Metalloproteinases as biomarker of tick-borne encephalitis and biomarkers of its severity

Abstract

The present invention provides a method for the in vitro diagnosis of tick-borne encephalitis in an individual, wherein : (a) the levels of metalloproteinase 14 (MMP- 14) and metalloproteinase 17 (MMP- 17) are determined in a cerebrospinal fluid sample from an individual, and (b ) the levels of metalloproteinases in the test sample as obtained in step (a) are compared with the levels of metalloproteinases MMP- 14 and MMP- 17 in a reference s amp 1e, wherein the reference sample is a sample from a healthy control individual; and wherein levels of these two metalloproteinases in the test sample which are higher than in the reference sample indicate that the individual suf fers from tick-borne encephalitis. The present invention further provides metalloproteinases including MMP- 14 and MMP- 17 for use as a diagnostic biomarker of tick-borne encephalitis, as a biomarker of blood-brain barrier damage in tick-borne encephalitis, as a biomarker for monitoring the course and / or severity of tick-borne encephalitis, as a biomarker for assessing the ef fectiveness of tick-borne encephalitis pharmacotherapy.

Description

[0001] Metalloproteinases as biomarker of tick-borne encephalitis and biomarkers of its severity

[0002] Field of invention

[0003] The present invention relates to the field of medicine, in particular to medical diagnostics, more specifically to the diagnosis of tick-borne encephalitis (TBE) with an indication of the severity of this disease in individual patients. In particular, the invention relates to a method for the in vitro diagnosis of tick-borne encephalitis and to metalloproteinases for use as a diagnostic biomarker of tick-borne encephalitis, a biomarker of blood-brain barrier damage in tick-borne encephalitis, a biomarker for monitoring the course and / or severity of tick-borne encephalitis, a biomarker for assessing the effectiveness of tick-borne encephalitis pharmacotherapy.

[0004] Background art

[0005] Tick-borne encephalitis (TBE) is a zoonotic infectious disease caused by a virus of the Flaviviridae family most commonly transmitted by ticks. It is an increasingly frequently diagnosed disease, especially in areas characterised by an increase in tick populations in recent years, which include central and eastern Europe, including Poland. In European countries, about 2,000-3,000 cases and hospitalisations related to TBE are registered annually [Riccardi N, et al. Tick-borne encephalitis in Europe: a brief update on epidemiology, diagnosis , prevention, and treatment . Eur. J. Intern. Med. 2019; 62:1-6] . TBE is also the most common viral form of neuroinfections occurring in Poland .

[0006] Clinical data show that the number of recorded tick bites in Europe, Asia and America is increasing every year. The high number of bites is accompanied by an increasing number of infections that occur as a result of infection with tick-borne pathogens , which is a huge problem because tick- borne diseases often cause long- lasting and burdensome complications , including permanent health problems ( especially neurological or mental ) and even death . At the same time , it is observed that a greater number of ticks are infected with bacteria and viruses , including several pathogens at the same time , which makes the identi fication of the pathogen very important in view of the possibility of rapid application of targeted pharmacotherapy . Ticks can be vectors of bacteria, viruses and parasites , but one of the pathogens most commonly transmitted by ticks is tick-borne encephalitis virus from the Flavi virus genus . The first symptoms of infection do not seem dangerous - fever and muscle aches , but then the central nervous system becomes infected - usually the meninges and in more severe cases the brain and spinal cord . The consequences are intractable headaches and symptoms associated with involvement of the above-mentioned structures - neck sti f fness , epileptic sei zures , paralysis , balance and consciousness disorders . Patients diagnosed with tick- borne encephalitis require hospitalisation, but the course of treatment depends not only on the severity of the disease , but also on individual personal characteristics . Some patients recover quickly, others die from the infection and a large proportion require long-term rehabilitation . Symptoms of TBE in di f ferent people , regardless of their age , sex or severity of the disease , may have a di f ferent picture , which can confuse medical doctors when deciding on therapy . The clinical picture o f TBE corresponds to aseptic meningitis and / or encephalitis . In the early stages of this disease , symptoms are nonspeci fic, while the health consequences can be long- lasting and even irreversible . The disease therefore poses a huge challenge for both diagnosticians and medical doctors . The problem of tick-borne encephalitis af fects not only humans , but also animals , especially pets and livestock, which can contribute to signi ficant losses , including economic losses , in these sectors .

[0007] Tick-borne encephalitis is a disease for which there is no speci fic treatment . The relatively late and unclear symptoms make diagnosis and therapy di f ficult and insuf ficiently ef fective . Due to the lack of unambiguous symptoms that distinguish TBE from other viral neuroinfections , the diagnosis of the disease requires confirmation by laboratory tests . The primary diagnostic method is currently the evaluation of the level of speci fic IgM and IgG class antibodies in serum and / or cerebrospinal fluid using immunoenzymatic assays (ELISA) , less commonly indirect immunofluorescence or haemagglutination inhibition tests .

[0008] However, the diagnostic methods used to date do not include analyses of the degree and mechanism of blood-brain barrier damage , which in relation to viral infection of the nervous system indicates the degree of penetration of the pathogen into the infected structures . Indeed, damage to the bloodbrain barrier opens the way for the pathogen to infect the human nervous system, thereby causing tick-borne encephalitis . It is therefore not possible to predict the further development of the infection, to introduce appropriately selected pharmacotherapy, to monitor its ef fectiveness and to assess the risk of possible complications and long-term side ef fects of tick-borne encephalitis .

[0009] Currently available diagnostic means and methods for TBE are therefore insuf ficient . There is , therefore , a need for quick, effective, specific and sensitive means of diagnosing TBE that allow not only the diagnosis of the disease, but also the assessment of its severity, the monitoring of the effectiveness of its treatment, as well as the assessment of the extent of blood-brain barrier damage. There is therefore a need to identify specific disease biomarkers and to provide means, including diagnostic biomarkers and kits, for the unambiguous and rapid diagnosis of TBE, for assessment of the extent and mechanism of blood-brain barrier damage, the severity of TBE and for the assessment of the efficacy of pharmacotherapy for the disease.

[0010] The objective of the invention is therefore to provide new, sensitive biomarkers, methods and means for the quick, reliable, specific and sensitive diagnosis of tick-borne encephalitis, for assessment of the severity and monitoring the effectiveness of treatment of the disease and predicting its potential complications.

[0011] Brief description of the invention

[0012] The above objectives have been achieved by the solutions claimed in the attached patent claims. Preferable variants of the invention are defined in the dependent claims.

[0013] The present invention provides a method for the in vitro diagnosis of tick-borne encephalitis in an individual, wherein : a) levels of metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) are determined in a cerebrospinal fluid sample from an individual, and b) the levels of metalloproteinases in the test sample as obtained in step (a) are compared with the levels of metalloproteinases MMP-14 and MMP-17 in a reference sample, wherein the reference sample is a sample from a healthy control individual; and wherein levels of these two metalloproteinases in the test sample higher than in the reference sample indicate that the individual is suffering from tick-borne encephalitis.

[0014] Preferably, in the method according to the invention, the level of a metalloproteinase selected from MMP-2, MMP-9 and MMP-16 is also additionally determined.

[0015] Preferably, in the method according to the invention the levels of at least two metalloproteinases selected from MMP-2, MMP-9, MMP-16, preferably MMP-2 and MMP-9, MMP-2 and MMP-16, MMP-9 and MMP-16, or MMP-2, MMP-9 and MMP-16, are additionally determined.

[0016] More preferably, in the method according to the invention, the levels of MMP-2, MMP-9 and MMP-16 are additionally determined .

[0017] Preferably, in the method according to the invention, the individual from whom the sample is derived is a human being .

[0018] Preferably, in the method according to the invention, the levels of metalloproteinases are determined by an ELISA method or by liquid chromatography coupled to mass spectrometry (LC-MS) , especially by LC-MS / MS.

[0019] The present invention further provides metalloproteinases including metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) for use as a diagnostic biomarker for tick-borne encephalitis.

[0020] Also the present invention provides metalloproteinases including metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) for use as a biomarker of blood-brain barrier damage in tick-borne encephalitis. The present invention further provides metalloproteinases including metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) for use as a for monitoring the course and / or severity of tick-borne encephalitis.

[0021] The present invention further provides metalloproteinases including metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) for use as a biomarker for assessing the effectiveness of the tick-borne encephalitis pharmacotherapy .

[0022] Preferably, the uses according to the invention further include the use (as an additional biomarker) of a metalloproteinase selected from MMP-2, MMP-9 and MMP-16.

[0023] Preferably, the uses according to the invention include the use of at least two metalloproteinases selected from MMP-2, MMP-9, MMP-16 as the additional biomarker.

[0024] More preferably, the applications according to the invention include the use as an additional biomarker of metalloproteinases: MMP-2, MMP-9 and MMP-16.

[0025] Detailed description of the invention

[0026] The inventions according to the present application are based on the analysis of the levels of metalloproteinases (MMPs) in a cerebrospinal fluid (CSF) sample from a tick- bitten patient with suspected tick-borne encephalitis (TBE) . Metalloproteinases are enzyme proteins naturally present in the cerebrospinal fluid of healthy individuals (without TBE) at very low levels, as their activity could break the continuity of the blood-brain barrier. According to the invention, it found that one of the most important proteins whose expression distinguishes the cerebrospinal fluid profile of patients with TBE from healthy individuals are metalloproteinases. These enzymes can enzymatically degrade the proteins of the intercellular matrix leading to their partial remodelling or complete damage, allowing the pathogen to invade the body's internal structures, including the nervous system. Increased levels and activity of metalloproteinases in the cerebrospinal fluid are known to accompany many diseases of the nervous system, such as dementia, multiple sclerosis and Alzheimer's disease. As a result of high levels and activity of metalloproteinases, signalling proteins and molecules in the blood flow uncontrollably into the cerebrospinal fluid. This is observed by high levels of protein content in the cerebrospinal fluid. According to the invention, it has been found that in patients with tick-borne encephalitis, the levels of these proteins are significantly, often many times, increased in the case of this disease compared to the levels of these proteins in reference samples, i.e. from healthy (control) individuals. The present inventors have found, for the first time, that in the case of tick-borne encephalitis, an infection caused by the tick-borne encephalitis virus (TBEV) , the levels of metalloproteinases are increased compared to the levels found in healthy individuals, which makes it possible to diagnose this disease and to assess its severity. More specifically, according to the invention, it has been found that increased levels of metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) in a test sample relative to a reference sample (from a healthy control individual) are indicative that the individual has tick-borne encephalitis. In other words, the detection of increased levels of metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) in the test sample relative to the reference sample allows a sensitive and specific diagnosis of tick- borne encephalitis. The levels of metalloproteinases 14 and 17 are respectively 3.5-fold and 7-fold higher in the cerebrospinal fluid of individuals with TBE than in healthy controls. The degree of increase of the aforementioned MMP-14 and MMP-17 levels is also indicative of the severity of TBE. Such increased levels of metalloproteinases contribute to the impairment of the blood-brain barrier functioning and the opening of a pathway for the virus to colonise the nervous system of an infected individual, e.g. a human being. As a result, the detection of increased levels of metalloproteinases, particularly MMP 14 and 17, can be a key parameter showing the severity of the infection and the severity of damage to the infected organism, including the brain. Therefore, extending the diagnosis of tick-borne encephalitis to the analysis of cerebrospinal fluid levels of metalloproteinases in individuals with distressing symptoms following a tick bite, particularly MMP 14 and 17, provides an important diagnostic tool to more effectively diagnose this disease, determining the severity of the infection and the extent of damage to the body (especially at the level of the central nervous system, and in particular the blood-brain barrier) , giving the clinician information to support an unambiguous diagnosis and, consequently, the possibility of quickly applying the most effective therapy in a given case and monitoring its effectiveness.

[0027] In the first aspect, the present invention provides a method for the in vitro diagnosis of tick-borne encephalitis in an individual, preferably a human being, wherein: (a) the levels of metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) are determined in a sample of cerebrospinal fluid from an individual, and (b) their levels in the test sample as obtained in step a) are compared with the levels of MMP-14 and MMP-17 in a reference sample , wherein the reference sample is a sample from a healthy control individual , wherein the determination of higher levels of MMP- 14 and MMP- 17 in the test sample compared to the levels of these metalloproteinases in the reference sample indicates that the individual is suf fering from tick-borne encephalitis .

[0028] The test individual is preferably a human being, but it can also be an animal in which a diagnosis of tick-borne encephalitis is needed, e . g . a domestic animal such as a dog or cat , or a farm animal . The reference sample according to the invention is a sample from a healthy control individual , i . e . a healthy individual without tick-borne encephalitis . For example , where the control individual is a human being, the reference sample is a sample from a healthy human being .

[0029] The determination of higher level s of metalloproteinases 14 and 17 in a test sample relative to a reference sample allows an ef fective diagnosis of TBE . On the other hand, the degree of increase in the levels of these metalloproteinases in the test sample relative to the reference level ( in a sample from an individual without TBE ) is a diagnostic indicator of disease progression ( severity) , allowing di f ferentiation between patients with varying degrees of disease severity, giving an idea of the patient ' s current disease state while at the same time providing an opportunity to predict further development of the infection and the risk of possible complications . In this way, even at the initial stages of diagnosis and treatment , it is possible to introduce an appropriately selected therapy, which would result in a more ef fective treatment process and, consequently, protect the patient from the long-term side ef fects of tick-borne encephalitis . Preferably, in the method according to the invention, the level of at least one additional metalloproteinase selected from: MMP-2, MMP-9 and MMP-16 can be determined. Preferably, in the method according to the invention, the level of at least two metalloproteinases selected from MMP-2, MMP-9, MMP-16 is additionally determined, preferably MMP-2 and MMP-9, MMP-2 and MMP-16, MMP-9 and MMP-16, or MMP-2, MMP-9 and MMP-16. More preferably, in the method according to the invention, the levels of MMP- 2, MMP-9 and MMP-16 are additionally determined. The determination of the levels of additional metalloproteinases allows increasing the sensitivity and specificity of the method according to the invention, confirming the degree of blood-brain barrier damage determined on the basis of the levels of metalloproteinases 14 and 17.

[0030] The level of metalloproteinase in a sample of cerebrospinal fluid, is determined according to the invention by any measurement method that allows quantitative determination of the aforementioned MMP . This method can be an ELISA method, which allows for a quick and simple determination and is relatively inexpensive. Another method can be a method using liquid chromatography coupled to mass spectrometry (LC-MS) , especially LC-MS / MS, which allows for highly accurate determination results.

[0031] In further aspects of the present invention, metalloproteinases are provided for use as a diagnostic biomarker for tick-borne encephalitis, a biomarker for blood-brain barrier damage in tick-borne encephalitis, a biomarker for monitoring the course and / or severity of tick-borne encephalitis, a biomarker for assessing the effectiveness of tick-borne encephalitis pharmacotherapy. In accordance with the present invention, it has been shown that the metalloproteinases MMP-14 and MMP-17 can be effectively used:

[0032] .as a diagnostic biomarker of TBE by determining higher levels of these metalloproteinases in a test sample relative to a reference sample,

[0033] .as a biomarker of blood-brain barrier damage,

[0034] .as a biomarker for monitoring the course and / or severity of tick-borne encephalitis,

[0035] .as a biomarker of potential neurological and psychiatric complications of TBE,

[0036] .as a biomarker for assessing the effectiveness of the tick-borne encephalitis pharmacotherapy.

[0037] In accordance with the present invention, it has also been shown that, in addition to the metalloproteinases MMP-14 and MMP-17, metalloproteinases selected from: MMP-2, MMP- 9 and MMP-16, especially MMP-2, MMP-9 and MMP-16 in combination, which further increases the diagnostic sensitivity and specificity according to the present invention. The levels of each of these metalloproteinases are higher than in healthy control individuals.

[0038] The advantages of the present invention are manifold. This is because the present invention allows for:

[0039] • unambiguous identification of the type of pathogen causing encephalitis,

[0040] • determination of the progression (severity) of the TBE and the degree of the blood-brain barrier damage,

[0041] • determination of the body's condition during infection with the virus causing tick-borne encephalitis,

[0042] • predicting the development of infection and the risk of complications, • application of appropriate pharmacotherapy for patients with tick-borne encephalitis .

[0043] The present invention, based on the analysis of MMP- 14 and - 17 metalloproteinase levels in the cerebrospinal fluid of an individual with suspected tick-borne encephalitis , therefore allows for a more ef ficient and quick speci fic diagnosis of this disease and provides more knowledge about the patient ' s condition, allowing for faster implementation of adequate pharmacotherapy and avoidance or minimisation of complications of the disease . By linking the levels of metalloproteinases in a patient ' s cerebrospinal fluid to the severity of the disease , knowledge of the levels of these proteins in individual patients also provides information about the degree of damage in the body caused by this infection . Faster and more extensive diagnosis , together with prompt and appropriate pharmacotherapy, means faster cure , fewer disease complications and deaths from the disease or its complications . In addition, monitoring the levels of the biomarker according to the invention over time allows monitoring the course of the disease and assessing the ef fectiveness of the pharmacotherapy provided . The inventions disclosed herein can therefore be used not only for the diagnosis of TBE , but also for monitoring the ef fectiveness of treatment (pharmacotherapy) of TBE . In this case , the sample to be tested is from the individual undergoing treatment and / or after treatment , wherein the levels of metalloproteinases in the sample from the patient are determined at predetermined intervals and compared with the corresponding data for that patient obtained earlier, i . e . at the stage of the diagnosis of TBE and / or at an earlier stage of its treatment . The change in the levels of the TBE biomarker according to the invention relative to the levels determined earlier will provide a diagnostic indicator allowing to determine whether the treatment applied is effective. The change in the levels of the biomarker according to the invention during treatment towards reference levels (in individuals without TBE) indicates the effectiveness of the TBE treatment. After successful treatment, the levels of the biomarker according to the invention should gradually decrease until they reach levels like those of an individual without TBE.

[0044] The present invention will now be illustrated in the following example and figure, which, however, are not intended to limit in any way the scope of the invention as defined in the patent claims. Unless otherwise indicated, all methods and parameters are those commonly used in the field to which the present invention belongs, and the equipment and reagents used are those recommended by their manufacturers .

[0045] Brief description of the figures

[0046] Figure 1 shows the levels of identified metalloproteinases (MMP-2, -9, -14, -16, -17) in the cerebrospinal fluid of patients with tick-borne encephalitis (TBE; n=20) compared to healthy individuals (Control; n=10) . Results obtained by proteomic analysis using the LC-MS / MS technique (QExactiveHF-Orbitrap) , presented as mean and normalised to Control taken as 100%. *- statistically significant differences at p<0.05.

[0047] Example

[0048] Collection of samples

[0049] The cerebrospinal fluid samples were collected from a group of 20 patients (seven females and 13 males) with a mean age of 40.5 years (range 23-53) treated at the Department of Infectious Diseases and Neuroinfections, Medical University of Bialystok. All patients were diagnosed with tick-borne encephalitis according to the guidelines of the European Academy of Neurology (EAN) , based on clinical symptoms, positive serology and lymphocytic pleocytosis in cerebrospinal fluid. Samples were also collected from 10 healthy individuals (four women and six men) with a mean age of 41.5 years (range 22-55) . The study was conducted in accordance with the Declaration of Helsinki and was approved by the Local Bioethics Committee of the Medical University of Bialystok (Poland) , No. R-I-002 / 169 / 2018. Written informed consent was obtained from all patients and healthy donors.

[0050] Protein digestion and proteomic analysis

[0051] Prior to protein digestion, samples were centrifuged at 3000 xg (4°C) and total protein concentration was measured using the Bradford assay. A sample volume containing 30 pg of protein was denatured by mixing with 8M urea. Proteins were then reduced with 1 , 4-dithiothreitol (DTT, 10 mM) and alkylated by incubation with iodoacetamide ( IAA, 50 mM) . To stop the alkylation, DTT was added again. After dilution (1:4, sample : ammonium bicarbonate, 25 mM) , samples were digested overnight (37°C) with trypsin (Promega, Madison, WI, USA) at a ratio of 1:50 ( trypsin : protein) . To stop trypsinisation, formic acid (FA, 10%) was added in an amount to ensure a final concentration in the samples of 0.1%. The resulting peptide mixture was evaporated under an inert gas atmosphere and frozen until direct analysis.

[0052] Thawed peptides were dissolved in 5% acetonitrile with 0.1% FA and separated using a high-performance liquid chromatography system (Ultimate 3000; Dionex, Idstein, Germany) on a pPAC 200 column ( PharmaFluidics , Ghent, Belgium) at a flow rate of 0.300 pl / min. The solvent gradient started after 3 min and increased to 60% eluent B (90% ACN + 0.03% FA) for 55 min. Eluent A contained 5% ACN with 0.1% FA. Eluted peptides were analysed using a Q Exactive HF mass spectrometer with an electrospray ionisation (ESI) source (Thermo Fisher Scientific, Bremen, Germany) .

[0053] Protein identification and quantification without iabeiiing

[0054] The data obtained were cross-referenced with the UniProtKB-SwissProt database (taxonomy: Homo sapiens, release 2021-02) using Proteome Discoverer 2.0 (Thermo Fisher Scientific, Seattle, WA, USA) . Peptide mass tolerance was set to 10 ppm, MS / MS mass tolerance was set to 0.02 Da, cysteine carbamidomethylation / carboxymethylation and methionine oxidation were set as dynamic modifications of amino acid residues. Quantification without protein labelling was carried out based on the intensity of precursor ion signals .

[0055] Metalloproteinase activity

[0056] MMP-2 and MMP-9 metalloproteinase activity was measured by zymography, with electrophoretic separation of samples in a 10% gelatin-containing gel (1 mg / ml) . MMP activity was induced by incubating the gel in Tris-HCl buffer (50 mM, pH 7.8) with CaC12 (5 mM) and Triton X-100 (0.1%) for 20 hours at 37 °C. The gel was then stained with Coomassie blue and densitometric analysis of bright bars on a dark background was performed using the Versa Doc System and Quantity One software (Bio-Rad Laboratories Inc., CA, USA) .

[0057] Statistical analysis

[0058] The results of the determination of individual protein levels were normalised ( logarithmised and scaled) using MetaboAnalyst 5.0 software (http: / / www.metaboanalyst.ca) which ensured a normal distribution of the results.

[0059] Resul ts

[0060] The results obtained clearly showed significant differences between the proteomic profile of the cerebrospinal fluid of patients with tick-borne encephalitis and the control group, which may be important in the course of the disease and its therapy.

[0061] The levels of metalloproteinases 2, 9, 14, 16 and 17, examined using a proteomic approach using advanced LC- MS / MS technology leading to the analysis of protein profiles in cerebrospinal fluid (CSF) , were found to be increased in patients diagnosed with tick-borne encephalitis compared to levels in reference samples, i.e. in the cerebrospinal fluid of 10 healthy individuals. The results are shown in Fig. 1. Particularly pronounced differences were found in the case of MMP 14 and MMP 17. The average result obtained for metalloproteinase 14 in samples from people with TBE was more than 3.5 times higher, while the level of metalloproteinase 17 was more than seven times higher compared to the respective levels of these metalloproteinases found in healthy individuals.

[0062] Correlation of proteomic results with clinical data

[0063] The results of the study showed a strong positive correlation between MMP-2 activity and total protein concentration in the cerebrospinal fluid (r=0.75) , which partly influenced each patient's length of recovery (hospitalisation) . In addition, protein concentration in the cerebrospinal fluid positively correlated with MMP-2 (r=0.62) and MMP-17 concentration (r=0.72) .

[0064] In addition, the ROC curve indicated that MMP-9 activity also differentiated patients with meningitis from patients with encephalitis at a cut-off of - 148 points (sensitivity -71.3%, specificity -100%, AUG - 0.86) . MMP-9 activity was higher in patients with encephalitis (Me = 1.5, min = 0.98, max = 1.69) than in patients with meningitis (Me = 1.1, min = 0.85, max = 1.48) , p<0.05. Patients with ataxia had significant differences in MMP-9 activity (with ataxia: Me = 1.55, min = 1.24, max = 1.69; without ataxia: Me = 1.08, min = 0.85, max =1.36) , and IL -1A (with ataxia: Me = - 0.08, min = -0.37, max = 0.61; without ataxia: Me = 0.99, min =-0.41, max =1.34) concentrations. MMP-9 activity correlated negatively with ALT (r=-0.69) and AST (r=- 0.57) , while MMP-17 concentrations showed a moderate positive correlation with ALT. There was also a moderate positive correlation between MMP-17 and cytosis of the cerebrospinal fluid (r=0.49) . MMP-17 showed a strong positive correlation with time from tick bite to onset of symptoms .

[0065] The results presented here indicate that tick-borne encephalitis-induced changes in the proteome of the cerebrospinal fluid are not only a result of the progression of the disease, but also an indicator of its course. More specifically, this applies to metalloproteinases, the expression and activity of which indicate the severity of the disease, the degree of damage to the blood-brain barrier and correspond to the length of patients' recovery. Moreover, differences in the expression / activity of specific metalloproteinases can also differentiate between the different clinical forms of tick-borne encephalitis, such as meningitis and encephalitis. The obtained results allow for more effective diagnosis and treatment of this disease, selection of supportive therapy and prediction of further individual patient fate. In summary, according to the invention, it has been unequivocally shown that the level s of metalloproteinases , especially metalloproteinases 14 and 17 , in patients with TBE are statistically signi ficantly increased relative to the values obtained in the cerebrospinal fluid of healthy individuals . According to the invention, it has furthermore been unequivocally shown that the levels of metalloproteinases 2 , 9 and 16 in patients with TBE are also statistically signi ficantly increased relative to the values obtained in the TBE of healthy individuals . This means that the metalloproteinases including MMP- 14 and MMP- 17 are suitable for use as an ef fective diagnostic biomarker of tick-borne encephalitis , as well as a biomarker of blood-brain barrier damage in tick-borne encephalitis , and are also suitable for use as an ef fective biomarker for monitoring the severity and / or course of tick-borne encephalitis and for assessing the ef fectiveness of tick-borne encephalitis pharmacotherapy . Metalloproteinases selected from MMP-2 , MMP- 9 and MMP- 16 are suitable for use as additional diagnostic biomarkers for TBE , biomarkers of blood-brain barrier damage in TBE , as well as biomarkers for monitoring the severity and / or course of TBE and for for assessing the ef fectiveness of TBE pharmacotherapy .

Claims

AMENDED CLAIMS received by the International Bureau on 19 January 2025 (19.01.2025)Patent claims1. A method for the rn vi tro diagnosis of tick-borne encephalitis in an individual, wherein:(a) the levels of metalloproteinase 14 (MMP-14) and metalloproteinase 17 (MMP-17) are determined in a cerebrospinal fluid sample from an individual, and(b) the levels of metalloproteinases in the test sample as obtained in step (a) are compared with the levels of metalloproteinases MMP-14 and MMP-17 in a reference s amp 1 e , wherein the reference sample is a sample from a healthy control individual ; wherein the levels of these two metalloproteinases in the test sample higher at least 3 times and 6 times for MMP-14 andMMP-17, respectively, than in the reference sample indicate that the individual is suffering from tick-borne encephalitis; wherein the level of the metalloproteinases is determined by an ELISA method or by liquid chromatography coupled to mass spectrometry (LC-MS) , especially by LC-MS / MS.

2. The method for the in vitro diagnosis of tick-borne encephalitis according to claim 1, wherein a level of a metalloproteinase selected from MMP-2, MMP-9 and MMP-16 is additionally determined.

3. The method for the in vitro diagnosis of tick-borne encephalitis according to claim 1 or 2, wherein the levels of at least two metalloproteinases selected from MMP-2, MMP-9, MMP-16, preferably MMP-2 and MMP-9, MMP-2 and MMP-16, MMP-9 and MMP-16, or MMP-2, MMP-9 and MMP-16, are additionally determined.

4. The method for the in vitro diagnosis of tick-borne encephalitis according to claim 3, wherein the levels of MMP-2,MMP-9 and MMP-16 are determined.

5. The method for the in vitro diagnosis of tick-borne encephalitis according to any one of claims 1 to 4, characterised in that the individual is a human being.AMENDED SHEET (ARTICLE 19)6. Use of metalloproteinases including MMP-14 and MMP-17 as a diagnostic biomarker in the in vitro diagnosis of tick- borne encephalitis.

7. Use of metalloproteinases including MMP-14 and MMP-17 as a biomarker in the in vitro method of diagnosis of bloodbrain barrier damage in tick-borne encephalitis.

8. Use of metalloproteinases including MMP-14 and MMP-17 as a biomarker in the in vitro method of monitoring the course and / or severity of tick-borne encephalitis.

9. Use of metalloproteinases including MMP-14 and MMP-17 e as a biomarker in the in vitro method of assessing the effectiveness of tick-borne encephalitis pharmacotherapy.

10. The use according to any one of claims 6 to 9, wherein a metalloproteinase selected from MMP-2, MMP-9 and MMP-16 is used as an additional biomarker.

11. The use according to any one of claims 6 to 10, wherein at least two metalloproteinases selected from MMP-2, MMP-9, MMP-16 are used as the additional biomarker.

12. The use according to claim 11, wherein the metalloproteinases MMP-2, MMP-9 and MMP-16 are used as the additional biomarker.AMENDED SHEET (ARTICLE 19)

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