WO2017149206A1

WO2017149206A1 - Method and a kit for detecting a combination of markers from liver-derived plasma extracellular vesicles (pev)

Info

Publication number: WO2017149206A1
Application number: PCT/FI2017/050145
Authority: WO
Inventors: Andreas Baur; Erkko YLÖSMÄKI
Original assignee: Evexys Biotech Oy
Priority date: 2016-03-04
Filing date: 2017-03-03
Publication date: 2017-09-08

Abstract

The present invention is directed to a method and a kit for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to Alzheimer's disease, schizophrenia, depression, melanoma, breast cancer, cancer survival,drug-induced immune suppression, allergy or cancer relapse. Among the markers relating to Alzheimer's disease are MMP-2, MMP-3, MMP-13,TIMP-1,TIMP-2, FGF-6,ICAM-1, IGFBP-6, MIP-β, and VEGF.

Description

Method and a kit for detecting a combination of markers from fiver-derived piasma extraceHluiar vesicles (pEV)

FIELD OF THE INVENTION

The present invention is based on the discovery that the liver not only reacts to the development of diseases, but induces acute and chronic diseases through a lasting release of large amounts of plasma extracellular vesicles (hereafter referred to as pEV). These liver pEV contain an array of around 60 proteins and peptides effectors (hereafter referred to as CCF: cytokine, chemokine, soluble factor; and MP: marker proteins), in different combinations and quantities. The composition of the CCF from this limited pool of markers differs markedly for different diseases and clinical stages of these diseases, including diseases that are diagnosed predominantly by clinical criteria, like Alzheimer's disease, depression (i.e. major depressive disorder) and chronic fatigue syndrome. Furthermore, the MP/CCF composition reflects the effects of treatments on a given disease and is detected far earlier than any other clinical signature (e.g. medical imaging) or plasma marker (e.g. tumor marker). Thus pEV-derived diagnosis is far superior to current blood-derived diagnostic assays.

Owing to the presence of a whole array of liver-derived MP and CCF, the present discovery allows the early, precise and semi-quantitative detection of acute and chronic diseases, different clinical stages and diseases predisposing conditions, for which only in advanced disease stages clinical criteria and conventional biomarkers become positive so far. Furthermore, these biomarker combinations allow the precise monitoring of a given disease and condition, which may reflect therapeutic success or failure, and may allow a biomarker-based prognosis.

BACKGROUND OF THE INVENTION

Extracellular vesicles (EV) are secreted by different types of regular cells and tumor ceils (lymphocytes, platelets, dendritic cells, neuron ceils, mast ceils, intestinal cell, macrophages, among others) with an assumed wide range of functions, including intercellular exchange of cellular components for example of enzymes, signaling proteins, cytokines, chemokine, soluble factors and ligands, micro-RNA, mRNA, non- coding RNA, endogenous retro-elements and DNA, With their cargo, EV were shown to manipulate the function of target cells, for example cause the secretion of chemokine and cytokines and induce angiogenesis and apoptosis. It is also believed that they transmit prion and mycobacterial disease. EV are found in abundance in all body fluids, in particular plasma, and ceil cultures supernatant ranging from approximately 40 nm to several hundred nanometer in diameter. EV are composed by a double lipid layer containing membrane proteins derived from the plasma membrane or cellular endosomes. This membrane surrounds their cargo of factors.

In summary, EV are viewed as conveyors of information that is represented by the multiplicity of factors contained in the membrane of the vesicles or in their lumen.

The wide range of factors contained in EV makes them a highly promising source of biomarkers. This fact is increasingly recognized and people have detected factors (platlet-derived immunoglobulins and complement factors) that are seemingly characteristic for a specific disease, like systemic sclerosis or rheumatoid arthritis (0stergaard et al., 2013). In a recent publication a single tumor exosome (derived from tumor tissue) -derived factor was identified (Glypican-1 ) that seems sufficient to predict the early development of pancreas and breast cancer (Melo et al., 2015). WO2014108480 discloses a method for in vitro detection and monitoring of a disease in a sample comprising a step of measuring enzymatic activity of at least one disease- associated protease in extracellular vesicles in the sample. In the method, protease- containing extracellular vesicles are enriched and purified within the sample prior to measuring enzymatic activity. The disease-associated protease is selected from the group consisting of matrix meta!loproteinases such as MMP2, MMP5, MMP9, ADAM10, ADAM 17, ADAM9 and ADAMS.

We discovered that by far the largest fraction of peripheral pEV is secreted by the liver. These liver vesicles originate from two subcellular sources. One source is the endosomal compartment of a cell, including multivesicular bodies (MVB), from which pEV are released that have characteristics of Exosomes (Tsg101 -dependent release and surface markers including CD83, CD81 , CD9, DC-SIGN, CD25). The function of these vesicles is likely to maintain homeostasis and survival of peripheral ceils (e.g. blood cells) not growing in cohesive tissue compartments, for example, by inducing anti- apoptosis.

The second type of liver vesicles, described here as relevant for the diagnosis of diseases, is released after stimulation of the innate sensing system in the liver: 1 ) physiologically, e.g. by activation through endogenous retro-elements or retroviruses, for example shed by tumor ceils, 2) upon microbial infection (bacteria, viruses, fungi), and 3) non-physiologically e.g. by environmental stimuli, e.g. aluminum. Hence, these pEV are part of an innate defense mechanism that is activated upon danger signals (e.g. microbes) that enter the blood stream. They are released in response to even very low amounts of danger signals and their function is to reach the periphery as soon as possible to efficiently neutralize the developing threat to the organism.

To exert their function pEV contain an array of defense factors including ADAM- and Matrixmetallo-Proteases, Cytokines, Chemokines, other soluble factors (e.g. soluble TNF receptors), acute phase proteins (e.g. haptoglobin) and micro RNAs. The composition of all of these factors depends on the specific stimulation of one or more of the innate sensors (as for example STING, AV5, RLR-fami!y) and the magnitude of the response correlates inversely with the disposal of the danger signals by nucleases and associated factors (e.g. TREX, SAMHD1 ). As the innate sensors are large in number, their response pattern is theoretically unlimited. Despite this enormous theoretical variability, there are patterns that are characteristic for a given disease condition or clinical stage, depending on the type and magnitude of a typical danger signal (e.g. viral infection). On the other hand the acute or chronic release of a certain type of pEV, which may be released non-physiologically, may induce the very clinical condition they diagnose. This is particularly true for chronic diseases like Alzheimer and autoimmune diseases. In summary we propose to use this biomarker pattern (including those factors that could be present but are absent) derived from a specific subset of pEV for the diagnosis and monitoring of acute and chronic diseases. These factors comprise a defined set of around 120 protein and peptide effector molecules (CCF) of the innate immune system, of which around 80 repeatedly appear in pEV of diseased patients in different combinations. Specific combinations of these factors are highly specific for a given disease as well as different stages of this disease. What sets our invention apart from other findings is the discovery that a defined, so far not described set of factors from a defined pool of markers is sufficient to characterize and discriminate many diseases and disease stages.

SUMMARY OF THE INVENTION

We have discovered that the liver reacts extremely sensitive to an array of danger signals by secreting huge amounts of plasma extracellular vesicles (pEV) containing around 120 protein (e.g. proteases) and peptide (e.g. cytokines) effectors to neutralize/combat the danger that has been sensed. We found that the specific content/composition of these effectors is easily assessed in plasma and reflects the nature of the danger signal as well as the disease it may cause. Furthermore we found, that chronic diseases are induced by a lasting release of specific subsets of pEV that may be non-physiological, containing a specific subset of CCF effectors. The characteristic content combination of factors in these pEV subsets allow to diagnose and monitor these diseases and their clinical stage. Thus, the assessment of pEV marker combinations allows the early and precise diagnosis of a whole array of acute and chronic diseases and disease conditions.

Accordingly, if is an aim of the present invention to provide a method for detecting a combination of markers from plasma extracellular vesicles. In one aspect of the invention, said markers are related to Alzheimer's disease and the method comprises the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject: ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles:

- at least one of the matrix metalloproteinases selected from the group consisting of: MMP-2, MMP-3, and MMP-13; - at least one of the tissue inhibitors of metalloproteinases selected from the group consisting of: TIMP-1 , and TIMP-2; and

- at least one of the markers selected from the group consisting of: FGF-8, !CA -1 , IGFBP-8, ΜΙΡ-3β, and VEGF.

It is another aim of the invention to provide a kit for use in the in vitro detection or diagnosis of markers related to Alzheimer's disease in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of:

- at least one of the matrix metalloproteinases selected from the group consisting of: MMP-2, MMP-3, and MMP-13;

- at least one of the tissue inhibitors of metalloproteinases selected from the group consisting of: TIMP-1 , and TIMP-2; and

- at least one of the markers selected from the group consisting of: FGF-6, !CAM-1 , IGFBP-8, ΜΙΡ-3β, and VEGF.

!n another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to schizophrenia, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the fo!io ving markers in the purified plasma extracellular vesicles: BDNF, CCL23, CNTF, CTAK, IL-1 R4, NT-3 and PARK8.

In another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to depression, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the following markers in the purified plasma extracellular vesicles: IGFBP3, IL-1 R4, MiP-1 a, Μ!Ρ-1 β, MMP9,

Oncostatin M, PARC, and TIMP-2.

!n another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to HIV, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the following markers in the purified plasma extracellular vesicles: Angiogenin, GMCSF, GRO, ICAM-1 , IL-6R, NAP- 2, RANTES, and TIMP-1. In another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to melanoma, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the fo!io ving markers in the purified plasma extracellular vesicles: FGF-8, ΜΙΡ-1 α, ΜΙΡ-1 β, EGF, Fas, !L-12p40, MCP-1 , and MiF.

In another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to breast cancer, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the following markers in the purified plasma extracelluiar vesicles: Angiogenin, Arcp30, ΜΙΡ-1 β, GCSF, NAP-2,

RANTES, IL-I Ra, IL-4, and IL-8.

In another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to cancer survival, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the following markers in the purified plasma extracelluiar vesicles: IGFBP-3, ΜΙΡ-3β, IFN-γ, IL-12p70, Arcp30, GCSF, NAP-2, RANTES, and VEGF. In another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to drug- induced immune suppression, the method comprising the steps of: i) purifying plasma extracelluiar vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the fo!io ving markers in the purified plasma extracellular vesicles: BDNF, GMCSF, MCSF, RANTES, FGF-9, IL-6R, TIMP-1 , PARC, ICAM-1 , and IL-6R.

In another aspect, the invention provides a method for defecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to allergies, as for example Type I and Type IV allergies (i.e. Type I and Type IV hypersensitivities), the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles:

- at ieast one or two of the following chemokines: AcrpSQ, NAP-2, RANTES

In another aspect, the invention provides a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to the monitoring the risk for cancer relapse, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles: - at Ieast one the following: Hck, CD83, PD-L1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a CCF analysis demonstrating the presence of CCF in pEV from patients with different diseases. (A-C) Protein arrays demonstrating the content of cytokines, chemokines and soluble factors (CCF) in pEV from healthy controls and patients with chronic but non-viremic HIV infection (with low and high CD4 count), Alzheimers disease and Depression.

FIG. 2 shows a CCF analysis after pEV purification from plasma pools (20 ml; 4 patients each) demonstrating the presence of CCF in pEV from patients with Melanoma in different clinical stages (IA - IV), and two individual patients after treatment (15 years after DC vaccination of two stage IV melanomas). Low risk: melanoma patients with a low risk for tumor relapse (around 5% in 5 years); high risk: melanoma patients with a high risk for relapse (30 - 80% in 5 years); tumor: patients having a tumor burden (stage III - IV; before operation). Long term survivor: patients after successful treatment with a cancer vaccine 15 years after they entered stage IV (around 90-95% relapse in 5 years).

FIG. 3 shows a CCF analysis demonstrating the presence of CCF in pEV from patients with breast cancer before and after operation (OP).

FIG. 4 shows a CCF analysis demonstrating the presence of CCF in pEV from patients with Type IV allergies.

(A-C) Bar diagram demonstrating the relative presence of indicated cytokines, chemokines and soluble factors (CCF) in pEV from individuals with Type-IV allergies. CCF values of both individuals were calculated in relation to healthy controls (set to 1 ).

DETAILED DESCRIPTION OF THE INVENTION

The relevant biomarkers are contained in the plasma membrane and lumen of pEV. They are bioactive in target cells and, in their composition, represent a specific and rapidly induced response of the innate immune system (in this instance the liver) to a great variety of danger signal that entered the bloodstream. Hence, assessment of these biomarkers allow an early and precise analysis of events that eventually will lead or have led to an acute disease. Examples for the development of an acute disease would be an infection (by viruses, bacteria, fungi) and the growth of a malignant or prema!ignant tumor.

The chronic and deregulated release of bioacfive pEV from the liver/innate immune system will lead to secondary effects in distant cell systems and organs that constantly ingest pEV, like for example the brain or endothelial cells. This discovery implies that the pEV biomarker not only reflect the onset of acute diseases but particularly chronic diseases that develop over years. Among those are neurodegenerative diseases (e.g. Alzheimer, Depression, Chronic Fatigue Syndrome, Schizophrenia), but also chronic infections (e.g. hepatitis viruses, HIV and Borrelia), and chronic autoimmune diseases (e.g. lupus erythematosus, dermatomyositis. Psoriasis, chronic polyarthritis, Sjogren^'s Syndrome, allergies etc.).

The assessment of pEV biomarkers is superior to the assessment of any other biomarkers because (1 ) they represent a specific reaction of the immune system on the development of a disease, (2) they consist of a whole array of markers with known effector functions and (3) the combinatorial signature of these markers in combination with their semi-quantitative measurement allows a highly discriminatory analysis of a given clinical situation/disease at a given time point and over a given time period.

The new analysis described here represents a significant advancement for the diagnosis and monitoring of acute and chronic diseases. The most important advancement lies in the fact that each disease and disease condition is characterized by the presence or absence of a whole combination of markers comprising approximately 50 to 100 individual factors/proteins or peptides.

Accordingly, the present invention is directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to

Alzheimer's disease, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles:

- at least one of the markers selected from the group consisting of: FGF-8, !CAM-1 , !GFBP-8, Μ!Ρ-3β, and VEGF.

Preferably, the presence or absence of at least the following markers is determined in step ii): MMP-2, MMP-3, MMP-13, TIMP-1 , TIMP-2, FGF-8, ICAM-1 , IGFBP-6, Μ!Ρ-3β, and VEGF. However, a person skilled in the art would understand that any selection of at least three or four of the listed markers would provide relevant results when used as such or as core biomarkers in the assay of the present invention.

Further, we have found that fragmented hyaluronic acid (fHA) such as low molecular weight hyaluronic acid (LMW-HA) is present in abundance on plasma extracellular vesicles from Alzheimer patients and other chronic diseases. Hyaluronic acid or hyaluronan (HA) is processed by hyaluronidases into a continuum of different sized fragmented HA (fHA) polymers, including low molecular weight HA (LMW-HA;

<120kDa). Whereas high molecular weight HA polymers (HMW-HA; > 400 kDa) predominate in most healthy tissues and have anti-inflammatory effects, fHA polymers predominate in sites of active inflammation. Accordingly, fHA is one of the preferred markers of the invention.

In an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): Angiogenin. BDNF, fHA, !FNy, MCSF, NAP-2, RANTES, ADAIV117, ADAM 10, IV1P9, y-secretase, Fas, FGF-9, GCSF, GRO, IGFBP-3, IL-1 R4, ΜΙΡ-1 β, TRAIL-R4, PARC, NT3, Haptoglobin, and ανβ3. In another embodiment, the presence or absence of at least one of the following further markers is also determined in step ii):

Eotaxin-1/ -2/ -3, LIGHT, SLF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTAK, l-TAC, TECK, Dfk, PDGF, PLGF, GDNF, EGF, EGFR, bFGF, FGF-7, GMCSF, i-309, IGFBP-1 , !GFBP-4, IGF-1 , !GF-1 sR, IGF-1 R, IL-1 R1 , IL-1 Ra, IL-2Ra, IL-1 a, fL-1 β, IL-3, !L~4, !L-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-13, IL-15, IL-16, IL-17, IL-6R, IL-12p40, IL-12p70, PDGF- BB, P!GF, TNF, TNF-β, sTNFR-i, sTNFR-ll, TRAIL-R3, GRO-a, Arcp30, ICAM-3, MCP- 2, CP-3, MCP-4, DC, !G, MIF, MP-15, MCP-1 , MSPa, IFN-a, !FN-β, BACE, P8, NT-4, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF-D, I- TAG, VEGF, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-β TGF-P3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, 08M, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

The present method may also comprise further steps of comparing the level of expression of the one or more listed biomarkers in the purified sample of plasma extracellular vesicles from the subject to a control value, such as a sample from a healthy control, and defecting the existence of Alzheimer's disease in the subject, wherein differences in expression of the one or more biomarkers in the sample relative to the control value for expression in a sample from a control indicate the existence of Alzheimer's disease. For instance, simultaneous presence of markers MMP-2, MMP-3, and MMP-13 and absence of markers TiMP-1 and TIMP-2 in plasma extracellular vesicles relate to Alzheimer's disease and indicate the existence of Alzheimer's disease in a subject. The present method thus may further comprise a step of monitoring the existence or status of Alzheimer's disease in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles.

Accordingly, the present method can also be used for monitoring the efficacy of any treatment of Alzheimer's disease.

The present method may also comprise a further step of treating a subject diagnosed by the previous steps of the method to suffer from Alzheimer's disease by administering to said subject a drug treating Alzheimer's disease. The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to Alzheimer's disease in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of:

- at least one of the matrix metailoproteinases selected from the group consisting of: MMP-2, MMP-3, and MMP-13;

- at least one of the tissue inhibitors of metailoproteinases selected from the group consisting of: TI P-1 , and T!MP-2: and

- at least one of the markers selected from the group consisting of: FGF-6, !CAM-1 , !GFBP-8, ΜΙΡ-3β, and VEGF. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample.

Schizophrenia The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to schizophrenia, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: BDNF, CCL23, CNTF, CTAK, IL-1 R4, NT-3 and PARK6.

Preferably, the presence or absence of at least the following markers is determined in step ii): BDNF, CNTF, CTAK, and IL-1 R4. More preferably, the presence or absence of at least the following markers is determined in step ii): BDNF, CCL23, CNTF, CTAK, IL- 1 R4, NT~3 and PARK8. However, a person skilled in the art would understand that any selection of at least four of the iisted markers would provide relevant results related to schizophrenia, when the markers are used as such or as core biomarkers in the assay of the present invention. In an embodiment, the presence or absence of at least one of the following further markers is determined in step ii):

Angiogenin, Eotaxin-1/ -21 -3, LIGHT, SLF, BMP-4, BMP-6, BLC, l-TAC, TECK, Dtk, PDGF, PLGF, GDNF, EOF, EGFR, bFGF, FGF-6, FGF-7, FGF-9, GMCSF, GCSF, fHA, CSF, 1-309, IGFBP-1 , IGFBP-3, IGFBP-4, IGFBP-8, IGF-1 , IGF-1 sR, IGF-1 R, IL-1 R1 , IL-1 Ra, IL-2Ra, IL-1 a, ΙΙ_-1 β, IL-3, IL- , IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-13, IL-15, IL- 18, IL-17, IL-6R, !L-12p40, IL-12p70, NAP-2, PDGF-BB, PIGF, TNF, TNF-β, sTNFR-l, sTNFR-ll, TRAIL-R3, TRAIL-R4, Fas, RANTES, GRO, GRO- , ArcpSO, ICAM-1 , SCA - 3, MCP-2, MCP-3, MCP-4, MDC, MIG, MIF, MP-15, ΜΙΡ-1 β, ΜΙΡ-3β, MCP-1 ,

MSPa, IFN-a, IFN-β, IFN-γ, TIMP-1 , TIMP-2, ADAM 10, ADAM 17, BACE, γ-secretase, MMP2, MMP3, MMP8, MMP9, MMP13, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, VEGF, Haptoglobin, ανβ3, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ , TGF-p3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axi, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands. The present method may also comprise further steps of comparing the level of expression of the one or more listed biomarkers in the purified sample of plasma extracellular vesicles from the subject to a control value, such as a sample from a healthy control, and detecting the existence of schizophrenia in the subject, wherein differences in expression of the one or more biomarkers in the sample relative to the control value for expression in a sample from a control indicate the existence of schizophrenia. The present method thus may further comprise a step of monitoring the existence or status of schizophrenia in a subject based on the presence or absence of one or more of the Iisted biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of schizophrenia.

The present method may also comprise a further step of treating a subject diagnosed by the previous steps of the method to suffer from schizophrenia by administering to said subject a drug treating schizophrenia.

The present invention is aiso directed to a kit for use in the in vitro detection or diagnosis of markers related to schizophrenia in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: BDNF, CCL23, CNTF, CTAK, IL-1 R4, NT-3 and PARK6. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample.

Depression

The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to depression (i.e. major depressive disorder), the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: SGFBP3, IL-1 R4, ΜΙΡ-1 α, ΜίΡ~1 β, MMP9,

Oncostatin M, PARC, and TiMP-2.

Preferably, the presence or absence of at least the following markers is determined in step ii): MiP-1 a, Oncostatin , PARC, and TiMP-2. More preferably, the presence or absence of at least the following markers is determined in step ii): IGFBP3, !L-1 R4, MIP- 1 a, ΜΙΡ-1 β, MMP9, Oncostatin M, PARC, and TIMP-2. However, a person skilled in the art would understand that any selection of at least four of the listed markers would provide relevant results related to depression, when the markers are used as such or as core biomarkers in the assay of the present invention. The present method thus may further comprise a step of monitoring the existence or status of depression in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of depression,

In an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): ADAM17, ADA 10, RANTES, TNF, !CAM-1 , FGF-9, fHA, NTS, M-CSF, NAP-2, EGFR, Arcp30, IGFBP-6, MIF, IL-12p40, and BDNF, In another embodiment, the presence or absence of at least one of the following further markers is also determined in step ii):

Angiogenin, Eotaxin-1/ -21 -3, LIGHT, SLF, BMP-4, BMP-6, BLC, l-TAC, TECK, Dtk, PDGF, PLGF, GDNF, EGF, bFGF, FGF-6, FGF-7, GMCSF, GCSF, 1-309, IGFBP-1 , IGFBP-4, IGF-1 , IGF-1 sR, IGF-1 R, IL-1 R1 , IL-1 Ra, IL-2Ra, IL-1 a, ΙΙ_-1 β, IL-3, !L-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-13, IL-15, IL-16, IL-17, IL-8R, IL-12p70, PDGF-BB, PIGF, TNF-β, sTNFR-l, sTNFR-ll, TRAIL-R3, TRAIL-R4, Fas, GRO, GRO-α, ICAM-3, MCP-2, MCP-3, CP-4, MDC, MIG, MP-15, ΜΙΡ-3β, MCP-1 , MSPa, IFN-a, !FN-β, IFN-γ, TIMP- 1 , BACE, γ-secretase, MP2, P3, P8, M P13, NT-4, TARC, uPAR,

Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, VEGF, Haptoglobin, ανβ3, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ , TGF^3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck, and Wnt-Ligands.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to depression in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: IGFBP3, IL-1 R4, ΜΙΡ-1 α, ΜΙΡ-1 β, MMP9, Oncostatin M, PARC, and TIMP-2. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample,

HHV

The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to HIV, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: Angiogenin, GMCSF, GRO, SCAM-1 , IL-6R, NAP-

2, RANTES, and TIMP-1.

Preferably, the presence or absence of at least the following markers is determined in step ii): GMCSF, GRO, 1L-6R, and ΤΊΜΡ-1 . More preferably, the presence or absence of at least the following markers is determined in step ii): Angiogenin, GMCSF, GRO, ICAM-1 , IL-6R, NAP-2, RANTES, and TIMP-1. However, a person skilled in the art would understand that any selection of at least four of the listed markers would provide relevant results related to HIV, when the markers are used as such or as core biomarkers in the assay of the present invention. The present method thus may further comprise a step of monitoring the existence or status of HIV infection in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of HIV infection.

!n an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): BDNF, FGF-9, ADAM10, ADAM17, MMP9, fHA, Haptoglobin, νβ3, TNF, sTNFR-l, I-309, IGFBP-1 , IL-2Ra, GCSF, and RANTES. In another embodiment, the presence or absence of at least one of the following further markers is determined in step ii):

Eotaxin-1/ -71 -3, LIGHT, SLF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, ! AC, TECK, Dtk, PDGF, PLGF, GDNF, EGF, EGFR, bFGF, FGF-8, FGF-7, MCSF, !GFBP-3, !GFBP-4, !GFBP-8, !GF-1 , !GF-1 sR, IGF-1 R, IL-1 R1 , IL-1 R4, IL-1 Ra, IL-1 a, IL-1 β, IL-3, IL-4, !L-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-13, IL-15, IL-16, IL-17, !L-12p40, IL-12p70, PDGF-BB, PIGF, TNF-β, sTNFR-li. TRAIL-R3, TRAIL-R4, Fas, GRO-α, Arcp30, ICAM- 3, CP-2, MCP-3, CP-4, DC, MIG, MIF, MP-15, ΜΙΡ-1 β, ΜΙΡ-3β, MCP-1 ,

MSPa, IFN-a, IFN-β, !FN-γ, TIMP-2, BACE, γ-secretase, MMP2, MMP3, MMP8, MMP13, NT-3, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, VEGF, SPPL3, Leptin, SDF-Ι α, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ , TGF^3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Ax!, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck, and Wnt-Ligands.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to HIV in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: Angiogenin, GMCSF, GRO, ICAM-1 , IL-6R, NAP-2, RANTES, and TIMP-1 . Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins.

Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample.

Melanoma

The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to melanoma, the method comprising the steps of:

I) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the fo!io ving markers in the purified plasma extracellular vesicles: FGF-8, ΜΙΡ-1 α, ΜΙΡ-1 β, EGF, Fas, !L-12p40, MCP-1 , and MiF.

Preferably, the presence or absence of at Ieast the following markers is determined in step ii): EGF, Fas, iL-12p40, MCP-1 , and MIF. More preferably, the presence or absence of at Ieast the following markers is determined in step ii): FGF-6, MIP-1 , MIP- 1 β, EGF, Fas, IL-12p40, MCP-1 , and MIF. However, a person skilled in the art would understand that any selection of at ieast four of the listed markers would provide relevant results related to melanoma, when the markers are used as such or as core biomarkers in the assay of the present invention. The present method thus may further comprise a step of monitoring the existence or status of melanoma in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of melanoma. In an embodiment, the presence or absence of at Ieast one of the following further markers is also determined in step ii): ADAM 10, MMP8, MMP9, MCSF, NAP-2, TNF, fHA, Arcp30, EGFR, GRO, IL-6R, Oncostatin M, sTNFR-l, TiMP-1 , BDNF, FGF-9, MIP- 3a, WNT-3a, RANTES, GCSF, ICAM-1 , IGFBP-3, VEGF, Haptoglobin, ανβ3, and !FNy.

In another embodiment, the presence or absence of at ieast one of the following further markers is also determined in step ii):

Angiogenin, Eotaxin-1/ -21 -3, LIGHT, SLF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, l-TAC, TECK, Dtk, PDGF, PLGF, GDNF, bFGF, FGF-7, GMCSF, 1-309, IGFB- 1 , !GFBP-4, IGFBP-6, IGF-1 , !GF-1 sR, IGF-1 R, IL-1 R1 , IL-1 R4, IL-1 Ra, IL-2Ra, IL-1 a, ΙΙ_-1 β, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-13, !L-15, IL-16, IL-17, IL-12p70, PDGF-BB, PIGF, TNF-β, sTNFR-ll, TRAIL-R3, TRAIL-R4, GRO-a, ICAM-3, MCP-2, MCP-3, MCP-4, MDC, MSG, MP-18, ΜΙΡ-3β, MSPa, !FN-a, !FN-β, TIMP-2, ADAM17, BACE, γ-secretase, MMP2, MMP3, MMP13, NT-3, NT-4, PARC, TARC, uPAR,

Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, SPPL3, Leptin, SDF-l a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ , TGF-p3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to melanoma in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: FGF-6, ΜΙΡ-Ι α, ΜΙΡ-1 β, EGF, Fas, IL-12p40, MCP-1 , and MIF. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample.

Breast cancer

The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to breast cancer, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject: ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: Angiogenin, Arcp30, ΜΙΡ-1 β, GCSF, NAP-2, RANTES, IL-1 Ra, !L-4, and SL-8. Preferably, the presence or absence of at least the following markers is determined in step ii): Angiogenin, IL-1 R<x, IL-4, and IL-8. More preferably, the presence or absence of at least the following markers is determined in step ii): Angiogenin, ArcpSO, ΜΙΡ-1 β, GCSF, NAP-2, RANTES, IL~1 Ra, IL-4, and IL-8. However, a person skilled in the art would understand that any selection of at least four of the listed markers would provide relevant results related to breast cancer, when the markers are used as such or as core biomarkers in the assay of the present invention. The present method thus may further comprise a step of monitoring the existence or status of breast cancer in a subject based on the presence or absence of one or more of the listed biomarkers in plasma

extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of breast cancer.

In an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): fHA, IFN-γ, IGFBP-3, !L-12p70, ΜΙΡ~3β, and VEGF.

In another embodiment, the presence or absence of at least one of the following further markers is determined in step ii):

Eotaxin-1/ -21 -3, LIGHT, SLF, BDNF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, I- TAG, TECK, Dtk, PDGF, PLGF, GDNF, EGF, EGFR, bFGF, FGF-6, FGF-7, FGF-9,

GMCSF, CSF, 1-309, IGFB-1 , IGFBP-4, IGFBP-6, IGF-1 , IGF-1sR, IGF-1 R, IL-1 R1 , IL- 1 R4, IL-2Ra, IL-1 a, Ι β, IL-3, IL-5, IL-8, IL-7, IL-10, IL-1 1 , IL-13, IL-15, IL-16, IL-17, IL- 6R, IL-12p40, PDGF-BB, PIGF, TNF, TNF-β, sTNFR-L sTNFR-ll, TRAIL-R3, TRAIL-R4, Fas, GRO, GRO-α, ICAM-1 , ICAM-3, MCP-2, MCP-3, MCP-4, MDC, MIG, MIF, MP-15, MCP-1 , MSPa, IFN-a, IFN-β, TIMP-1 , TIMP-2, ADAM 10, ADAM 17, BACE, γ-secretase, BACE, MMP2, MMP3, MMP8, MMP9, MMP13, NT-3, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF-D, i-TAC, VEGF,

Haptoglobin, ανβ3, SPPL3, Leptin, SDF~1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-β Ι , TGF-p3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b- NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck, and Wnt-Ligands.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to breast cancer in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: Angiogenin, ArcpSO, ΜΙΡ-1 β, GCSF, NAP-2, RANTES, IL-1 Ra, !L-4, and IL-8. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample. Cancer survival

The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to cancer survival, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: IGFBP-3, Μ!Ρ-3β, IFN-γ, IL-12p70, Arcp30, GCSF, NAP-2, RANTE8, and VEGF, Preferably, the presence or absence of at least the following markers is determined in step ii): !FN-γ, IGFBP-3, IL-12p70, ΜΙΡ~3β, and VEGF. More preferably, the presence or absence of at least the following markers is determined in step ii): !GFBP-3, ΜΙΡ-3β, IFN-γ, iL~12p70, Arcp30, GCSF, NAP-2, RANTES, and VEGF. However, a person skilled in the art would understand that any selection of at least four of the listed markers would provide relevant results related to cancer survival, when the markers are used as such or as core biomarkers in the assay of the present invention. The present method thus may further comprise a step of monitoring the existence or status of cancer in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of cancer.

In an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): Angiogenin. EGF, Fas, FGF-8, fHA, MCP-1 , MiF. MiP-1 a, ΜΙΡ-1 β, IL-12p40, IL-1 Ra, !L-4, BDNF, EGFR, ADA 10, MMP8, MP9, FGF- 9, MCSF, TNF, GRO, IL-6R, MIP-3a, Oncostatin M, sTNFR-l, TIMP-1 , WNT-3a, ICAM- 1 , Haptoglobin, ανβ3 and IL-8.

In another embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): Eotaxin-1/ -21 -3, LIGHT, SLF, BDNF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, I- TAC, TECK, Dtk, PDGF, PLGF, GDNF, EGFR, bFGF, FGF-7, FGF-9, GMCSF, MCSF, 1-309, IGFB-1 , IGFBP-4, IGFBP-6, IGF-1 , !GF-1 sR, IGF-1 R, IL-1 R1 , IL-1 R4, IL-2Ra, !L- 1 a, Ι β, IL-3, IL-5, IL-6, IL-7, IL-10, IL-1 1 , IL-13, IL-15, !L-16, IL-17, IL-6R, PDGF-BB, P!GF, TNF, TNF-β, sTNFR-ll, TRAIL-R3, TRAIL-R4, GRO, GRO-a, ICAM-3, MCP-2, MCP-3, MCP-4, MDC, MIG, MP-15, MCP-1 , MSPa, IFN-a, !FN-β, TIMP-2, ADAM 10, ADAM 17, γ-secretase, BACE, MMP2, MMP3, MMP8, MMP9, MMP13, NT-3, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, G!TR, GITR-Ugand, VEGF, VEGF-D, l-TAC, SPPL3, Leptin, SDF-Ι α, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF- β1 , Τ6Ρ-β3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

The present method may also comprise further steps of comparing the level of expression of the one or more listed biomarkers in the purified sample of plasma extracellular vesicles from the subject to a control value, such as a sample from a healthy control, a cancer patient or a cancer survivor, and detecting the probability of cancer survival in the subject, wherein differences in expression of the one or more biomarkers in the sample relative to the control value indicate the probability of cancer survival.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to cancer survival in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: IGFBP-3, ΜΙΡ-3β, IFN-γ, IL-12p70, Arcp30, GCSF, NAP-2, RANTES, and VEGF. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample. Drug-induced immune suppression

The present invention is also directed to a method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to drug- induced immune suppression, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: BDNF, GMCSF, MCSF, ANTES, FGF-9, IL-6R, TIMP-1 , PARC, ICAM-1 , and IL-8R. Preferably, the presence or absence of at least the following markers is determined in step ii): BDNF, GMCSF, MCSF, RANTES, FGF-9, IL-8R, TIMP-1 , PARC, ICAM-1 , and IL-6R.

The present method thus may further comprise a step of monitoring the existence or status of a drug-induced immune suppression in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles.

Accordingly, the present method can also be used for monitoring the efficacy of any treatment of the drug-induced immune suppression.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to drug-induced immune suppression in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: BDNF, GMCSF, MCSF, RANTES, FGF-9, IL-6R, TIMP-1 , PARC, ICAM-1 , and IL-6R. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample. Allergy

In one aspect of the invention, said markers are related to allergies, as for example Type I and Type IV allergies, and the method comprises the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject: ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles:

- at least one or two of the following chemokines: AcrpSO, NAP-2 and RANTES.

Preferably, at least the presence or absence of the following markers is determined in step ii): AcrpSO, NAP-2 and RANTES; or AcrpSO and NAP-2: or AcrpSO and RANTES; or NAP-2 and RANTES.

In an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii):

ANG, ANGPT2, AREG, Axl, b-NGF, bFGF, BLC, BMP-4, BMP-6, BTC, CCL-28, CCL23, CNTF, Dtk, EGF-R, EGFP, ENA78, Eotaxin-1/-2/-3, FGF-7, Fractalkine, Ftl-3Ligand, fHA, GCP-2, GDNF, GITR, GITR-Ligand, GMCSF, GRO-a, Has2, Has3, HCC-4, HGF, I- 309, l-TAC, ICAM-3, IGF-1 , IGF-1 R, IGF-1 sR, IGFBP-1 , IGFBP-4, IL-10, IL-1 1 , IL- 12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17, IL-1 a, IL-1 β, IL-1 R1 , IL-1 Ra, IL-2Ra, IL-3, IL- 4, IL-5, IL-6, IL-6R, IL-7, IL-8, Leptin, LIGHT, MCP-1 , MCP-2, MCP-3, MCP-4, MDC, MIF, MIG, MMP8, MP-15, MSPa, IFN-a, IFN-β, BACE, NT-4, OncostatinM, OPG, OSM, Osteoprotegerin, PDGF, PDGF-BB, PIGF, PLGF, SDF-1 a, sgp130, SLF, SPPL3, sTNFR-l, sTNFR-ll, TARC, TECK, TGF-βΙ , TGF-p3, THPO, TNF, TNF-β, TRAIL-R3, uPAR, VEGF, VEGF-D, Wnt-Ligands, XCL-1

The present method may also comprise further steps of comparing the level of expression of the one or more listed biomarkers in the purified sample of plasma extracellular vesicles from the subject to a control value, such as a sample from a healthy control, and detecting the existence of allergies in the subject, wherein differences in expression of the one or more biomarkers in the sample relative to the control value for expression in a sample from a control indicate the existence of allergies, as for example Type-I and Type-IV allergies. For instance, the simultaneous presence of markers RANTES, NAP-2 and Acrp30 and the absence of other CCF, except BDNF. The present method thus may further comprise a step of monitoring the existence or status of an allergy in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of allergy. The present method may also comprise a further step of treating a subject diagnosed by the previous steps of the method to suffer from allergies by administering to said subject a drug or vaccine against allergies, as for example Type-I and Type-IV allergies.

The present invention is also directed to a kit for use in the in vitro detection or diagnosis of markers related to Type-I and Type-IV allergies in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of the following markers: Acrp30, NAP-2, RANTES. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. Preferably, the antibodies are immobilized on a solid support. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample. Risk for cancer relapse

The present invention is also directed to a method for detecting the presence or absence of a combination of markers from plasma extracellular vesicles, wherein said markers are related to risk for cancer relapse, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles: - at least one the following: Hck, CD83, PD-L1 .

Preferably, the presence or absence of the following markers is determined in step ii): Hck, CD83 and PD-L1 .

Preferably, the presence or absence of the following further markers is determined in step ii): EGF, Fas, IL-12p40, MCP-1 , and MIF. More preferably, the presence or absence of the following further markers is determined in step ii): EGF, Fas, FGF-6, IL- 12p40, MCP-1 , MIF, MIP-1 a, and ΜΙΡ-1 β. However, a person skilled in the art would understand that any selection of at least four of the listed markers would provide relevant results related to cancer relapse, when the markers are used as such or as core biomarkers in the assay of the present invention. The present method thus may further comprise a step of monitoring the existence or status of a cancer in a subject based on the presence or absence of one or more of the listed biomarkers in plasma extracellular vesicles. Accordingly, the present method can also be used for monitoring the efficacy of any treatment of cancer. In an embodiment, the presence or absence of at least one of the following further markers is also determined in step ii): ADAM10, Arcp30, BDNF, EGFR, FGF-9, fHA, GCSF, GRO, ICAM-1 , IFNy, IGFBP-3, IL-6R, MCSF, MIP-3a, MMP8, MMP9, NAP-2, Oncostatin M, RANTES, sTNFR-l, TIMP-1 , TNF, VEGF, and WNT-3a.

In another embodiment, the presence or absence of at least one of the following further markers is also determined in step ii):

ADAM 17, Angiogenin, ANGPT2, AREG, ανβ3, AxL b-NGF, BACE, bFGF, BLC, BMP-4, BMP-6, BTC, CCL-28, CCL23, CNTF, CTACK, Dtk, ENA78, Eotaxin-1/-2/-3, FGF-7, Fractalkine, Ft!-3 Ligand, γ-secretase, GCP-2, GDNF, GITR, GITR-Ligand, GMCSF, Haptoglobin, Has2, Has3, HCC-4, HGF, I-309, l-TAC, ICAM-3, !FN-a, IFN-b, IGF-1 , IGF- 1 R, IGF-l sR, IGFB-1 , IGFBP-4, IGFBP-8, IL-10, IL-1 1 , IL-12p70, IL-13, IL-15, IL-16, IL- 17, !L-1 a, ΙΙ_-1 β, IL-1 R1 , IL-1 R4, !L-1 Ra, IL-2Ra, !L-3, IL-4, IL-5, IL-6, IL-7, IL-8, Leptin, LIGHT, MCP-2, MCP-3, MCP-4, MDC, MIG, ΜΙΡ-3β, MMP13, MMP2, MMP3, MP-15, MSPa, , NT-3, NT-4, OPG, OSM, Osteoprotegerin, PARC, PDGF, PDGF-BB, PIGF, PLGF, SDF-1 a, sgp130, 8LF, SPPL3, sTNFR-ll, TARC, TECK, TGF-βΙ , TGF-p3, THPO, TIMP-2, TNF-β, TRAIL-R3, TRAIL-R4, uPAR, VEGF-D, Wnt-ligands, and XCL-1.

The present invention is also directed to a kit for use in the in vitro detection or monitoring or diagnosis of markers related to cancer relapse in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous defection of the following markers: Hck, PD-L1 and CD83. Said means in said kit can be antibodies specifically recognizing the listed biomarker proteins. The kit may also comprise means for the purification of plasma extracellular vesicles from a blood or plasma sample.

The present method may also comprise a further step of treating a subject diagnosed by the previous steps of the method to suffer from cancer relapse by administering to said subject a drug or other treatment against cancer treatment.

Control samples

The above disclosed methods for the detection of combination biomarkers relating to schizophrenia, depression, HIV, melanoma, breast cancer, or allergies may also comprise further steps of comparing the level of expression of the one or more listed biomarkers in the purified sample of plasma extracellular vesicles from the subject to a control value, such as a sample from a healthy control, and detecting the existence of schizophrenia, depression, HIV, melanoma, breast cancer, or allergies in the subject, wherein differences in expression of the one or more biomarkers in the sample relative to the control value for expression in a sample from a control indicate the existence or status of the disease.

The above disclosed methods may also comprise a further step of treating a subject diagnosed by the previous steps of the method to suffer from schizophrenia, depression, HIV, melanoma, breast cancer, or allergies by administering to said subject a drug treating the diagnosed disease or condition.

Procedure of pEV isolation

Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the separation of pEV using techniques of differential centrifugation and immunoseparation. It will be appreciated that modification of detail may be made without departing from the scope of the invention. In order to measure biomarkers in pEV, they have to be separated from other plasma components and concentrated into a small volume. Any method allowing concentration and separation of pEV is both suitable and required to assess pEV biomarkers. Suitable methods are for example: differential ultracentrifugation, uitracentrifugation in combination with gradient fractionation, antibody coupled matrices (e.g. beads or filters), exchange/spin-column based methods and pEV~binding resins.

Preferably, the pEV are separated from the plasma of an individual or animal model. Techniques for the separation of plasma from blood will be clear to the skilled reader. However, suitable fluids for use as a biological sample in the present method include bodily fluids such as blood, ascites and urine and growth medium in which ceils are cultured in vitro. The subject from which blood plasma is taken for preparation of pEV may be any animal. Suitable animals include primates, preferably higher primates such as chimpanzees and also humans. Most preferably, pEV are prepared from human patients.

It has been discovered that the purification of pEV from 50μί-15ml plasma allows the preparation of sufficient quantities of biomarkers for assessment. Preferably, pEV are prepared by differential centrifugation, according to the technique of Raposo et al. (1996).

According to one embodiment of the invention, the assessment of pEV-derived biomarkers comprises the sequential steps of centrifuging plasma obtained from an individual to give a pellet that is enriched in pEV, and isolating said biomarkers from said pEV. For pEV purification patient plasma samples are diluted 1 :1 with PBS and centrifuged for 30 min at 2,000 g, 45 min at 12,000 g and ultra-centrifuged for 2 h at 1 10,000 g. Pellets are resuspended in 10 ml PBS and centrifuged at 1 10,000 g for 1 h. Pellets are finally resuspended in 100 μΙ PBS and considered as EV preparations. As an alternative purification method, plasma samples may be incubated with beads coated with antibody that recognizes marker molecules on the surface of pEV, For example, anti-avp3 or anti-ADAM10 antibodies may be used in this respect. As the skilled reader will appreciate, magnetic beads, such as those manufactured by Dynabeads, Dynal, Oslo, Norway, or polystyrene beads (for example, those made by Pierce) are particularly suitable in this embodiment of the invention. Other alternatives for the purification of pEV include the use of sucrose density gradients or organelle electrophoresis (Tuip et a!., 1994).

Biomarker assessment

In order to measure their biomarker content, the concentrated pEV should be resuspended in a lysis buffer. Preferably this is done in a small volume adjusted to the requirements of the readout assay (for example 5-1 ΟΟμΙ for antibody-coated filters from Ray Biotech or for dot blot analysis, or antibody coated beads for FACS analysis (e.g. from BioLegend) or for miRNA quantification by the Nanostring technology microRNA microarray by Agilent.

One technique that is suitable for analyzing pEV protein/peptide content is by SDS- PAGE and Western blotting, using antibodies directed against proteins/peptides that are contained in pEV particles. Antibodies directed against ADAM- or Matrix metal!o- proteases are particularly suitable. Binding of these primary antibodies to pEV can be assessed using, for example, labelled secondary antibodies that bind to the primary antibodies. For example, anti-ADA 17 monoclonal antibody can be used as the primary antibody, whilst a labelled anti-mouse IgG can be used as the secondary antibody.

Alternatively, any assay system that identifies/recognizes individual proteins or peptides in a concentrated pEV preparation is suitable to assess the biomarker composition. Ideally this assessment is performed in a quantitative or semiquantitative manner to judge the relative magnitude/concentration of a signal/biomarker. Conventional methods to recognize these components are specific antibodies immobilized for example on a filter surface (e.g. protein array from Ray Biotech) or a plastic bead and assessed by FACS analysis (e.g. multiplex array from Biolegend). The following CCF and combinations thereof are particularly suitable for the diagnosis and monitoring of acute and chronic diseases:

Angiogenin, BDNF, EGF, EGFR, FGF-8, FGF-9, fHA, GMCSF, GCSF, MCSF, 1-309, IGFB-1 , !GFB-3, !GFBP-3, !GFBP-8, !GF-SSR, IL-1 R4, IL-1 Ra, IL-3, !L-4, !L~6, IL-6R, IL- 8, !L-12p4Q, !L-12p7Q, NAP-2, PDGF-BB, PiGF, TNF, sTNFR-i , TRAIL-R4, Fas, RANTES, GRO, Arcp30, ICAM-1 , MIF, ΜΙΡ-1 α, ΜΙΡ-1 β, Μ!Ρ-3β, MCP-1 , SP , IFN- γ, ΤΙΜΡ-1 , TIMP-2, ADAM 10, ADAM 17, γ-secretase, BACE, CCL23, CNTF, CTAK, MMP2, MMP3, MMP8, MMP9, MMP13, NT-3, PARC, PARK6, Oncostatin M, Osteoprotegerin, GITR-Ligand, VEGF, S-TAC, VEGF, Haptoglobin, ανβ3, 8PPL3, Leptin, PD-L1 , CC83, Has3, Has2 and Hck.

Certain combinations of the markers listed above characterize a specific disease in comparison to healthy individuals and other diseases. The absence of certain markers increases the discrimination in between different diseases, e.g Depression vs Alzheimer or tumor control vs. tumor relapse. In addition, the pEV miRNA profile adds additional diagnostic specificity for a given analysis and disease.

The following combinations of CCF and MP markers, as disclosed in the Examples, are of high diagnostic value if at least 70% are present in a given sample, or at least 3-8 of the key markers, marked in bold.

Having now generally described the invention, the same will be more readily understood by reference to the following Examples, which is provided by way of illustration and is not intended as limiting.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. EXAMPLES

Isolation and purification of EV EV purification was performed essentially as described previously (Muratori et al., 2009; Thery et al, 2009). Briefly, supernatants were collected after 48 h and centrifuged for 20 min at 2,000 g, 30 min at 10,000 g and ultra-centrifuged for 1 h at 100,000 g. Pellets were resuspended in 35 mi PBS and centrifuged at 100,000 g for 1 h. Pellets were resuspended in 100 μΙ PBS and considered as EV preparations.

For EV purification from patient samples, 30 ml blood plasma was diluted with 30 ml PBS and centrifuged for 30 min at 2,000 g, 45 min at 12,000 g and ultra-centrifuged for 2 h at 1 10,000 g. Pellets were resuspended in 30 ml PBS and centrifuged at 1 10,000 g for 1 h. Pellets were again resuspended in 100 μΙ PBS and considered as EV preparations. For further purification, EV were diluted in 2 ml of 2.5 M sucrose, 20 mM Hepes/NaOH, pH 7.4 and a linear sucrose gradient (2 - 0,25 M sucrose, 20 mM Hepes/NaOH pH 7.4) was layered on top of the EV suspension. The samples were then centrifuged at 210,000 g for 15 h. Gradient fractions were collected and the refractive index was determined. Each fraction was diluted in 10 mi PBS and ultra-centrifuged for 1 h at 1 10,000 g. Pellets were solubilized in SDS sample buffer or resuspended in 100 μΙ PBS and analyzed by immunoblotting or Cytokine/Chemokine/soluble Factor (CCF) protein array. The biomarker target names are as listed in the user manual of Human Cytokine Array Q840 (RayBiotech Inc.). Rests Its

Example (1 ) (Figure 1 ): Non-viremic HIV infection with high or low immune activation vs healthy controls.

High immune a ctivation : High levels of TNF, GRO, ICAM-1 , IL-6R, sTNFR-l and TIMP-1 ; in addition I-3G9, !GFBP-1 , IL-2 a; absence of GCSF. Low immune activation; GCSF, Low levels of INF, GRO, ICAM-1 , IL-8R, sTNF -l, and TIMP-1 . High levels of RANTES, no 1-309, !GFBP-1 , !L-2Ra. GCSF.

Healthy Control: BDNF, NAP-2, GRO. Example (2): (Figure 1 ): Alzheimer vs. age matched control.

Alzheimer: MMP2, MMP3, MMP9, MMP13, Fas, !GFBP-3, IGFBP-6, ΜΙΡ-1 β, ΜΙΡ~3β, TRAILR4, VEGF, PARC, NTS, Angiogenin, ICAWt-1 , low BDNF and FGF-6.

Age-matched Control: BDNF, TNF, FGF-9, TIMP-1 , TIIVIP-2 and low ICAM-1 . Example (3): Depression vs. healthy control (as above).

Depression: MMP9, TIMP-2, ADAM17, ADAM 10, RANTES, TNF, ICAM-1 , IL-1 R4, FGF- 9, NTS, low BDNF, IGFBP3, WUP-1a_s W!IP-Ιβ, Oncosiatin M, and PARC.

Example (4): Drug-induced immune suppression (DIS) (e.g. after transplantation) vs. healthy control. DIS: low BDNF, GMCSF, MCSF, low RANTES, FGF-9, IL-6R, TIMP-1 , PARC.

Example (5) (Figure 2): Cancer (Melanoma) patients with tumor load (tumor patients), or without tumor load (after primary tumor resection and with low or high risk for tumor relapse), and patients with tumor load (clinical stage IV) 15-20 years after successful immunotherapy (long term survivors), all compared to healthy controls (as above).

Melanoma Tumor patients: ADAM 10, MMP9, EGF, FGF-6, MCP-1 , MCSF, NAP-2, TNF, Arcp30, EGFR, Fas, G O_s IL-12p40, IL-6R, MIF, ΜΙΡΊα, ΜΙΡ-1β, Oncostafin M, sTNFR.

Melanoma high risk patients: ADAM 10, WIMPS, EGF, FGF-6, NAP-2, TNF, Arcp30, EGFR, MIF, ΜΙΡ-ί , ΜΙΡ-1β, Oncostatin M, sTNFR, TIMP-1 , IF y, low !L-12p40.

Melanoma low risk patients: ADA1VI1 Q, ArcpSQ, MMP8

Long term survivors : ADAM 10, NAP-2, RANTES, ArcpSO, low EGFR, low Fas, low EGFR, GCSF, ICAM-1 , IGFBP-3, IL-1 p40, VEGF.

Example (6) (Figure 4): Type-IV allergy vs healthy controls. Type-iV allergy: High levels of RANTES, NAP-2, AcrpSQ:

Healthy Control: BDNF

Example (7): Tumor relapse vs. tumor control

Tumor relapse or imminent Tumor relapse: Hck, PD-L1 , CD83; in addition: CCF present in cancer patients, e.g. ADAM 10, MMP9, EGFP, FGF-6, MCP-1 , MCSF, NAP-2, TNF, ArcpSO, EGFR, Fas, GRO, IL-12p40, IL-8R, MIF, MIP-1 α, ΜΙΡ-Ι β, Oncostatin M, sTNFR.

Tumor Control; CCF present in cancer patients, e.g. ADAM 10, MMP9, EGFP, FGF-6, MCP-1 , MCSF, NAP-2, TNF, ArcpSO, EGFR, Fas, GRO, IL-12p40, IL-6R, MIF, MIP-1 a, MIP-1 β, Oncostatin M, sTNFR.

REFERENCES

Meio, Sonia A., Linda B. Luecke, Christoph Kahiert, Agustin F. Fernandez, Seth T. Gammon, Judith Kaye, Valerie S. LeBleu, Elizabeth A. Mittendorf, Juergen Weitz, Nuh Rahbari, Christoph Reissfelder, Christian Pilarsky, Mario F. Fraga, David Piwnica- Worms and Raghu Kaliuri (2015), Giypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523,177-182 uratori C, et aL "Massive secretion by T cells is caused by HIV Nef in infected ceils and by Nef transfer to bystander cells," Cell Host. Microbe 6(3), 218 (2009). 0stergaard, O., Nielsen, C. T., iversen, L. V., Tanassi, J. T., Knudsen. S., Jacobsen, S. and Heegaard, N. H. H. (2013), Unique Protein Signature of Circulating icropartic!es in Systemic Lupus Erythematosus. Arthritis & Rheumatism, 65: 2680-2690

Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1 161 -72. Thery, C, M. Ostrowski. and E. Segura, "Membrane vesicles as conveyors of immune responses," Nat Rev. Immunol. 9(8), 581 (2009).

Tulp, A., Verwoerd, D., Dobberstein, B., Ploegh, H. L., and Pieters, J. (1994). Isolation and characterization of the intracellular MHC class Π compartment. Nature 369, 120- 126.

Claims

1. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to Alzheimer's disease, the method comprising the steps of: I) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles:

- at least one of the markers selected from the group consisting of: FGF-6, !CAM-1 , !GFBP-8, ΜΙΡ-3β, and VEGF. 2. The method according to claim 1 , wherein the presence or absence of at least the following markers is determined in step ii): MMP-2, MMP-3, MMP-13, TIMP-1 , TIMP-2, FGF-8, !CAM-1 , IGFBP-8, ΜΙΡ-3β, and VEGF.

3. The method according to claim 1 or 2, wherein the presence or absence of at least one of the following further markers is determined in step ii): Angiogenin. BDNF, fHA, IFNy, MCSF, NAP-2, RANTES, ADAM 17, ADAM 10, MMP9, γ-secretase, Fas, FGF-9, GCSF, GRO, IGFBP-3, IL-1 R4, ΜΙΡ-1 β, TRAIL-R4, PARC, NTS, Haptoglobin, and ανβ3.

4. The method according to any one of claims 1 - 3, wherein the presence or absence of at least one of the following further markers is determined in step ii): Eotaxin-1/ -21 -3, LIGHT, SLF, BMP-4, BMP-8, BLC, CCL23, CNTF, CTAK, !-TAC, TECK, Dtk, PDGF, PLGF, GDNF, EGF, EGFR, bFGF, FGF-7, GMCSF, 1-309, IGFBP-1 , !GFBP-4, IGF-1 , !GF~1 sR, SGF-1 R, IL-1 R1 , IL-1 Ra, IL-2Ra, ll_-1 a, ΙΙ_-1 β, SL-3, IL-4, IL-5, !L-6, IL-7, !L-8, !L-10, IL-1 1 , IL-13, IL-15, !L-16, IL-17, IL-6R, IL-12p40, !L-12p70, PDGF- BB, P!GF, TNF, TNF-β, sTNFR-i, sTNFR-ll, TRAIL-R3, GRO-a, Arcp30, !CAM-3, MCP- 2, MCP-3, MCP-4, MDC, iG, M!F, MP-15, MCP-1 , MSPa, IFN-a, IFN-β, BACE, MMP8, NT-4, TARC, uPAR, Oncostatin M, Osteoproiegerin, G!TR, GITR-Ligand, VEGF-D, i- TAC, VEGF, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ Τ6Ρ-β3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

5. The method according to any one of claims 1 - 4, wherein the simultaneous presence of markers MMP-2, MMP-3, and MMP-13 and absence of markers T!MP-1 and TIMP-2 in plasma extracellular vesicles relate to Alzheimer's disease.

6. A kit for use in the in vitro detection of markers related to Alzheimer's disease in purified plasma extracellular vesicles, wherein said kit comprises means for

simultaneous detection of:

- at least one of the matrix meta!loproteinases selected from the group consisting of: MMP-2, MMP-3, and MMP-13;

- at least one of the markers selected from the group consisting of: FGF-6, !CAM-1 , !GFBP-8, ΜΙΡ-3β, and VEGF.

7. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to schizophrenia, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject: ii) determining the presence or absence of at ieast four of the folio ving markers in the purified plasma extracellular vesicles: BDNF, CCL23, CNTF, CTAK, IL-1 R4, NT-3 and PARK8.

8. The method according to claim 7, wherein the presence or absence of at Ieast the following markers is determined in step ii): BDNF, CNTF, CTAK, and IL-1 R4.

9. The method according to claim 8 or 7, wherein the presence or absence of at Ieast the following markers is determined in step ii): BDNF, CCL23, CNTF, CTAK, IL-1 R4, NT- 3 and PARK6.

10. The method according to claim any one of claims 8 - 10, wherein the presence or absence of at Ieast one of the following further markers is determined in step ii):

Angiogenic Eotaxin-1/ -21 -3, LIGHT, SLF, B P-4, B P-6, BLC, l-TAC, TECK, Dtk, PDGF, PLGF, GDNF, EOF, EGFR, bFGF, FGF-6, FGF-7, FGF-9, fHA, GMCSF, GCSF, MCSF, 1-309, IGFBP-1 , IGFBP-3, IGFBP-4, !GFBP-8, IGF-1 , IGF-1 sR, SGF-1 R, IL-1 R1 , IL-I Ra, IL-2Ra, IL-1 a, Ι β, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, !L-1 1 , IL-13, IL-15, IL- 16, IL-17, SL-6R, !L-12p40, !L-12p70, NAP-2, PDGF-BB, P!GF, TNF, TNF-β, sTNFR-l, sTNFR-ll, TRAIL-R3, TRAIL-R4, Fas, RANTES, GRO, GRO-a, Arcp30, ICAM-1 , ICAM- 3, MCP-2, MCP-3, MCP-4, MDC, M!G, MIF, MP-15, ΜΙΡ-1 β, ΜΙΡ-3β, MCP-1 ,

MSPa, IFN-a, IFN-β, IFN-γ, TIMP-1 , TIMP-2, ADAM 10, ADAM 17, BACE, γ-secretase, MMP2, MMP3, MMP8, MMP9, MMP13, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, VEGF, Haptoglobin, νβ3, SPPL3, Lepfin, SDF-Ι α, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ , Τ6Ρ-β3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axi, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

1 1 . A kit for use in the in vitro detection of markers related to schizophrenia in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at Ieast four of the following markers: BDNF, CCL23, CNTF, CTAK, IL-1 R4, NT-3 and PARK8.

12. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to depression, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracelluiar vesicles: IGFBPS, IL-1 R4, ΜίΡ~1 α, ΜΙΡ-1 β, MMP9, Oncostatin M, PARC, and TIMP-2.

13. The method according to claim 12, wherein the presence or absence of at least the following markers is determined in step ii): MIP-1 a, Oncostatin M, PARC, and TIMP-2, 14. The method according to claim 12 or 13, wherein the presence or absence of at least the following markers is determined in step ii): IGFBPS, IL-1 R4, ΜίΡ-1 α, MiP-Ι β, MMP9, Oncostatin M, PARC, and TIMP-2.

15. The method according to any one of claims 12 - 14, wherein the presence or absence of at least one of the following further markers is determined in step ii):

ADAM 17, ADAM 10, RANTES, TNF, iCAM-1 , fHA, FGF-9, NTS, M-CSF, NAP-2, EGFR, ArcpSO, IGFBP-6, MIF, !L~12p40, and BDNF.

16. The method according to any one of claims 12 - 15, wherein the presence or absence of at least one of the following further markers is determined in step ii):

Angiogenin, Eotaxin-1/ -2/ -3, LIGHT, SLF, BMP-4, BMP-8, BLC, l-TAC, TECK, Dtk, PDGF, PLGF, GDNF, EGF, bFGF, FGF-6, FGF-7, GMCSF, GCSF, I-309, IGFBP-1 ,

IGFBP-4, IGF-1 , IGF-1sR, IGF-1 R, IL-1 R1 , IL-1 Ra, IL-2Ra, IL-1 a, ΙΙ_-1 β, IL-3, !L-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-13, IL-15, IL-16, IL-17, IL-6R, IL-12p70, PDGF-BB, PIGF, TNF-β, sTNFR-i, sTNFR-ll, TRAIL-R3, TRAIL-R4, Fas, GRO, GRO-a, ICAM-3, MCP-2, MCP-3, MCP-4, MDC, MIG, MP-18, ΜΙΡ-3β, MCP-1 , MSPa, IFN-a, !FN-β, IFN-y, TIMP- 1 , BACE, γ-secretase, MMP2, MMP3, MMP8, MMP13, NT-4, TARC, uPAR,

Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, VEGF, Haptoglobin, ανβ3, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ Τ6Ρ-β3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck, and Wnt-Ligands.

17. A kit for use in the in vitro detection of markers related to depression in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: IGFBP3, IL-1 R4, ΜΙΡ-1 α, ΜΙΡ-1 β, MIV1P9, Oncostatin M, PARC, and ΤΊ Ρ-2.

18. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to HIV, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: Angiogenin, GMCSF, GRO, ICAM-1 , IL-6R, NAP- 2, RANTES, and TIMP-1. 19. The method according to claim 18, wherein the presence or absence of at least the following markers is determined in step ii): GMCSF, GRO, IL-8R, and TIMP-1.

20. The method according to claim 18 or 19, wherein the presence or absence of at least the following markers is determined in step ii): Angiogenin, GMCSF, GRO, ICAM-1 , IL-6R, NAP-2, RANTES, and TIMP-1. 21. The method according to any one of claims 18 - 20, wherein the presence or absence of at least one of the following further markers is determined in step ii): BDNF, FGF-9, ADAM 10, ADAM 17, MMP9, fHA, Haptoglobin, ανβ3, TNF, sTNFR-l, I-309, IGFBP-1 , IL-2Ra, GCSF, and RANTES.

22. The method according to any one of claims 18 - 21 , wherein the presence or absence of at least one of the following further markers is determined in step ii): Eotaxin-1/ -21 -3, LIGHT, SLF, BMP-4, BMP-8, BLC, CCL23, CNTF, CTACK, !-TAC, TECK, Dtk, PDGF, PLGF, GDNF, EGF, EGFR, bFGF, FGF-8, FGF-7, MCSF, IGFBP-3, !GFBP-4, !GFBP-8, IGF-1 , !GF~1 sR, SGF-1 R, IL-1 R1 , IL-1 R4, IL-I Ra, IL-1 a, IL-Ι β, IL-3, !L-4, IL-5, IL-6, IL-7, IL-8, IL-10, !L-1 1 , IL-13, IL-15, IL-16, !L-17, IL-12p40, IL-12p70, PDGF-BB, P!GF, TNF-β, sTNFR-l!, TRAIL-R3, TRAIL-R4, Fas, GRO-α, Arcp30, ICAM- 3, MCP-2, MCP-3, MCP-4, MDC, M!G, MIF, MP-15, ΜΙΡ-1 β, ΜΙΡ-3β, MCP-1 ,

MSPa, IFN-a, IFN-β, !FN-γ, TIMP-2, BACE, γ-secretase, MMP2, MMP3, MMP8, MMP13, NT-3, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, VEGF, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-βΙ , ΤΟΡ-β3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axi, OPG, 08M, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck, and Wnt-Ligands.

23. A kit for use in the in vitro deiection of markers related to HIV in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: Angiogenin, GMCSF, GRO, ICAM-1 , IL-6R, NAP-2, RANTES, and TIMP-1 .

24. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to melanoma, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: FGF-6, ΜΙΡ-1 α, ΜΙΡ-1 β, EGF, Fas, IL-12p40, MCP-1 , and MIF.

25. The method according to claim 24, wherein the presence or absence of at least the following markers is determined in step ii): EGF, Fas, IL-12p40, MCP-1 , and MIF. 28. The method according to claim 24 or 25, wherein the presence or absence of at least the following markers is determined in step ii): FGF-8, ΜΙΡ-1 α, ΜΙΡ-1 β, EGF, Fas, IL-12p40, MCP-1 , and MIF.

27. The method according to any one of claims 24 - 28, wherein the presence or absence of at least one of the following further markers is determined in step ii):

ADAM 10, fHA, MMP8, MP9, CSF, NAP-2, TNF, Arcp30, EGFR, GRO, IL-6R, Oncostatin M, sTNFR-l, TIMP-1 , BDNF, FGF-9, MiP~3a, WNT-3a, RANTES, GCSF, ICAM-1 , IGFBP-3, VEGF, Haptoglobin, ανβ3, and IFNy.

28. The method according to any one of claims 24 - 27, wherein the presence or absence of at least one of the following further markers is determined in step ii):

Angiogenin, Eotaxin-1/ -21 -3, LIGHT, SLF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, !-TAC, TECK, Dtk, PDGF, PLGF, GDNF, bFGF, FGF-7, GMCSF, 1-309, !GFB- 1 , !GFBP-4, IGFBP-8, IGF-1 , IGF-1 sR, IGF-1 R, IL-1 R1 , IL-1 R4, IL-1 Ra, !L-2Ra, !L-1 a, !L-Ι β, IL-3, !L~4, IL-5, !L-8, !L-7, IL-8, IL-10, IL-1 1 , !L-13, !L-15, IL-16, IL-17, IL-12p70, PDGF-BB, P!GF, TNF-β, sTNFR-ll, TRAIL-R3, TRAIL-R4, GRO-a, ICAM-3, MCP-2, MCP-3, CP-4, MDC, MSG, MP-18, ΜΙΡ-3β, MSPa, !FN-a, !FN-β, TIMP-2, ADAM17, BACE, γ-secretase, MMP2, MMP3, MMP13, NT-3, NT-4, PARC, TARC, uPAR,

Osteoprotegerin, G!TR, G!TR-Ligand, VEGF, VEGF-D, S-TAC, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF-β TGF~p3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axi, OPG, GSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

29. A kit for use in the in vitro detection of markers related to melanoma in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: FGF-6, iP-1 a, ΜΙΡ-1 β, EGF, Fas, !L- 12p40, MCP-1 , and MIF.

30. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to breast cancer, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at ieast four of the fo!io ving markers in the purified plasma extracellular vesicles: Angiogenin, Arcp30, ΜΙΡ~1 β, GCSF, NAP-2,

ANTE8, IL-I Ra, IL-4, and IL-8.

31 . The method according to claim 30, wherein the presence or absence of at Ieast the following markers is determined in step ii): Angiogenin, !L-1 Ra, IL-4, and IL-8.

32. The method according to claim 30 or 31 , wherein the presence or absence of at least the following markers is determined in step ii): Angiogenin, Arcp30, Μ!Ρ-1 β, GCSF, NAP-2, RANTES, IL-1 Ra, IL-4, and IL-8.

33. The method according to any one of claims 30 - 32, wherein the presence or absence of at Ieast one of the following further markers is determined in step ii): IFN-γ, IGFBP-3, IL-12p70, fHA, ΜίΡ~3β, and VEGF.

34. The method according to any one of claims 30 - 33, wherein the presence or absence of at Ieast one of the following further markers is determined in step ii):

Eotaxin-1 / -2/ -3, LIGHT, SLF, BDNF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, I- TAG, TECK, Dtk, PDGF, PLGF, GDNF, EGF, EGFR, bFGF, FGF-6, FGF-7, FGF-9,

GMCSF, MCSF, 1-309, IGFB-1 , IGFBP-4, IGFBP-6, IGF-1 , IGF-1 sR, IGF-1 R, IL-1 R1 , IL- 1 R4, IL-2Ra, IL-1 a, IL-Ι β, !L-3, IL-5, IL-8, !L~7, IL-10, IL-1 1 , IL-13, IL-15, !L-16, IL-17, IL- 6R, !L-12p40, PDGF-BB, PIGF, TNF, TNF-β, sTNFR-l, sTNFR-ll, TRAIL-R3, TRAIL-R4, Fas, GRO, GRO-a, ICAM-1 , SCAM-3, CP-2, CP-3, CP-4, MDC, MIG, MIF, MP-15, MCP-1 , MSPa, IFN-a, IFN-β, TIMP-1 , TIMP-2, ADAM10, ADAM 17, BACE, γ-secretase, BACE, MMP2, MMP3, MMP8, MMP9, MMP13, NT-3, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF-D, l-TAC, VEGF,

Haptoglobin, ανβ3, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractaikine, TNF-β, TGF-β Ι , TGF^3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axi, OPG, GSM, b- NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck, and Wnt-Ligands.

35. A kit for use in the in vitro detection of markers related to breast cancer in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: Angiogenic Arcp30, ίΡ-Ι β, GCSF, NAP-2, RANTES, IL-1 Ra, IL-4, and IL-8. 36. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to cancer survival, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: IGFBP-3, ΜίΡ~3β, IFN-γ, !L-12p70, Arcp30,

GCSF, NAP-2, RANTES, and VEGF.

37. The method according to claim 38, wherein the presence or absence of at least the following markers is determined in step ii): IFN-y, !GFBP-3, IL-12p70, ΙΡ-3β, and VEGF, 38. The method according to claim 38 or 37, wherein the presence or absence of at least the following markers is determined in step ii): IGFBP-3, ΜΙΡ-3β, IFN-γ, IL-12p70, Arcp30, GCSF, NAP-2, RANTES, and VEGF.

39. The method according to any one of claims 38 - 38, wherein the presence or absence of at least one of the following further markers is determined in step ii): Angiogenin, EGF, Fas, FGF-8, fHA, MCP-1 , MIF, ΜΙΡ-1 α, ΜΙΡ-1 β, IL-12p4Q, IL-1 Ra, IL- 4, BDNF, EGFR, ADAM 10, MMP8, MP9, FGF-9, MCSF, TNF, GRO, IL-8R, MiP-3a, Oncostatin M, sTNFR-l, TIMP-1 , WNT-3a, ICAM-1 , Haptoglobin, ανβ3 and IL-8.

40. The method according to any one of claims 36 - 39, wherein the presence or absence of at least one of the following further markers is determined in step ii): Eotaxin-1/ -21 -3, LIGHT, SLF, BDNF, BMP-4, BMP-6, BLC, CCL23, CNTF, CTACK, I- TAC, TECK, Dtk, PDGF, PLGF, GDNF, EGFR, bFGF, FGF-7, FGF-9, GMCSF, MCSF, 1-309, IGFB-1 , IGFBP-4, IGFBP-6, IGF-1 , IGF-1 sR, IGF-1 R, IL-1 R1 , IL-1 R4, IL-2Ra, IL- 1 a, IL-Ι β, IL-3, IL-5, IL-6, IL-7, IL-10, IL-1 1 , IL-13, IL-15, !L-16, IL-17, IL-6R, PDGF-BB, P!GF, TNF, TNF-β, sTNFR-ll, TRAIL-R3, TRAIL-R4, GRO, GRO-a, ICAM-3, MCP-2, MCP-3, MCP-4, MDC, MIG, MP-15, MCP-1 , MSPa, IFN-a, !FN-β, TIMP-2, ADAM 10, ADAM 17, γ-secretase, BACE, MMP2, MMP3, MMP8, MMP9, MMP13, NT-3, NT-4, PARC, TARC, uPAR, Oncostatin M, Osteoprotegerin, GITR, GITR-Ligand, VEGF, VEGF-D, l-TAC, SPPL3, Leptin, SDF-1 a, Ftl-3 Ligand, ANG, Fractalkine, TNF-β, TGF- β1 , Τ6Ρ-β3, GCP-2, XCL-1 , ENA78, THPO, ANGPT2, AREG, Axl, OPG, OSM, b-NGF, sgp130, BTC, HCC-4, CCL-28, HGF, Hck and Wnt-Ligands.

41 . A kit for use in the in vitro detection of markers related to cancer survival in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: IGFBP-3, ΜΙΡ-3β, IFN-y, IL-12p70, Arcp30, GCSF, NAP-2, RANTES, and VEGF.

42. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to drug-induced immune suppression, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least four of the following markers in the purified plasma extracellular vesicles: BDNF, GMCSF, MCSF, RANTES, FGF-9, IL-8R, TiMP-1 , PARC, iCAM-1 , and IL-6R.

43. The method according to claim 42, wherein the presence or absence of at least the following markers is determined in step ii): BDNF, GMCSF, MCSF, RANTES, FGF-9, IL-

6R, TIMP-1 , PARC, ICAM-1 , and IL-8R.

44. A kit for use in the in vitro detection of markers related to drug-induced immune suppression in purified plasma extracellular vesicles, wherein said kit comprises means for simultaneous detection of at least four of the following markers: BDNF, G CSF, MCSF, RANTES, FGF-9, IL-8R, TIMP-1 , PARC, ICAM-1 , and IL-6R. 45. Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to allergies such as Type-! and Type-IV allergic reactions, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of the following markers in the purified plasma extracellular vesicles:

- at least one or two of the chemokines RANTES, AcrpSQ and NAP-2.

46. The method according to claim 45, wherein at least the presence or absence of the following markers is determined in step ii): RANTES, Acrp30 and NAP-2. 47. The method according to claim 45 or 48, wherein the presence or absence of at least one of the following further markers is determined in step ii):

ANG, ANGPT2, AREG, Axl, b-NGF, bFGF, BLC, BMP-4, BMP-6, BTC, CCL-28, CCL23, CNTF, Dtk, fHA. EGF-R, EGFP, ENA78, Eotaxin-1/-2/-3, FGF-7, Fractalkine, Ftl- 3Ligand, GCP-2, GDNF, GITR, GITR-Ligand, GMCSF, GRO-a, Has2, Has3, HCC-4, HGF, I-309, l-TAC, ICAM-3, IGF-1 , IGF-1 R, IGF-1sR, IGFBP-1 , IGFBP-4, IL-10, IL-1 1 , IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17, IL-1 a, IL-1 β, IL-1 R1 , IL-1 Ra, IL-2Ra, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, Leptin, LIGHT, MCP-1 , MCP-2, MCP-3, MCP-4, MDC, MIF, MIG, MMP8, MP-15, MSPa, IFN-a, IFN-β, BACE, NT-4, OncostatinM, OPG, OSM, Osteoprotegerin, PDGF, PDGF-BB, PIGF, PLGF, SDF-1 a, sgp130, SLF, SPPL3, sTNFR-l, sTNFR-ll, TARC, TECK, TGF-βΙ , TGF-p3, THPO, TNF, TNF-β, TRAIL-R3, uPAR, VEGF, VEGF-D, Wnt-Ligands, XCL-1

48. A kit for use in the in vitro detection of markers related to allergies such as Type-I and Type-IV allergic reactions in purified plasma extracellular vesicles, wherein said kit comprises means for detection of:

- at least one of the chemokines selected from the group consisting of: RANTES, Acrp30 and NAP-2.

49. Use of a kit in the in vitro detection of markers related to allergies such as Type-I and Type-IV allergic reactions in purified plasma extracellular vesicles, wherein said kit comprises means for detection of the chemokines selected from the group consisting of: RANTES, Acrp30 and NAP-2. 50. The use according to claim 49, wherein said kit comprises means for detection of the chemokines RANTES, Acrp30 and NAP-2.

51 . Method for detecting a combination of markers from plasma extracellular vesicles, wherein said markers are related to risk of cancer relapse, the method comprising the steps of: i) purifying plasma extracellular vesicles from a biological sample obtained from a subject; ii) determining the presence or absence of at least one of the following markers in the purified plasma extracellular vesicles: Hck, CD83 and PD-L1 .

52. The method according to claim 51 , wherein the presence or absence of at least the following further markers is determined in step ii): EGF, Fas, IL~12p40, MCP-1 , and M!F.

53. The method according to claim 51 or 52, wherein the presence or absence of at least the following further markers is determined in step ii): FGF-6, fHA, ΜΙΡ-1 α, ΜΙΡ-1 β, EGF, Fas, IL-12p40, MCP-1 , and MIF.

54. The method according to claim any one of claims 51 - 53, wherein the presence or absence of at least one of the following further markers is determined in step ii): ADAM 10, ADA 17, Angiogenic ANGPT2, Arcp30, AREG, ανβ3, Axi, b-NGF, BACE, BDNF, bFGF, BLC, BMP-4, BMP-6, BTC, CCL-28, CCL23, CNTF, CTACK, Dtk, EGFR, ENA78, Eotaxin-1/-2/-3, FGF-7, FGF-9, Fractaikine, Ftl-3 Ligand, γ-secretase, GCP-2, GCSF, GDNF, G!TR, G!TR-Ligand, G CSF, GRO, Haptoglobin, Has2, Has3, HCC-4, HGF, I-309, !-TAC, ICAM-1 , ICAM-3, IFN-a, !FN-b, IFNy, IGF-1 , IGF-1 R, IGF-1 sR,

IGFB-1 , !GFBP-3, !GFBP-4, !GFBP-8, IL-10, IL-1 1 , IL-12p70, !L-13, IL-15, IL-16, !L-17, IL-1 a, !L-Ι β, IL-1 R1 , IL-1 R4, IL-1 Ra, IL-2Ra, !L-3, IL-4, !L-5, IL-6, IL-6R, !L-7, IL-8, Leptin, LIGHT, MCP-2, MCP-3, MCP-4, MCSF, MDC, MIG, M!P-3a, ΜΙΡ-3β, MMP13, MMP2, MMP3, MMP8, MMP9, MP-15, SPa, , NAP-2, NT-3, NT-4, Oncostatin M, OPG, OS , Osteoprotegerin, PARC, PDGF, PDGF-BB, PIGF, PLGF, RANTES, SDF-1 a, sgp130, SLF, SPPL3, sTNFR-L sTNFR-ll, TARC, TECK, TGF-βΙ , TGF- 3, THPO, TIMP-1 , T! P-2, TNF, TNF-β, TRAIL-R3, TRAIL-R4, uPAR, VEGF, VEGF-D, WNT-3a, Wnt-Ligands, and XCL-1.

55. A kit for use in the in vitro detection of markers related to risk of cancer relapse in purified plasma extracellular vesicles, wherein said kit comprises means for detection of at least one of the following markers: Hck, CD83, PD-L1 .

58. Use of a kit in the in vitro detection of markers related to risk of cancer relapse in purified plasma extracellular vesicles, wherein said kit comprises means for defection of at least one of the following markers: Hck, CD83, PD-L1 .