FIELD OF THE INVENTION:

The invention relates to the field of medicine, and more particularly to the detection and of Brucella infection and the stratification of the patients.

BACKGROUND OF THE INVENTION:

Brucell spp. are Gram-negative facultative intracellular coccobacilli. They are the causative agent of brucellosis, a worldwide re-emerging zoonosis, affecting livestock (bovine for B. abortus, ovine and caprine for B. melitensis, swine for B. suis and wildlife with serious economic loss, as Brucella infected animals undergo spontaneous abortion and infertility ¹ ' ³ . Brucellosis can be transmitted to humans via contaminated food or infected aerosol particles and discloses a broad spectrum of clinical manifestations ⁴ ’ ⁵ . Acute human brucellosis is characterized by undulant febrile illness, which, if left untreated as often the case, leads to spreading of bacteria in various tissues. This ends up in a chronic disease with severe symptoms, such as osteoarthritis, spondylitis, endocarditis and neurological disorders ^1,2 . B. melitensis is the most frequent cause of human brucellosis ¹ . Due to its high infectivity, Brucella has been classified as a potential warfare agent ⁶ and is manipulated in BSL3 only. Brucella spp. have the ability to enter, survive and replicate within phagocytic and non-phagocytic cells and as such evade host immune system defence mechanisms ⁶ ' ¹⁰ . These properties may account for human brucellosis particularities ¹¹ .

Dendritic cells (DC) have been widely studied in brucellosis and demonstrated to be important for the induction of immune responses as well as providing a safe replication niche for the bacterium ¹² ' ¹⁸ . DCs play a central role in the induction of both innate and adaptive immune responses by activating T cells during the course of Brucella infection ¹² ' ^14,18 . However, Brucella controls DC maturation and TLR signalling by amongst others expressing two Brucella effector proteins, BtpA and BtpB, to counteract the protective Thl immune response ^13,19 and harbouring a non-canonical LPS ^17,20,21 . In vitro, infection ith Brucella leads to moderate activation of mouse DCs as illustrated by intermediate expression levels of costimulatory molecules and cytokine secretion ^13,22,23 . In vivo, DCs promote Brucella dissemination when macrophages are depleted. DCs are also required to control bacterial growth through secretion of pro-inflammatory cytokines such as interleukin- 12 (IL- 12) or enzyme like iNOS ^12,14 . Our recent transcriptomics study identified SLAMF 1 as a strongly upregulated gene in human DCs stimulated with the Brucella 01,2 cyclic glucan (C0G) ²⁴ ’ ²⁵ . SLAMF1 (or SLAM, CD 150) is a cell surface receptor belonging to the signalling lymphocyte activation molecule family (SLAMF) of receptors, itself a member of the Immunoglobulin (Ig) superfamily ²⁶ ’ ²⁷ . SLAMF1 is expressed on haematopoietic cells only and constitutes a self-ligand that triggers T cell activation ²⁸ ' ³⁰ . SLAMF 1 has been involved in immune responses against various pathogens ⁴³ . SLAMF 1, identified as the entry receptor for Measles virus, controls DC and T cell responses during the course of viral infection ³² ’ ³³ . In macrophages, SLAMF 1 also binds E. coll outer membrane proteins OmpC and OmpF, thus regulating bacterial phagosome function and killing bacteria ³⁴ ' ³⁶ . SLAMF 1 plays a dual role as both an activator and an inhibitor of the immune system during tuberculosis and T. cruzi infections ³⁷ ' ⁴⁰ . The role of SLAMF 1 in brucellosis has not been investigated thus far.

Here, the inventors show that in human brucellosis expression of SLAMF 1 as SLAMF7, SLAMF8, SH2D1A, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E is specifically upregulated at the gene level in blood leukocytes of acute phase patients only, whereas other serum biomarkers define the chronic stage of the disease.

SUMMARY OF THE INVENTION:

The present invention relates to the detection of Brucella infection and the stratification of the patients (those with acute phase or chronic phase of the infection). In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Brucella is a facultative intracellular pathogenic bacterium responsible for brucellosis, a worldwide re-emerging zoonotic disease affecting wildlife and livestock. Brucella can be transmitted to humans via contaminated food or infected aerosol particles, thereby leading to undulant fever followed by chronic devastating multi-organ inflammation if untreated. By RNA-seq analysis of blood of patient cohorts from Spain and Macedonia, the inventors have identified SLAMF1 together with SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes and IL-9, IL-31, TNF0 and IL- 22, IL-la, VEGF-D, CXCL10, 0NGF proteins as biomarkers of the acute phase of Brucella infection in humans. In addition, cytokine analysis led the inventors to define a set of four serum biomarkers (IL-10, IL1RA, CXCL1 and IL-7) for Brucella chronicity. SLAMF1, 7 and 8 are immune cell receptors expressed notably at the surface of dendritic cells (DC), lymphocytes and haematopoietic stem cells and involved in the control of immune responses.

Method for diasnosins brucellosis

Accordingly, the present invention relates to a method for diagnosing brucellosis in a subject, comprising the steps of i) determining in a sample obtained from a patient, the expression level of at least three markers selected in the group consisting in SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PDL1, LAG3, LY6E genes and IL-9, IL-31, TNF p, IL-22, IL-la, VEGF-D, CXCL10, pNGF, IL-10, CXCL1, IL1RA, and IL-7 proteins; ii) comparing the expression of the markers determined at step i) with a reference value; and iii) concluding that the patient has brucellosis when the expression level of the markers determined at step i) are higher than the reference value.

In some embodiments, the method of the present invention is performed in vitro or ex vivo

In some embodiment, brucellosis is in its acute phase, and the expression level of at least three markers selected in the group consisting in SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2 PDL1, LAG3, LY6E, and NKG7 genes and IL- 9, IL-31, TNF P, IL-22, IL-la, VEGF-D, CXCL10, PNGF proteins are determined in the step i).

In some embodiment, brucellosis is in its acute phase, and the expression level of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 markers selected in the group consisting in SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2 PDL1, LAG3, LY6E, and NKG7 genes and IL-9, IL-31, TNF p, IL-22, IL-la, VEGF-D, CXCL10, PNGF proteins are determined in the step i).

Thus, in some embodiment, the present invention relates to a method for diagnosing acute phase of brucellosis in a subject, comprising the steps of i) determining in a sample obtained from the patient, the expression level of at least three markers selected in the group consisting m SLAMFl, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2 PDL1, LAG3, LY6E, and NKG7 genes and IL-9, IL-31, TNF p, IL-22, IL-la, VEGF-D, CXCL10, PNGF proteins; ii) comparing the expression of the markers determined at step i) with a reference value; and iii) concluding that the patient has acute phase of brucellosis when the expression level of the markers determined at step i) are higher than the reference value

In some embodiment, brucellosis is acute phase of brucellosis, and the expression level of at least three markers selected in the group consisting in SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, PDL1, LAG3, LY6E, and NKG7 genes are determined in the step i).

In other words, the invention relates to a method for diagnosing acute phase of brucellosis in a subject, comprising the steps of i) determining in a sample obtained from the patient, the expression level of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 genes selected in the group consisting m SLAMFl, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E, ii) comparing the expression of the genes determined at step i) with a reference value, and iii) concluding that the patient has an acute phase of brucellosis when the expression levels determined at step i) are higher than the reference value.

In some embodiment, brucellosis is in its acute phase of brucellosis and the expression level of SLAMF1 and at least two markers selected in the group consisting in SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes and IL-9, IL-31, TNFp, IL-22, IL-la, VEGF-D, CXCL10, pNGF proteins are determined in the step i).

In some embodiment, the present invention relates to a method for diagnosing acute phase of brucellosis in a subject, comprising the steps of i) determining in a sample obtained from the patient, the expression level of SLAMF1 and at least two markers selected in the group consisting in SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes, and IL-9, IL-31, TNFp, IL-22, IL-la, VEGF-D, CXCL10, PNGF proteins ii) comparing the expression of the markers determined at step i) with a reference value and iii) concluding that the patient has an acute phase of brucellosis when the expressions level determined at step i) are higher than the reference value.

In some embodiment, brucellosis is in its acute phase, and the expression level of SLAMF1 gene, and at least two genes selected in the group consisting in SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E are determined in the step i).

In some embodiment, brucellosis is in its acute phase, and the expression level of SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes are determined in the step i).

Thus, in some embodiment, the invention relates to a method for diagnosing acute phase of brucellosis in a subject, comprising the steps of i) determining in a sample obtained from a patient, the expression level of SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes, ii) comparing the expression level o SLAMFl, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes determined at step i) with a reference value and iii) concluding that the patient has an acute phase of brucellosis when the expression level of SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PD-L1, LAG3 and Ly6E genes determined at step i) are higher than the reference value.

In some embodiment, brucellosis is in its acute phase, and the expression level of at least three markers selected in the group consisting in IL-9, IL-31, TNFP, IL-22, IL-la, VEGF- D, CXCL10 and 0NGF proteins are determined in the step i).

Thus, the invention relates to a method for diagnosing acute phase of brucellosis in a subject, comprising the steps of i) determining in a sample obtained from the patient, the expression level of 3, 4, 5, 6, 7 or 8 markers selected in the group consisting in IL-9, IL-31, TNFP, IL-22, IL-la, VEGF-D, CXCL10 and PNGF proteins, ii) comparing the expression of the markers determined at step i) with a reference value and iii) concluding that the patient has an acute phase of brucellosis when the expression level determined at step i) is higher than the reference value.

In some embodiment, brucellosis is in its acute phase, and the expression level of IL-9, IL-31, TNFP proteins are determined in the step i).

Thus, the invention relates to a method for diagnosing acute phase of brucellosis in a subject, comprising the steps of (i) determining in a sample obtained from the patient, the expression level of IL-9, IL-31, and TNFP, ii) comparing the expression of the markers determined at step i) with a reference value and iii) concluding that the patient has an acute phase of brucellosis when the expression level determined at step i) is higher than the reference value.

In some embodiment, brucellosis is in its chronic phase and the expression level of 3 or 4 markers selected in the group consisting in IL-10, CXCL1, IL1RA and IL-7 are determined in the step i).

In some embodiment, brucellosis is in its chronic phase, and the expression level of IL- 10, CXCL1, IL IRA and IL-7 are determined in the step i).

Thus, the present invention relates to a method for diagnosing chronic phase of brucellosis in a subject, comprising the steps of i) determining in a sample obtained from the patient, the expression level of IL-10, CXCL1, IL1RA, and IL-7, ii) comparing the expression of the markers determined at step i) with a reference value and iii) concluding that the patient has a chronic phase of brucellosis when the expression level determined at step i) is higher than the reference value.

As used herein, the term “subject” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the term “subject” refers to a human and more particular, to a human afflicted with brucellosis.

As used herein, the term “brucellosis” refers to a bacterial zoonosis caused by Brucella, a genus of gram-negative bacteria that behave as facultative intracellular pathogens of ruminants, swines, canids, camelids and several forms of wildlife. These bacteria are extremely infectious, and humans acquire brucellosis readily from animals and their products. There are several highly homologous Brucella species (Moreno et al., Vet.Microbiol.90,209-227,2002; Chain et al., Infect.Immun. 73,8353-8361,2005) among which B. abortus preferentially infects cattle, B. melitensis sheep and goats, and B. suis swine and wildlife. These three species are the main cause of human brucellosis. Sheep may also be infected by B. ovis. Patients with acute, uncomplicated brucellosis usually recover in 2 to 3 weeks, even without treatment. Some go on to subacute, intermediary, or chronic disease. Patients were divided into three groups according to their history, symptoms and clinical presentation time. As used herein, the term “acute phase” refers to brucellosis that it is of short duration (i.e with symptoms less than 3 months) and, as a corollary of that, of recent onset. Sooner the acute phase is treated, chronic phase can be prevented.

As used herein, the term “chronic phase” refers patients whom exhibited symptoms more than one year).

As used herein and according to all aspects of the invention, the term “sample’ denotes blood, serum, fresh whole blood, peripheral-blood, peripheral blood mononuclear cell (PBMC), lymph sample. In particular embodiment, the sample is a blood sample, and more particularly serum or peripheral blood mononuclear cell (PBMC).

As used herein, the term “serum” has its general meaning in the art and refers to liquid part of the blood after the coagulation.

As used herein, the term “SLAMF1” for “Signaling lymphocytic activation molecule 1”, refers to a gene coding for SLAMF1 protein, also known as “CD150", which is modulating the activation and differentiation of a wide variety of immune cells and thus are involved in the regulation and interconnection of both innate and adaptive immune response. Its Entrez reference is 6504. As used herein, the term “SLAMF7” for “Signaling lymphocytic activation molecule 1”, refers to a gene coding for SLAMF7 protein, also known as “19A; CS1; CD319; CRACC", which is modulating the activation and differentiation of a wide variety of immune cells and thus are involved in the regulation and interconnection of both innate and adaptive immune response. Its Entrez reference is 57823.

As used herein, the term “SLAMF8” for “Signaling lymphocytic activation molecule 1”, refers to a gene coding for SLAMF8 protein, also known as “BLAME; CD353; SBBI42", which is modulating the activation and differentiation of a wide variety of immune cells and thus are involved in the regulation and interconnection of both innate and adaptive immune response.. Its Entrez reference is 56833.

As used herein, the term “PDL1” for “Programmed death-ligand 1”, refers to a gene coding for PD-L1 protein, also known as “CD274; B7-H; B7H1; PD-L1; hPD-Ll; PDCD1L1; PDCD1LG1 ", which is an immune inhibitory receptor ligand that is expressed by hematopoietic and non-hematopoietic cells. Its Entrez reference is 29126.

As used herein, the term “LAG3” for "Lymphocyte-activation protein 3”, refers to a gene coding for LAG3 protein, also known as “CD223", which is modulating the activation and differentiation of immune cells, such as T and B cells, and thus involved in the regulation adaptive immune response. Its Entrez reference is 3902.

As used herein, the term “LY6E” for “Lymphocyte antigen6 family member molecule E”, refers to a gene coding for SLAMF1 protein, also known as “RIGE; SCA2; RIG-E; SCA- 2; TSA-1", which is modulating the activation and differentiation of a wide variety of immune cells and thus involved in the regulation and interconnection of both innate and adaptive immune response. Its Entrez reference is 4061.

As used herein, the term “SH2D1A” for “SH2 domain-containing 1A” refers to a gene coding for protein that plays a major role in the bidirectional stimulation of T and B cells and is associated to SLAM signaling. Its Entrez reference is 4068.

As used herein, the term “C1QC” for “Complement Clq Chain” refers to a gene coding for the C-chain polypeptide of serum complement subcomponent Clq, which associate with Clr and Cis to yield the first component of the serum complement system. It entrez referenc is 714.

As used herein, the term “EOMES” for “Eomesodermin” refers to a gene coding for T- box brain protein 2 (Tbr2), a member of a conserved protein family that shares a common DNA- binding domain, the T-box. Its Entrez reference is 8320. As used herein, the term “TBX21” for “T-box transcription factor TBX21” refers to a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. T-box genes encode transcription factors involved in the regulation of developmental processes. Its Entrez reference is 30009.

As used herein, the term “GZMK” for “Granzyme K” refers to a member of a group of related serine proteases from the cytoplasmic granules of cytotoxic lymphocytes. Cytolytic T lymphocytes (CTL) and natural killer (NK) cells share the remarkable ability to recognize, bind, and lyse specific target cells. Its Entrez reference is 3003.

As used herein, the term “GZMH” for “Granzyme H” refers to a gene encoding a member of the peptidase SI family of serine proteases. This protein is reported to be constitutively expressed in the NK (natural killer) cells of the immune system and may play a role in the cytotoxic arm of the innate immune response by inducing target cell death and by directly cleaving substrates in pathogen-infected cells. Its Entrez reference is 2999.

As used herein, the term “AIM2” for “Absent in melanoma 2” refers to a gene encoding the interferon-inducible protein AIM2 which contributes to the defence against bacterial and viral DNA. Its Entrez reference is 9447.

As used herein, the term “NKG7” for “Natural Killer Cell Granule Protein 7” refers to a gene on human chromosome 9 that is expressed in natural killer cells and T cells. Its Entrez reference is 4818.

As used herein, the term “IL-9” for “Interleukine 9” is a pleiotropic cytokine (cell signalling molecule) belonging to the group of interleukins. IL-9 is produced by variety of cells like mast cells, NKT cells, Th2, Thl7, Treg, ILC2, and Th9 cells in different amounts. Its Entrez reference is 3578 and its Uniprot reference is P15248.

As used herein, the term “IL-31” for “Interleukine 31” is is an inflammatory cytokine that helps trigger cell-mediated immunity against pathogens. It has also been identified as a major player in a number of chronic inflammatory diseases, including atopic dermatitis. Its Entrez reference is 386653 and its Uniprot reference is Q6EBC2.

As used herein, the term “TNFP” for “tumor necrosis factor-beta”, also known as “lymphotoxin-alpha”, is a protein that in humans is encoded by the LTA gene. Belonging to the hematopoietic cell line, LT-a exhibits anti-proliferative activity and causes the cellular destruction of tumor cell lines. Its Entrez reference is 4049 and its Uniprot reference is P01374.

As used herein, the term “IL-22” for “Interleukin 22” is an a-helical cytokine. IL-22 binds to a heterodimeric cell surface receptor composed of IL-10R2 and IL-22R1 subunits. Its entrez reference is 50616 and its Uniprot reference is Q9GZX6. As used herein, the term “IL- la” for “Interleukin 1 alpha” also known as hematopoietin 1 is a cytokine of the interleukin 1 family that in humans is encoded by the ILIA gene. Its entrez reference is 3552 and its Uniprot reference is P01583.

As used herein, the term “VEGF-D” for “Vascular Endothelial Growth Factor D” is a member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family and is active in angiogenesis, lymphangiogenesis, and endothelial cell growth. Its entrez reference is 2277 and its Uniprot reference is 043915.

As used herein, the term “PNGF” for “Beta-Nerve Growth Factor” is a member of the NGF-beta family and encodes a secreted protein which homodimerizes and is incorporated into a larger complex. Its entrez reference is 4803 and its Uniprot reference is P01138.

As used herein, the term “IL- 10” for “Interleukin 10”, also known as “human cytokine synthesis inhibitory factor (CSIF)”, is an anti-inflammatory cytokine. In humans, interleukin 10 is encoded by the IL10 gene. IL-10 signals through a receptor complex consisting of two IL- 10 receptor- 1 and two IL- 10 receptor-2 protein. Its Entrez reference is 3586 and its Uniprot reference is P22301.

As used herein, the term “CXCL10” for “chemokine (C-X-C motif) ligand 10”, also known as interferon gamma-induced protein 10 (IP- 10), is a small cytokine belonging to the CXC chemokine family that is secreted by several cell types in response to IFN-y. In humans, this protein is encoded by the gene CxcllO. Its Entrez reference 3627 and its Uniprot reference is P02778.

As used herein, the term “CXCL1” for “chemokine (C-X-C motif) ligand 1”, also known as FSP, GRO1, GROa, MGSA, MGSA-A, NAP-3 or SCYB1, is a small cytokine belonging to the CXC chemokine family that was previously called GRO1 oncogene, GROa, KC, neutrophil-activating protein 3 (NAP-3) and melanoma growth stimulating activity, alpha (MGSA-a). In humans, this protein is encoded by the gene Cxcll. Its Entrez reference 2919 and its Uniprot reference is P09341.

As used herein, the term “IL1RA” for “interleukin 1-receptor antagonist (IL1-RA)”, also known as IL1-RN, IRAP, or ICIL-1RA, is a protein that in humans is encoded by the IL1RN gene. IL-IRA is an agent that binds non-productively to the cell surface interleukin-1 receptor (IL-1R), the same receptor that binds interleukin 1 (IL-1), preventing IL-1 from sending a signal to that cell. Its Entrez reference 3557 and its Uniprot reference is Pl 8510.

As used herein, the term “IL-7” for “interleukin 7” is a protein that in humans is encoded by the IL7 gene. IL-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. It is also produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells, but is not produced by normal lymphocytes. Its Entrez reference 3574 and its Uniprot reference is Pl 3232.

As used herein, the term "expression level" refers, e.g., to a determined level of expression of gene of interest or protein of interest. The expression level of expression indicates the amount of expression product in a sample. The expression product of a gene of interest can be the nucleic acid of interest itself, a nucleic acid transcribed or derived therefrom, or the a polypeptide or protein derived therefrom.

Measuring the expression level of the genes listed above can be done by measuring the gene expression level of these genes and can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labelled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labelled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colourless substance into a coloured substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5 ',5" dibro mopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5 -[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDaminofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).

Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.

Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe- specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210). In another embodiment, the expression level is determined by metabolic imaging (see for example Yamashita T et al., Hepatology 2014, 60: 1674-1685 or Ueno A et al., Journal of hepatology 2014, 61 : 1080-1087).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the response of antipsychotic treatment, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, TBP and ABLP This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

Measuring the expression level of the proteins listed above can be done by measuring the gene expression level of these proteins and can be performed by a variety of techniques well known in the art.

Typically protein expression level may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS), flow cytometry, mass cytometry or ELISA performed on the sample.

In the present application, the “level of protein” or the “protein level expression” means the quantity or concentration of said protein. In particular embodiment, the protein is expressed at the cell surface for markers whose function is linked to their correct plasma membrane expression or total expression for markers whose function is not limited to membrane expression. In still another embodiment, the “level of protein” means the quantitative measurement of the proteins expression relative to a negative control.

Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresismass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Particularly, a mass spectrometry-based quantification methods may be used. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches [DeSouza and Siu, 2012], Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labeling or proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, a quantification based on extracted ion chromatogram (EIC) and then profile alignment to determine differential level of polypeptides.

Particularly, a mass spectrometry-based quantification method particularly useful can be the use of targeted mass spectrometry methods as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data independent acquisition (DIA) and sequential window acquisition of all theoretical mass spectra (SWATH) [Moving target Zeliadt N 2014 The Scientist;Liebler Zimmerman Biochemistry 2013 targeted quantitation pf proteins by mass spectrometry; Gallien Domon 2015 Detection and quantification of proteins in clinical samples using high resolution mass spectrometry. Methods v81 pl 5-23 ; Sajic, Liu, Aebersold, 2015 Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications. Proteomics Clin Appl v9 p 307-21], Particularly, the mass spectrometry-based quantification method can be the mass cytometry also known as cytometry by time of flight (CYTOF) (Bandura DR, Analytical chemistry, 2009).

Particularly, the mass spectrometry-based quantification is used to do peptide and/or protein profiling can be use with matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF), surface-enhanced laser desorption/ionization time of flight (SELDI-TOF; CLINPROT) and MALDI Biotyper apparatus [Solassol, Jacot, Lhermitte, Boulle, Maudelonde, Mange 2006 Clinical proteomics and mass spectrometry profiling for cancer detection. Journal: Expert Review of Proteomics V3, 13, p311-320 ; FDA K130831],

Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins of the invention. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins of the invention.

In some embodiments, the reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the score in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the score in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured expression levels of the gene(s) in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE- ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is determined by carrying out a method comprising the steps of a) providing a collection of samples; b) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding subject (i.e. the duration of the survival); c) providing a serial of arbitrary quantification values; d) determining the expression levels of different genes for each sample contained in the collection provided at step a) so as to calculate the score as described above; e) classifying said samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for the score that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said score that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the patients from which samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g). For example the score has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to the determined score. Sample 1 has the highest score and sample 100 has the lowest score. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding subject, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the score corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value.

Such reference values of expression level may be determined for any marker defined above.

In another aspect, the invention relates to an in vitro method for determining the patient’s susceptibility to relapse from an acute phase of brucellosis in a subject, comprising a step of (i) determining in a sample obtained from the patient, the protein expression level of IL- 22, IL- la, CXCL10 and 0NGF, ii) comparing the expression of the markers determined at step i) with a reference value and iii) concluding that the patient has a risk to relapse from an acute phase of brucellosis when the expression level determined at step i) is higher than the reference value.

As used herein, the term “risk to relapse” has its general meaning in the art and refers to the recurrence is the recurrence of signs and symptoms of illness, with or without the presence of bacteria in blood, after the period of treatment. Relapse is major problem in therapy ofbrucellosis.

In another particular embodiment, a step of communicating the result to the patient may be added to all the methods of the invention. The result can be a result about the diagnostic of acute or chronic brucellosis.

Method for treatins and monitorins brucellosis

Accordingly, subject identified with a poor prognosis according to the invention can be administered therapy, for example systematic therapy. Subject identified with a malignancy tumor according to the invention can also be administered therapy, for example systematic therapy. Thus, the invention relates to a method for treating brucellosis in a patient in need thereof comprising administering a therapeutically effective amount of classical treatment of brucellosis when the patient is diagnosed with brucellosis according to the invention.

In some embodiment, brucellosis is acute phase of brucellosis

The invention relates to a method for treating acute phase of brucellosis in a patient in need thereof comprising administering a therapeutically effective amount of classical treatment of brucellosis when the patient is diagnosed with acute phase of brucellosis according to the invention.

The invention also relates to a method for treating chronic phase of brucellosis in a patient in need thereof comprising administering a therapeutically effective amount of a compound selected from the group consisting in: IL-10 inhibitor, CXCL1 inhibitor, IL1RA inhibitor and IL-7 inhibitor.

In some embodiment, the patient has been diagnosed with chronic phase of brucellosis according to the invention.

The term “inhibitor” as used herein, refers to an agent that is capable of specifically inhibit the expression or activity of a target molecule, i.e IL-10, CXCL1, IL1RA or IL-7. An “expression inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.

The inhibitor can be a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

As used herein, the term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

As used herein, the term “aptamers” refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique. In a particular embodiment, the inhibitor is a neutralizing antibody. In a particular, the inhibitor is an intrabody having specificity for target molecule, i.e IL-10, CXCL1, IL1RA or IL-7. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of target molecule, i.e. IL- 10, CXCL1, IL IRA or IL-7. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Antisense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell.

As used herein, the term “anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of target mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of target proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

As used herein, the term “IL- 10 inhibitor” refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of IL- 10. In a particular embodiment, the IL- 10 inhibitor is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide

In particular embodiment, the IL- 10 inhibitor is an anti-IL-10 neutralizing antibody.

As used herein, the term “CXCL1 inhibitor” refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of CXCL1.

In a particular embodiment, the CXCL1 inhibitor is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

In particular embodiment, the CXCL1 inhibitor is an anti-CXCLl neutralizing antibody. Thus, in particular, the invention relates to a method for treating chronic phase of brucellosis in a patient in need thereof comprising administering a therapeutically effective amount of an anti-CXCLl neutralizing antibody.

As used herein, the term “IL IRA inhibitor” refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of IL1RA. In a particular embodiment, the IL1RA inhibitor is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

In particular embodiment, the IL IRA inhibitor is an anti-ILIRA neutralizing antibody.

As used herein, the term “IL-7 inhibitor” refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of IL-7. In a particular embodiment, the IL-7inhibitor is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

In particular embodiment, the IL-7 inhibitor is an anti-IL-7 neutralizing antibody.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.

As used herein, the term “classical treatment of brucellosis” has its general meaning in the art and refers to any compound, natural or synthetic, used for the treatment of brucellosis.

Example of compounds used for the treatment of brucellosis include antibiotics such as tetracyclines, rifampicin, aminoglycosides, ciprofloxacin, streptomycin, doxycycline, co- trimoxazole and gentamicin.

The invention relates to a therapeutic composition comprising a compound according to the invention for use in the treatment of brucellosis in a patient diagnosed with brucellosis as described above.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like. Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Kit of the invention

The present invention includes a kit for performing the method of the present invention comprising means for determining the level of SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7 PDL1, LAG3, LY6E, IL-9, IL-31, TNFp, IL- 22, IL-la, VEGF-D, CXCL10, pNGF, IL-10, CXCL1, IL1RA, and/or IL-7 expression in a sample.

Thus, a further object of the invention is a kit suitable for diagnosing brucellosis comprising:

At least a means for determining the expression level of SLAMF1, SLAMF7, SLAMF8, SH2D1A, C1QC, EOMES, TBX21, GZMK, GZMH, AIM2, NKG7, PDL1, LAG3, LY6E, IL-9, IL-31, TNF/3, IL-22, IL-la, VEGF-D, CXCL10, [3NGF, IL-10, CXCL1, IL1RA, and/or IL-1 expression in a sample obtained from a subject, Instructions for use.

As used herein, the term “means for determining” denotes all physical means which are able to bind to the different markers. For example, means for determining the markers may be an antibody against a marker coupling with a signalling system.

Typically the kit may include primers, probes, an antibody, or a set of antibodies. In a particular embodiment, the antibody or set of antibodies are labelled. The kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Expression of SLAMF1 marks acute phase patients. A. Volcano plots representing overall gene expression changes observed in acute relapse (right) and chronic (left) infected groups. Each point represents a gene, the dashed vertical lines indicate log2 foldchanges of -1 and +1, and the dashed horizontal line indicates a false discovery rate (FDR) of 0.01. Genes displaying statistically significant differences in expression compared with Controls are marked in black. B, B’. The median expression of selected differentially expressed genes is shown. X-axis: control (n = 36), acute treated (n = 54), acute with relapse (n = 6), chronic (n = 12). Y-axis: log2 residual gene expression counts after regressing out the effect of leukocyte proportions. C. Multivariate analysis (OPLS-DA) loading plot of cytokine and chemokine secretion (white circle) measured by Luminex in the sera of the three different groups of patients, acute (n = 54), acute with relapse (n = 6) and chronic (n = 12), together with Controls (n = 36) (black square). Variables near each other are positively correlated; variables opposite to each other are negatively correlated. Variables closer to dots corresponding to “acute, acute relapse, chronic” or “controls” dots (i.e. with the largest absolute loading values) are higher in the corresponding populations. P=5.34218e-006.

Figure 2. In a mouse model of brucellosis, neutrophils are recruited to peritoneal cavity and omentum from the onset to the chronic phase of infection by Brucella abortus. A. C57BL/6 mice were injected i.p. with 106 CFU B. abortus or mock-injected with PBS. At 2, 8, and 30 days, peritoneal fluid and omentum were collected. Individualized cells were then analyzed by flow cytometry. Normalized absolute numbers of omental and peritoneal Ly6G+ cells at the indicated time-points are shown, each symbol representing one animal. 2-5 mice per group, n= 3. Mean ± SD from pooled data. B. Normalized absolute numbers of peritoneal Ly6G+ cells expressing Scal+, PD-L1+ or both markers together with PD-L1 and Seal individual mean fluorescence intensity (MFI) at 2, 8 and 30 days post-infection. Each symbol represents one animal. 2-5 mice per group, n= 3. Mean ± SD from pooled data (represented by a vertical bar for MFI). *, P<0.05; **, P< 0.01; ***, P<0.001; ****, P<0.0001; ns, non significant. Mann Whitney test.

Figure 3. The early recruitment of Ly6G+ and PD-Ll+Scal+ Ly6G+ cells in the omentum and peritoneal cavity is triggered by Brucella LPS independently of its core, which is in contrast required for persistence of these cells at late time-points. Omentum and peritoneal fluid were collected at 4h, 12h, 24h, 48h and 72h after i.p. challenge with Ba- wt, Ba-wadC or E.coli (Ec) LPS, or mock-PBS injection, and individualized cells analyzed by flow cytometry. A. Kinetics of omental and peritoneal Ly6G+ neutrophils are shown. Mean ± SD from pooled data of normalized absolute numbers. Significant differences from Ba-wt LPS are displayed. On the right panels, Ly6G+ cells, with each symbol representing one animal, are shown at 4h, 48h and 72h. Mean ± SD from pooled data of normalized absolute numbers. 2-5 mice per group, n= 3. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Absence of P value, non-significant. Multiple comparison Kruskal Wallis ANOVAtest, followed by variance analysis with the Dunn’s test. B. Kinetics of Ly6G+ macrophages expressing Seal, PD-L1 or both surface markers. Mean ± SD from pooled data of normalized absolute numbers and significant differences from Ba-wt LPS are shown. Individual points, with each symbol representing one animal, are presented at 48h post-challenge at the right of each kinetics. Mean ± SD from pooled data of normalized absolute numbers. 2-5 mice per group, n= 3. *, P .05; **, P .01; ***, P .001; ****, PO.OOOL Absence of P value, non-significant. Multiple comparison Kruskal Wallis ANOVA test, followed by variance analysis with the Dunn’s test. C. Representative confocal microscopy image of an omentum collected 48h after i.p. injection of Ba-wt LPS in a C57BL/6 mouse, and stained for Ly6G (magenta, neutrophils), CD64 (yellow, macrophages) and CD3 (cyan, T cells). Scale bar, 100 pm. 2-3 mice per group, n= 3.

Figure 4. CXCL1 mediates recruitment of neutrophils to the omentum. A. Serum, peritoneal fluid and omentum were collected from C57BL/6 mice 24h or 48h after i.p. challenge with Ba-wt LPS or mock-PBS injection. CXCL1 and CCL2 cytokines were measured by ELISA. Mean ± SD of normalized absolute numbers. Pooled data. 3 mice per group, n= 2. *, P<0.05; ***, P0.001; ****, PO.OOOL Absence of P value, non-significant. Sidak's multiple comparison test. B. Representative plots of Ly6C+ monocytes secreting CXCL1+ from omenta collected 48h after challenge of C57BL/6 mice with Ba-wt LPS or PBS, incubated with brefeldin A for 3h and then analyzed by flow cytometry. C. Flow cytometry analysis of omenta collected 48h after challenge of C57BL/6 mice with Ba-wt or E.coli (Ec) LPS, or mock-PBS and treated as desccribed in B. Normalized absolute numbers of Ly6C+CXCLl+ , F4/80+CXCL1+ and Ly6G+CXCLl+ cells, with each symbol representing one animal. Mean ± SD from pooled data of normalized absolute numbers. 2-3 mice per group, n= 3. *, PO.05; ****, PO.OOOL Absence of P value, non-significant. Multiple comparison Kruskal Wallis ANOVA test, followed by variance analysis with the Dunn’s test.

Table 1. P values of different clinical group comparisons for main regulated genes in our human brucellosis cohort. PBMC were isolated using Ficoll from blood of healthy volunteers or brucellosis patients, and RNA sequencing performed as described in Materials and Methods. Number of patients enrolled in each clinical group: Control, n = 36; Acute treated, n = 54; Acute with relapse, n = 6; Chronic, n = 12. On selected genes, groups were analysed with the non-parametric Krukal Wallis test followed by variance analysis with the Post-hoc Dunn test. P values <0.05 were considered as significant. P values >0.05 are shown in italics.

EXAMPLE:

Material & Methods

Ethics

As regards human PBMCs, blood was taken from healthy volunteers acquired under protocols approved by the Institutional Review Board (IRB) of the University General Hospital of Albacete, Albacete, Spain, the University Clinic for Infectious Diseases and Febrile Conditions, Skopje, Macedonia and the Benaroya Research Institute, Seattle, USA.

Human study design Patients were enrolled for clinical studies from 2010 to 2015 at the University General Hospital of Albacete, Albacete, Spain and the University Clinic for Infectious Diseases and Febrile Conditions, Skopje, Macedonia, following a procedure established by the Benaroya Research Institute, Seattle, USA. Adults seeking medical care for acute fever (less than 8 days); who were positive for n -Brucella Ig in their serum were invited to participate. Healthy, age- matched controls were adults visiting hospital for routine vaccinations or simple fractures. On admission, a trained nurse applied a standardized questionnaire to gather demographic and clinical data. Whole blood samples were collected in EDTA and Tempus tubes from each adult, on or shortly after admission. One set of EDTA tubes were sent to the routine clinical laboratory for complete blood counts and the other set was used to prepare serum, which was partly frozen at -80°C and sent to the CIML on dry ice for cytokine dosage. Tempus tubes were frozen at - 20°C and shipped to the Benaroya Research Institute on dry ice. Blood samples were transported to the laboratory within 1 hour. In total data were collected from 56 healthy controls and 145 brucellosis patients with a range of treatment outcomes. Patient disease was classified based on previous history of Brucella infection and progression of symptoms during treatment. Gene expression data was collected via RNAseq at initial visits.

RNA sequencing

Reagents

Antibodies used in flow cytometry are the following, F4/80-BV785 (cloneBM8), CD86- FITC (clone GL-1), CD15O/SLAMF1-PE/Cy7 (clone TC15-12F12.2), CD80-PE/Cy5 (clone 16-10A1), CD40-Alexa 647 (clone 3/23), MHC II (I-A/I-E)-AF700 (clone M5/114.15.2), CDl lc-APC/ Cy7 (clone MI/70), CD19-APC-Cy7 (clone N418), NKl.l-APC-Cy7 (clone PK136), CD3-BV650 (clonel7A2), CD8-BV711 (clone 53-6.7), CD69PE-Cy7 (clone Hl.2F3), XCR1-Bv421 (clone ZET), CD317/Bst2, CDl lc-PE-Cy7 (clone N418), CD172a-SIRPa-APC (clone 84) were all purchased from BioLegend. CD4-eFluor450 (clone RM4-5), CD3 eFluor450, CD317/Bst2-PE-eFluor610 (clone eBio927) were purchased from eBioscience. CD1 Ib-PE (clone MI/70) was purchased from BD Biosciences.

Cell Culture

BMDC were prepared from 6-8 week-old C57BL/6J or Slamfl' ¹ ' female femurs and tibias as previously described ¹⁵ . Briefly, bone ends were cut off and bone marrow was flushed with RPMI 1640 (Gibco, Life Technologies) supplemented with 5% FCS, 100 lU/mL Penicillin, 100 pg/mL Streptomycin and 50 pM 2-mercaptoethanol. Red blood cells were removed by 1 min exposure to IxRBC lysis buffer solution (eBioscience). 3x10 ⁶ cells were seeded onto 6-well plates in 5 mL medium containing 0.8 % supernatant of the J558L GM-CSF producing cell line. Medium was changed at day 2.5 and GM-DCs were ready to use at day 5. Cells were mock-treated or stimulated with E. coli LPS (100 ng/mL), B. abortus C0G (10 pg/mL) or infected for 16 h. Cells were collected for flow cytometry and supernatant was kept at -80°C for cytokine dosage.

RNA extraction and RT

Total RNA were extracted from infected BMDC using RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. cDNAs were generated by using Quantitech Reverse Transcription Kit (Qiagen) according to manufacturer’s recommendations using 300 ng of RNA as a template.

Cytokine dosage

Cytokines and chemokines from patient sera were analysed by Luminex. Cytokine profiles (TNFa, IFNy, and IL-6) in BMDC culture supernatants were analysed by cytometric beads assay (BD Biosciences, Mouse Inflammation kit).

Flow cytometry

Cells were stained for 20 min at 4°C with the antibodies listed above, then washed once in 2% FCS in IxPBS and once in IxPBS. Infected cells were then fixed for 20 min in 3 % PF A at 22°C. Events were collected on flow cytometry using a or FACS LSRII UV or Fortessa (BD Biosciences) and analysis was performed on FACS DIVA and FlowJo softwares (BD Biosciences).

Statistical analysis

Statistical analyses were done using with the GraphPad Prism software. When comparing groups of our human cohort, Kruskal-Wallis rank sum test was performed followed by variance analysis with the Post-hoc Dunn test on group comparisons. For mouse data, Brown-Forsythe ANOVA test followed by variance analysis with the Welch’s test were performed, otherwise indicated in the figure legend. Mantel Cox test was used for survival curve. All values are expressed as mean ± standard deviation. Differences between values were considered significant at p<0.05,* (/?<0.05; **, <0.001; ***, P<0.0001). All experiments were performed at least three times in triplicate otherwise indicated.

• EXAMPLE 1

Results

We postulated that human patients infected with Brucella would elicit distinct patterns of gene expression in their peripheral blood according to their clinical outcome and that these gene expression profiles might be used to define immune responses associated with this pathogen and disease stages. To test this assumption, we performed RNA sequencing (RNA- seq) on whole blood samples collected from adult patients exhibiting at their first visit acute infection (n = 54), acute infection with relapse (i.e. patients who developed disease relapse while treated) (n = 6), chronic infection (n = 12) as well as from healthy control subjects (n = 36). Transcript profiling by RNA-seq of blood peripheral leukocytes of this patient cohort allowed for quantitative unbiased analysis of all expressed genes. To identify infection-specific effects on patient gene expression, we performed differential gene expression analysis and assigned genes to gene network modules using weighted gene correlation network analysis (WGCNA) (data not shown). Expression of individual genes clearly separated clinical groups: Chronic patients displayed an overall gene expression similar to healthy controls with no significant difference; Acute patients and Acute patients with relapse presented numerous transcriptional changes relative to healthy controls. Interestingly Acute patients with relapse showed larger magnitude modifications than Acute ones. Up-regulated genes were enriched for response to Interferon (IFN) gamma, cytolysis, T cell proliferation and cell cycle; down- regulated genes included genes involved in B cell proliferation. The Volcano plot in 1A highlights the enhanced phenotype of the Acute with relapse patient group versus the Chronic one. Figures IB, IB’ and Table 1 detail significant variations in expression of selected genes. SLAMF1 gene expression was significantly upregulated in peripheral blood leukocytes of Acute patients compared to healthy controls or to Chronic group; this rise in SLAMF1 expression was further exacerbated in the Acute with relapse group. Again no significant differences in SLAMF1 expression were observed between Chronic group and healthy controls. The SH2D1A gene which encodes the cytoplasmic adapter for SLAM family members followed the same pattern of expression, as did C1QC, encoding the first component of serum complement, or genes coding for proteins involved in THI response (such as those of the transcription factors EOMES, TBX21/T-BET), innate immune response like granzymes, GZMK and GZMH, inflammasome component AIM2, and NKG7, expressed in T cells and NK cells. Expression profiles of the CD19 and SSH2 genes were shown as examples of respectively invariant and down-regulated genes upon human Brucella Acute or Acute with relapse infection.

We also quantified in patient sera the levels of cytokines/chemokines potentially related to their immune response to Brucella infection. Then, we tested the correlations between cytokine and gene expression levels together with their variations amongst patient groups by using the OPLS-DA (Orthogonal Partial Least-Squares Discriminant Analysis) model created from the entire cohort by SIMCA (Figure 1C). A number of serum cytokines appeared to be responsible for the discrimination between groups (P=5.34218e-006?). In particular, Acute patients secreted significantly more IL-9, IL-31 and TNF0, all pro-inflammatory cytokines, Acute with relapse patients, IL-22, IL-la, VEGF-D, CXCL10/IP-10, 0NGF; and chronic patients, IL-10, CXCLl/GROa, IL1RA, IL-7. Overall, variations in levels of serum secreted cytokines between groups diverge from that of differentially leukocyte expressed genes. In conclusion, we characterized for the first time the transcript pattern of peripheral blood leukocytes from brucellosis patients and correlated it to the levels of cytokines in their serum. Determinants that discriminate between acute and chronic patients were also identified, amongst which SLAMF1 appeared as a good marker of acute Brucella infection in humans.

• EXAMPLE 2

Neutrophils and PD-L1+/ Sca-1+ neutrophils are recruited to the spleen, peritoneal fluid and omentum during the chronic phase of B. abortus infection in a mouse model

Neutropenia is observed in one-third of human patients with brucellosis (Colmenero et al., 1996) and we found that one of the main chemokines to attract neutrophils (Capucetti et al., 2020; McDonald and Kubes, 2010), CXCL1, is upregulated in the serum of chronically infected patients (Figure 2A). To reconcile this apparent contradiction, we postulated that neutrophils might be recruited to organs at the chronic phase of Brucella infection. We used an intraperitoneal (i.p.) murine model of infection to determine the kinetics of neutrophil mobilization along the course of Brucella abortus infection in C57BL/6 mice. Numbers of Ly6G+ neutrophils were determined at 2, 8 and 30 days post-infection (p.i.) in the spleen, peritoneal fluid and omentum, as we have shown that this organ plays a major role in this model (Gonzalez-Espinoza et al., in preparation, 2021). At the onset of B. abortus infection (2 days p.i.), neutrophils significantly accumulated in the spleen, peritoneal fluid and omentum (Figure 2A) and even more at 8 days p.i.. At the chronic phase of infection (30 days p.i.), recruitment of neutrophils in peritoneal fluid, omentum and spleen persisted. Overall high levels of neutrophils are observed in the omentum and spleen along the course of B. abortus infection including the chronic phase.

Accumulated evidence suggests that neutrophils dampen adaptative immunity during B. abortus infection (Barquero-Calvo et al., 2013). We wondered whether they express suppressive phenotype markers as described for other pathogenic contexts like HIV or Staphylococcus aureus (Bowers et al., 2014; Park et al., 2020). We thus checked the expression of the PD-L1 and Sca-1 markers on peritoneal neutrophils from infected mice. The PD-L1 marker alone was significantly upregulated on neutrophils from day 2 until 30 days p.i.. Sca- 1+ cells and expression on neutrophils was almost unaltered (Figure 2B).

However, when focusing on the coexpression of PD-L1 and Sca-1 markers, a pattern similar to that observed in monocytes and macrophages was obtained with an increase in the absolute numbers of PD-Ll/Sca-1 coexpressing neutrophils from the onset to the chronic phase of infection. In conclusion, B. abortus infection induces an early and constant influx of neutrophils in the peritoneal fluid and omentum that express immunosuppressive markers, suggesting an essential role in chronic stages of infection.

Late PD-Ll+/Sca-1+ neutrophil recruitment in omentum is B. abortus core LPS- dependent

Endotoxic LPS has been known for long to drive neutrophil recruitment (Chignard and Balloy, 2000; Shahin et al., 1987). In order to get insight into the mechanisms underlying the persistent influx of neutrophils with an inhibitory phenotype triggered by B. abortus infection in the mouse model, we compared the kinetics of recruitment and immune status of neutrophils in the peritoneal fluid and omentum of i.p. injected mice with three different types of LPSs (Figure 3). We used the LPS purified from the wild-type B. abortus (Ba-wt) strain, or from a mutant of B. abortus with a mannosyltransferase (wadC) gene deletion (Ba-wadC), which exhibit a defective outer core, or the classical LPS from E. coli (Ec). The Ba-wadC and Ec LPSs efficiently potentiated dendritic cell and T cell activation, unlike the Ba-wt LPS (Zhao et al., 2018).

Ba-wt or Ba-wadC LPS challenge generated an early accumulation of neutrophils in the peritoneal fluid and omentum of mice in contrast to Ec LPS (Figure 3A). Similar neutrophil counts were observed for both Ba-wt and Ba-wadC LPSs 4 hours after injection, suggesting that this rapid recruitment is independent of the outer core of Brucella LPS. At 24h postinjection, all LPSs drove high and comparable levels of neutrophils in both omentum and peritoneal fluid. From 48h post-injection onward, neutrophil increase was exclusive to Ba-wt LPS, indicating that this late neutrophil recruitment and maintenance is a feature of wild-type Brucella LPS structure and involves its outer core component (Figure 3, right panel).

As found for neutrophils from B. abortus infected mice, all LPSs induced expression of PDL-1+ and Sca-1+ on neutrophils but not with the same kinetics. Injection of any of the three LPSs in mice produced similar influx of PD-Ll+/Sca-1+ neutrophils in the peritoneal fluid and omentum from 4h to 24h post-treatment (Figure 3B). However, the PD-Ll+/Scal+ neutrophil mobilization observed 48 hours after injection was exclusive to Ba-wt LPS. Indeed both Ba- wadC and Ec LPS elicited a downregulation of these immunosuppressive markers at late stimulation time-points (Figures 3B and 3C).

To visualize the spatial localization of neutrophils in omentum, we analyzed by confocal microscopy the omentum of mice collected 48h post-injection with PBS or Ba-wt LPS (Data not shown). While no neutrophils (Ly6G+) and few CD64+ cells (monocyte derived macrophages) were visible in the mock PBS condition, Ba-wt LPS treatment led to a high recruitment of neutrophils around the omental milky spots (MS), defined by the presence of T cells (CD3+) (Data not shown). This increase in neutrophils at 48h coincides with an influx of macrophages in omentum and an augmented size of omental milky spots compared to that observed in the PBS mock-injected mice (Data not shown).

Altogether these results demonstrate that the Ba-wt LPS is responsible for the fast mobilization of neutrophils exhibiting a PD-Ll+/Sca-1+ phenotype to the omentum and that their persistence at late phase depends on its outer core component.

CXCL1 mediates the omental recruitment of neutrophils elicited by Ba-wt LPS

Neutrophils are rapidly mobilized from the bone marrow to the blood and tissues by chemoattractants. CXCL1 is one of the major chemokines involved in neutrophil recruitment, and monocyte chemoattractant protein- 1 MCP-1/CCL2, the main monocyte chemoattractant, has also been shown to recruit neutrophils in very peculiar situation (Capucetti et al., 2020). To determine if these molecules might contribute to the persistent neutrophil recruitment observed in mice infected with B. abortus or injected with Ba-wt LPS at the chronic late phase, we analyzed by ELISA the levels of CXCL1 and CCL2 in the serum, peritoneal fluid and omentum collected 24 or 48 hours after injection of mice with PBS or Ba-wt LPS (Figure 4A). A significant increase in these two chemokines was detected in serum collected 24h post-injection of mice with Ba-wt LPS. The high and decreasing levels of CCL2 cytokine in the serum, peritoneal fluid and omentum reflected the successive considerable influx of circulating monocytes triggered by Ba-wt in the blood (data not shown), peritoneal fluid and finally omentum (Gonzalez-Espinoza et al., in preparation, 2021). All CCL2 values declined at 48h post-LPS injection.

Instead, the CXCL-1 chemokine levels were significantly upregulated in serum at 24h post-LPS injection only, unchanged in the peritoneal fluid whatever time-point considered, and significantly increased in omentum at both 48h and 72h post-LPS administration. Although the total concentration of CXCL-1 at 48h post-LPS injection in serum was higher, the fold of induction of this chemokine in serum at 48h versus the PBS or the 72h conditions was 3.1 and 3.2, whereas in omentum it reached 5.6 and 6.6 respectively. This indicated a higher and constant CXCL-1 secretion in omentum from 48h onward. These data also revealed the existence of a gradient of CXCL1 from blood to omentum.

To define which omental myeloid cells accounted for the secretion of CXCL-1, we next performed a flow cytometry analysis of omentum collected 48h after injection of mice with Ba- wt, Ec LPS or PBS (Figures 4B and 4C). Intracellular staining for CXCL-1 revealed after Ba- wt LPS stimulation a significant production of CXCL-1 from Ly6C+ monocytes and autocrine secretion in Ly6G+ neutrophils, with negligible contribution of omental F4/80hiCDl Ibhi macrophages. Of note, omental cells from Ec LPS-injected mice barely produced CXCL1 (Figure 4C), explaining the low influx of neutrophils observed in Figure 3A at 48h after treatment.

Collectively, we demonstrated that the production of the CXCL-1 chemokine by omental monocytes, neutrophils and stromal cells is associated with the retention and accumulation of neutrophils in omentum at late time-points after Ba-wt LPS exposure.

Discussion

Here, we have recapitulated the first blood transcriptional signature of human brucellosis using RNA seq that discriminates acute brucellosis from chronic brucellosis and healthy individuals. We show that this unbiased sequencing approach is robust in distinguishing in patients the acute phase, treated or with relapse, from the chronic phase of brucellosis, and that this latter presents an overall gene expression very close to that of uninfected controls. This situation explains why, if acute brucellosis is not diagnosed but left untreated as often the case, it is hard to recognise chronic Brucella infection when peripheral inflammatory symptoms appear. However, thanks to the comparative analysis of blood transcriptome and cytokine profiling in our clinical cohort, we have identified a number of molecules specific to each disease stage. As such, SLAMF1, a molecule overexpressed in several inflammation-related diseases (including infection, arthritis and pulmonary allergy), has been identified as a marker of the acute phase of brucellosis in humans together with other classical markers of infection. The SH2D1A gene, which codes for the SLAMF1 adaptor protein (SAP), varies accordingly to SLAMFL A series of genes encoding other molecules shown to be involved in brucellosis were also overexpressed in Brwce/Za-infected human blood, such as C1QC, encoding the first component of serum complement ⁴¹ , or genes coding for proteins involved in THI response ^42,43 (such as those of the transcription factors EOMES, TBX21/T-BET), innate response like Granzymes, GZMK and GZMH ⁴⁴ or an inflammasome component AIM2 ^45,46 . As expected, Acute patients secreted in their serum significantly more pro-inflammatory cytokines, in particular TNF-a, IL-9 and IL-31, while Acute with relapse patients presented an exacerbated phenotype with more serum IL-10 together with IL-22, VEGF-D, CXCL10/IP-10 and 0NGF. IL-22, a T-cell derived cytokine structurally related to IL-10, which protects against tissue destruction caused by exacerbated immune response, has been reported to be induced in murine T cells by IL-9, which itself is involved in the induction of TH2 cell type immune response ⁴⁷ . This cytokine being significantly elevated in Acute blood patients it might explain the rise in IL-22 when relapse occurs. Moreover, the upregulation of these cytokines indicates an important contribution of the TH2 response in human acute brucellosis, as confirmed by that of VEGF-D, shown to elicit TH2 responses in DC through binding to its receptor VEGFR3 ⁴⁸ , and ofNGF, which inhibits TLR-mediated inflammation in human monocytes ⁴⁹ . CXCL10/IP-10, a pan-marker of viral or bacterial infection ⁵⁰ and an IFN- stimulated gene, like AIM2, is induced in bone marrow derived murine macrophages via the STING pathway upon Brucella infection ⁴⁵ ’ ⁵¹ , suggesting an important role of the IFN- type I in human brucellosis. Chronic brucellosis, although silent at the transcriptional level, is marked by the presence in patient serum of classical anti-Tnl cytokines like IL- 10 and IL IRA, as well as of two molecules known as a neutrophil and pro-angiogenic chemokine for CXCL1/KC, and as a key pro-B and T cell differentiating cytokine for IL-7. Since Brucella elicits premature cell death of human neutrophils without inducing proinflammatory phenotypic changes ⁵² ’ ⁵³ , elevated blood CXCL1 levels might account for the neutropenia described in chronically-infected brucellosis patients ⁵⁴ . High levels of both IL-10 and IL1RA explain the low activation transcriptional signature of chronic patients and are thought to trigger an anergic state supporting persistence of bacteria ⁵⁵ . As regards Brucella, IL- 10 plays indeed an essential role in its maintenance in vivo notably by favouring escape of Brucella from the late endosome compartment in macrophages ⁵⁶ ’ ⁵⁷ . Interestingly genetic polymorphism in the IL1RA gene has been linked to susceptibility to brucellosis ⁵⁸ . High serum levels of IL-7 in the Chronic patient group likely reflected an impaired T cell sensitivity to IL-7 as reported recently for tuberculosis patients ⁵⁹ and is not related to IL-7 function itself. These data highlight specific features of the chronic phase of human brucellosis and infer that combined detection of IL-10, IL1RA, CXCL1 and IL-7 should form a unique and promising bio-marker serum signature for identifying Brucella chronicity.

Collectively, these results disclose distinctive hallmarks of human brucellosis that are of the utmost importance for improving its diagnosis. They open up new avenues of detection of all phases of this disease with blood identification of SLAMF1 as an acute brucellosis biomarker and a set of four serum biomarkers (IL-10, IL1RA, CXCL1 and IL-7) for chronic brucellosis.

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