WO2014142646A1 - Q-fever diagnostic - Google Patents

Abstract

The present invention relates to a method for diagnosing the Q-fever status in a subject, the method comprising the steps of: (a) obtaining a sample from said subject, (b) contacting said sample with a source of a Coxiella burnetiiantigen and (c) determining the profile of expression levels of both a pro-inflammatory cytokine such as IFN-γ and a T lymphocyte growth factor such as IL-2 in said sample at the end of step (b).

Description

Q-fever diagnostic

Field of the invention

The present invention relates to the field of diagnostics and therapeutics. It provides a method for diagnosing Q-fever status in a subject, using the determination of a proinflammatory cytokine and a T lymphocyte growth factor.

Background of the invention

Q-fever is a systemic infection caused by the intracellular bacterium Coxiella burnetii. The infection is common in animals, especially livestock, and transmitted to humans mainly through the airborne route. The disease presentation varies widely, ranging from asymptomatic infection, acute febrile illness most often with pneumonia, chronic complicated Q-fever (mainly with endocarditis or vascular infection) and post-Q-fever fatigue.

Recently The Netherlands was faced with the most extensive outbreak of Q-fever, related to goat farms. Since 2007, an increasing number of human cases have occurred. The diagnosis is based on a history of exposure, clinical examination and on PCR and serology. Due to the difficulty in culturing Coxiella burnetii in the laboratory, the variation in the antibody response, and the difficulties to standardize serology, the laboratory diagnosis is often not easy. Quite some weight is being given to the phase dependent antibodies, where phase 1 antibodies are seen in chronic infection and the phase 2 antibodies are related to acute infection. With accumulating experience in complicated cases in the current Dutch epidemic and especially in its aftermath, we have experienced that the results of the serology may be equivocal and often not helpful in the diagnosis.

If diagnosed in the acute stage, the disease can be cured with a relatively short course of the antibiotic doxycyclin, but chronic Q-fever is a much more difficult-to-treat infection. Chronic Q-fever has a high mortality rate. A special situation is Q-fever in pregnancy, in which the risk for the unborn and the management is not entirely clear.

A special area where the diagnostic tool is critical is the Q-fever vaccination of humans. Currently, patients are screened with serology and a skin test, and only if both tests are negative, vaccination is regarded a safe procedure. The skin test is not easy to perform and laborious. Its sensitivity and specificity are unknown.

Recently (WO2012/023852), a method for diagnosing Q-fever has been disclosed consisting of the assessment of the expression level of a pro-inflammatory cytokine as IFNy. The current method of the invention has the advantage of being more sensitive and more specific than previous known methods such as the one described in WO 2012/023852. There is a need for a specific and sensitive method for diagnosing Q-fever in a subject, circumventing all the drawbacks of existing methods. In addition, the current tests (with the possible exception of the skin test) do not assess the state of specific T lymphocyte immunity, which is considered to be needed for cure of the infection. Description of the invention

Diagnosis method

In a first aspect, the invention relates to a method for diagnosing Q-fever status in a subject, the method comprising the steps of:

(a) obtaining a sample from said subject,

(b) contacting said sample with a source of a Coxiella burnetii antigen and,

(c) determining the profile of expression or expression level of both a proinflammatory cytokine and a T lymphocyte growth factor in said sample at the end of step (b).

A pro-inflammatory cytokine is a cytokine that is able to promote systemic inflammation. A pro-inflammatory cytokine is preferably selected from the group consisting of IL-Ιβ, T Fa or IFNy, IL-6, and IL-17. IFNy is a preferred proinflammatory cytokine.

A T lymphocyte growth factor is a protein that promotes proliferation of T cells.

A T lymphocyte growth factor is preferably selected from the group consisting of: IL-2, IL-2 family members (i.e. IL-2, IL-4, IL-7, IL-15), IL-10, TGF .

Preferably a T lymphocyte growth factor is IL-2. The inventors surprisingly discovered that in patients with chronic Q fever discriminative profiles or discriminative expression profiles of a pro-inflammatory cytokine and of a T lymphocyte growth factor are found:

a detectable expression level or an increase of the expression level of a pro-inflammatory cytokine (preferably IFNy) and a low or undetectable expression level of a T lymphocyte growth factor (preferably IL-2) and/or

a high ratio between the expression levels of said pro-inflammatory cytokine (preferably IFNy) and said T lymphocyte growth factor (preferably IL-2). Elevated production levels of a pro-inflammatory cytokine (preferably IFNy) and of a T lymphocyte growth factor (preferably IL-2) were found in past Q fever and cured Q fever. Lower production levels of a such a pro-inflammatory cytokine and T lymphocyte growth factor were found in acute Q fever and post Q fever fatigue. In uninfected individuals the levels are generally undetectable

It is critical to assess chronic Q-fever as early as possible in order to be able to optimize the chances of curing said subject. In the context of the invention, the expressions "profile", "expression profile" or "profile of expression" may be replaced by " expression level" or " production level". In the context of the invention, "diagnosing Q-fever status" preferably means that a diagnosis is reached in: subjects with chronic Q-fever.

Subjects who have not been infected with Coxiella burnetii will not produce these cytokines in vitro. As soon as a detectable expression level or an increase of the expression level of a pro-inflammatory cytokine and a low or undetectable expression level of a T lymphocyte growth factor or a high ratio between the expression levels of said pro-inflammatory cytokine (preferably IFNy) and said T lymphocyte growth factor (preferably IL-2) have been detected in step c), chronic Q-fever has been diagnosed in a subject. In this context, acute Q-fever infection means recently acquired infection caused by Coxiella burnetii. "Recently" may mean at least 1, 2, 3, 4, 5, 6, 7 days or longer but shorter than 30 days.

In this context, chronic Q-fever means that the illness caused by Coxiella burnetii exists since 30 days or more.

In this context subjects with past Q fever are individuals that have had acute Q fever and do not have symptoms which occur in chronic Q fever.

In this context subjects with cured Q fever are individuals that have had Q fever and have been treated for the infection.

In this context subjects that received Q fever vaccine are individuals that received Q fever vaccine and in whom the state of cellular immunity has to be assessed.

In this context, post-Q-fever fatigue means severe fatigue after infection caused by Coxiella burnetii.

A symptom/parameter associated with acute Q-fever infection is fever, pneumonia or hepatitis.

In the context of the invention, a subject may be a human being or an animal. The animal may be goat. The diagnosis method may be applied as often as necessary in a subject. If the subject is a human being, the subject may be a person suspected to have a high risk of having or developing chronic Q-fever, for example due to the fact that this subject lives in a region wherein Q-fever is common such as the Netherlands, France, United kingdom, Australia and developing countries with many sheep and goats. In an embodiment, it is not known whether a subject has been infected with a Q-fever bacterium Coxiella burnetii . If the subject is an animal, e.g., a goat, the invention is used to make the diagnosis of infection caused by Coxiella burnetii in the animal. In an embodiment, a subject to be diagnosed using a method of the invention has not been vaccinated with a vaccine against Q-fever.

In a preferred method, chronic Q-fever is diagnosed when step (c) leads to the finding of:

- a detectable or increased expression level of a pro-inflammatory cytokine (preferably IFNy) and a low or undetectable expression level of a T lymphocyte growth factor (preferably IL-2) and/or - a high ratio between the expression levels of said pro-inflammatory cytokine and said T lymphocyte growth factor.

Optionally in a method of the invention, one may compare the profile or the expression level of a pro-inflammatory cytokine and of a T lymphocyte growth factor as determined in step (c) with reference values for said expression levels or profiles, the reference values preferably being the average value for said expression levels or profiles in a control sample. In the context of the invention, "a reference value" for the profiles or the expression level of said pro-inflammatory cytokine and T lymphocyte growth factor is preferably the average value for said expression levels or profiles in a control sample. A control sample may be derived from a control subject or from control subjects or from the culture medium used for step (b). A control subject may be a subject who does not live in a region at risk or who does not have animal contact. "A reference value" may mean that no pro-inflammatory cytokine and/or no T lymphocyte growth factor is detectable. A reference value may be obtained from a seropositive subject. A seropositive subject is a subject having anti-C. burnetii antibodies (IgG antiphase I or II titre > 1 :32) without a serologic profile suggesting chronic Q fever infection (IgG anti-phase I titre < 1 : 1,024) and without symptoms of chronic Q fever infection as earlier defined herein.

The assessment of the profile or the expression levels of said pro-inflammatory cytokine and said T lymphocyte growth factor may be directly realised at the protein expression level (quantifying the amount of said proteins), and/or indirectly by quantifying the amount of nucleotide sequences encoding said pro-inflammatory cytokine and said T lymphocyte growth factor. The skilled person will understand that it is possible to isolate multiple isoforms of a pro-inflammatory cytokine (preferably IFN-γ) and of a T lymphocyte growth factor (preferably IL-2) depending on the subject or species to be tested. A preferred nucleotide acid sequence encoding IL-2 comprises or consists of SEQ ID NO: 1. A preferred corresponding IL-2 amino acid sequence comprises or consist of SEQ ID NO: 2. A preferred nucleotide acid sequence encoding IFN-γ comprises or consists of SEQ ID NO:3. A preferred corresponding IFN-γ amino acid sequence comprises or consists of SEQ ID NO:4.

In a preferred embodiment, a T lymphocyte growth factor comprises or consists of IL- 2. A preferred IL-2 is:

represented by an amino acid sequence comprising at least 60%, 70%, 80%, 90%, 95%, or 100% identity with SEQ ID NO:2 and/or

encoded by a nucleotide acid sequence which has at least 60%, 70%, 80%, 90%, 95%, or 100% identity with SEQ ID NO : 1.

In another preferred embodiment, a nucleotide acid sequence encoding IL-2 has:

- at least 60%, 70%, 80%, 90%, 95%, or 100% identity with SEQ ID NO: 1 and/or encodes an amino acid sequence of IL-2 that has at least 60%, 70%, 80%, 90%, 95%), or 100%) identity with an amino acid sequence encoded by SEQ ID NO:2.

In a preferred embodiment, a pro-inflammatory cytokine comprises or consists of IFNy. More preferably, IFN-γ is :

represented by an amino acid sequence comprising at least 60%, 70%, 80%,

90%, 95%, or 100% identity with SEQ ID NO:4 and/or

encoded by a nucleotide acid sequence which has at least 60%, 70%, 80%, 90%, 95%, or 100% identity with SEQ ID NO:3.

In another preferred embodiment, a nucleotide acid sequence encoding IFN-γ. has:

- at least 60%, 70%, 80%, 90%, 95%, or 100% identity with SEQ ID NO: 3 and/or encodes an amino acid sequence of IFN-γ that has at least 60%, 70%, 80%, 90%), 95%), or 100%) identity with an amino acid sequence encoded by SEQ ID NO:4.

Identity is later herein defined. The quantification of the amount of a nucleotide sequence encoding a pro-inflammatory cytokine and/or a T lymphocyte growth factor (preferably IFN-γ and/or IL-2) is preferably performed using classical molecular biology techniques such as (real time) PCR, arrays or northern analysis. In this embodiment, a nucleotide sequence encoding said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) as described herein means a messenger RNA (mRNA). Alternatively, according to another preferred embodiment, in the diagnosis method the expression level of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) is determined directly by quantifying the amounts of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2). Quantifying a polypeptide amount may be carried out by any known technique. Preferably, a polypeptide amount is quantified using a molecule that specifically binds to said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2). Preferred binding molecules are selected from: an antibody, which has been specifically raised for recognizing said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN- γ and/or IL-2), any other molecule which is known to specifically bind said proinflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2). Such antibody could be used in any immunoassay known to the skilled person such as western blotting, or ELISA (Enzyme-Linked Immuno Sorbent Assay) or FACS (Fluorescence Activated Cell Sorting) using latex beads. The preparation of an antibody is known to those skilled in the art. In an embodiment, the expression level of IL-2 is detectable as T-cell growth factor using T-cell lines, such as Jurkat T-cells and a suitable ELISA. Even more preferably, the presence of IL-2 is assessed as carried out in the experimental data. A short explanation of methods that could be used to prepare antibodies is later herein given. In the context of the invention, any other molecule known to bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) may be a nucleic acid, e.g. a DNA regulatory region, a polypeptide, a metabolite, a substrate, a regulatory element, a structural component, a chaperone (transport) molecule, a peptide mimetic, a non-peptide mimetic, or any other type of ligand. Mimetic is later herein defined. Examples of molecules known to bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN- γ and/or IL-2), include a receptor of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) such as the IFN-γ receptor, the IL-2 receptor, an antibody directed against said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2). Binding of said pro- inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) to a second binding molecule may be detected by any standard methods known to those skilled in the art. Suitable methods include affinity chromatography co- electrophoresis (ACE) assays and ELISA. The skilled person will understand that alternatively or in combination with the quantification of a nucleic acid sequence encoding said pro-inflammatory cytokine and/or T lymphocyte growth factor and/or a corresponding polypeptide (preferably IFN-γ and/or IL-2), the quantification of a substrate of a corresponding polypeptide or of any compound known to be associated with a function or activity of a corresponding polypeptide or the quantification of a function or activity of a corresponding polypeptide using a specific assay is encompassed within the scope of the diagnosis method of the invention. For example, trans-activation of a target gene by said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) or a molecule which is able to bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) can be determined and quantified, e.g., in a transient transfection assay in which the promoter of the target gene is linked to a reporter gene, e.g., P- galactosidase or luciferase. Such evaluations can be done in vitro or in vivo or ex vivo. In a method of the invention, a sample from a subject is used. A method of the invention is therefore an in-vitro or ex-vivo method. A sample preferably comprises or consists of a fluid obtained from a subject. More preferably, a fluid comprises or consists of or is selected from: urine, blood, spinal cord fluid, saliva, semen, or bronchoalveolar lavage. A preferred fluid is, comprises, consists of or is derived from blood. Blood may be used as whole blood or diluted before being further used. The dilution may be 1 :4, 1 :5 or 1 :6 in culture medium or a buffered solution.

In a method of the invention, said obtained sample of step (a) is subsequently contacted with a source of a Coxiella burnetii antigen. The choice of the antigen may be dependent on prevalent Coxiella burnetii strains in different areas of the world. Preferably Coxiella burnetti Nine miles is being used. The contact may have a duration of 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 30, 48, 60, 70, 80, 90, 93, 96, 100, 1 10 hours, or more. Preferably the contact has a duration of 4-96 hours, or 20-50 hours, or 24 hours, or 48 hours. This contact step may be a culture step in a culture medium such as RPMI 1640. A source of a Coxiella burnetii antigen may mean that a whole Coxiella burnetii cell is being used. In a preferred embodiment, a whole Coxiella burnetii cell is heat- inactivated or formalin fixated. Heat-inactivated could be replaced by heat-killed. The skilled person knows how to obtain heat-inactivated or formalin fixated Coxiella burnetii cells. Heat-inactivated cells are preferably prepared by heating at 95, 96, 97, 98 or 99°C for 20, 25 or 30 minutes. More preferably heat-inactivated cells are prepared by heating at 99°C for 30 minutes. Formalin fixated cells may be obtained by contacting the cells with formalin for 40, 50, 60 minutes. More preferably, cells are contacted or transferred to 4% formalin for one hour. Subsequently cells are washed several times with PBS (Phosphate Buffered Saline) buffer. Alternatively, part of a Coxiella burnetii cell may be used. A part of a Coxiella burnetii cell is preferably an antigenic part thereof. Said part comprises or consists of an antigen. An antigen may be a protein, a digest of the protein and/or a fragment thereof, which may be in a purified form or may be comprised within a crude composition, preferably of biological origin, such as a lysate, sonicate, extract or fixate of a Coxiella burnetii. Said extract may be a cytosolic or a nuclear extract. Alternatively, an antigen may be chemically synthesized or enzymatically produced in vitro. The source of a protein, or fragment thereof as antigen, may also be a nucleic acid encoding said, or fragment thereof, from an RNA or DNA template. In a preferred embodiment, a source of a Coxiella burnetii antigen is a whole Coxiella burnetii cell or an antigen from said cell. The use of a whole Coxiella burnetii cell is attractive and preferred above the use of a part of a Coxiella burnetii cell for at least two reasons. The use of a whole Coxiella burnetii cell is easier and cheaper for the skilled person. There is no need to identify and subsequently synthetize suitable parts (i.e. antigenic parts) of a Coxiella burnetii cell. In addition, by using a whole Coxiella burnetii cell, all potential suitable parts (i.e. all antigens) of a Coxiella burnetii cell are present. The diagnostic method is therefore expected to be far more sensitive and efficient than a corresponding diagnostic method carried out using a given antigen. Very promising results were obtained using heat-inactivated whole Coxiella burnetii cell.

Subsequently, the profile or the expression levels of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) is determined in said sample at the end of the contact step of step (b). In a preferred embodiment, at the end of the contact step, the supernatant is isolated by centrifugation and the nucleotide sequences or the proteins of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) are determined by a skilled person using known methods. The centrifugation may be at 1200 rpm at 4°C. Alternatively, one may add a detergent to the sample at the end of step b). Several detergents could be used such as Triton X 0.1 %. Adding a detergent is attractive since it is expected that no centrifugation step is needed. One may determine the expression level of said proinflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) cytokine in the sample comprising said detergent, which is also called a cell lysate.

In a more preferred diagnosis method, chronic Q-fever is diagnosed when step c) leads to the finding of:

a detectable expression level or an increase of the expression level of a pro-inflammatory cytokine (preferably IFNy) and a low or undetectable expression level of a T lymphocyte growth factor (preferably IL-2) and/or

- a high ratio between the expression levels of said pro-inflammatory cytokine (preferably IFNy) and said T lymphocyte growth factor (preferably IL-2).-

The finding of a high ratio (ratio IFNY/IL-2) is discriminative by comparison to the value of said ratio in control subjects and in control samples. Such a control is preferably a seropositive control as earlier defined. The value of said ratio in such controls is lower than 1 1 such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 10, 9. Detection of the expression level or profile of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) and/or of their corresponding nucleotide sequences (or steady state levels of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2)) is preferably defined as being a detectable expression level or profile or a detectable change of the expression level or profile of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) and/or of their corresponding nucleotide sequences (or steady state levels of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) or any detectable activities thereof or detectable change in a biological activities thereof) using a method as defined earlier on as compared to the expression profile of of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) and/or of corresponding nucleotide sequences (or steady state levels of the corresponding encoded pro- inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) in a control subject or in a control. Such a control is preferably a seropositive control as earlier defined. According to a preferred embodiment, a detection or an increase or a change of activity of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) is quantified using a specific mRNA assays for the genes encoding said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2).

Preferably, an increase of the expression level of a nucleotide sequence encoding said pro-inflammatory cytokine (preferably IFN-γ ) means an increase of at least 5% of the expression level of said nucleotide sequence using PCR.

Preferred primers used for the IFN-γ PCR are identified as Forward Primer

5'-CTCTTGGCTGTTACTGCCAGG-3' (SEQ ID NO:5) ; and Reverse Primer 5'-CTCCACACTCTTTTGGATGCT-3' (SEQ ID NO:6).

More preferably, an increase of the expression level of a nucleotide sequence means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.

A low or undetectable profile or expression level of a T lymphocyte growth factor (preferably IL-2) preferably means that using PCR, the expression level of a nucleotide sequence encoding said T lymphocyte growth factor (preferably IL-2) is not detectable or the Ct value is 35 or higher.

Preferred primers used for the IL-2 PCR are identified as Forward Primer:

5'-TCCTGTCTTGCATTGCACTAAG-3' (SEQ ID NO: 7) and Reverse primer 5'- CATCCTGGTGAGTTTGGGATTC-3 ' (SEQ ID NO: 8) .

Preferably, an increase of the expression level of said pro-inflammatory cytokine (preferably IFN-γ ) means an increase of at least 5% of the expression level of said proinflammatory cytokine (preferably IFN-γ) using western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of said polypeptide means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.

Preferably, an increase of an activity of said pro-inflammatory cytokine (preferably IFN-γ) means an increase of at least 5% of the polypeptide activity using a suitable assay. More preferably, an increase of the polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. In a most preferred diagnostic method, chronic Q-fever is diagnosed when the detection or comparison leads to the finding of a profile or an expression level of proinflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) comprising or consisting of:

- a detectable level or an increase of the level of expression of a pro-inflammatory cytokine (preferably IFN-γ) or an increase or a detection of the expression level of a nucleotide sequence encoding said cytokine (preferably IFN-γ), said detection or increase being detected at the level of the amino acid sequence of said cytokine (preferably IFN-γ), more preferably an increase of at least 5% of the expression level of said cytokine (preferably IFN-γ) using ELIS A as defined herein And

- a low or undetectable level of expression of a T lymphocyte growth factor (preferably IL-2) or a low or undetectable expression level of a nucleotide sequence encoding said T lymphocyte growth factor (preferably IL-2), assessed at the level of the amino acid sequence of said T lymphocyte growth factor (preferably IL-2), more preferably a low or undetectable expression level of said T lymphocyte growth factor (preferably IL-2) using ELIS A as defined herein, and/or

- a high ratio between the expression level of said pro-inflammatory cytokine and T lymphocyte growth factor (preferably high IFNy/IL-2). A high ratio of said pro-inflammatory cytokine and said T lymphocyte growth factor, preferably means that said ratio (pro-inflammatory cytokine/T lymphocyte growth factor) is increased, elevated compared to corresponding ratio of a reference value. A reference value is preferably from a seropositive control as earlier defined herein. Preferably a high ratio is a ratio of 11 or at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. More preferably, a high ratio of IFNy/IL-2 is an IFNy/IL-2 ratio of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. Preferably the concentration of each of said proinflammatory cytokine and T lymphocyte growth factor is assessed in pg/ml. This ratio is calculated using standard immunoassays, preferable as those used in the experimental part.

The method of the invention is attractive since the diagnosis is reached with more certainty. It has a clear added value to the serology that is often equivocal. Furthermore, this method is non-invasive, simple, reproducible, sensitive, specific, and time and cost efficient. It further allows to start a treatment against Q-fever at an early (preferably before the apparition of any symptom).

Assay device

In a second aspect, an assay device is provided for diagnosing Q-fever status or chronic Q-fever in a subject, wherein said device comprises molecules which specifically bind to either of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2). This device may be used in a diagnosis method of the invention. Any subject or physician could use this device at office/home, repeat the use of such device as often as necessary.

The type of molecules that are known to specifically bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) have already been earlier described herein. In a preferred embodiment, molecules which specifically bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) and which are present in the device are antibodies. These antibodies are preferably derived from different species so that they can be recognized by second antibodies that recognize these species and that are labelled by conjugation to physically detectable distinct labels. More preferable the antibodies, which specifically bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) are labelled by conjugation to physically detectable distinct labels.

In a preferred embodiment, an assay device is a lateral flow test strip also known as dipstick, preferably, though not necessarily, encased in a housing, designed to be read by the subject, and the assay consists of sandwich immunoassays. Such devices are impregnated with reagents that specifically indicate the presence of the given molecules, here said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) by changing colours upon contact with a sample. Preferred subject's samples have already been defined herein. The antibodies are preferably labelled by conjugation to physically detectable distinct labels, and upon contacting with a sample containing said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) form complexes. Said antibody- pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) - complexes, such antibody-IL-2 complex and antibody-IFN-γ complex, which are immobilized on the solid support, are detectable by virtue of the distinct labels. A test strip may then be inserted into a reader, where signals from said labels in the complexes are measured.

Alternatively, the antibodies that bind said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) are not labelled by conjugation to physically detectable distinct labels, but are derived form different species. When they form complexes, they are contacted with second antibodies that recognize either of the complexes by virtue of their species specificity. The complexes are immobilized on a solid support within the device. The second antibodies specifically capture either said antibody-pro-inflammatory cytokine complex (preferably antibody-IFN-γ complex) or antibody-T lymphocyte growth factor complex (preferably antibody-IL-2 complex) to form an antibody-pro-inflammatory cytokine sandwich complex (preferably an IFN-y-antibody sandwich complex) or an antibody-T lymphocyte growth factor sandwich complex (preferably an IL-2-antibody sandwich complex), and the resulting complexes, which are immobilized on the solid support, are detectable by virtue of the distinct labels of the second antibodies. A test strip may then be inserted into a reader, where signals from said distinct label in the antibody-pro-inflammatory cytokine sandwich complex (preferably antibody-IFN-γ- antibody sandwich complex) and from said distinct label in the antibody-T lymphocyte growth factor sandwich complex (preferably antibody-IL-2-antibody sandwich complex is measured. Alternatively, a test strip could be inserted into the reader prior to addition of the sample. Alternatively and according to a preferred embodiment, the presence of said pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2) are visualised by a subject as changes of colour of at least a part of a device. Dipsticks are usually made of paper or cardboard. Usually additional molecules are present in a device as a positive or negative control. A typical positive control could be an antibody recognizing a molecule which is known to be present in a sample to be tested. A typical negative control could be an antibody recognizing a molecule which is known to be absent in a sample to be tested.

Method

In a further aspect, the invention relates to a method for treating chronic Q-fever in a subject, the method comprising the steps of: (a) obtaining a sample from said subject,

(b) contacting said sample with a source of a Coxiella burnetii antigen,

(c) determining the profile of expression or expression level of both a proinflammatory cytokine and a T lymphocyte growth factor in said sample at the end of step (b), and

(d) wherein a subject with a low or undetectable level of expression of a T lymphocyte growth factor and a low or undetectable expression level of a nucleotide sequence encoding said T lymphocyte growth factor and/or a high ratio between the expression level of said pro-inflammatory cytokine and T lymphocyte growth factor (preferably high IFNy/IL-2) as determined at the end of step (c) is being administered a treatment against Q-fever.

Each of the steps (a), (b), (c) and part of step (d) have been extensively explained in the section dedicated to the first aspect of the invention.

A treatment against Q-fever may be long term administration of an antibiotic (preferable doxycycline) drug and chloroquin.

Such a treatment is intended to cure or chronically suppress the infectionof said subject after at least one week, one month, six month of treatment. This could be assessed by serology, negativity for PCR of Coxiella, normalization of inflammatory parameters, normalization of imagistic tests (i.e. PET-scan), absence of clinical symptoms and/or by normalization of the IFNy/IL-2 ratio towards a lower value than the value measured in said subject at the onset of the treatment, preferably towards value lower than 11 such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 10, 9. In this context, "lower than" may mean 5% lower than, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower than or more.

Sequence identity

"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid or two nucleic acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof. Part thereof may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Val; Leu to He or Val; Lys to Arg, Gin or Glu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to He or Leu.

Antibodies

Some aspects of the invention concern the use of antibodies or antibody- fragments that specifically bind to either of a pro-inflammatory cytokine and/or T lymphocyte growth factor (preferably IFN-γ and/or IL-2). Methods for generating antibodies or antibody-fragments that specifically bind to such polypeptides are described in e.g. Harlow and Lane (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and WO 91/19818; WO 91/18989; WO 92/01047; WO 92/06204; WO 92/18619; and US 6,420, 113 and references cited therein. The term "specific binding," as used herein, includes both low and high affinity specific binding. Specific binding can be exhibited, e.g., by a low affinity antibody or antibody-fragment having a Kd of at least about 10^"4 M. Specific binding also can be exhibited by a high affinity antibody or antibody-fragment, for example, an antibody or antibody-fragment having a Kd of at least about of 10^"7 M, at least about 10^"8 M, at least about 10^"9 M, at least about 10^"10 M, or can have a Kd of at least about 10^"11 M or 10^"12 M or greater. General

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a method or an assay device as defined herein may comprise additional step(s), respectively component(s) than the ones specifically identified, said additional step(s), respectively component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Figure legends:

Figure 1. Cburnetii induced IFN-γ production is overall higher in chronic Q fever patients than in seropositive and seronegative controls.

Net IFN-γ production (stimulated minus unstimulated) is shown after 24 hours incubation of whole blood with phytohemagglutinin (PHA) or Cburnetii specific antigens (Q-vax and C.&.-NM). Median ± IQR are shown. Mann- Whitney U test was used to compare medians, ns = not significant, *** P < 0-001. Figure 2. Cburnetii induced IFN-γ production to discriminate between chronic Q fever patients and seropositive controls.

(A) Receiver operator characteristics (ROC) curve of the Cburnetii NM stimulated IFN-γ production after 24 hours in chronic Q fever patients versus seropositive controls. The AUC is 0-8664 (95% CI; 0-7933 to 0-9395; O-0001). The arrow indicates the optimal cut-off of IFN-γ production (500 pg/mL) with best combination of sensitivity and specificity. (B) The individual IFN-γ production is shown for all seropositive controls (n=135) and chronic Q fever patients (n=28), plotted against the IgG anti-C. burnetii phase I antibodies. Each dot indicates one individual. The horizontal line indicates the cut-off at 500 pg/ml. The vertical line distinguishes chronic Q fever patients (closed circles) from controls (open circles).

Figure 3. Cburnetii induced other cytokines in chronic Q fever patients and controls.

Cytokines IFN-γ, IL-2, IL-Ιβ, T F-a, IL-10 and IL-IRa in 48 hours C.b. NM stimulated blood are shown for a selection of seronegatives (n=18), seropositives (n=18) and proven chronic Q fever patients (n=16). Horizontal lines indicate the median value of the respective population. The dotted line represents the lower detection limit of the assay. Mann- Whitney U test was used to compare medians, ns = not significant, * P <0-05, ** P <0-01, *** P <0-001.

Figure 4. The ratio of Cburnetii induced IFN-γ and IL-2 to discriminate between chronic Q fever patients and seropositive controls.

The IFN-γ / IL-2 ratio was calculated by dividing IFN-γ production (pg/mL) by IL-2 production (pg/mL) in 24 hours Cburnetii stimulated whole blood. All patients (n=28) and all seropositive controls with IFN-γ production > 32 pg/mL (n=102) were included. (A) Receiver operator characteristics (ROC) curve of the IFN-γ / IL-2 ratio in chronic Q fever patients versus seropositive controls. The AUC is 0-8813 (95% CI; 0-8135 to 0-9472). (B) The individual IFN-γ / IL-2 ratio is shown for chronic Q fever patients and seropositive controls, plotted against the IgG anti-C. burnetii phase I antibodies. Each dot indicates one individual. The horizontal line indicates a IFN-y/IL-2 ratio cut-off at 11. The vertical line distinguishes chronic Q fever patients (closed circles) from controls (open circles).

(C) IFN-γ plotted against IL-2 production shows patients (black dots) and seropositive controls (open dots). All patients (n=28) and all seropositive controls with IFN-γ production > 32 pg/mL (n=102) were included. The horizontal line indicates the cut-off of IFN-γ at 500 pg/mL. The sloping line indicates the cut-off IFN-γ / IL-2 ratio at 11.

Tables

Table 1. Clinical features of chronic Q fever study patients

number Sex Age Focus of IgG anti- IgG anti- PCR Duration

(yrs) infection phase I " phase II " serum/ antibiotic

(reciprocal) (reciprocal) plasma" treatment

(months)" male 82 Prosthetic 8192 16384 positive

valve

male 72 Vascular 8192 32768 positive

prosthesis

female 55 Vascular 16384 65536 positive

prosthesis

male 69 Valvuloplasty 16384 16384 positive male 70 Vascular 4096 8192 negative

prosthesis

male 68 Vascular 1024 2048 negative

prosthesis

male 68 Vascular 32768 16384 negative

prosthesis

male 30 Vascular 131072 131072 positive 0

prosthesis

female 77 Prosthetic 32768 32768 positive 0

valve

10 male 69 Vascular 8192 8192 negative 0

prosthesis

11 male 86 Vascular 16384 8192 negative 1

prosthesis

12 male 66 Prosthetic 131072 131072 positive 1

valve

13 male 64 Aortic 8192 8192 negative

aneurysm

14 male 58 Prosthetic 2048 4096 negative

valve

15 male 72 Biovalve 2048 2048 negative

16 female 60 Aortic 8192 8192 negative

aneurysm

17 male 47 Vascular 32768 32768 positive prosthesis

18 male 73 Vascular 8192 8192 negative 7 prosthesis

19 female 65 Biovalve 8192 8192 positive 8

20 male 72 Vascular 8192 4096 negative 9 prosthesis

21 male 83 Aortic 2048 2048 negative 13 aneurysm

22 female 66 Vascular 2048 2048 negative 18 prosthesis

23 male 58 Vascular 8192 32768 negative 18 prosthesis

24 male 80 Prosthetic 1024 4096 positive 20 valve

25 male 69 Aortic 4096 4096 negative 20 aneurysm

26 male 68 Aortic 1024 512 negative 20 aneurysm

27 female 56 Vascular 2048 2048 negative 24 prosthesis

28 male 51 Prosthetic n.a.^a n.a. negative 26 valve

a at the moment of bloodsampling

b not available, CFT IgG anti-phase I > 160 and anti-phase II > 640

Table 2. Demographics of the total study population.

Mean ±SD age (yr) No. (%) male Chronic Q fever patients 66-2 ± 11-8 22 (78-6)

(n=28)

Seropositive controls (n=135) 60-8 ± 15- 1 105 (77-8)

Seronegative controls (n=908) 63-6 ± 14-0 559 (61-6)

Examples

Materials and methods

Study population

Chronic Q fever patients were recruited from participating hospitals. At the time of diagnosis, all patients had IgG anti-C. burnetii phase I titre > 1 : 1,024 (in the absence of acute infection), with either a positive C burnetii PCR in serum and/or tissue or signs of endocarditis as defined by the modified Duke criteria, or evidence of vascular

(prosthetic) infection on PET/CT or ultrasound. They fulfilled the criteria for proven chronic Q fever of the Dutch consensus group on chronic Q fever (Wegdam-Blans MC, et al) .

Individuals screened in the Dutch Q fever vaccination campaign from January to April 2011, as previously described, were used as controls (Schoffelen T et al). They all had pre-existing valvular or vascular risk factors for chronic Q fever.

Control individuals were classified as seronegative if serological testing and the Q- vax® skin test (CSL, Australia) were both negative. Controls were classified as seropositive when serological tests showed anti-C. burnetii antibodies (IgG anti-phase I or II titre > 1 :32) without a serologic profile suggesting chronic Q fever infection (IgG anti-phase I titre < 1 : 1,024), and without symptoms of chronic Q fever infection.

The study was approved by the Medical Ethical Committee Arnhem-Nijmegen and written informed consent was obtained from all subjects.

Measurement of CburneHi-sptcific antibodies and detection of Cburnetii DNA

IgG-antibodies against Cburnetii phase I and phase II were measured by

immunofluorescence assay (IF A). IFA was performed in three different laboratories connected to the participating hospitals; all used the same commercially available IFA test (Focus Diagnostics, Cypress, CA, USA) with comparability between laboratories within close range, as previously reported (Herremans T, et al).

Cburnetii DNA in blood (serum or EDTA) and tissue was performed in the

laboratories using the same real-time PCR based targeting IS 1111 (Tilburg JJ et al).

In vitro whole blood stimulation Venous blood was drawn into 5 mL endotoxin-free lithium-heparin tubes (Vacutainer System, BD Biosciences) and samples were processed within 12 hours.

Aliquots of 0.5 mL undiluted whole blood were incubated in a closed tube at 37°C for 24 hours. In another format, aliquots of blood were diluted 1/5 in RPMI 1640 (Dutch modification, supplemented with glutamax (2 mM), pyruvate (1 mM) and gentamicin (1 mg/mL)) and plated out 1 mL/well in a 24 well tissue culture plate at 37 °C and 5% C0₂ for 48 hours. C.burnetii Nine Mile (NM) RSA 493 phase I, a well-described reference strain (Seshadri R et al), was used in a concentration of 10⁷

microorganisms/mL for stimulation. Q-vax vaccine, containing formaldehyde- inactivated C.burnetii Henzerling strain phase I, was used separately in a concentration of 100 ng/mL. The mitogen phytohemagglutinin (PHA, Sigma-Aldrich, St. Louis, USA) was used as a positive control. One aliquot was incubated with only culture medium as a negative control. After incubation, blood cultures were centrifuged after which supernatants were stored at -20°C until assayed.

Cytokine measurements

IFN-γ concentration was measured using a commercial ELISA kit (Pelikine compact, Sanquin, Amsterdam, the Netherlands) as previously described Schoffelen T et al). TNF-a, IL-Ιβ, IL-IRa, IL-2, IL-4, IL-5, IL-6, IL-10 were measured using a multiplex beads assay (Merck Millipore, Billerica, MA, USA). IL-12p70, IL-23 and IL-18 were measured simultaneously in a magnetic beads multiplex assay (Merck Millipore) according to the manufacturer's instructions.

Statistical analysis

Graphpad Prism (Graphpad software Inc., version 5) was used to analyze the data. Cytokine results were displayed as individual values or expressed as medians with interquartile range. The Mann- Whitney £/-test was used to determine the differences between groups. Spearman's rho correlation coefficient was used to calculate correlation. A receiver operator characteristic (ROC) curve was constructed, and the area under the curve (AUC) was estimated to assess the discriminative performance of measuring (a ratio of) cytokines. In all analyses, P < .05 was considered significant. Results

Patients and controls

From January 2011 until January 2012, blood samples were obtained from 28 patients with proven chronic Q fever infection. At the time of blood sampling, ten patients had not yet started antibiotic treatment. The median duration of antibiotic therapy among patients on treatment (n=18) was 7.5 months (range 1-26 months). Patient

characteristics are shown in Table 1.

As controls, blood samples from individuals with risk factors for chronic Q fever participating in the Q fever pre-vaccination screening from January to April 2011 were obtained (Schoffelen T et al). In total, 908 individuals were seronegative, suggesting no previous contact with C. burnetii; 135 individuals were seropositive with IgG anti-phase I below 1 : 1,024 and thus diagnosed with previous Q fever. None of the seropositive controls had detectable titres of IgM antibodies without IgG antibodies, suggesting the absence of acute Q fever. As can be seen from the demographic data shown in Table 2, patients and controls were similar in terms of age range and gender, although the seronegative controls were significantly more males.

Interferon-gamma production in 24hrs-stimulated whole blood

We measured the net IFN-γ production (stimulated - unstimulated) in undiluted whole blood incubated for 24 hrs with PHA, Q-vax or C. burnetii NM in all patients and controls (figure 1). PHA induced IFN-γ production did not differ between patients and controls. In contrast, both C.burnetii-antigens induced significantly more IFN-γ in chronic Q fever patients (median 151 pg/mL and 2486 pg/mL by Q-vax and C. burnetii NM respectively) than in seropositive controls (3 pg/mL and 120 pg/mL) ( O-001) and seronegative controls (0 and 0-0 pg/mL) (PO-001). ROC curves for C. burnetii induced IFN-γ for chronic Q fever patients versus seropositive controls were made to establish optimal cut-offs for IFN-γ production. C. burnetii NM stimulated IFN-γ production had an accuracy (AUC) of 0-8664 (95% CI; 0-7933 to 0-9395, PO-0001) (figure 2a); Q-vax stimulated IFN-γ production had a smaller AUC (0-8484, 95% CI; 0-7639 to 0-9330, P<0-0001) (not shown). Therefore, further analyses were performed only with C. burnetii NM stimulated samples. As can be seen in figure 2b, there was a considerable overlap between IFN-γ production in seropositive controls and chronic Q fever patients. Choosing 500 pg/mL as optimal cut-off, 75-0 % (21/28) of the chronic Q fever patients had a value above this cut-off, and 17-8% (24/135) of the seropositive individuals.

Interestingly, the height of IgG antibody titres showed significant correlation with the amount of IFN-γ produced. Spearman's rho for the correlation between IFN-γ and IgG anti-phase I in the total of seropositive controls and chronic Q fever patients, was 0-3069 ( O-001).

Further analyses of IFN-γ production did not show significant differences between untreated (n=10) and treated (n=18) chronic Q fever patients (R=0-105), neither did duration of treatment (in months) correlate with IFN-γ production ( =0-367).

Cytokine profiles in 48hrs Cburnetii Nine Mile-stimulated diluted blood

Next, we investigated whether the production of TNF-a, IL-Ιβ, IL-IRa, IL-4, IL-5, IL- 6, IL-10, IL-12p70, IL-23, IL-18 and IL-2 would help to discriminate between chronic Q fever patients and seropositive controls, both groups having high IFN-γ production. Because not all cytokines were expected to be detectable in the first 24 hrs, the 48 hrs Cburnetii NM stimulated diluted blood samples were used. The complete cytokine profile measured in a subset of samples of 16 chronic Q fever patients (obtained in the first ten months of the study), 18 seropositive controls with substantial (>32 pg/mL) IFN-γ production in 24 hrs, and 18 seronegative controls with low IFN-γ production, were compared. Figure 3 shows the results for IFN-γ, IL-2, IL-Ιβ, TNF-a, IL-10 and IL-IRa. Not surprisingly, the median IFN-γ production in this subset did not differ significantly between chronic Q fever and the selected seropositive individuals. Most interestingly, the production of IL-2 was significantly lower in chronic patients (median of 5-0 pg/mL) than in these seropositive controls (median of 39-5 pg/mL) ( O-001). The production of the pro-inflammatory monocyte-derived cytokines IL-Ιβ and TNF-a was high in both chronic Q fever patients and in seropositive individuals, but not significantly different ( =0·236 and R=0- 158 respectively). However, both were significantly higher than the production in seronegative controls. The same pattern was seen for the production of IL-6 (not shown).

The anti-inflammatory cytokine IL-10 showed the same pattern as IL-Ιβ and TNF-a: its production was not different in chronic Q fever patients compared to seropositive controls. However, chronic Q fever patients had significantly higher IL-10 production than seronegative individuals (P<0-01). IL-IRa did not differ between the groups. The C.burnetii NM stimulated production of IL-4, IL-5, IL-18 and IL-23 remained below detection limit in all tested patients and controls. IL-12p70 levels were very low and therefore lacked any power of discrimination. IFN-y IL-2 ratio for diagnosis of chronic Q fever

In the subset described, the cytokine profile of patients with chronic Q fever shows high IFN-γ and low IL-2 production, whereas seropositive controls with substantial IFN-γ production show high IL-2.

Following this finding, we decided to assess IL-2 production in all chronic Q fever patients and all 102 seropositive controls with IFN-γ production > 32 pg/mL. The IFN- y/IL-2 ratio was calculated for each individual by dividing IFN-γ production by IL-2 production (both in pg/mL) (figure 4). The AUC of the ROC-curve for chronic Q fever patients versus seropositive controls was 0-8873 (95% CI; 0-7983 to 0-9762,

O-OOOl). In 22/28 (78-6%) chronic Q fever patients, the ratio was above 11, compared to only 6/102 (5-9%) seropositive controls with a IFN-γ production > 32 pg/mL and 6/135 (4-4%) of all seropositives. So, the number of high values in the seropositive controls diminished by 75% (from 24 to 6), at cut-off 11 for IFN-y/IL-2 ratio compared to the cut-off 500 pg/mL for IFN-γ production alone (figure 4c).

No correlation was found between the IFN-Y/IL-2 ratio and the duration of antibiotic treatment in patients (.Ρ=0·44). However, patients with positive PCR on C.burnetii

DNA in serum or plasma at the moment of blood sampling (n=10), had a significantly higher IFN-y/IL-2 ratio than patients with negative PCR (n=18) (median IFN-Y/IL-2 ratio of 92-9 and 15-9 respectively, <0·01).

Discussion

In the present study, we showed that chronic Q fever patients exhibit a distinct

C. burnetii induced cytokine production profile compared to control individuals. A combination of a high IFN-γ and low IL-2 production appears to characterize chronic Q fever infection, while individuals with a previous infection had both high IFN-γ and high IL-2 production. The production of monocyte-derived pro-inflammatory cytokines IL-Ιβ, T F-α and IL-6 was high both in patients with chronic Q fever as well as in individuals with a cleared infection and did not discriminate between both groups. Similarly, the pattern for the anti-inflammatory cytokines IL-10 and IL-IRa was not significantly different in chronic Q fever patients and in individuals with a past infection.

All 28 patients included in this study had a proven chronic Q fever infection. We deliberately chose to obtain an indisputable study population, by not including patients with uncertain diagnosis, i.e. a serologic profile of chronic Q fever without a positive PCR or focus of infection by diagnostic imaging.

Interestingly, we found that chronic Q fever patients display high C.burnetii-specific IFN-γ production while it is commonly assumed that chronicity of Q fever infection is due to T-lymphocyte unresponsiveness and impaired IFN-γ production (Koster FT et al, Izzo AA et al). We show here that C.burnetii-specific IFN-γ production in chronic Q fever patients is even higher than in individuals with past C. burnetii infection. This is in accordance with the findings of Limonard et al, who recently found higher numbers of IFN-γ positive cell in three chronic Q fever patients compared to nine convalescent controls, using a Coxiella ELISPOT assay (Limonar GJ et al). Apparently, this higher IFN-γ production in peripheral blood cells is not sufficient to activate Cburnetii- infected monocytes/macrophages at the site of infection, to such an extent that the infection is cleared.

In our population, the measurement of IFN-γ production alone has a sensitivity of 75% and a specificity of 82% at a cut-off of 500 pg/mL to discriminate proven chronic Q fever infection from seropositive controls. Notably, some controls had IFN-γ production that overlapped with chronic Q fever patients. The high IFN-γ production in these individuals, with low serological titres without signs of chronic Q fever, is intriguing. IFN-γ production by nonspecific PHA stimulation was not significantly higher (data not shown). We presume that the high C.burnetii-specific IFN-γ production may follow from ongoing exposure of T-cells by non- viable bacterial residues (Marnion BP et al, Sukocheva OA et al), or by the persistence of intracellular bacteria (Raoult D et al). The latter explanation suggests that these individuals, in spite of a low serological titre, are at risk for developing chronic Q fever disease.

The measurement of the production of monocyte-derived pro-inflammatory cytokines did not aid to differentiate between chronic Q fever patients and individuals previously exposed to C. burnetii. Nevertheless, both groups had higher production than naive controls. From this it can be concluded that restimulation with C. burnetii in primed individuals leads to enhanced production of cytokines by monocytes. We did not detect enhanced production of anti-inflammatory cytokines in chronic Q fever patients, albeit that the production of IL-10 in chronic Q fever patients was higher than in seronegative controls. This is in line with previous reports that describe overproduction of IL-10 by unstimulated peripheral blood mononuclear cells of patients with Q fever endocarditis compared to healthy controls (Capo C, et al, Honstettre A et al) However, these studies also claim low IL-10 in individuals with Q fever without chronic evolution. IL-10 was also found to induce C. burnetii replication in human monocytes and neutralization of this cytokine inhibited bacterial replication in monocytes from patients with Q fever endocarditis (Ghigo E et al). Thus, IL-10 may be important in the development of chronic Q fever, but we did not find that the measurement of C. burnetii-speciiic IL-10 production could be used as a diagnostic marker.

IL-2 production was found to be significantly lower in chronic Q fever patients than in seropositive controls. This distinct pattern of IL-2 production might reflect the type of T cells involved in the C. burnetii-specific immune responses. We assume that in chronic infection, the ongoing inflammatory response is accompanied by increased numbers of circulating C. burnetii-specific effector T-cells which produce

predominately IFN-γ and low amounts of IL-2 upon activation. In seropositive controls, it is probably the central memory T-cell that dominates and produces mainly IL-2 (Lanzavecchia A et al). We showed that the ratio of IFN-γ to IL-2 production is more specific than IFN-γ production alone (96% vs 82%), and has a slightly higher sensitivity (79% vs 75%) to distinguish patients with a chronic Q fever infection from seropositive controls. The relative high specificity of the IFN-y/IL-2 ratio suggests that seropositive individuals with a ratio above the cut-off, merit thorough follow-up for the presence of chronic Q fever. Ten of the 28 patients were not yet on treatment with antibiotics at the moment of bloodsampling. The absence of a significant difference in cytokine profile between untreated and the treated individuals suggests that the cytokine profile observed is not affected by the use of antibiotics. Still IFN-Y/IL-2 ratio seems to be related to the load of C. burnetii, since patients with a positive C. burnetii PCR in serum or plasma have a significantly higher ratio than patients with negative PCR. Longitudinal studies will be needed to assess the applicability of∑FN-y/IL-2 ratio in follow-up during treatment of patients with chronic Q fever.

Reference list

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interleukin-10 and transforming growth factor beta by peripheral blood mononuclear cells in Q fever endocarditis. Infect Immun 1996; 64(10): 4143-7.

Herremans T, Hogema BM, Nabuurs M, et al. Comparison of the performance of IF A, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment. Diagn Microbiol Infect Dis 2013; 75(1): 16-21.

Honstettre A, Imbert G, Ghigo E, et al. Dysregulation of cytokines in acute Q fever: role of interleukin-10 and tumor necrosis factor in chronic evolution of Q fever. J Infect Dis 2003; 187(6): 956-62.

Izzo AA, Marmion BP. Variation in interferon-gamma responses to Coxiella burnetii antigens with lymphocytes from vaccinated or naturally infected subjects. Clin Exp Immunol 1993; 94(3): 507-15.

Koster FT, Williams JC, Goodwin JS. Cellular immunity in Q fever: specific

lymphocyte unresponsiveness in Q fever endocarditis. J Infect Dis 1985; 152(6): 1283-9.

Lanzavecchia A, Sallusto F. Dynamics of T lymphocyte responses: intermediates, effectors, and memory cells. Science 2000; 290(5489): 92-7.

Limonard GJ, Thijsen SF, Bossink AW, Asscheman A, Bouwman jj. Developing a new clinical tool for diagnosing chronic Q fever: the Coxiella ELISPOT. FEMS Immunol Med Microbiol 2012; 64(1): 57-60.

Marmion BP, Sukocheva O, Storm PA, et al. Q fever: persistence of antigenic nonviable cell residues of Coxiella burnetii in the host—implications for post Q fever infection fatigue syndrome and other chronic sequelae. OJ 2009; 102(10): 673- 84. Raoult D. Host factors in the severity of Q fever. Ann N Y Acad Sci 1990; 590: 33-8.

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Seshadri R, Paulsen IT, Eisen JA, et al. Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci USA 2003; 100(9): 5455-60. Sukocheva OA, Marmion BP, Storm PA, Lockhart M, Turra M, Graves S. Long-term persistence after acute Q fever of non-infective Coxiella burnetii cell components, including antigens. OJ 2010; 103(11): 847-63.

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Claims

Claims

1. A method for diagnosing Q-fever status in a subject, the method comprising the steps of:

(a) obtaining a sample from said subject,

(b) contacting said sample with a source of a Coxiella burnetii antigen and

(c) determining the profile or expression level of both a pro-inflammatory cytokine and a T lymphocyte growth factor , in said sample at the end of step (b).

2. A method according to claim 1, wherein chronic Q-fever is diagnosed when step (c) leads to the finding of:

a detectable expression level or an increase of the expression level of a proinflammatory cytokine and a low or undetectable expression level of a T lymphocyte growth factor and/or

- a high ratio between the expression levels of said pro-inflammatory cytokine and said T lymphocyte growth factor .

3. A method according to claim 1 or 2, wherein:

-said pro-inflammatory cytokine is selected from the group consisting of: IL-1-β, IL-6, IL-17, T Fa and ΠΤΝΓγ, and

-said T lymphocyte growth factor is selected from the group consisting of: IL-2, IL- 2 family members, IL-10, TGF .

4. A method according to claim 3, wherein said pro-inflammatory cytokine is IFNy and said T lymphocyte growth factor is IL-2.

5. A method according to any one of claim 1 to 4, wherein the expression level of said pro-inflammatory cytokine and said T lymphocyte growth factor are determined by directly quantifying the amount of said pro-inflammatory cytokine and said T lymphocyte growth factor and/or indirectly by quantifying the amount of said nucleotide sequence encoding said pro-inflammatory cytokine and said T lymphocyte growth factor.

6. A method according to any one of claims 1 to 5, wherein said source of a Coxiella burnetii antigen is a heat-inactivated or formalin-fixed whole Coxiella burnetii cell.

7. A method according to any one of claims 1 to 6, wherein chronic Q-fever can be distinguished from acute Q-fever infection, past Q fever, cured Q fever, post Q-fever fatigue and no Q fever.

8. A method according to any one of claims 1 to 7, wherein the sample is a fluid obtained from the subject.

9. A method according to claim 8, wherein the fluid is selected from blood or spinal cord fluid.

10. An assay device for diagnosing Q-fever status or chronic Q-fever in a subject, wherein the device comprises molecules that specifically bind to either of said proinflammatory cytokine and said T lymphocyte growth factor.

11. A device according to claim 10, wherein said molecules are antibodies.

12. A device according to claim 10 or 11, wherein the device is a lateral flow test strip.

13. A method for treating chronic Q-fever in a subject, the method comprising the steps of:

(a) obtaining a sample from said subject,

(b) contacting said sample with a source of a Coxiella burnetii antigen,

(c) determining the profile of expression or expression level of both a proinflammatory cytokine and a T lymphocyte growth factor in said sample at the end of step (b), and

(d) wherein a subject with a low or undetectable level of expression of a T lymphocyte growth factor and a low or undetectable expression level of a nucleotide sequence encoding said T lymphocyte growth factor and/or a high ratio between the expression level of said pro-inflammatory cytokine and T lymphocyte growth factor as determined at the end of step (c) is being administered a treatment against Q-fever.