CROSS-REFERENCE TO PRIOR APPLICATIONS

This application is the U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2015/077045, filed on Nov. 19, 2015, which claims the benefit of European Patent Application No. 14193886.0, filed on Nov. 19, 2014. These applications are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the means and methods for the detection of bacterial infections, methods discriminating between viral and bacterial infections, methods of stratifying patients for subsequent treatment and further diagnostic purposes and methods to monitor antibiotherapy. The present invention is based on the detection of specific epitopes of human neutrophil lipocalin (HNL) using specific binding agents.

BACKGROUND OF THE INVENTION

New antibiotic resistance mechanisms emerge and spread globally threatening the ability to treat common infectious diseases, resulting in death and disability of individuals who until recently could continue a normal course of life. Without effective anti-infective treatment, many standard medical treatments will fail or turn into very high risk procedures (http://www.who.int/mediacentre/factsheets/fs194/en/; Antimicrobial resistance—Fact sheet No. 194, updated April 2014).

In 2012, there were about 450,000 new cases of multidrug-resistant tuberculosis (MDR-TB). Extensively drug-resistant tuberculosis (XDR-TB) has been identified in 92 countries. MDR-TB requires treatment courses that are much longer and less effective than those for non-resistant TB. There are high proportions of antibiotic resistance (ABR) in bacteria that cause common infections (e.g. urinary tract infections, pneumonia, bloodstream infections) in all regions of the world. A high percentage of hospital-acquired infections are caused by highly resistant bacteria such as Methicillin-ResistantStaphylococcus aureus(MRSA) or multidrug-resistant Gram-negative bacteria. Treatment failures due to resistance to treatments of last resort for gonorrhea (third-generation cephalosporins) have now been reported from 10 countries. Gonorrhea may soon become untreatable as no vaccines or new drugs are in development. Patients with infections caused by drug-resistant bacteria are generally at increased risk of worse clinical outcomes and death, and consume more healthcare resources than patients infected with the same bacteria that are not resistant.

As an example, the death rate for patients with serious infections caused by common resistant bacteria treated in hospitals can be about twice that of patients with infections caused by the same non-resistant bacteria. For example, people with MRSA, another common source of severe infections in the community and in hospitals, are estimated to be 64% more likely to die than people with a non-resistant form of the infection.

Various modes of behaviors favor the development of AMR. For example, antibiotics are extensively used for prophylaxis in animal breeding and livestock industry, e.g. to increase weight gain. This leads to development of resistant bacteria in these animals, their transmission to humans working with these animals or consumers buying and using products from affected animals or farms. Another problem is the contamination of the environment surrounding animal farm. Further, the development of AMR may also result from the careless prescription and distribution of antimicrobial drugs, e.g. antibiotics, to patients that do not need these medicaments, for example because the severity of the illness does not justify the use of antibiotics, or the illness is not due to bacterial infection. Additionally, antibiotics are freely available as OTC drugs in several countries. Patients frequently do not use these antibiotics for a sufficiently long period of time, they do not use the right antibiotics for their respective illnesses, or they do not properly discard remaining antibiotics, but release it to the environment, e.g. by flushing them down the toilet.

There is an increasing need in the field to discriminate between bacterial and viral infections and to administer antibiotics only to patients with confirmed bacterial infections. It is one objective of the present invention to provide means and methods for this purpose.

Human neutrophil lipocalin (HNL) (also named neutrophil gelatinase-associated lipocalin (NGAL)), is a ubiquitous glycoprotein originally isolated from human neutrophils and localized in their specific granules. HNL/NGAL exists as a 25-kD monomer, or as a 45-kD disulfide-linked homodimer, and it is covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulfide bridge as a 135-kD heterodimeric form (Cai et al., Clin J Am Soc Nephrol. December 2010; 5(12): 2229-2235). The amino acid sequence of HNL is shown in SEQ ID NO: 1 corresponding to the protein sequence found in www.uniprot.org/uniprot/P80188, in particular referring to isoform 1.

The discriminatory power of HNL in the discrimination between acute viral and acute bacterial infections showed variations in sensitivities and specificities between 75-94%. The lower figures are obviously not satisfactory in a clinical setting and the goal of the development should be an HNL assay that reliably rules-out bacteria as the cause of the acute infections in order to reduce the need to treat the infection with antibiotics. A secondary goal should be a reliable identification of those acute infections in need of antibiotics treatment.

Measurement of HNL (human neutrophil lipocalin) in serum or plasma can be used to discriminate acute infections caused by virus from those caused by bacteria as disclosed in EP 0 756 708 B1.

It is an object of the invention to provide new binding agents specifically recognizing HNL and to provide methods and uses of such binding agents, e.g. in the detection of bacterial infections, the selection of the appropriate treatment of patients, and the like.

Another objective of the invention is the monitoring of septic patients undergoing antibiotherapy e.g. to help to determine efficacy of treatment and time of cessation of antibiotics through repeated testing of sample of patients at different timepoints

SUMMARY OF THE INVENTION

The present invention provides means for and methods of detecting HNL in a sample. These means are binding agents, e.g. antibodies or derivatives thereof that are characterized by their binding regions (e.g. the CDR regions) as well as their capacities to bind specifically to HNL. The binding agents of the present invention can be used in the specific detection of HNL, and permit distinguishing between bacterial and viral infections. In some embodiments, the amount of HNL as a parameter is suitable for the identification of bacterial infections and/or distinguishing bacterial from viral infections and can be used in diagnosis of disease, prognosis of clinical outcomes, or in the monitoring of the course of anti-bacterial treatments. The correct diagnosis of an acute bacterial infection permits prescribing antibiotics appropriately to a subject from whom a sample is subjected to analysis of the presence or absence or quantity of HNL, e.g. in blood samples or urine samples.

In embodiments of the invention, the biological sample can be pretreated with a neutrophil activator to improve the detection of HNL and further improve the herein disclosed methods. In preferred embodiments the neutrophil activator is an N-formyl peptide, more preferably the tri-peptide fMLP. In further embodiments, the neutrophil activator is Protein A, or may be a combination of fMLP and Protein A. The present invention further envisages that the neutrophil activator is Lipopolysaccharide (LPS), platelet-activating factor, an unmethylated CpG oligodinucleotide, or tumor necrosis factor (TNF). Thus, according to specific embodiments of the present invention, the neutrophil activator may be any combination of two or more of the elements selected from the group consisting of fMLP, Protein A, Lipopolysaccharide (LPS), platelet-activating factor, an unmethylated CpG oligodinucleotide, and tumor necrosis factor (TNF).

In one embodiment of the invention, a polypeptide, e.g. a recombinantly produced polypeptide, comprising an amino acid sequence corresponding to amino acid 83 through 154 of HNL, or monomeric or dimeric HNL purified from human plasma/serum may be used in the production of a binding agent specifically recognizing HNL. The polypeptide can be used to immunize an animal, obtain antibody producing cells, produce hybridoma cells, harvest antibodies, optionally elucidate their sequence and further optionally produce recombinant antibodies, characterize these antibodies for their capacity to specifically bind HNL, and use these antibodies in the diagnosis of bacterial infections by determination of the HNL level in a sample. The antibodies may be modified as described herein below.

In embodiments of the invention, methods for detecting or diagnosing a bacterial and/or viral infection, or permitting the discrimination between viral and bacterial infections are provided, characterized in that the HNL-level in a sample is measured using a binding agent specifically binding to amino acids of epitopes of HNL that are exposed on the inner and outer rim surface as depicted inFIG. 1, wherein these epitopes preferably comprise amino acids 83 to 88 (preferably amino acids 82 to 102) and/or 141 to 156 of HNL depicted in SEQ ID NO. 1. In additional embodiments, these epitopes further comprise amino acids 51 to 76 and/or 113 to 132 of HNL depicted in SEQ ID NO: 1.

Thus, in some embodiments the binding agents disclosed herein, and the methods wherein said binding agents are used, recognize specifically amino acids comprised by amino acids 51 to 76 and/or 82 to 102 and/or 113 to 132 and/or 141 to 156 of HNL (SEQ ID NO: 1). In a particularly preferred embodiment a binding agent is capable of specifically binding to a polypeptide epitope of HNL, wherein said polypeptide epitope comprises amino acids 141 to 156 of HNL as defined in SEQ ID NO: 1, or wherein said polypeptide epitope is a conformational epitope comprised by the peptide in SEQ ID No: 26.

In preferred embodiments of the present invention, the binding agent binds to HNL that is at least predominantly produced by neutrophils.

In some embodiments of the present invention, the binding agent disclosed and used herein binds to a region of HNL that is comprised by the sequence depicted in SEQ ID NO: 26.

In some embodiments of the present invention, the binding agent comprises a HNL epitope-specific binding region comprising at least one, two, three, four, five, or six amino acid sequences depicted in SEQ ID Nos: 8 to 13, 14 to 19, and 20 to 25, or a functional derivative thereof.

In another embodiment of the invention a method for diagnosing an infection, e.g. a bacterial infection, or re-testing a bacterial infection, e.g. in the course of treatment with antibiotics, is provided, or differentiating a bacterial infection from a viral infection characterized in that a sample, e.g. a sample obtained from a subject suspected to have a bacterial or viral infection is analyzed, is analyzed for the presence of the HNL-level using a binding agent specifically binding to the sequence depicted in SEQ ID NO: 26, or as defined herein above. Said method may comprise the steps:a) Incubating a sample in the presence of said binding agent;b) Optionally washing off unbound sample material;c) Measuring the level of HNL in a sample from a subject suspected to have a bacterial or viral infection;d) Comparing the level of HNL measured in step c) with one or more control samples, optionally obtained from(i) healthy subjects,(i) subjects known to have a bacterial infection, and(iii) subjects known to have a virus infection, and/oroptionally comparing the level of HNL measured in step c) with one or more normalized control values indicative for healthy subjects, subjects with a virus infection, and/or subjects with a bacterial infection.

In a specific embodiment, the method comprises a further step of diagnosing a bacterial infection when the level of HNL in step c) is significantly higher than the level detected in control samples (i) of healthy subjects and (iii) of patients known to have a viral infection.

In another aspect the present invention relates to a method of ruling out a bacterial infection in a subject comprising:a) measuring the polypeptide concentration of HNL in a sample obtained from a subject using a binding agent as defined above, or a diagnostic composition or kit as defined above; andb) ruling out a bacterial infection for the subject if the polypeptide concentration of HNL determined in step (a) is lower than a pre-determined first threshold value.

In a further aspect, the present invention provides a method of ruling out a viral infection in a subject comprising:a) measuring the polypeptide concentration of HNL in a sample obtained from a subject using a binding agent as defined above, or a diagnostic composition or kit as defined above; andb) ruling out a viral infection for the subject if the polypeptide concentration of HNL determined in step (a) is higher than a pre-determined first threshold value.

In still another aspect the present invention relates to a method of ruling in a bacterial infection in a subject comprising:a) measuring the polypeptide concentration of HNL in a sample obtained from a subject using a binding agent as defined above, or a diagnostic composition or kit as defined above; andb) ruling in a bacterial infection for the subject if the polypeptide concentration of HNL determined in step (a) is higher than a pre-determined first threshold value.

In yet another aspect, the present invention provides a method of ruling in a viral infection in a subject comprisinga) measuring the polypeptide concentration of HNL in a sample obtained from a subject using a binding agent as defined above, or a diagnostic composition or kit as defined above; andb) ruling in a viral infection for the subject if the polypeptide concentration of TRAIL determined in step (a) is lower than a pre-determined first threshold value.
In one embodiment, the above method of ruling out a bacterial infection or ruling in a viral infection further comprisesa) measuring the polypeptide concentration of TRAIL in a sample obtained from a subject; andb) ruling out a bacterial infection or ruling in a viral infection for the subject if the polypeptide concentration of TRAIL determined in step (a) is higher than a pre-determined first threshold value.
In another embodiment, the above method of ruling out a viral infection or the method of ruling in a bacterial infection further comprisesa) measuring the polypeptide concentration of TRAIL in a sample obtained from a subject; andb) ruling in a bacterial infection or ruling out a viral infection for the subject if the polypeptide concentration of TRAIL determined in step (a) is lower than a pre-determined first threshold value.

In a further aspect the present invention relates to a method of distinguishing between a bacterial infection and a viral infection in a subject comprising:a) measuring the polypeptide concentration of HNL using a binding agent as defined above or a diagnostic composition or kit as defined above, and of CRP, optionally of TRAIL, in a sample obtained from a subject;b) applying a pre-determined mathematical function on the concentrations of HNL and CRP and optionally of TRAIL, to compute a score;c) comparing the score to a predetermined reference value.

In yet another aspect the present invention provides a method of distinguishing between a bacterial or mixed infection, and a viral infection in a subject comprising:a) measuring the polypeptide concentration of HNL using a binding agent as defined above, or a diagnostic composition or kit as defined above, and of CRP, optionally of TRAIL, in a sample obtained from a subject;b) applying a pre-determined mathematical function on the concentrations of HNL and CRP and optionally of TRAI, to compute a score;c) comparing the score to a predetermined reference value.

According to a further aspect, the invention provides a method of providing a treatment recommendation for a subject comprising:a) measuring the polypeptide concentration of HNL in a sample obtained from a subject using a binding agent as defined above, or a diagnostic composition or kit as defined above; andb) recommending that the subject receives an antibiotic treatment if the polypeptide concentration of HNL determined in step (a) is higher than a pre-determined threshold value;c) recommending that the patient does not receive an antibiotic treatment if the polypeptide concentration of HNL determined in step (a) is lower than a predetermined threshold value; ord) recommending that the patient receive an anti-viral treatment if the polypeptide concentration of HNL determined in step (a) is lower than a pre-determined threshold value.

In one embodiment, the method of providing a treatment recommendation for a subject further comprises in step a) additionally measuring the polypeptide concentration of TRAIL in a sample obtained from a subject; andb) recommending that the subject receives an antibiotic treatment if the polypeptide concentration of HNL determined in step (a) is higher than a pre-determined threshold value and if the concentration of TRAIL determined in step (a) is lower than a pre-determined threshold value;c) recommending that the patient does not receive an antibiotic treatment if the polypeptide concentration of HNL determined in step (a) is lower than a predetermined threshold value and if the polypeptide concentration of TRAIL determined in step (a) is higher than a predetermined threshold value; ord) recommending that the patient receive an anti-viral treatment if the polypeptide concentration of HNL determined in step (a) is lower than a pre-determined threshold value and if the polypeptide concentration of TRAIL determined in step (a) is higher than a predetermined threshold value.

According to another aspect, the present invention relates to a method of providing a diagnostic test recommendation for a subject comprising:a) measuring the polypeptide concentration of HNL in a sample obtained from a subject using a binding agent as defined above, or a diagnostic composition or kit as defined above; andb) recommending testing the sample for the presence of bacteria if the polypeptide concentration of HNL determined in step (a) is higher than a pre-determined threshold value; orc) recommending testing the sample for the presence of a virus if the polypeptide concentration of HNL determined in step (a) is lower than a pre-determined threshold value.

According to one embodiment, the method of providing a diagnostic test recommendation for a subject, further comprises in step a) additionally measuring the polypeptide concentration of TRAIL in a sample obtained from a subject; andb) recommending testing the sample for the presence of bacteria if the polypeptide concentration of HNL determined in step (a) is higher than a pre-determined threshold value and if the concentration of TRAIL determined in step (a) is lower than a pre-determined threshold value;c) recommending testing the sample for the presence of a virus if the polypeptide concentration of HNL determined in step (a) is lower than a predetermined threshold value and if the polypeptide concentration of TRAIL determined in step (a) is higher than a predetermined threshold value.

The present invention further provides, in another aspect, a method of ruling out an infectious disease, preferably a bacterial or viral disease, in a subject comprising:a) measuring the polypeptide concentration of HNL using a binding agent as defined above, or a diagnostic composition or kit as defined above, and the polypeptide concentration of one or more polypeptides selected from the group consisting of TRAIL, IP10, ILIRa or Mac-2BP in a sample obtained from a subject;b) applying a pre-determined mathematical function on the concentrations of the polypeptides measured to compute a scorec) comparing the score to a predetermined reference value.

In yet another aspect, the present invention relates to a method of identifying the type of infection, preferably a bacterial or viral infection, in a subject is provided, comprising:a) measuring the polypeptide concentration of HNL using a binding agent as defined above, or a diagnostic composition or kit as defined above and the levels of a first polypeptide determinant selected from the group consisting of TRAIL, ILIRA, IP 10, Mac-2BP, B2M, BCA-1, CHI3L1, Eotaxin, IL1a, MCP, CD62L, VEGFR2, CHP, CMPK2, CORO1C, EIF2AK2, ISG15, RPL22L1, RTN3, CD112, CD134, CD182, CD231, CD235A, CD335, CD337, CD45, CD49D, CD66A/C/D/E, CD73, CD84, EGFR, GPR162, HLA-A/B/C, ITGAM, NRG1, RAP1B, SELI, SPINT2, SSEA1, IgG non-specific bound molecules, IL1, I-TAC and TNFR1 in a sample obtained from a subject; andb) measuring the levels of a second determinant selected from the group consisting of(i) the polypeptide determinants TRAIL, IL1RA, IP 10, Mac-2BP, B2M, BCA-1, CHI3L1, Eotaxin, IL1a, MCP, CD62L, VEGFR2, CHP, CMPK2, CORO1C, EIF2AK2, ISG15, RPL22L1, RTN3, CD112, CD134, CD182, CD231, CD235A, CD335, CD337, CD45, CD49D, CD66A/C/D/E, CD73, CD84, EGFR, GPR162, HLA-A/B/C, ITGAM, NRG1, RAP1B, SELI, SPINT2, SSEA1, IgG nonspecific bound molecules, IL1, I-TAC and TNFR1;(ii) the polypeptide determinants IFITM3, IFIT3, EIF4B, IFIT1, LOC26010, MBOAT2, MX1, OAS2, RSAD2, ADIPOR1, CD15, CD8A, IFITM1, and IL7;(iii) the polypeptide determinants CRP, SAA, TREM-1, PCT, IL-8, TREM-1 and IL6; or(iv) the non-polypeptide determinants Age, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), neutrophil % (Neu (%)), lymphocyte % (Lym (%)), monocyte % (Mono (%)), Maximal temperature, Time from symptoms, Creatinine (Cr), Potassium (K), Pulse and Urea;c) comparing the levels of HNL, first and second determinants to a reference value thereby identifying the type of infection in the subject wherein the measurement of the first and/or second determinant increases the accuracy of the identification of the type of infection over the measurement of HNL.

In a further aspect, the present invention relates to a method of identifying the type of infection, preferably a bacterial or viral infection, in a subject comprising:a) measuring the polypeptide concentration of HNL using a binding agent as defined above, or a diagnostic composition or kit as defined above, and the levels of one or more polypeptide determinants selected from the group consisting of ABTB1, ADIPOR1, ARHGDIB, ARPC2, ATP6V0B, Clorf83, CD15, CES1, CORO1A, CSDA, EIF4B, EPSTI1, GAS 7, HERC5, IFI6, KIAA0082, IFIT1, IFIT3, IFITM1, IFITM3, LIPT1, IL7R, ISG20, LOC26010, LY6E, LRDD, LTA4H, MAN1C1, MBOAT2, NPM1, OAS2, PARP12, PARP9, QARS, RAB13, RAB31, RAC2, RPL34, PDIA6, PTEN, RSAD2, SART3, SDCBP, SMAD9, SOCS3, TRIM22, SART3, UBE2N, XAF1, ZBP1, CRP and MX1 in a sample obtained from a subject; andb) comparing the levels of HNL and of the one or more polypeptide determinants to a reference value thereby identifying the type of infection in the subject.

In one embodiment, the method of identifying the type of infection further comprises measuring one or more non-polypeptide determinants, selected from the group consisting of Age, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), neutrophil % (Neu (%)), lymphocyte % (Lym (%)), monocyte % (Mono (%)), Maximal temperature, Time from symptoms, Creatinine (Cr), Potassium (K), Pulse and Urea.

In another embodiment, the level of the determinants absolute neutrophil count (ANC) and neutrophil % (NEU(%)) are used to normalize the level of HNL.

In another embodiment of the invention methods of stratifying subjects into those having a having a bacterial disease and those not having a bacterial disease is provided comprising the above steps, and optionally comprising the step of therapeutically administering antibiotics to subjects identified as being infected by bacteria.

In some embodiments of the invention the sample is selected from the group consisting of blood (i.e. whole blood) or fractions thereof, e.g. serum, plasma, and/or urine, cerebrospinal fluid (CSF), bone marrow, saliva, and sputum.

Locations where the tests disclosed herein may be used and the methods described may be performed are intensive care units (ICU), hospitals, particularly emergency departments, neonatal departments, GP/physicians premises, pharmacies, community hospitals, and level 1 and level 2 settings.

In another embodiment the invention provides a device for the diagnosis of bacterial infections, said device comprising at least a compartment, e.g. a contact area, that comprises the herein described binding agents. The device may be a test strip with immobilized binding agents on at least (a part) of its surface, particles carrying immobilized binding agents on at least (a part) of its surface, etc. The surface may be three-dimensional, e.g. particles with a partially porous surface (layer) may carry the above binding agents. It is important that the device comprises a contact area that can be exposed to a sample that is analyzed, e.g. a sample that is suspected to contain HNL. In embodiments of the device of invention, the determination of the level of HNL is possible. In further embodiments, the device may be connected to other devices, e.g. those suitable to take a blood sample from a subject suspected to have a bacterial infection, or it may be connected to a computer or apparatus permitting to analyze and measure the interaction between the binding agents disclosed herein and HNL that is present in a sample. The interaction may be measured using a physical, chemical or biological signal that is specifically generated upon binding of HNL and the herein described binding agent(s). The interaction may produce a measurable signal, e.g. a chromogenic signal, a fluorogenic signal, a spectroscopically measurable signal, a change in ionization, a change in conductivity and the like when compared with controls, where no interaction has occurred.

In a specific embodiment, the device as mentioned above additionally comprises a binding agent for one or more of the additional polypeptide determinants mentioned above, wherein said reactions are indicative for an interaction of the polypeptide determinants, and which further optionally permits determining the level of said polypeptide determinants in said sample.

In a specific embodiment the present invention relates to method as defined herein above, which further comprise measuring the level of C-reactive protein, and/or of TRAIL, and/or of procalcitonin, and/or of CD64, and/or determining the number of white blood cells and/or determining the number of neutrophils.

In another embodiment the invention provides a method of producing an antibody comprising the step of culturing antibody-producing cells obtained from an animal previously exposed to an antigen comprising the sequence in SEQ ID NO: 26, and providing the antibodies, optionally further modifying the obtained antibodies. Preferably, the antibodies comprise a binding region comprising at least one, two, three, four, five, or six amino acid sequences depicted in SEQ ID Nos: 1 to 6, 7 to 12, and 13 to 18, or a functional derivative thereof.

The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein may be contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document.

In addition to the foregoing, the invention can include, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs herein. For example, certain aspects of the invention that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the invention. Also, aspects described as a genus or selecting a member of a genus, should be understood to embrace combinations of two or more members of the genus.

It should be understood that while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment may also be described using “consisting of or “consisting essentially of language.

