidase: insect group II (Api m 2), lysozyme: chicken group IV (Gal d 4) (described by Bufe A (1998) Int Arch Allergy Immunol).

[0114] Enzymatic activity can be measured by an in vitro reaction by incubating the purified protein with a substrate and performing a kinetic measurement of the substrate and / or product concentration. In the case of phospholipase (e.g., Api m 1), the substrate may be diC6 thio-PM (racemic 2,3-bis(hexanoylthio)propyl-1-phosphomethanol lithium salt) or diC6 thio-PC (1,2-didecanoylthio-1,2-dideoxy-glycero-3-phosphosphocholine), and the conversion of the substrate to the product can be measured using a spectrophotometer at 405 nm. Protease activity (e.g., Der p 1, Lol p 1) can be evaluated by incubating the purified protein at 37°C for several hours and evaluating its stability, for example, by Western blot.

[0115] Infectious agent The present invention finds particular application in the detection of antigen - specific B cells where the antigen is derived from an infectious agent such as a pathogen, for example a virus, bacterium, fungus, protozoan or parasite (or any pathogen or infectious agent described herein). Thus, the present invention can be applied to determine the state of infection and immunity against pathogens. The pathogens are not limited to human pathogens and include other animal pathogens such as bovine, ovine, canine, feline pathogens.

[0116] Exemplary viruses include those associated with or causing respiratory conditions or diseases. The antigen - specific B cells can be specific to a particular viral protein, such as a nucleocapsid protein or a spike protein, or a domain within those proteins. Exemplary viral proteins and domains include those defined herein, including in the examples.

[0117] The virus can be selected from coronavirus, influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus, cytomegalovirus (CMV), Epstein - Barr virus (EBV), varicella - zoster virus (VZV), dengue virus, rhinovirus, herpes simplex virus and enterovirus. More preferably, the virus is coronavirus or influenza. Even more preferably, the virus is severe acute respiratory syndrome coronavirus (SARS - CoV), Middle East respiratory syndrome coronavirus (MERS - CoV), or severe acute respiratory syndrome coronavirus 2 (SARS - CoV - 2), and most preferably, SARS - CoV - 2.

[0118] Representative bacteria are Clostridium tetani and Corynebacterium diphtheria, or the bacteria that cause tetanus and diphtheria.

[0119] The antigen can be derived from any pathogen described herein and can be referred to as a pathogen - derived antigen.

[0120] Exemplary antigens (e.g., pathogen-derived antigens) from various viral or bacterial infectious agents are listed in Table 1.

[0121] Any reference to "allergen" as used herein can be a reference to "antigen" unless the context otherwise indicates.

[0122] Autoantigen Autoimmune diseases are broadly classified into two categories: organ-specific diseases and systemic diseases. The exact etiology of systemic autoimmune diseases has not been identified. In contrast, organ-specific autoimmune diseases are associated with specific immune responses involving B and T cells that target an organ and thereby induce and maintain a chronic state of local inflammation. Examples of organ-specific autoimmune diseases include type 1 diabetes, myasthenia gravis, thyroiditis, and multiple sclerosis. In each of these conditions, one or a few autoantigens, including insulin, acetylcholine muscle receptor, thyroid peroxidase, and myelin basic protein, respectively, have been identified.

[0123] An autoimmune reaction is directed against the subject's own cells or tissues, more particularly, "autoantigens", i.e., antigens (proteins) that are naturally present within the subject. In this mechanism, the autoantigen is recognized by B and / or T cells that activate the immune system to attack the tissue containing the autoantigen.

[0124] Exemplary self - antigens and diseases associated therewith include thyroid diseases: thyroglobulin, thyroid peroxidase, and TSH receptor; type 1 diabetes: insulin (pro - insulin), glutamic acid decarboxylase (GAD), tyrosine phosphatase IA - 2, heat shock protein HSP65, pancreatic islet - specific glucose 6 - phosphatase, catalytic subunit - related protein (IGRP); adrenalitis: 21 - OH hydroxylase; polyendocrine syndrome: 17 - α hydroxylase, histidine decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase; gastritis and pernicious anemia: H+ / K+ ATPase, intrinsic factor; multiple sclerosis: myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP); myasthenia gravis: acetylcholine receptor; eye diseases: retinol - binding protein (RBP); inner ear diseases: type II and type IX collagen; celiac disease: tissue transglutaminase; inflammatory bowel disease: pANCA, histone H1 protein; and atherosclerosis: heat shock protein HSP60 are included.

[0125] Detectable label The method of the present invention involves the detection of immunoglobulins specific for a given allergen or antigen, such as IgE and IgG, using a recombinant allergen or antigen linked to a detectable label.

[0126] As used herein, the term "detectable" refers to the generation or change of a signal that can be detected directly or indirectly by any means of observation or measurement. Typically, a detectable response is the generation of a signal where the fluorophore is inherently fluorescent. Alternatively, a detectable response is an optical response that results in a change in the wavelength distribution pattern or the intensity of absorbance or fluorescence, or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0127] As used herein, the term "label" refers to a chemical moiety or protein that binds to a recombinant or synthetic allergen or antigen and is directly or indirectly detectable (e.g., due to its spectral properties, conformation, or activity) when used in the methods described herein.

[0128] The detection label linked to the allergen / antigen may be a fluorescent dye. Suitable fluorescent labels are known in the art and include fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor 488, Alexa fluor 647, Alexa fluor 710, Alexa fluor 405, cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), pacific blue (PacB), horizon violet 450 (HV450), pacific orange (PacO), horizon-V500 (HV500), Krome Orange, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 711 (BV711), Brilliant Violet 785 (BV785), Brilliant Ultraviolet 395 (BUV395), Brilliant Ultraviolet 496 (BUV496), Brilliant Ultraviolet 737 (BUV737), Orange Cytognos (OC)515, quantum dots, and conjugates thereof (e.g., PE / Cy5, PE / Cy5.5, PE / Cy7, PerCP / Cy5.5, APC / Cy7, APC-H7, APC-Alex750, PE-Texas Red, PE-Dazzle, PE-CF594) bound to PE, APC, or PerCP, or any additional compatible fluorescent dye or fluorescent dye tandem, etc.

[0129] Appropriate labels can be directly or indirectly linked to recombinant allergens / antigens through the use of appropriate tags. In a preferred embodiment, the detectable label is linked to streptavidin. Fluorescent dye reagents are useful in panel reactivity assays where two or more defined pools of allergens / antigens are each conjugated to a different fluorescent dye and added to a sample. Multiple allergens / antigens can be tested at once, enabling multiplexing from a single blood draw. Blood samples are taken from subjects suspected of having an allergy or hypersensitivity to the test allergens.

[0130] Sample Any biological sample known or suspected to contain cells presenting IgG (e.g., B cells) or IgE (e.g., basophils) is intended for use in the present invention.

[0131] The term sample, as used herein, includes blood samples, but also includes hematopoietic biological samples such as lymph, leukocyte products, bone marrow, etc., and this term also includes derivatives and fractions of such fluids. Blood samples are taken from any site, for example by venipuncture. Blood samples are typically about 1 - 100 mL of whole blood, i.e., 10 5 ~10 7 nucleated blood cells and may be treated with anticoagulants such as heparin, EDTA, citrate, acid citrate dextrose, or citrate phosphate dextrose, as is known in the art.

[0132] The sample may be a body fluid, such as a blood sample as described above. Alternatively, the sample may be a tissue sample. The sample may include body fluids and tissue samples.

[0133] Blood samples can be whole blood, cerebrospinal fluid, peripheral blood mononuclear cells (PBMC), cord blood, purified or sorted cell populations, or body fluids. Body fluids include lymph, semen, nasal secretions, bronchial secretions, alveolar fluid, cerebrospinal fluid, endolymph, synovial fluid, pleural effusion, pericardial fluid (pericardial effusion), menstrual fluid, or combinations thereof.

[0134] The tissue sample may be selected from tonsils, lymph nodes, bronchi, nose or intestine or skin biopsies. Preferably, the tissue sample is processed to form a single cell suspension. Formation of the single cell suspension can be performed via a mesh filter for tonsils, thymus or lymph nodes. Alternatively, formation of the single cell suspension may be via tissue digestion followed by use of a mesh through filter.

[0135] Specifically, in relation to a biological sample known or suspected to contain B cells or basophils, the sample can be blood, bone marrow or lymphoid tissue. The tissue may be selected from tonsil, lymph node, bronchus, nose or intestinal biopsies. Alternatively, the blood sample can be a whole blood sample, a leptomeningeal sample, a peripheral blood mononuclear cell (PBMC) sample, cord blood, a purified or sorted cell population or a body fluid. Body fluids include samples from the group consisting of lymph amniotic fluid, nasal secretions, bronchial secretions, alveolar fluid, endolymph, pericardial fluid (pericardial fluid), peritoneal fluid, breast milk, or combinations thereof.

[0136] Samples can be taken from any mammal including primates. In particular, humans, rats, more particularly mice, horses, cows, sheep, pigs, dogs, cats, etc. Whole blood can be withdrawn from the sample using any acceptable procedure. Use of whole blood allows detection of effector cells such as eosinophils and basophils. Alternatively, the blood sample may be resuspended in a solution that selectively lyses red blood cells, such as ammonium chloride-potassium chloride; ammonium oxalate, etc.

[0137] An advantage of the present invention is that the sample requires no pretreatment and can be performed on the sample with minimal subsequent processing. However, in some situations, the sample can be subjected to processing such as dilution, concentration, filtration in a buffered medium, or other overall processing without disruption of allergen / antigen-binding cells. The processing can also include removal of cells by various techniques, including centrifugation, use of Ficoll-Hypaque, panning, affinity separation, use of an antibody specific for one or more markers present as surface membrane proteins on the cell surface, or other techniques that provide enrichment of white blood cells. For example, if the sample is diluted due to its large volume, the sample may require concentration or centrifugation to enable the addition of a smaller amount of recombinant allergen / antigen.

[0138] The recombinant allergen / antigen described above can be added directly to the whole blood sample. The amount of allergen / antigen required to bind to a specific cell subset is determined empirically by performing a test assay. This amount can vary depending on the affinity of the allergen / antigen and the density of specific binding partners, such as member-bound Ig or Ig bound to surface Fc receptors. Incubate the cells and allergen / antigen for a time sufficient for the allergen / antigen to bind to either membrane-bound Ig or Ig bound to surface Fc receptors. This incubation time is typically at least about 10 minutes, 1 hour or less, usually 30 minutes or less.

[0139] After incubating the sample with a recombinant allergen or antigen linked to a detectable label, the sample may be incubated with one or more molecules for detection of immune cells. This molecule binds to an immune cell marker and enables, for example, visual detection. Typically, the molecule is bound to a detectable label or is itself a detectable label. For example, the molecule may be a fluorescent dye, an antibody, a nucleotide probe, or an enzyme that results in the production of a substrate.

[0140] Alternatively, this molecule is linked to a tag that facilitates binding to a detectable label. For example, the tag can bind non-covalently to a detectable label or can form a covalent interaction with a detectable label. Suitable tags are known in the art and are described herein. The molecule is preferably an antibody that detects a marker of interest, and the detectable label is preferably a fluorescent dye.

[0141] Prior to contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types, the sample may be contacted with a recombinant or synthetic allergen / antigen linked to a detectable label. Alternatively, the sample may be contacted with a recombinant allergen / antigen linked to a detectable label after or simultaneously with contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types.

[0142] Detection method Detection of immunoglobulins and / or immune cells, particularly B cells or basophils, that bind to a recombinant or synthetic allergen / antigen linked to a detectable label can be performed by flow cytometry or microscopy. These methods are performed as known in the art. The use of flow cytometry or microscopy may be used in conjunction with other cell phenotyping agents.

[0143] As an alternative to fluorescence detection, the allergen or antigen may be multimerized, for example, by tetramerization with streptavidin conjugated to an isotopically pure element for mass cytometry analysis. Using a CyTOF (cytometry by time of flight) instrument, cells are sprayed and sent through an argon plasma that ionizes the metal-conjugated antibody. The metal signal is then analyzed by a time-of-flight mass spectrometer (e.g., Spitzer et al (2016) Cell 165(4):780-9).

[0144] When a sample bound to a fluorescent dye selection reagent is used, flow cytometry or microscopy can be used to detect the presence of immune cells labeled with an antigen / allergen conjugate. Such methods are carried out as known in the art. This method provides detection of either allergen / antigen-specific Ig bound to immune cells or Ig expressed on the cell surface (e.g., B cells and basophils). In non-allergic patient samples, the number of allergen-binding cells in the sample may be low, which is due to the low number of cells in the starting population. In contrast, in test samples from allergic patients, the number of allergen-binding cells present in the sample is typically around 20%, and in some cases can reach up to 90%. Purity can be evaluated by various methods. Conveniently, flow cytometry may be used in combination with a light-detectable reagent specific for cell surface markers expressed by white blood cells. For the diagnosis of allergy, a concentrated cell population is analyzed for the presence of allergen-binding cells, such as allergen-binding basophils.

[0145] In allergic patients, at least about 50% of the allergen-binding cells are cells such as basophils, and can reach up to 90% of the allergen-binding cells. In non-allergic patients, less than about 10% of the allergen-binding cells are basophilic. A positive diagnosis of allergy to a specific allergen is made when the basophil population is increased compared to a control sample. The number of basophils can be at least about twice that of normal non-allergic donors in samples tested similarly, and can be about 10 times the number of basophils in the control sample. Allergen / antigen-binding cells from a concentrated cell population, particularly B cells from human donors, can be used to produce allergen / antigen-specific antibodies. B cells can be immortalized through infection with Epstein-Barr virus, fusion with myeloma cell lines, transfection with transforming retroviruses, etc. Alternatively, B cells can be sorted into single cell wells, the heavy and light chains amplified, and sequenced. Antibodies from either EBV-transformed cells or those produced via recombinant means can be screened by conventional methods such as ELISA, RIA, SPR, etc. to determine the allergen specificity of B cells that produce monoclonal IgE or IgG of particular interest for the production of test reagents, etc.

[0146] Detection of immune cells The present invention relates, in part, to the diagnosis of a subject having an allergic reactivity or sensitivity to an allergen. In particular, the method of the present invention facilitates the detection of IgE-coated basophils in whole blood without the need for in vitro activation by an allergen. Further, the present invention also relates, in part, to determining the effectiveness of allergen immunotherapy (AIT) or allergy immunotherapy. In particular, the method of the present invention facilitates the detection of allergen / antigen-specific B cells through the binding of their Ig surface receptors to recombinant allergen / antigen. Comparison of the proportion of allergen-specific B cells having IgG will provide an indicator of the effectiveness of allergen therapy in a subject.

[0147] The method of the present invention further comprises contacting the sample with a molecule that enables the identification of one or more, preferably two or more, immune cell types. This molecule binds to an immune cell marker and enables visual detection. Typically, the molecule is either conjugated to a detectable label or is itself a detectable label. For example, the molecule may be a fluorescent dye, an antibody, a nucleotide probe, or an enzyme that results in the production of a substrate. Alternatively, the molecule is linked to a tag that facilitates binding to a detectable label. For example, the tag may bind non-covalently to a detectable label or may form a covalent interaction with a detectable label. Suitable tags are known in the art and are described herein.

[0148] For example, the molecule is an antibody that detects a marker of interest and the detectable label is a fluorescent dye. Suitable fluorescent dyes are known in the art and are described herein. Several fluorescently conjugated antibodies against different phenotypic markers on immune cells can be added to the sample to facilitate the detection and discrimination of different cell types. Preferably, the sample is contacted with a panel of fluorescent dye-conjugated antibodies under conditions appropriate for antibody binding to each antigen.

[0149] The sample may be contacted with all the antibodies simultaneously, i.e., a cocktail, mixture, or composition of antibodies with or without recombinant allergens / antigens. However, it may be appropriate to add the antibodies in two or more steps. For example, a two-step incubation may be performed if both surface membrane and intracellular staining are required. In such cases, surface membrane staining is first performed, followed by fixation and permeabilization to facilitate cytoplasmic staining. In either case, unlabeled antibodies can be used, but multiple incubation and washing steps may be required. Preferably, multiplex staining is generally not preferred in routine diagnostic tests.

[0150] CD represents cluster names and is a nomenclature for the identification of specific cell surface antigens defined by monoclonal antibodies. Antibodies against the markers shown are commercially available from various companies, including Becton Dickinson (BD) Biosciences, Dako, Beckman Coulter, CYTOGNOS, Caltag, Pharmingen, Exbio, Sanquin, Invitrogen, and others.

[0151] Preferably, the antibody is provided with a detectable label that enables separate detection and quantification by flow cytometry. A number of detectable fluorescent dye labels are known in the art. For example, a panel of differentially labeled antibody reagents includes fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor 488, Alexa 647, Alexa 710, Alexa fluor 405, cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), Pacific Blue (PacB), Horizon Violet 450 (HV450), Pacific Orange (PacO), Horizon-V500 (HV500), Krome Orange, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 711 (BV711), Brilliant Violet 785 (BV785), Brilliant Ultraviolet 395 (BUV395), Brilliant Ultraviolet 496 (BUV496), Brilliant Ultraviolet 737 (BUV737), Orange Cytognos (OC)515, quantum dots, and combinations of compatible fluorescent dyes selected from their conjugates (e.g., PE / Cy5, PE / Cy5.5, PE / Cy7, PerCP / Cy5.5, APC / Cy7, APC-H7, APC-Alex750, PE-Texas Red, PE-Dazzle, PE-CF594) bound to PE, APC or PerCP or any additional compatible fluorescent dye or fluorescent dye tandem. Fluorescent dye-labeled antibodies can be used in the recombinant allergen / antigen, method or kit of the present invention, can be prepared according to routine techniques known in the art, or can be obtained via various commercial sources.

[0152] In one example, the antibody is conjugated to (1) Pacific Blue (PacB), Brilliant Violet 421 (BV421), or Horizon V450; (2) Pacific Orange (PacO), Horizon V500 (HV500), BV510, Khrome Orange (KO), or OC515; (3) Horizon BB515, fluorescein isothiocyanate (FITC), or Alexa488; (4) phycoerythrin (PE); (5) peridinin chlorophyll protein / cyanine 5.5 (PerCP-Cy5.5), PerCP, or PE-TexasRed; (6) phycoerythrin / cyanine 7 (PE-Cy7); (7) allophycocyanin (APC), or Alexa647; and (8) allophycocyanin / hilite 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750, or Alexa700.

[0153] In another example, the antibody is conjugated to (1) Brilliant Violet 421; (2) Brilliant Violet 510 (BV510); (3) Brilliant Violet 650 (BV650); (4) Brilliant Violet 786 (BV786); (5) fluorescein isothiocyanate (FITC); (6) peridinin chlorophyll protein / cyanine 5.5 (PerCP-Cy5.5); (7) phycoerythrin (PE); (8) phycoerythrin / cyanine 7 (PE-Cy7); (9) allophycocyanin (APC); and (10) allophycocyanin / H7 (APC-H7), APC-C750, or APC-Alexa750.

[0154] Any suitable phenotypic marker can be used to identify the cells of interest. For example, basophils can be identified using pan-basophil markers. Suitable basophil markers are CD63, IgE, 2D7 antigen, CD117, CD124, CD203c, CD200R3 or FcεRIα. For example, B cells can be identified using pan-B cell markers. Suitable B cell markers are CD19, CD20, CD79a or CD22. Most preferably, it is the CD19 antigen.

[0155] To further characterize the immune cells of interest, most preferably basophils and B cells, it may be useful to include them in a panel of fluorescently labeled antibodies. For example, the antibodies are reactive with markers for the characterization of memory B cells, preferably markers selected from the group consisting of CD23, CD40, CD80, CD86, CD180, TACI, CD200, CD73 and CD62L. TCL1 binds to IgE and may appear as non-specifically stained IgE+ naive B cells, suitable for intracellular staining and discrimination between immature / naive B cells and memory B cells, or for the exclusion of FcεRII / CD23+ B cells (e.g., CD23). The panel may also include one or more antibodies useful for the characterization of plasmablasts, for example, antibodies against the CD20 or CD138 antigens. Further, additional antibodies may be selected to characterize activated basophils: for example, CD123, HLA-DR, CXCR3 and / or IgE.

[0156] For example, the sample is subjected to multi-color flow cytometry, gated for lymphocytes based on forward scatter and side scatter, and typically followed by the exclusion of cell doublets and multiplets in a forward scatter-pulse area vs forward scatter-pulse height bivariate dot plot according to conventional criteria.

[0157] Method for producing recombinant protein The allergens or antigens described herein can be recombinant or synthetic.

[0158] The present invention provides an expression vector and a host cell transformed to express the nucleic acid of the present invention. Exemplary methods, vectors, and host cells are described in the Examples.

[0159] The nucleic acid sequence encoding the recombinant allergen or antigen of the present invention, or at least one fragment or portion thereof, may be expressed in bacterial cells (e.g., E. coli), insect cells (baculovirus), yeast, or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or human embryonic kidney cells (HEK 293T)). Appropriate expression vectors, promoters, enhancers, and other expression control elements can be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Other suitable expression vectors, promoters, enhancers, and other expression elements are known to those skilled in the art. Expression in mammalian, yeast, or insect cells leads to partial or complete glycosylation of the recombinant material and the formation of any interchain or intrachain disulfide bonds. Vectors suitable for expression in yeast include YepSec1 (Baldari C et al., (1987), EMBO J., 6(1):229-234); pMFa (Kurjan and Herskowitz (1982), Cell, 30(3):933-943); JRY88 (Schultz LD et al. (1987) Gene, 54(1):113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.). These vectors are freely available. Baculovirus and mammalian expression systems are also available. For example, the baculovirus system is commercially available for expression in insect cells (PharMingen, San Diego, Calif.), while the pMSG vector is commercially available for expression in mammalian cells (Pharmacia, Piscataway, N.J.).

[0160] After isolation, the nucleic acid is inserted operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of DNA) or for expression in a cell-free system or in cells.

[0161] As used herein, the term "promoter" should be interpreted in its broadest context and includes, for example, TATA boxes or initiator elements required for accurate transcription initiation, including transcriptional regulatory sequences of genomic genes, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers and silencers) that change the expression of the nucleic acid in response to development and / or external stimuli or in a tissue-specific manner. In this context, the term "promoter" is also used to describe recombinant, synthetic or fusion nucleic acids, or derivatives, that confer, activate or enhance the expression of a nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance the expression of the nucleic acid and / or to change its spatial and / or temporal expression.

[0162] As used herein, the term "operably linked" means that a promoter is positioned relative to a nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0163] Many vectors are available for expression in cells. Vector components generally include, but are not limited to, one or more of the following: signal sequences, sequences encoding proteins (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. Those skilled in the art will know suitable sequences for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0164] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-α promoter (EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; hybrid regulatory elements including the CMV enhancer / β-actin promoter or its immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0165] Representative promoters suitable for expression in yeast cells (e.g., yeast cells selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae, and S. pombe) include, but are not limited to, the ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0166] Means for introducing an isolated nucleic acid or an expression construct containing the same into cells for expression are known to those skilled in the art. The techniques used for a given cell depend on known successful techniques. Means for introducing recombinant DNA into cells include, among others, microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes (e.g., by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA)), DNA uptake mediated by PEG, electroporation, and particle bombardment (e.g., by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA)).

[0167] Host cells used for producing proteins can be cultured in various media depending on the cell type used. Commercially available media such as Ham’s Fl0 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0168] Isolation of Proteins Methods for isolating proteins are known in the art and / or described herein.

[0169] When the recombinant allergen / antigen is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants. Alternatively or additionally, the supernatant may be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0170] Recombinant allergens / antigens prepared from cells may be purified, for example, using ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known and described in the art.

[0171] Those skilled in the art also know that the protein may be modified to include a tag (e.g., a polyhistidine tag (e.g., a hexahistidine tag), or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag) to facilitate purification or detection. The resulting protein is then purified using methods known in the art such as affinity purification. For example, a protein containing a hexahis tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexahis tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively or additionally, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0172] Nucleotide or amino acid sequence The present invention also contemplates modified forms of the recombinant allergens / antigens of the present invention that contain one or more conservative amino acid substitutions as compared to the sequences described herein. In some examples, the recombinant allergen / antigen contains 10 or fewer, for example, 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue has been replaced with an amino acid residue having a similar side chain and / or hydrophobicity and / or hydrophilicity.

[0173] Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydrophilicity indices are described, for example, in Kyte and Doolittle (1982) J. Mol. Biol., 157(1):105-132 and hydrophilicity indices are described, for example, in U.S. Patent No. 4,554,101.

[0174] Preferred nucleic acids encode recombinant allergens / antigens having at least about 50% homology, more preferably at least about 60% homology, and most preferably at least about 70% homology to the recombinant allergens / antigens of the present invention. Nucleic acids encoding recombinant allergens / antigens having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology to the recombinant allergens / antigens of the present invention are also within the scope of the present invention. Homology refers to sequence similarity between two recombinant allergens / antigens or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. When a position in the sequences being compared is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0175] The present invention also contemplates non-conservative amino acid changes. For example, of particular interest is the substitution of a charged amino acid with another charged amino acid and a neutral or positively charged amino acid. In some instances, the recombinant allergen / antigen comprises 10 or fewer, such as 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0176] In one example, mutations occur within the Ig region that binds to the recombinant allergen / antigen of the present invention. In another example, the mutations occur within the non-Ig binding portion of the allergen / antigen of the present invention.

[0177] Exemplary methods for generating mutant forms of the recombinant allergen / antigen include the following: · Mutagenesis of DNA (Thie et al., (2009), Methods Mol. Biol. 525:309-322) or RNA (Kopsidas et al., (2006) Immunol. Lett. 107(2):163-168; Kopsidas et al. (2007) BMC Biotechnology, 7:18; and WO 1999 / 058661 pamphlet); · Introducing a nucleic acid encoding a polypeptide into mutagenized cells, such as XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene); · DNA shuffling (e.g., as disclosed in Stemmer, (1994) Nature 370(6488):389-91); · Site-directed mutagenesis, e.g., as described in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995); · The H34Q mutation for Api m 1 (as described by Forster E et al. (1995) J Allergy Clin Immunol. 95(6):1229-35)); · The H104V mutation for Lol p 1 (as described by Grobe K et al. (2002) Eur J Biochem, 269(8):2083-92)); and · The Y98F mutation for the HA protein of the H1N1 influenza strain to prevent sialic acid binding (as described by Whittle JR et al. (2014)). Flow cytometry has revealed that H5N1 vaccination induces cross-reactive stem-directed antibodies from multiple Ig heavy chain lineages. Journal of virology. 88(8):4047-4057.

[0178] Exemplary methods for determining the biological activity of the mutant recombinant allergens / antigens of the present invention will be apparent to those skilled in the art and / or are described herein (e.g., recombinant allergens / antigens). For example, methods for determining allergen / antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0179] Monitoring the success of allergy treatment The method of the present invention includes monitoring or determining the success of allergy immunotherapy. For example, monitoring the therapeutic efficacy of anti-IgE therapy or allergy immunotherapy (oral / subcutaneous). Allergy immunotherapy can be allergen-specific or non-allergen-specific (e.g., omalizumab).

[0180] In particular, the present invention relates to monitoring the efficacy of allergy immunotherapy. Allergy immunotherapy (also called desensitization therapy, immunotherapy desensitization, hyposensibilization, or allergen immunotherapy) includes immunotherapy for allergic diseases in which patients are gradually inoculated with increasing amounts of allergens for the purpose of inducing immune tolerance. It also includes other treatments that are not allergen-specific, for example, treatments that reduce sensitivity to allergens by targeting the IgE Fc region (e.g., omalizumab). Allergen-specific immunotherapy is the only treatment strategy for treating the underlying cause of allergic diseases. This can reduce the need for drug therapy, reduce the severity of symptoms, or completely eliminate hypersensitivity. Allergens can be administered sublingually (under the tongue), by injection under the skin (subcutaneous), or in some cases, intradermally.

[0181] The immune system of an individual suffering from an allergy misinterprets a normally harmless substance as a causative agent and initiates the production of IgE. This is called the "primary antibody response". The IgE produced during this response binds to similar types of cells called basophils in the bloodstream and mast cells in the tissues. When a person encounters the allergen again, these basophils and mast cells that were bound to the IgE release histamine, prostaglandins, and leukotrienes, which cause inflammation in the surrounding tissues and result in allergic symptoms. Immunotherapy by repeated exposure to a specific allergen via any of the sublingual, subcutaneous, intradermal, on-skin, or intralymphatic routes brings about desensitization to the allergen and thus reduces the overall symptoms of the allergy and the use of symptomatic treatment. The exact mechanism is not fully understood, but it is recognized that immunotherapy causes a modification of the immune system. This modification brings about changes in IgE synthesis and the production of IgE-blocking antibodies, thereby reducing the allergic response of the immune system to a specific allergen. There is also a shift from a Th2 response to regulatory T cells. Such molecular mechanisms of immunotherapy can be partially interpreted as the induction of allergen-specific IgG to neutralize the allergen instead of the induction of allergen-specific IgE.

[0182] As will be understood by those skilled in the art, the method of the present invention is well-suited to monitor any quantitative changes in allergen-specific B cells in biological samples of patients suffering from allergies and / or undergoing allergy immunotherapy. For this purpose, IgG-expressing cells (which may be memory B cells and / or plasma cells) are easily identified and quantified by staining with the allergen of interest, which is provided with a detectable label such as a fluorescent dye. Accordingly, a method for monitoring the therapeutic efficacy of allergy immunotherapy is provided. This method involves analyzing a subset of memory B cells and plasma cells in a biological sample isolated from a subject undergoing said immunotherapy (oral / subcutaneous) using the above-described procedures herein for detecting allergen IgG+ memory B cells and / or IgG+ plasma cells. This procedure involves determining the allergen specificity of the IgG+ memory B cell population and / or IgG+ plasma cell population by contacting with the fluorescent dye-conjugated allergen of interest, and further includes correlating the amount of IgG+ memory B cells and / or IgG+ plasma cells with disease diagnosis and / or classification, wherein an increase in the number of allergen-specific IgG+ memory B cells and / or IgG+ plasma cells compared to pre-treatment values indicates that the treatment is successful.

[0183] A positive response to allergy immunotherapy can be any one or more of the following (where the first sample is before immunotherapy and the second sample is during or after immunotherapy): An increase in the total number or proportion of IgG-expressing B cells in the second sample relative to the first sample; An increase in the ratio of IgG:IgE-expressing B cells in the second sample relative to the first sample; or An increase in the total number or proportion of IgG2 and / or IgG4-expressing B cells in the second sample relative to the first sample; or A decrease in the total number or proportion of basophils having bound IgE in the second sample relative to the first sample.

[0184] An increase or decrease in the total number or proportion of Ig can be determined by an increase or decrease in the intensity of a detectable label, e.g., the amount of allergen / antigen-binding detectable label molecule bound per cell.

[0185] Kit A kit may be provided for practicing the present invention. For example, the kit may include a recombinant allergen / antigen conjugated to a detectable label as described herein, and optionally, one or a panel of other antibodies that phenotypically determine the cells of interest. Optionally, the kit of the present invention is packaged with instructions for use in the methods described herein.

[0186] Still further aspects of the present invention relate to a diagnostic kit comprising reagents for performing the methods disclosed herein. In one embodiment, it is a diagnostic assay kit for diagnosing and / or classifying and / or monitoring the therapeutic efficacy of a disease or condition associated with a change (production) in IgG level and / or IgE specificity. This kit includes a panel of fluorescent dye-conjugated antibodies against IgM, IgA, IgG, IgD, and IgE; antibodies against B cell markers and an antibody against the CD38 antigen. Preferably, the B cell marker is the CD19, CD20, CD79a, or CD22 antigen, more preferably the CD19 antigen. This kit may also include a fluorescent dye-conjugated CD27 antibody. Each antibody may be conjugated to a distinct fluorescent dye to allow for separate detection by flow cytometry.

[0187] The kit may further include any additional reagents, buffers, or devices for use in the methods of the present invention. For example, the kit may include reagents for creating a standard curve, reagents for calibrating a flow cytometer, positive controls, negative controls, etc. Aspects of the present invention Aspects of the present invention are further described in the following clauses: [Clause 1] A method for determining allergic reactivity in a subject, the method comprising: providing a sample from the subject, and Contacting the sample with the allergen linked to a detectable label under conditions that permit binding of the allergen to IgE molecules present in the sample; determining the binding of the allergen to IgE molecules in the sample by detecting the label, comprising: The method, wherein the detection of the label indicates that the subject is allergic. [Item 2] The method according to item 1 above, wherein the allergen is a recombinant allergen. [Item 3] The method according to item 1 above, wherein the allergen is a synthetic allergen. [Item 4] The method according to item 1 above, wherein the allergen is a natural allergen. [Item 5] The method according to any one of items 1 to 4 above, wherein the sample is a whole blood sample. [Item 6] The method according to any one of items 1 to 5 above, wherein the IgE molecules are present on the surface of cells. [Item 7] The method according to item 6 above, wherein the cells are immune cells. [Item 8] The method according to item 7 above, wherein the immune cells are basophils, eosinophils, mast cells or B cells. [Item 9] The method according to any one of items 6 to 8 above, further comprising removing cells present in the blood sample that do not have IgE molecules bound to the allergen linked to a detectable label on their surfaces. [Item 10] The method according to any one of items 1 to 9 above, wherein the allergen linked to a detectable label that is not bound to IgE is removed. [Item 11] The method according to any one of items 1 to 10 above, further comprising contacting the sample with a molecule that permits identification of two or more immune cell types. [Item 12] The method according to any one of items 1 to 11 above, further comprising contacting the sample with a molecule that distinguishes basophils from other cells. [Item 13] The method according to any one of items 1 to 11 above, wherein the detection of the label is performed by flow cytometry. [Item 14] The method according to any one of items 1 to 13 above, wherein the allergen is a food-based allergen. [Item 15] The method according to any one of items 1 to 13 above, wherein the allergen is an airborne allergen or an environmental allergen. [Item 16] The method according to any one of items 1 to 13 above, wherein the allergen is an animal allergen. [Item 17] The method according to any one of items 1 to 13 above, wherein the allergen is a plant allergen. [Item 18] The method according to any one of items 1 to 13 above, wherein the allergen is an arthropod allergen. [Item 19] The method according to item 18 above, wherein the allergen is an insect allergen. [Item 20] The method according to item 18 above, wherein the allergen is a myriapod allergen. [Item 21] The method according to item 18 above, wherein the allergen is a spider-like allergen. [Item 22] The method according to item 18 above, wherein the allergen is a crustacean allergen. [Item 23] The method according to any one of items 1 to 22 above, wherein the allergen is an enzyme. [Item 24] The method according to item 23 above, wherein the enzyme is modified so as to reduce its activity. [Item 25] The method according to item 24 above, wherein the modification is a point mutation or cleavage. [Item 26] The method according to any one of items 1 to 25 above, wherein the sample is contacted with two or more allergens each conjugated to a different detectable label. [Item 27] The method according to any one of items 1 to 26 above, wherein the allergen is conjugated to a tag that facilitates binding to the detectable label. [Item 28] The method according to item 27 above, wherein the tag is Bir-A. [Item 29] The method according to item 28 above, wherein the Bir-A is biotinylated. [Item 30] The method according to any one of items 1 to 29 above, wherein the detectable label is fluorescent. [Item 31] The method according to any one of items 1 to 30 above, wherein the detectable label is conjugated to streptavidin. [Item 32] A method for determining the effectiveness of allergy immunotherapy in a subject, the method comprising: providing a first sample obtained from the subject prior to receiving allergy immunotherapy; providing a second sample obtained from the subject after receiving allergy immunotherapy; contacting the first and second samples from the subject with a recombinant or synthetic allergen conjugated to a label under conditions that allow binding of the allergen to Ig molecules on the surface of B cells present in the samples; determining binding of the allergen to Ig molecules on the surface of B cells present in the first and second samples by detecting the label; an increase in the total number or proportion of IgG-expressing B cells in the second sample relative to the first sample indicates the effectiveness of the allergy immunotherapy in the subject; an increase in the ratio of IgG:IgE-expressing B cells in the second sample relative to the first sample indicates the effectiveness of the allergy immunotherapy in the subject; or A method wherein an increase in the total number or proportion of IgG2 and / or IgG4-expressing B cells in the second sample relative to the first sample indicates the effectiveness of the allergy immunotherapy in the subject. [Item 33] The method according to item 32 above, further comprising contacting the first and second blood samples with a molecule that enables identification of B cells expressing IgM, IgA, IgG, IgD, and IgE. [Item 34] The method according to item 32 or 33 above, wherein the blood sample is a whole blood sample. [Item 35] The method according to any one of items 32 to 34 above, wherein an allergen linked to a detectable label not bound to an Ig molecule is removed. [Item 36] The method according to any one of items 32 to 35 above, further comprising contacting the sample with a molecule that enables identification of two or more immune cell types. [Item 37] The method according to any one of items 32 to 36 above, further comprising contacting the sample with a molecule that differentiates B cells from other cells. [Item 38] The method according to any one of items 32 to 37 above, wherein detection of the label is performed by flow cytometry or CyToF. [Item 39] The method according to any one of items 32 to 38 above, wherein the allergen is a food-based allergen. [Item 40] The method according to any one of items 32 to 38 above, wherein the allergen is an airborne allergen or an environmental allergen. [Item 41] The method according to any one of items 32 to 38 above, wherein the allergen is an animal allergen. [Item 42] The method according to any one of items 32 to 38 above, wherein the allergen is a plant allergen. [Item 43] The method according to any one of items 32 to 38 above, wherein the allergen is an arthropod allergen. [Item 44] The method according to item 43 above, wherein the allergen is an insect allergen. [Item 45] The method according to item 43 above, wherein the allergen is a myriapod allergen. [Item 46] The method according to item 43 above, wherein the allergen is an arachnid allergen. [Item 47] The method according to item 43 above, wherein the allergen is a crustacean allergen. [Item 48] The method according to any one of items 32 to 47 above, wherein the allergen is an enzyme. [Item 49] The method according to item 48 above, wherein the enzyme is modified so as to reduce its activity. [Item 50] The method according to item 49 above, wherein the modification is a point mutation or cleavage. [Item 51] The method according to any one of items 32 to 50 above, wherein the sample is contacted with two or more allergens each linked to a different detectable label. [Item 52] The method according to any one of items 32 to 50 above, wherein the allergen is linked to a tag that facilitates binding to a detectable label. [Item 53] The method according to item 52 above, wherein the tag is Bir-A. [Item 54] The method according to item 53 above, wherein the Bir-A is biotinylated. [Item 55] The method according to any one of items 32 to 54 above, wherein the detectable label is fluorescent. [Item 56] The method according to any one of items 32 to 55 above, wherein the detectable label is linked to streptavidin. [Item 57] The method according to any one of items 1 to 56 above, wherein the sample is contacted with two or more allergens each conjugated to a different detectable label. [Item 58] A recombinant polypeptide comprising an amino acid sequence encoding an allergen and a tag, wherein the tag facilitates ligation to a detectable label. [Item 59] The recombinant polypeptide according to item 58 above, wherein the tag is a Bir-A tag. [Item 60] The recombinant polypeptide according to item 59 above, wherein the Bir-A tag is biotinylated. [Item 61] The detectable label is linked via any one of streptavidin and its derivatives, avidin and its derivatives, biotin, immunoglobulins, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments and their derivatives, the leucine zipper domain of AP-1, jun, fos, hexa his, hexa hat glutathione S-transferase, glutathione affinity, calmodulin-binding peptide, Strep-tag, cellulose-binding domain, maltose-binding protein, S-peptide-tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2 tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope, carbohydrates, lipids and proteins, a lectin that mediates binding to various compounds including proteins, Con A or WGA and tetranectin or protein A and protein G. The recombinant polypeptide according to any one of items 58 to 60 above. [Item 62] The recombinant polypeptide according to any one of items 58 to 61 above, wherein the detectable label is a fluorescent tag. [Item 63] The recombinant polypeptide according to any one of items 58 to 62 above, wherein the allergen is a food-based allergen. [Item 64] The recombinant polypeptide according to any one of items 58 to 62, wherein the allergen is an airborne allergen or an environmental allergen. [Item 65] The recombinant polypeptide according to any one of items 58 to 62, wherein the allergen is a plant allergen. [Item 66] The recombinant polypeptide according to any one of items 58 to 62, wherein the allergen is an animal allergen. [Item 67] The recombinant polypeptide according to any one of items 58 to 62, wherein the allergen is an arthropod allergen. [Item 68] The method according to item 67, wherein the allergen is an insect allergen. [Item 69] The method according to item 67, wherein the allergen is a myriapod allergen. [Item 70] The method according to item 67, wherein the allergen is a spider-like allergen. [Item 71] The method according to item 67, wherein the allergen is a crustacean allergen [Item 72] The recombinant polypeptide according to any one of items 58 to 71, wherein the allergen is an enzyme. [Item 73] The recombinant polypeptide according to item 72, wherein the enzyme is modified so as to reduce its activity. [Item 74] The recombinant polypeptide according to item 73, wherein the modification is a point mutation or cleavage. [Item 75] A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to any one of items 58 to 74. [Item 76] A vector comprising the nucleic acid according to item 75. [Item 77] A host cell comprising the nucleic acid according to item 75 or the vector according to item 76. [Item 78] A kit for use in the method according to any one of items 1 to 57 above, comprising one or more recombinant allergens, a detectable label, and instructions for use, buffer, and / or control samples. [Item 79] A method for detecting antigen-specific B cells in a subject, the method comprising: providing a sample from the subject; contacting the sample with the antigen linked to a detectable label under conditions that allow binding of the antigen to Ig molecules on the surface of B cells present in the sample; determining the binding of the antigen to the Ig molecules in the sample by detecting the label, wherein the detection of the label indicates that the subject has antigen-specific B cells. [Item 80] The method according to item 79 above, wherein the antigen is derived from a vaccine. [Item 81] The method according to item 79 above, wherein the antigen is derived from a pathogen or infectious agent. [Item 82] The method according to item 81 above, wherein the antigen is derived from a virus, bacterium, fungus, protozoan, or parasite. [Item 83] The method according to item 82 above, wherein the antigen is virus-derived. [Item 84] The method according to item 83 above, wherein the virus is associated with or causes a respiratory condition or disease. [Item 85] The method according to item 83 or 84 above, wherein the antigen is a nucleocapsid protein or spike protein, or a domain within a nucleocapsid protein or spike protein. [Item 86] The method according to item 85 above, wherein the antigen is derived from SARS-CoV-2. [Item 87] The virus can be selected from measles, polio, coronavirus, influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus, cytomegalovirus (CMV), Epstein - Barr virus (EBV), varicella - zoster virus (VZV), dengue virus, rhinovirus, herpes simplex virus, and enterovirus. More preferably, the virus is coronavirus or influenza. Even more preferably, the virus is severe acute respiratory syndrome coronavirus (SARS - CoV), Middle East respiratory syndrome coronavirus (MERS - CoV), or severe acute respiratory syndrome coronavirus 2 (SARS - CoV - 2), the method according to item 86 above. [Item 88] The method according to item 87 above, wherein the virus is SARS - CoV - 2. [Item 89] The method according to item 82 above, wherein the infectious agent is a bacterium, preferably Clostridium tetani or Corynebacterium diphtheria. [Item 90] The method according to item 79 above, wherein the factor is related to a self - antigen.

[0188] [Table 1]

[0189] [Table 2]

[0190] [Table 3]

[0191] [Table 4]

[0192]

Table 5

[0193]

Table 6

[0194]

Table 7

[0195]

Table 8

[0196]

Table 9

[0197]

Table 10

[0198]

Table 11

[0199]

Table 12

[0200]

Table 13

[0201]

Table 14

[0202]

Table 15

[0203]

Table 16

[0204]

Table 17

[0205]

Table 18

[0206]

Table 19

[0207]

Table 20

[0208]

Table 21

[0209]

Table 22

[0210]

Table 23

[0211]

Table 24

[0212]

Table 25

[0213]

Table 26

[0214]

Table 27

[0215]

Table 28

[0216]

Table 29

[0217]

Table 30

[0218]

Table 31

[0219]

Table 32

[0220]

Table 33

[0221]

Table 34

[0222]

Table 35

[0223]

Table 36

[0224]

Table 37

[0225]

Table 38

[0226]

Table 39

[0227]

Table 40

[0228]

Table 41

[0229]

Table 42

[0230]

Table 43

Example

[0231] Example 1: Design, protein production, purification and tetramerization of recombinant antigen constructs Overview The inventors used recombinant proteins to introduce modifications that inhibit enzymatic activity and remove the toxicity of the antigen being tested. Further, peptide sequences were added to enable efficient purification (6-His tag) and target biotinylation (Figure 1). The protein production pipeline includes the step of producing the protein in the most relevant cell expression system and the step of secreting these to ensure that the protein structure and post-translational modifications are as similar as possible to the native protein (Figure 2).

[0232] Recombinant proteins produced by the methods described herein have been demonstrated to be immunogenic. Recombinant HA proteins from influenza strains A / Michigan / 200 / 2019 (H1N1) and B / Phuket / 3073 / 2013 (B lineage) were recognized by IgG in the sera of vaccinated subjects. Further, recombinant phospholipase A2 (Api m 1) and grass pollen allergen (Lol p 1) were able to induce IgE-mediated basophil activation in allergen-sensitized subjects.

[0233] Staining of immune cells was achieved by biotinylation of the recombinant protein and tetramerization using fluorescently labeled streptavidin. The advantage of this system is to utilize target biotinylation that has little or no effect on the rest of the protein, and the tetramer should increase the binding activity for binding Ig (either binding to the cell surface or expressed on the cell surface). Indeed, all antigen tetramers tested showed a high fluorescence signal, and antigen-binding cells were clearly distinguishable from non-binding cells by flow cytometry.

[0234] Materials and Methods Design of Recombinant Antigen Constructs For robust detection of cells expressing or binding allergen-specific or antigen-specific immunoglobulins (Ig), the inventors developed the following set of criteria for the design and generation of recombinant antigens or allergens: 1. The protein must be non-pathogenic, non-toxic, and have no enzymatic activity in order to minimize risks to researchers and prevent cell death in the target assay; 2. The protein should fold naturally and, if applicable, should include post-translational modifications to ensure the presence of conformational epitopes; 3. The protein must be purified without affecting its protein structure; 4. The protein must contain tags to facilitate binding in order to enable multimerization and / or (fluorescent) detection (Figure 1).

[0235] To measure the vaccine response against tetanus (Clostridium tetani) and diphtheria (Corynebacterium diphtheriae), recombinant forms of the toxins were created with modifications to prevent toxicity. For tetanus toxin, the 451 amino acid C-terminal domain of the heavy chain (TTC) was generated (as described in Fairweather NF et al. (1986) J Bacteriol 165(1):21-7), and for diphtheria toxin, a single amino acid substitution (G52E) was introduced to generate the non-toxic CRM197 variant (as described in Giannini G et al. (1984) Nucleic acids Res 12(10):4063-9 and Malito E et al. (2012) Proc Natl Acad Sci USA, 109(14):5229-34). Both constructs included a leader sequence (MIKFLSALILLLVTTAAQA) to target production to the periplasm of Escherichia coli (E. coli) (as described by Goffin P et al. (2017) Biotechnol J, 12(7)). Furthermore, both constructs included a C-terminal 6-histidine (6-His) and a 15-residue BirA substrate peptide (BSP) (LHHILDAQKMVWNHR) (as described in Schatz PJ (1993) Biotechnology(NY), 11(10):1138-43). The constructs were codon-optimized for E. coli and cloned into the pET30 vector (Novagen).

[0236] To measure the vaccine response to trivalent and quadrivalent influenza vaccines, recombinant hemagglutinin (HA) antigens from the A / Michigan / 45 / 2015 (AM15), A / Brisbane / 02 / 2018 (AB18), and B / Phuket / 3073 / 2013 (BP13) strains were generated. All constructs contained a C-terminal AviTag (GLNDIFEAQKIEWHE) BirA target sequence and a 6-His tag (as described in Whittle JR et al. (2014) J Virol, (8):4047-57, Wheatley AK et al. (2016), Sci Resp 25;6:26478, and Liu Y et al. (2019) Nat Commun, 18;10(1):324). The constructs contained their native leader sequences for secretion. Point mutations were introduced into AM15, AB18 (Y98F), and PB13 (T139G) to prevent specific binding to sialic acid on the cell surface. The constructs were codon-optimized for humans (Homo sapiens) and cloned into the pCR3 vector (Invitrogen) for expression in 293Expi cells.

[0237] To measure allergic sensitization to insect venom, aeroallergens, and food allergen venom, the major allergen components were recombinantly produced from bee venom (Api m 1), rye grass pollen (Lol p 1 and Lol p 5), timothy grass pollen (Phl p 1), house dust mite (Der p 2), cat dander (Fel d 1), peanut (Ara h 2), and black tiger shrimp (Pen m 1). The sequences were obtained from the WHO / IUIS allergen nomenclature database (http: / / www.allergen.org / ), and the nucleotide sequences can be found in GenBank: X16709.1 for Api m 1 and M57474.1 for Lol p 1.

[0238] All constructs except Fel d 1 contained an Api m 1 N-terminal leader sequence for extracellular production and either a C-terminal or N-terminal AviTag and 6-His sequence. Enzyme activity was impaired by introduction of point mutations H34Q for Api m 1 (as described in Forster E et al. (1995) J Allergy Clin Immunol, 95(6):1229-35) and H104V for Lol p 1 and Phl p 1 (as described in Grobe K et al. (2002) Eur J Biochem 269(8):2083-92). All constructs except Fel d 1 were codon-optimized for Spodoptera frugiperda (fall armyworm) and cloned into the pFastBac vector (Thermo Fisher Scientific) before being incorporated into Bacmid for baculovirus production. Fel d 1 was produced using its native leader sequence and C-terminal AviTag and 6His tag, codon-optimized for Homo sapiens, and cloned into the pCR3 vector (Invitrogen) for expression in 293Expi cells (as for the influenza constructs).

[0239] Protein production, purification, and tetramerization Bacterial constructs were inserted into BL21(DE3)pLysE Escherichia coli (E. coli) cells by heat shock, and single colonies were cultured overnight (16 h) at 37 °C in 10 ml of Luria-Bertani (LB) broth containing kanamycin and chloramphenicol antibiotics. These 10 ml cultures were then transferred to 1 L cultures and grown at 37 °C for 4–7 h to an OD 600Growth was allowed to proceed until. Subsequently, protein production was induced by the addition of 1 mM IPTG, and the bacteria were cultured at 27 °C overnight (16 hours). Bacterial pellets were collected, and the periplasmic fraction was prepared by osmotic shock at 4 °C. First, the pellet was thoroughly resuspended in 30 mM Tris-HCl, 20% sucrose solution at pH 8 for 10 minutes. Subsequently, the cells were pelleted and resuspended in 5 mM MgSO4 for 10 minutes. After the final pelleting, the supernatant was collected for protein purification.

[0240] The viral construct was transfected into 293Expi cells (Thermo Fischer Scientific) using Expifectamine and cultured in suspension in Opti-MEM I reduced serum medium (Gibco) on a shaker at 37 °C for 5 days. Thereafter, the culture supernatant was collected.

[0241] The insect and plant allergen constructs were incorporated into a Bacmid encoding baculovirus. The Bacmid was transfected into Sf21 cells and cultured at 27 °C. The supernatant from the infected Sf21 culture was clarified by centrifugation.

[0242] Since all protein constructs contained a 6-His tag, they were purified from the bacterial periplasm preparation, as well as from the 293Expi and Sf21 culture supernatants, through retention on a cobalt column. The supernatant was gravity-fed into a 25 ml column packed with 4 ml of Talon NTA-cobalt-agarose beads (Clontech). The beads were washed with PBS, and the protein was eluted with PBS (pH 8.5) containing 200 mM imidazole. The eluate was dialyzed against 10 mM TRIS, pH 7.5.

[0243] All recombinant proteins contained a BSP tag for biotinylation and were biotinylated by incubating overnight at room temperature with 2.5 μg / ml of BirA enzyme in 10 mM TRIS containing 62.5 mM biotin-HCl, 12.5 mM ATP, 12.5 mM MgOAc, and 62.5 μM D-biotin. The proteins were dialyzed against PBS. To tetramerize our antigen, we first determined the required volume of streptavidin by calculating a 4:1 molar ratio of antigen:streptavidin. The volume of streptavidin-fluorophore conjugates (PE, APC, BUV395, BUV737, all obtained from BD Biosciences) was added stepwise at 1 / 10 th of the antigen every 5 minutes and repeated 10 times at room temperature to tetramerize the antigen.

[0244] Western blotting Baculovirus-containing Sf21 supernatant, 293T Expi cell supernatant, and Escherichia coli (E. coli) periplasm preparation were mixed with 6X reducing buffer or non-reducing buffer (0.1 M Tris-HCl (pH 6.8), 0.2% bromophenol blue, and 20% glycerol, and the reducing buffer contains 4% SDS and 50 mM DTT). The reducing samples were heated at 85 °C for 10 minutes. Protein samples were loaded onto a 4-15% Mini-PROTEAN® TGX Stain-Free gel (Bio-Rad) and separated at 200 V for 30 minutes. Proteins were transferred onto a PVDF membrane (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was probed with mouse anti-His (clone: 27471001, category #18594, GE Healthcare), followed by goat anti-mouse IgG HRP (clone: NA9310N; category #D614011, Cell Signaling Technology). The PVDF membrane was developed using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences), and chemiluminescence was detected using a ChemiDoc Imager (Bio-Rad).

[0245] Enzyme-linked immunosorbent assay (ELISA) ELISA plate wells were coated with recombinant protein, blocked with 2% bovine serum albumin in PBS (Sigma Aldrich, Darmstadt, Germany), and incubated with serial dilutions of serum samples. Bound IgE was detected using rabbit polyclonal anti-hIgE (Dako), followed by goat anti-rabbit IgG HRP (Bio-Rad). ELISA was developed using TMB (Thermo Scientific), and the reaction was stopped with 1 M HCl. Absorbance (OD 450 nm) was measured using a FLUOstar Optima plate reader (BMG Labtech).

[0246] Test participants Healthy adult controls were registered in the low-risk reference value study (Monash University project 2016-0289) and consented to the collection of basic demographics (age, gender, history of immunological and hematological diseases) and the provision of 40 ml of blood. A second blood sample was taken 4 weeks after vaccination from those who indicated that they had received the 2019 influenza vaccine of their own free will.

[0247] Results Recombinant antigens were produced using BSP and 6-His peptide tags as described in the Methods section. Recombinant non-toxic tetanus toxin (TTC) and diphtheria toxin (CRM197) constructs were produced in Escherichia coli (E. coli) and purified from the periplasm before purification on a cobalt column. Western blot using anti-His antibody (Figure 3) showed the presence of the protein in the periplasm preparation and concentration after purification. When performed under non-reducing conditions, two bands of approximately 45 and 50 kDa were seen for TTC, while only the 50 kDa band was seen under reducing conditions. Thus, there appears to be one product with potentially two modified conformations. The product was slightly shorter than predicted based on the sequence (57 kDa), suggesting cleavage of the leader peptide. For CRM197, only one band of approximately 60 kDa was seen under both non-reducing and reducing conditions, which was also slightly shorter than predicted based on the sequence alone (64 kDa).

[0248] HA proteins from one A-type (AM15, A / Michigan / 45 / 2015) and one B-type (BP13, B / Phuket / 3073 / 2013) influenza strain were produced using AviTag and 6-His peptide tags in 293T expi cells and purified from the supernatant on a cobalt column. Western blot using anti-His antibody under non-reducing conditions showed single bands of approximately 65 kDa (AM15) and approximately 75 kDa (BP13) (Figure 4A). These were close to the predicted size of 64 kDa for both, indicating that the proteins were produced as monomers.

[0249] To examine whether the product is specifically recognized by IgG in the sera of vaccinated individuals, the inventors determined the levels of HA-specific IgG against each of the HA proteins in 16 individuals before and after vaccination with the 2019 quadrivalent seasonal influenza vaccine (Afluria-Quad (Seqirus), Influvac tetra (Mylan Health) or Fluquadri (Sanofi-Aventis)) previously vaccinated in 2018. All individuals had detectable levels of IgG against HA from AM15 (range, 2.6 - 29 μg / ml) and BP13 (range, 3.1 - 14 μg / ml) before 2019 vaccination, and these specific IgG levels were significantly higher at 4 weeks after booster vaccination (both p < 0.01; paired t-test, Figures 4B and 4C).

[0250] Using a baculovirus expression system, seven major allergens from bee venom (Api m 1), rye grass (Lol p 1, Lol p 5), timothy grass pollen (Phl p 1), house dust mite (Der p 2), peanut (Ara h 2) and black tiger shrimp (Pen m 1) were produced in Sf21 insect cells. The major cat dander allergen, Fel d 1, was produced in 293T expi cells. The constructs contained an AviTag and a 6-His peptide tag and were purified from the culture supernatant using a cobalt column. The recombinant proteins were evaluated by Western blotting using an anti-His antibody and showed bands of the expected size or slightly larger due to post-translational modifications (e.g., glycosylation) (Figure 5). For Lol p 1, a single non-reduced product of approximately 70 kDa and a single reduced product of approximately 42 kDa were observed. This suggests that Lol p 1 is produced as a dimer, which is in good agreement with the prediction of its native arrangement based on the structure of the highly homologous Phl p 1 allergen (Protein Data Bank entry: 1N10 (Flicker S et al. (2006) J Allergy Clin Immunol 117(6):1336 - 43).

[0251] Example 2: Detection of Allergen Sensitization by Staining of Basophils and Flow Cytometry Detection Overview The inventors have demonstrated the ability to detect antigen-specific Ig on soluble Ig-binding cells (e.g., basophils). The inventors demonstrated that basophil staining is highly specific for allergen-sensitized subjects since the signal for allergens was more than twice higher than that of streptavidin alone. None of the non-sensitized controls showed a signal exceeding 1.6-fold. Thus, allergen straining can be utilized for laboratory testing of allergen sensitization. Importantly, since this method utilizes multicolor flow cytometry, the test can be easily multiplexed with several allergens conjugated to different fluorescent fluorophores, allowing several allergens to be tested within a single tube.

[0252] Since only 2 - 3 fluorescence channels are required to detect basophils, depending on the flow cytometer used, there are many available options for the fluorescent conjugate. For example, with a 3-laser, 8-parameter FACS Canto, there are 5, or with a 5-laser LSR Fortessa X-20, up to 14 parameters, etc. Fluorescent-labeled streptavidin is readily available and can be easily implemented to conjugate to recombinant proteins (PE, APC, BUV395, and BUV737).

[0253] Basophil staining offers the opportunity to perform component-resolved diagnosis (CDR) in a single sample tube and thus has many advantages over the standard basophil activation test (BAT), which can only read for a single allergen (mix) per tube. Thus, allergen-tetramers with different fluorescent dyes can be combined in a single staining cocktail to analyze allergen sensitization and cross-reactivity.

[0254] Materials and Methods Test Participants Patients with wasp venom, wheat allergy, giant Japanese hornet, house dust mite, cat dander, peanut and / or shrimp were recruited from the Alfred Health Allergy Clinic after obtaining informed consent regarding blood collection and medical information (Alfred Health, Ethics Projects #514 / 13 and #509 / 11). Allergen sensitization was defined as positive by prick test and / or RAST. Patients provided 40 ml of blood once before the start of allergen immunotherapy and, if applicable, once 2 - 4 weeks after (wasp venom allergy) or 4 months after (wheat allergy) allergen immunotherapy.

[0255] This study was conducted in accordance with the principles of the Helsinki Declaration and approved by the local Human Research Ethics Committee.

[0256] Flow cytometry of basophils The absolute number of white blood cells was determined within 24 hours of blood sampling in Vacutainer containing EDTA (BD Biosciences) using a lysis - non - washing method. 50 μl of whole blood was added to a TruCount tube (BD Biosciences) together with 20 μl of antibody cocktail to stain CD3, CD4, CD8, CD16, CD45 and CD56. After incubation at room temperature for 15 minutes, 500 μL of 0.155 M NH4Cl was added to lyse red blood cells for 15 minutes. Subsequently, the mixture was stored at 4°C in the dark and acquired by flow cytometer within 2 hours.

[0257] To identify circulating basophils, whole blood was incubated with anti - CD123 - BV605 (6H6; BioLegend) and anti - IgE - FITC (goat anti - human; Thermo Fisher). Red blood cells were lysed and washed with ammonium chloride solution (NH4Cl 154 mM, KHCO3 10 mM, EDTA 1 mM). The remaining cells were resuspended in wash buffer containing 7AAD (BD Biosciences) to identify live cells. Samples were stored at 4°C in the dark and analyzed by flow cytometry within 2 hours.

[0258] Activation of basophils To stimulate circulating basophils, whole blood was incubated at 37 °C for 10 minutes in stimulation buffer (Hepes 20 mM, NaCl 133 mM, KCl 5 mM, CaCl2 7 mM, CaCl2 3.5 mM, BSA 1 mg / ml, rIL-3 2 ng / ml, Heparin 20 μl / ml, pH 7.4). Basophils were activated with streptavidin-fluorophore conjugate (0.5 μg / ml) or allergen tetramer (1 μg / ml) at 37 °C for 20 minutes. Activation was stopped by incubating on ice for 5 minutes. Serum was removed by washing with cold wash buffer (Hepes 20 mM, NaCL 133 mM, KCl 5 mM, EDTA 0.27 mM, pH 7.3). Basophil activation was tested by flow cytometry using the above-described markers for defined basophils (anti-CD123, anti-IgE) and positivity for surface CD63 using anti-CD63-PE (H5C6; BD Biosciences).

[0259] Flow cytometer setup All flow cytometry was performed on three instruments within our flow core facility with a nearly identical setup for the four shared lasers (BD LSRII and BD LSRFortessa) or five lasers (BD LSRFortssa X-20). Instrument setup and calibration were performed using the standardized EuroFlow SOP (as described in Kalina T et al. (2012) Leukemia 26(9):1986 - 2010) with in-house optimization for three additional fluorescence channels (V610, V710, and YG610) (as described in Edwards ESJ et al (2019), Front Immunol. 10:2593).

[0260] Data analysis and statistics All data were analyzed using the FACS DIVA v8.0.1 (BD Biosciences) and FlowJo v10 software package (FlowJo, LLC). Statistical analysis was performed using the non-parametric Mann-Whitney U test. The statistical analysis of the sampling distribution was evaluated by the chi-square test. For all tests, p < 0.05 was considered significant.

[0261] Results Initial tests of antigens and allergens To investigate whether recombinant allergens can be recognized by IgE and mediate allergic responses, the inventors measured their ability to activate basophils using a flow cytometry basophil activation test (BAT) (as described in Hemmings O et al. (2018) Curr Allergy Asthma Rep, 18(12):77). After activating basophils in whole blood in vitro with either streptavidin-APC, allergen-streptavidin-APC, or an allergen extract, the basophils were processed and analyzed by flow cytometry. Samples were analyzed by electronically gating for high expression of CD123 and IgE (Figure 6A). Activated basophils were defined as being positive for surface expression of CD63 (as described in Hemmings O et al. (2018) Curr Allergy Asthma Rep 18(12):77).

[0262] Twenty subjects with wasp venom hypersensitivity and 24 non-allergic controls' fresh whole blood samples were tested using the BAT assay. For all samples tested, streptavidin-APC alone (negative control) did not result in CD63 surface expression (Figure 6B). Basophils from all patients were activated by the Api m 1 tetramer, but there was no activation in controls. Similarly, blood basophils from 50 subjects with rye grass pollen hypersensitivity and 20 controls were stimulated with streptavidin-APC and Lol p 1 tetramer (Figure 6C). For all subjects, CD63 expression was hardly observed with streptavidin-APC alone, but with the Lol p 1 tetramer, the frequency of CD63+ basophils was high in patients but not observed in controls.

[0263] Collectively, these results indicate that the inventors' recombinant antigen production pipeline has the ability to produce proteins recognized by IgG and IgE antibodies and immunologically active.

[0264] Detection of allergen sensitization via fluorescent antigen staining of basophils The BAT assay functions based on the main principle that basophils in the blood bind to high levels of soluble IgE via high-affinity FcεRI on their surface. These soluble IgE have diverse specificities for antigens, and in the case of allergic patients, the IgE molecules on the surface of basophils include those specific for their particular allergen (as outlined in Hoffmann HJ et al. (2015), Allergy 70(11):1393 - 405). Therefore, during in vitro incubation, these IgE bind to the allergen, and when multiple molecules bind, they crosslink via FcεRI and activate basophils. This is a highly sensitive clinical test but has certain limitations: 1. Basophils can become non-responsive by storing blood for more than 4 hours and under suboptimal conditions, so handle with care; Since the BAT at 2.1 times only reads the response of the whole allergen extract, it is impossible to distinguish information regarding specific allergen sensitization, and additional tests for each specific allergen will be necessary.

[0265] The specificity of basophil activation is based on the presence of allergen-specific IgE bound to its FcεRI. Therefore, the inventors determined whether their recombinant allergens (Api m 1 and Lol p 1) could specifically bind to the basophils of allergen-sensitized individuals upon tetramerization with allophycocyanin (APC)-labeled streptavidin. Whole blood from non-sensitized controls and allergen-sensitized individuals was incubated with either an antibody cocktail and streptavidin-APC or an allergen-streptavidin-APC conjugate. Following flow cytometric gating of CD123+IgE+ basophils (Figure 6A), the fluorescence intensity of the APC signal was determined in these cells (Figures 7A and 7B). In non-sensitized controls, the median fluorescence intensities of streptavidin-APC, (Api m 1)4-APC, and (Lol p 1)4-APC were very similar, around 10 - 60, and the allergen-tetramer signal was typically 1.1 - 1.5 times higher than the streptavidin-APC signal (Figures 7C and 7D). In contrast, the fluorescence intensity of the allergen tetramer was much higher in the basophils of allergen-sensitized patients. All fluorescence signals were higher for Api m 1 in almost all patients with wasp venom allergy and Lol p 1 in almost all patients with rye grass pollen allergy. The signal intensity was 2 - 1000 times higher than the streptavidin-APC signal on the basophils of the same patients. Therefore, the allergen tetramer specifically stains the basophils of sensitized patients. Importantly, sensitization can be determined by the signal intensity or by the fold difference relative to streptavidin staining only. This is shown by receiver operator characteristic (ROC) curves with extremely high areas under the curve (AUC) of 10,000 for Api m 1 and 9980 for Lol p 1 to separate sensitized from non-sensitized individuals (Figures 7C and 7D).

[0266] Recombinant allergen tetramers of six other allergens were generated, similar to Api m 1 for bee venom and Lol p 1 for rye grass pollen (Lol p 5, Phl p 1, Der p 2, Fel d 1, Ara h 2, Pen m 1). After incubation of basophils from patients with relevant allergen sensitization (Figure 8A), all six allergen tetramers specifically stained basophils with a 2- to 10-fold increase in signal intensity compared to streptavidin alone (Figures 8B - G).

[0267] Example 3: Evaluation of allergen desensitization by B cell staining and flow cytometry detection Overview The present inventors demonstrated the ability to detect antigen-specific surface Ig-expressing cells (B cells).

[0268] B cell staining with fluorescent antigens can be multiplexed. Detection of antigen-specific B cells can be used to evaluate the humoral immune response in multiple situations. In the case of allergy, it can complement the findings of allergen sensitization observed by basophil staining.

[0269] The present invention enables the monitoring of changes in the B cell compartment over time after treatment with allergen immunotherapy (AIT). This can be measured as a measure of desensitization success through a shift towards more IgG-expressing B cells, particularly IgG2 and IgG4-expressing B cells.

[0270] In addition to allergy, the response to infection and vaccination can be monitored in the B cell compartment. Similarly, changes in the use of Ig isotypes and IgG subclasses can reflect a successful primary or booster response. Furthermore, measurement of antigen-specific B cells may provide a means to examine the vaccination response in individuals receiving Ig replacement therapy (IgRT). This is because in these individuals, serum IgG measurement reflects the donor IgG composition rather than the host response.

[0271] Materials and methods Flow cytometry of B cells To immunophenotype circulating allergen-specific B cells, peripheral blood mononuclear cells (PBMCs) were incubated with both the PE-conjugated variant and the APC-conjugated variant of the same allergen tetramer. Furthermore, an antibody cocktail containing CD3-BV711 (UCHT1), CD19-PE-Cy7 (SJ25C1), CD27-BV421 (M-T271), anti-IgD-PE-CF594 (IA6-2), anti-IgG-BV786 (G18-145; all from BD Biosciences), CD38-APC-Cy7 (HIT2), CD123-BV605 (6H6), anti-IgM-BV510 (MHM088; all from BioLegend), anti-IgE-FITC (goat anti-human; Thermo Fisher), and Fixable Viability Stain 700 (BD Biosciences) was added. After incubation at room temperature for 15 minutes, the PBMCs were washed with PBS containing 0.2% BSA. The samples were stored at 4°C in the dark and then data acquisition was performed with a flow cytometer within 2 hours.

[0272] To immunophenotype circulating influenza HA-specific B cells, PBMCs were incubated with both BUV395-binding and BUV737-binding variants of the same HA antigen tetramer. Additionally, an antibody cocktail containing anti-CD3-BV711 (UCHT1), anti-CD19-PE-Cy7 (SJ25C1), anti-CD21-BV711 (B-ly4), anti-CD27-BV421 (M-T271; all from BD Biosciences), anti-CD38-APC-Cy7 (HIT2), anti-IgD-PerCP-Cy5.5 (IA6-2), anti-IgM-BV510 (MHM088; all from BioLegend), anti-IgA-PE-Vio615 (REA1014; Miltenyi Biotec), anti-IgG1-PE (SAG1), anti-IgG2-FITC (SAG2), anti-IgG2-PE (SAG2), anti-IgG3-FITC (SAG3), anti-IgG4-APC (SAG4; all from Cytognos), and Fixable Viability Stain 700 (BD Biosciences) was added. After incubation for 15 minutes at room temperature, the PBMCs were washed with PBS containing 0.2% BSA. The samples were stored at 4°C in the dark and then data acquisition was performed on a flow cytometer within 2 hours.

[0273] Flow Cytometer Setup All flow cytometry was performed on three instruments within our flow core facility with a nearly identical setup for four lasers (BD LSRII and BD LSRFortessa) or five lasers (BD LSRFortssa X-20). Instrument setup and calibration were performed using standardized EuroFlow SOPs as previously described (Kalina T et al. (2012) Leukemia 26(9):1986-2010) and in-house optimization for three additional fluorescence channels (V610, V710, and YG610) as described (Edwards ESJ et al., (2019), Front Immunol. 10:2593).

[0274] Data analysis and statistics All data were analyzed using FACS DIVA v8.0.1 (BD Biosciences) and the FlowJo v10 software package (FlowJo, LLC). Statistical analysis was performed using the non-parametric Mann-Whitney U test. The statistical analysis of the sampling distribution was evaluated by the chi-square test. For all tests, p < 0.05 was considered significant.

[0275] Results B cells each have surface Ig molecules with specificities. Theoretically, within the pool of naive B cells, a small fraction will have specificities for foreign proteins such as allergens and vaccine antigens. Furthermore, in individuals exposed to these proteins, a memory B cell population is expected to exist. Finally, in allergic patients who produce soluble IgE against an allergen, there may be a fraction of B cells that express allergen-specific IgE. To test this, the inventors combined ex vivo staining of B cell antibody cocktails with their fluorescent antigen tetramers. Since fluorophores such as phycoerythrin (PE) and APC are large proteins to which antibodies can be formed, incubations were always performed using two protein conjugates. After electronic gating on single live cells in whole blood, CD19+ B cells were defined (Figure 9A), and the fraction of antigen tetramer-positive cells among all B cells was determined. As expected, PE-specific cells and APC-specific cells were present among the B cells of wasp venom-sensitized individuals (Figure 9B). These were also evident in both antigen tetramer staining and staining with streptavidin alone. However, in antigen tetramer staining, there was a small fraction of 0.27% that was double-positive for the Api m 1 tetramer. This fraction was almost completely absent in staining with streptavidin alone. Similarly, within all B cells of grass pollen allergy patients, a fraction of Lol p 1 was clearly identified. Therefore, the Api m 1 and Lol p 1 allergen tetramers can be utilized to specifically stain allergen-specific B cells.

[0276] When examining Lol p 1-specific memory B cells, it was revealed that grass pollen allergy patients had an increased frequency of IgG+ Bmem compared to non-allergic controls (Figure 10A). This was at the expense of IgM+ Bmem. In repeated sampling after four months, no changes were observed in patients under standard treatment outside the pollen season (Figure 10B), but patients who had received sublingual immunotherapy (SLIT) for four months had significantly higher fractions of IgM+ Bmem and lower IgG+ Bmem. Thus, SLIT is associated with a change in the allergen-specific Bmem compartment to a profile similar to that seen in non-sensitized controls (Figure 10A).

[0277] Using a more extensive antibody cocktail, the inventors were able to further immunophenotype Api m 1-specific B cells in wasp venom-sensitized individuals before and 2 - 4 weeks after ultra-rush allergen-immunotherapy (AIT). At both times, before and after AIT, the Api m 1-specific fraction consisted of both naive (CD27-IgM+) and memory (CD27+) B cells. Importantly, within the memory cell fraction, there appeared to be a shift from dominant IgM expression to IgG expression (Figure 11). This may indicate a repeated immune response due to repeated allergen exposure and could be an indicator of treatment success.

[0278] Example 4: Monitoring of vaccination response Similar to the recombinant allergen-tetramers, the inventors also performed immunophenotyping of vaccine antigen-specific B cells. B cells from healthy adults after booster vaccination with the 2019 quadrivalent vaccine were immunophenotyped with an antibody cocktail and two recombinant HA tetramers derived from AM15 (Figure 12A). Using either a combination of PE- and APC-conjugates, or a combination of BUV395- and BUV737-conjugates, HA-specific B cells were clearly detected in less than 1% of total B cells. Within the HA-specific B cells, multiple subsets were identified. Specifically, using Ig isotype and IgG subclass antibodies, memory B cells expressing IgG1, IgG2, IgG3, IgG4, and IgA could be separated. Such partitioning may be useful in the context of evaluating booster vaccination as repeated exposure to the antigen is associated with an increase in the use of IgG2 at the expense of IgG1 and IgG3.

[0279] Immunophenotyping of vaccine-antigen specific B cells is shown in Figure 12. Stepwise gating of CD3+ T cells and CD19+ B cells (left panel), followed by dual discrimination of AM15-specific B cells is shown in Figure 12A. Subsetting of total B cells to distinguish naive (IgM+CD27-) from memory B (mem) cells (all others; left panel), followed by separation of IgM / IgD+ non-switched Bmem from IgM- / IgD-switched Bmem within switched Bmem, IgG1, IgG2, IgG3, IgG4, and IgA-expressing B cells can be distinguished as shown in Figure 12B. Subsetting of AM15-specific B cells by a similar approach as B is shown in Figure 12C. Influenza booster vaccination resulted in an increase in the number of AM15-specific Bmem cells, and through detailed immunophenotyping, these were evaluated to be specifically involved in IgG1+ Bmem as shown in Figure 12D. Statistics, Mann-Whitney U test; * , p<0.05; ** , p<0.01.

[0280] Example 5: Monitoring of immune responses against viral infection To measure the immune response against the coronavirus, recombinant nucleocapsid and spike protein antigens were generated from SARS-CoV and SARS-CoV2. All constructs contained a C-terminal AviTag (GLNDIFEAQKIEWHE) BirA target sequence and a 6-His tag. The constructs contained an Ig leader sequence (MVLSLLYLLTALPGILS) or a Fel d 1 leader sequence (MRGALLVLALLVTQALG) for secretion. Wild-type nucleocapsid proteins from SARS-CoV and SARS-CoV2, and a mutant from SARS-CoV2 (pos 256-261 KKPRQK→GGPRQG) to improve protein stability (Array Nos. 115 and 116) were generated (Table 1). The full extracellular region of the spike protein, as well as the S1 and S1B domains, were generated. The full-length spike protein contained mutations to prevent cleavage between S1 and S2 (pos 682-685 RRAR→SGA G ) (Array Nos. 117 and 118) (Walls et al. (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein, Cell 181, 281-292), and contained mutations to improve the stability of the S2 domain (986-987 KV→PP) (Array Nos. 117 and 118) (Table 1).

[0281] Detection of recombinant nucleocapsid and S1B proteins from SARS-CoV2 is shown in Figure 13. As shown in Figure 13A, Western blot using anti-His detection antibody for nucleocapsid (left panel) and S1B (right panel). After concentrating the 6His-tag containing proteins on a cobalt loaded retention column, the culture supernatant of sf21 cells was loaded. The calculated molecular weights of the reduced proteins are approximately 48 kDa (N) and approximately 30 kDa (S1B). Abbreviations: NR, non-reduced; R, reduced. Figure 13B shows serum IgG specific for the antigen, determined by ELISA using the antigen for capture. Thirty-six historical samples of healthy adults previously vaccinated with influenza vaccine (sampled in 2019 and the first quarter of 2020), and serum samples from 4 - 20 individuals after or following recovery (convalescence) from COVID-19 were performed. Statistics, Mann-Whitney U test.

[0282] Detection of B cells with surface Ig specific for the SARS-CoV2 nucleocapsid protein in patients after recovery from COVID-19 is shown in Figure 14. Specifically, Figure 14A is a 2D plot showing B cells from non-infected controls stained with BUV395-binding and BUV737-binding nucleocapsid tetramers (left plot) and BV480-binding and BV650-binding S1B tetramers. Figure 14B shows a 2D plot of B cells from COVID-19 convalescent patients stained with the same NCP and S1B tetramers, showing distinct populations for both that were mutually exclusive (right plot) (left plot and middle plot).

[0283] Figure 16 shows the immunophenotyping of COVID19-specific B cells. Stepwise gating of CD3+ T cells and CD19+ B cells (left panel), followed by dual discrimination of COVID-19 nucleocapsid (NCP)-specific B cells and COVID-19 S1B-specific B cells within the same flow cytometry staining are shown in Figures 16B and 16C. Subsetting of total B cells within the same staining can be identified to distinguish non-switched B cells (IgD+) from Ig class-switched memory B (mem) cells (IgD-) within switched Bmem, IgG1, IgG2, IgG3, IgG4 and IgA-expressing B cells (Figure 16A). Subsetting of S1B-specific B cells within the same flow cytometry staining by a similar approach as in A is shown in Figures 16B, C.

[0284] The antibody response to SARS-CoV2 infection can be measured 4-7 days after symptom onset and peaks around 20 days. This is observed in our patient cohort using both S1B and NCP proteins as targets (Figure 17A). The subsequent decline in levels may make it more difficult to detect previous infections. In contrast, antigen-specific memory B cells in the blood do not show a decrease after 20 days post-infection (Figure 17B). In fact, total S1B-specific and total NCP-specific Bmem tend to increase over time, up to at least about 140 days. This increase was more prominent in IgG+ Bmem for both S1B-specific Bmem and NCP-specific Bmem, while IgM+ Bmem remained at similar levels (Figures 17C, D).

[0285] Example 6: Multiplex allergen staining for detecting allergen sensitization Multiplex allergen staining was performed using the recombinant allergen tetramer ("Cytobas") to detect allergen sensitization on blood basophils. Using a 7-color flow panel, blood basophils (CD123+IgE+) and plasmacytoid dendritic cells (pDC; CD123+IgE-) were identified using monoclonal antibodies CD123 and anti-IgE, and the expression levels of IgE specific for Ara h 2, Fel d 1, Lol p 1, Lol p 5, and Api m 1 were determined for these cells (Figure 15A).

[0286] The expression levels of IgE and specific IgE against 5 allergens on basophils and pDCs from 6 individuals previously diagnosed with one form of allergy were measured (Figure 15B). Patients 1 and 2 were diagnosed with rye grass pollen allergy, patients 3 and 4 with wasp venom allergy, patient 5 with cat dander allergy, and patient 6 with peanut allergy. Sensitization can be quantified using the ratio of the median fluorescence intensity (MFI) of basophils (bold font) to that of pDCs (normal font). This would obviate the need for a second staining with streptavidin to be used as a negative control.

[0287] Multiparameter basophil staining (CytoBas) shows the potential for differential diagnosis of allergen sensitization using molecular components (component-resolved diagnosis; CRD) with flow cytometry. This approach has advantages over current basophil activation tests (BAT) as it does not require in vitro stimulation. Furthermore, washed cells from fresh whole blood or fresh or frozen PBMCs can be used. Finally, CytoBas can multiplex allergen components in a single flow cytometry tube without the need for serial dilutions.

[0288] CRD is currently performed using microchip technology, which has proven difficult to implement widely in routine diagnosis. Flow cytometry is a standard test in many pathology laboratories, facilitating the easy implementation of CytoBas.

Claims

**Claim 1** An in vitro method for determining allergic reactivity in a subject, the method comprising: providing a sample obtained from the subject; contacting the sample with one or more recombinant or synthetic allergens conjugated to a detectable label under conditions that permit binding of the one or more allergens to IgE molecules present on the surface of immune cells in the sample; determining the binding of the one or more allergens to IgE molecules present on the surface of immune cells in the sample by detecting the label, wherein detection of the label indicates that the subject is allergic reactive, wherein the sample is a whole blood sample, a washed whole blood sample, a cerebrospinal fluid sample, a cord blood sample or a peripheral blood mononuclear cell (PBMC) blood sample. A method. **Claim 2** The method according to claim 1, wherein the immune cells are basophils, eosinophils, mast cells or B cells. **Claim 3** An in vitro method for determining the efficacy of allergy immunotherapy in a subject, the method comprising: providing a first sample obtained from the subject before receiving allergy immunotherapy; providing a second sample obtained from the subject after receiving allergy immunotherapy; contacting the first and second samples from the subject with one or more recombinant or synthetic allergens conjugated to a detectable label under conditions that permit binding of the one or more allergens to Ig molecules on the surface of B cells present in the samples; determining the binding of the one or more allergens to Ig molecules on the surface of B cells present in the first and second samples by detecting the label; wherein in the method, an increase in the total number or proportion of IgG-expressing B cells in the second sample relative to the first sample indicates the efficacy of the allergy immunotherapy in the subject; or an increase in the ratio of IgG:IgE-expressing B cells in the second sample relative to the first sample indicates the efficacy of the allergy immunotherapy in the subject; or an increase in the total number or proportion of IgG2 and / or IgG4-expressing B cells in the second sample relative to the first sample indicates the efficacy of the allergy immunotherapy in the subject; wherein in the method, The sample is a whole blood sample, a washed whole blood sample, a leptomeningeal blood sample, a cord blood sample or a peripheral blood mononuclear cell (PBMC) blood sample, Method.

4. The method further comprising contacting the first and second blood samples with a molecule that enables the identification of B cells expressing IgM, IgA, IgG, IgD and IgE, the method according to claim 3.

5. The method according to any one of claims 1 to 4, wherein the one or more allergens are animal allergens or plant allergens.

6. The method according to any one of claims 1 to 5, wherein the one or more allergens are selected from insect allergens, myriapod allergens, arachnid allergens or crustacean allergens.

7. The method according to any one of claims 1 to 6, wherein the one or more allergens are enzymes and / or the one or more allergens are enzymes modified to reduce their activity.

8. The method according to any one of claims 1 to 7, wherein the one or more allergens are linked to a tag that facilitates binding to the detectable label.

9. The method according to any one of claims 1 to 8, wherein the sample is contacted with two or more allergens each linked to a different detectable label.

10. A recombinant polypeptide for use in the method according to any one of claims 1 to 9, comprising an amino acid sequence encoding a recombinant allergen and a tag, wherein the recombinant allergen is linked to the tag, and the tag facilitates linkage to a detectable label.

11. An in vitro method for detecting antigen-specific B cells in a subject, the method comprising: providing a sample obtained from the subject; contacting the sample with one or more recombinant or synthetic antigens linked to a detectable label under conditions that allow binding of the one or more antigens to Ig molecules on the surface of B cells present in the sample; determining the binding of the one or more antigens to Ig molecules on the surface of B cells in the sample by detecting the label, wherein the detection of the label indicates that the subject has antigen-specific B cells; A method wherein the sample is a whole blood sample, a washed whole blood sample, a leptomeningeal blood sample, a cord blood sample or a peripheral blood mononuclear cell (PBMC) blood sample.

12. The method according to claim 11, wherein the sample is contacted with two or more allergens each conjugated to a different detectable label.

13. The method according to claim 11 or 12, wherein the one or more antigens are conjugated to a tag that facilitates binding to the detectable label.

14. The method according to any one of claims 11 to 13, wherein the one or more antigens are vaccine-derived, related to self-antigens, or derived from a pathogen or infectious agent.

15. The one or more antigens are related to a respiratory condition or disease or are derived from a virus that causes a respiratory condition or disease; and / or the method according to claim 14, wherein the one or more antigens are derived from a virus selected from measles, polio, coronavirus, influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), dengue virus, rhinovirus, herpes simplex virus, enterovirus, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

16. The method according to claim 15, wherein the one or more antigens are a nucleocapsid protein or a spike protein, or a domain within a nucleocapsid protein or a spike protein; and / or the virus is SARS-CoV-2.

17. An isolated nucleic acid or vector comprising a nucleotide sequence encoding the recombinant polypeptide according to claim 10.

18. An isolated host cell comprising the nucleic acid or vector according to claim 17.

19. The method according to any one of claims 11 to 16, wherein the Ig molecule is IgM, IgD, IgA, IgG or IgE.

20. The method according to claim 8 or 13, wherein the detectable label is linked via any one of the group consisting of streptavidin and its derivatives, avidin and its derivatives, biotin, immunoglobulins, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments and their derivatives, the leucine zipper domain of AP-1, jun, fos, hexa his, hexa hat glutathione S-transferase, glutathione affinity, calmodulin-binding peptide, Strep-tag, cellulose-binding domain, maltose-binding protein, S-peptide-tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2 tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope, lectin, Con A, WGA, tenascin, protein A and protein G.

21. The method according to any one of claims 1 to 9, 11 to 16, 19 and 20, further comprising contacting the sample with a molecule that enables the identification of two or more immune cell types, and / or further comprising contacting the sample with a molecule that distinguishes B cells or basophils from other cells.

22. A kit for use in the method according to any one of claims 1 to 9, 11 to 16 and 19 to 21, comprising one or more recombinant allergens or antigens, a detectable label, and instructions for use, buffer, and / or a control sample.

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